Chitosan polysaccharide-based nanoparticles for oral insulin delivery

Introduction

Over the last decades, the interest of the pharmaceutical therapy utilizing nanotechnology in diabetes
treatment has been dramatically increased. Different types of insulin-loaded nanoparticles as oral
delivery systems have been developed, such as natural polymeric nanoparticles, synthetic polymeric
nanoparticles, solid lipid nanoparticles, liposomes, and nanoemulsions, as well as inorganic
nanoparticles . These nanoscale delivery systems aim to endure difficulties and provide a convenient
route of insulin delivery. Compared to other nanoparticles, the natural polymeric nanoparticles
exhibited higher biocompatibility and biodegradability, greater safety, and better storage and
physiological stability.

Barriers in GI track for efficient oral insulin delivery

Significant challenges still remain for developing effective insulin-loaded nanoparticles for oral delivery,
due to some physiological barriers in GI tract, which can be classified as chemical, enzymatic and
physical barriers.

-Chemical barrier

The first challenge for nanoparticles is the stability over a wide range of pH variation in GI tract.
Nanoparticles reach stomach from mouth in less than 1 min where they encounter a sudden reduction
in pH from 6.8 to 1.2, followed by transition from highly acidic environment to slightly basic
environment (pH 6.5 to 8.0) in small intestine. The mean transit time in stomach and residence time in
small intestine is around 2.5 h and 3–4 h, respectively. Studies have shown that the dissociation of
nanoparticulate structure could happen due to the deprotonation or protonation of particle
components (such as polysaccharides, proteins or lipids) in different pH environment, resulting in the
burst release and degradation of encapsulated bioactive compounds . Hence, good GI-stability of
nanoparticles across a wide range of pH is crucial to protect insulin throughout the entire GI transition
against chemical degradation and improve its oral bioavailability.
-Enzymatic barrier

The enzymatic degradation is another challenge for oral delivery of insulin. The susceptibility of
nanoparticles to various digestive enzymes in GI tract poses a major obstacle to achieving desired
efficacy for oral delivery. The decomposition of lipid- and protein- based nanoparticles begins in the
stomach by gastric lipase and pepsin, respectively . The degradation continues in small intestinal lumen
by pancreatic enzymes, including lipase, trypsin, α-chymotrypsin, elastase, and carboxypeptidases A and
B . Even though the decomposition of polysaccharide based nanoparticles does not happen in stomach,
their nanostructure may disassemble due to weakened electrostatic interactions with other
polyelectrolytes. As a result, a rapid release of insulin from carrier to harsh GI conditions may occur due
to degradation of polymer network and hence disassembly of nanoparticulate structure.

-Physical barrier

After nanoparticles overcome the chemical and enzymatic degradation and successfully transit to the
intestinal lumen, they will be exposed to the intestinal epithelium layer covering the surface of the GI
tract, which is a collection of enterocytes, goblet cells, Paneth cells and microfold cells, constituting
another major barrier before absorption. Goblet cells are able to produce a mucus layer that covers and
protects the underlying epithelium. The mucus layer is a negatively charged viscous mixture containing
mucins, antiseptic enzymes, immunoglobulins and inorganic salts, and thus it is capable to trap those
molecules with high molecular weight and low permeability (such as insulin), as well as nanoparticles
possessing strong positive charge and hydrophobic nature in surface properties.
Chitosan

Preparation methods

Chitosan is a linear polysaccharide derived from chitin via deacetylation process. It is composed of
randomly distributed D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit),
it is made by treating chitin shells of shrimp, crustaceans, with alkaline substances like sodium
hydroxide. Due to the desirable physicochemical properties, such as biodegradability, non-toxicity, good
mucoadhesion, abundant renewable sources and low cost, chitosan and its derivatives have been widely
used for food, medicine and pharmaceutical applications, especially the development of nanoscale
delivery systems

As the unique cationic polysaccharide, chitosan is able to interact with polyanions leading to the
spontaneous formation of nanoparticles, which is generally referred as ionic gelation or polyelectrolyte
complexation method. In general, chitosan-based nanoparticles are prepared by allowing chitosan or its
derivatives to react with various anionic polyelectrolytes, among which the most common ones include
tripolyphosphate (TPP) alginate, poly (g-glutamic acid)] and lecithin .

Although most preparation methods are based on this principle, the experimental protocols vary with
different polyelectrolytes and chitosan derivatives, and five commonly reported protocols to prepare
chitosan-based nanoparticles for insulin oral delivery are.

First and the most commonly used protocol is to dissolve insulin in hydrochloric acid and mixed with
anionic polymer, to which mixture is added to chitosan solution with mild stirring to form core-shell
nanoparticles via electrostatic interactions. For instance, insulin was first mixed with alginate to form the
ionic polyelectrolytes and then the mixture was reacted with chitosan to form nanoparticles. The as-
prepared nanoparticles had a particle size of 100–200 nm and insulin loading efficiency of 15.7%.

Second, in order to improve the solubility of chitosan in aqueous phase and facilitate the transportation
in GI tract, various water soluble chitosan derivatives, i.e., N-trimethylated chitosan (TMCS) ,
carboxymethyl chitosan (CMCS) , deoxycholic acid chitosan (DCS) , and methoxy poly(ethylene glycol)
amine grafted chitosan (PEG-g-CS) , have been studied and applied in preparation of nanocomplexes.
DCS, for instance, is a water-soluble chitosan derivative obtained by grafting deoxycholic acid onto
chitosan backbone using 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-
hydroxysuccinimide (NHS). DCS was premixed with insulin and intermediate nanoparticles were formed
by adjusting pH to 6.21, followed by adding aqueous poly (γ-glutamic acid) (γ-PGA) into the mixture.
The nanocomplexes with a particle size of 226.1 nm and insulin loading efficiency of 33.0% was
obtained.
Third, as a negatively charged peptide, insulin can also act as an anionic polymer and react with
positively charged chitosan or its derivatives directly to form self-assembled nanoparticles via
electrostatic interactions. prepared self-assembled nanoparticles with small particle size of 210 nm and
high insulin loading efficiency of 39.2% by directly mixing equal volume of chitosan and insulin solutions
Although the preparation method is simple, the nanoparticulate structure of these nanoparticles formed
by directly mixing chitosan and insulin may be very vulnerable to environmental changes.

Alternative fabrication methods have been also used in other studies

Developing a selfnanoemulsifying insulin delivery system by mixing propylene glycols diester, porcine
insulin and chitosan together with the presence of surfactant (cremophor EL) and co-solvent (glycerol
and DMSO) .

In another study , a water/oil/water (W/O/W) double emulsion was prepared from poly (lactic-co-
glycolic acid) (PLGA) and folic acid modified chitosan (FA-CS) . Typically, the insulin solution was added
into oil phase (ethyl acetate oil containing PLGA and insulin) to fabricate phase one of W/O emulsion.
Then, W/O emulsion was added into water phase containing poly (vinyl alcohol) (PVA) and folic acid-
chitosan conjugate to form W/O/W double emulsion with a particle size of 252.4 nm and insulin loading
efficiency of 6.8%.

Apart from acting as a major material in the preparation of nanoparticles, chitosan has also been
chemically conjugated on the surface of nanoparticles as a coating material to enhance the chemical and
enzymatic stability, as well as the mucoadhesion . To further facilitate the absorption of nanoparticles by
receptor mediated endocytosis, several epithelial cell membrane targeted ligands, such as cell-
penetration peptides (CPP), vitamin B12 , and L-valine , can be further grafted on chitosan coating.

Due to the functional groups on the backbone of chitosan, including amino/acetamido group, primary
and secondary hydroxyl groups at C-2, C-3 and C-6 positions , the grafting process could be easily
achieved. For instance, in a recent study , TMCS was synthesized by methylation of amine groups of
chitosan with methyl iodide, followed by conjugation with CPP using EDC/ NHS . Subsequently, insulin-
loaded amphiphilic dodecylaminegraft-g-polyglutamic acid (PGA-g-DA) micelles were coated with as
prepared TMCS conjugate to form nanoparticles with a particle size of 184.5 nm and loading efficiency
of 12.0%.
Mechanisms to overcome multiple barriers

The in vitro insulin release study is a common method to evaluate the chemical and enzymatic stability
of chitosan-based nanoparticles. Typically, insulin loaded nanoparticles are

- first incubated in simulated gastric fluid (SGF, pH 1.2–2.0) with pepsin, followed by transferring to
simulated intestinal fluid (SIF, pH 6.5–7.4) with pancreatin, mimicking the real fate of insulin in GI tract.

The possible mechanism of insulin release is the disassembly of matrix network due to the weakened
electrostatic interactions by the variation of pH and strong ionic strength in GI tract . It is known that
chitosan is highly soluble under acidic environment in stomach due to the protonation of amine groups.

As a result, severe swelling or dissociation of chitosan-based nanoparticles could occur, leading to the
burst release of insulin.

On the contrary, chitosan becomes deprotonated and water-insoluble under basic environment in small
intestine, consequently weakening the polymers network and leading to the disruption of nanoparticles.
In order to overcome this drawback, some approaches have been applied involving modification of
chitosan and chemical cross-linking to strengthen the polymer network.

A nanoparticle delivery system based on CMCS and phenylboronic acid was synthesized in an attempt
to improve the stability in GI tract. Phenylboronic acid was used as a hydrophobic and glucose-sensitive
unit. Then Insulin was loaded into the nanoparticles via electrostatic interactions with CMCS and the
hydrophobic reactions with phenylboronic acid. The insulin release study was carried out in different pH
phosphate buffer solution containing different concentration of glucose. The release percentage in SGF
was only 16.6%, indicating an inhibitory effect on the release of insulin at low pH

Authors claimed that the sustained release profile in gastric condition was attributed to the protonated
carboxyl groups on CMCS restricting the swelling of nanoparticles.

In another approach, a hydrophobic polymer was applied as a coating on the surface of insulin-loaded
chitosan/TPP nanoparticles to ensure its integrate structure in GI tract. The insulin release percentages
in pH 1.2 and 6.8 medium were approximately 10 and 40%, respectively.

In a recent study prepared the nanoemulsions for oral delivery of insulin using PLGA (poly lactic-co
glycolic acid) and folic acid-modified chitosan. PLGA was used as a pharmaceutical excipient to associate
with insulin in ethyl acetate oil and form the oil phase, while chitosan act as a positive charged
polyelectrolyte in water phase. The results of insulin release in vitro demonstrated that only 32.2 and
34.9% of insulin were released from nanoemulsions after 6 h incubation in SGF and SIF, respectively.
More than 60% of insulin was found intact with the existence of digestive enzymes and remained in the
nanoemulsions, while only about 10% free insulin (not encapsulated) was detected after incubation in
simulated GI fluids. After oral administration of insulin-loaded nanoemulsions to the diabetic rats, more
prolonged hypoglycemic activity was observed compared to subcutaneous injection of insulin.
A dramatic increase of insulin oral bioavailability by 14-fold was noticed when comparing the insulin-
loaded nanoemulsions to free insulin solution at equivalent dosage of oral administration

Chitosan is one of the few biopolymers with known mucoadhesive property and TJs-opening capability,
and several recently published studies focused on the elaboration of underlying cellular mechanisms by
which chitosan nanoparticles facilitated the transepithelial transport of insulin. The transepithelial
electrical resistant value (TEER) of Caco-2 monolayer’s (heterogeneous human epithelial colorectal
adenocarcinoma) is a well-studied indicator to the permeability across the paracellular barrier .

The significant reduction of TEER of epithelial monolayers suggests the opening of TJs and the passage of
nanoparticles via paracellular route

Other studies prepared a calcium phosphate nanoparticles coated with vitamin B12-chitosan conjugate
and sodium alginate. The nanoparticles coated with native chitosan and sodium alginate were used as
control. Vitamin B12 has been reported as a ligand for intrinsic factor (IF) receptor present on the ileum
wall, which initiates the IF receptor mediated transepithelial transport. By comparing the TEER reduction
after Caco-2 cells were treated by vitamin B12 grafted-chitosan coated nanoparticles with control,
authors claimed that modification of chitosan with vitamin B12 was necessary to simultaneously
improve TJs-opening capability and epithelial transport of nanoparticles. Notably, the enhanced
TJsopening capability was ascribed to the better solubility of B12-graftedchitosan/alginate nanoparticles
under neutral pH, whereas the greater epithelial transport was attributed to additional receptor
mediated endocytosis via IF receptor .

Animal study further indicated that insulin loaded vitamin B12 grafted-chitosan/alginate nanoparticles
(at the dose of 50 IU/kg) exhibited sustained hypoglycemic effects up to 12 h compared to subcutaneous
administered insulin (at the dose of 5 IU/kg). Although many evidences have shown that mucoadhesive
property of chitosan contributes to the high affinity of nanoparticles with mucus layer and permeability,
controversially, a recent study concluded that this property may actually lead to the retention of
nanoparticles within mucus layer and limit the access to epithelial surface.

Other studies] prepared novel TMCS-insulin nanoparticles via self-assembly which was further coated
with a hydrophilic N-(2-hydroxypropyl) methacrylamide copolymer (pHPMA) derivative on the surface.

In mucus diffusion study, the confocal laser scanning microscope (CLSM) images showed large
aggregates of uncoated nanoparticles and largely overlaid signals with mucus. In comparison, the
nanoparticles with pHPMA coating could diffuse through the mucus and get access to the underlying
epithelial cells more efficiently, indicating that pHPMA molecules increased the mucus permeability by
reducing the interaction between nanoparticles and mucin. The dissociation of pHPMA coating
happened after crossing mucus, allowing the intact insulin-TMCS nanoparticles to interact with αvβ3
integrin receptors on cell membrane. According to the TEER measurement and CLSM results, the
transient and reversible TJs opening was caused by chitosanmediated conformation change of αvβ3
integrin receptor, filamentous actin (F-actin) reorganization and claudin-4 down regulation. This
mechanism was also confirmed

in another study , in which the dephosphorylation of claudin-4 was observed, followed by translocation
of claudin-4 from cell membrane to cytosol and TJs opening after oral administration of CMCS-based
nanoparticles.

In another study, a PLGA-monomethoxy-PEG micelles coated with targeting ligand FQSIYPpIK (FQS)
peptide-modified TMCS was prepared , which enhanced paracellular transport across Caco-2 cells which
over-expressed integrin αvβ3 receptor. In addition, the pharmacokinetics study showed that the oral
bioavailability of insulin encapsulated in as-prepared nanoparticles was 25-fold higher than that of free
insulin solution. Besides, particle size also plays an important role in transepithelial transport via
paracellular pathway.

In a recent study, prepared chitosan/TPP nanoparticles with different sizes (45 and 115 nm) and
explored the size-dependent effects on insulin delivery by both in vitro cellular and in vivo animal
models. Both nanoparticles were able to induce a fast reduction of TEER in Caco-2 cell monolayers
within 2 h indicative of effective opening of TJs. However, the apparent permeability (Papp) value of
insulin in smaller nanoparticles was significantly higher than that of larger nanoparticles, revealing that
the insulin transportation through Caco-2 monolayers was size dependent. In addition, the smaller
nanoparticles exhibited significantly better amelioration on blood glucose level through oral
administration in type I diabetes rat model than the larger nanoparticles .

Apart from paracellular pathway, some strategies have been exploited to fabricate novel nanoparticles
aiming to improve the transepithelial transport via transcellular pathway. Grafting different cell
membrane targeting ligands onto the surface of nanoparticles or chitosan coating is a commonly used
approach.
Some studies have modified TMCS with a goblet cell-targeting peptide (CSK), which was then applied as
polymeric coating on the surface of insulin-loaded dodecylamine-graft-g-polyglutamic acid micelles. The
results of cellular uptake by Caco-2/E12 co-cultures revealed that an energy-dependent active transport
process was mediated by the specific affinity of CSK peptide, leading to the increased intracellular
uptake of nanoparticles and thus the enhanced transcellular permeation of insulin .

To further specify the endocytotic pathway, different endocytosis inhibitors were used, including
chlorpromazine, filipin, nystatin, and amiloride. They claimed that both clathrin-dependent and
caveolae-dependent endocytosis were involved in the uptake of nanoparticles. In addition, this type of
nanoparticles exhibited a prolonged hypoglycemic response with relative bioavailability of 7.05% after
oral administration in diabetic rats.

Folic acid has also been reported as a ligand for cell membrane and promoting the endocytosis of
nanoparticles by folate receptor. In a recent study, insulin-loaded nanoparticles were prepared with
PLGA and folic acid modified chitosan by ionic gelation . The improved intracellular delivery of
nanoparticles via folate-mediated endocytosis was confirmed by CLSM .

In another study, a novel deoxycholic acidmodified chitosan nanoparticles has been proved to enhance
the cellular internalization via apical sodium-dependent bile acid transporter (ASBT)-mediated
endocytosis .

In order to further study the mechanism of intracellular trafficking and basolateral release of insulin,
stimulated emission depletion microscope (STED) was employed to visualize the intracellular interaction
between nanoparticles and immunofluorescence stained endosomes and lysosomes

. It is known that the endosomal trafficking is a complex intracellular process involving collection of
internalized cargoes into vesicles, sortation, dissociation, and maturation of vesicles in endosomes and
lysosomes . The entrapped nanoparticles in vesicles can be digested by enzymes in the late endosomes
and lysosomes . Therefore, nanoparticles need to be engineered to escape from the endolysosomal
network for insulin oral administration. Through STED microscopy, they found that deoxycholic acid-
modified chitosan nanoparticles were located outside of endosomes and lysosomes, indicating the
effective endolysosomal escaping. This could be explained by the fact that deoxycholic acid is able to
perturb endolysosomal membrane structure due to its detergent properties. Moreover, according to the
pharmacokinetic studies, a remarkable oral bioavailability of 15.9% for insulin carried by deoxycholic
acid-modified chitosan nanoparticles was achieved in type I diabetic rats.
Future prospects and conclusions

Overcoming multiple barriers in GI tract remains the major challenge for oral delivery of insulin. Thus,
different insulin encapsulated polysaccharides-based nanoparticles have been used to protect insulin
from chemical and enzymatic degradation in stomach and small intestine, facilitate the mucus
permeation, improve the absorption of insulin by either TJs opening (paracellular pathway) or target
receptormediated endocytosis of epithelium cells (transcellular pathway), and finally achieve the
sustained hypoglycemia effect in diabetic animal model. Based on animal studies in recent literature,
there is still a large amount of insulin (N50% encapsulated insulin) released from nanocarriers before
reaching the absorption site, thereby reducing the portion of insulin administrated orally reaches the
blood stream to control over elevated blood glucose. In addition, more research efforts should focus on
understanding the mucus permeation of engineered nanoparticles. Some emerging evidence suggests
that mucus is not a diffusion barrier for insulin, which is in opposite to the traditional theory. Thus,
better understanding on the physicochemical properties of nanoparticles delivery system is needed for
improving mucus permeation in small intestine.

Furthermore comprehensive studies concerning the mechanism of intracellular trafficking and

basolateral release of insulin from nanoparticles are needed. Although different targeting ligands have
been used in facilitating the transepithelial transport of insulin, few studies demonstrated the
absorption of intact insulin-loaded nanoparticles into epithelium cells which is an ideal way to protect
and delivery encapsulated insulin, and the conclusive mechanism of exocytosis of nanoparticles is not
established yet. Moreover, by taking advantage of glucose-responsive materials, many types of insulin-
loaded nanoparticles that are able to release insulin at elevated blood glucose level have been
synthesized. Nevertheless, most of those glucose-responsive materials are usually obtained from
inorganic matter (such as silica) or synthetic polymers via rather complex and tedious synthesis process,
which may inevitably lead to high cost and potential toxicity. How to engineer naturally-occurring
polysaccharides with glucose-responsive property through facile and green chemistry could be very
challenging, but it definitely deserves further investigation in the future to potentially open new avenues
for safe and effective delivery of insulin via oral administration.