Niosomes modified with a novel pH-responsive coating (mPEG-OA) enhance the antibacterial and anti-biofilm activity of vancomycin against methicillin-resistant Staphylococcus aureus

Surface functionalization of nanoparticles has shown potential in enhancing the efficacy of antibiotic-loaded nanosystems against drug-resistant bacteria. The objective of this study was to synthesize and characterize an acid-cleavable pH-responsive polymer from methoxy polyethylene glycol and oleylamine (mPEG-OA) to surface modify vancomycin (VCM)-loaded niosomes and to evaluate their antibacterial and anti-biofilm effectiveness against methicillin-resistant Staphylococcus aureus (MRSA). The novel mPEG-OA-coated niosomes were biocompatible, hemocompatible with size, polydispersity index, and zeta potential of 169.2 ± 1.6 nm, 0.21 ± 0.01 and −0.82 ± 0.22 mV, respectively. Under acidic conditions, mPEG-OA-coated niosomes exhibited a pH-responsive and sustained VCM release profile and in vitro antibacterial activity than non-coated niosomes and bare VCM. mPEG-OA-coated niosomes showed a significant reduction in biofilm formation at pH 6 compared to pH 7.4 (p = 0,0119). The in vivo efficacy of mPEG-OA-coated niosomes in the BALB/c mice skin infection model showed a 9.9-fold reduction in MRSA load compared to bare VCM. Histomorphologically, the mPEG-OA-coated niosomes group displayed the lowest bacterial load, tissue swelling, and inflammation. The results of this study demonstrate the potential of novel pH-responsive mPEG-OA-derived polymer coating to enhance bacterial killing kinetics, and antibacterial and anti-biofilm efficacies over conventional antibiotic and non-functionalized nano delivery systems.


Introduction
Infections due to methicillin-resistant Staphylococcus aureus (MRSA) constitute one of the principal causes of morbidity and mortality in community-acquired and nosocomial infections [1].According to the Centers for Disease Control and Prevention (CDC) in the United States, the mortality rate of MRSA infection has surpassed Parkinson's disease, acquired immune deficiency syndrome (AIDS), and murder mortality [2].Moreover, patients with MRSA infections are 64% more likely to die than patients infected with antibiotic-sensitive bacteria [3].MRSA is a serious pathogen and has been reported to cause an estimated 100,000 deaths worldwide in 2019 [4].MRSA bacteria is a pan-resistant microorganism even to newer drugs [5].This alarming trend poses significant challenges in the effective treatment and management of MRSA infections, necessitating urgent attention and the development of innovative strategies to combat this growing public health threat [6].Therefore, it is imperative to explore and implement newer strategies to address this critical public health concern and bridge this gap effectively.
One of the strategies employed to combat antimicrobial resistance is the application of nanotechnology in designing novel drug delivery systems that protect, potentiate and make them sensitive to resistant bacteria [7,8].Antibiotic nanoencapsulation enhances its pharmacokinetic and pharmacodynamic properties [9].One such nanosystem includes niosomes, which are bilayer-structured nanovesicles of cholesterol and a non-ionic surfactant [10,11].The use of cholesterol as a lipid part in niosomes has been reported to impact the stability, rigidity, permeability control, biocompatibility, and encapsulation efficiency of the niosomes [12][13][14].Nonionic surfactants are employed because they are more biocompatible compared to their ionic counterparts, exhibit lower toxicity profiles compared to ionic surfactants, and have the ability to form stable vesicles due to their structure and amphiphilic nature [15].Additionally, non-ionic surfactants promote passive targeted drug delivery, reduce phagocytic uptake and prolong niosomes systemic circulation [16].Thus the formed niosomes have been found to have advantages such biocompatibility, high loading capacity for hydrophilic and hydrophobic drugs, capability to be easily functionalized, and low cost of production [17].Therefore, these properties have made niosomes a suitable carrier for the delivery of antibiotics compared to conventional drug dosage forms.
Even though nano-based approaches have superior antibacterial activity than conventional antibiotics, there are still current challenges, including short circulation times, non-specific and sub-optimal distribution, and targeting of infection sites.Moreover, nano-carrier stability, potential toxicity, and adequate tissue distribution and penetration raise concerns, particularly for long-term administration [18,19].In response to these challenges, there is a growing need to further engineer nanosystems in order to overcome the aforementioned limitations while simultaneously enhancing the targeting of bacterial and infection sites.
Surface modification enhances nanosystems' therapeutic index by improving drug delivery.It enables sitespecific release, better pharmacokinetics, and prolonged nano-carrier lifespan while improving immune compatibility [20].The surface of antibiotic-loaded nanoparticles has been modified with different coating materials, including polymers, enzymes, antibiotics, peptides, and cell membranes, and applied for macrophage-targeted delivery [21], ocular antibiotic bioavailability [22], and toxin neutralization [23].In addition, the surface modification technique has been applied to prolong systemic antibiotic circulation and release, which can preferentially enhance antibiotic accumulation and uptake by pathogenic bacteria [24,25].Apart from surface modification niosomes have been engineered with biomaterials that impact stimuli responsiveness to changes such as changes in pH [25].Bacterial infection sites often exhibit an acidic pH due to Inflammatory response as a result of the invading bacteria, that triggers cells release proinflammatory compounds [26] and bacterial metabolites , such as lactic acid or acetic acid that can accumulate at the infection site [27] and disruption of the normal physiological balance of the affected tissue that leads to a decrease in oxygen supply and increased production of carbon dioxide and all these result can lower the pH of the infection site and the surrounding tissue [28].
Through niosome surface modification, pH-responsiveness has been achieved through functionalization with coatings that are acid-labile, have pH-degradable linkers or functionalization with coatings that protonates under acidic conditions that, thereby , enhancing the performance of the nano delivery system [29,30].pH responsive coated systems improve antibiotic-targeted and controlled drug delivery to enhance antibacterial efficacy with reduced systemic and off-target toxicity [31].Whilst pH-responsive, polymer-coated niosomes have been explored for the delivery of anti-cancer drugs [32,33], no study to date has explored a pH-responsive polymer for coating of niosomes to enhance antibacterial and antibiofilm activity.
In this study, we report for the first time the synthesis of a novel acid-cleavable pH-responsive polymer from methoxy polyethylene glycol and Oleylamine (mPEG-OA) to be employed in the surface modification of vancomycin (VCM)-loaded niosomes.OA has been reported to enhance the antibacterial activity of antibioticloaded nanoparticles [34], but no study has reported OA for the coating of nanoparticles.mPEG polymer coating has been reported to promote nanosystems biocompatibility and shield the surface of the coated nanosystems from aggregation and opsonization, which enhances the longevity of the coated nanosystems [9,35,36].Owing to the advantages mentioned above, the rationale for the surface modification of VCM-loaded niosomes with mPEG-OA polymer is to maximize the compatibility and therapeutic efficacy of conventional niosomes.Furthermore, it provides additive therapeutic outcomes by minimizing the required effective dose and the dose-dependent adverse effects of bare VCM.In addition, the cleavage of the hydrophobic and hydrophilic portion of mPEG-OA coating amphiphile at acidic conditions potentially enhances the hydrophilicity of the niosomes, leading to the swelling of the nanosystem and increased VCM release at the infection site.
Moreover, the advantages of pH-responsiveness due to cleavage of the acid-labile bond between the mPEG and OA will result in charge-inversion of the niosomes from negative to positive, which is advantageous in enhancing binding to the anionic bacterial surface compared to non-coated and bare VCM.Herein, the synthesis and characterization of mPEG-OA and the formulation of novel mPEG-OA-coated niosomes loaded with VCM are reported.In addition, in vitro biosafety, drug release, and antibacterial studies of mPEG-OAcoated niosomes against Staphylococcus aureus and MRSA mPEG-OA-coated, compared with non-coated and bare VCM are reported in this study.Furthermore, the in vivo antibacterial activity and histomorphology studies of mPEG-OA surface-modified niosomes against MRSA were also evaluated.

Synthesis of mPEG-OA Schiff base
A previously reported method was used for synthesis of mPEG-OA Schiff base [38].Briefly, mPEG-CHO (1.0 g, 0.195 mmol), OA (0.067 g, 0.25 mmol), and TEA (70 μl, 0.5 mmol) were dissolved in 10 ml of dimethyl sulfoxide (DMSO) solution under a nitrogen atmosphere (Scheme 1).The reaction mass was refluxed under stirring for 24 h, then dialyzed with a dialysis membrane having 3500 molecular weight cutoffs.The product was then freeze-dried, resulting in a yellowish-white powder with a yield of 75.4%.FT-IR,1H NMR, and 13 C NMR characterization were as follows: FT-IR (S2, supplementary materials).1H-NMR (CDCl3) 0.

Preparation of mPEG-OA-coated niosomes
Niosomes containing VCM were prepared using the thin-film hydration method [39].Cholesterol, non-ionic surfactants, and DCP ((1:1:0.05)molar ratios) were dissolved in an organic solvent mixture (chloroform: methanol 1:1, v/v) in a round-bottomed flask.Then the solvents were evaporated using a rotary vacuum evaporator at 60 °С, and a thin film was formed on the inside wall of the round bottom flask.After that, the film was stored in a vacuum desiccator overnight to remove the residue of the solvents.The dried film was then hydrated for 1 h with 10 ml of the VCM solution (1 mg mL −1 ) at 60 °С using a rotary vacuum evaporator.Subsequently, the hydrated niosomes were sonicated at pulse on 3 sec and pulse off 2 sec for 10 min at 30% amplitude using an Omni sonic rupture 400 Ultrasonic Homogenizer (Kennesaw, GA 30144, USA).Several surfactants were screened to obtain an optimum formulation size, polydispersity index (PDI), zeta potential (ZP), and drug entrapment.For niosomes surface coating, the mPEG-OA polymer solution was added in equivalent volume to the optimized niosomes dropwise under continuous stirring (600 rpm) for 2 h at room temperature [40].

Particle size, PDI, ZP and morphology studies
The average size, PDI, and ZP of mPEG-OA-coated niosomes were measured using dynamic and electrophoretic light scattering method with a Zetasizer Nano ZS90 (Malvern Instruments, UK).mPEG-OAcoated VCM niosomes were diluted with phosphate-buffered saline (PBS) (pH 7.4, 6, and 4.5) before the measurements.All the experiments were performed in triplicate at 25 °C.The morphology of the mPEG-OAcoated niosomes was studied via High-Resolution Transmission Electron Microscope (HRTEM, JEOL 2100).Samples were placed on an HRTEM grid, stained with 1% uranyl acetate, and viewed after drying.HRTEM was operated at an accelerating voltage of 100 kV [41].

Entrapment efficiency (EE %) study
The ultrafiltration method was used to determine EE %.Briefly, mPEG-OA-coated VCM niosomes were centrifuged at 2500 RPM for 30 min using Amicon ® Ultra-4 centrifugal filter tubes (molecular weight cutoff 10 kDa).The amount of VCM in the supernatant was quantified using High-Pressure Liquid Chromatography (HPLC) (Shimadzu, Japan) with UV detection at 280 nm.The analysis was carried out using a mobile phase of 0.1% trifluoroacetic acid in water (85%) and acetonitrile (15%) at a flow rate of 1 ml min −1 , which was pumped through Nucleosil 100-5 C18 column (150 mm × 4.6 mm) [42].The amount of the drug was determine via equation y = 81624× + 16173 and the regression coeffient was R 2 = 0.9996 The following formula quantified EE %: Weight of VCM added 100 1

Thermal profile analysis study
The thermal characteristics of mPEG-OA-coated niosomes, non-coated niosomes, bare VCM, DCP, cholesterol, and their physical mixture were analyzed using a differential scanning calorimeter (DSC) (Shimadzu DSC-60, Japan).Samples (2 mg) were placed in aluminum pans and hermetically sealed (for the formulation and the physical mixture the contents were in similar ratios as the optimum formular).The runs were conducted over a temperature range of 25 °C up to 300 °C.The heating rate was 10 °C/min under a constant nitrogen flow (20 ml min −1 ) [43].

Protein adsorption study
Adsorption of human serum albumin (HSA) on mPEG-OA-coated and non-coated niosomes was investigated to study the affinity between HSA blood protein and the surface of the niosomes.Niosomal samples were incubated with 1 ml of HSA (400 μg mL −1 ) and stirred for 2 h at 37 °C.The unabsorbed HSA were then removed by centrifugation using a K241R Medium Prime centrifuge (Centurion Scientific, UK) at 6000 rpm for 20 min at 4 °C.The surface charge of mPEG-OA-coated and non-coated niosomes was analyzed by Zetasizer Nano ZS90 (Malvern Instruments, UK) [44].Furthermore, the adsorption behavior of human serum albumin (HSA) on both mPEG-OA-coated and non-coated niosomes was explored using the Microscale Thermophoresis technique with the use of a Monolith [45,46].NT.115 instrument (Nano Temper Technologies, Germany) following a literature reported protocol The HSA was labeled using the Monolith protein labeling kit (RED-NHS 2nd generation) following the manufacturer's instructions.The labeled HSA was adjusted to a concentration of 45 nM using an assay buffer of phosphate-buffered saline (PBS) pH 7.4 supplemented with 0.05% v/v tween 20.Next, 10 μl of labeled HSA was introduced into 10 μl of 16 serial dilutions of ligands (mPEG-OA-coated and noncoated niosomes).Subsequently, the protein-ligand complex underwent a 15-minute incubation period in the dark.Followed by transferring the complexes into Standard Treated Capillaries (Nano Temper Technologies, Germany) and analyzed using the Monolith NT.115 instrument.The measurements were conducted at a temperature of 25 •C, utilizing low excitation and 20% MST power.To assess the ligands binding affinities to HSA, the dissociation constant (Kd) was determined using MO-Affinity analysis software, version 2.1.3,developed by Nano Temper Technologies (Germany).

In vitro biosafety of mPEG-OA-coated niosomes 2.8.1. Hemolysis study
The hemolytic activity of mPEG-OA-coated niosomes was assessed following a previous method reported in the literature [47,48].In brief, fresh sheep blood samples were washed with PBS (pH 7.4).This washing step was repeated 3 times and then centrifuged for 5 min.Samples of mPEG-OA-coated niosomes were serially diluted with PBS to prepare a concentration range between 0.05 to 0.5 mg mL −1 .Then 1.8 ml of each sample was mixed with 0.2 ml of the red blood cell suspension and incubated at 37 °C for 30 min.The intact red blood cells were removed by centrifugation, and the amount of hemoglobin released by the damaged cells in the supernatant of each sample was determined using a Spectrostar Nano microplate reader at 405 nm.The negative and positive controls were prepared by adding 0.2 ml of the red blood cell suspension to 1.8 ml of PBS and distilled water, respectively, to obtain 0% and 100% hemolysis.The experiment was performed in triplicate, and the percentage of hemolysis was then calculated according to the following equation.

Cytotoxicity and cell viability study
The biosafety of the mPEG-OA-coated VCM niosomes was assessed using the MTT assay [49]on Henrietta Lack's cervical cancer (HeLa), Michigan Cancer Foundation-7 (MCF-7) breast cancer, and human embryonic kidney 293 (HEK-293) cell lines [50].Briefly, 2.5 ×10 3 of each cell line were seeded in 100 μl of (EMEM for MCF-7, DMEM for HEK-293, and HeLa cells) in a 96-well plate with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin-fungizone and 1% L-glutamine and incubated in a humid atmosphere containing 5% CO 2 at 37 °C for 24 h.Subsequently, a series of dilutions of mPEG-OA-coated VCM niosomes (20, 40, 60, 80, and 100 μg mL −1 ) was added to each well, and cells were incubated.After 24 h of incubation, the medium was replaced with fresh MTT solution and incubated for another 4 h.Following the supernatant aspiration, DMSO was added to solubilize the formazan crystals.The absorbance was measured at 570 nm using a microplate reader (Spectrostar nano, Germany).The percentage of viable cells was calculated using equation (3): A570 nm treated cells A570 nm untreated cells 100 3 2.9.In vitro drug release study The in vitro drug release studies of VCM from non-coated niosomes and mPEG-OA-coated niosomes were performed using the dialysis release method [51].In brief, dialysis bags (pore size cut off:14 000 Da) containing 2 ml of the tested formulations were placed in 40 ml of the release medium (PBS pH 7.4 and 6) and kept in a shaking incubator at 37 °C.At a predetermined time, 3 ml of the release medium were collected and replaced with fresh PBS allowing the sink condition to be maintained.VCM content was quantified using the HPLC method mentioned above in section 2.5.

Release kinetics modeling study
The drug release data were then quantitatively analyzed using the DDSolver program, an Excel add-in via fitting to various kinetics models including zero-order, first-order, Higuchi, Hixson-Crowell, Weibull and Korsmeyer-Peppas, while the release mechanism was characterized using Korsmeyer-Peppas exponent (n) [52].

Antibacterial efficacy studies 2.11.1. In vitro antibacterial activity study
The antibacterial activity of blank niosomes, bare VCM, non-coated niosomes, and mPEG-OA-coated niosomes were evaluated using the broth dilution method against Staphylococcus aureus and MRSA (Rosenbach ATCC 700699) at pH 6 and 7.4 [53].Bacteria were cultured in nutrient broth and incubated at 37 °C in a shaker incubator (100 RPM).Then, the concentration of the bacteria was adjusted to 0.5 McFarland standard (DEN-1B densitometer, Latvia).Bacterial suspensions were diluted to achieve 5 × 10 5 colony-forming units (CFU)/mL concentration.Serial dilutions of mPEG-OA-coated niosomes, non-coated niosomes, blank niosomes, and bare VCM were prepared in Mueller-Hinton broth (MHB pH 6 and 7.4) in a 96-well plate, followed by the addition of bacterial suspensions and incubation in a shaking incubator (24 h, 37 °C, 100 RPM).Following incubation, the samples were spotted in triplicate on MHA plates and incubated (24 h, 37 °C), and the minimum inhibitory concentration (MIC) values were determined.

In vitro anti-MRSA biofilm activity study
The inhibition of MRSA biofilm formation by mPEG-OA-coated niosomes was quantified using a microtiter plate assay [54].An overnight culture of MRSA was adjusted to a concentration of 0.5 McFarland's Standard, then the bacteria were cultured in MHB pH 7.4 and 6 and pipetted into 96-well plates (100 μl/well).After static incubation for 10 days at 37 °C, the plates were washed with PBS (pH 7.4 and 6) to remove the unattached bacterial cells.100 μl of bare VCM solution and non-coated and coated niosomes formulations were then added to the wells and incubated at 37 °C for 24 h.After that, the plates were washed and dried for 15 min, followed by staining with 0.1% (w/v) crystal violet solution, and kept at 25 °C for 15 min.The wells were then washed with PBS pH 7.4 and 6 and solubilized with 30% acetic acid.The absorbance was read at 550 nm using a Spectrostar Nano microplate reader.The % biofilm eradication was quantified according to the following equation.

Time killing assay against MRSA
The time-killing analysis of bare VCM, non-coated, and coated niosomes was performed using the plate colony count method [55].MRSA cultures were prepared by diluting an overnight culture in nutrient broth and incubating.Thereafter, bacteria cultures were diluted with sterile PBS (pH 7.4 and 6) to obtain 5 × 105 CFU ml −1 concentrations.MRSA bacteria was added to Bare VCM, non-coated and coated niosomes at a concentration of 10 times greater than MICs and then placed in the shaking incubator at 37 °C.100 μl of each sample was removed at the specific intervals of 0, 2, 4, 6, 8, 12, and 24 h and plated in triplicate.The number of colonies was counted after 24 h.

In vivo antibacterial activity
To further study the antibacterial effect of mPEG-OA-coated niosomes in-depth, the in vivo antibacterial study was performed in an experimental mice model of MRSA skin infection according to the protocol developed by our group [56,57] and approved by the Animal Research Ethics Committee (AREC) of the University of KwaZulu-Natal (UKZN) (ethical approval number: AREC/010/020D).BALB/c mice (male, 20-25 g) were obtained from the UKZN Biomedical Resources Unit.The back hair of the mice was shaved carefully and disinfected with ethanol (70%).Twenty-four hours later, the mice were inoculated intradermally with 50 μl of MRSA bacteria (1.5 × 10 8 CFU ml −1 ).The mice were then divided into 4 groups (n = 4), the first group received 50 μl of saline (negative control group), the second group received 50 μl of VCM (48.75 μg K −1 g −1 ) (positive control group), the third and fourth groups of uncoated niosomes and coated niosomes received 50 μl containing 24.78 μg K −1 g −1 of VCM at the same site of infection.The mice were housed under observation with the following conditions: a standard 12 h light: dark cycle, temperature (19 °C-23 °C), relative humidity (55 ± 10%), and adequate ventilation for 48 h.Then the mice were euthanized via isoflorane inhalation, and the infected skin was excised and homogenized in 5 ml of PBS (pH 7.4).The tissue homogenates were serially diluted in PBS (pH 7.4), and 100 μl from each dilution were spotted in triplicate on MHA.The number of CFU per ml was counted after incubation for 24 h at 37 °C.
For histomorphological analysis, freshly harvested skin samples were excised from the infection site and fixed in 10% buffered formalin until further processing.The samples were subsequently dehydrated using ethanol and mounted in paraffin wax blocks.The wax blocks were sectioned using a microtome (Leica RM2235, Leica Biosystems, Germany).The tissue sections were collected on slides, dried, and stained with hematoxylin and eosin (H&E) using standard procedures.The sections were examined and captured with a Nikon E400 light microscope fitted with a Nikon Digital Sight-Fi2 camera (Nikon Instrument Group, Melville, NY).

Statistical analysis
All experiments were carried out in triplicate, and the data were presented as the mean ± standard deviation.Statistical analysis was performed using one-way analysis of variance (ANOVA), followed by Bonferroni's multiple comparison tests.The differences were considered statistically significant with p values <0.05.All graphs and statistical tests were conducted using GraphPad Prism ® software (Graph Pad Software Inc., Version 6, San Diego, CA, [58].

Synthesis and characterization of mPEG-OA
The pH-sensitive Schiff bases of mPEG and OA were synthesized in a two-step syntheses scheme (scheme 1).First, FBA was coupled to mPEG-5000 via an esterification reaction with DMAP and DCC as catalysts.The resulting mPEG-FBA was purified by dissolving in DCM and subsequent precipitation in anhydrous diethyl ether.The precipitate was collected, dried, and characterized.FT-IR confirmed the formation of the mPEG-FBA ester with a new peak appearing at 1720.48 cm −1 , corresponding to the ester bond wave number (figure S1, supplementary material).The ester formation was further confirmed with the proton NMR spectrum of the product that had peaks at 4.49 ppm, 7.2-7.9ppm, and 10.09 ppm (figure S1, supplementary material) that corresponds to protons adjacent to the ester bond, aromatic, and aldehyde protons, respectively.The reaction's second step involved forming the Schiff base between mPEG-FBA and OA.The reaction involved reflux of mPEG-FBA and OA in DMSO with few drops of TEA.The Schiff base formed via the formation of an imine bond between the aldehyde of mPEG-FBA and the primary amine group of OA.The structure was confirmed with the appearance of the peak at 1626.96 cm −1 in the IR spectrum, proton NMR peak at 8.16 ppm, and disappearance of the peak at 10.09 ppm, which indicate the formation of an imine from the aldehyde bond (figure S2, supplementary material).The imine bond formed is acid-sensitive that breaks the amphiphile, resulting in the release of the drug in acidic environments.

Preparation and characterization of mPEG-OA coated VCM niosomes
In this study, we successfully prepared VCM-loaded niosomes from cholesterol and various surfactants (Span 80, Span 60, and Span 20) using the thin-film hydration method; and optimization was considered to select the optimum formulation (table 1).The use of different types of surfactants influences the size, PDI, and ZP of nanopartilces due to their surface charges and varied abilities to interact with and stabilize the lipid components during nanoparticle formation.Niosomal formula composed of cholesterol and Span 20 of a 1:1 molar ratio was considered the formula of choice for further characterization studies.Increasing the HLB (hydrophiliclipophilic balance) value of the nonionic surfactants tend to have reduced the size of the niosomes.Span 20 (HLB value of 8.6) formed niosomes with particle sizes of 170 nm compared to 315 nm and 311 nm for span 80 (HLB 4.7) and 60 (HLB = 4.3) respectively.Surfactants with high HLB value facilitate the production of smaller lipidic nanoparticles by improving emulsification efficiency, enhancing lipid solubilization, providing dispersion stability, and reducing interfacial tension [59].The size, PDI, and ZP of the optimal VCM-loaded niosomes were 170.2 ± 2.5 nm, 0.27 ± 0.01, and −8.01 ± 2.3 mV, respectively, while EE % was found to be 52.5 ± 2.04%.These results were comparable to previously reported VCM-loaded niosomes, and in both studies, the addition of Tween 40( HLB 15) as a co-surfactant resulted in a smaller size [60,61].
Following mPEG-OA coating, the size of the niosomes increased from 169 nm at pH 7.4 to 190.7 nm and 255.4 nm at pH 6 and 4.5, respectively (table 2).The resulting pH-responsive increase in the average size is due to the cleavage of the acid-labile bond between the mPEG and OA.This pH-dependent increase in size is a result of the cleavage of the acid-labile bond between the mPEG and OA components of the niosomes.In an acidic environment, the acid-catalyzed cleavage of Schiff bases occurs.This cleavage process involves the addition of a proton (H + ) to the imine nitrogen, forming a positively charged intermediate.Subsequently, a nucleophilic attack occurs at the carbon atom adjacent to the nitrogen, leading to the breakage of the bond between mPEG and OA.As a result of this cleavage reaction, the mPEG-OA-coated niosomes undergo structural changes, causing an expansion in their size.The cleavage of the bond disrupts the compact conformation of the nanosystems, resulting in the exposure of their hydrophobic regions.This exposure might lead to the aggregation of niosomes and an increase in their average size [62].The formation of the amine after cleavage in acidic media resulted in the the surface charge of mPEG-OA coated niosomes switching from negative at pH 7.4 to positive 7.53 mV and 17.7 mV at pH 6 and 4.5, respectively.This confirmed the cleavage of the mPEG-OA acid-labile bond (table 2).This property is advantageous for enhancing binding to the anionic bacterial surface [18].
The microscopic visualization of mPEG-OA-coated niosomes using HRTEM showed smooth rounded vesicles, as shown in figure 1.

Thermal profile analysis
Differential scanning colorimetry can detect the phase transition and change in enthalpy of nanoparticles upon heating; therefore, it is helpful as an additional tool to support drug entrapment studies [63].The thermal characteristics of mPEG-OA-coated niosomes, non-coated niosomes, bare VCM, DCP, cholesterol, and their physical mixture were analyzed.As shown in figure 2, the DSC thermogram of mPEG-OA, DCP, bare VCM, and cholesterol showed endothermic peaks at 64.27 °C, 80.11 °C, 112.14 °C, and 161.21 °C, respectively.The physical mixture of the niosomal components showed nearly the same thermal behavior with slight shifts of their respective peaks.The DSC thermogram of non-coated niosomes did not exhibit any peak.However, the DSC thermogram of mPEG-OA-coated niosomes showed an endothermic peak at 52.6 °C, which might be attributed to the mPEG-OA polymer.The disappearance of the endothermic peak of VCM in the DSC thermogram of mPEG-OA-coated niosomes and non-coated niosomes indicated the successful entrapment of VCM into the niosomes in the amorphous form [45].Moreover, the absence of the endothermic peak of VCM in the niosome formulations indicates the transition of the drug from a crystalline structure to an amorphous one.Amorphous structures typically exhibit higher solubility compared to crystalline forms, presenting a notable advantage of drug entrapment within the niosomes [64].

Protein adsorption study
The interaction of nano-carriers with blood proteins is critical in determining the fate of the nano-carriers as they promote the identification and uptake of nano-carriers by the phagocytic system.Consequently, they are cleared from blood circulation.It has been established that modification of nano-carriers with PEG confers the ability to prevent opsonization, enhance nano-carriers pharmacokinetics and reduce clearance by phagocytosis [36,44].Therefore, the adsorption of HSA protein on mPEG-OA-coated and non-coated niosomes was investigated to study the affinity between the niosomes' surface and blood protein.As shown in figure 3, the adsorption of HSA protein significantly shifted the ZP of non-coated niosomes to more negative values of  −13.33 mV (p = 0.0005).In comparison, the mPEG-OA-coated niosomes had no significant shift in ZP of −3.2 mV following the adsorption of HSA protein (p = 0.0641).Likewise, the MST investigations have shown that the non-coated niosomes exhibit a greater binding affinity to HSA in comparison to the mPEG-OA-coated niosomes.The dissociation constant (Kd) values are an indicator of the affinity between the ligand and the target.Lower Kd value is an indicative of a stronger binding affinity, while larger values suggest a lower degree of binding [45,46].Based on the findings, it was seen that the non-coated niosomes exhibited a Kd value of 1.44 μM, which was much lower (by 15 folds) in comparison to the mPEG-OA-coated niosomes that had a Kd value of 21.33 μM. Figure 4, illustrates a relative view of the fraction bound between non-coated and mPEG-OA-coated niosomes to HSA.The figure shows that the non-coated niosomes exhibit a larger fraction bound at various ligand concentrations compared to the mPEG-OA-coated niosomes.
These results from ZP and MST measurements confirm the ability of mPEG-OA polymer coating to reduce the adsorption of HSA protein on niosomes surface by steric repulsion, which diminishes the phagocytic recognition and uptake, resulting in enhanced biodistribution of the surface-modified niosomes.More importantly, this polymer will improve the immune compatibility of the parenterally administrated nanocarriers, which is advantageous in designing and developing long-circulating nano-delivery systems.

3.5.
In vitro mPEG-OA-coated niosomes biosafety studies 3.5.1.Hemolysis study To assess the potential interactions between mPEG-OA-coated niosomes and blood components, a hemolysis assay was performed.This assay involved subjecting the niosomes to a simulated blood environment to evaluate their impact on red blood cells (RBCs).The results obtained from the hemolysis assay showed hemolytic activity, ranging from 0.07% to 0.17% (figure 5).The negligible hemolytic activity suggests that the mPEG-OA-coated  niosomes do not induce significant damage or disruption to RBC membranes.This finding is pivotal in confirming the hemocompatibility of the niosomal systems and highlights their potential as biocompatible carriers for various biomedical applications [65].

Cytotoxicity and cell viability study
To further assess the biosafety of mPEG-OA-coated niosomes, cell viability studies were evaluated using HEK-293, MCF-7, and HeLa cell lines by the MTT assay.Cells were incubated with different concentrations of the test material, and the percentage of cell viability is illustrated in figure 6.At all concentrations tested, the cell viability of all cell lines was found to be more than 90%, and there were no dose-dependent trends observed.These results confirmed the biocompatibility of mPEG-OA-coated niosomes, as a percentage of cell viability above 75% is considered noncytotoxic [66].Thus, this system is a suitable candidate for nano antibiotic delivery.

In vitro drug release and kinetics studies
Vancomycin release profiles from mPEG-OA-coated niosomes and non-coated niosomes were investigated at pH 7.4 and 6 (figure 7).At pH 7.4, the cumulative VCM release from mPEG-OA-coated niosomes and noncoated niosomes was statistically non-significant during the 48-h study period (p = 0.591).55.3% of VCM was released from coated niosomes, while 59.3% of VCM was released from non-coated niosomes at the same time interval as shown in figure 7(A).These results agree with antibiotic-loaded niosomes that have been applied to provide consistent, controlled release, which depends mainly on cholesterol content and type of non-ionic surfactant [67,68].
However, at pH 6, the VCM release rate from mPEG-OA-coated niosomes was higher than from non-coated niosomes.61.44% of VCM was released from mPEG-OA-coated niosomes in 48 h, while only 26.8% of VCM was released from non-coated niosomes at the same time interval (figure 7(B)).This data confirmed that VCM release is significantly dependent on the mPEG-OA-coating under acidic pH conditions (p = 0.0152).The drug release of VCM from mPEG-OA-coated niosomes at pH 6 was slightly higher than pH 7.4, as shown in figure 7(C).As per the hypothesis of the study it was expected a faster VCM release from mPEG-OAcoated niosomes at acidic conditions compared to pH 7.4, may be due to the cleavage of the hydrophobic and hydrophilic portion of mPEG-OA coating amphiphile [69].While some drug release enhancement may have occurred due to the cleavage of mPEG-OA, at acidic pH, niosomes have been reported to be stable nanostructures that do not disassemble easily under stimuli response that is due to surface modification compared to other vesicular systems like liposomes [70].However, this phenomenon of continued stability characteristics, makes them valuable carriers for antibacterial delivery.This controlled release behavior ensures that a significant amount of VCM remains encapsulated within the niosomes, preventing the premature release and potential wastage of the drug in non-infected areas.Moreover, it minimizes the exposure of healthy tissues to the drug, reducing the likelihood of unnecessary side effects.Secondly, it ensures a higher concentration of VCM at the infection site for a prolonged period where it is needed most, improving its antibacterial efficacy.Thus the nanosystem can help to maximize the therapeutic effect of VCM while minimizing potential harm to surrounding healthy tissues [71] thus reducing side effects.
Understanding release kinetics is significant in the development of a sustained release nanosystem.The release data of mPEG-OA-coated niosomes were fitted to zero order, first order, Higuchi, Hixson-Crowell, Korsmeyer-Peppas, and Weibull models for kinetic analysis.The Weibull model appeared to be the best fit kinetic model for describing the release from mPEG-OA-coated and non-coated niosomes at both pH conditions, as it had the highest regression coefficient values of 0.9601 and 0.9528 at pH 7.4, while at pH 6, the highest values were 0.9605 and 0.9629.On the other hand, the root means square error (RMSE) were 3.7527 and 3.5423 at pH 7.4, 2.7702, and 1.8068 at pH 6 for coated and non-coated niosomes, respectively.
The Weibull parameter (β) values were 0.497 and 0.448 at pH 7.4 and 0.622 and 0.352 at pH 6 for coated and non-coated niosomes, respectively.This data implies that VCM release from coated and non-coated niosomes followed Fickian diffusion as β values were lower than 0.75.Moreover, the diffusional exponent (n) of the Korsmeyer-Peppas model was below 0.5 for coated and non-coated niosomes at both pH conditions, confirming that VCM release was controlled by Fickian diffusion across niosomes bilayer depending on VCM concentration [72,73].

Determination of MICs
The broth dilution method was used to investigate the MICs of bare VCM, non-coated, and coated niosomes against Staphylococcus aureus and MRSA.Blank niosomes exhibited no antibacterial activity against both bacterial strains at pH 7.4 and 6.Encapsulation of VCM in non-coated and coated niosomes showed better antibacterial activity against both bacterial strains than bare VCM at both pHs (table 3).The enhanced antibacterial activity can be linked to the unique permeability of niosomes.Niosomes are composed of nonionic surfactants, which form bilayer structures resembling the lipid bilayers found in cell membranes.This similarity allows niosomes to interact readily with bacterial cell membranes.Upon contact with bacteria, niosomes fuse with the bacterial cell membrane, facilitating the delivery of the loaded drug directly to the bacterial interior [10,74].Furthermore, the interaction between niosomes can also be endocytosis by the bacterial cells.In this case, niosomes are taken up by the bacteria and are released within the bacterial cytoplasm, allowing VCM to exert its antibacterial effects directly at the site of action [75].The combination of these desired traits i.e. permeability, membrane fluidity, and fusogenic properties resulted in an efficient and targeted delivery system for the delivery of VCM.By leveraging the unique characteristics of niosomes, the antibacterial activity of VCM was significantly potentiated, enabling it to eliminate the bacteria more effectively compared to the bare drug.
Furthermore, mPEG-OA-coated niosomes showed enhanced and extended antibacterial activity for 72 h compared to non-coated niosomes at both pHs.At pH 7.4, mPEG-OA-coated niosomes and non-coated niosomes had the same MIC against Staphylococcus aureus.However, mPEG-OA-coated niosomes exhibited prolonged antibacterial activity for 72 h, while non-coated niosomes lost their antibacterial activity after 24 h.The extended duration of antibacterial activity ensures that a sufficient concentration of vancomycin is maintained at the infection site for a more extended period [76].This enhanced exposure to the antibiotic increases the likelihood of effectively eliminating the bacteria, leading to improved therapeutic outcomes.The prolonged antibacterial activity of mPEG-OA-coated could help minimize the risk of bacterial regrowth during treatment, reducing the likelihood of developing antibiotic-resistant strains.This is crucial in the context of the global challenge of antibiotic resistance as the extended duration of antibacterial activity ensures that a sufficient concentration of VCM is maintained at the infection site for a more extended period.This enhanced exposure to the antibiotic increases the likelihood of effectively eliminating the bacteria, leading to improved therapeutic outcomes and complete clearance of bacteria thus eliminating the chance of resistant strains emerging [77].
For MRSA, mPEG-OA-coated niosomes resulted in a 2-fold reduction in MIC compared to non-coated niosomes.The antimicrobial activity of mPEG-OA-coated niosomes was enhanced at pH 6, as evidenced by a 2-fold decrease in the minimum inhibitory concentration (MIC) against Staphylococcus aureus, as well as prolonged antibacterial activity against MRSA when compared to non-coated niosomes (table 3).This enhanced antimicrobial activity of the mPEG-OA-coated niosomes at pH 6, compared to non-coated niosomes is crucial because pH 6 is often associated with infection sites and inflammatory microenvironments, such as abscesses, which are typical in MRSA infections [78].The enhanced activity at pH 6 can be attributed to a smart mechanism involving the cleavage of the Schiff base between the mPEG and OA components of the niosomes.At neutral pH, the Schiff base linkage is relatively stable, maintaining the structural integrity of the niosomes and providing controlled drug release.However, when the pH drops to 6, which is more acidic, the Schiff base linkage becomes more susceptible to cleavage.This cleavage results in pH responsiveness, that leads to the change in the surface charge of the mPEG-OA-coated niosomes from negative to positive in acidic milieu similar to bacterial infection microenvironment.This positive charge on the niosome surface may have enhanced its binding affinity to negatively charged bacteria, such as MRSA.This electrostatic attraction aids stronger interactions between the niosomes and the bacterial cell membranes, leading to more effective delivery of VCM to the MRSA cells [79].
The extended antibacterial activity could be due to the prolonged release of VCM from mPEG-OA-coated niosomes resulting in longer-lasting antibacterial efficacy [80].An additional factor that might have potentiated the antibacterial activity of mPEG-OA-coated niosomes compared to non-coated niosomes is the inherent antibacterial activity of OA and its ability to disrupt and enhance bacterial cell membrane penetration [34,81].
Coated niosomal formulation exhibited a non-significant difference in MICs values against both bacterial strains at both pHs (p = 0.3408).To date, there is no complete understating of the effects of surface modification interactions.It can be anticipated that the subtle changes to the surface of coated nanoparticles can modulate their interactions with bacteria.These results collectively revealed that mPEG-OA-coated niosomes possess higher and sustained antimicrobial activity than non-coated and bare VCM.Thus, administration of VCM via mPEG-OA-coated niosomes can reduce the optimal effective dose of VCM and minimize its dose-dependent adverse effects compared to its conventional dosage forms and non-coated niosomal systems.These enhanced sustained antibacterial activity findings concur with previous studies of VCM encapsulated in nanovesicles [60,82].

In vitro anti-biofilm activity study
Biofilms, which are complex communities of bacteria encased in a protective matrix, pose a significant challenge in the treatment of bacterial infections.The crystal violet assay was applied to assess the efficacy of mPEG-OAcoated niosomes for the reduction of the biomass of biofilms.At pH 7.4, mPEG-OA-coated niosomes formulation displayed the highest efficiency of MRSA biofilm eradication with a 4-fold reduction in the MRSA biofilm biomass.In contrast, non-coated niosomes had a 3.4-fold reduction in the MRSA biofilm biomass compared to bare VCM (figure 8).The superior performance of mPEG-OA-coated niosomes implies that the coating enhances the biofilm eradication capabilities of biofilms, by facilitating better interaction with the biofilm structure which results in the increased penetration of the drug through the protective matrix of the biofilms.This significant reduction indicates the potent antibiofilm activity of the coated niosomes, highlighting their potential as a promising strategy to combat biofilm-related infections.
At pH 6, MRSA biofilm treated with mPEG-OA-coated niosomes showed a 15-fold reduction in the biofilm biomass compared to a 5.8-fold reduction in the biofilm biomass treated with non-coated niosomes than bare VCM (figure 8).The enhancement of the antibiofilm activity may be attributed to the small size of the nanoscale vesicles, that allows them to readily interact with the biofilm structure thus enabling them to penetrate the inner layers of the biofilm more effectively [83].While fusogenic properties of niosomes enables them to fuse with the biofilm's extracellular matrix, allowing them to deliver vancomycin, directly to the bacterial cells embedded within the biofilm which enhances their interaction and penetration into the MRSA biofilm inner layers [10,84].
In addition, mPEG-OA-coated niosomes formulation displayed better efficiency against MRSA biofilm with 1.2-fold reduction (pH 7.4) and 2.6-fold reduction in the biofilm biomass (pH 6) compared to non-coated niosomes.Moreover, mPEG-OA-coated niosomes showed significant anti-MRSA biofilm activity at acidic conditions (p = 0,0119), with a 32.46% reduction in the MRSA biofilm biomass at pH 6 compared to a 4.74% reduction in the MRSA biofilm biomass at pH 7.4, respectively (figure 8).The enhancemement of mPEG-OAcoated niosomes antibiofilm activity is the pH-responsive property which enhances the accumulation to the acidic environment inside biofilm compared to the pH of 7.4.Furthermore, under acidic conditions, such as those found within the biofilm and at infection sites, the mPEG-OA coating undergoes a surface charge switch to positive.This change in surface charge enables the positively charged niosomes to interact more strongly with the negatively charged bacterial cell surfaces.The multifaceted and targeted approach for eliminating biofilms by disrupting bacterial cell membranes, interfering with the biofilm matrix, and delivering antimicrobial agents directly to the bacterial cells might be the reason for enhanced activity [6].

Time killing assay against MRSA
The rates of MRSA killing by bare VCM, non-coated, and coated niosomes at pH 7.4 and 6 are illustrated in figure 9.At pH 7.4, mPEG-OA-coated niosomes showed rapid bactericidal kinetics compared to bare VCM, whereas non-coated niosomes had slower killing kinetics with 24 h.However, at pH 6, bare VCM eradicated 19.4% within 12 h and eliminated MRSA bacteria within 24 h.In contrast, non-coated niosomes and mPEG-OA-coated niosomes eliminated the bacteria within 8 h and 6 h, respectively.These results showed the faster bactericidal kinetics of mPEG-OA-coated niosomes than VCM therapy and non-coated niosomes at pH 7.4 and acidic conditions.In addition, the killing kinetics of mPEG-OA-coated niosomes confirmed that it effectively eradicated MRSA infections quicker than the non-coated niosomes at both pHs.mPEG-OA-coated niosomes eliminated 59.1% MRSA within 24 h compared to 13.8% clearance of non-coated at the same time interval at pH 7.4.However, in acidic conditions, mPEG-OA-coated niosomes eliminated the bacteria within 6 h, whereas non-coated niosomes took 8 h to eliminate MRSA bacteria.The charge-reversible property of the surface of mPEG-OA-coated niosomes to positive charge improved the electrostatic attraction with negatively charged bacterial cell surfaces at acidic infection sites.Therefore, mPEG-OA-coated niosomes significantly eliminated MRSA at faster killing rates at acidic conditions than pH 7.4 (p = 0.0210).
The rapid generation time of bacteria accounts for the rapid progression of the infectivity bacteria.Conventional VCM therapy is associated with poor pharmacokinetic properties, slow time-dependent antibacterial activity, and poor cell penetration, accounting for high rates of VCM therapy failure and toxicity [85,86].Therefore, this study offers an effective nano-delivery system for improving bacterial killing kinetics of antibiotics with long-term release and activity compared with conventional therapy and non-modified nanoparticles.

In vivo antibacterial activity
As mPEG-OA-coated niosomes demonstrated superior performance for in vitro antibacterial activity, killing kinetics, and antibiofilm efficacy compared to the non-coated formulation, it was further subjected to in vivo studies against MRSA skin infection mouse model to confirm it's in vivo performance.The average MRSA CFU of the untreated group was 71000 CFU/mL and was reduced to 42000 CFU/mL, 40000 CFU/mL, and 30000 CFU/mL after treatment with bare VCM, uncoated niosomes, and mPEG-OA-coated niosomes respectively as shown in figure 10. mPEG-OA-coated niosomes significantly inhibited MRSA CFU compared to uncoated niosomes (p = 0.0049), bare VCM (p = 0.0068), and untreated (p = 0.0006) groups.These results signify the potential mPEG-OA-coated niosomes application to overcome infections induced by MRSA bacteria while minimizing VCM drug dose and adverse effects to improve patient compliance.Surface modification of the niosomes with the pH-responsive acid cleavable moiety (mPEG-OA) can enhance VCM pharmacokinetics, release, and accumulation at the acidic infection sites.On the other hand, surface charge-switching to positive is advantageous in improving VCM release and promoting the binding affinity to the anionic bacterial surface in an acidic environment.The results from both in vitro and in vivo studies highlight the promising potential of mPEG-OA-coated niosomes as a novel approach for treating MRSA infections.The surface modifications of niosomes provide targeted drug delivery, and enhanced antibacterial activity, thereby offering a potential   niosomes group, there were minimal signs of tissue swelling and inflammation (figure 11(D)) and the number of white blood cells present were markedly reduced in this sample (figure 11(H)).
Multiple components of microorganisms are identified as foreign to the body and thereby induce an immune and inflammatory response when they gain access to tissue.The magnitude of these response processes is modulated depending on the bacterial load in the tissue.If the infection is confined to a local site with a small bacterial load, it will cause a controlled inflammatory response and recruit immune cells [87,88].The greater the bacterial load within a tissue, the greater the inflammatory and immune response.Classic indicators of acute inflammation include redness, heat, and swelling [88].Tissue thickness has been previously utilized to evaluate swelling and inflammation of skin [89,90].Additionally, cellular infiltration is an essential characteristic of skin inflammation.The prevalent cell types that infiltrate the area are leukocytes and neutrophils.These cell populations perform a vital function in the progression of the inflammatory reaction and thereby contribute to tissue swelling [91,92].The magnitude of this cellular response is dependent on the bacterial load in the tissue.
The histomorphological evaluations correlate directly with the findings of the in vivo anti-MRSA activity (figure 8).The histomorphological indicators of inflammation observed relate to the number of bacteria isolated in these samples.The untreated control group displayed the highest bacterial load and histomorphological tissue swelling and inflammation signs.Comparatively, the VCM-HCl group showed a significantly reduced bacterial load, and a corresponding reduction in tissue swelling and indicators of inflammation was observed in this sample.The -uncoated niosomes group displayed an intermediate bacterial load and presented the mild tissue swelling and inflammation histomorphologically.The mPEG-OA-coated niosomes group displayed the lowest bacterial load and presented the least tissue swelling and inflammation histomorphologically.These histomorphological findings further validate the antimicrobial superiority of the mPEG-OA-coated niosomes group.

Conclusion
In this study, a novel pH-responsive polymer was successfully synthesized, characterized, and applied for the surface modification of VCM-loaded niosomes to treat MRSA infections.mPEG-OA-coated niosomes displayed pH-responsive sustained VCM release, which resulted in enhanced in vitro and in vivo anti-MRSA activity compared to the non-coated niosomes and bare VCM therapy.In addition, the designed mPEG-OA polymer reduced the adsorption of HSA protein on the niosomes surface, which is advantageous in prolonging blood circulation and preventing opsonization and uptake by the phagocytic cells.In addition to the enhanced biocompatibility and hemocompatibility of the mPEG-OA coated niosomal formulation, mPEG-OA polymer also improved the killing kinetics and antibiofilm activity of mPEG-OA coated niosomes against MRSA, proposing an alternative approach to restoring conventional VCM therapy efficacy.This research is influential and opens an avenue for the applications of responsive coating materials in improving the antibacterial spectrum of antibiotic nano-drug delivery systems, aiming to reduce dose-dependent adverse reactions and toxicity to improve patient compliance and antibiotic therapy outcomes.

Scheme 1 .
Scheme 1. Synthesis of mPEG-OA Schiff base: Step one involved the esterification of FBA (CHO) to mPEG.The formed mPEG-CHO was then refluxed under nitrogen with OA to form the mPEG-OA Schiff base.

Figure 3 .
Figure 3.The influence of HSA protein adsorption on ZP of mPEG-OA-coated (blue) and non-coated (red) niosomes (A) before and (B) after adsorption of HSA protein to niosomes surface (n = 3).

Figure 4 .
Figure 4.A comparative illustration of the fractions bound of non-coated and mPEG-OA-coated niosomes to HSA.The data is given in the form of mean ± standard deviations.The fraction bound of non-coated niosomes to HSA is significantly greater than that of mPEG-OA-coated niosomes to HSA (p-value < 0.05).

Table 1 .
The effect of various non-ionic surfactants on particle size, PDI, and ZP of different uncoated niosomal formulations.

Table 2 .
The effect of pH disparity on Particle size, PDI, and ZP of mPEG-OA-coated niosomes.