Journal of Drug Delivery Science and Technology 76 (2022) 103706

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Journal of Drug Delivery Science and Technology

journal homepage: www.elsevier.com/locate/jddst

Red blood cell membrane-camouflaged vancomycin and chlorogenic

acid-loaded gelatin nanoparticles against multi-drug resistance infection
mice model
Zul Kamal a, b, 1, Jing Su a, 1, Weien Yuan a, Faisal Raza a, Liangdi Jiang a, Yichen Li a,
Mingfeng Qiu a, *
a
School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China
b
Department of Pharmacy, Shaheed Benazir Bhutto University, Sheringal, Pakistan

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.

1. Introduction efficacy of most of antibiotics like methicillin, penicillin, oxacillin,

According to the global burden of antimicrobial resistance (AMR),
estimated by the institute of health metric and evaluation (IHME), there
were 4.95 million deaths worldwide with bacterial-AMR. They were
mostly associated with Escherichia coli, followed by Staphylococcus
aureus, Streptococcus pneumonia, Klebsiella pneumoniae, Pseudomonas
aeruginosa and Acinetobacter baumannii. According to another estima­
tion, only MRSA killed more than 100 000 people globally due to
resistance emergence [1]. MRSA is mostly involved in skin and soft
tissue associated with more serious invasive syndromes such as severe
sepsis, pneumonia, and endocarditis [2]. The health problems associated
with MRSA is its biofilm formation, especially in implant material like
catheters and prosthetic devices, which acts as barrier to antimicrobial
agents, shielding colonies from environmental variability and retain the
amoxicillin and cephalopsorins [3,4]. The last line defense against
MRSA are vancomycin and linizolide which also declining their efficacy
each year due to resistance. Different therapeutic strategies like stew­
ardship, combination therapy (antibiotics, natural product, polymer
etc), and drug delivery system (DDS) etc., have been adopted to main­
tain antibiotics efficacy against MDR strains. Thus, the critical needs
diverge to innovate conventional antibiotics with modern therapeutic
technologies [5,6].
Among various nanotechnology-based antibacterial DDS [7,8], toxin
neutralization and infection micro-environment responsiveness through
RBCM coating nanosystem have gained more traction, especially against
MRSA strains. Though, membrane surface proteins, such as receptors
and transporters, are now an important drug targets and a major focus
for new drug development [9]. The RBCM coated polymeric

* 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.
nanoparticles (gelatin, PLGA, hydrogel, etc.) which shows responsive­
ness at infection site. In situ, the exotoxins secrete pore-forming toxins
(PFTs) and gelatinase enzymes, which permeate the RBCM and hydro­
lyze the polymeric nanocore respectively to release drug [6,10–13]. Hu
et al. demonstrate that PLGA nanoparticles camouflaged with RBCM can
serve as toxin “nanosponge”, absorbing pore-forming toxins [5]. Lin
et al. propose validated biomimetic visualization selenium antibacterial
nanosystem for synergism in mice MRSA infection model, but its
application was only limited to one spot in skin infection mice model, as
well, large number of characterizations were required [8]. Chin et al.
propose RBCM nanosponge that can neutralize the bacterial virulence
factors in MRSA condition like sepsis and bacteremia [7]. Li et al. state
the exotoxin-microenvironment responsiveness and on-demand anti­
biotic delivery of vancomycin in-vitro [3]. Zhang et al. propose RBCM
coated redox-responsive crosslinker nanogel embedded vancomycin for
antivirulence and responsive delivery system against MRSA [10].
Similarly, Xinyi et al., also propose RBCM camouflaging tedizolid
phosphate PLGA copolymer nanosystem against MDR strains, but the
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
uncertainties regarding its target specificity, quantification, and release
[15].
The characteristics of MRSA is to form a biofilm on biotic and abiotic
surfaces which retain the efficacy of most clinically available antibiotics
[16]. As we did mention, vancomycin declined its therapeutic effects
against MRSA each year due to high-level of resistance. So in this study,
vancomycin and a polyphenol compound chlorogenic acid were loaded
in combination for the first time to prepared SGNPs nanoparticles, which
were then coated with RBCM to obtained VC-SGNPs-R. Chlorogenic acid
is abundant in fruits, vegetables and Chinese herbs, possesses anti-MRSA
biofilm effects through its quorum sensing effects, which mostly
potentiate various pathogenic traits, including biofilm formation
[17–20]. This combination may show synergistic effects against MRSA
biofilm and cell wall synthesis.
The VC-SGNPs-R surface characterization was confirmed for mem­
brane retaining and cell uptake, which help in immune escaping, sus­
tained release and prolong circulation. The in vitro anti-MRSA activities
were firstly studied through colony forming unit (CFU) and optical
density measurement (OD600 nm), while its morphological changes were
observed through AFM. Similarly, for the in vivo wound healing and
MRSA eradication, the leg and skin infection mice model were devel­
oped. VC-SGNPs-R were administered intravenously (I.V), which were
then followed for FMT tissue quantification, biodistribution and wound
healing, and accurately measured through IVIS live imaging and pho­
tographed in both mice infection model. The aim of this study was to
develop a promising biomimetic nanosystem-coated RBCM delivery
system for on-demand delivery, immune evading, prolong circulation
and synergistic effects of vancomycin in a combination with a natural
compound of chlorogenic acid against MDR pathogens.
2. Material and methods
2.1. Material, cell and animals
Vancomycin (HPLC ≥99%, Meilun Biologics, Dalian, China),
chlorogenic acid (≥95%, titration), bovine bone type-B (~250 g,
Bloom), sodium metabisulfite (HPLC ≥99%) and dialysis bag of
8000–14000 MW (Sigma-Aldrich, US), glutaraldehyde solution (50%
wt. in H2O), ethylene diamine tetra acetic acid (EDTA, HPLC ≥99.5%,
Innochem Beijing, China), PBS, DAPI (2-(4-Amidinophenyl)-6-indole­
carbamidine dihydrochloride, 5 mg/mL) and SDS-PAGE gel quick
preparation kit were obtained from Beyotime Biotechnology (Shanghai,
China). RAW 264.7, 293T, and LO2 cell lines were purchased from Cell
Culture Center of Institute of Basic Medical Sciences, Chinese Academy
of Medical Sciences (Beijing, China), cell counting kit (CCK-8), and
tryptic soy broth (TSB) were obtained from Beyotime Institute of
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Journal of Drug Delivery Science and Technology 76 (2022) 103706
Biotechnology (Shanghai, China). Culture plates (96-well, Corning
Company), methicillin-resistant Staphylococcus aureus (MRSA) USA
300 strains were provided by Dr. Mingkai Li Lab, Department of Phar­
macology, School of Pharmacy, the Fourth Military Medical University,
Xi’an city, China on special request. Fluorescein isothiocyanate (FITC,
10 mg, Tianjin Biolite Biotech Co, Ltd, Tianjin, China), 1,1′ -dioctadecyl-
3,3,3′ ,3′ -tetramethylindotricarbocyanine iodide, (DiR, 10 mg, Dalian
Meliun Biotechnology Co., Ltd, Dalian, China), fetal bovine serum (FBS)
and Dulbecco’s Modified Eagle Medium: (Nutrient Mixture F-12 DMEM/
F12) were from Gibco Company (New York State, USA). DAPI dihy­
drochloride and MTT were from Beyotime Biotechnology (Shanghai,
China). Heparin was purchased from Sinopharm Chemical Reagent Co.,
Ltd. (Shanghai, China). All reagents were of analytical grade. Specific-
pathogen free male SD mice were supplied by Shanghai Jiesijie Exper­
imental Animal Co., Ltd. The animal experiments were carried out
following the Guidelines for Care and Use of Laboratory Animals of
Shanghai Jiao Tong University and approved by the Animal Ethics
Committee (Number: A2018021).
2.2. SGNPs preparations and drug loading
SGNPs were prepared using the desolvation method [6,21], a 100 mL
of gelatin solution (0.5% w/v, pH 6.0) was prepared by stirring the solid
gelatin and ultrapure water at 50 ◦ C/1000 rpm. Then 20 mg, each of V
and C in combinations were added to the filtered solution (at a ratio of
2:2:10) and were kept on bath sonication for proper mixing and com­
ponents miscibility. To induce the desolvation process, 20 mL of acetone
was added, until to obtained a faint turbidity. Finally, glutaraldehyde
aqueous solution (10% v/v, 500 μL) was added to the gelatin solution
with continuous stirring at 1000 rpm for 2 h. An aqueous sodium met­
abisulphite solution (1.6% w/v, 8 mL) was added to stop the cross­
linking. The nanoparticle-formed solution was dialyzed overnight to
remove the unloaded free V and C, and were lyophilized to form
VC-SGNPs nanoparticles. The FITC-labeled VC-SGNPs were prepared for
macrophage uptake research (methodology 2.6), adding 400 μL of FITC
solution (50 μg/mL) into 600 μL 1 × PBS, which were then added in 1:1
to VC-SGNPs.
2.3. Preparation of RBCM derived vesicles
The RBCM derived vesicles were prepared by the hypotonic hemo­
lysis method [21,22]. The whole blood was collected from SD rats and in
tube containing 1.5 mg anticoagulant EDTA per mL of blood. The whole
blood was centrifuged at 2000 rpm for 5 min at 4 ◦ C to remove plasma.
Blood plasma, white blood cells, platelets, and the buffy coat were dis­
carded. The resulting RBCs were washed 3 times with ice-cold 1 × PBS
following the removal of PBS. Then, add 0.2 mM EDTA for hemolysis
along with 10 × PBS and repeated 3 times. The internal hemoglobin
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
vesicles were stored at − 80 ◦ C.
2.4. RBCM coating and encapsulations efficiency
RBCM coating of VC-SGNPs were carried out according to the pre­
viously published method [3,13]. VC-SGNPs nanocore were added to
light pink RBCM suspension with 1:1 ratio and mixed together. The
mixture was sonicated at 4 ◦ C for 2 min for membrane electroporation
and coating. Then centrifuged at 7000 rpm for 5 min at 4 ◦ C, supernatant
was discarded and VC-SGNPs-R was collected. The loading content
(DLC) and encapsulation efficiency (DLE) of V and C in VC-SGNPs were
determined by RP-HPLC (Supplementary Infromation, S1) and calcu­
lated as following.
W2 − W1
DLC (%) = × 100 (1)
W3
Z. Kamal et al.
W2 − W1
DLE (%) = × 100 (2)
W2
where W1 is the weight of free drug, W2 is the weight of initial added
drug, and W3 is the total gathered SGNPs.
VC-SGNPs-R stability studies were carried as per [23,24] published
procedure. The stability of size, PDI and pH of VC-SGNPs and
VC-SGNPs-R nanosystem were investigated. The samples were kept for
stability under − 20 ◦ C, 4 ◦ C and room temperature for 15 days.
2.5. Characterization
The morphology and size of SGNPs, VC-SGNPs and VC-SGNPs-R were
examined using transmission electron microscope (TEM). The research
was carried out on Tecnai G2 spirit biotwin biology TEM at accelerating
voltage of 120 keV. The zeta potential, size distribution and PDI were
measured by dynamic light scattering (DLS) method, while protein
retaining of RBCM was confirmed through SDS-PAGE. Fluorescence
spectra was measured on laser confocal fluorescence microscopy (CLSE).
Biofilm disruptions were confirmed through atomic force microscopy
(AFM), where its quantifications were affirmed through multifunctional
microplate reader. Similarly, fluorescence molecular tomography and
biodistribution were studied, while tissue homogenization quantifica­
tion was completed. Drug release study was confirmed through RP-
HPLC and LC-MS/MS.
2.6. SDS-PAGE for protein confirmation
Membrane protein contents were measured with bicinchoninic acid
assay (BCA) through sodium dodecyl sulfate polyacrylamides (SDS-
PAGE) gel electrophoresis experiments [6,10,21]. SGNPs and VC-SGNPs
were taken as a blank group. SGNPs-R was centrifuged for 30 min at the
speed of 14 000 rpm to remove unbound RBCM and subsequently
treated with SDS to solubilize the membrane proteins. BCA protein
quantification kit was used to determine and adjust protein concentra­
tion in each sample equally. Mixed 25 μL of loading buffer with 100 μL of
all the samples (RBCM, SGNPs, VC-SGNPs and VC-SGNPs-R). For protein
denaturation, all mixtures were boiled in water for 5 min at 95 ᵒC. Gel
plate was loaded with 15 μL of each sample and protein marker (2 μL)
was added into another separate loading slot for gel electrophoresis. The
gel piece was stained in dark with Coomassie Brilliant Blue at RT for 30
min and kept overnight on for proper decolorization. The gel was
recorded by photographing under the Tanon 2500 Gel Imaging Analysis
System.
2.7. Macrophage cells uptake and internalization
The immune escaping capability of VC-SGNPs-R was examined
through anti-phagocytosis against RAW 264.7 cell lines [6,21,25,26].
These macrophage cells were cultured in DMEM medium (10% FBS) and
seeded in a 24-well plate with a density of 105 cells per well. Hydrophilic
FITC fluorophore, (green color, excitation/emission = 492 nm/520 nm)
were used to dye VC-SGNPs (50 μg/mL) which is then added into the
macrophage culture medium dyed with Hoechst (excitation/emission =
350 nm/461 nm) with blue-cyan fluorescent. The macrophage uptake
and internalization of VC-SGNPs and VC-SGNPs-R were examined after
incubated with macrophage cells for 30 min and then thoroughly
washed with PBS. The fluorescence quantification by macrophage cells
were imaged with CLSEM, through 100 oil-immersion objective lenses at
488 nm.
2.8. mRNA expression for inflammatory cytokines cascading’s
Ex-vivo analysis for tissue necrotic factor (TNF-ɑ), pro-inflammatory
cytokines interleukin (IL-2 and IL-6), and anti-inflammatory mediator’s
interleukin (IL-10) cytokines were analyzed against VC, VC-SGNPs,
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Journal of Drug Delivery Science and Technology 76 (2022) 103706
SGNPs-R, and VC-SGNPs-R for their pro-inflammatory and anti-
inflammatory cascading’s through quantitative real-time PCR (Q-
PCR). Total mRNAs were extracted from RAW 264.7 cells through the
method [14], using Trizol reagent (Supplementary Infromation, S3). The
mRNA expressions were analyzed on a 1% agarose gel stained with
ethidium bromide and conducted in the light cycler quantitative PCR
apparatus using the SYBR green master mix. The results were presented
as a fold increase relative to the expression of housekeeping gene glyc­
eraldehyde 3-phosphate dehydrogenase (GAPDH). The following primer
pairs were used, such as TNF-α, forward, 5′ -
TCTTCTGCCTGCTGCACTTT-3′ , and reverse, 5′ - ATGAGGTA­
CAGGCCCTCTGA -3’; IL-2, forward, 5′ AACTCACCAGGATGCTCACA-3’;
IL-6, forward, 5′ - CCACCGGGAACGAAAGAGAA-3′ , and reverse, 5′ -
CAGCTCTGGCTTGTTCCTCA-3’; IL-10, forward, 5′ -
GTGAAAACAAGAGCAAGGCCG-3′ , and reverse, 5′ - GCCACCCT­
GATGTCTCAGTTT-3’ [27,28].
2.9. MRSA exotoxins neutralization study
The hemolytic activity of exotoxins secreted by MRSA can evaluate
the neutralization ability of the VC-SGNPs-R. A 10 mL overnight MRSA
culture (12 h incubation, with 0.5 OD600 nm (~1.5 × 106 CFU/mL)) was
centrifuged (4500 g for 30 min), and the supernatant was passed
through a 0.2 μm filter membrane. Than 1 mL of the filtered supernatant
was incubated at 37 ◦ C for 30 min with VC-SGNPs-R (400 μL, 25 μg/mL).
Subsequently, 100 μL of whole RBCs was added to the above mixture,
incubate at 37 ◦ C for 1 h to examine the bacterial exotoxins hemolysis.
Through centrifugation (4500 g, 15 min) the supernatant was collected,
and the degree of hemolysis was judged from hemoglobin release. The
absorbance for percent hemolysis can be measured at 540 nm. TSB
medium and 1% Triton X-100 served as a negative and positive control
respectively [8].
2.10. In vitro drug release
The cumulative release of VC-SGNPs-R was performed through
dialysis cellulose membrane (MW cut off 8k-14 kDa) method. To ensure
thorough wetting, the dialysis membranes were soaked overnight in the
dissolution media. Then 1 mL of VC, VC-SGNPs, VC-SGNPs-R were
placed in their respective dialysis bag, and the two ends of the bag were
tied and fixed by clamps. The dialysis bag was submerged into a beaker
containing 10 mL of PBS (pH 7.4) and kept at 37 ± 0.5 ◦ C, stirred
magnetically at 100 rpm. At preset intervals, the samples were with­
drawn with instant replacement of equal volumes of the fresh PBS to
uphold sink condition. The samples were passed through 0.22 μm sy­
ringe filters and the released content was determined by RP-HPLC [3].
In the same way the responsive release of VC-SGNPs-R was exposed
to MRSA bacterial strain for cumulative release over time. In order to
demonstrate the release of V and C from RBCM coated DDS in the
presence and without MRSA exotoxins (triggered by gelatinase and
pore-forming toxins), was determined by LC-MS/MS [6,10,12].
VC-SGNPs-R derived vesicles were kept in Tris buffer (50 mM NaCl, 20
mM Tris HCl, 0.05% Brij-35, 5 mM CaCl2) in a pH 7.4, along with and
without MRSA exotoxins, and the release of V and C at different time
intervals were analyzed [8]. It should be noted that Tris within a buffer
range of pH 7–9, harmonized with physiological pH and increase cell
membrane permeability in most of the organisms [29].
2.11. In vitro anti-MRSA activity assay
The antibacterial assays of RBCM coated DDS were previously re­
ported on gelatinase positive and negative bacteria [5,9]. In this work,
MRSA USA 300 strains was selected only as a PFTs secreting and
gelatinase-positive bacteria for all anti-MRSA assays. MRSA was
cultured in a fresh TSB medium for 12 h at 37 ◦ C on a shaker bed (220
rpm). Minimum inhibitory concentration and minimum bactericidal
Z. Kamal et al.
concentration of V and C were determined as per guidelines of clinical
and laboratory standards institute using tetrazolium microplate assay in
U-shaped 96-well plates, [3,8,30]. Then synergism in VC (combination)
was determined through fractional inhibitory concentration (FIC)
checkerboard dilution method (Supplementary Infromation, S4). The
FIC index was calculated using the first turbid column and row, while for
its determination using the formula, FIC index = FIC V + FIC C =
[V]/MIC V + [C]/MIC C, where [V], MIC V, FIC V, are the concentrations,
MIC and FIC of vancomycin respectively, while [C], MIC C, FIC C are
represented in the same fashion for chlorogenic acid [21,31]. The MICs
of V, C and all relative concentrations were used in various biological
activities of VC-SGNPs-R, VC-SGNPs, SGNPs, VC (with and without
RBCM). The bacterial kinetics viability, at different sample concentra­
tions 1/16 MIC, 1/8MIC, 1/4MIC, 1/2MIC, MIC, 2MIC μg/mL, and MIC
alone were determined at 0, 0.3, 1, 2, 4, 6, 8, 12, 18 h time intervals. The
bacterial culture in log phase was diluted with fresh medium and then
20 μL of bacteria OD600nm of 0.5 McFarland standard (equivalent to 1.5
× 106 CFU/mL) were added to 96-well plate. PBS, SGNPs, VC-SGNPs
and VC-SGNPs-R were added to 96-well plate separately and cultured
overnight, then were measured through multi-function microplate
reader under OD600nm. The antibacterial effects was also shown by the
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
applied to the TSA plate, then cultured at 37 ◦ C overnight. CFU/mL
quantification was evaluated through colony counting method using
image j-software 1.52v version. The groups treated with PBS were used
as control. All experiments were carried out in triplicates.
2.12. Quantification of biofilm biomass
Anti-biofilm activity of V and C (free drugs, SGNPs, and RBCM
coated samples) was determined against MRSA using the procedure of
24-well microtiter plate [22]. The bacterial suspension from overnight
culture was prepared with suspension turbidity of 0.7 OD600 nm (~109
CFU/mL). For tested samples, two-fold serial dilutions were prepared in
100 μL TSB (supplemented with 1% glucose) and added to the wells.
Then adding 60 μL of above bacterial suspension followed by 40 μL of
fresh TSB with 1% glucose to make the final inoculum of 6 × 107
CFU/mL in every well. The final concentrations of the tested samples
were ranged from 1/16MIC,1/8MIC, 1/4MIC, 1/2MIC, MIC, 2MIC
μg/mL. After incubation for 24 h at 37 ◦ C, the planktonic cells were
removed from each well with PBS and fixed the biofilms for 15–30 min.
The biofilm with crystal violet (0.1% wt/vol) was stained for 10–15 min
and rinsed thoroughly with water until the negative control wells
appeared colorless. For biofilm quantification, 200 μL of ethanol (95%)
was added to the crystal violet stained wells and measured at 595 nm
(A595). The effect of tested samples was also examined based on these
pre-formed biofilms. Biofilm quantification was assessed through
percent biofilm inhibition, calculated using the formula, % biofilm in­
hibition = [OD in control – OD of tested samples]/OD in control ×
100%.
2.13. Biofilm morphological changes
The tested samples from biofilm quantification research were sub­
jected to AFM for morphological changes. 10 μL polylysine (was added
on freshly cleaved mica slide and dried at room temperature. Withdrawn
5–10 μL from the positive control (105 CFU) and tested samples from
biofilm microtiter plates wells were applied on prepared polylysine mica
slides, dried, and subjected to AFM analysis for morphological changes.
2.14. FMT-IVIS live imaging and biodistribution studies
Successful quantification of VC-SGNPs-R at infection sites and tissue
biodistribution were carried out with minor modification in mice
infection model [8,10]. Four types of infection model (Model 1: 1a,1b,
4
Journal of Drug Delivery Science and Technology 76 (2022) 103706
neck infection. Model 2: 2a, 2b, thigh infection. Model 3: 3a,3b, right leg
infection. Model 4: 4a, 4b), left leg infection were established with male
SD mice along with negative control (Cta, Ctb). The mice were anes­
thetized with pentobarbital and the hair on the thighs, neck, and back
was removed. Then fresh culture of MRSA (108 CFU/mL, 50 μL) were
inoculated into the 8 mm knife cut injuries of neck and thigh regions
while in legs it was injected intraplantarly in the dorsal surface of the
right and left hind paw on Day 1. From Day 1 to Day 5, the mice were
kept for incubation period at 37 ◦ C for 24 h observations. When in­
fections were fully developed, VC-SGNPs-R-DiR (coated with DiR
labeled fluorescence RBCM, Ex/Em = 748/780 nm) was injected
intravenously via tail, for 5 consecutive days. FMT were measured both
in IVIS live imaging as well quantified for infection-microenvironment
responsiveness in all respective models.
For fluorescence biodistribution and quantification, the respective
mice were sacrificed after IVIS imaging. All vital organs including the
infection sites were extracted and homogenized in normal saline (0.1 g
of tissue/mL). Then it was centrifuged, the supernatant was collected
and quantified at 748 nm/780 nm. VC-SGNPs-R-DiR content in each
organ was expressed as a percentage of the VC-SGNPs-R-DiR injected
dose per gram of tissue, denoted as % ID/g.
2.15. In vivo anti-MRSA activity for inflammation healing and wound
repairing
The MRSA infection model for neck and right leg were selected for in
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
administered IV into respective groups along with 1 × PBS (negative
control). Wound healing in skin infection model size and inflammation
in leg infection model were assessed and recorded on a daily routine
basis during the therapy (Supplementary Information, S5). For colony-
forming units (CFUs), 10 μL of the samples collected from leg and skin
infection were diluted in 103-fold of 1 × PBS, then 20 μL was applied to
the TSA plate, and cultured at 37 ◦ C overnight, and were evaluated for
CFU/mL quantification. The group treated with PBS was used as a
negative control, while 1 × PBS-MRSA acted as a positive control. For
biodistribution, inflammation healing and wound repairing, infection-
microenvironment responsiveness, and on-demand antibiotic delivery
of VC-SGNPs-R-DiR in comparison with SGNPs-R-DiR (act as a control),
FMT was carried out through IVIS Lumina imaging system, during the
treatment period on Day 5, 6, 7, 8 and 10 consecutive days.
While for % ID/g of tissues, mice were sacrificed after 24, 48, 72, 96,
and 144 h, and homogenized in normal saline and quantified at 748/
780 nm. The anti-MRSA activity of VC-SGNPs-R, like wound healings,
CFU, FMT and tissue quantification was photographed, observed, and
co-related with each other as well with negative and positive control.
The previously published method was modified according to designed
experiments and therapeutic goal achievement [5,22].
2.16. Biocompatibility and toxicological studies
The biocompatibility and toxicology of VC-SGNPs-R DDS with mice
body physiology like tissues, blood, and vital organs were carried out [3,
8,25,32]. Both leg and skin infection model were weighed on a daily
basis during treatment, and the changes in body weight were recorded.
Similarly, acute toxicities were also observed during the entire treat­
ment plan. Phlebitis, blood cross match and hemolysis test were per­
formed for safe I.V administration. As VC-SGNPs-R was injected through
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
used as negative and positive control respectively (Supplementary
Infromation, S6). For cytotoxicity’s and cyto-compatibility, MTT assay
was carried out against human embryonic kidney (HEK) 293T and liver
LO2 cell lines. These cells were seeded in 96-well plates [3], 104 cells in
each well were treated with different concentrations of
Z. Kamal et al. Journal of Drug Delivery Science and Technology 76 (2022) 103706

VC-SGNPs/VC-SGNPs-R (2MIC, MIC). Then, 10 μL MTT solution (5%) 3. Results

was added to each well, and incubation was continued for 3 h. The
cytotoxicity’s were measured at OD545nm. Respective mediums were
served as a negative control while untreated cells were used as a positive
control, where the proliferation rate was set to 100%. Cells viability
were calculated according to the formula, viability (%) = ODt/ODn ×
100%, OD: optical density (absorbance), ODt: absorbance of the
experimental group, ODn: absorbance of the blank control. After treat­
ment with VC-SGNPs-R DDS (with and without membrane), the mice
were euthanized, their main organs were collected and stained for his­
tological analysis (Supplementary Information, S7) through hematoxy­
lin and eosin staining (H&E).
2.17. Statistical analysis
All data were presented as the ±standard error means (SEM). Graphs
were plotted and appropriate statistically significant differences were
assessed by one-way, two-way ANOVA, Tukey tests, Danette test,
Sidak’s multiple comparisons test, linear regressions, and co-relations
using graph pad prism software 8.4.2 (679) version. The differences
were significant statistically when * indicates p < 0.05, ** indicates p <
0.01, and *** indicates p < 0.001, ****p < 0.0001, where ns indicates
non-significant.
3.1. VC-SGNPs-R preparation and characterization
Fig. 1 illustrated the overall procedure of RBCM vesicles derivation,
SGNPs preparation, drug loading and its administration to infection sites
for VC-SGNPs-R delivery and synergism. Surface characterization,
coating morphology, size and surface zeta potentials were confirmed
through TEM (Fig. 2A I, II & III) and DLS (Fig. 2B). The successful RBCM
coating were shown in TEM images, which depicts spherical and uni­
form size approximation of ~41.32 ± 3.06 nm for SGNPs, ~51.80 ±
3.22 nm for VC-SGNPs, ~77.78 ± 2.50 nm for VC-SGNPs-R. The
retaining of membrane protein was also confirmed through SDS-PAGE
protein bands, which was well preserved in comparison with natural
RBCM bands, SGNPs, VC-SGNPs (Fig. 2E). Similarly, the zeta (ζ) po­
tential of core SGNPs and VC-SGNPs were − 7.19 ± 1.17 mV, 7.43 ±
1.34 mV respectively and became − 25.63 ± 0.99 mV after being coated
with RBCM (Fig. 2C). DLE and DLC of V and C were carried out on the
basis of total surface area (tSA) calculations (Supplementary Informa­
tion, S2) of VC-SGNPs, tSA VC-SGNPs = 0.84 × 1012μm2, and calculated as
per procedure mentioned [8]. Highest DLE of V reached to 83. 95% with
DLC 13.85%, while C was 81.22% and 13.85% in a V:C:SGNPs ratio of
2:2:10, which were used in all in vivo and in vitro experiments. Fig. 2D
shows different V, C and SGNPs ratios (1:1:10, 2:2:10 and 5:5:10), and
their DLE and DLC efficiencies. The stability test of VC-SGNPs-R (size
and PDI) at different temperature and pH were shown in Fig. 3A, B, 3C
and its pH variation were shown in Fig. 3D.

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.)

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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

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Z. Kamal et al.
cytokines like TNF-ɑ, IL-2 and IL-6 and anti-inflammatory mediator IL-
10 (Fig. 4C, D, 4E, 4F) respectively.
3.3. Exotoxins neutralization and relative hemolysis
RBCM acts as a nanosponge, absorb exotoxins on its surface, which
disrupts the membrane and hydrolyze the gelatin core, releasing the
internal contents in infection-microenvironment, as shown in (Fig. 5A
and B). The anti-hemolytic activity further affirmed the exotoxins ca­
pabilities of pore formations and membrane eruptions. The relative
hemolysis was shown according to the content of hemoglobin released
from RBCs in the positive control (distilled water), negative control
(PBS) and with MRSA (Fig. 5C and D). The releasing of RBCs hemoglobin
contents provides a logical basis for the release profile of V and C from
VC-SGNPs-R DDS in an infection-microenvironment.
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Journal of Drug Delivery Science and Technology 76 (2022) 103706
Fig. 4. Macrophage cells uptake and inflammatory
cytokines cascades studies. A) Confocal laser scanning
electron microscopy images of macrophage (RAW
264.7 cells) cells uptake and internalization (scale
bar = 10 μm), B) Fluorescence intensities (FL) of
SGNPs, VC-SGNPs, SGNPs-R and VC-SGNPs-R, fluo­
rescence dye FITC (50 μg/mL, emit green color) Ex/
Em = 492/520 nm used to labeled nanoparticles,
while nucleus of macrophage cells were stained with
Hoechest (1 mg/mL, 0.5–5 μm used, emit blue-cyan
fluorescent light) Ex/Em = 350/461 nm, analyzed
through ordinary comparison, one-way ANOVA, (n =
4,mean ± SEM ****p < 0.05), C), D) & E) Pro-
inflammatory cytokines cascades of SGNPs-R, VC-
SGNPs-R, VC and VC-SGNPs on mRNA expressions
(extracted from RAW 264.7 cell lines) of TNF-ɑ, IL-2
and IL-6, F) Effects against Anti-inflammatory cyto­
kine IL-10, (n = 3,mean ± SEM, ****p < 0.0001, two-
way ANOVA Tukey’s multiple comparisons test). (For
interpretation of the references to color in this figure
legend, the reader is referred to the Web version of
this article.)
3.4. In vitro drug release from VC-SGNPs-R
The in-vitro cumulative release of V and C from VC-SGNPs-R were
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
tively, while it took 96 h in VC-SGNPs-R. Similarly, 100% cumulative
release of C from VC and VC-SGNPs were 2 h and 8 h respectively, where
it was 89.46% in 96 h from VC-SGNPs-R. In responsive release study,
VC-SGNPs-R were subjected to MRSA exotoxins contains gelatinase and
PFTs, and Tris buffer (pH 7.4) for 48 h. Within 6 h, 85.46% ± 2.88 of V
was released in the presence of bacterial toxins, while in Tris buffer the
on-demand responsive release were 6.20% ± 2.86 (Fig. 5G). Similarly,
the responsive release of C was 85.36% ± 2.91 (with MRSA) and 12.12%
± 2.30 (without MRSA) within 6 h, as shown in Fig. 5H.
Z. Kamal et al.
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
The entrapment of V and C combination exerts synergistic effects,
which were determined through checkerboard dilution assay (Fig. 6A).
MIC of V was 16 μg/mL, while that of C was 512 μg/mL, where in
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
C combinations. This combination was used for all in vitro and in vivo
experiments on VC (free drug combination), VC-SGNPs (entrapped in
gelatin nanoparticles) and VC-SGNPs-R (RBCM coated SGNPs). The
percent biofilm inhibitions were higher for VC-SGNPs-R, VC-SGNPs and
VC in comparison with SGNPs and TSB as a negative control (Fig. 6B).
These biofilm quantifications were further morphologically observed on
AFM through height sensor and 3D images. MRSA and SGNPs treated
sample shows AFM height sensors of 666.4 nm and 756.8 nm respec­
tively, while they were 4.5 nm, 34.7 nm, 35.7 nm for VC, VC-SGNPs, VC-
SGNPs-R respectively (Fig. 6C). The anti-MRSA concentration-depen­
dent and time-dependent (0, 30 min, 1–18 h) viability were higher at
MIC concentrations for VC, VC-SGNPs and VC-SGNPs-R nanoparticles in
comparison with control (Fig. 6D and E). The VC-SGNPs-R treated MRSA
samples showed decreased optical densities and colony formations
(Fig. 6F and G). The TSA plates further affirmed the anti-MRSA effects of
VC-SGNPs-R at MIC and 1/16MIC (Fig. 6H).
3.6. FMT quantification and tissue biodistribution
The FMT of VC-SGNPs-R-DiR through IVIS live imaging were quan­
tified in all infection sites of leg and skin infection model in comparison
with control (Fig. 7A). The average radiant efficiency [p/s/cm2/sr]/
[μW/cm2] in each mice model infection sites were calculated from the
region of interest (ROI), properly encircled (white), in association with
control mice (Fig. 7 A1-A4). The increase in radiant efficiency at ROIs
indicates the accumulation of VC-SGNPs-R-DiR in infection sites, which
affirmed our hypothesis of its responsiveness in infection-
microenvironment.
Fig. 7B and C shows the biodistribution of VC-SGNPs-R-DiR to
various tissues in all control and infection mice models. Percent injected
dose per gram of tissue (% ID/g) during 24 h was confirmed through the
homogenization of vital organ and infected sites collection for fluores­
cence quantification. Fig. 7D demonstrates fluorescence % ID/g of the
neck and skin mice infection model in comparison with negative control
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
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Journal of Drug Delivery Science and Technology 76 (2022) 103706
were further evaluated for responsive drug release, wound healing and
anti-MRSA activities.
3.7. In vivo anti-MRSA activity for inflammation healing and wound
repairing
After 5 days of incubation in an optimum condition, the right
inflamed leg infection model and wound size of 2.21 mm in skin infec­
tion model was developed (Fig. 8A and B). On Day 6, VC-SGNPs-R-DiR,
SGNPs-R-DiR, VC, VC-SGNPs, and 1 × PBS (control) were administered
via tail vein, inflammation and wound healing were observed with
physical measurements and FMTs IVIS live imaging for 5 consecutive
days. As the size were reduced in both inflammation and wound
repairing with VC-SGNPs-R treated models, which is an indication of
VC-SGNPs-R anti-MRSA responsiveness in an infection-
microenvironment (Fig. 8C and D). No effects on body weight in all
infection models were observed during the treatment (Fig. 8E and F).
The MRSA colonies formations were also reduced in all infection
models treated with VC (free drug combination for reference), VC-
SGNPs and VC-SGNPs-R nanoparticles, as in comparison with SGNPs
treated and positive control (Fig. 9A and B), while CFU/mL calculation
were mentioned in Fig. 9C. Linear regression of correlations among in
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.
The healing of inflammations and wound repairing in leg and skin
infection models treated with fluorescence labeled SGNPs-R and VC-
SGNPs-R, were tracked through FMTs IVIS live imaging (Fig. 10A and
B) respectively. The high radiant efficiency in the bright field (BF) with
fluorescence showed prominent inflammation, and wound size in
SGNPs-R treated mice model, which can be seen in the BF as well. In
comparison with VC-SGNPs-R treated mice model, the low radiant ef­
ficiencies indicate infection healing and wound repairing (Fig. 10C and
D). The fluorescence labeled nanoparticles were quantified in various
tissues, as well in infection sites for biodistribution. The ex-vivo fluo­
rescence intensities of %ID/g of infection tissue (leg and skin) and
various organ reflectance images were shown in Fig. 10E and F, while
quantification were presented in Fig. 10G and H. These fluorescence
intensities and reflectance were compared with control mice model
(Fig. 10I).
3.8. Biocompatibility and toxicology
As VC-SGNPs-R were I.V injected, so it is necessary that it should be
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.

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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.

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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.

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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).

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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
acute toxicities, the survival ratio were 100% during the treatment
schedule (Fig. 11A and B), which affirmed its non-lethality. The histo­
logical examination conducted through H & E staining for the vital or­
gans (heart, lungs, kidneys, liver etc.) of infection model treated with
VC-SGNPs-R nanoparticles showed no toxicological observations as in
comparison with normal mice (Fig. 12A). It illustrates high tissue
biocompatibility and safe use of VC-SGNPs-R. Cyto-compatibility &
toxicities at various concentration by VC-SGNPs-R were checked at
normal cell lines, 293T cells (kidneys) and LO2 cells (liver) shows 95%
of viability at highest concentration (Fig. 12B), while Figs. S4A and S4B
(Supplementary Information) show cytotoxicities at different concen­
trations against kidney and liver cell lines. Fig. 12C shows regression co-
relation analysis of HEK 293T and LO2 after 24 h treatment with VC-
SGNPs-R. As VC-SGNPs-R were directly injected into blood, so it is
necessary that it should be compatible. Hemolysis at various concen­
tration (100–500 μL, control) showed no obvious hemolysis (Fig. 12D),
while that of DI water showed 100% hemolysis after 0, 3, 24 h. Optical
microscopic images of hemolysis and blood compatability of VC-SGNPs-
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.

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Z. Kamal et al.
(Supplementary Information). The hemoglobin released were used for
relative hemolysis, which is measured from the absorbance through the
microplate reader (Fig. 12E).
4. Discussions
The VC-SGNPs-R and its characterization revealed a successful
translocation to the surface nanoparticles and homogeneous dispersion.
Herein, RBCM gives an extra thickness of ~8.69 ± 1.4 nm membrane,
though, usually its thickness varies 5–10 nm [3,25]. It was affirmed that
the majority of endogenous and surface protein retained during the
preparation of VC-SGNPs-R nanoparticles. The hydrodynamic particle
size distribution measured by DLS was relatively higher due to water
film on the outside of the nanoparticle molecules, as in comparison with
TEM in which the water film is removed on drying [14]. The average size
on DLS of unloaded SGNPs were 114.63 ± 2.3 nm, loaded VC-SGNPs
182.93 ± 3.98 nm and VC-SGNPs-R 190.53 ± 3.4 nm along with PDI
of 0.07 ± 0.09, 0.08 ± 0.09 and 0.11 ± 0.07 respectively. Enhanced
antimicrobial efficacy is associated with quantum size of the nano­
particles, which increase the surface areas [26]. The alteration in surface
charge potential may be due to the membrane charge shielding with its
lower negative potential to gelatin core with higher negative potential
[10,33]. Similarly, higher ζ-potential magnitude exhibits stable sus­
pension, which is due to a larger electrostatic repulsion among particles.
DLE & DLC of the nanosystem in reference with per unit weight of the
gelatin nanoparticles provides sustained therapeutic released.
Similarly, escaping the immune system is the primary core concept of
VC-SGNPs-R DDS for a prolonged sustained release. As antibiotic accu­
mulation at infection sites and rapid elimination by the immune system
e.g. mononuclear phagocytic system severely minimizing the release of
nanoparticles in bacteria-microenvironment infection site thus it can
impede antibacterial efficacy, drug delivery and bioavailability [10,34].
The responsive release of VC-SGNPs-R at infection sites may also avoid
the premature drug release [35,36]. Thus, the innate features of
macrophage cells help VC-SGNPs-R to escape the immune system, pro­
long systemic circulation [37] and avoid the inflammatory cytokines
responses [27,28]. Recently, RBCM coated polymeric nanoparticles
have been investigated as a new class of biomimetic material in DDS,
which attain a fabricated stealth surface retains from natural RBCs,
exhibit low immunogenicity, reduce opsonization, escape phagocytosis
and prolong the drug half-life in plasma [38,39].
After incubation with macrophage cells, SGNPs/VC-SGNPs emit
green radiations, while no fluorescence was observed with SGNPs-R or
VC-SGNPs-R depicted immune escaping. Similarly, mRNA inflammatory
cytokine folde expressions were not observed in the cells treated with
SGNPs-R or VC-SGNPs-R, which showed no apparent inflammatory
cascades and immunological responses [3,8,10,32–34,40,41]. The safe
elimination of pathological agents has been reported using RBC surface
loading via injection of hetero-conjugates binding to complement re­
ceptor [35,36], where fusion binding like recombinant therapeutics
proteins to the RBCs markedly prolong circulations, enhance potency
and reduce systemic toxicities [42–45].
The MRSA exotoxins helps in tissue invasion, penetration, evasion of
host defenses and survival under extreme conditions in various patho­
gens. The hemolysin (α, β and γ) released by these exotoxins can degrade
erythrocytes and leukocytes membrane via binding to ADAM10 recep­
tor, disintegrin and matrix metalloproteinase (MMP-2 & MMP-9) [3,46].
It may causes pore formation and permeability, which may cause Ca2+
influx/K+ efflux, which can disrupts homeostasis of the host cells and
thus lead to cell death [47]. MRSA and other gram-positive bacteria also
used gelatinase, an extracellular proteolytic enzyme as a virulence factor
that dissolves the connective tissue of the host cells which helps in
infection invasion. Gelatinase causes the hydrolysis of gelatin into
polypeptides and then breakdown its complex structure into monomeric
amino acids [38,48–50]. This phenomenon was used for the lysis of
VC-SGNPs-R DDS released in MRSA-infection-microenvironment. The
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Journal of Drug Delivery Science and Technology 76 (2022) 103706
RBCM nanosponging attract the PFTs onto its surface while the gelati­
nases (proteases) hydrolyze the SGNPs core, while releasing V and C at
infection site. RBCM also protects the hydrophilic surface polymeric
core and retained drug molecules in polymeric gelatin nanoparticles for
prolonging circulation and sustained release [51].
The combination of V and C and it’s in vitro synergistic effects against
MRSA, time-concentration dependent viability and biofilm inhibitions
in VC-SGNPs-R, VC-SGNPs and VC were similar as in comparison with
SGNPs and negative control. In comparisons, VC-SGNPs-R nanoparticles
are different from other combinations in respects with its RBCM coating,
immune evading, extending circulation and drug releases in the pres­
ence of exotoxins. These studies were further justified through in vivo
studies in infection mice models.
In order to achieve an efficient level of target specificity respon­
siveness in infection, tumor, and inflammation-microenvironment, these
RBCM coated nanoparticles should extravasates from circulating blood
and concentrate in certain tissues either non-specifically or specifically
attached to a molecular sites or undergoing cellular internalization [10].
VC-SGNPs-R quantification at infection-microenvironment in both mice
infection models (leg and skin). The degradation of gelatin nano-core
through PFTs and gelatinase enzymes (secreted by exotoxins), triggers
the release of V and C at infection sites. The variation in FMT quantifi­
cation in an infected and non-infected groups exhibits that VC-SGNPs-R
showed a long-lasting, stronger fluorescence signals at infection sites
that depleted with the inflammation healing and wound repairing pro­
cess progressively. Similarly, SGNPs-R accumulates at infection sites,
but no healing expositions can be seen. FMTs and tissue biodistribution
revealed that VC-SGNPs-R was highly quantified in liver and spleen. The
physical examinations of leg and skin infections in comparisons with
FMTs, justified that VC-SGNPs-R nanoparticles quantified at infection
sites released V and C in infection-microenvironment for anti-MRSA
activities.
Successful in vitro and in vivo studies demand a safe and biocom­
patible determination of VC-SGNPs-R. In the last few decade, diverse
strategies are adopted to loaded drugs and nanocarriers to the surface as
well as to the inner core of RBC. Their pharmacokinetics, bio­
compatibilities, systemic toxicology, local adverse effects, adherence to
endothelial cells, formation of on toward cargo complexes, destruction,
elimination and clearance are the potential risks and challenges of
RBCM DDS [52–56]. Biodistribution of VC-SGNPs-R to all major organs
like, liver, spleen, kidney, lung, heart, brain showed normal tissue
structure in H & E staining, as well as no-hemolysis with blood after I.V
administrations. The RBCM make it compatible with blood and other
tissues. Kidney and liver, which are the major organs for clearance and
metabolism, are biocompatible with VC-SGNPs-R nanoparticles both in
vitro and in vivo studies. All these observations, strengthens its biosafety,
compatibility and toxicology aspects. Pharmacokinetic studies have
been reported on RBCM coating nanoparticles previously [10,26,51],
which showed in situ sustained release, that provided a prolong circu­
lation and immune escaping. Thus the overall in vitro, in vivo safe and
biocompatible profile of VC-SGNPs-R nanoparticles and its on-demand
antibiotic delivery at infection-microenvironment could be a prom­
ising candidate in biomedical application against MDR strains.
5. Conclusion
We developed VC-SGNPs-R DDS for V and C loaded in combination
to supramolecular gelatin nanoparticles for anti-MRSA effects in mice
infection model. The limitations in the previous studies reported [3,7,8,
10,11] on RBCM coated nanoparticles and MRSA virulence factor neu­
tralizations were highlighted and extended to a new treatment
approach. Here, in VC-SGNPs-R, the RBCM coating escape the immune
system, significantly prolong the systemic circulation and quantified at
infection-microenvironment for on-demand and responsive release of V
and C combine delivery. The successful in vitro, time-concentration
dependent anti-MRSA effects, biofilm inhibitions, depletion in colonies
Z. Kamal et al.
formations and optical densities were confirmed and subjected through
in vivo, mice-infection models (leg and skin). Declination in the fluo­
rescence quantification of VC-SGNPs-R at infection sites, inflammation
healing and wound repairing indicate its infection-microenvironment
responsive release of V and C for synergistic effects against MRSA.
VC-SGNPs-R has been proved to have low toxicity, good biocompati­
bility with tissues, blood and vital organs, which make it a safe DDS.
Moreover, an excellent in vitro and in vivo results indicate that drug
combination strategies in VC-SGNPs-R nanoparticles can inhibit multi­
drug resistance infections through synergistic effects. Thus, these
infection-microenvironment responsiveness and on-demand antibiotic
delivery platform are expected to be an alternative approach in future
nanotechnology to treat MDR bacteria.
CRediT authorship contribution statement
Zul Kamal: Contributed in the experimental study, including the
investigation, methodology, data curation, formal analysis of data,
research administration, and drafting the original manuscript. Jing Su:
Conceptualization, writing, reviewing, editing and formal analysis,
Faisal Raza: Participated in vivo studies and infection mice model
development, Liangdi Jiang: Assist and participated in characterization
and biodistribution studies, Yichen Li: Help in manuscript revision and
contents formatting, Weien Yuan: Take part in research administration
and revising the manuscript. Mingfeng Qiu: As corresponding authors,
acquired the funding, participated in the study conceptualization, data
analysis, reviewing and editing the manuscript, and finalized this
manuscript. All authors have read and agreed to the published version of
the manuscript.
Institutional review board statement
The study was conducted according to the guidelines of the decla­
ration of Helsinki, and approved by the Animal Ethics Committee of
Shanghai Jiao Tong University (No. A2018021).
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper. I further assure that this work is neither
published nor under consideration elsewhere.
Data availability
Data will be made available on request.
Acknowledgment
This research was funded by the National Major Science and Tech­
nology Projects of China, 2018ZX09711003-008-002, National Natural
Science Foundation of China, 81973487and Shanghai Association for
Science and Technology, 18ZR1419700. Beside it, we are highly
thankful to Professor Bo Zhao Lab, School of Pharmacy, Shanghai Jiao
Tong University for assisting all anti-MRSA activities, Asad Farooqi (East
China Normal University) for pro & pre-inflammatory cytokine studies
primer designing’s.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jddst.2022.103706.
15
Journal of Drug Delivery Science and Technology 76 (2022) 103706
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