Professional Documents
Culture Documents
Red Blood Cell Membrane-Camouflaged Vancomycin and Chlorogenic
Red Blood Cell Membrane-Camouflaged Vancomycin and Chlorogenic
A R T I C L E I N F O A B S T R A C T
Keywords: Misuse of antibiotics leads to the emergence of multidrug-resistant (MDR) bacterial infection globally. Vanco
Multidrug resistance infections mycin (V), the so-called last line defense against methicillin resistance Staphylococcus aureus (MRSA), declines its
Vancomycin therapeutic effect each year due to high-level of resistance. Stewardships, drug-combination, development of
Chlorogenic acid
antibiotic delivery and biomimetic systems are the current practical strategies to reduce the resistance emer
On-demand antibiotic delivery
gence. In this study, red blood cell membrane (RBCM) and bacterial exotoxins-responsive supramolecular gelatin
Red blood cell membrane (RBCM)
Gelatin nanoparticles nanoparticles (SGNPs) loading vancomycin and chlorogenic acid (C) are used to construct the VC-SGNPs-R drug
delivery system to inhibit MRSA infection both in vitro and in vivo. RBCM coating evade the immune system,
prolong circulation and neutralize exotoxins in an infection-microenvironment. Moreover, excellent in vivo
inflammation healing, wound repairing and MRSA eradication were confirmed by leg and skin infection mice
model. The intense fluorescence molecular tomography (FMT) measurement of VC-SGNPs-R, tissue quantifica
tion and successful wound healing shows its capability for on-demand antibiotic delivery and responsiveness in
an infection-microenvironment.
This approach develops a biomimetic drug delivery system of VC-SGNPs-R with long circulation and syner
gistic effect against MDR bacteria.
* Corresponding author. School of Pharmacy, Shanghai Jiao Tong University, Rd. 800, Minhang District, Shanghai, 200240, China.
E-mail address: mfqiu@sjtu.edu.cn (M. Qiu).
1
Zul Kamal and Jing Su contributed equally to this work.
https://doi.org/10.1016/j.jddst.2022.103706
Received 30 April 2022; Received in revised form 9 August 2022; Accepted 11 August 2022
Available online 23 August 2022
1773-2247/© 2022 Elsevier B.V. All rights reserved.
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
nanoparticles (gelatin, PLGA, hydrogel, etc.) which shows responsive Biotechnology (Shanghai, China). Culture plates (96-well, Corning
ness at infection site. In situ, the exotoxins secrete pore-forming toxins Company), methicillin-resistant Staphylococcus aureus (MRSA) USA
(PFTs) and gelatinase enzymes, which permeate the RBCM and hydro 300 strains were provided by Dr. Mingkai Li Lab, Department of Phar
lyze the polymeric nanocore respectively to release drug [6,10–13]. Hu macology, School of Pharmacy, the Fourth Military Medical University,
et al. demonstrate that PLGA nanoparticles camouflaged with RBCM can Xi’an city, China on special request. Fluorescein isothiocyanate (FITC,
serve as toxin “nanosponge”, absorbing pore-forming toxins [5]. Lin 10 mg, Tianjin Biolite Biotech Co, Ltd, Tianjin, China), 1,1′ -dioctadecyl-
et al. propose validated biomimetic visualization selenium antibacterial 3,3,3′ ,3′ -tetramethylindotricarbocyanine iodide, (DiR, 10 mg, Dalian
nanosystem for synergism in mice MRSA infection model, but its Meliun Biotechnology Co., Ltd, Dalian, China), fetal bovine serum (FBS)
application was only limited to one spot in skin infection mice model, as and Dulbecco’s Modified Eagle Medium: (Nutrient Mixture F-12 DMEM/
well, large number of characterizations were required [8]. Chin et al. F12) were from Gibco Company (New York State, USA). DAPI dihy
propose RBCM nanosponge that can neutralize the bacterial virulence drochloride and MTT were from Beyotime Biotechnology (Shanghai,
factors in MRSA condition like sepsis and bacteremia [7]. Li et al. state China). Heparin was purchased from Sinopharm Chemical Reagent Co.,
the exotoxin-microenvironment responsiveness and on-demand anti Ltd. (Shanghai, China). All reagents were of analytical grade. Specific-
biotic delivery of vancomycin in-vitro [3]. Zhang et al. propose RBCM pathogen free male SD mice were supplied by Shanghai Jiesijie Exper
coated redox-responsive crosslinker nanogel embedded vancomycin for imental Animal Co., Ltd. The animal experiments were carried out
antivirulence and responsive delivery system against MRSA [10]. following the Guidelines for Care and Use of Laboratory Animals of
Similarly, Xinyi et al., also propose RBCM camouflaging tedizolid Shanghai Jiao Tong University and approved by the Animal Ethics
phosphate PLGA copolymer nanosystem against MDR strains, but the Committee (Number: A2018021).
study was limited only to the skin infections model [14]. In most of the
above studies, lack of biodistribution and FMT studies still left many 2.2. SGNPs preparations and drug loading
uncertainties regarding its target specificity, quantification, and release
[15]. SGNPs were prepared using the desolvation method [6,21], a 100 mL
The characteristics of MRSA is to form a biofilm on biotic and abiotic of gelatin solution (0.5% w/v, pH 6.0) was prepared by stirring the solid
surfaces which retain the efficacy of most clinically available antibiotics gelatin and ultrapure water at 50 ◦ C/1000 rpm. Then 20 mg, each of V
[16]. As we did mention, vancomycin declined its therapeutic effects and C in combinations were added to the filtered solution (at a ratio of
against MRSA each year due to high-level of resistance. So in this study, 2:2:10) and were kept on bath sonication for proper mixing and com
vancomycin and a polyphenol compound chlorogenic acid were loaded ponents miscibility. To induce the desolvation process, 20 mL of acetone
in combination for the first time to prepared SGNPs nanoparticles, which was added, until to obtained a faint turbidity. Finally, glutaraldehyde
were then coated with RBCM to obtained VC-SGNPs-R. Chlorogenic acid aqueous solution (10% v/v, 500 μL) was added to the gelatin solution
is abundant in fruits, vegetables and Chinese herbs, possesses anti-MRSA with continuous stirring at 1000 rpm for 2 h. An aqueous sodium met
biofilm effects through its quorum sensing effects, which mostly abisulphite solution (1.6% w/v, 8 mL) was added to stop the cross
potentiate various pathogenic traits, including biofilm formation linking. The nanoparticle-formed solution was dialyzed overnight to
[17–20]. This combination may show synergistic effects against MRSA remove the unloaded free V and C, and were lyophilized to form
biofilm and cell wall synthesis. VC-SGNPs nanoparticles. The FITC-labeled VC-SGNPs were prepared for
The VC-SGNPs-R surface characterization was confirmed for mem macrophage uptake research (methodology 2.6), adding 400 μL of FITC
brane retaining and cell uptake, which help in immune escaping, sus solution (50 μg/mL) into 600 μL 1 × PBS, which were then added in 1:1
tained release and prolong circulation. The in vitro anti-MRSA activities to VC-SGNPs.
were firstly studied through colony forming unit (CFU) and optical
density measurement (OD600 nm), while its morphological changes were 2.3. Preparation of RBCM derived vesicles
observed through AFM. Similarly, for the in vivo wound healing and
MRSA eradication, the leg and skin infection mice model were devel The RBCM derived vesicles were prepared by the hypotonic hemo
oped. VC-SGNPs-R were administered intravenously (I.V), which were lysis method [21,22]. The whole blood was collected from SD rats and in
then followed for FMT tissue quantification, biodistribution and wound tube containing 1.5 mg anticoagulant EDTA per mL of blood. The whole
healing, and accurately measured through IVIS live imaging and pho blood was centrifuged at 2000 rpm for 5 min at 4 ◦ C to remove plasma.
tographed in both mice infection model. The aim of this study was to Blood plasma, white blood cells, platelets, and the buffy coat were dis
develop a promising biomimetic nanosystem-coated RBCM delivery carded. The resulting RBCs were washed 3 times with ice-cold 1 × PBS
system for on-demand delivery, immune evading, prolong circulation following the removal of PBS. Then, add 0.2 mM EDTA for hemolysis
and synergistic effects of vancomycin in a combination with a natural along with 10 × PBS and repeated 3 times. The internal hemoglobin
compound of chlorogenic acid against MDR pathogens. contents were removed at 13 200 rpm for 10 min at 4 ◦ C, and the RBCM
derived vesicles were collected as pink pellets. The resulting RBCM
2. Material and methods vesicles were stored at − 80 ◦ C.
2.1. Material, cell and animals 2.4. RBCM coating and encapsulations efficiency
Vancomycin (HPLC ≥99%, Meilun Biologics, Dalian, China), RBCM coating of VC-SGNPs were carried out according to the pre
chlorogenic acid (≥95%, titration), bovine bone type-B (~250 g, viously published method [3,13]. VC-SGNPs nanocore were added to
Bloom), sodium metabisulfite (HPLC ≥99%) and dialysis bag of light pink RBCM suspension with 1:1 ratio and mixed together. The
8000–14000 MW (Sigma-Aldrich, US), glutaraldehyde solution (50% mixture was sonicated at 4 ◦ C for 2 min for membrane electroporation
wt. in H2O), ethylene diamine tetra acetic acid (EDTA, HPLC ≥99.5%, and coating. Then centrifuged at 7000 rpm for 5 min at 4 ◦ C, supernatant
Innochem Beijing, China), PBS, DAPI (2-(4-Amidinophenyl)-6-indole was discarded and VC-SGNPs-R was collected. The loading content
carbamidine dihydrochloride, 5 mg/mL) and SDS-PAGE gel quick (DLC) and encapsulation efficiency (DLE) of V and C in VC-SGNPs were
preparation kit were obtained from Beyotime Biotechnology (Shanghai, determined by RP-HPLC (Supplementary Infromation, S1) and calcu
China). RAW 264.7, 293T, and LO2 cell lines were purchased from Cell lated as following.
Culture Center of Institute of Basic Medical Sciences, Chinese Academy
W2 − W1
of Medical Sciences (Beijing, China), cell counting kit (CCK-8), and DLC (%) = × 100 (1)
W3
tryptic soy broth (TSB) were obtained from Beyotime Institute of
2
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
3
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
concentration of V and C were determined as per guidelines of clinical neck infection. Model 2: 2a, 2b, thigh infection. Model 3: 3a,3b, right leg
and laboratory standards institute using tetrazolium microplate assay in infection. Model 4: 4a, 4b), left leg infection were established with male
U-shaped 96-well plates, [3,8,30]. Then synergism in VC (combination) SD mice along with negative control (Cta, Ctb). The mice were anes
was determined through fractional inhibitory concentration (FIC) thetized with pentobarbital and the hair on the thighs, neck, and back
checkerboard dilution method (Supplementary Infromation, S4). The was removed. Then fresh culture of MRSA (108 CFU/mL, 50 μL) were
FIC index was calculated using the first turbid column and row, while for inoculated into the 8 mm knife cut injuries of neck and thigh regions
its determination using the formula, FIC index = FIC V + FIC C = while in legs it was injected intraplantarly in the dorsal surface of the
[V]/MIC V + [C]/MIC C, where [V], MIC V, FIC V, are the concentrations, right and left hind paw on Day 1. From Day 1 to Day 5, the mice were
MIC and FIC of vancomycin respectively, while [C], MIC C, FIC C are kept for incubation period at 37 ◦ C for 24 h observations. When in
represented in the same fashion for chlorogenic acid [21,31]. The MICs fections were fully developed, VC-SGNPs-R-DiR (coated with DiR
of V, C and all relative concentrations were used in various biological labeled fluorescence RBCM, Ex/Em = 748/780 nm) was injected
activities of VC-SGNPs-R, VC-SGNPs, SGNPs, VC (with and without intravenously via tail, for 5 consecutive days. FMT were measured both
RBCM). The bacterial kinetics viability, at different sample concentra in IVIS live imaging as well quantified for infection-microenvironment
tions 1/16 MIC, 1/8MIC, 1/4MIC, 1/2MIC, MIC, 2MIC μg/mL, and MIC responsiveness in all respective models.
alone were determined at 0, 0.3, 1, 2, 4, 6, 8, 12, 18 h time intervals. The For fluorescence biodistribution and quantification, the respective
bacterial culture in log phase was diluted with fresh medium and then mice were sacrificed after IVIS imaging. All vital organs including the
20 μL of bacteria OD600nm of 0.5 McFarland standard (equivalent to 1.5 infection sites were extracted and homogenized in normal saline (0.1 g
× 106 CFU/mL) were added to 96-well plate. PBS, SGNPs, VC-SGNPs of tissue/mL). Then it was centrifuged, the supernatant was collected
and VC-SGNPs-R were added to 96-well plate separately and cultured and quantified at 748 nm/780 nm. VC-SGNPs-R-DiR content in each
overnight, then were measured through multi-function microplate organ was expressed as a percentage of the VC-SGNPs-R-DiR injected
reader under OD600nm. The antibacterial effects was also shown by the dose per gram of tissue, denoted as % ID/g.
dilution coating TSA plate method. The MRSA suspension from well was
diluted 103-fold in 1 × PBS, and 100 μL of the diluted solution was 2.15. In vivo anti-MRSA activity for inflammation healing and wound
applied to the TSA plate, then cultured at 37 ◦ C overnight. CFU/mL repairing
quantification was evaluated through colony counting method using
image j-software 1.52v version. The groups treated with PBS were used The MRSA infection model for neck and right leg were selected for in
as control. All experiments were carried out in triplicates. vivo antibacterial activity. After the incubation period, on Day 7, VC, VC-
SGNPs, SGNPs-R, and VC-SGNPs-R (MIC conc, 100 μL, 2 mg/kg) were
2.12. Quantification of biofilm biomass administered IV into respective groups along with 1 × PBS (negative
control). Wound healing in skin infection model size and inflammation
Anti-biofilm activity of V and C (free drugs, SGNPs, and RBCM in leg infection model were assessed and recorded on a daily routine
coated samples) was determined against MRSA using the procedure of basis during the therapy (Supplementary Information, S5). For colony-
24-well microtiter plate [22]. The bacterial suspension from overnight forming units (CFUs), 10 μL of the samples collected from leg and skin
culture was prepared with suspension turbidity of 0.7 OD600 nm (~109 infection were diluted in 103-fold of 1 × PBS, then 20 μL was applied to
CFU/mL). For tested samples, two-fold serial dilutions were prepared in the TSA plate, and cultured at 37 ◦ C overnight, and were evaluated for
100 μL TSB (supplemented with 1% glucose) and added to the wells. CFU/mL quantification. The group treated with PBS was used as a
Then adding 60 μL of above bacterial suspension followed by 40 μL of negative control, while 1 × PBS-MRSA acted as a positive control. For
fresh TSB with 1% glucose to make the final inoculum of 6 × 107 biodistribution, inflammation healing and wound repairing, infection-
CFU/mL in every well. The final concentrations of the tested samples microenvironment responsiveness, and on-demand antibiotic delivery
were ranged from 1/16MIC,1/8MIC, 1/4MIC, 1/2MIC, MIC, 2MIC of VC-SGNPs-R-DiR in comparison with SGNPs-R-DiR (act as a control),
μg/mL. After incubation for 24 h at 37 ◦ C, the planktonic cells were FMT was carried out through IVIS Lumina imaging system, during the
removed from each well with PBS and fixed the biofilms for 15–30 min. treatment period on Day 5, 6, 7, 8 and 10 consecutive days.
The biofilm with crystal violet (0.1% wt/vol) was stained for 10–15 min While for % ID/g of tissues, mice were sacrificed after 24, 48, 72, 96,
and rinsed thoroughly with water until the negative control wells and 144 h, and homogenized in normal saline and quantified at 748/
appeared colorless. For biofilm quantification, 200 μL of ethanol (95%) 780 nm. The anti-MRSA activity of VC-SGNPs-R, like wound healings,
was added to the crystal violet stained wells and measured at 595 nm CFU, FMT and tissue quantification was photographed, observed, and
(A595). The effect of tested samples was also examined based on these co-related with each other as well with negative and positive control.
pre-formed biofilms. Biofilm quantification was assessed through The previously published method was modified according to designed
percent biofilm inhibition, calculated using the formula, % biofilm in experiments and therapeutic goal achievement [5,22].
hibition = [OD in control – OD of tested samples]/OD in control ×
100%. 2.16. Biocompatibility and toxicological studies
2.13. Biofilm morphological changes The biocompatibility and toxicology of VC-SGNPs-R DDS with mice
body physiology like tissues, blood, and vital organs were carried out [3,
The tested samples from biofilm quantification research were sub 8,25,32]. Both leg and skin infection model were weighed on a daily
jected to AFM for morphological changes. 10 μL polylysine (was added basis during treatment, and the changes in body weight were recorded.
on freshly cleaved mica slide and dried at room temperature. Withdrawn Similarly, acute toxicities were also observed during the entire treat
5–10 μL from the positive control (105 CFU) and tested samples from ment plan. Phlebitis, blood cross match and hemolysis test were per
biofilm microtiter plates wells were applied on prepared polylysine mica formed for safe I.V administration. As VC-SGNPs-R was injected through
slides, dried, and subjected to AFM analysis for morphological changes. I.V route, hemolytic activity was evaluated by measuring hemoglobin in
the supernatant at OD450nm. RBCs in 0.1 M PBS buffer and DI water were
2.14. FMT-IVIS live imaging and biodistribution studies used as negative and positive control respectively (Supplementary
Infromation, S6). For cytotoxicity’s and cyto-compatibility, MTT assay
Successful quantification of VC-SGNPs-R at infection sites and tissue was carried out against human embryonic kidney (HEK) 293T and liver
biodistribution were carried out with minor modification in mice LO2 cell lines. These cells were seeded in 96-well plates [3], 104 cells in
infection model [8,10]. Four types of infection model (Model 1: 1a,1b, each well were treated with different concentrations of
4
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
Fig. 1. Hypothetical illustration of preparation procedure of VC-SGNPs-R nanosystem for eradication of methicillin resistance Staphylococcus aureus (MRSA) infection
in mice model through its immune escaping capability, on-demand antibiotic delivery, synergism and infection-microenvironment responsiveness mechanism. RBC
= Red blood cell, RBCM = Red blood cell membrane, SGNP = Supramolecular gelatin nanoparticle, V = vancomycin, C = chlorogenic acid, VC-SGNPs = vancomycin-
chlorgenic acid-supramolecular gelatin nanoparticles, VC-SGNP-R = RBCM coated drug delivery system. (For interpretation of the references to color in this figure
legend, the reader is referred to the Web version of this article.)
5
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
Fig. 2. Surface characterization, A) TEM images show the core-membrane structure I: VCSGNPs-R, RBCM coated nanoparticles (scale bar = 100 nm), II: RBCM
coated single nanoparticle (scale bar = 50 nm), III: VC-SGNPs without RBCM (scale bar = 100 nm), samples were prepared by contacting the nanoparticle droplets
with copper grids for 30 s, removing the excess droplets, and staining by phototungstic acid for 10 s before the TEM analysis, B) Hydrodynamic diameter (nm)
measured through dynamic light scattering and polydispersity index (PDI) in red color. C) ζ potential of SGNPs, VC-SGNPs, VC-SGNPs-R. D) Percent drug loading
efficiency (DLE) and drug loading contents (DLC) with a weight ratio of vancomycin and chlorogenic acid to SGNPs 1:1:10, 2:2:10, 5:5:10 respectively. E) VC-SGNPs-
R SDS-PAGE of membrane protein retaining in comparison with natural RBCM, SGNPs and VC-SGNPs. (For interpretation of the references to color in this figure
legend, the reader is referred to the Web version of this article.)
Fig. 3. VC-SGNPs-R nanoparticles stability studies. A) 、B) and C) DSL size and PDI stability at 4ᵒC, − 20ᵒC and room temperature respectively. D) VC-SGNPs and VC-
SGNPs-R pH stabilities.
3.2. Internalization and uptake of macrophage cells uptake and internalized by the macrophage cells, as shown in CLSE
visualized Fig. 4A, while Fig. 4B demonstrates fluorescence intensities.
The RBCM coated nanoparticles (SGNPs-R, VC-SGNPs-R) evade the The compatibility of these nanoparticles with the macrophage cells were
immune system, where SGNPs and VC-SGNPs without membrane were further subjected to the mRNA expressions (folds) of pro-inflammatory
6
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
cytokines like TNF-ɑ, IL-2 and IL-6 and anti-inflammatory mediator IL- 3.4. In vitro drug release from VC-SGNPs-R
10 (Fig. 4C, D, 4E, 4F) respectively.
The in-vitro cumulative release of V and C from VC-SGNPs-R were
3.3. Exotoxins neutralization and relative hemolysis observed in (Fig. 5E and F) respectively. The maximum cumulative
release of V from VC and VC-SGNPs were 95% in 3 h and 9 h respec
RBCM acts as a nanosponge, absorb exotoxins on its surface, which tively, while it took 96 h in VC-SGNPs-R. Similarly, 100% cumulative
disrupts the membrane and hydrolyze the gelatin core, releasing the release of C from VC and VC-SGNPs were 2 h and 8 h respectively, where
internal contents in infection-microenvironment, as shown in (Fig. 5A it was 89.46% in 96 h from VC-SGNPs-R. In responsive release study,
and B). The anti-hemolytic activity further affirmed the exotoxins ca VC-SGNPs-R were subjected to MRSA exotoxins contains gelatinase and
pabilities of pore formations and membrane eruptions. The relative PFTs, and Tris buffer (pH 7.4) for 48 h. Within 6 h, 85.46% ± 2.88 of V
hemolysis was shown according to the content of hemoglobin released was released in the presence of bacterial toxins, while in Tris buffer the
from RBCs in the positive control (distilled water), negative control on-demand responsive release were 6.20% ± 2.86 (Fig. 5G). Similarly,
(PBS) and with MRSA (Fig. 5C and D). The releasing of RBCs hemoglobin the responsive release of C was 85.36% ± 2.91 (with MRSA) and 12.12%
contents provides a logical basis for the release profile of V and C from ± 2.30 (without MRSA) within 6 h, as shown in Fig. 5H.
VC-SGNPs-R DDS in an infection-microenvironment.
7
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
Fig. 5. In vitro release studies, A) & B) Multi-VC-SGNPs-R and Uni-VC-SGNPs-R TEM images of gelatinase and PFTs of RBCM degradation and gelatin core hydrolysis
respectively (scale bar = 50 nm), C) & D) Percent relative hemolysis and in vitro experiment of hemoglobin release in 1: positive control (distilled water), 2: negative
control (PBS) and 3: MRSA suspension (n = 8, mean ± SEM, ****p < 0.05), E) Vancomycin cumulative release, F) Chlorogenic acid cumulative release, G) & H)
Responsive release of vancomycin and chlorogenic acid respectively.
3.5. Anti-MRSA assays and synergism were further evaluated for responsive drug release, wound healing and
anti-MRSA activities.
The entrapment of V and C combination exerts synergistic effects,
which were determined through checkerboard dilution assay (Fig. 6A). 3.7. In vivo anti-MRSA activity for inflammation healing and wound
MIC of V was 16 μg/mL, while that of C was 512 μg/mL, where in repairing
checkerboard dilutions that was 2 and 128 μg/mL respectively. Its FIC
index were 0.3 which is lesser than 0.5, it indicates synergisms in V and After 5 days of incubation in an optimum condition, the right
C combinations. This combination was used for all in vitro and in vivo inflamed leg infection model and wound size of 2.21 mm in skin infec
experiments on VC (free drug combination), VC-SGNPs (entrapped in tion model was developed (Fig. 8A and B). On Day 6, VC-SGNPs-R-DiR,
gelatin nanoparticles) and VC-SGNPs-R (RBCM coated SGNPs). The SGNPs-R-DiR, VC, VC-SGNPs, and 1 × PBS (control) were administered
percent biofilm inhibitions were higher for VC-SGNPs-R, VC-SGNPs and via tail vein, inflammation and wound healing were observed with
VC in comparison with SGNPs and TSB as a negative control (Fig. 6B). physical measurements and FMTs IVIS live imaging for 5 consecutive
These biofilm quantifications were further morphologically observed on days. As the size were reduced in both inflammation and wound
AFM through height sensor and 3D images. MRSA and SGNPs treated repairing with VC-SGNPs-R treated models, which is an indication of
sample shows AFM height sensors of 666.4 nm and 756.8 nm respec VC-SGNPs-R anti-MRSA responsiveness in an infection-
tively, while they were 4.5 nm, 34.7 nm, 35.7 nm for VC, VC-SGNPs, VC- microenvironment (Fig. 8C and D). No effects on body weight in all
SGNPs-R respectively (Fig. 6C). The anti-MRSA concentration-depen infection models were observed during the treatment (Fig. 8E and F).
dent and time-dependent (0, 30 min, 1–18 h) viability were higher at The MRSA colonies formations were also reduced in all infection
MIC concentrations for VC, VC-SGNPs and VC-SGNPs-R nanoparticles in models treated with VC (free drug combination for reference), VC-
comparison with control (Fig. 6D and E). The VC-SGNPs-R treated MRSA SGNPs and VC-SGNPs-R nanoparticles, as in comparison with SGNPs
samples showed decreased optical densities and colony formations treated and positive control (Fig. 9A and B), while CFU/mL calculation
(Fig. 6F and G). The TSA plates further affirmed the anti-MRSA effects of were mentioned in Fig. 9C. Linear regression of correlations among in
VC-SGNPs-R at MIC and 1/16MIC (Fig. 6H). vitro and in vivo CFU/mL, in both skin and leg infection model treated
with VC-SGNPs-R were shown in Fig. 9D and E respectively.
3.6. FMT quantification and tissue biodistribution The healing of inflammations and wound repairing in leg and skin
infection models treated with fluorescence labeled SGNPs-R and VC-
The FMT of VC-SGNPs-R-DiR through IVIS live imaging were quan SGNPs-R, were tracked through FMTs IVIS live imaging (Fig. 10A and
tified in all infection sites of leg and skin infection model in comparison B) respectively. The high radiant efficiency in the bright field (BF) with
with control (Fig. 7A). The average radiant efficiency [p/s/cm2/sr]/ fluorescence showed prominent inflammation, and wound size in
[μW/cm2] in each mice model infection sites were calculated from the SGNPs-R treated mice model, which can be seen in the BF as well. In
region of interest (ROI), properly encircled (white), in association with comparison with VC-SGNPs-R treated mice model, the low radiant ef
control mice (Fig. 7 A1-A4). The increase in radiant efficiency at ROIs ficiencies indicate infection healing and wound repairing (Fig. 10C and
indicates the accumulation of VC-SGNPs-R-DiR in infection sites, which D). The fluorescence labeled nanoparticles were quantified in various
affirmed our hypothesis of its responsiveness in infection- tissues, as well in infection sites for biodistribution. The ex-vivo fluo
microenvironment. rescence intensities of %ID/g of infection tissue (leg and skin) and
Fig. 7B and C shows the biodistribution of VC-SGNPs-R-DiR to various organ reflectance images were shown in Fig. 10E and F, while
various tissues in all control and infection mice models. Percent injected quantification were presented in Fig. 10G and H. These fluorescence
dose per gram of tissue (% ID/g) during 24 h was confirmed through the intensities and reflectance were compared with control mice model
homogenization of vital organ and infected sites collection for fluores (Fig. 10I).
cence quantification. Fig. 7D demonstrates fluorescence % ID/g of the
neck and skin mice infection model in comparison with negative control 3.8. Biocompatibility and toxicology
mice. Fig. 7E is the percent injected dose per g of infected legs (right, left
hind-limbs) with normal fore limbs along with normal mice. These FMTs As VC-SGNPs-R were I.V injected, so it is necessary that it should be
8
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
Fig. 6. In vitro anti-MRSA activities and biofilm morphological changes, A) Vancomycin-chlorogenic acid checkerboard synergism, FIC index: ≤ 0.5 = synergism, 0.5
to 4.0 = additive or indifferent, < 4.0 = antagonism, B) Biofilm percent inhibitions of SGNPs, VC, VC-SGNPs, VC-SGNPs-R nanoparticles after 18 h of incubation, C)
3D image and height sensor of atomic force microscope biofilm morphological changes against SGNPs, VC, VC-SGNPs, VC-SGNPs-R in comparison with positive
control (scale bar = 1 μm), D) Heat map MRSA viability after 18 h of incubation, E) MRSA viability in comparison with negative control, F) OD 595 nm measurements
of SGNPs, VC, VC-SGNPs, VC-SGNPs-R against MIC respectively, G) CFU/mL calculations, H) TSA plates growth observations of SGNPs, VC, VC-SGNPs, VC-SGNPs-R
against MIC and 1/16MIC concentrations, (n = 3, **p < 0.01, ***p < 0.001, ****p < 0.0001), two-way ANOVA, Tukey’s multiple comparisons test.
9
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
Fig. 7. In vivo fluorescence molecular tomography image and quantification in MRSA mice infection model. A) FMT IVIS live imaging intensity in all infection models
after treated with VC-SGNPs-R-DiR labeled, Ex/Em = 748/780 nm, Model 1: neck infection, mouse 1a, 1b, Model 2: thigh infection, mouse 2a, 2b, Model 3:right leg
(hind) infection, mouse 3a, 3b, Model 4: left leg (hind) infection, mouse 4a, 4b, Control: Cta, Ctb, A1-A4) Average radiant efficiency [p/s/cm2/sr]/[μW/cm2]
calculated from region of interest (ROI) in each mice infection models in comparison with control, Note: total average radiant efficiency in each mouse = 3.82 × 109
± 2.04 × 109 [p/s]/[μW/cm2], B) & C) FMT organ and infection sites quantification in percent of injected dose/gm of tissue (% ID/g of tissue) in control and all
models respectively, D) Average % ID/g of tissue FMT quantification in skin infection mice model, E) Average % ID/g of tissue FMT quantification in leg infection
mice model, (**p < 0.01, ***p < 0.001, ****p < 0.0001), two-way ANOVA, Tukey’s, Sidak’s and Dunnett multiple comparisons test.
10
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
Fig. 8. In vivo anti-MRSA activity of leg inflammation healing and wound repairing of VC-SGNPs-R nanoparticles, A) & B) Right leg and skin infection model (n = 4
per group) respectively, Day 1: culture inoculation (50 μL, 108 CFU/mL), Day 1–5: incubation period (37 ᵒC), Day 6–10: treatment with VC-SGNPs-R, C) CFU/mL in
both leg and skin infection model treated with VC-SGNPs-R during the whole treatment schedule (day 1–10), D) Changes in leg inflammation and wound size (mm)
both in leg and skin infection model treated with VC-SGNPs-R, E) & F) Body weight (g) observations in both leg and skin infections model during the treatment with
SGNPs-R, VC-SGNPs-R and 1 × PBS (control). (**p < 0.01, ***p < 0.001, ****p < 0.0001), two-way ANOVA, Tukey’s multiple comparisons test.
Fig. 9. A) & B) Anti-MRSA activity of inflammation healing (leg infection model) and wound repairing (skin infection model) respectively, and its in vivo, CFU/mL
observations, (n = 4 per group), treated with a) SGNPs, b) VC, c) VC-SGNPs, d) VC-SGNPs-R in comparison with positive and negative control (− /+), C) CFU/mL
graphical observations (n = 3, mean ± SEM, ****p < 0.0001), two-way ANOVA, Tukey’s multiple comparisons test, D) & E) Linear regression of correlation among in
vitro and in vivo anti-MRSA activity of VC-SGNPs-R treated leg and skin infections mice model respectively.
11
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
Fig. 10. In vivo IVIS fluorescence molecular tomography imaging of inflammation healing (leg infection model) and wound repairing (skin infection model), A)
treatment with SGNPs-R-DiR nanoparticle (without drugs), B) treatment with VC-SGNPs-R-DiR nanoparticles (with drugs), C) Radiance or average radiant efficiency
(p/s/cm2/sr) of SGNPs-R and VC-SGNPs-R in leg infection model, (total radiant efficiency, SGNPs-R = 2.72 × 1010 ± 2.36 × 1010, VC-SGNPs-R = 1.38 × 109 ± 1.70
× 109 [p/s]/[μW/cm2], D) Radiance or average radiant efficiency (p/s/cm2/sr) of SGNPs-R and VC-SGNPs-R in skin infection, (total radiant efficiency, SGNPs-R =
3.58 × 109 ± 2.02 × 109), VC-SGNPs-R = 1.29 × 1010 ± 2.05 × 1010 [p/s]/[μW/cm2], E) Ex-vivo organ reflectance imaging of leg infection mice model, F) Ex-vivo
organ reflectance imaging of skin infection mice model, G) & H) % ID/g of tissue fluorescence quantification in leg and skin infection model treated with SGNPs-R
and VS-SGNPs-R nanoparticles respectively, I) % ID/g of tissue fluorescence quantification in normal mice without infection (control).
12
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
Fig. 11. Acute toxicities (survival %) of VC-SGNPs-R nanoparticles, A) leg infection mice model, B) Skin infection mice model, (n = 6 per group, comparison of
survival curves was analyzed through Log-rank (Mantel-Cox) test, p = ns).
safe, non-toxic and biocompatible with major organs and tissues. In of viability at highest concentration (Fig. 12B), while Figs. S4A and S4B
acute toxicities, the survival ratio were 100% during the treatment (Supplementary Information) show cytotoxicities at different concen
schedule (Fig. 11A and B), which affirmed its non-lethality. The histo trations against kidney and liver cell lines. Fig. 12C shows regression co-
logical examination conducted through H & E staining for the vital or relation analysis of HEK 293T and LO2 after 24 h treatment with VC-
gans (heart, lungs, kidneys, liver etc.) of infection model treated with SGNPs-R. As VC-SGNPs-R were directly injected into blood, so it is
VC-SGNPs-R nanoparticles showed no toxicological observations as in necessary that it should be compatible. Hemolysis at various concen
comparison with normal mice (Fig. 12A). It illustrates high tissue tration (100–500 μL, control) showed no obvious hemolysis (Fig. 12D),
biocompatibility and safe use of VC-SGNPs-R. Cyto-compatibility & while that of DI water showed 100% hemolysis after 0, 3, 24 h. Optical
toxicities at various concentration by VC-SGNPs-R were checked at microscopic images of hemolysis and blood compatability of VC-SGNPs-
normal cell lines, 293T cells (kidneys) and LO2 cells (liver) shows 95% R at various concentration were shown in Figs. S2A and S2B and Fig. S3
Fig. 12. Tissue biocompatibility and toxicology, A) H & E staining histological photos of liver, spleen, kidney lungs, heart and brain of mice before and after
treatment with SGNPs, VC, VC-SGNPs and VC-SGNPs-R and its biocompatibility, heathy mice were served as a control, B) Percent cell viability of VC-SGNPs-R
nanoparticles in various concentrations against HEK 293T (kidney cells) and LO2 (liver cells), C) Regression co-relation analysis of HEK 293T and LO2 after 24 h
treatment with VC-SGNPs-R, D) Blood compatability and hemolysis, 1–5: 100, 200, 300, 400, 500 μL of VC-SGNPs-R respectively, 6: normal saline, 7: DI water
(positive control) after 0, 3 and 24 h observations, E) Absorbance of relative hemolysis, (n = 8, mean ± SEM, ****p < 0.0001), two-way ANOVA, Tukey’s multiple
comparisons test.
13
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
(Supplementary Information). The hemoglobin released were used for RBCM nanosponging attract the PFTs onto its surface while the gelati
relative hemolysis, which is measured from the absorbance through the nases (proteases) hydrolyze the SGNPs core, while releasing V and C at
microplate reader (Fig. 12E). infection site. RBCM also protects the hydrophilic surface polymeric
core and retained drug molecules in polymeric gelatin nanoparticles for
4. Discussions prolonging circulation and sustained release [51].
The combination of V and C and it’s in vitro synergistic effects against
The VC-SGNPs-R and its characterization revealed a successful MRSA, time-concentration dependent viability and biofilm inhibitions
translocation to the surface nanoparticles and homogeneous dispersion. in VC-SGNPs-R, VC-SGNPs and VC were similar as in comparison with
Herein, RBCM gives an extra thickness of ~8.69 ± 1.4 nm membrane, SGNPs and negative control. In comparisons, VC-SGNPs-R nanoparticles
though, usually its thickness varies 5–10 nm [3,25]. It was affirmed that are different from other combinations in respects with its RBCM coating,
the majority of endogenous and surface protein retained during the immune evading, extending circulation and drug releases in the pres
preparation of VC-SGNPs-R nanoparticles. The hydrodynamic particle ence of exotoxins. These studies were further justified through in vivo
size distribution measured by DLS was relatively higher due to water studies in infection mice models.
film on the outside of the nanoparticle molecules, as in comparison with In order to achieve an efficient level of target specificity respon
TEM in which the water film is removed on drying [14]. The average size siveness in infection, tumor, and inflammation-microenvironment, these
on DLS of unloaded SGNPs were 114.63 ± 2.3 nm, loaded VC-SGNPs RBCM coated nanoparticles should extravasates from circulating blood
182.93 ± 3.98 nm and VC-SGNPs-R 190.53 ± 3.4 nm along with PDI and concentrate in certain tissues either non-specifically or specifically
of 0.07 ± 0.09, 0.08 ± 0.09 and 0.11 ± 0.07 respectively. Enhanced attached to a molecular sites or undergoing cellular internalization [10].
antimicrobial efficacy is associated with quantum size of the nano VC-SGNPs-R quantification at infection-microenvironment in both mice
particles, which increase the surface areas [26]. The alteration in surface infection models (leg and skin). The degradation of gelatin nano-core
charge potential may be due to the membrane charge shielding with its through PFTs and gelatinase enzymes (secreted by exotoxins), triggers
lower negative potential to gelatin core with higher negative potential the release of V and C at infection sites. The variation in FMT quantifi
[10,33]. Similarly, higher ζ-potential magnitude exhibits stable sus cation in an infected and non-infected groups exhibits that VC-SGNPs-R
pension, which is due to a larger electrostatic repulsion among particles. showed a long-lasting, stronger fluorescence signals at infection sites
DLE & DLC of the nanosystem in reference with per unit weight of the that depleted with the inflammation healing and wound repairing pro
gelatin nanoparticles provides sustained therapeutic released. cess progressively. Similarly, SGNPs-R accumulates at infection sites,
Similarly, escaping the immune system is the primary core concept of but no healing expositions can be seen. FMTs and tissue biodistribution
VC-SGNPs-R DDS for a prolonged sustained release. As antibiotic accu revealed that VC-SGNPs-R was highly quantified in liver and spleen. The
mulation at infection sites and rapid elimination by the immune system physical examinations of leg and skin infections in comparisons with
e.g. mononuclear phagocytic system severely minimizing the release of FMTs, justified that VC-SGNPs-R nanoparticles quantified at infection
nanoparticles in bacteria-microenvironment infection site thus it can sites released V and C in infection-microenvironment for anti-MRSA
impede antibacterial efficacy, drug delivery and bioavailability [10,34]. activities.
The responsive release of VC-SGNPs-R at infection sites may also avoid Successful in vitro and in vivo studies demand a safe and biocom
the premature drug release [35,36]. Thus, the innate features of patible determination of VC-SGNPs-R. In the last few decade, diverse
macrophage cells help VC-SGNPs-R to escape the immune system, pro strategies are adopted to loaded drugs and nanocarriers to the surface as
long systemic circulation [37] and avoid the inflammatory cytokines well as to the inner core of RBC. Their pharmacokinetics, bio
responses [27,28]. Recently, RBCM coated polymeric nanoparticles compatibilities, systemic toxicology, local adverse effects, adherence to
have been investigated as a new class of biomimetic material in DDS, endothelial cells, formation of on toward cargo complexes, destruction,
which attain a fabricated stealth surface retains from natural RBCs, elimination and clearance are the potential risks and challenges of
exhibit low immunogenicity, reduce opsonization, escape phagocytosis RBCM DDS [52–56]. Biodistribution of VC-SGNPs-R to all major organs
and prolong the drug half-life in plasma [38,39]. like, liver, spleen, kidney, lung, heart, brain showed normal tissue
After incubation with macrophage cells, SGNPs/VC-SGNPs emit structure in H & E staining, as well as no-hemolysis with blood after I.V
green radiations, while no fluorescence was observed with SGNPs-R or administrations. The RBCM make it compatible with blood and other
VC-SGNPs-R depicted immune escaping. Similarly, mRNA inflammatory tissues. Kidney and liver, which are the major organs for clearance and
cytokine folde expressions were not observed in the cells treated with metabolism, are biocompatible with VC-SGNPs-R nanoparticles both in
SGNPs-R or VC-SGNPs-R, which showed no apparent inflammatory vitro and in vivo studies. All these observations, strengthens its biosafety,
cascades and immunological responses [3,8,10,32–34,40,41]. The safe compatibility and toxicology aspects. Pharmacokinetic studies have
elimination of pathological agents has been reported using RBC surface been reported on RBCM coating nanoparticles previously [10,26,51],
loading via injection of hetero-conjugates binding to complement re which showed in situ sustained release, that provided a prolong circu
ceptor [35,36], where fusion binding like recombinant therapeutics lation and immune escaping. Thus the overall in vitro, in vivo safe and
proteins to the RBCs markedly prolong circulations, enhance potency biocompatible profile of VC-SGNPs-R nanoparticles and its on-demand
and reduce systemic toxicities [42–45]. antibiotic delivery at infection-microenvironment could be a prom
The MRSA exotoxins helps in tissue invasion, penetration, evasion of ising candidate in biomedical application against MDR strains.
host defenses and survival under extreme conditions in various patho
gens. The hemolysin (α, β and γ) released by these exotoxins can degrade 5. Conclusion
erythrocytes and leukocytes membrane via binding to ADAM10 recep
tor, disintegrin and matrix metalloproteinase (MMP-2 & MMP-9) [3,46]. We developed VC-SGNPs-R DDS for V and C loaded in combination
It may causes pore formation and permeability, which may cause Ca2+ to supramolecular gelatin nanoparticles for anti-MRSA effects in mice
influx/K+ efflux, which can disrupts homeostasis of the host cells and infection model. The limitations in the previous studies reported [3,7,8,
thus lead to cell death [47]. MRSA and other gram-positive bacteria also 10,11] on RBCM coated nanoparticles and MRSA virulence factor neu
used gelatinase, an extracellular proteolytic enzyme as a virulence factor tralizations were highlighted and extended to a new treatment
that dissolves the connective tissue of the host cells which helps in approach. Here, in VC-SGNPs-R, the RBCM coating escape the immune
infection invasion. Gelatinase causes the hydrolysis of gelatin into system, significantly prolong the systemic circulation and quantified at
polypeptides and then breakdown its complex structure into monomeric infection-microenvironment for on-demand and responsive release of V
amino acids [38,48–50]. This phenomenon was used for the lysis of and C combine delivery. The successful in vitro, time-concentration
VC-SGNPs-R DDS released in MRSA-infection-microenvironment. The dependent anti-MRSA effects, biofilm inhibitions, depletion in colonies
14
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
formations and optical densities were confirmed and subjected through References
in vivo, mice-infection models (leg and skin). Declination in the fluo
rescence quantification of VC-SGNPs-R at infection sites, inflammation [1] A.R. Collaborators, Articles Global burden of bacterial antimicrobial resistance in
2019, a systematic analysis 6736 (2022), https://doi.org/10.1016/S0140-6736
healing and wound repairing indicate its infection-microenvironment (21)02724-0.
responsive release of V and C for synergistic effects against MRSA. [2] D. Oliveira, A. Borges, M. Simões, Staphylococcus aureus toxins and their
VC-SGNPs-R has been proved to have low toxicity, good biocompati molecular activity in infectious diseases, Toxins (Basel) 10 (2018), https://doi.org/
10.3390/toxins10060252.
bility with tissues, blood and vital organs, which make it a safe DDS. [3] L.L. Li, J.H. Xu, G. Bin Qi, X. Zhao, F. Yu, H. Wang, Core-shell supramolecular
Moreover, an excellent in vitro and in vivo results indicate that drug gelatin nanoparticles for adaptive and ‘on-demand’ antibiotic delivery, ACS Nano 8
combination strategies in VC-SGNPs-R nanoparticles can inhibit multi (2014) 4975–4983, https://doi.org/10.1021/nn501040h.
[4] Y. Guo, T. Xu, C. Bao, Z. Liu, J. Fan, R. Yang, S. Qin, Design and synthesis of new
drug resistance infections through synergistic effects. Thus, these norfloxacin-1,3,4-oxadiazole hybrids as antibacterial agents against methicillin-
infection-microenvironment responsiveness and on-demand antibiotic resistant Staphylococcus aureus (MRSA), Eur. J. Pharm. Sci. 136 (2019), 104966,
delivery platform are expected to be an alternative approach in future https://doi.org/10.1016/j.ejps.2019.104966.
[5] C.M.J. Hu, R.H. Fang, J. Copp, B.T. Luk, L. Zhang, A biomimetic nanosponge that
nanotechnology to treat MDR bacteria.
absorbs pore-forming toxins, Nat, Nanotechnol (2013), https://doi.org/10.1038/
nnano.2013.54.
CRediT authorship contribution statement [6] G. Qing, X. Zhao, N. Gong, J. Chen, X. Li, Y. Gan, Y. Wang, Z. Zhang, Y. Zhang,
W. Guo, Y. Luo, X.J. Liang, Thermo-responsive triple-function nanotransporter for
efficient chemo-photothermal therapy of multidrug-resistant bacterial infection,
Zul Kamal: Contributed in the experimental study, including the Nat. Commun 10 (2019) 1–12, https://doi.org/10.1038/s41467-019-12313-3.
investigation, methodology, data curation, formal analysis of data, [7] Y. Chen, Y. Zhang, M. Chen, J. Zhuang, R.H. Fang, W. Gao, L. Zhang, Biomimetic
research administration, and drafting the original manuscript. Jing Su: nanosponges suppress in vivo lethality induced by the whole secreted proteins of
pathogenic bacteria, Small 15 (2019) 1–9, https://doi.org/10.1002/
Conceptualization, writing, reviewing, editing and formal analysis, smll.201804994.
Faisal Raza: Participated in vivo studies and infection mice model [8] A. Lin, Y. Liu, X. Zhu, X. Chen, J. Liu, Y. Zhou, X. Qin, J. Liu, Bacteria-responsive
development, Liangdi Jiang: Assist and participated in characterization biomimetic selenium nanosystem for multidrug-resistant bacterial infection
detection and inhibition, ACS, Nano 13 (2019) 13965–13984, https://doi.org/
and biodistribution studies, Yichen Li: Help in manuscript revision and 10.1021/acsnano.9b05766.
contents formatting, Weien Yuan: Take part in research administration [9] L.A. Stoddart, S.J. Briddon, Advances in the application of fluorescence correlation
and revising the manuscript. Mingfeng Qiu: As corresponding authors, spectroscopy to study detergent purified and encapsulated membrane proteins, Int.
J. Biochem. Cell Biol. 146 (2022), https://doi.org/10.1016/j.biocel.2022.106210.
acquired the funding, participated in the study conceptualization, data [10] Y. Zhang, J. Zhang, W. Chen, P. Angsantikul, K.A. Spiekermann, R.H. Fang,
analysis, reviewing and editing the manuscript, and finalized this W. Gao, L. Zhang, Erythrocyte membrane-coated nanogel for combinatorial
manuscript. All authors have read and agreed to the published version of antivirulence and responsive antimicrobial delivery against Staphylococcus aureus
infection, J. Control. Release 263 (2017) 185–191, https://doi.org/10.1016/j.
the manuscript.
jconrel.2017.01.016.
[11] X. Wu, Red Blood Cell Membrane-Camouflaged Tedizolid Phosphate-Loaded PLGA
Institutional review board statement Nanoparticles for Bacterial-Infection Therapy, 2021.
[12] M. Otto, Staphylococcus aureus toxins, Curr. Opin. Microbiol. 17 (2014) 32–37,
https://doi.org/10.1016/j.mib.2013.11.004.
The study was conducted according to the guidelines of the decla [13] J. Su, R. Zhang, Y. Lian, Z. Kamal, Z. Cheng, Y. Qiu, M. Qiu, Preparation and
ration of Helsinki, and approved by the Animal Ethics Committee of characterization of erythrocyte membrane-camouflaged berberine hydrochloride-
Shanghai Jiao Tong University (No. A2018021). loaded gelatin nanoparticles, Pharmaceutics 11 (2019), https://doi.org/10.3390/
pharmaceutics11020093.
[14] T. Ogura, A. Tsuchiya, T. Minas, S. Mizuno, Methods of high integrity RNA
Declaration of competing interest extraction from cell/agarose construct, BMC Res. Notes 8 (2015) 1–8, https://doi.
org/10.1186/s13104-015-1627-5.
[15] Y. Shi, Z. Li, R. Chen, J. Zhang, X. Hu, C. He, Q. Su, H. Ma, H. Ren, M. Qian, S. Cui,
The authors declare that they have no known competing financial W. Jiang, Immethridine, histamine H3-receptor (H3R) agonist, alleviated
interests or personal relationships that could have appeared to influence experimental autoimmune encephalomyelitis via inhibiting the function of
the work reported in this paper. I further assure that this work is neither dendritic cells, Oncotarget 8 (2017) 75038–75049, https://doi.org/10.18632/
oncotarget.20500.
published nor under consideration elsewhere. [16] M.M. Konai, J. Haldar, Fatty acid comprising lysine conjugates: anti-MRSA agents
that display in vivo efficacy by disrupting biofilms with No resistance
Data availability development, Bioconjug. Chem. 28 (2017) 1194–1204, https://doi.org/10.1021/
acs.bioconjchem.7b00055.
[17] L. Wang, C. Bi, H. Cai, B. Liu, X. Zhong, X. Deng, T. Wang, H. Xiang, X. Niu,
Data will be made available on request. D. Wang, The therapeutic effect of chlorogenic acid against Staphylococcus aureus
infection through sortase A inhibition, Front, Microbiol. 6 (2015) 1–12, https://
Acknowledgment doi.org/10.3389/fmicb.2015.01031.
[18] A. Karunanidhi, R. Thomas, A. Van Belkum, V. Neela, In vitro antibacterial and
antibiofilm activities of chlorogenic acid against clinical isolates of
This research was funded by the National Major Science and Tech stenotrophomonas maltophilia including the trimethoprim/sulfamethoxazole
nology Projects of China, 2018ZX09711003-008-002, National Natural resistant strain, Biomed Res. Int 2013 (2013), https://doi.org/10.1155/2013/
392058.
Science Foundation of China, 81973487and Shanghai Association for [19] Z. Lou, H. Wang, S. Zhu, C. Ma, Z. Wang, Antibacterial activity and mechanism of
Science and Technology, 18ZR1419700. Beside it, we are highly action of chlorogenic acid, J. Food Sci. 76 (2011), https://doi.org/10.1111/j.1750-
thankful to Professor Bo Zhao Lab, School of Pharmacy, Shanghai Jiao 3841.2011.02213.x.
[20] G. Li, X. Wang, Y. Xu, B. Zhang, X. Xia, Antimicrobial effect and mode of action of
Tong University for assisting all anti-MRSA activities, Asad Farooqi (East chlorogenic acid on Staphylococcus aureus, Eur. Food Res. Technol 238 (2014)
China Normal University) for pro & pre-inflammatory cytokine studies 589–596, https://doi.org/10.1007/s00217-013-2140-5.
primer designing’s. [21] C.L. Sy, T.S. Huang, C.S. Chen, Y.S. Chen, H.C. Tsai, S.R. Wann, K.S. Wu, J.K. Chen,
S.S.J. Lee, Y.C. Liu, Synergy of β-lactams with vancomycin against methicillin-
resistant Staphylococcus aureus: correlation of disk diffusion and checkerboard
Appendix A. Supplementary data methods, J. Clin. Microbiol. 54 (2016) 565–568, https://doi.org/10.1128/
JCM.01779-15.
[22] B. Choi, W. Park, S. Bin Park, W.K. Rhim, D.K. Han, Recent trends in cell
Supplementary data to this article can be found online at https://doi.
membrane-cloaked nanoparticles for therapeutic applications, Methods 177 (2020)
org/10.1016/j.jddst.2022.103706. 2–14, https://doi.org/10.1016/j.ymeth.2019.12.004.
[23] X. Que, J. Su, P. Guo, Z. Kamal, E. Xu, S. Liu, J. Chen, M. Qiu, Study on preparation,
characterization and multidrug resistance reversal of red blood cell membrane-
camouflaged tetrandrine-loaded PLGA nanoparticles, Drug Deliv 26 (2019)
199–207, https://doi.org/10.1080/10717544.2019.1573861.
15
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706
[24] J. Su, G. Liu, Y. Lian, Z. Kamal, X. Que, Y. Qiu, M. Qiu, Preparation and [41] C.M.J. Hu, L. Zhang, S. Aryal, C. Cheung, R.H. Fang, L. Zhang, Erythrocyte
characterization of erythrocyte membrane cloaked PLGA/arsenic trioxide membrane-camouflaged polymeric nanoparticles as a biomimetic delivery
nanoparticles and evaluation of their: in vitro anti-tumor effect, RSC Adv 8 (2018), platform, Proc. Natl. Acad. Sci. U, S. A 108 (2011) 10980–10985, https://doi.org/
https://doi.org/10.1039/c8ra01417e. 10.1073/pnas.1106634108.
[25] S. Ye, F. Wang, Z. Fan, Q. Zhu, H. Tian, Y. Zhang, B. Jiang, Z. Hou, Y. Li, G. Su, [42] R. Carnemolla, C.H. Villa, C.F. Greineder, S. Zaitsev, K.R. Patel, M.A. Kowalska, D.
Light/pH-Triggered biomimetic red blood cell membranes camouflaged small N. Atochin, D.B. Cines, D.L. Siegel, C.T. Esmon, V.R. Muzykantov, Targeting
molecular drug assemblies for imaging-guided combinational chemo-photothermal Thrombomodulin to Circulating Red Blood Cells Augments its Protective Effects in
therapy, ACS Appl. Mater. Interfaces 11 (2019) 15262–15275, https://doi.org/ Models of Endotoxemia and Ischemia-Reperfusion Injury, 2016, pp. 1–11, https://
10.1021/acsami.9b00897. doi.org/10.1096/fj.201600912R.
[26] D.R. Perinelli, L. Fagioli, R. Campana, J.K.W. Lam, W. Baffone, G.F. Palmieri, [43] S. Zaitsev, M.A. Kowalska, M. Neyman, R. Carnemolla, S. Tliba, B. Ding,
L. Casettari, G. Bonacucina, Chitosan-based nanosystems and their exploited A. Stonestrom, D. Spitzer, J.P. Atkinson, M. Poncz, D.B. Cines, C.T. Esmon, V.
antimicrobial activity, Eur. J. Pharm. Sci. 117 (2018) 8–20, https://doi.org/ R. Muzykantov, Targeting recombinant thrombomodulin fusion protein to red
10.1016/j.ejps.2018.01.046. blood cells provides multifaceted thromboprophylaxis 119 (2016) 4779–4786,
[27] J.S. Cho, J. Zussman, N.P. Donegan, R.I. Ramos, C. Nairy, D.Z. Uslan, Y. Iwakura, S. https://doi.org/10.1182/blood-2011-12-398149.The.
I. Simon, A.L. Cheung, R.L. Modlin, J. Kim, L.S. Miller, NIH Public Access 131, [44] S. Zaitsev, D. Spitzer, J. Murciano, B. Ding, S. Tliba, M.A. Kowalska, O.A. Marcos-
2011, pp. 907–915, https://doi.org/10.1038/jid.2010.417.Noninvasive. contreras, A. Kuo, V. Stepanova, J.P. Atkinson, M. Poncz, D.B. Cines, V.
[28] J.C. Garrelts, J.D. Peterie, Vancomycin and the ‘red man’s syndrome’, N. Engl, R. Muzykantov, Sustained thromboprophylaxis mediated by an RBC-targeted pro-
J. Med. 312 (1985) 245. http://europepmc.org/abstract/MED/3155563. urokinase zymogen activated at the site of clot formation, Blood 115 (2016)
[29] R.T. Irvin, T.J. MacAlister, J.W. Costerton, Tris(hydroxymethyl)aminomethyl 5241–5249, https://doi.org/10.1182/blood-2010-01-261610.The.
buffer modification of Escherichia coli outer membrane permeability, J. Bacteriol [45] M.H. Polymeropoulos, H. Xiao, D.S. Rath, C.R. Merril, P.R. Hoban, A.M. Kelsey,
145 (1981) 1397–1403, https://doi.org/10.1128/jb.145.3.1397-1403.1981. Dinucleotide repeat polymorphism at the human cysteine-proteinase inhibitor
[30] D.F. Basri, R. Khairon, Pharmacodynamic interaction of quercus infectoria galls pseudogene (CSTP1) Hinfl polymorphism within the 3 ’ untranslated region of the
extract in combination with vancomycin against MRSA using microdilution candidate Wilms tumour gene, Nucleic Acids Res. 19 (1990) 4637.
checkerboard and time-kill assay, Evidence-Based Complement. Altern, Med 2012 [46] C. Kong, H.M. Neoh, S. Nathan, Targeting Staphylococcus aureus toxins: a
(2012), https://doi.org/10.1155/2012/493156. potential form of anti-virulence therapy,, Toxins (Basel) 8 (2016) 1–21, https://
[31] C. Santiago, K.H. Lim, H.S. Loh, K.N. Ting, Inhibitory effect of Duabanga doi.org/10.3390/toxins8030072.
grandiflora on MRSA biofilm formation via prevention of cell-surface attachment [47] A. Gupta, M. Biyani, A. Khaira, Vancomycin nephrotoxicity: myths and facts, neth,
and PBP2a production, Molecules 20 (2015) 4473–4482, https://doi.org/10.3390/ J. Med 69 (2011) 379–383.
molecules20034473. [48] M.S. Lener, 乳鼠心肌提取 HHS public access, physiol, Behav 176 (2016) 139–148,
[32] M. Gao, C. Liang, X. Song, Q. Chen, Q. Jin, C. Wang, Z. Liu, Erythrocyte-membrane- https://doi.org/10.1128/microbiolspec.GPP3-0039-2018.Staphylococcus.
enveloped perfluorocarbon as nanoscale artificial red blood cells to relieve tumor [49] A. Karmakar, P. Dua, C. Ghosh, Biochemical, Molecular Analysis, Of
hypoxia and enhance cancer radiotherapy, Adv. Mater 29 (2017) 1–7, https://doi. Staphylococcus aureus clinical isolates from hospitalized patients, Can. J. Infect.
org/10.1002/adma.201701429. Dis. Med. Microbiol. 2016 (2016), https://doi.org/10.1155/2016/9041636.
[33] R.H. Fang, B.T. Luk, C.M.J. Hu, L. Zhang, Engineered nanoparticles mimicking cell [50] X. Du, W. Wang, C. Wu, B. Jia, W. Li, L. Qiu, P. Jiang, J. Wang, Y.Q. Li, Enzyme-
membranes for toxin neutralization, Adv. Drug Deliv. Rev. 90 (2015) 69–80, responsive turn-on nanoprobes for in situ fluorescence imaging and localized
https://doi.org/10.1016/j.addr.2015.04.001. photothermal treatment of multidrug-resistant bacterial infections, J. Mater. Chem.
[34] L. Rao, R. Tian, X. Chen, Cell-membrane-Mimicking nanodecoys against infectious B 8 (2020) 7403–7412, https://doi.org/10.1039/d0tb00750a.
diseases, ACS Nano 14 (2020) 2569–2574, https://doi.org/10.1021/ [51] Q. Xia, Y. Zhang, Z. Li, X. Hou, N. Feng, Red blood cell membrane-camouflaged
acsnano.0c01665. nanoparticles: a novel drug delivery system for antitumor application, Acta Pharm.
[35] R, H.L. and E.L.W. R P Taylor, C J Reist, W M Sutherland, A Otto, in vivo binding Sin. B. 9 (2019) 675–689, https://doi.org/10.1016/j.apsb.2019.01.011.
and clearance of circulating antigen by bispecific heteropolymer-mediated binding [52] D.C. Pan, J.W. Myerson, J.S. Brenner, P.N. Patel, A.C. Anselmo, S. Mitragotri,
to primate erythrocyte complement receptor.,, J. Immunol 148 (1992) 2462–2468. V. Muzykantov, Nanoparticle properties modulate their attachment and effect on
[36] M.A. Lindorfer, C.S. Hahn, P.L. Foley, R.P. Taylor, Heteropolymer-mediated carrier red blood cells, Sci. Rep. (2018) 1–12, https://doi.org/10.1038/s41598-
clearance of immune complexes via erythrocyte CR1: mechanisms and 018-19897-8.
applications, Immunol. Rev. 183 (2001) 10–24, https://doi.org/10.1034/j.1600- [53] V.R. Muzykantov, Drug Delivery Carriers on the Fringes : Natural Red Blood Cells
065X.2001.1830102.x. versus Synthetic Multilayered Capsules, 2013, pp. 1–4.
[37] W. Chen, K. Zeng, H. Liu, J. Ouyang, L. Wang, Y. Liu, H. Wang, L. Deng, Y.N. Liu, [54] J.S. Brenner, D.C. Pan, J.W. Myerson, O.A. Marcos-contreras, C.H. Villa, P. Patel,
Cell membrane camouflaged hollow prussian blue nanoparticles for synergistic H. Hekierski, S. Chatterjee, J. Tao, H. Parhiz, K. Bhamidipati, T.G. Uhler, E.
photothermal-/chemotherapy of cancer, Adv. Funct. Mater 27 (2017), https://doi. D. Hood, R.Y. Kiseleva, V.S. Shuvaev, T. Shuvaeva, M. Khoshnejad, I. Johnston,
org/10.1002/adfm.201605795. J. V Gregory, J. Lahann, T. Wang, E. Cantu, W.M. Armstead, S. Mitragotri,
[38] X.W. Wang, P.R.C. School of Pharmacy, Changzhou University, Changzhou V. Muzykantov, Red blood cell-hitchhiking boosts delivery of nanocarriers to
213164,. Jiangsu, M. by X. Wang, and J.W., Jiawei Wang, Lin Qiu, Cheng Wang, chosen organs by orders of magnitude, Nat, Commun (2018), https://doi.org/
Xiaoling Lei, Pengfei Cui, Shuwen Zhou, Donghui Zhao, Xinye Ni*, Pengju Jiang*, 10.1038/s41467-018-05079-7.
Gelatinase-Responsive Photothermal Nanotherapy Based on Au Nanostars [55] C.H. Villa, D.C. Pan, I.H. Johnston, C.F. Greineder, L.R. Walsh, E.D. Hood, D.
Functionalized with Antimicrobial Peptides for Treating Staphylococcus aureus B. Cines, M. Poncz, D.L. Siegel, V.R. Muzykantov, Biocompatible coupling of
Infectionse, ACS Appl. Nano Mater. 5 (2022) 8324–8333. doi:https://doi.org/ therapeutic fusion proteins to human erythrocytes 2 (2018), https://doi.org/
10.1021/acsanm.2c01390. 10.1182/bloodadvances.2017011734.
[39] C. Li, X. Wang, R. Li, X. Yang, Z. Zhong, Y. Dai, Q. Fan, Y. Lin, R. Zhang, T. Liang, [56] Z. Zhao, A. Ukidve, V. Krishnan, A. Fehnel, D.C. Pan, Y. Gao, J. Kim, M.A. Evans, A.
Y. Ye, M. Zhou, Resveratrol-loaded PLGA nanoparticles functionalized with red Mandal, J. Guo, V.R. Muzykantov, S. Mitragotri, Systemic tumour suppression via
blood cell membranes as a biomimetic delivery system for prolonged circulation the preferential accumulation of erythrocyte-anchored chemokine-encapsulating
time, J. Drug Deliv. Sci. Technol 54 (2019), 101369, https://doi.org/10.1016/j. nanoparticles in lung metastases, Nat. Biomed. Eng. (n.d.). doi:10.1038/s41551-
jddst.2019.101369. 020-00644-2.
[40] W. Gao, C.M.J. Hu, R.H. Fang, B.T. Luk, J. Su, L. Zhang, Surface functionalization
of gold nanoparticles with red blood cell membranes, Adv. Mater 25 (2013)
3549–3553, https://doi.org/10.1002/adma.201300638.
16