Advanced Materials - 2024 - Li - Intestinal Delivery of Probiotics Materials Strategies and Applications

Intestinal Delivery of Probiotics: Materials, Strategies, and Applications
Chengcheng Lia,*, Zi-Xi Wangb, Huining Xiaoc, Fu-Gen Wub,*
a
International Innovation Center for Forest Chemicals and Materials and Jiangsu Co-
Innovation Center for Efficient Processing and Utilization of Forest Resources, Nanjing
Forestry University, Nanjing 210037, China
b
State Key Laboratory of Digital Medical Engineering, Jiangsu Key Laboratory for
Biomaterials and Devices, School of Biological Science and Medical Engineering, Southeast
University, Nanjing 210096, China
c
Department of Chemical Engineering, University of New Brunswick, Fredericton, New
Brunswick, E3B 5A3, Canada
*Corresponding authors:
Chengcheng Li, E-mail: lichengcheng@njfu.edu.cn
Fu-Gen Wu, E-mail: wufg@seu.edu.cn
This article has been accepted for publication and undergone full peer review but has not been through the
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and the Version of Record. Please cite this article as doi: 10.1002/adma.202310174.
This article is protected by copyright. All rights reserved.

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Abstract: Probiotics with diverse and crucial functions and properties have attracted broad
interest from many researchers, who adopt intestinal delivery of probiotics to modulate the
gut microbiota. However, the major problems faced for the therapeutic applications of
probiotics are the viability and colonization of probiotics during their processing, intake, and
subsequent delivery to the gut. The challenges of simple oral delivery (stability,
controllability, targeting, etc.) have greatly limited the use of probiotics in clinical therapies.
Nanotechnology can endow the probiotics to be delivered to the intestine with improved
survival rate and increased resistance to the adverse environment. Additionally, the progress
in synthetic biology has created new opportunities for efficiently and purposefully designing
and manipulating the probiotics. In this article, we present a brief overview of the types of
probiotics for intestinal delivery, the current progress of different probiotic encapsulation
strategies, including the chemical, physical, and genetic strategies and their combinations,
and the emerging single-cell encapsulation strategies using nanocoating methods. The action
mechanisms of probiotics that are responsible for eliciting beneficial effects are also briefly
discussed. Finally, we discuss the therapeutic applications of engineered probiotics, and the
future trends toward developing engineered probiotics with advanced features and improved
health benefits.
Keywords: probiotic, intestinal delivery, encapsulation, synthetic biology, therapy
2
1. Introduction
The intestinal microbiome and the host comprise the intestinal microecosystem of the
human body with over 100 trillion bacteria in the mammalian gut.[1,2] The number of
microorganisms in the human gut is about 10 times more than the sum of human cells.[3] The
intestinal symbiotic microorganisms, including bacteria, archaea, and fungi, have attracted
much attention for their benefits to the host.[4] For example, they supply essential nutrients,
metabolize indigestible compounds and drugs, inhibit opportunistic pathogen colonization by
competing for limited nutrients or direct killing, stimulate immune responses, provide
intestinal barrier, and even contribute to the intestinal architecture.[1,5–8] The balance of
intestinal microbiome is closely related to various diseases of the human body, such as
obesity, diabetes, metabolic diseases, inflammatory bowl disease (IBD), atherosclerosis,
cancer, psychiatric disorders, and SARS-CoV‑2 infection.[9–12] The perturbation of the
intestinal flora by the endogenous and external factors like food intake, antibiotic therapy,
and pathogen invasion disturbs the microecological balance, leading to the disorder of the
host functional system.[13]
Based on the beneficial functions of the human microbiota, selecting specific microbial
species, which are recognized as probiotics with putative health-promoting capacities is
highly desirable.[14] Probiotics are live microorganisms that, when administered with
sufficient amounts, can endow the host with several health benefits.[15] They have been
reported to function in various fields such as functional foods, nutraceuticals, and
medicine.[16,17] The research about probiotics covers various fields, like microbiology, food
processing, nutriology, physiology, toxicology, medicine.[16,17] Generally, probiotics are
3
orally administered in the forms of nutraceuticals, functional foods, drugs, and dietary
supplement, which are commercially available.[16,18–20] The probiotics are expected to provide
beneficial effect to the host via regulating intestinal microecology, producing functional
metabolites, neutralizing dietary carcinogens, inducing cytokine production, and preventing
pathogens.[21] However, the stability of probiotics is seriously concerned for targeted colon
delivery when administered orally.[16] It is necessary to maintain the high viability of
probiotics during gastrointestinal delivery to promote their efficacy. Probiotics require
protection against various stress factors during processing, storage, and digestion, such as pH,
oxygen, and temperature.[22] Moreover, to achieve the health effects, probiotics must survive
in the harsh gastric environment with characteristics such as enzymatic degradation, low pH,
and the antimicrobial activity of bile salts, remain metabolically active, compete with other
bacteria, and be controllably released in a large enough quantity at the target site to confer
beneficial health effects.[21] At the same time, probiotics should effectively colonize the
intestine by attaching efficiently to the gut epithelium.[21] Microbial encapsulation is a
promising strategy for protection and efficient delivery of probiotics to their target site in an
orally administration manner.[22] The most widely used encapsulation strategy is the
microencapsulation by embedding the probiotics within a protective matrix before being
delivered, which can enhance the viability of probiotics during administration and
storage.[23,24] However, the microencapsulation strategy faces some drawbacks, such as the
leakage of probiotics and low in vivo delivery efficiency. In recent years, progress has been
made in developing alternative encapsulation strategies to cope with the existing challenges,
and nanocoating strategy for individual cell encapsulation has emerged as a viable
alternative.[25]
Probiotics have been applied in clinic, including the prevention and treatment of
gastrointestinal infections, IBD, and allergic diseases and the use as adjuvants in vaccination.
4
Probiotic or probiotic-based delivery systems have also led to a synergy of interdisciplinary
research for various biomedical applications. Although several action mechanisms of
probiotics against diseases have been illustrated,[26,27] including excluding or inhibiting
pathogens, protecting intestinal epithelial barrier,[28,29] and modulating host immune
responses,[30] a better understanding of how probiotics interact with host cells is necessary for
their optimized application in clinic.[31] Moreover, the encapsulation strategies of probiotics
should be continuously improved for enhancing their intestinal delivery efficiency and
promoting their clinical application.
Here, we will comprehensively summarize the advances in the intestinal delivery of

probiotics for the diagnosis and therapy of various diseases, including cancer, enteric disease,
immune imbalance, and infectious disease from the aspects of definition, type, action
mechanism, clinical trial, encapsulation strategy, probiotic-derived materials, therapeutic
application, as well as the synthetic biology for probiotics (Figure 1). In detail, we will first
overview the definition and types of intestinal delivery probiotics. Second, we will introduce
the various action mechanism of probiotics, including inhibiting the growth of pathogens,
protecting the intestinal epithelial barrier, and regulating the immunne system. Third, based
on the beneficial and therapeutic effects, many probiotic-associated products, including
probiotics, synbiotics, microecologics, and LBPs (Live Biotherapeutic Products) have been
put into use or undergone clinical trials. Fourth, the encapsulation of probiotics by biofilms,
lipids, emulsions, nanocoatings, cell membranes, hydrogels, films, microspheres, and
prebiotics will be summarized. Fifth, the probiotic-derived oral delivery materials, such as
probiotic spore-based materials,[32] minicells from probiotics,[33] and the dead probiotics will
also be discussed in this review. Sixth, the therapeutic applications of probiotics in
biomedicine are introduced.[34] Seventh, we also demonstrate the potential of synthetic
biology as a revolutionary technology in probiotic intestinal delivery. Finally, the current
5
challenges regarding the intestinal delivery of probiotics and future prospects in this field are
proposed. We hope that this review can have implications for the further research on
designing and developing the clinically applicable probiotics and promote their biomedical
applications.
Figure 1. Scheme illustrating the types of probiotics and probiotic-derived materials

(including probiotic spores, minicells of probiotics, and inactive probiotics), probiotic action
mechanisms, encapsulation strategies, therapeutic applications (including cancer
6
immunotherapy and infected wound healing), and synthetic biology-based modification of
probiotics.
2. Types and Encapsulation Materials of Intestinal Delivery Probiotics
2.1. Live Probiotics
Elie Metchnikoff first reported that the longevity of residents in Bulgaria was due to the
probiotics in their daily drinking yogurt rather than their diet structure. However, the
relationship between yogurt consumption and longevity has not been scientifically proven up
to now. Since the probiotic theory was put forward, the definition of probiotics has undergone
many revisions along with the increasing awareness of probiotics. Currently, the consensus
definition of probiotics is “live microorganisms that, when administered with sufficient
amounts, confer a health benefit on the host.[35] In the current consensus definition of
probiotics, the function mechanism of probiotics is not limited to “contributing to the
intestinal microbial balance” or “improving the properties of the indigenous microflora”, but
also involves other function types, which open the door to a wider range of probiotic
possibilities, encouraging innovation in the field.[36]
Many currently used probiotics are from the genera Lactobacillus[37] and
Bifidobacterium[38,39] (Table 1), which belong to the conventional probiotics.[40] In recent
years, researchers also frequently use other types of lactic acid bacteria,[41‒49] including
Lactococcus, Pediococcus, Streptococcus, and Enterococcus for achieving health-promoting
performance. Besides the conventional probiotics, some nonconventional probiotics currently
available in the marketplace include Bacillus spp.,[50‒56] Saccharomyces cerevisiae (S.
cerevisiae),[57‒59] Escherichia coli (E. coli),[60‒75] Weissella spp,[76] Clostridium butyricum (C.
7
butyricum),[77] and enterococci.[40] Some typical probiotics used for biomedical applications
include Lactobacillus rhamnosus (L. rhamnosus),[78] Lactobacillus acidophilus (L.
acidophilus),[79,80] Lactobacillus reuteri (L. reuteri),[81] Lactobacillus casei (L. casei),[82]
Lactococcus lactis (L. lactis),[83] Lactobacillus helveticus (L. helveticus),[8] Lactobacillus
plantarum (L. Plantarum),[84‒86] Lactobacillus rhamnosus (L. rhamnosus),[87] Bacillus subtilis
(B. subtilis),[88,89] and Lactobacillus paracasei subsp. Paracasei (L. paracasei subsp.
Paracasei),[90,91] Bifidobacterium breve (B. reuteri),[1] Bifidobacterium,[92–94] Streptococcus
thermophilus,[95] Pediococcus pentosaceus.[96] Although many of them are derived from the
gut of healthy individuals or traditional fermented foods, nowadays, the newly explored
resident microorganisms in human are considered by researchers as live biotherapeutics,
identified as next-generation probiotics (NGPs), such as Akkermansia,[97,98] Faecalibacterium
prausnitzii (F. prausnitzii),[99,100] and Bacteroides fragilis (B. fragilis).[101,102]
2.2. Inactivated Probiotics
Probiotics are often claimed to be effective in improving health by modulating the gut
microflora, promoting the host digestion, and relieving gastrointestinal symptoms. But
generally speaking, these probiotics are alive. How about the functionalities of the inactivated
ones? Some recent studies suggest that inactivated probiotics may also offer benefits.[103] The
inactivated probiotics possess many advantages, such as strong resistance to the gut
environment, capacity to be combined with antibiotics, ease of storage.[103] Such
microorganisms or their metabolites are called paraprobiotics.[104] A growing number of
studies have shown that postprobiotics have the favorable clinical outcomes in the treatment
of a variety of diseases. Depommier et al. found that oral administration of live and
pasteurized Akk bacteria showed similar effects to overweight and obesity, and dead bacteria
8
performed better than live bacteria.[105] In this study, oral administration of the pasteurized
inactivated Akk increased the insulin sensitivity and decreased insulinemic response and total
plasma cholesterol in patients by approximately 30% without eliciting any adverse responses.
The body weight, fat mass, and hip circumference in the patients were also decreased after
oral administration of the inactivated Akk. Moreover, neither the live nor the inactivated Akk
influenced the gut microbiome. Another study found that both live and heat-killed
Faecalibacterium prausnitzii showed an antiasthmatic effect by modulating the gut
microbiota composition and producing the SCFAs (short chain fatty acids).[106] Even heat-
killed L. paracasei D3-5 showed anti-aging effect by promoting intestinal mucin production
through its cell wall component, lipoteichoic acid, which reduces age-related intestinal
leakage and inflammation.[91] Similarly, the live and heat-killed Lactobacillus reuteri (L.
reuteri) showed similar effects on Galleria mellonella (G. mellonella). However, only live L.
reuteri significantly increased the hemocyte density in G. mellonella.[107] Although studies
about the postprobiotics are increasing, there are still some challenges to be faced and solved
for better use of the inactivated probiotics, which have been proposed by de Almada et al.[104]
More importantly, the underlying action mechanisms of the postprobiotics should be
carefully elucidated. The comparison of the postprobiotics with the live probiotics should be
performed to better understand the action mechanism of the postprobiotics. However, most of
the current researches about the postprobiotics lack the comparison with the live ones.
Although some recent researches have showed that some types of inactive probiotics
exhibited some activities, not all types of probiotics can possess specific functions after
inactivation treatment. Moreover, to retain the complete cell structure and the original
properties of the inactivated probiotics, it needs strict and special technology. So, some
probiotics that must be used in the form of alive bacteria should be encapsulated to improve
the intestinal delivery rate.[108] For example, live probiotics can enhance the immunity,
9
improve the lactose intolerance, and transfer to and colonize the gut.[109] To sum up, in
addition to having the same beneficial effect, the live and inactivated probiotics still have
different functions in promoting human health. Deep investigations about the action
mechanisms and functions of the live and inactivated probiotics, and the comparisons
between them after administration in clinical trials should be considered.
Table 1. Probiotic types, their encapsulation materials, and the applications.
Type Species Application Encapsulation Reference

material
Conventional Lactobacillus Treating colitis Alginate [80]

acidophilus microcapsules
Lactobacillus casei Enhancing both Soybean protein- [82]

probiotic viability and based microparticles
tolerance against
simulated
gastrointestinal fluids
(SGF)
Lactobacillus. Enhancing the storage Pickering high [85,88]

plantarum and gastrointestinal internal phase
passage viability emulsions (HIPEs);
Biofilms
Lactobacillus Enhancing Exfoliated

rhamnosus bentonite/alginate
resistance of probiotics [87,78]
nanocomposite
to gastric pH and
hydrogel; Hyaluronic
realizing the
acid-based hydrogel
10
on-demand release of
probiotics;
Ameliorating enteritis
Lactobacillus Wound Methacrylate- [34]

reuteri modified hyaluronic
healing
acid (HA)
Bifidobacterium Photothermal cancer Indocyanine [93]

bifidum immunotheranostics
green (ICG)
encapsulating
Cremophor EL
(CRE) nanoparticles
Bifidobacterium Protection against Protamine-assisted [92]

breve gastrointestinal fluids SiO2 nanoparticle
and antibiotics
yolk−shell packing,
alginate gelation, and
zinc-salt based
metal–organic
framework (ZIF-8)
mineralization
Streptococcus Enhancing stability and Endogenous [95]

thermophilus function production of
hyaluronan
Lactococcus lactis Improving gastric acid Thiolated oxidized [108]

resistance and adhesive konjac glucomannan
colonization (sOKGM)
microspheres
11
Pediococcus Clostridium difficile Alginate–starch [96]
pentosaceus infection (CDI) microgels
treatment
Non- Saccharomyces – Nanostructured silica [59]

conventional
matrices
Escherichia coli Treating intestinal Silk fibroin; Tannic [21,71–

mucositis; Treating acid, Eudragit 74]
gastrointestinal
L100; Dopamine
(GI) tract-related (DA), Chitosan;
diseases; Treating Nanozyme; Cell
colitis membrane
Bacillus Treating metabolic Alginate [89]
syndrome
Clostridium Regulating gut Dextran [32]

butyricum
microbiota and
suppressing colon
cancer
Next- Akkermansia Improving the survival Xanthan and gellan [86]

generation muciniphila rate gum matrix
Faecalibacterium Improving the survival Lipid [99]

prausnitzii rate
2.3. Live Biotherapeutic Products (LBPs)
Since the first LBP guideline was published in the United States in 2016, LBPs have
been developed and introduced to treat some diseases or conditions.[110] In recent years,
12
several products have already obtained related clinical results and have been approved by
Food and Drug Administration (FDA) or some relevant national institutions (Table 2). For
over several decades, LBPs have been proved to be effective and useful for various diseases.
The FDA has approved five LBPs: Rebyota (RBX2660),[111] SER-109,[112] CP101,[113]
SYNB1934,[114] and SYNB1353 (Table 2).[115] Currently, a series of probiotic-based food
products (e.g., yogurt, Actimel, Biobalance Support, and Yakult, et al.) and medical products
(Inner health, VSL#3, and Medilac-Vita, etc.) have been developed. VSL#3,[116] which
contains 3 bifidobacteria, 4 lactobacilli, and 1 Streptococcus thermophilus strain, is one of the
famous LBPs. They can help people improve the gut health and immune function. In addition
to the above-mentioned LBPs approved by FDA, many other LBPs from these companies are
currently undergoing the clinical stage.[117–124] Recently, some researchers have observed that
some probiotic products do not meet their claimed active counts, which may be due to the
inefficient encapsulation methods leading to the decreased strain viability.[125] Thus,
appropriate engineering strategies, including the physical, chemical, and genetic methods
should be considered and adopted for better clinical use and product manufacturing.
Table 2. LBPs in clinical trials.
Comp Produ Admini FD NCT number Pha Diseases/ Reference or website link
any cts stration A se condition
name appr s
oval
13
[111]
[117]
[112]
[118]

GvHD
rCDI
rCDI
rCDI
Pha
Pha
Pha
Pha
1b
se
se
se
se
3
14
NCT03244644
NCT02981316
NCT03183128
NCT04995653
Yes
Yes
/
/
stration
capsule
capsule
admini
Rectal
Oral
Oral
Oral
WST)
(RBX
2660)
(toget
(toget
SER-
SER-
Nestl
Reby
B-X)
RBX
CAR
7455
(VO
with
with
109
155
her
her
ota
e)
Rebiot
ix Inc.
peutic
Thera
Seres
s
[119]
[113]

rCDI
UC
Pha
Pha
2b
se
se
15
NCT02618187
NCT03497806
Yes
/
capsule
Oral
Oral
peutic
(toget
micro
based
CP10
spore
SER-
Nestl
biom
thera
with
287
her
(a
e)
1
e
-
peutic
Thera
Finch
s
Vedan VE30 Oral / NCT03788434 Pha rCDI https://www.vedantabio.co
ta 3 se m/clinical-trials/ve303 and
Biosci (toget 2 https://clinicaltrials.gov/se
ences, her arch?term=NCT03788434
Inc. with
CAR
B-X)
VE70 / MDROs https://www.vedantabio.co
7 infection m/pipeline/ve707
(toget
her
with
CAR
B-X)
VE20 Oral / NCT05370885 Pha UC https://www.vedantabio.co
2 capsule se m/pipeline/ve202 and
(work 2 https://clinicaltrials.gov/se
with arch?term=NCT05370885/
Johns
on)
VE80 Oral / NCT04208958 Pha Advanced https://www.vedantabio.co
0 se or m/clinical-trials/ve800 and
(toget 1/2 metastatic https://clinicaltrials.gov/se
her cancer arch?term=NCT04208958
16
with
BMS)
VE41 Oral / NCT03936998 Pha Food https://www.vedantabio.co
6 se allergies m/clinical-trials/ve416 and
1/2 https://clinicaltrials.gov/se
arch?term=NCT03936998
4D MRx Oral / NCT03637803 Pha Tumor [120]
pharm 0518 se
a 1/2
Moon MNC Oral / IN Tumor [121]
Biotec -168 D
h of
MP
Synlo SYN Oral / NCT04629170 Pha Enteral [122]
gic B880 se hyperoxal
Thera 2 1 uria
peutic
SYN Oral Yes NCT04984525/ Pha Phenylket [114]
s
B193 NCT04534842 se onuria
4 1/2
17
SYN Oral Yes NCT05462132 Pha HCU [123]
B135 se
3 1
Xbiom XBI- Oral / NCT05001360 IN GvHD [124]
e 302 capsule D
of
FD
Guang SK08 / Pha IBS https://www.zypharm.com.
zhou (B. se cn/en/products
Zhiyi fragil 3
Biotec is) (Ch
h ina)
SK08 / Pha UC https://www.zypharm.com.
(B. se cn/en/products
fragil 2
is) (Ch
ina)
SK08 / IN Tumor https://www.zypharm.com.
(B. D cn/en/products
fragil
is)
comb
ined
18
with
PD-1
SK10 / IN CID https://www.zypharm.com.
D cn/en/products
Abbreviations: CID: chemotherapy-associated diarrhea, FDA: Food and Drug

Administration, GvHD: graft-versus-host disease, HCU: homocystinuria, IBS: irritable bowel
syndrome, IND: investigational new drug, MDROs: multidrug-resistant organisms, PD-1:
programmed death-1, rCDI: recurrent Clostridium difficile infection, UC: ulcerative colitis.
2.4. Encapsulation Materials
The selection of encapsulation materials is important for improving the intestinal delivery
efficiency of the probiotics due to the adverse effect of the harsh gut environment and the
special demands of humans on the targeted release of probiotics in the intestine and the high
colonization rate. The encapsulation strategies for probiotics range from the
microencapsulation to the nanoencapsulation.[72,80] The encapsulation materials can be the
natural materials and the synthetic materials with different properties. For example,
polysaccahrides,[80] proteins,[82] lipids,[85] cell membranes,[21] and biofilms,[88] which belong
to the natural materials, and Eudragit[72] and nanozymes,[74] which belong to the synthetic
materials, can all be used to encapsulate probiotics as the protective materials (Table 1). They
can also be modified to realize the targeted release in response to pH of the GIT, or the
enzyme produced in the gut environment, such as the pH-responsive materials (Eudragit
L100, sodium alginate, Eudragit S100, etc.) and enzyme-responsive materials (chitosan).
19
Meanwhile, some materials can avoid the clearance by the immune system, such as the
biofilm and cell membrane (red blood cell membrane and yeast membrane, etc.). However,
the cell membrane-encapsulated probiotics still face the problem of limited tolerance to the
gastrointestinal environment.
2.4.1. Natural Encapsulation Materials
Polysaccahrides are commonly used encapsulation materials for probiotics. For instance,
sodium alginate is a common choice for the encapsulation of probiotics due to its low cost,
biocompatibility, and accessible, and pH-responsive property. The large content of carboxyl
groups on its molecular chain interacts with divalent cations (eg., Ca2+) to form a porous gel
via ionic crosslinking to encapsulate probiotics.[96] The carboxylate ion (‒COO‒) can be
changed to ‒COOH to form alginic acid under the acidic condition, preventing the early
release of probiotics. Pectin is another suitable encapsulation material for the targeted
delivery of probiotics via the specific enzymatic hydrolysis-targeted colon release system due
to their good adhesion property to the mucin in mucus.[126] The carboxyl groups on pectin can
interact with Ca2+ to form a gel for encapsulating probiotics, which is similar to sodium
alginate. Besides the above commonly used polysaccahride materials, hyaluronic acid (HA),
which has been approved as a new raw food material in China, Japan, and South Korea is
also commonly used to encapsulate probiotics.[127] It possesses many promising
characteristics for encapsulating probiotics, such as biocompatibility, modifiability, high
viscosity, self-cross-linking property, molecular weight tunability, and molecule
designability. Chitosan is also a promising coating material for probiotics due to its
biocompatible, biodegradable, renewable, mucoadhesive, film-forming, and modifiable
properties. In addition, chitosan can be degraded by the action of enzymes produced by gut
20
microorganisms, such as lysozyme and cellulases.[128] However, chitosan is easy to dissolve
in the acidic environments, resulting the unexpected release of probiotic in the stomach. So, it
is often combined with other materials to prevent the early release of probiotics in the
stomach and efficiently release probiotics in the colonic conditions. Moreover, due to its
antibacterial ability, chitosan is commonly used as a coating or shell for enhancing the
strength of capsules. In addition to the above mentioned polysacchride materials for probiotic
encapsulation, sOKGM, xanthan, gellan, lactic acid bacteria-derived polysaccharides, and
dextran are also used for encapsulating probiotics.[129‒132]
Proteins are also commonly used to construct an encapsulating matrix to protect

probiotics from the harsh environments. Silk fibroin (SF) is an especially promising
candidate due to its attractive features, such as biocompatibility, biodegradability, low cost,
and ease of processing. SF can realize the single probiotic encapsulation by self-assembling.
In addition to the protective effect, SF nanocoating can maintain the probiotic proliferation
ability. Moreover, SF has antiinflammatory activity, synergistically enhancing the therapeutic
effect of the encapsulated probiotics on intestinal mucositis.[71] In addition, soybean protein
isolate (SPI), soybean protein concentrate, whey protein, and whey protein concentrate
(WPC) have also been used to encapsulate probiotics.
Lipids can also be used as a coating material for encapsulating probiotics. The most
important advantage by using lipids to encapsulate probiotics is that they cannot be degraded
in the gastric fluid due to the lack of lipases in this condition, realizing a sufficient protective
effect to probiotics. Triglycerides, oils, fatty acids, waxes, and other lipid-based emulsions
can also be used as encapsulation materials.[132]
Polyphenols are natural compounds that are abundant in plants with biocompatibility,
biodegradability, and a diverse range of physiological benefits.[133] Phenolic compounds
21
containing catechol or gallic acid functional groups can form hydrogen bonds, covalent
bonds, and/or π‒π interactions with different substrates, resulting in good adhesion property.
The coordination-driven metal‒phenolic networks has found extensive uses across multiple
fields. Based on their above mentioned features, polyphenols can be used to encapsulate
ptobiotics. For example, the assembly of tannic acid and FeIII to form TA‒FeIII layer to coat
EcN. Phenolic compounds can form protective coatings on individual cells via oxidative
polymerization in the presence of probiotic cells.[134]
2.4.2. Synthetic Encapsulation Materials
In addition to the natural materials for encapsulating probiotics, there are some synthetic
materials that can also be used to coat probiotics. Eudragit is a pH-responsive material
formed by polymerization of methacrylate monomers and methacrylic acid monomers in
different ratios (e.g., Eudragit L100 (approved by FDA for pharmaceutical use) and Eudragit
S100 (approved by FDA for pharmaceutical use) at a 1:1 and 1:2 ratios of methacrylic acid
and methacrylate monomers, respectively). The protective effect of Eudragit products on
probiotics comes from the carboxyl groups, which can form carboxylate with alkali or amine
and can form a water-insoluble membrane in water or dilute acids to resit the gastric juice.
Nanozymes can be designed to mimic natural antioxidant enzymes for treating reactive
oxygen species (ROS)-related inflammation due to its high ROS-scavenging ability, high
stability in harsh environments, and excellent biocompatibility.[135] Based on these features,
Pt nanozymes were used to encapsulate EcN to yield Pt-Lipid@EcN, which can significantly
reduce the ROS level and exert a robust antiinflammatory effect.[136] In addition to the ROS-
scavenging ability of the nanozymes, other functions like the response ability to the tumor
microenvironment (TME), are also required for nanozymes to encapsulate probiotics for
22
realizing therapeutic accuracy and efficacy. The peroxidase-like (POD-like) artificial
enzymes were thus prepared by Cao et al. to modify probiotics to achieve switchable control
of probiotic therapy.[137] In addition, nanofibers fabricated from polyvinyl alcohol (PVA) and
polyethylene oxide (PEO), and some organic or inorganic nanoparticles, can also be used for
encapsulating probiotics.
3. Evaluation of Probiotics by Clinical Data Analysis
Many clinical trials have been successfully carried out using probiotics or probiotics in
combination with other drugs to treat different diseases, including diarrhea, constipation,
diabetes, irritable bowel syndrome, obesity, irritable bowel disease, colitis, Alzheimer
disease, diabetes, and Parkinson disease. Moreover, currently, there are still many probiotic-
based treatments undergoing clinical trials. Taking IBD as an example, in the clinical process,
30 studies were completed, 5 studies were active but not recruiting, and 7 studies are
terminated (Table 3). In addition, a series of probiotic-based medicines are now
commercially available in some countries (e.g., probiotics for 60+ Years Capsules, Double
Strength Probiotic Capsules, and JSHealth Vitamins Probiotic, etc., registrated as medicine in
Australia (https://www.tga.gov.au/search?keywords=probiotic&submit=Search)). In the past
few years, more and more academic and nutrient supplement companies are devoted to the
studies in the probiotic field. Some of them, like Bifodan, Bayo, Cohanson, Danone, Raman,
Nestlé, and Probi have made some investments in clinical trials. More than 1000 completed
probiotic clinical studies were registered on Clinicaltrials.gov and/or the World Health
Organization's International Clinical Trials Registry Platform (ICTRP), covering more than
700 different diseases and conditions. Among the clinical studies, 25 studies are in early
phase 1 and the numbers of studies in phases 1, 2, 3, and 4 are 75, 159, 121, and 81,
23
respectively. In addition, 789 studies are not applicable. In the clinical trials, L. rhamnosus
GG (LGG) (about 78 studies) and Bifidobacterium animalis (about 136 studies) are the most
frequently studied probiotic strains.
Table 3. Clinical trials by using probiotics for managing IBD.
NCT Phas
Study title Diseases/conditions Interventions Sponsor
Number e
A Study of
Moderate
to Severe
Inflammat
ory Bowel
Disease,
NCT048
Including IBD/UC/CD Takeda
73700
Ulcerative
Colitis
(UC) and
Crohn's
Disease
(CD)
Food
Supplemen
tation With Dietary
VSL#3 as supplemement: VSL

NCT009
a Support UC VSL#3/Dietary Pharmace NA
51548
to Standard supplemement: uticals
Pharmaceu Placebo
tical
Therapy in
24
Ulcerative
Colitis
Fondazio
ne
COVID-19 Policlinic
Dietary
Pneumonia o
NCT057 supplemement:
and Gut Inflammation Universit NA
81945 probiotics/Standar
Inflammati ario
d therapy
on Agostino
Gemelli
IRCCS
Oligofruct
ose-
Enriched
Inulin for
the Dietary Universit
NCT020
Treatment UC supplemement: y of NA
93767
of Mild to Synergy1 Alberta
Moderate
Active
Ulcerative
Colitis
Treatment
of
Ulcerative Drug: Hvidovre
NCT017 Colitis Ciprofloxacin/Diet Universit Phas
UC
72615 with ary supplemement: y e4
Ciprofloxa EcN Hospital
cin and E.
coli Nissle
25
Evaluation
of Safety, Dietary
Tolerance supplemement:
and Effects Milk with Assistanc
on the probiotic and e
NCT007 Intestinal Preterms with gestational age prebiotic Publique
NA
11633 Flora of a ranging from 30 to 35 weeks activities/Dietary -
New supplemement: Hôpitaux
Fermented Milk without de Paris
Milk for probiotic and
Preterm prebiotic activities
Infants
Effects of
Oral
Probiotic
Supplemen
tation on
Late onset neonatal
the IBSS
NCT020 sepsis/necrotizing
Clinical Probiotic/Placebo Biomed NA
73214 enterocolitis/infant, very low
Status of S.A.
birth weight
Very-Low-
Birth-
Weight
Preterm
Neonates
Clinical
Trial on
the Effects Dietary
NCT012 Bai, Julio
of Celiac disease supplemement: NA
57620 M.D.
Bifidobacte Probiotic
rium
infantis in
26
Active
Celiac
Disease
Effect of
Gut
Microbiota
and Fecal Chang
Clinical infection/microbial
NCT038 Inflammat Gung
colonization/gastroenteritis/infl
56138 ory Marker Memorial
ammatory response/probiotics
on Hospital
Childhood
Gastroente
ritis
Effect of
Yogurt
Added
with Yogurt added with
NCT011 Bifidobact bifidobacteria and Instituto Phas
IBD
73588 eria and soluble fiber Lala e3
Soluble (YBF)
Fiber on
Bowel
Function
Treatment
of Behavioral:
Ulcerative Odense
Administration of
NCT003 Colitis Universit
UC probiotic (L. NA
74725 with a y
rhamnosus and L.
Combinati Hospital
acidophilus)
on of
Lactobacill
27
us
rhamnosus
and
Lactobacill
us
acidophilu
s
Influence
of
Adiposity
King
and Other
NCT056 Observational, Saud
Factors on Obesity/metabolic syndrome
64321 case-control study Universit
the Gut
y
Microbiota
Compositi
on
A
Randomize
d
Controlled
Trial of
VSL#3 for Universit
NCT001 the Drug: Probiotic - Phas
CD/IBD y of
75292 Prevention VSL#3 e3
Alberta
of
Endoscopi
c
Recurrence
Following
Surgery for
28
Crohn's
Disease
Dietary Izmir
Kefir and supplemement: Katip
NCT039 MS/obesity/hypertension/hyper
Metabolic Kefir/Dietary Celebi NA
66846 lipidemias/insulin resistance
Syndrome supplemement: Universit
Milk y
VSL#3
Versus
Placebo in
NCT001 Maintenan Drug: Orphan Phas
CD
14465 ce of VSL#3/Placebo Australia e4
Remission
in Crohn’s
Disease
Effect of
Probiotic
Supplemen
tation on
the
Drug: Probiotic Phas
Immune Universit
NCT042 formula e
System in UC y of
23479 capsule/Drug: 2/Ph
Patients Jordan
Placebos ase3
with
Ulcerative
Colitis in
Amman,
Jordan
NCT041 Lactobacill UC chronic mild/UC chronic San

Dietary Phas
02852 us moderate Giovanni
supplemement: L. e
rhamnosus Addolora
29
GG rhamnosus GG ta 1/Ph
(ATCC ATCC 53103 Hospital ase 2
53103) in
Mild-
moderately
Active UC
Patients
Probiotic
Treatment
of
Biological:
Ulcerative
NCT035 Trichuris suis ParaTech Phas
Colitis UC chronic moderate
65939 ova/Biological: A/S e2
with
Placebo
Trichuris
Suis Ova
(TSO)
Additive
Effect of
Probiotics
(Mutaflor)
Drug: EcN Kangbuk
NCT049 in Patients Phas
UC (Mutaflor)/Drug: Samsung
69679 with e4
Placebo Hospital
Ulcerative
Colitis on
5-ASA
Treatment
Synbiotic
Treatment Drug: Synbiotic Universit
NCT003
in Crohn's CD (Synergy I/B. y of NA
05409
Disease longum) Dundee
Patients
30
Synbiotic
Treatment
Synbiotic Universit
NCT008 of
UC (Synergy1/B. y of NA
03829 Ulcerative
longum) Dundee
Colitis
Patients
Trial on
Profermin Phas
Nordisk
NCT011 and Profermin/Fresubi e
UC Rebalanc
93894 Fresubin in n 2/Ph
e A/S
Ulcerative ase 3
Colitis
Biological:
FMT in
biologically active
Ulcerative Virginia
NCT020 human fecal Phas
Colitis- UC associated pouchitis O.
49502 microbiota/Proced e2
Associated Shaffer
ure:
Pouchitis
sigmoidoscopy
Treatment
with
Lactobacill
us
rhamnosus Odense
and Behavioral:
NCT003 Universit
Lactobacill CD Administration of NA
74374 y
us probiotic
Hospital
acidophilu
s for
Patients
with
Active
31
Colonic
Crohn's
Disease.
Effect of
VSL#3 on
Dietary Children'
Intestinal
supplemement: s Mercy
NCT009 Permeabilit Phas
CD VSL#3/Dietary Hospital
44736 y in e3
supplemement: : Kansas
Pediatric
Placebo City
Crohn's
Disease
An
Evaluation
of
Probiotic
Dr.
in the Dietary
NCT037 Liyana
Clinical CRC supplemement: NA
82428 Zaharudd
Course of Probiotic/Placebo
in
Patients
with
Colorectal
Cancer
Use of Dietary
Oral supplemement:Oxa
Phas
Probiotics drop/Dietary
NCT005 Nephrolithiasis/hyperoxaluria/C Mayo e
to Reduce supplemement:
87041 D Clinic 1/Ph
Urinary Agri-King
ase 2
Oxalate Synbiotic
Excretion (AKSB)/Placebo
32
Faecal
Calprotecti
n as a
Potential
The
Non-
Royal
invasive
Wolverha
NCT027 Inflammat
IBD/CD/UC/pregnancy mpton
78464 ory Marker
Hospitals
in
NHS
Pregnancy
Trust
and
Inflammat
ory Bowel
Disease
Personalize
d
Behavioral:
“Alberta”
Alberta anti-
Diet for Universit
NCT020 inflammatory
Prevention UC y of NA
93780 diet/Behavioral:
of Relapse Alberta
Canada’s Food
in
Guide Diet
Ulcerative
Colitis
Effect of
the 1: Freeze-dried
Consumpti probiotics provided
NCT016 on of a in capsule (150 Danone
Probiotic CD NA
98970 mg); 2- excipients Research
Strain on (150 mg) in
the capsule (control)
Prevention
of Post-
33
Operative
Recurrence
in Crohn's
Disease
Interest of
Propioniba
cterium
freudenreic Rennes
Probiotics in the
NCT024 hii for the Universit
UC form of cheese NA
88954 Treatment y
portion
of Mild to Hospital
Moderate
Ulcerative
Colitis
Fecal
Transplant
Biological: Fecal Meir
NCT023 ation for Phas
IBD microbial l Medical
91012 Inflammat e1
transplantation Center
ory Bowel
Disease
Lactobacill
us
acidophilu
s and Drug: Lactobacilus Hvidovre
NCT002 Bifidobacte acidophilus & Universit Phas
rium UC
68164 Bifidobacterium y e2
animalis animalis/lactis Hospital
subsp.
lactis,
Maintenan
ce
34
Treatment
in
Ulcerative
Colitis
Probiotic
Administar
tion to
Mothers of
Preterm Tel-Aviv
NCT008 Infants to Sourasky
Necrotizing enterocolitis/sepsis Drug: Probiotics NA
35874 Prevent Medical
Necrotizin Center
g
Enterocolit
is and
Sepsis
The Role
of
Synbiotics
in
Reducing
Surgical wound Beth
Post-
NCT001 infection/cystitis/bacteremia/pn Drug: Synbiotic Israel Phas
Operative
64099 eumonia/enterocolitis and 2000 Medical e4
Infections
pseudomembranous Center
in Patients
Undergoin
g Cardiac
Surgery: A
Pilot Study
35
PRObiotic
VSL#3 for
Maintenan
ce of
Dietary
Clinical
Supplement: VSL
NCT034 and
UC VSL#3/Drug: Pharmace NA
15711 Endoscopi
Mesalamine/Drug: uticals
c
Placebo
REMission
in
Ulcerative
Colitis
Combined
Nutritional
Dietary
Therapies
supplement: A: 1 AB
NCT034 for the
UC dosis/Dietary Biotics, NA
44311 Treatment
supplement: B: 2 SA
of
dosis
Ulcerative
Colitis
Effect of Omega-3 fatty

an Anti- acids/antioxidants/
National
inflammato Cervical cancer/uterine cervical soluble
Institute
NCT039 ry Diet on neoplasm/pelvic inflammatory fiber/probiotics/lac
of NA
94055 Patients disease/radiation toxicity/diet tose
Cancerol
With modification restriction/fiber
ogía
Cervical restriction/fat
Cancer restriction
NCT048 The Dietary

CD Hvidovre NA
42149 Effects of supplement:
Universit
Bifidobact Bif195
36
erium capsules/Dietary y
Breve supplement: Hospital
Bif195 for Placebo capsules
Small
Intestinal
Crohn’s
Disease
The Effect
of VSL#3
Probiotic
Preparation
on the Bile Dietary
Charles
Acid supplement:
NCT017 Universit
Metabolis CD/UC VSL#3 (Original NA
65439 y, Czech
m in De Simone
Republic
Patients formulation)
With
Inflammat
ory Bowel
Disease
Oslo
Boosting Dietary
NCT042 Universit Phas
Biologics UC/IBD supplement:
41029 y e1
in UC Idoform Travel
Hospital
Effect of a Dietary
Probiotic supplement: Massach
NCT032 Mixture on Probiotic usetts
the Gut IBD/CD/UC NA
66484 mixture/Dietary General
Microbiom supplement: Hospital
e and Placebo
Fatigue in
37
Patients
with
Quiescent
Inflammat
ory Bowel
Disease
Data from: https://clinicaltrials.gov/
Abbreviations: CD: crohn disease, CRC: colorectal cancer, IBD: inflammatory bowel
diseases, MS: metabolic syndrome, NA: not applicable, UC: ulcerative colitis.
Although many probiotics have been proved to be effective as an adjuvant therapy for
achieving health-promoting performance, some clinical results reveal that some probiotics are
ineffective because of the different patient characteristics, the antigenic property of the
probiotics, and deficiency of prebiotics. In addition, the low survival and the poor
colonization rate may also contribute to the failure in clinical trials. Moreover, there are some
obstacles regarding the manufacture and commercialization of food products or therapeutics
containing probiotics, as well as the maintanence of the bioavailability of probiotics. For a
probiotic to become a commercial product, careful consideration should be given to how to
maintain the viability and stability of the probiotic. Moreover, the improvement of the
functions like targeting, colonization, and immunomodulation of the probiotics should be
considered. Currently, probiotics in the clinical trials or commercial products are mostly
freeze-dried powders or encapsulated in oral capsules, given to patients in a certain amount.
These methods just provide simple protective effects for probiotics, and some capsules or
powders will result in early release of probiotics after oral administration, reducing the
bioavailability of these probiotics. Therefore, engineering probiotics with some appropriate
38
methods and materials will promote the delivery efficiency as well as contribute to the
positive effect in clinical results and the commercialization of probiotic products. In recent
years, great progress has been made in engineering probiotics with different methods and
materials for efficient delivery of probiotics with the help of synthetic biology and
nanotechnology.
4. Engineered Probiotics for Intestinal Delivery
4.1. Physical Engineering of Probiotics
4.1.1. Biofilm Encapsulation
In nature, microorganisms are frequently encased in a secreted thick and dense self-
synthesized extracellular polymeric matrix mainly composed of polysaccharides, proteins,
and extracellular DNA. Such a complex microorganism-containing matrix is termed biofilm,
which can survive in different extreme conditions and combat physical and chemical
detriments such as removal by adverse environments, displacement by physical forces,
exposure to antibiotics, and the elimination by the host immune system.[138-140] Biofilm is a
double-edged sword toward global public health. For the pathogenic microorganisms,
biofilms can be inhibited or disrupted to prevent the development of antimicrobial
resistance.[141,142] However, for the probiotics, biofilm provides a new strategy for probiotic
modification due to their protection effect. Probiotic biofilm is considered to be the fourth
generation of probiotics and is recognized as the innovative probiotic encapsulation
strategy.[143] Biofilms can help the bacteria attach to a surface, prevent their removal by a
flowing fluid, and protect bacteria from the host immune system and antibiotics by
preventing the penetration.[139,140] Inspired by the excellent resistance of biofilms to harsh
39
environments, probiotics with biofilm-forming capacity or those coated with biofilms can
endow probiotics with excellent adhesion and resistance abilities in the transplanted gut
microbiota.[144,145] Alginate is an anionic polysaccharide commonly isolated from seaweed
composing of guluronic acid and mannuronic units, and it is a crucial extracellular polymeric
component of Pseudomonas aeruginosa (P. aeruginosa) biofilms.[146] Li et al. designed an
intelligent alginate-encapsulated probiotic to combine tobramycin for completely eradicating
the widely implicated bacteria in chronic wounds, such as cocultured methicillin-resistant
Staphylococcus aureus and P. aeruginosa (Figure 2a).[147] The biofilm-inspired encapsulation
of probiotics with alginate enables the coadministration of probiotics and antibiotics by
conferring temporary protection of probiotics from antibiotics. In another study, Yahav, et al.
developed a novel cultivation system that enabled the incorperation of L. plantarum NCIBM
12422 into the biofilm produced by biofilm-forming B. subtilis via increasing the production
of the extracellular matrix by B. subtilis cells (Figure 2b).[148] Results showed that the L.
plantarum cells that were encapsulated in the extracellular matrix produced by B. subtilis
showed higher survivability than the planktonic L. plantarum cells. In addition to
encapsulating probiotics with biofilm components or with the help of other biofilm-producing
bacteria, self-coating with biofilms provides a simple and effective approach to improve the
resistance and adhesion capacities of probiotics. Wang et al. developed a self-coating strategy
with biofilms produced by bacteria themselves (Figure 2c).[144] The gut probiotic B. subtilis
(BS) adopted two mutually exclusive lifestyles: biofilm formation and flagellum-mediated
swimming motility. When BS was cultured on solid minimal salts glycerol glutamate (MSgg)
plates, they formed a robust biofilm with 8.9 × 109 colony-forming units (CFUs) per gram.
The authors subsequently prepared BCBS (the individual biofilm-coated BS) by
homogenizing the biofilms. In vivo study showed that the biofilm-self-coated B. subtilis
demonstrated extraordinary GIT tolerance and mucoadhesion. The oral bioavailability of the
biofilm-coated probiotics increased by 125-fold and the intestinal colonization showed a 17-
40
fold increment than the uncoated one. Different from other encapsulation strategies that only
provide a transient coating before probiotic division, self-produced biofilms enable the
probiotics to be coated by extracellular matrix (ECM) during growth, which can provide a
durable effect for probiotic protection and mucoadhesion.
Figure 2. Biofilm or biofilm-inspired encapsulation of probiotics. a) Scheme illustrating the

action mechanism of probiotic–antibiotic coadministration in pathogen removal. Reproduced
from ref.[147] with permission from Wiley-VCH. Copyright 2018. b) Confocal microscopic
images showing the dual-species biofilm formed by co-cultuing B. subtilis (expressing GFP
(green fluorescent protein) or CFP (cyan fluorescent protein)) and L. plantarum, and the SEM
41
images showing the B. subtilis cells, the L. plantarum cells, and the mixed cells of B. subtilis
and L. plantarum that were grown in the dual-species biofilm. Reproduced from ref.[148] with
permission from Taylor & Francis. Copyright 2018. c) Design of biofilm self-coating strategy
for the delivery of gut microbiota and characterization of the bacteria after biofilm self-
coating. Reproduced from ref.[144] with permission from American Association for the
Advancement of Science. Copyright 2020.
4.1.2. Lipid Coating
Low bioavailability and insufficient retention in the GIT for probiotics that play a role in
dealing with diseases are the main problems limiting the clinical application of probiotics.
Despite many strategies that have been used for oral delivery of probiotics to ensure the
sufficient survival rate, enough colonization, and proliferation of probiotics in the GI tract,
new strategies with simple preparation and separation procedures are still demanded to
address these limitations for developing oral bacterial therapeutics.[147] Lipids are one kind of
building blocks used to construct food-grade delivery systems, and are commonly used to
encapsulate the probiotics due to their biocompatibility, biodegradability, and nutrient
properties.[149] Cao et al. used lipid membrane to coat probiotics through a simple yet highly
efficient strategy via biointerfacial supramolecular self-assembly.[150] In their study, EcN was
first vortexed with the biocompatible lipid dioleoylphosphatydic acid (DOPA) and
cholesterol in calcium phosphate buffer (Figure 3a). After 15 min, the lipid membrane was
coated on the negatively charged bacterial surface. The lipid-coated EcN exhibited
significantly improved survival rate and nearly unchanged viability and bioactivity in the
harsh environmental conditions, including strong acid, base, antibiotics, and ethanol, thus
exhibiting the enhanced efficiencies in oral delivery and colitis therapy. Such a simple yet
42
highly efficient strategy has been demonstrated to be applicable for other probiotics like B.
subtilis. Moreover, in Salmonella typhimurium (STm)-induced colitis mouse model, the ratio
of EcN/STm in the feces of the mice coadministrated with lipid membrane-coated bacteria
(LCB) was 25 to 8000 times higher than that in the feces of the mice treated with uncoated
EcN. This study provides a facile and efficacious biointerfacial supramolecular self-assembly
strategy for generating lipid-coated probiotics to largely enhance the survival rate of
probiotics without influencing their viability and bioactivity, which may be important for
treating intestine- and metabolism-related diseases.
Moreover, lipids can also be used to develop probiotic delivery systems by forming
emulsions, such as oil-in-water (O/W), water-in-oil-in-water (W1/O/W2), or water-in-oil
(W/O) emulsions. Qin et al. developed WPI (whey protein isolate)/EGCG ((−)-
epigallocatechin-3-gallate) covalent conjugates to stabilize Pickering high internal phase
emulsions (HIPEs) for encapsulating the probiotic (L. plantarum) powder (Figure 3b).[85]
Encapsulation of L. plantarum powder with the HIPEs could increase the viable cell number
after 14 days of storage and GIT digestion. In addition to HIPEs, double emulsion was also
used to encapsulate probiotics. A pH-sensitive W1/O/W2 double emulsion carrier based on
an alginate-Ca-EDTA (ethylene diamine tetraacetic acid) system was used for colon-targeted
delivery of L. plantarum.[151] In another study, Ding et al. developed the W1/O/W2 double
emulsions, which were encapsulated by Ca-alginate hydrogel beads (ACGs) for intestine-
targeted delivery of Lactobacillus reuteri (Figure 3c).[152] The probiotics encapsulated in the
emulsions showed a sustained release in simulated intestinal fluid (SIF) and a high bacterial
viability.
43
Figure 3. Lipid-coated probiotics. a) Scheme illustrating the preparation of the lipid

membrane-encapsulated EcN. Reproduced from ref.[150] with permission from Nature
Publishing Group. Copyright 2019. b) Encapsulation of L. plantarum with pickering HIPEs.
Reproduced from ref.[85] with permission from Elsevier. Copyright 2021. c) Scheme
44
illustrating the preparation of carboxymethyl konjac glucomannan-chitosan nanogel-
stabilized W1/O/W2 double emulsions loaded with probiotics and the coating process of the
double emulsions by alginate hydrogel beads. Reproduced from ref.[152] with permission from
Elsevier. Copyright 2022.
4.1.3. Nanocoating
Combining probiotics and other therapeutic agents is highly needed to develop oral
microecologics with improved survival rate and therapeutic effect. Therapeutic nanocoating
is a versatile strategy to coat probiotics for enhanced oral delivery and therapy.[129] Inspired
by the strong protective effect and therapeutic effect of silkworm cocoons, Hou et al. used
silk fibroin as a therapeutic nanocoating agent to coat EcN by a layer-by-layer deposition
method to protect EcN from intraluminal insults of the GIT, and afford a synergistic
antiinflammatory outcome (Figure 4a).[71] In this system, silk fibroin self-assembled onto the
surface of EcN by transforming a random coil to β-sheet conformation to form core/shell
structure. After coating, the survival rate of EcN increased by ~52-fold in simulated gastric
fluid (SGF) and the colonization of the coated EcN in mouse intestinal tract showed 5.8-fold
increment compared with the uncoated EcN. Moreover, this strategy also exhibited a
synergistically enhanced therapeutic effect in treating intestinal mucositis. This work
represents a typical example that fully demonstrates the combination of the therapeutic silk
fibroin and probiotics through a simple coating strategy, thus providing a new alternative for
improving the bioavailability and therapeutic efficacy of oral microecologics.
However, the application of probiotics for therapeutic purposes often suffers from
severe side effects arising from their living characteristics, such as translocation into other
tissues during intestinal delivery. For example, translocation of intestinal symbiotic bacteria
45
may destroy the intestinal epithelial barrier and cause bacterial infection. Thus, to avoid
potential side effects, it is of great importance to tailor probiotics to perform precisely in the
right place at the right time. Feng et al. described an intelligent nanocoating strategy that can
allow on-demand bacterial reactivation by responding to gastrointestinal environments
(Figure 4b).[153] The authors used an anionic copolymer, enteric polymer Eudragit L100-55
(dissolved at pH > 5.5) to decorate EcN by a three-step method. EcN coated with L100-55
(EcN-L) was deactivated and could arrive at the intestine without inducing gastric attacks
after oral administration. In the intestine, EcN-L could respond to gastrointestinal
environments and be reactivated by a pH-triggered decoating to perform its precise bacteria-
mediated biofunctions. In the Salmonella-infected intestinal inflammation animal model,
when treated with EcN-L, the relative abundances of some essential beneficial bacteria were
significantly increased, and the colonization of pathogenic microorganisms in the infected
intestine was efficiently decreased. This strategy of targeted delivery of bacteria provides an
important means for the preparation of bacteria-mediated intelligent microecologics.
However, in most clinical cases, antibiotics targeting pathogens are often combined with
probiotics for treating gut disorders. The killing action of antibiotics toward microorganisms
is nonspecific, resulting in the elimination of beneficial gut microbiota as well as pathogens.
The dramatically reduced beneficial gut flora leads to intestinal microecological disorder,
which can be connected with many diseases, including allergic reactions, inflammation, AAD
(antibiotic-associated diarrhea), obesity, neurological disorders, and even stress or anxiety.
To avoid the consequences brought by the antibiotics, a facile, safe, and effective coating
strategy with broad-spectrum protection effect from antibiotics should be designed. Pan et al.
designed a single-cell coating by using tannic acid (TA) and ferric ion (FeIII), protecting
bacteria from the effect of antibiotics (Figure 4c‒e).[154] To prepare the armored probiotics,
EcN, Lactobacillus casei (L. casei) ATCC393T, or CVS Health Probiotic Capsule (CVS
HPC) suspended in PBS solutions were mixed with FeCl3•6H2O and tannic acid solutions,
46
respectively. The formed nanoarmor protected probiotics from six clinically relevant
antibiotics via the multiple interactions between the FeIII-TA nanoarmor and antibiotics for
enhancing the survival rate of probiotics in the gastrointestinal tracts of patients administered
with antibiotics, producing a long-time microenvironment with a low antibiotic concentration
around the probiotics and preventing probiotics from being harmed. This protective effect
persists even after the probiotics divided and broke through the protective shell. The freeze-
dried armored probiotics could recover quickly under the proper culture conditions. The
authors encapsulated the armored probiotics in enteric-soluble capsules to avoid the damage
of stomach acid, and to realize the successful delivery of probiotics to the intestine and
colonization in the intestinal environment. The animal experiments showed that the armored
probiotics could successfully colonize the intestinal tract of AAD rats despite the continuous
use of antibiotics, and significantly reduce AAD resulting from the treatment of levofloxacin.
Meanwhile, the polyphenols also displayed antiinflammatory and antioxidant effects. This
strategy by using nanoarmor for coating probiotics represents a robust platform to improve
the potency of therapeutic bacteria in the GIT of a patient receiving antibiotics and to avoid
the negative effects of antibiotics in the GIT. Recently, engineering functional systems with
the biointerfaces of living cells is an emerging strategy, Centurion et al. developed a living
cell-mediated catalytic process for phenolic oxidation, inducing the formation of protective
nanoshells on individual probiotic cells (Figure 4f‒h).[155] In this process, when catechol-
containing compounds including DA and plant polyphenols such as pyrocatechol (PC) and
caffeic acid (CA) were separately exposed to probiotics L. helveticus PABTM-LZ-R-5 (LH),
(L. rhamnosus PABTM-LRGG (LR), and L. plantarum PABTM-Lp3a (LP)) in mild alkaline
conditions, the polyphenols were oxidized to quinone by manganese (an essential nutrient) of
the probiotic cells, and then assembled on the individual probiotic surfaces to form phenolic
coatings. The phenolic coatings of probiotic cells were in a nanometer scale, showing
excellent biodegradability and biocompatibility. The encapsulated probiotics displayed an
47
improved gastric tolerance and enhanced adhesion onto the intestinal epithelial cells. Finally,
the coated probiotics exhibited a high antioxidant activity, which reduced the risk of diseases
by decreasing the oxidative stress. This work provides a unique strategy for improving the
probiotic delivery using the cell-mediated assembly of phenolic polymers for probiotic
nanoencapsulation with protective effect and additional functions.
Figure 4. Nanocoating of probiotics. a) Scheme depicting the decoration of bacteria with a

silk fibroin by biointerfacial self-assembly for synergistically enhanced biotherapy.
48
Reproduced from ref.[71] with permission from Elsevier. Copyright 2021. with permission
from Wiley-VCH. Copyright 2021. b) A triggerable nanocoating of EcN. b: Reproduced from
ref.[153] with permission from Elsevier. Copyright 2021. c) Natural polyphenol-based
nanoarmor (single-cell coating) for protecting probiotics from antibiotics in the GIT. d)
Confocal images of armored EcN. e) TEM images of naïve or armored EcN, L. casei, and
CVS HPC. c‒e) Reproduced from ref.[154] with permission from Nature Publishing Group.
Copyright 2022. f) Structures of the phenolic precursors. g) Probiotic cell-mediated oxidation
and polymerization of catechol compounds for in situ nanoencapsulation of probiotics. h)
Probiotic bacteria pathway, including enhanced gastric acid resistance, enhanced adhesion,
and antioxidant properties. f‒h) Reproduced from ref.[155] with permission from from Wiley-
VCH. Copyright 2022.
4.1.4. Cell Membrane Coating of Probiotics
Owing to the challenges faced by synthetic functionalization strategies, such as

replicating the collective functions of biological systems and overcoming their
foreignness,[156] cell membrane coating has emerged as a promising probiotic delivery system
since 2011.[157] Cell membrane is composed of a mixture of lipids, proteins, and
carbohydrates, and it has been recently used in probiotic coating.[158,159] Cao et al. reported a
strategy for generating stealth bacteria through camouflaging with cell membrane, which
were termed cell membrane-coated bacteria (CMCB) by simply extruding erythrocyte
membranes with bacteria (Figure 5a,b).[158] In this coating process, the probiotic EcN
expressing mCherry (red) was coated with erythrocyte membrane. Since erythrocyte
membrane has low immunogenicity, long circulating property, and antiphagocytic nature, the
CMCB displayed decreased body clearance, lowered inflammatory reaction, and therefore
49
fewer side effects. In addition, the authors revealed that the growth and bioactivity of the
coated bacteria were not significantly influenced (Figure 5b). The in vivo experiments
confirmed that the cell membrane-coated EcN exhibited improved blood reservation and
biocompatibility. This study also demonstrated the potential of CMCB (FITC-affinity anti-
CD47-antibody labeling) to act as efficient tumor imaging agents. Besides EcN, other
bacteria with different shapes including spherical Staphylococcus aureus (S. aureus) and
elliptic Enterococcus faecalis (E. faecalis) could be simply coated with erythrocyte
membranes. Based on its unique characteristics, like simple operation, wide applicability,
fewer side effects, higher blood reservation, etc., such a bacterial coating strategy provides an
efficient tool for realizing bacteria-based diagnosis, bioimaging, and therapy applications in
various biomedical fields. In another study, Lin et al. used physical coextrusion to achieve
individually camouflaged EcN coated with a yeast membrane (YM) to increase the
accumulation of probiotics in Peyer’s patches (PPs) following oral administration (Figure
5c,d).[159] Specifically, the authors mechanically squeezed yeast cells with glass beads to
prevent the potential degradation of β-glucan to prepare YMs. Then, EcN was fused with
YMs by physical extrusion to generate YM-coated EcN (EcN@YM). Owing to the presence
of embedded β-glucan on YMs, after oral administration, EcN@YM could be absorbed by
microfold cells (M cells) located in the intestinal epithelium through dectin-1 receptor-
mediated phagocytosis. The in vivo experiments showed that EcN@YM could maintain the
homeostasis of the gut microbiota when encountering environmental insults. This work
develops a new approach to maintain the composition and function of the gut flora under
external stimuli and developing innovative bacteria-mediated therapeutics for treatment of
diseases.
50
Figure 5. Cell membrane coating of probiotics. a) Schematic illustration of the fabrication of
CMCB by extruding bacteria with erythrocyte membranes. b) TEM images of the uncoated
bacteria and CMCB (scale bar, 1 μm). a,b): Reprinted from Ref.[158] with permission from
Nature Publishing Group. Copyright 2019. c) Scheme illustrating the encapsulation of
probiotics by YMs (EcN@YM) via an extrusion method. d) Scheme showing that EcN@YM
enhanced the resistance of EcN against gastric fluid and the delivery of living EcN into PPs
51
through M cells and promoted robust mucosal immune responses for maintaining the gut
homeostasis. c,d) Reprinted from Ref.[159] with permission from American Association for the
Advancement of Science, Copyright 2021.
4.1.5. Hydrogel Encapsulation of Probiotics
Hydrogels are soft, three-dimensional networks, which can trap a large amount of
water.[160] They can be generally formed via both covalent crosslinking of polymers and
noncovalent interactions such as electrostatic interaction and hydrogen bonding between
polymers.[161] The hydrogels possess many excellent biological properties and functionalities
depending on the structures and properties of the building blocks they are composed of, such
as tunable fluidity and stiffness, stimuli-responsive degradation, and the interactions between
them, such as surface adhesion, and self-healing property, which facilitate their applications
as substrates for cell culture, templates for tissue engineering, and effective vectors for drug
and cell delivery.[162,163] Recently, hydrogels are considered as a safe and effective strategy to
load probiotics for disease treatments.[164–166] As a naturally derived linear polysaccharide,
alginate has been extensively investigated and applied for probiotic encapsulation due to its
mild gelling conditions upon contact with divalent cations like calcium chloride.[144] Zhang et
al. reported a sodium alginate (SA)/cellulose nanofiber (CNF) gel macrosphere (ACM) for
encapsulation of L. plantarum (Figure 6a).[167] In this study, 2 wt% SA solution and 2 wt%
2,2,6,6,-tetramethyl-1-piperidinyloxy radical (TEMPO)-oxidized CNF suspension in different
ratios were mixed and L. plantarum was added into the mixtures. Then the mixtures were
extruded into a CaCl2 solution (0.2 M) with a syringe to form pH-responsive gel
macrospheres containing L. plantarum. The obtained ACMs showed excellent stability in
SGF, which can protect the encapsulated L. plantarum in acidic environment. When the
52
ACMs were treated with the neutral SIF, suitable composition ratios of the ACMs were
dissolved, and L. plantarum was released. In addition to the resistance to the extreme
environmental conditions, such as extreme temperature, acidity, or alkalinity, etc., the
therapeutic effect of the local administration of living bacteria is often limited by several
other challenges, such as the limited space and environment for bacterial growth, the
diffusion of some genetically modified bacteria, and the lack of extracellular matrix-
mimicking environment for providing efficient growth factors for probiotic growth.[75] To
solve these problems, Lu et al. prepared a novel thermoresponsive heparin-poloxamer (HP)
hydrogel for loading living L. lactis to modulate the wound microenvironment and promote
the angiogenesis in a highly dynamic and temporal manner.[75] In this study, L. lactis NZ9000
was genetically modified by transforming a plasmid encoding VEGF (vascular endothelial
growth factor) under an inducible promoter PnisZ. Then, a hybrid living hydrogel
(HP@LL_VEGF) was fabricated by incorporating HP and engineered L. lactis that secreted
VEGF. The synthesized HP@LL_VEGF hydrogel could effectively regulate the wound
microenvironment by simultaneously and steadily releasing a higher amount of VEGF to
promote the angiogenesis in diabetic wounds.
In addition to VEGF playing a vital role in diabetic wound healing, NO plays a crucial
role in regulating the oxidative stress and intestinal inflammation after burn injury. Thus,
designing formulations with targeted NO response may be an effective strategy for
preventing the mucosal damage and improving the outcome of IBD.[164] Based on this feature
of NO, Wang et al. developed a kind of NO-responsive hydrogel microsphere loaded with
probiotics for treating IBD (Figure 6b).[164] In this study, γ-PGA (poly-γ-glutamic acid) was
first reacted with GMA (glycidyl methacrylate) to form γ-PGA-GMA under pH 4.5. Then, the
NO-responsive crosslinking agent APD (N,N-(2-amino-1,4-phenylene)diacrylamide) was
obtained by the reaction between acryloyl chloride and 2-nitrobenzene-1,4-diamine in
53
tetrahydrofuran (THF) and N,N-diisopropylethylamine (DIPEA) at 0 °C. Afterwards, γ-PGA-
GMA and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator were

dissolved thoroughly in APD solution and then exposed to visible light irradiation with a
wavelength of 405 nm for 30 s to obtain the NO-responsive γ-PGA hydrogel microcapsules
(NRPM). Finally, L. acidophilus was encapsulated into the hydrogel microspheres via droplet
microfluidics with the polydimethylsiloxane (PMX-200) as the continuous phase and 5%
DOWSIL RSN-0749 as the emulsifier. After encapsulation of L. acidophilus with the γ-PGA
microgel, most of the probiotics survived successfully with a high survival rate of 89.67%
and 93.67% after treatment with artificial SGF and SIF for 3 h, respectively. However, the
survival rate of free cells significantly reduced to 0% and 61.60% after a 3 h exposure to SGF
and SIF, respectively. Moreover, the hydrogel microspheres could respond to NO and
controllably release probiotics to sustain the intestinal mechanical barrier and regulate the
intestinal flora balance. The above results demonstrate that NRPM (NO-responsive γ-PGA
hydrogel microcapsule) is a promising approach to improve the efficiency of orally
administered probiotics in treating colonic IBD.
In another study, Mei et al. fabricated a L. rhamnosus-loaded hyaluronate-adipic

dihydrazide/aldehyde-terminated Pluronic F127/fucoidan hydrogel (HPF@L.rha) for
improving the antibacterial effect of the fabricated hydrogel to promote the healing of
superbacteria-infected wounds (Figure 6c).[166] In this research, HA was first modified with
adipic dihydrazide, obtaining the hyaluronate-adipic dihydrazide (HA-ADH), and then HA-
ADH interacted with PF127-CHO through the Schiff-base reaction to obtain the HPF@L.rha
hydrogel. Finally, L. rhamnosus and sulfated polysaccharide fucoidan (FD) were
incorporated into the HA-ADH solution to obtain HPF@L.rha. The developed HPF@L.rha
exhibited a decreased gelation time, improved mechanical strength, and remarkable self-
healing and liquid-absorption capacities. More importantly, their antibacterial effect against
54
P. aeruginosa was greatly increased in a dose-dependent manner. Additionally, in vitro
evaluation showed that the prepared HPF@L.rha with appropriate probiotic concentrations
possessed satisfactory blood compatibility and cytocompatibility. Further, the hydrogel
incorporated with probiotics remarkably inhibited P. aeruginosa infection and inflammation
in vivo compared with the HPF hydrogel, promoting the superbacteria-infected wound repair,
showing that the hydrogel combined with probiotics was comparable to commercial
Prontosan gel. This work provides an efficient bioinspired platform that combines the
biomimicking hydrogel and probiotic for superbug-infected wound management.
55
Figure 6. Hydrogel encapsulation of probiotics. a) Schematic illustration of intestinal

delivery of probiotics using a pH-responsive gel macrosphere. Reprinted from Ref.[167] with
permission from American Chemical Society. Copyright 2018. b) Scheme showing the
production process and design concept of the proposed intestine-targeted NRPM. Reprinted
from Ref.[164] with permission from from Wiley-VCH. Copyright 2022. c) Preparation of the
56
injectable, self-healing, and probiotics-loaded hydrogel to promote superbacteria-infected
wound healing. Reprinted from Ref.[166] with permission from American Chemical Society.
Copyright 2022.
4.1.6. Layer-by-Layer Encapsulation
In recent years, owing to the rapid development of assembly techniques, layer by-layer
(LbL) self-assembly strategy has emerged as one promising alternative approach for probiotic
encapsulation due to its simple manipulation but high versatility and tunability.[168] Based on
this strategy, multilayer shells with controllable compositions and structures can be fabricated
to meet the needs of a variety of biomedical applications. For multilayer encapsulation, a
suitable coating thickness is needed to ensure a negligible impact on the motility and
bioactivity of decorated probiotics. The coating thickness can be controlled by the layer
number of a polyelectrolyte multilayer shell and the LbL approach realizes the encapsulation
of living probiotics by using a minimal quantity of polymers.[169] Anselmo et al. successfully
encapsulated the probiotic B. coagulans (BC) using two biodegradable polysaccharides,
cationic chitosan and anionic alginate via the LbL self-assembly method (Figure 7a).[170] In
this system, chitosan (CHI) and alginate (ALG) with the opposite charges were alternately
coated on a BC cell via electrostatic interactions for up to three bilayers to obtain the
individually encapsulated BC cells. Anselmo et al. further proved that the LBL encapsulation
can lead to probiotic aggregation, which is beneficial for their intestinal colonization and
growth (Figure 7b).[170]
In another study, Feng et al. reported a double-layered vehicle for encapsulating

probiotics (Figure 7c).[171] In this system, the pH-responsive polymer, sodium alginate (SA)
was selected as the wall material for probiotic encapsulation via the one-step coaxial
57
electrospinning procedure. The L. plantarum cell pellet suspension and PVA solutions served
as the core components, and the SA and PVA solutions served as the shell solution. The core
and shell solutions were electrospinned to obtain the electrospun fiber containing L.
plantarum. The SEM (scanning electron microscopy) result showed that a beaded fiber
morphology with increased width was observed for the electrospun fiber by adding probiotic
cells into the spinning solution, leading to local widening of the electrospun fiber (Figure 7d)
and exhibiting an obvious core–shell structure (Figure 7e). In addition, the encapsulation
with core–shell electrospun fiber enhanced the tolerance of L. plantarum to simulated
gastrointestinal conditions. Furthermore, the encapsulated L. plantarum cells showed
improved thermal stability compared with the free cells. This double-layered vehicle not only
provides an alternative approach for encapsulating probiotics, but further promotes the
application of electrospinning technique in the biomedical field. In addition, Liu reported a
double-layer coating strategy that adopted tannic acid (TA) and enteric L100 for EcN
encapsulation.[72] The encapsulated EcN showed robust resistance against the harsh external
environment of the GIT, and the strong mucoadhesive capability of the TA layer improved
their intesitinal retention time, resulting in considerable therapeutic effect against colitis.
58
59
Figure 7. Multilayer encapsulation of probiotics. a) Scheme depicting the layer-by-layer
(LbL) encapsulation of B. coagulans (BC) using chitosan and alginate as coating agents. b)
Brightfield images and SEM images of (i,iii) uncoated BC and (ii,iv) LbL-(CHI/ALG)2-BC.
a,b) Reprinted from Ref.[170] with permission from Wiley-VCH. Copyright 2016. c) Scheme
showing the preparation of double-layered encapsulation of L. plantarum with electrospun

fiber. d,e) SEM and TEM images of the SA/PVA/L. plantarum fiber mat prepared via the
method shown in (c). c‒e) Reprinted from Ref.[171] with permission from Elsevier. Copyright
2020.
4.1.7. Film Encapsulation
Owing to the serious negative effect of antibiotic resistance on human health, new
antibiotic-free methods are urgently needed to treat and prevent bacterial infections.[172]
Nowadays, probiotics have been widely investigated and have shown high effectiveness in
many different fields, including treatment of chronic diseases such as inflammatory bowel
disease (IBD) through modulating the gut microbiota.[173] Some kinds of probiotics like
Lactobacilli strains have already become a promising alternative way to treat bacterial
infection especially the antibiotic-resistant micoorganisms due to their antimicrobial and
antibiofilm activities and the ability to accelerate the healing process.[174,175] Based on these
properties of probiotics, Sabio et al. prepared a probiotic cellulose for treating severe skin
infections and chronic wounds.[172] In this work, the authors first cocultured the Acetobacter
xylinum (Ax) suspension and the probiotic (Lactobacillus fermentum (Lf) or Lactobacillus
gasseri (Lg)) suspensions at a volume ratio of 1:1 in 1 mL of Hestrin-Schramm (HS) medium
and aerobic condition at 30 °C. The thick cellulose film containing Ax and the probiotics (Lf
or Lg) obtained after 3 days of culture was referred to as bacterial cellulose (BC) in this work.
60
At this time, the probiotics (Lf or Lg) were located at the bottom of the BC film because that
anaerobic condition was optimal for facultative anaerobic probiotics (Figure 8a, b).
Afterwards, HS medium was replaced by Man-Rogosa-Sharp (MRS) (5 mL) and BC was
anaerobically incubated at 37 °C for 48 h. The MRS medium was replaced every 24 h. After
48 h of culturing in MRS medium, probiotic-cellulose (Lf- or Lg-cellulose) complexes were
obtained. Microscopic observation showed that the probiotics extensively grew and invaded
the entire cellulose matrix with the increased incubation time in the anaerobic condition
(Figure 8a, b). Importantly, most entrapped probiotics were alive, and they migrated and
colonized throughout the entire cellulose matrix after 48 h. The in vitro antibacterial
experiments showed that both Lf- and Lg-cellulose produced inhibition zones for both S.
aureus (SA) and P. aeruginosa (PA). However, BC+Lf or BC+Lg (BC-based materials
prepared by an adsorption-incubation method) did not inhibit the growth of the pathogens to
the same extent as Lf- and Lg-cellulose performed. Moreover, the growth of MRSA was
inhibited by both Lf- and Lg-cellulose. Although BC+Lf and BC+Lg also showed some
inhibitory effects, the inhibition zones were smaller than those found for Lf- and Lg-cellulose.
In summary, fabricating the probiotic cellulose is a simple method for developing a new
antibiotic-free antibacterial biomaterial, which shows excellent antibacterial activities against
the two most active pathogens (i.e., SA and PA) in severe skin infections and chronic wounds.
Furthermore, the probiotic celluloses (Lf- and Lg-cellulose) were found to be effective in
inhibiting the proliferation of methicillin-resistant SA (MRSA). At the same time, the wound
healing ability of BC makes probiotic cellulose an alternative to antibiotics for the treatment
of topical infections, including severe and hard-to-heal pathogen-infected chronic wounds. In
addition, novel cellulose acetate (CA) and poly(vinyl alcohol) (PVA) hybrid fibers were also
used to encapsulate the probiotics via angled dual-nozzle electrospinning (Figure 8c).[176]
Here, CA enhanced the probiotic stability under the gastric condition and PVA protected
probiotics from the toxic solvent during the electrospinning process. In the simulated
61
digestive system, the survival rate of free EcN cells decreased to 0 within 100 min, whereas
EcN encapsulated in PVA/CA film survived with a final count of 3.9 log CFU/mL (from an
initial count of 7.8 log CFU/mL). In another study, Feng et al. encapsulated L. plantarum
with nanofibers prepared by electrospinning fructooligosaccharides (FOS) and PVA.[177] The
L. plantarum cells encapsulated in the FOS/PVA/L. plantarum nanofilm showed a high
viability and was reduced by only 0.04‒2.71 log CFU/mL compared with the initial probiotic
count. When the treated temperature increased to 70 °C, the number of probiotic was still
above 6 log CFU/mL; however, the number of free probiotics was significantly reduced. This
study provides an effective strategy for encapsulating probiotics using fibers to protect the
probiotics under the gastric condition.
Figure 8. Film encapsulation of probiotics. a) Graphical illustration of the preparation of the

probiotic cellulose. b) Confocal fluorescence images of BC cocultured with Acetobacter
xylinum (Ax) and Lactobacillus fermentum (Lf) under aerobic and anaerobic conditions. The
bacteria in A‒D include fibrous Ax and short bacilliform Lf; the bacteria in G‒J include only
the shorter bacilliform Lf bacteria. a,b) Reprinted from Ref.[172] with permission from
62
Elsevier. Copyright 2021. c) Probiotics encapsulated within CA and PVA hybrid fibers.
Reprinted from Ref.[176] with permission from Elsevier. Copyright 2021.
4.1.8. Microsphere Encapsulation (Microencapsulation)
Microencapsulation is an effective method for protecting probiotics from the gastric acid
and intestinal bile salt environments. Liu et al. designed a thiolated oxidized konjac
glucomannan (sOKGM) microsphere with pH responsive and mucoadhesive properties
(Figure 9a‒c).[108] First, the OKGM polymer was prepared by TEMPO oxidation of the
konjac glucomannan (KGM) polymer, and then cysteine molecules were conjugated to the
carboxyl groups on OKGM through amide formation to yield sOKGM (Figure 9a). Then, the
sOKGM microsphere was prepared by the double crosslinking via the disulfide bond
formation and carboxyl‒Fe3+ coordination using an inverse emulsion method, and the
probiotics, L. lactis NZ9000 (NZ9000) with red fluorescence was encapsulated during the
crosslinking process (Figure 9b,c). The survival rate of NZ9000 after being encapsulated
with sOKGM microsphere was increased in SGF compared with the zero-survival rate of
naked probiotics. Moreover, the mucoadhesion and colonization of probiotics at the intestine
were also enhanced. This work provides an effective probiotic oral delivery system for
improving the mucus-adhesion property and promoting the release of probiotics from the
sOKGM microspheres when exposed to the intestinal pH condition, suggesting the potential
of the sOKGM microsphere as a mucoadhesive delivery vehicle.
In another work, sporopollenin exine capsules (SECs) were also used to encapsulate
pobiotics (Figure 9d).[178] Here, the authors used silica particles as pressing microprobes to
open the SECs, and then L. casei cells were loaded into the opened SECs by ultrasound bath
(60 W) for 30 s. Then, the encapsulated L. casei would generate enough pressure through
63
proliferation, leading to the burst and release of the active cells. In all, this study provides not
only an encapsulation method for improving the survival rate of probiotics in harsh
conditions, but also a bioreactor for probiotic to efficiently proliferate before being released,
following the SEC’s burst.
It is known that the symbiotic relationship between probiotics and prebiotics can
promote the growth of probiotics. Encapsulation of more than one type of probiotics with
prebiotics may be more efficient towards treating the intesitinal diseases.[179,180] Chen et al.
constructed living materials with dual probiotic system, including Lactobacillus rhamnosus
GG (LGG) and Lactobacillus delbrueckii subsp. bulgaricus (LDB) for effective management
of cholestatic drug-induced liver injury (DILI) (Figure 9e).[181] The outer enteric polymer
coated on the constructed living materials showed pH-triggered dissolution property,
resulting in the release of LGG, which could decrease hepatic bile acid synthesis through
activating intestinal FXR-FGF-15 signaling pathway, and promote BA excretion via
enhancing the richness of BSH (bile salt hydrolase)-active gut commensal microbes. This
dual probiotics system exhibits great potential for treating cholestatic DILI.
64
Figure 9. Microsphere encapsulation of probiotics. a) Design of the sOKGM oral carrier with
mucoadhesive property for probiotics. b) SEM photographs of the frozen sOKGM
microspheres. c) Confocal microscopic images of the three-dimensional (3D) distribution of
NZ9000 (red) in the sOKGM microsphere (bright field). a‒c) Reprinted from Ref.[108] with
permission from Wiley-VCH. Copyright 2020. d) SEM images of SECs obtained for each
tableting technique. ⅰ) directly compressed SECs, ⅱ) mixture of SECs and the untreated
pollen shells at the ratio of 1:1, ⅲ) mixture of SECs and the spherical silica particles.
Reprinted from Ref. [178] with permission from Wiley-VCH. Copyright 2021. e) Scheme
depicting the fabrication process of dual probiotic system by hierarchically assembling LDB
and LGG into Ca2+-coordinated polymer microspheres. Reprinted from Ref.[181] with
permission from Wiley-VCH. Copyright 2022.
65
4.1.9. Prebiotic Encapsulation of Probiotics
Prebiotics have been suggested to be able to enhance the beneficial effect of the oral
administration of live probiotics towards various diseases including certain infectious
disorders, IBDs, diarrheal illnesses, and irritable bowel syndrome.[179] Designing strategies to
apply the synergistic effect of prebiotics and probiotics is a promising way to treat
metabolism-related diseases. Zhao et al. developed a dual-core microcapsule that
encapsulated Lactobacillus and B. subtilis in separate compartments (Figure 10a).[89] The
probiotics after microcapsulation showed acid resistance and unchanged probiotic activity.
Such a dual-core microcapsule could improve fat metabolism, regulate inflammatory
reaction, and restore the intestinal barrier function, thus contributing to the treatment of
metabolic syndrome in vivo. In another study, Zheng et al. constructed a safe microbiota-
modulating material by combining probiotics and prebiotics. The spores of C. butyricum,
which were characterized to be able to enrich in the tumor tissue were coated with prebiotic
dextran by host–guest interaction mediated by AD (adamantine) and β-CD (β-cyclodextrin)
(Figure 10b‒d).[32] After oral administration, the as-prepared prebiotic-encapsulated
probiotic spores (spores-dex) could be specifically enriched in colon cancer and they
fermented the dextran in situ to produce SCFAs with anticancer capacity. Moreover, spores-
dex enhanced the abundance of SCFA-producing bacteria, and remakably increase the overall
richness of gut microbiota. Tumor growth was inhibited by up to 89% and 65%, respectively
when treated with the capecitabine (Cape)- or diclofenac (DC)-loaded spores-dex in
subcutaneous and orthotopic tumor models. This work provides a safe strategy to regulate gut
microbiota, and provides a promising avenue for treating various gastrointestinal diseases.
66
67
Figure 10. Prebiotics-encapsulated probiotics. a) Scheme illustrating the preparation of
probiotics-loaded dual-core prebiotic microcapsules for management of the metabolic
syndrome by enhancing the intestinal barrier function. Reprinted from Ref.[89] with
permission from American Chemical Society. Copyright 2020. b) Prebiotics-encapsulated
probiotics for regulating the gut microbiota and suppressing colon cancer. c) Effects of
various polymers, including polyethyleneimine (PEI), polyethylene glycol (PEG),
polyvinylpyrrolidone-iodine (PVP-I), HA, polyacrylic acid (PAA), and dextran, on the
growth of C. butyricum. d) Schematic illustration of the combination of probiotics and
prebiotics through host–guest chemistry. b–d) Reprinted from Ref.[32] with permission from
Wiley-VCH. Copyright 2022.
4.1.10. Other Strategies
Besides the above summarized encapsulation strategies and their contributions to

probiotics, there are also some other encapsulation methods, such as nanozyme
encapsulation,[182] tannic acid and mucin coating,[183] and in situ modification of gut
bacteria,[184,185] etc. Understanding the implementation processes and action mechanisms of
these strategies will help us to design more functional probiotics that can be delivered
through the gut.
It has been reported that platinum (Pt) nanozyme can be used as an antioxidant in the
treatment of ROS-related inflammation due to their high ROS-scavenging ability similar to
native enzymes, such as superoxide dismutase (SOD) and catalase (CAT).[182] Moreover, the
Pt nanozyme had good biocompatibility and was highly stable in harsh environments. Given
these features of Pt nanozyme, Zhao et al. introduced a versatile and facile approach for
decorating individual probiotics (EcN) with nanozyme coatings (forming Pt-Lipid@EcN),
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and the resultant Pt-Lipid@EcN could synergistically enhance the biotherapy of the colonic
inflammatory disease associated with excess ROS (Figure 11a).[136] In this system, Pt NPs
were first embedded in the mPEG-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-
N-methoxy-poly(ethylene glycol)) lipid membrane, and EcN was further coated with the Pt–
lipid membrane through vortexing to obtain Pt-Lipid@EcN. The growth behavior of the Pt-
lipid-coated EcN cells was not significantly influenced except that the logarithmic growth
phase was delayed for the coated EcN. The nanocoating-decorated EcN cells showed higher
in vivo and in vitro survival rates compared to the free EcN cells when exposed to the SGF.
Moreover, the colonization rate of the EcN in the intestinal tract was significantly elevated. In
the murine ulcerative colitis (UC) model, the reduction of histological injury and
inflammatory effects were observed, and the epithelial barrier homeostasis in the intestine
was also improved. In another work, a super probiotic (EcN@TA-Ca2+@Mucin) coated with
tannic acid and mucin via a layer-by-layer technology was developed (Figure 11b).[183] In
this platform, the EcN showed significant resistance to the harsh environment of the GIT and
displayed strong adhesiveness to the intestine via the interaction of mucin with mucus, which
enhanced the mucus layer colonization and growth of probiotics in the intestine without
removing the coating. EcN@TA-Ca2+@Mucin could also scavenge ROS to downregulate
inflammation in inflammatory bowel diseases. Overall, this is a powerful platform for
improving the intestinal colonization of probiotics by regulating the pathological
microenvironment, and it provides an important solution for utilizing the intestinal
colonization of probiotics to treat a variety of diseases.
Chemical, physical, and biological modifications of probiotics with enhanced functions

can be a powerful therapeutic strategy against various diseases by regulating the gut
microbiota. However, such regulation approaches towards the gut microbiota can not
precisely regulate the specific bacterial functions without destroying the microbiota and/or
69
host physiology. To solve this problem, widely available tools are needed, especially the ones
that can regulate the specific bacterial functions without while minimizing the disruption to
nontargeted genes, microbes, and host physiology. Hsu et al. reported a noninvasive strategy
for in situ regulating the bacterial gene expression, thus modifying the bacterial functions in
the gut via oral delivery (Figure 11c–e).[184] First, they engineered temperate phage λ
expressing a nuclease-deactivated Cas9 (dCas9) to specifically repress a targeted E. coli gene,
when they were in vitro or colonized the gut (Figure 11c). Subsequently, they encapsulated
the engineered temperate phage λ with a layer-by-layer (LbL) assembly method to protect the
phage during oral delivery by forming a thin polymer film (Figure 11d). When this
engineered and encapsulated temperate phage λ entered the bacterial-dense large intestine,
the cargo release was triggered due to the microbiota-specific tunable release mechanism
(Figure 11e). This work provides an efficient tool for the precise in situ regulation of the
bacterial gene expression in the mammalian gut with a single oral dose.
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Figure 11. Nanozyme and mucin encapsulation of probiotics. a) Pt-lipid-coated EcN for the
treatment of ulcerative colitis. Reprinted from Ref.[136] with permission from Royal Society of
Chemistry. Copyright 2022. b) Tannic acid- and mucin-coated EcN (EcN@TA-
Ca2+@Mucin) for biotherapy. Reprinted from Ref.[183] with permission from American
Chemical Society. Copyright 2022. c) Construction of temperate phage expressing dCas9,
trcrRNA, and crRNA. d) Scheme illustrating the encapsulation strategy of temperate phage.
e) Oral delivery of engineered temperate phage for in situ modification of gut bacteria. c–e)
Reprinted from Ref.[184] with permission from Nature Publishing Group. Copyright 2020.
Collectively, the conventional physical engineering strategies, like microencapsulation,

which can protect the probiotics from the harsh gastrointestinal environments often realize
the bulk encapsulation of probiotics. Such strategies are relatively simple and safe due to the
fact that they do not need extra chemical agents. However, there exist some drawbacks for
this strategy, including low encapsulation efficiency, early release due to the high porosity,
operation pressures (e.g., heat, dehydration, and ice crystal damage), high cost, and time-
consuming process. Recently, some new physical coating strategies have been developed,
such as the LBL,[170] biofilm,[144] and cell membrane approaches,[158] which can realize single
cell encapsulation. Compared with the conventional and bulk encapsulation strategies, single
cell encapsulation can overcome the issues with conventional microencapsulation or bulk
encapsulation strategies for probiotic delivery and provides some unique features, including
in vivo gastric resistance, enhanced colonization, and potential therapeutic effect in treating
the related diseases. However, such methods for encapsulating probiotics still have some
drawbacks. For example, the LBL method is time-consuming and cumbersome, which
impedes its use for large-scale production. Biofilm or cell membrane encapsulation usually
leads to a very limited survival rate of probiotics in gastric and intestinal juice. Meanwhile,
not all the probiotics can form biofilms. Moreover, the biofilm life cycle is limited because
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the bacteria in the biofilm will grow and the new bacterial cells have no suitable conditions to
form new biofilms. Taken together, the improvement in the encapsulation efficiency of
probiotics as well as the enhancement of the resistance of the biofilm or cell membrane
coatings to the gastric juice and bile acids will certainly advance the biomedical applications
of probiotics.
4.2. Chemical Engineering Approach
The bacterial surface is composed of diverse macromolecules, including peptidoglycan,

lipopolysaccharide, teichoic acid, protein, and lipid, which provide a range of functional
groups and endow the cell surface with negative charges.[14] The functional groups including
hydroxyl, carboxyl, amine, and sulfhydryl groups allow for bacterial cell surface modification
with different molecules through various physicochemical methods. Vargason et al.
successfully modified the live therapeutic bacteria (LTB) including Escherichia coli (EC),
Lactobacillus casei (LC), and B. coagulans (BC), and the probiotic consortia with synthetic
adhesions to allow specific interactions between receptor and target surfaces.[186] Then, biotin
was conjugated to the surface of probiotics using N-hydroxysuccinimide ester (NHS) to react
with ubiquitous primary amines on bacterial surfaces and form amide bonds (Figure 12a).
Streptavidin-linked monoclonal antibodies were further attached to biotinylated bacteria to
enable their adhesion in the GIT for improved biotherapy.
In addition, Liu reported a double-layer coating strategy that adopted tannic acid (TA)
and enteric L100 for EcN encapsulation.[72] The encapsulated EcN showed robust resistance
against the harsh external environment of the GI tract, and the strong mucoadhesive
capability of the TA layer improved their intesitinal retention time, resulting in considerable
therapeutic effect against colitis.
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Moreover, improving the colonization of LTBs in the GIT[187] or other treatment sites
(e.g., tumor region)[188] is important for probiotics to perform their function. Vargason et al.
reported a modular platform that can rapidly modify the surface of LTBs to enable the
specific interactions between the receptor and target surfaces (Figure 12b).[187] For
improving adhesion, synthetic adhesins (SAs) were conjugated to the probiotic surface. This
platform enables rapid formation of an intestinal niche, leading to an increased maximum
concentration and a 20% improvement in total LTB exposure.
In addition, bioorthogonal click chemistry has become one of the most widely used
approaches for biomedical applications since the early 2000s due to its high selectivity and
biocompatibility.[189] Bioorthogonal chemistry involves the chemical reactions that can occur
inside the living organisms and does not disturb the native biochemical processes or
negatively affect cell metabolism or viability. In addition, metabolic decoration of bacteria,
like amino acid-based metabolic decoration has recently become one of the most powerful
approaches for gut microbe-related research.[169,190] By combining the bioorthogonal click
chemistry and the metabolic decoration technologies, Song et al. developed a bacterial
delivery strategy using a click chemistry method for enhancing the probiotic colonization in
the intestine (Figure 12c).[191] First, azido decoration of gut inhabitants was performed by
metabolically incorporating azido-modified D-alanine (N3-DAA) into the peptidoglycan
(PGN). Probiotics were then modified with dibenzocyclooctyne (DBCO) through the
formation of amide bonds. Subsequently, the bioorthogonal reactions between probiotics and
the gut inhabitants of mice happened after orally administrating DBCO-decorated probiotics
for longer intestinal reservation. The in vitro and in vivo experiments all demonstrated the
significantly improved intestinal colonization efficiency of probiotics induced by the
conjugation of functionalized probiotics and gut inhabitants via a bioorthogonal reaction.
DBCO-modified C. butyricum also showed enhanced intestinal colonization efficiency and
73
more efficient therapeutic effect in dextran sodium sulfate (DSS)-induced colitis mice. This
work provides a promising strategy for enhanced delivery and intestinal colonization of
probiotics for treating intestine-related diseases. In another study, Luo et al. developed an in
situ chemical reaction-mediated bacterial covalent localization of mucous layer (Figure 12d,
e).[192] In this study, the authors first transformed the primary amino residues on the surface of
diverse bacteria to free thiols via an imidoester reaction to afford thiolated probiotics that
were able to spontaneously link with poly(disulfide)s-enriched mucous layer via a dynamic
thiol–disulfide exchange reaction. Owing to the ability to form covalent bonds, the surface-
thiolated bacteria show advantages to target and colonize the mucin-enriched tissues. They
subsequently took EcN as an example to perform the microbiota transplantation. They found
that oral delivery of bioorthogonal EcN generated a notably promoted remission of
inflammation related to jejunal mucositis. This strategy for modification of bacteria with a
reactive surface provides a robust method to prepare functional living bioagents.
In addition, Yang et al. reported an oral “Super Gut Microorganism (SGM)” to modulate
microbiome composition for the prevention and treatment of colitis (Figure 12f,g).[193] In this
system, the authors used tannic acid and poloxamer 188 (F68, intravenous excipients) to
encapsulate EcN by a self-assemblilng method for enhancing intestinal colonization and
modulating the gut microbiota. In DSS-induced colitis mice, SGM showed an excellent
antiinflammatory effect and the strong capacity to restore intestinal barrier functions and
prompt the balance of gut flora. In Salmonella enterica serovar Typhimurium (STm) colitis
mice, probiotics treated with SGM resulted in a 6.8-fold reduction of STm compared with the
uncoated probiotics, showing the improved efficiency of antiinflammation in the prevention
of STm colitis. This work provides a new strategy for creating novel probiotic systems to
treat and prevent microbiome-related diseases. Cao al. synthesized an iron-based single-atom
catalyst by pyrolysis of Fe-containing metal–organic framework (Fe@MOF) (Figure
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12h).[194] Then they used the linker of boronic acid–poly(ethylene glycol) (C18–PEG-B) to
bind the anaerobic Bifidobacterium longum (B. longum) probiotic and iron single-atom
catalyst (Fe SA) to form the artifcial enzyme-modifed B. longum (BL@B-SA50), showing
extraordinary stability in the GIT and excellent ROS-scavenging ability. In vivo experiments
proved that BL@B-SA50 displayed a superior efficacy in treating UC and Crohn’s disease
(CD), as well as regulating dysbiosis.
To summarize, chemical modification of probiotics is effective for single cell

encapsulation, which can protect them from the harsh environments, thereby improving their
survival rate. In addition, chemical modification can improve the colonization rate of
probiotics in the intestine, extend the intestinal retention time of probiotics, and achieve
satisfactory treatment outcome of the related diseases. The controllable release of probiotics
can also be achieved after chemical modification by endowing probiotics with stimuli-
responsive properties. Moreover, extra functional substances can be introduced into
probiotics by chemical conjugation to endow them with enhanced functions to improve the
therapeutic effect and expand the scope of application. However, there are still some room for
progress of the effective chemical engineering strategy, such as developing more
biocompatible materials and tender reaction process that do not affect the bacterial cell
activity for chemical modification, reducing the utilization of chemical reagents.
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Figure 12. Chemical engineering of probiotics. a) Scheme of LBP surface biotinylation of
live biotherapeutic products. Reprinted from Ref.[186] with permission from American
Chemical Society. Copyright 2020. b) Scheme of NHS chemistry for bioconjugation of biotin
to primary amines on the probiotic surface. Reprinted from Ref.[187] with permission from
Wiley-VCH. Copyright 2020. c) Scheme depicting the bioorthogonal conjugation of the
delivered probiotics with gut inhabitants through click chemistry. Reprinted from Ref.[191]
with permission from American Chemical Society. Copyright 2022. d) Scheme showing the
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preparation process of surface-thiolated bacteria. e) Bonding of surface-thiolated bacteria
with poly(disulfide)s-abundant mucin. d,e). Reprinted from Ref.[192] with permission from
Nature Publishing Group. Copyright 2022. f) Preparation of SGM encapsulated with TA and
F68. g. Scheme illustrating the EcN expressing GFP and the staining of the bacterial
membranes with rhodamine B. f,g). Reprinted from Ref.[193] with permission from Elsevier.
Copyright 2022. h. Scheme displaying the construction of BL@B-SA50 (ⅰ), its ROS-
scavenging mechanism (ⅱ), and the community composition of the gut microbiota in mice
with different treatments (ⅲ). Reprinted from Ref.[194] with permission from Nature
Publishing Group. Copyright 2023.
4.3. Probiotic Spore-Based Materials for Delivery of Probiotics
Oral administration is a common way for the direct delivery of probiotics, requiring the
protection of bacteria from the strong acidic environment in stomach and the harsh
environment in GI tract to allow for bacteria to preserve a certain amount of live probiotics to
reach intestinine and keep the proliferation capability after reaching the desired site.[195]
Spores, a dormant form of microorganisms, are extremely stable and resistant to harsh
environments, such as the strong acidity, extreme temperature, desiccation, and toxic
chemicals. Spores are capable of surviving the low pH of the gastric barrier and reaching the
intestine intact. After decomposing the hydrophobic protein coat of spores, they can
germinate to metabolically active vegetative cells by utilizing certain nutrients in the
intestine. Thus, probiotic spores themselves can be used for probiotic delivery due to their
ability to resist against the strongly acidic environment of the stomach and their capacity to
germinate to form probiotics or consume oxygen in the intestine.[195,196]
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Song et al. constructed an oral autonomous NPs generator based on the spores of
Bacillus cagulans (BC) in vivo (Figure 13a).[196] The obtained spores were conjugated with
hyaluronic acid (HA) or deoxycholic acid (DA) to obtain Spore-HA and Spore-DA.
Secondly, the doxorubicin (DOX) and sorafenib (SOR) were coloaded on the Spore-HA and
Spore-DA by electrostatic adsorption to produce an autonomous NPs generator
(DOX/SOR/Spore-DA/HA). DOX/SOR/Spore-DA could pass through the GIT and colonized
rapidly under the protection of spores. Subsequently, a large number of DOX/SOR/Spore-DA
NPs were generated in the GI tract microenvironment during the regermination process of the
DOX/SOR/Spore-DA in the intestinal regions. Then the autonomously formed NPs could
enhance the stability of drugs in the GIT and overcome the multibiological barriers of
intestinal epithelium through the bile acid pathway, thereby increasing the basolateral drug
release and exhibiting remarkable antitumor effect in vivo. In another research of their group,
they constructed a multifunctional spore coat nanomaterial (CN) by a mechanical force
extrusion method. Subsequently, the CN-coated probiotics were fabricated by anchoring CN
to the surface of the probiotics, which exhibited resistance to the harsh intestinal environment
with the protection of CN (Figure 13b).[197] After reaching the intestine, the CN-coated
probiotics grew rapidly, and then colonize the intestine and inhibited the pathogenic bacteria.
Spores can also be used to load natural plant-derived anticancer and antiinflammatory
agents, like curcumin (CUR). Yin et al. used Bacillus spores as oral carriers to load curcumin
for the therapy of colon cancer (Figure 13c).[198] In this system, curcumin was linked
covalently to the outer coat protein of B. coagulans spore to form SPORE-CUR by a N,N’-
dicyclohexylcarbodie (DCC)/4-dimethylaminepyridine (DMAP) method. Subsequently,
folate (FA) was incorporated covalently to SPORE-CUR to afford SPORE-CUR-FA by the
DCC/DMAP method. This strategy significantly enhances the oral bioavailability of
curcumin and improves the capacity of curcumin to eliminate colon cancer cells. In another
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study, Chen et al. decorated curcumin on the surface of B. subtilis spores and encapsulated
the decorated spores into the cysteine-modified konjac glucomannan microspheres for
treating the colitis.[199] In this system, the germination of the spores by fermenting the konjac
glucomannan consumed the oxygen and produced SCFAs to regulate the gut microbiota and
thus treat the colitis. In addition, probiotic spores can be employed as antigen carriers or
vaccine platforms.[200] Peng et al. constructed a Bacillus amyloliquefaciens spore@zeolitic
imidazolate framework-8 (ZIF-8) vaccine platform (Figure 13d).[201] The ovalbumin (OVA)
was encapsulated in the ZIF-8 shell as a model antigen to form a spore@OVA@ZIF-8 (SOZ)
composite, which could enhance the immune efficacy of spores in several ways.
In conclusion, bacterial spores possess various advantages in the extreme environments

compared with the bacterial vegetative cells that are difficult to survive in the extremely
acidic environment of the stomach after oral administration. Moreover, the bacterial spore
surface is hydrophobic though they contain many functional groups, like amine or carboxyl
groups, which can be functionalized with different therapeutic agents. Otherwise, the high
specific surface area of the spore can facilitate the adsorption and attachment of the
therapeutic agents. The encouraging results achieved by previous studies suggest that the
bacterial spores can be developed to be versatile platforms for therapeutic application or
delivery of therapeutic agents across the GI tract.
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Figure 13. Probiotic spore-based materials for intestinal delivery. a) Scheme illustrating the
fabrication and the intestinal transepithelial transport mechanism of DOX/SOR/Spore-DA
NPs. Reprinted from Ref.[196] with permission from Wiley-VCH. Copyright 2019. b) Scheme
showing preparation of CN and CN-coated probiotics, and the action mechanism of CN-
coated probiotics for treating colitis. Reprinted from Ref.[197] with permission from Wiley-
VCH. Copyright 2021. c) Preparation of the Bacillus spore-based oral carrier loaded with
curcumin (SPORE-CUR-FA) for cancer therapy. Reprinted from Ref.[198] with permission
from Elsevier. Copyright 2018. d) Scheme illustrating the preparation of the
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spore@OVA@ZIF-8 composite and its use for inducing humoral and cellular immunity
responses. Reprinted from Ref. [201] with permission from Wiley-VCH. Copyright 2022.
4.4. Genetic Engineering of Probiotics for Enhancing the Intestinal Delivery Efficiency
Genetic engineering has been widely adopted for unraveling and remodeling microbial
genomes for altering the bacterial features and functionalities,[202] and is becoming easier
with advances in the gene editing tools and synthetic biology. “Rewriting” or even
“completely designing” genomes is becoming a reality. For example, CRISPR-Cas (clustered
regularly interspaced short palindromic repeats-CRISPR-associated proteins) is becoming an
efficient gene-editing tool to manipulate bacterial genome due to the flexibility, high
efficiency, and precisely targeted gene-editing property of CRISPR-Cas system.[203] Synthetic
biology has developed rapidly and shown great application prospects in medicine,
manufacturing, energy, and other fields.[204–206] Synthetic biology enables probiotics to be
reprogrammed by genetic engineering to perform a variety of new tasks and produce more
finely tuned bioactive molecules for desirable purposes.[207]
There are two main approaches to construct engineered bacteria, one is plasmid
overexpression, and the other is gene integration. With the help of genetic engineering
strategies (Figure 14a), probiotics can be endowed with various extra functions by
expressing desired genes that encode exogenous proteins (including antibodies and enzymes),
antibiotics, antigens, and peptides. The introduced exogenous components can be expressed
on the bacterial cell surface or secreted into the surroundings to perform several functions,
such as targeting, adhesion to the intestinal epithelium, and synthesis of specific metabolites
(Figure 14b) to facilitate site-specific targeting, improve the delivery efficiency and gut
duration of probiotics, and enhance the therapeutic efficacy of probiotics against a variety of
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diseases, including cancer, metabolic disorders, inflammation, microbial infections, and
chronic wounds.[207–210] By virtue of genetic engineering approaches, dynamic and
programmable properties and biomimetic features can also be integrated into bacteria.
Moreover, probiotics can be engineered to act as living therapeutics through the synthetic
biology technology to ensure the efficiency of living therapeutics under harsh gastric
environments and endow them with new functions to treat human diseases by secreting
desired metabolites, such as protein drugs and enzymes, specifically at the disease site for
realizing enhanced therapeutic effect and decreased side effects.
Figure 14. Genetic engineering of probiotics for different purposes, such as realizing cancer
cell targeting, enhancing the in vivo colonization ability, and secreting functional metablites.
a) Scheme illustrating the process for genetic engineering of probiotics. b) Probiotic
transformant with various functions like targeting, colonization, self-encapsulation, and
biosynthesis of specific metabolites .
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Benefiting from the recent advances in gene editing approaches and synthetic biology,
genetically engineered probiotics or probiotic-based living therapeutic systems have achieved
great progress and shown potential in the clinic. Duraj-Thatte et al. genetically engineered the
nonpathogenic E. coli PQN4 to produce a mucoadhesive hydrogel by synthesizing curli fibers
to encapsulate the bacterial cells to improve the intestinal residence time by protecting
bacterial cells in situ and promoting mucoadhesion of the bacteria (Figure 15a).[211] In this
system, the hydrogel can self-renew and grow under proper conditions. Moreover, this
hydrogel can be customized to interact with different GIT tissues selectively. This platform
not only provides a strategy for promoting mucoadhesion of the bacteria but also benefits the
development of bacteria-based therapeutic delivery systems. In addition to promoting the
mucus adhesion of probiotics for improving the survival rate of probiotics, improving the
colonization efficiency of probiotics is another strategy for promoting intestinal delivery of
probiotics. Moreover, genetically engineering probiotics to promote the probiotics’
competition with other intestinal pathogenic bacteria is also useful. For, example, surface
layer protein (SlpA) mediates the bacterial attachment to the intestinal mucus layer
contributing to the intestinal colonization. Vedantam et al. genetically engineered
Lactobacillus casei strain 334 and Lactobacillus acidophilus strain 4356 by expressing a
host-cell binding fragment of the C. difficile adhesin SlpA to improve the colonization
efficiency of the probiotics and exert the therapeutic effect on CDI.[212] In another study,
Wang et al. genetically engineered EcN by introducing a light-controlled adhesion protein
module to improve the precise spatiotemporal colonization ability of EcN in the intestinal
tract by secreting autotransporter 43 adhesins antigen (AG43) (Figure 15b).[213] In this
system, the authors first constructed a recombinant EcN strain by expressing AG43 protein.
Afterwards, they designed upconversion microgels (UCMs) that can convert near-infrared
(NIR) light into local visible blue light to activate recombinant EcN strain.
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Besides the genetic engineering strategies that can produce specific metabolites, surface
modification that can control the interaction of probiotics with the environment is attracting
significant attention. Harimoto et al. constructed the synthetic gene circuits to realize the
microbial surface modification to dynamically control the interactions of probiotics with their
surrounding environment (Figure 15c–e).[214] In this work, the authors constructed a plasmid
with a kfiC gene that encoded a glycotransferase of GlcNAc, and the constructed plasmids
were expressed in EcN ΔkfiC genome with various copy numbers of kfiC under the control of
the lac promoter, which could be activated with the small-molecule inducer isopropyl-b-D-
thiogalactopyranoside (IPTG) for tight regulation of capsular polysaccharide (CAP)
production. The EcN ΔkfiC transformed with a low copy number plasmid exhibited complete
immunity against the lytic bacteriophage ΦK1-5, and could realize inducible modulation of
CAP on cellular surface. This dynamic delivery strategy provides a programmable
encapsulation system to ameliorate the therapeutic performance of living engineered bacteria
for cancer.
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that are genetically engineered by expressing curli fibers (inset: orange and red symbols) in a
Figure 15. Genetic engineering of probiotics. a) Scheme showing the cultivation of bacteria
85
liquid culture medium and the enrichment of bacterial cells on a filter by vacuum filtration.
Sodium dodecyl sulfate (SDS) treatment leads to rapid gelation and maintains the viability of
embedded bacteria, obtaining “Live Gel”, while the “SDS + guanidinium chloride (GdmCl)”
treatment results in “Cell-Free Gel”. The SEM images show the microstructures of the “Live
Gel” and the “Cell-Free Gel”. Reprinted from Ref.[211] with permission from Wiley-VCH.
Copyright 2019. b) Scheme illustrating the NIR light-activated recombinant EcN that is
trapped in AG43 living bio-glue and can resist the harsh environment after oral
administration and scheme showing the recombinant EcN that can secret TGF-β1 for
therapeutic application in colitis mice. Reprinted from Ref.[213] with permission from
Elsevier. Copyright 2021. c) Engineering of the CAP biosynthesis pathway for tunable and
dynamic surface modulation of EcN via synthetic gene circuits. d) Programmable CAP
system for enhancing the probiotic delivery. e) CAP system for controlling the bacterial
translocation among tumors. c‒e) Reprinted from Ref.[214] with permission from Nature
Publishing Group. Copyright 2022.
To sum up, the genetic engineering strategies of probiotics can customize probiotics
with different application purposes, such as improving their characteristics, making them
more resistant to harsh environments, secreting protective materials, and producing effective
metabolites to improve the survival rates and functional effects. Besides, the genetic
engineering strategies can adopt a modular design strategy to endow probiotics with more
independent functions. Due to the different characteristics of each module, this modular
design can facilitate subsequent functional evaluation and performance optimization.
Moreover, the genetically engineered probiotics can be combined with other strategies to
further improve their intestinal delivery, colonization rate, and targeting effect. However,
such genetic engineering strategies for modifying probiotics still have some drawbacks. First,
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the gene transfer can occur at a high frequency among the genetically engineered probiotics
and gut microbes. Thus, probiotics should be genetically engineered to be auxotrophic but not
integrated with antibiotic resistance markers to enable biocontainment, preventing any
potential risk of spreading antibiotic resistance genes into surrounding environments and
other gut microorganisms. Second, stability of the genetically engineered strain should be
considered, as the gene mutation may occur when the probiotics are cultured for multiple
passages, resulting in the loss of original functions.
4.5. Combination of Genetic and Chemical Engineering Strategies
Combining the genetic approaches and the chemical engineering strategies is a typical
way to enhance the delivery efficiency and therapeutic effect of probiotics. For instance,
Zhou et al. coated the genetically engineered EcN overexpressing catalase and superoxide
dismutase with chitosan, sodium alginate, and effective biofilms via a layer-by-layer
electrostatic self-assembly strategy to yield ECN-pE(C/A)2 to improve the survival rate of
EcN in the gastric acid and bile salts in the GI tract.[173] Results showed that ECN-pE(C/A)2
could effectively relieve inflammation, repair epithelial barriers in the colon, modulate the
gut microbiota, and elevate the intestinal flora richness and diversity (Figure 16a). This work
may have implications for the development of therapeutic proteins by genetically engineering
the probiotics for treating intestinal-related diseases. In another research, Chen et al.
constructed a biohybrid probiotic microrobot with magnetic, hypoxia, and thermal
sensitivities and expressed an internal fluorescent protein in the above probiotic for targeted
cancer treatment (Figure 16b).[215] In this platform, the NDH-2 (type II NADH (nicotinamide
adenine dinucleotide)-dehydrogenase) or mCherry/NDH-2 gene was first cloned into
pBV220 expressing vectors under the thermally sensitive promoter TcI to obtain pBV220-
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NDH-2 and pBV220-mCherry/NDH-2 plasmids via homologous recombination. The
obtained plasmid was transformed into EcN to obtain EcNmCherry/NDH‑2. Then, magnetic
nanocubes (20 nm) modified with citric acid were coated on the surface of the genetically
engineered probiotics via amide bond formation by the EDC (1-ethyl-3(3-
dimethylaminopropyl-carbodiimide hydrochloride)/NHS (N-hydroxysuccinimide) chemistry
to obtain the bacteria-hybrid microrobot (EcN@MNP). The EcN@MNP presented triple
perception, including magnetothermal perception to trigger target gene biosynthesis, spatial
magnetic control, and hypoxia perception for precise targeting, and it may become an
intelligent microrobot system for biomedicine applications.
Lu et al. developed a heparin-poloxamer thermoresponsive hydrogel as a living delivery

system to load the engineered living Lactococcus (Figure 16c–e).[75] In this work, L. lactis
NZ9000 was genetically engineered by transforming a plasmid encoding vascular endothelial
growth factor (VEGF) under the inducible promoter PnisZ (Figure 16c). Then the engineered
probiotics were incorporated into the heparin-poloxamer thermoresponsive hydrogel, which
was fabricated by conjugating the heparin with the mono amine-terminated poloxamer
(Figure 16d–f). This living system could produce VEGF and protect it for promoting the
diabetic wound healing by increasing proliferation, migration, and angiogenesis, as well as
secreting lactic acid to promote the macrophage polarization. Meanwhile, the nisin produced
by L. lactis NZ9000 exerted an antibacterial effect to inhibit the growth of bacteria without
influencing its own growth.
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Figure 16. Combination of genetic and chemical engineering strategies for modification of
probiotics. a) Preparation of genetically engineered EcN that can produce CAT and SOD, and
the encapsulation of EcN with sodium alginate and chitosan by the layer-by-layer strategy.
Reprinted from Ref.[173], with permission from Nature Publishing Group. Copyright 2022. b)
Construction and characterization of the engineered bacteria-hybrid microrobot,
EcNmCherry/NDH‑2. Reprinted from Ref.[215], with permission from American Chemical Society.
89
Copyright 2022. c) Engineered L. lactis living hydrogel to accelerate angiogenesis in a
diabetic wound. d) Scheme showing the preparation of the engineered L. lactis for
continuously secreting VEGF under the induction of nisin. e) SEM image of engineered L.
lactis encapsulated inside the HP hydrogel. Scale bar: 1 µm. f) 3D confocal images showing
the distribution and growth of LL_mCherry at 3 and 24 h. c–f) Reprinted from Ref.[75] with
permission from Wiley-VCH. Copyright 2021.
Collectively, we have reviewed the recent advances in the engineering strategies of

probiotics for improving the intestinal delivery efficiency from the aspects of physical
engineering, chemical engineering, genetic engineering, and their combination strategies,
which have been representatively summarized in Table 4. Compared with the physical
encapsulation methods, the chemical modification methods need extra chemical agents,
which may result in high cost, environmental problems, potential toxicity to the probiotics,
and threat to human health. The genetic engineering strategies can achieve multifunctional
modifications of probiotics, but the limited genetic engineering platforms and the unclear
genomic information of some probiotics are challenges for their wide applications. Therefore,
there is still a need to continuously explore appropriate encapsulation strategies, develop
suitable encapsulation materials and genetic engineering platforms, and achieve a deep
understanding of probiotic genomic information to better utilize probiotics for product
development and clinical translation.
5. Specific Opportunities for Improving LBP Delivery in Humans to Overcome Clinical

Trial Failures
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LBPs have been potentially demonstrated as an emerging therapeutic approach based on
the current clinical trials. Although FDA has approved several LBP products,[111‒115] the
success clinical uses of LBPs still face several challenges, like the survival and stability of the
LBPs as they transit from oral cavity to the intestine, low adherence in the intestine, inability
to penetrate the intestinal mucosal barrier, metabolite safety of LBPs in the intestine, and
biocontainment of the genetically engineered LBPs, etc. To solve the above challenges and
shorten the gap between clinical trials and approved LBP products, LBP formulations,
specific delivery materials, delivery strategies, and even the equipments used for producing
engineered LBPs in bulk must be developed to ensure the viability of LBPs under harsh
gastric environments and the suitable residence time in the GIT after oral delivery. We thus
introduced some possible opportunities for improving LBP delivery in humans to overcome
clinical trial failures as follows.
First, in the LBP production process, the selection of probiotics with desired functions
and characteristics is difficult, limiting the development rate of LBPs. Therefore, strategies
based on new technologies to identify the desired LBPs should be conducted to promote the
selection speed of microorganisms used in LBP products, like multi-omics-based strategies,
computational modeling of the microbial community, and mathematical models.[216]
Integration of artificial intelligence (AI) and machine learning (ML) into the microbial strain
discovery and LBP design processes will greatly help to predict the functions of probiotics in
LBPs and discover the colonization-promoting genes, speeding the selection process of
probiotics used in LBPs. AI can also help researchers to design the combination of probiotics
in LBPs. In addition, some spore-forming probiotics should be considered in LBPs due to
their long-term shelf-lives and excellent harsh environmental resistance ability.
Second, for LBPs, from manufacturing process to the patient’s end use, due to the use of
live bacterial cells, they go through a number of steps that can affect the reproducibility and
91
efficacy of LBPs in every batch and bring about some unexpected adverse effects. Thus, the
LBP producer must control the fermentation conditions to ensure the viability of LBPs after a
series of treatment and the phenotypic and genetic consistency of LBPs in each batch.
Moreover, stability of LBPs during transportation or storage should be assessed to
determine the activity loss rate of LBPs and confirm the shelf life and recommended dose of
LBPs. For genetically engineered LBPs, evaluation of the genetic stability (e.g., genetic drift)
of LBPs is required, as genetic drift may occur in the genetically engineered strains, leading
to a loss of LBP efficacy or even safety issues. More importantly, government regulation
should be strengthened to ensure the quality and effectiveness of LBPs.
Third, LBPs must be delivered to the target site in a viable and active state to enhance
the delivery efficacy of LBPs and ensure their therapeutic efficacy. The delivery approaches
should be suitable for simplifying the storage conditions, the transportation process, and the
patients’ usage. For early developed LBPs, they are used in the form of lyophilized bacterial
strains, which face the low survival rate due to the adverse gastrointestinal environments.
Afterwards, the lyophilized bacterial strains are encapsulated in capsules or tablets, which
face the earlier release of strains and degradation of the bacterial metabolites. Currently,
microcapsules are frequently used to encapsulate LBPs, which solve the above problems.
However, the targeted delivery ability and satisfactory stability control are not provided by
the current microcapsule encapsulating strategies, which still limits the clinical success of
LBPs. Therefore, developing an efficient delivery system for LBPs is highly desirable.
Similar to probiotics, LBPs contain living organisms and are therefore uniquely sensitive
to external stresses such as enzymes, acids, bile salts, and oxygen when they are orally
administered, hence reducing the delivery efficiency of LBPs. Thus, similar to probiotics,
LBPs should be physically, chemically, or genetically engineered to improve their delivery
efficiency via establishing a protection shell against the harsh environment, providing the
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intestine-targeting ability, increasing the intestinal colonization rate, and prolonging the
intestinal duration during the oral administration process. Strategies for encapsulating
probiotics may also be suitable for LBPs, especially those composed of a single microbial
strain due to the simple cultivation and production process of LBPs. For example, physical
encapsulation strategies, including self-coating by forming biofilms, which increases the
mucoadhesion and bioavailability of living organisms,[144] layer-by-layer encapsulation,[173]
lipid encapsulation,[185] film encapsulation,[172] etc., are all effective approaches. For multi-
strain LBPs, due to the complex microorganism composition, the encapsulating strategy may
become complicated due to the unique characteristics of each microorganism. To overcome
the problems faced by multi-strain LBPs, specific strategies should be developed and
adopted. For example, zonal embedding and sequential release of each microorganism may
be effective. Different microorganisms in one system will interfere with each other; however,
if they can be embedded in different zones in one encapsulation system, the unique properties
of each microorganism will not be influenced, which can ensure the specific functions of
each microorganism in LBPs. Moreover, when LBPs are delivered to the intestine, they may
not need to be released at the same time because some metabolites produced by one microbial
strain will affect the viability and function of other microbial strains, and hence the
encapsulation strategies that can realize the sequential release seem to be useful to enable
LBPs to maximize their effectiveness.
Chemical engineering approaches are also applicable to LBPs. For example,

modification of LBPs with synthetic adhesins (SAs) that bind to the receptors of target
surfaces can improve the adhesion, colonization, and efficacy of LBPs.[186] In addition, due to
its high selectivity and biocompatibility, bioorthogonal chemistry has been used to
chemically engineer probiotics to enhance the probiotic colonization.[191] Similarly, due to the
93
similarity between probiotics and LBPs, it is expected that bioorthogonal chemistry can also
be adopted to modify LBPs for improving the intestinal colonization of LBPs.
Genetically engineered strategies are also suitable and have been employed for the
production of several clinically used LBPs. However, most existing genetically engineered
LBPs use E. coli or Lactococcus species as the model microorganisms because there are well-
defined genetic engineering tools to modify the genomes of the E. coli or Lactococcus
species and regulate transcription. In comparison, there are few synthetic biology toolkits that
can be applied to the other strains, like the obligate anaerobes that naturally reside in the
human intestine. Therefore, new LBP chassis should be developed for constructing new
genetically engineered LBPs to ensure the viability of LBPs under harsh gastric environments
and endow them with new functions to enhance the therapeutic effect of LBPs, promoting the
success of clinical trials. Moreover, to avoid the concern about the transgenosis, the LBPs are
usually genetically engineered to be auxotrophic to realize biocontainment. In addition,
combinations of the genetic engineering strategies and other encapsulation strategies to
improve LBP delivery are emerging, which has been introduced in section 4.5.
6. Intestinal Delivery of Engineered Probiotics for Different Applications
6.1. Treatment of Inflammatory Bowel Disease (IBD)
Intestinal health has become a hot research topic and is considered to influence the host
health. Human gut microflora plays important roles in maintaining the intestinal health. In the
health body, gut microflora maintains a steady state and is responsible for regulating human
metabolism, maintaining physiological activities, and keeping homeostasis of the GIT and the
barrier function of intestinal mucosa. Human gut microflora targets gastrointestinal via the
94
specific delivery pathway to exert the gut-regulating effect or gut-derived effects in humans.
Intestinal microorganisms can participate in the body’s metabolism process to decompose the
nutrient substances, promote the nutrient absorption, provide various nutriments (e.g.,
vitamins, SCFAs, amino acids, and peptides, and decompose hazadous substances), maintain
the integrity of intestinal epithelium, and participate in human energy metabolism. They also
function in immunomodulation. Once the balance of GIT is broken, dysbiosis happens and
human will suffer from a variety of intestine-related diseases, such as intestinal barrier
dysfunction, IBD, cardiovascular and cerebrovascular diseases, cancer, and diabetes.
Intestinal delivery of probiotics can ameliorate microecological imbalance, and can exert
therapeutic effects against some intestine-related diseases.[36,217]
IBD is one of the infectious diseases caused by the disorder of the gut microbiota.
Probiotics can inhibit pathogen colonization and proliferation in the GI tract and facilitate the
healing of the mucosal barrier to treat IBD. For example, fecal microbiota transplantation
(FMT) has shown great potential in treating IBD in the clinic with limited side effects. To
enhance the treatment efficacy of probiotics for IBD, and improve the survival rate and the
colonization ability of probiotics in intestine, some researchers use prebiotics that possess
ideal biocompatibility and are beneficial for the growth of gut microbiota to encapsulate the
probiotics. For example, Zheng et al. used chemically modified prebiotic dextran to
encapsulate the commercial C. butyricum for suppressing colon cancer.[32] In this system, the
spores of C. butyricum with the capacity to spontaneously enrich in the tumor tissue were
selected. For encapsulation of the probiotics, adamantane (AD) was first modified on the
surface of bacteria through EDC/NHS chemistry. After the simple mixing, dextran grafted
with β-cyclodextrin (β-CD) (β-CD-Dex) was assembled on the surface of bacteria. The
animal experiments showed that the dextran coating prolonged the intestinal retention of
spores. In conclusion, prebiotic encapsulation for oral delivery of probiotics is a highly safe
95
strategy for regulating gut microbiota, and provides a promising platform for treating various
intestinal diseases.
Praveschotinunt et al. developed an engineered EcN which could create a fibrous matrix
to promote gut epithelial integrity in situ for alleviating IBD. The fibrous matrix consisted of
curli nanofibers displaying trefoil factors (TFFs) to promote intestinal barrier function and
epithelial restitution (Figure 17a).[65] The engineered EcN could secrete the curli-fused TFFs
via the in situ self-assembly of the modified curli fibers. The animal results demonstrated that
the engineered EcN greatly decreased the weight loss of mice, reduced proinflammatory
cytokine levels, prevented colon length reduction, and displayed superior mucosal healing
and immunomodulation in a dextran sodium sulfate (DSS)-induced colitis model.
In addition to the above method that uses protection materials for maintaining the
epithelial integrity to alleviate IBD, directly producing substances with biotherapeutic effect
is another strategy for treating IBD. Liu et al. prepared ROS-scavenging nanoparticles and
conjugated the nanoparticles onto the surface of EcN, which was coated with a
polynorepinephrine (NE) layer to enhance the bacteriotherapy for IBD (Figure 17b–d).[218]
They first prepared a polymer of hyaluronic acid–poly(propylenesulfide) (HA-PPS) and
fabricated ROS-scavenging nanoparticles (HPN) that could effectively scavenge ROS. Then,
HPN were conjugated onto the surface of EcN that was coated with a polynorepinephrine
(poly-NE) layer to yield HPN-NE-EcN. The obtained HPN-NE-EcN exhibited increased
resistance to extreme environment, enhanced retention time in the intestine, and improved
survival rate after oral delivery. In vivo experiments proved that HPN-NE-EcN substantially
enhanced the prophylactic and therapeutic efficacies of IBD and showed gut microbiota
regulation ability, contributing to the alleviation of IBDs. Chua et al. constructed a
genetically engineered probiotic EcN that could produce a type III interferon that can drive
the immunomodulation for treating IBD (Figure 17e).[219] In this work, a nitric oxide (NO)
96
responsive IFNL1 (interferon lambda 1) expression cassette was integrated into the genome
of EcN using phage λ Red recombinase to obtain EcN-gIFNL1. The engineered EcN strain
showed an excellent antiinflammatory effect, demonstrating the potential of utilizing such an
engineered EcN strain as a live biotherapeutic agent for IBD immunotherapy.
Moreover, to exert the effect of probiotics, the adequate amount and activity of
probiotics delivered to the gut must be ensured. Enormous efforts have been made to improve
the viability and colonization of orally administrated probiotics in the intestine. Scott et al.
developed a unique self-tunable probiotic platform for the treatment of IBD and potentially
other inflammation-driven pathologies by combining directed evolution and synthetic biology
approaches in a yeast probiotic (Figure 17f).[220] In this platform, the human G protein-
coupled receptors (GPCRs) were expressed in Saccharomyces cerevisiae, which resulted in
up to a 1,000-fold increase in extracellular adenosine triphosphate (eATP) sensitivity. The
engineered yeast strain could sense a proinflammatory molecule, eATP, and produce a
proportional self-regulated response by secreting the apyrase for depleting eATP and promote
the production of the immunosuppressive adenosine. The engineered self-tunable yeast
ameliorated intestinal inflammation in IBD mouse model, and showed great potential in the
treatment of IBD. This work highlights that synthetic materials can provide living cells with a
nonevolutionary way to expand their functions.
97
98
Figure 17. Engineered probiotics for IBD treatment. a) Production of probiotic-associated
therapeutic curli hybrids (PATCH) from engineered EcN. Reprinted from Ref.[64] with
permission from Nature Publishing Group. Copyright 2019. b) Scheme depicting the
preparation of HPN-NE-EcN. c,d) Mechanism for IBD treatment. b–d) Reprinted from
Ref.[218] with permission from American Association for the Advancement of Science.
Copyright 2022. e) Mechanism of the engineered EcN producing a type III interferon IFNL1
for IBD immunotherapy. Reprinted from Ref.[219] with permission from permission from
American Chemical Society. Copyright 2022. f) Construction of the genetically engineered
eATP-responsive Saccharomyces cerevisiae strain, and hematoxylin and eosin (H&E)
staining results of chemically induced colitis treated with ethanol or different yeast strains.
Reprinted from Ref.[220] with permission from permission from Nature Publishing Group.
Copyright 2021.
6.2. Treatment of Microbial Infections
Bacterial infection has always been a serious global threat to human health and life.[221]
Several types of antibiotics have been developed, and are considered to be the final solution
to deal with bacterial infection for a long period.[222] However, owing to the overuse and
misuse of antibiotics, antimicrobial resistance issue has emerged as a challenging issue that
makes the diseases caused by bacterial infections more troublesome.[223] Even worse, the
discovery of new antibiotics fails to fully curb the rampant spread of drug-resistant
pathogens.[222] Moreover, another serious threat to patients lies in the possibility that drug-
resistant microorganisms adhere to the surface of implants and medical devices to promote
the formation of biofilms,[224] which are recalcitrant to be removed.
99
Preventing multidrug-resistant (MDR) bacteria-related infection and promoting
osseointegration are urgently desired for orthopedic implants. Engineered probiotics provide
a promising approach since such probiotic-based therapies can be developed with high
specificity without accompanying damage to the gut microbiota of the host. Hwang et al.
reported a microbe-based antimicrobial strategy that detected and targeted infectious
pathogens.[225] In this system, the authors developed an engineered EcN expressing an
antibiofilm enzyme encoding gene for inhibiting the growth of P. aeruginosa in vivo. In this
work, an antibiofilm enzyme dispersin B (DspB) was expressed in EcN to obtain EcN SED
with antibiofilm activity. In the P. aeruginosa- infected Caenorhabditis elegans (C. elegans)
and mouse models, the engineered EcN showed prophylactic and therapeutic activities. This
work provides a effective probiotic-based platform via engineering strategy in the
prophylactic and therapeutic fields against gut infections. In another study, Drolia et al.
reported bioengineered Lactobacillus probiotics (BLP) constructed from L. casei by
expressing the Listeria adhesion protein (LAP) from a nonpathogenic Listeria (Listeria
innocua (L. innocua)) and a pathogenic Listeria (Lm) (Figure 18a‒d).[226] Here, the lap ORF
(2.6 kb) was cloned into the Lactobacillus expression vector, pLP401T. Then, LAP was
expressed on the surface of the vancomycin-resistant L. casei wild-type ATCC334 (LbcWT)
strain. The mouse experiments proved that BLP prevented lethal Listeria monocytogenes (L.
monocytogenes) infection in mice, and the colonization and mucosal adhesion of the BLP
strains in the intestine were increased compared with those of the WT strain, and the
increased colonization was maintained despite Lm infection. Moreover, the coaggregation of
both the BLP strains (probed with anti-LAP mAb (an antibody against Hsp60)) with Lm
(probed with ant-Lm pAb) on the colonic villi and the absence of Lm strains in the lamina
propria were verified by immunostaining (Figure 18a). In addition, BLP formed increased
biofilms in monoculture (BLP alone, approximately 3-fold) or in coculture with Lm (BLP +
Lm, 1:1 ratio, ~3.5-fold) and restricted Lm cells at the lumen and epithelial surface, which
100
might contribute to the coaggregation of Lm strains (Figure 18b). Additionally, fluorescent
observation also confirmed the localization of Lbc aggregates on the surface of epithelial
cells in the BLP-treated mice while in LbcWT-treated mice, Lbc was mostly restricted to the
luminal mucus layer (Figure 18c). Three plausible mechanisms were put forward by the
authors (Figure 18d), including competitive exclusion, restored intestinal barrier function,
and immunomodulation. In another study, the same group developed an EcN that expressed
microcin H47 under the induction of tetrathionate (Figure 18e).[227] The engineered strain
could inhibit the growth of Salmonella in anaerobic conditions in the presence of
tetrathionate. Koh et al. designed a genetic circuit bearing a genetically encoded sensor,
amplifier, and actuator, enabling the conversion of the conjugated bile salts to the
unconjugated forms through the hydrolase-mediated modulation strategy.[228] The engineered
EcN inhibited the germination of endospores of C. difficile, prevented the growth of C.
difficile vegetative cells, and further significantly reduced CDI (Clostridium difficile
infection) in mouse model, as proved by a 100% viability and improved clinical outcomes.
101
Figure 18. Genetically engineered probiotics for the treatment of microbial infection. a)
Micrographs showing the stained colonic villi of mice treated with LbcWT or BLP for 10
days. b) Evaluation results of the BLP biofilm formation in different culture conditions by
crystal violet staining. c) Quantitative results of the L. casei cells attached to the epithelial
cells obtained from fluorescent in-situ hybridization (FISH) images. d) Scheme displaying the
mechanism of BLP-mediated protection against listeriosis. a‒d) Reprinted from Ref.[226] with
permission from Nature Publishing Group. Copyright 2020. e) Engineered probiotic for
inhibiting the growth of Salmonella via tetrathionate-induced production of microcin H47.
Reprinted from Ref.[227] with permission from American Chemical Society. Copyright 2018.
6.3. Cancer Immunotherapy
Cancer immunotherapy plays a revolutionized role in the field of oncology by

prolonging the survival time of patients with rapidly fatal cancers. Chung et al. engineered a
Pediococcus pentosaceus strain that encoded the therapeutic protein P8 fused to a secretion
signal peptide and a complementation system for colorectal cancer therapy (Figure 19a).[229]
The tumor volume was significantly reduced and the tumor growth was also remarkably
inhibited when the synthetic probiotic was used for colorectal cancer (CRC) therapy.
Moreover, the synthetic probiotic modulated gut microbiota and restored the normal intestinal
microecology. Correlation analysis showed that specific bacterial taxa, such as Akkermansia
or Turicibacter may be closely related to eubiosis or dysbiosis. This work provides stable and
effective synthetic probiotics as cell-based designer biopharmaceutical agents for treating
CRC and ameliorating impaired microbiota.
In another study, Yue et al. modified the symbiotic bacteria to express a specific tumor
antigen fused with the protein cytolysin A on the surface of outer membrane vesicles (OMVs)
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of the commensal bacteria (Figure 19b).[230] Oral coadministration of the genetically
engineered E. coli and arabinose led to the production of OMVs, which crossed the intestinal
epithelium into the lamina propria to stimulate dendritic cell maturation. In vivo study
showed that OMVs containing the tumor antigens inhibited tumor growth and protected the
animals from tumor recovery. This strategy of producing OMVs in situ by genetically
engineering commensal bacteria is also promising for developing other oral vaccines and
therapeutics.
Collectively, benefiting from the rapid advances in synthetic biology and the deep
understanding of the probiotic genome information, genetically engineered probiotics with
unique and combinatorial functionalities as therapeutic agents for cancer immunotherapy
become possible.
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Figure 19. Genetically engineered probiotics for cancer immunotherapy. a) Modular design
Reprinted from Ref.[229] with permission from Nature Publishing Group. Copyright 2021. b)
of a lactic acid bacterium-based drug delivery system for realizing optimal P8 productivity.
104
Genetically engineered bacteria-derived OMVs for oral tumor immunotherapy. Reprinted
from Ref.[230] with permission from Nature Publishing Group. Copyright 2022.
To sum up, we have reviewed the recent advances in the engineering strategies for
improving the intestinal delivery efficiency of probiotics and the applications of the
engineered probiotics through oral administration in Sections 4 and 5, as shown in Table 4.
Numerous encapsulation strategies developed for intestinal delivery of probiotics have been
proved to be effective in enhancing the therapeutic effect of probiotics. For the four
introduced encapsulation strategies, including the physical, chemical, genetic engineering,
and the combination strategies, each strategy owns its inherent advantages and disadvantages
depending on the specific application conditions.
Table 4. Probiotic encapsulation and their applications.
Probiotic Probiotic engineering Bacteria Application of the Reference
engineering approach engineered probiotics
type
Chemical Biotin–streptavidin Lactobacillus Improving the [187]
engineering conjugation casei, biotherapy ability of
Escherichia probiotics
coli, and
Bacillus
coagulans
105
Tannic acid (TA) and EcN Improving the [72]
enteric L100 coating intestinal retention
time and the
therapeutic effect of
the probiotics against
colitis
Self-assembly coating Escherichia [193]
with tannic acid and coli
poloxamer 188
Bioorthogonal click Clostridium Enhancing the [191]
chemistry butyricum colonization
efficiency and
therapeutic effect of
the probiotics against
colitis
Physical Biofilm encapsulation Lactobacillus Improving the [147]
engineering plantarum
antibiotic resistance
and adhesion capacity
of probiotics
Bacillus Improving the [144]
subtilis survival rate and the
106
mucoadhesion ability
of the probiotics
Lipid coating Escherichia Improving the [150]
coli survival rate and the
bioactivity of the
probiotics in the harsh
environmental
conditions
Lactobacillus Improving the [85]
plantarum survival rate of the
probiotics
Lactobacillus Colon-targeted [151]
plantarum release
reuteri survival rate of the
probiotics
Cell membrane Escherichia Diagnosis, [158]
encapsulation coli bioimaging, and
therapy applications
Nanomaterial coating Escherichia Improving the [71]
coli bioavailability and
107
therapeutic efficacy of
the probiotics
Escherichia Resisting to [154]
coli, antibiotics and
Lactobacillus reducing AAD
casei
Lactobacillus Improving the gastric [155]
Rhamnosus tolerance and
and enhancing the
Lactobacillus adhesion ability of the
plantarum probiotics
Hydrogel Lactobacillus Improving the [167]
encapsulation plantarum survival rate of the
probiotics
Lactobacillus Promoting the [75]
lactis angiogenesis in
diabetic wounds
acidophilus therapeutic effect of
orally administered
probiotics in patients
suffering from colonic
IBD
108
Lactobacillus Promoting the healing [166]
rhamnosus of superbacteria-
infected wounds
LBL encapsulation Bacillus Enhancing the [170]
coagulans intestinal colonization
and increasing the
growth rate of the
probiotics
plantarum survival rate of the
probiotics
Film encapsulation Lactobacillus Facilitating the [172]
fermentum or antibacterial activity
Lactobacillus
gasseri
Nanofibers Lactobacillus Improving the [177]
encapsulation plantarum survival rate of the
probiotics
Microsphere Lactobacillus Enhancing the [108]
Encapsulation lactis mucoadhesion and
colonization of
probiotics
109
casei survival rate of
probiotics
Lactobacillus Treating cholestatic [181]
delbrueckii DILI
subsp.
bulgaricus and
Lactobacillus
rhamnosus GG
Prebiotic Lactobacillus Resisting to acid [89]
Encapsulation and Bacillus without changing the
subtilis probiotic activity
The spores of Inhibiting tumor [32]
Clostridium growth
butyricum
Nanozyme Escherichia Improving the [136]
encapsulation coli survival rate of the
probiotics
Genetic Surface modification Escherichia Regulating capsular [214]
engineering by expression of a coli polysaccharide (CAP)
kfiC gene encoding a production for
glycotransferase of anticancer application
110
GlcNAc) in the
genome of EcN
In vivo modification Escherichia Antitumor application [67]
of EcN by expression coli
of Tum 5-p53 fusion
protein in the bacterial
genome
Expression of CAT Escherichia Alleviating [173]
encoding gene in EcN coli intratumoral hypoxia
and inhibiting the
tumor growth
Deleting the arginine Escherichia Antitumor application [231]
repressor gene (ArgR) coli
from EcN and
integrating ArgAfb into
the genome of EcN
Combined Overexpression of Escherichia Relieving [173]
strategy catalase and coli inflammation,
superoxide dismutase repairing epithelial
in EcN (forming barriers in the colon,
ECN-pE) and then modulating the gut
coating ECN-pE with microbiota, and
chitosan and sodium elevating the
alginate via a layer-
111
by-layer electrostatic intestinal flora
self-assembly strategy richness and diversity
Expression of NDH-2 Escherichia Targeted cancer [215]
(type II NADH - coli treatment
dehydrogenase) or
mCherry/NDH-2 gene
in the genome of EcN
and coating the
engineered EcN with
magnetic nanocubes
Expression of vascular Lactobacillus Promoting [75]
endothelial growth lactis angiogenesis in a
factor (VEGF) and diabetic wound
encapsulation of the
engineered bacteria
with the heparin-
poloxamer
thermoresponsive
hydrogel
7. Conclusions and Outlook
Probiotics show great potential as therapeutic agents to regulate the intestinal

environment, maintain the homeostasis of the intestine, and regulate the body immunity to
112
fight against various diseases due to their diverse properties and functions. However, the
survival rate and activity of probiotics are greatly reduced after oral administration, affecting
their beneficial and therapeutic effects in vivo. Although numerous fundamental studies that
employ probiotic engineering to solve the above problems have been reported, moving the
biomedical applications of probiotics or probiotic-based materials from lab to clinic seems a
difficult task at present, possibly due to the fact that some important properties of probiotics
are still not fully discovered and complete clinical trials are not conducted. As an overview of
the intestinal delivery of probiotics, this review not only summarizes the previous studies of
probiotics from the aspects of the types and encapsulation materials of intestinal delivery
probiotics, modification strategies for improving the intestinal delivery rate, clinical analysis
of probiotics, and applications involving the intestinal delivery of engineered probiotics, but
also proposes some unsolved problems of the current studies and puts forward several future
research directions in this field.
Although there have been huge advancements in probiotic intestinal delivery and the
application of engineered probiotics in the past years, some unsolved issues still exist and
need to be addressed. (1) Discovering and developing new types of probiotics that are more
suitable for intestinal delivery are still necessary for overcoming the complex human
gastrointestinal situation. Future studies should focus on expanding the range of candidate
probiotics essential for suiting customers/patients’ specific needs. (2) The physiological
stability, encapsulation efficiency, active targeting ability, and traceability of the current
encapsulated probiotics are still not satisfactory. For this reason, new encapsulation
biomaterials, such as the extracellular vesicles, microbial spores, fungal cell walls,
microalgae shells, etc., and the advanced strategies, such as microfluidics, biomeneralization,
bioprinting (3D or 4D printing) technique should be further developed to satisfy the different
purposes. (3) Co-encapsulation of antibiotics and probiotics is very challenging due to that
113
most probiotics are susceptible to antibiotics and cannot survive coadministration. Although
there have been some genetic strategies that aim to endow probiotics with antibiotic
resistance capacity to overcome this problem, more efficient and safe methods for co-
encapsulation of probiotics and antibiotics still need to be explored in the future. Therefore,
future encapsulation strategies that can locate probiotics and antibiotics in different zones
within the same encapsulation system are needed. (4) Owing to the lack of sophisticated
quantitative and high-throughput approaches, designing engineered LBPs for therapeutic
applications is still difficult. The utilization of ML and integration of AI help to rapidly
identify the functions and properties of probiotics from large-scale whole-genome primary
sequences, greatly reducing the experimental workload and accelerating the design and
formulation process of LBPs before experiments. (5) The lack of the gene editing tools and
unclear genome information of most probiotics still represent the main obstacles for
preparing genetic engineering probiotics. Currently, most genetic engineering strategies focus
on EcN due to its clear genome information and the presence of multiple gene editing tools.
A few genetic engineering approaches are performed on L. reuteri, L. lactis, and B. longum.
Thus, mining more genome information of other probiotics and implementing efficient and
robust genetic manipulation strategies for other species of probiotics are highly desired for
obtaining various functional probiotics with improved intestinal delivery efficiencies. (6) The
bioactivity and effectiveness evaluations of the engineered probiotics after experiencing the
manufacturing and oral ingestion process are still not enough. New evaluation strategies and
government regulations should be developed. In addition, the time of oral administration of
the engineered probiotics is not carefully considered by researchers. Commonly, in scientific
experiments, the engineered probiotics are often applied immediately after fabrication.
However, in the clinic, a long transportation and storage time is usually required before use
by patients, which greatly limits the clinical translation of the engineered probiotics. Thus, it
is necessary to develop appropriate preservation approaches for the engineered probiotics. (7)
114
To enable the widespread application of the engineered probiotics, it is important to develop
suitable large-scale production techniques and choose easily processable and cost-effective
encapsulation materials to prepare safe and stable encapsulated probiotics. Moreover, the
biosafety and in vivo fate of the encapsulation materials are also crucial for realizing
successful clinical translation of encapsulated probiotics, as the encapsulation materials
influence the function of probiotics and interact with the blood proteins and cells, normal
tissue cells, and immune system. Hence, it is desirable to evaluate the detailed interaction
processes between probiotics and the encapsulation materials, and the foreign body responses
of humans to the encapsulation materials.
Despite the above mentioned challenges, it is expected that engineered probiotics will
play a vital role in future disease treatments. If the aforementioned problems can be solved,
intestinal delivery of engineered probiotics will provide researchers and doctors with
powerful solutions to overcome some longstanding scientific and technical challenges in the
biomedical field. It is also hoped that this review will inspire future researchers to develop
more probiotic engineering strategies and engineered probiotic-containing LBPs and promote
their successful clinical translation for combating various diseases.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (32101916)
and the Natural Science Foundation of Jiangsu Province (BK20200619). The Figure 1 and
Figure 14 in this article were created with the help of BioRender (www.biorender.com).
Conflict of Interest
115
The authors declare no conflict of interest.
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Author Biography
Dr. Chengcheng Li is an associate professor of College of Light Industry and Food

Engineering at Nanjing Forestry University, China. She received her Ph.D. degree from
Southeast University in 2018. Her main research interests are natural nanomaterials and
probiotic delivery.
136
Mr. Zi-Xi Wang received his B.S. degree from Southeast University in 2022. Currently, he
is a master student under the guidance of Prof. Fu-Gen Wu at School of Biological Science
and Medical Engineering, Southeast University. His research interest is mainly about the
fabrication of antibacterial materials for wound healing applications.
137
Dr. Huining Xiao, a professor in Chemical Engineering at University of New Brunswick
(UNB) and the Fellow of Canadian Academy of Engineering (FCAE), obtained his Ph.D.
degree in Chemical Engineering at McMaster University in Canada in 1995. Before joining
the UNB in 2001, he was a tenured lecturer at University of Manchester in the UK from 1996
to 2001. His research interests mainly cover the cellulose or nanofiber materials, antibacterial
and antiviral polymers, green packaging materials, and bioadsorbents and responsive
hydrogels.
Dr. Fu-Gen Wu is a full professor of Biomedical Engineering at Southeast University,

Nanjing, China. He obtained his B.S. and Ph.D. degrees from Tsinghua University in 2006
and 2011, respectively. After a postdoctoral period at University of Michigan-Ann Arbor, he
joined Southeast University in 2013. His main research interests are nanodrugs and
biomaterials.
138
A comprehensive review on the progress of intestinal delivery of probiotics for the diagnosis
and therapy of various diseases is provided from the aspects of definition, type, action

Graphic abstract
139
mechanism, encapsulation strategy, probiotic-derived materials, therapeutic application, as
well as the synthetic biology for probiotics. The current limitations and future research

directions in the intestinal delivery of probiotics are also discussed.
140

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Intestinal Delivery of Probiotics: Materials, Strategies, and Applications

Chengcheng Lia,*, Zi-Xi Wangb, Huining Xiaoc, Fu-Gen Wub,*

Chengcheng Li, E-mail: lichengcheng@njfu.edu.cn

Fu-Gen Wu, E-mail: wufg@seu.edu.cn

This article is protected by copyright. All rights reserved.

Keywords: probiotic, intestinal delivery, encapsulation, synthetic biology, therapy

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This article is protected by copyright. All rights reserved.

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Here, we will comprehensively summarize the advances in the intestinal delivery of

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Figure 1. Scheme illustrating the types of probiotics and probiotic-derived materials

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2. Types and Encapsulation Materials of Intestinal Delivery Probiotics

2.1. Live Probiotics

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2.2. Inactivated Probiotics

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Table 1. Probiotic types, their encapsulation materials, and the applications.

Type Species Application Encapsulation Reference

Conventional Lactobacillus Treating colitis Alginate [80]

Lactobacillus casei Enhancing both Soybean protein- [82]

Lactobacillus. Enhancing the storage Pickering high [85,88]

Lactobacillus Enhancing Exfoliated

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Lactobacillus Wound Methacrylate- [34]

Bifidobacterium Photothermal cancer Indocyanine [93]

Bifidobacterium Protection against Protamine-assisted [92]

Streptococcus Enhancing stability and Endogenous [95]

Lactococcus lactis Improving gastric acid Thiolated oxidized [108]

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Non- Saccharomyces – Nanostructured silica [59]

Escherichia coli Treating intestinal Silk fibroin; Tannic [21,71–

Bacillus Treating metabolic Alginate [89]

Clostridium Regulating gut Dextran [32]

Next- Akkermansia Improving the survival Xanthan and gellan [86]

Faecalibacterium Improving the survival Lipid [99]

2.3. Live Biotherapeutic Products (LBPs)

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Table 2. LBPs in clinical trials.

any cts stration A se condition

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Biosci (toget 2 https://clinicaltrials.gov/se

ences, her arch?term=NCT03788434

VE70 / MDROs https://www.vedantabio.co

VE20 Oral / NCT05370885 Pha UC https://www.vedantabio.co

2 capsule se m/pipeline/ve202 and

VE80 Oral / NCT04208958 Pha Advanced https://www.vedantabio.co

(toget 1/2 metastatic https://clinicaltrials.gov/se

her cancer arch?term=NCT04208958

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VE41 Oral / NCT03936998 Pha Food https://www.vedantabio.co

6 se allergies m/clinical-trials/ve416 and

4D MRx Oral / NCT03637803 Pha Tumor [120]

Moon MNC Oral / IN Tumor [121]

Synlo SYN Oral / NCT04629170 Pha Enteral [122]

gic B880 se hyperoxal

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Xbiom XBI- Oral / NCT05001360 IN GvHD [124]

Guang SK08 / Pha IBS https://www.zypharm.com.

Chengcheng Lia,, Zi-Xi Wangb, Huining Xiaoc, Fu-Gen Wub,