pH-responsive chitosan nanoparticles from a novel twin-chain anionic amphiphile for controlled and targeted delivery of vancomycin

Article abstract of DOI:10.1016/j.colsurfb.2017.07.049

The design and synthesis of novel pH-responsive nanoantibiotics is an emerging research area to address the antibiotic resistance crisis. The purpose of this study was therefore to synthesize a new anionic gemini surfactant (AGS) that could result in the formulation of pH-responsive chitosan nanoparticles (CSNPs) to treat methicillin-resistant Staphylococcus aureus (MRSA) infections. The coupling of oleic acid with 2,2-dimethyl-5,5-bis(hydroxymethyl)-1,3-dioxane and subsequent deprotection followed by a reaction with succinic anhydride and sodium bicarbonate yielded AGS. Critical micelle concentration (CMC) was determined using conductometry and in vitro cytotoxicity was performed using a MTT assay. Vancomycin loaded CSNPs containing AGS (DL_CSSNPs) were prepared by ionotropic gelation of chitosan with pentasodium tripolyphosphate. CSNPs were characterized for size, polydispersity index (PDI), zeta potential (ZP), entrapment efficiency, surface morphology, in vitro drug release and in vitro antibacterial activity (at pH 6.5 and 7.4). Results from the in vitro antibacterial activity were further supported by an in vivo study using a mice skin infection model. The CMC of AGS was found to be 1.3?mM/L and it was non-toxic. The DL_CSSNPs were spherical with size, PDI and ZP of 220.57?±?5.9?nm, 0.299?±?0.004 and 21.9?±?0.9?mV respectively. An increase in the vancomycin release from the DL_CSSNPs was observed at pH 6.5 compared to pH 7.4. The minimum inhibitory concentration values at pH 6.5 and 7.4 against MRSA were 7.81 and 62.5?μg/ml respectively. In vivo antibacterial activity showed that the MRSA burden in mice treated with DL_CSSNPs was reduced by almost 8-fold compared to those treated with pure vancomycin.

Full text of DOI:10.1016/j.colsurfb.2017.07.049

Accepted Manuscript
Title: pH-responsive chitosan nanoparticles from a novel
twin-chain anionic amphiphile for controlled and targeted
delivery of vancomycin
Authors: Rahul S. Kalhapure, Mahantesh Jadhav, Sanjeev
Rambharose, Chunderika Mocktar, Sanil Singh, Jwala
Renukuntla, Thirumala Govender
PII:
S0927-7765(17)30461-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.colsurfb.2017.07.049
COLSUB 8712
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date:
Revised date:
Accepted date:
23-4-2017
23-6-2017
21-7-2017
Please cite this article as: Rahul S.Kalhapure, Mahantesh Jadhav, Sanjeev Rambharose,
Chunderika Mocktar, Sanil Singh, Jwala Renukuntla, Thirumala Govender, pH-
responsive chitosan nanoparticles from a novel twin-chain anionic amphiphile
for controlled and targeted delivery of vancomycin, Colloids and Surfaces B:
Biointerfaceshttp://dx.doi.org/10.1016/j.colsurfb.2017.07.049
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pH-responsive chitosan nanoparticles from a novel twin-chain anionic amphiphile for
controlled and targeted delivery of vancomycin
a,†
a
a
a
Rahul S. Kalhapure* , Mahantesh Jadhav , Sanjeev Rambharose , Chunderika Mocktar ,
b
c
a
Sanil Singh , Jwala Renukuntla , Thirumala Govender*
aDiscipline of Pharmaceutical Sciences, School of Health Sciences, University of KwaZulu-
Natal, Private Bag X54001 Durban 4000, South Africa.
b
Biomedical Resource Unit, University of KwaZulu-Natal, Westville Campus, Durban 4000,
South Africa.
c
School of Pharmacy, The University of Texas at El Paso, 500 W. University El Paso Texas,
7
9968, USA.
*
Corresponding Author: Private Bag X54001 Durban, 4000, KwaZulu-Natal, South Africa.
Tel: 00 27 31 260 7358, Fax: 0027 31 260 7792
E-mail addresses: govenderth@ukzn.ac.za (T. Govender), rahul.kalhapure@rediffmail.com;
kalhapure@ukzn.ac.za (R.S. Kalhapure).
†Author’s present address: School of Pharmacy, The University of Texas at El Paso, 500 W.
University El Paso Texas, 79968, USA. Email address: rkalhapure@utep.edu
1

Graphical abstract
Novel anionic gemini surfactant based vancomycin loaded chitosan nanoparticles releasing
drug at bacterial infection site.
Highlights




Synthesis and characterization of novel anionic gemini surfactant (AGS).
Vancomycin loaded pH-responsive chitosan nanoparticles (CSSNPs) using AGS.
An increase in vancomycin release from CSSNPs at pH 6.5 compared to pH 7.4.
Enhanced in vitro and in vivo anti-MRSA activity of CSSNPs.
Abstract
The design and synthesis of novel pH-responsive nanoantibiotics is an emerging research
area to address the antibiotic resistance crisis. The purpose of this study was therefore to
synthesize a new anionic gemini surfactant (AGS) that could result in the formulation of pH-
responsive chitosan nanoparticles (CSNPs) to treat methicillin-resistant Staphylococcus
aureus (MRSA) infections. The coupling of oleic acid with 2,2-dimethyl-5,5-
bis(hydroxymethyl)-1,3-dioxane and subsequent deprotection followed by a reaction with
succinic anhydride and sodium bicarbonate yielded AGS. Critical micelle concentration
(CMC) was determined using conductometry and in vitro cytotoxicity was performed using a
2

MTT assay. Vancomycin loaded CSNPs containing AGS (DL_CSSNPs) were prepared by
ionotropic gelation of chitosan with pentasodium tripolyphosphate. CSNPs were
characterized for size, polydispersity index (PDI), zeta potential (ZP), entrapment efficiency,
surface morphology, in vitro drug release and in vitro antibacterial activity (at pH 6.5 and
7
.4). Results from the in vitro antibacterial activity were further supported by an in vivo
study using a mice skin infection model. The CMC of AGS was found to be 1.3 mM/L and it
was non-toxic. The DL_CSSNPs were spherical with size, PDI and ZP of 220.57±5.9 nm,
0
.299±0.004 and 21.9±0.9 mV respectively. An increase in the vancomycin release from the
DL_CSSNPs was observed at pH 6.5 compared to pH 7.4. The minimum inhibitory
concentration values at pH 6.5 and 7.4 against MRSA were 7.81 and 62.5 µg/ml respectively.
In vivo antibacterial activity showed that the MRSA burden in mice treated with
DL_CSSNPs was reduced by almost 8-fold compared to those treated with pure vancomycin.
Keywords: anionic gemini surfactant; vancomycin, pH-responsive; chitosan nanoparticles;
targeted delivery; methicillin-resistant S. aureus; mice skin infection model; antibacterial.
1
. Introduction
Great efforts are being made to synthesize new surfactants due to their wide
application in biological, industrial, pharmaceutical and cosmetic areas [1, 2]. Gemini
surfactants, also known as dimeric surfactants, have attracted special attention in the
past few years [3-6]. The structural features of gemini surfactants include two
hydrophobic chains, two polar head groups, and a spacer linker at or near the head
groups [1]. Compared to conventional monomeric surfactants, gemini surfactants
exhibit low critical micelle concentration (CMC), an enhanced surface activity, better
foaming, wetting, and dispersing properties [3, 7] The application of gemini
3

surfactants has been explored in gene and various other drug delivery systems due to
their remarkable properties [3, 8-10]. As with conventional surfactants, the gemini
surfactants can be classified into cationic, anionic and non-ionic surfactants. Anionic
gemini surfactants (AGSs) reported to date are mainly sulfate and sulfonate and they
play a critical role in the development and application of gemini surfactants [1, 11].
Despite their interesting properties such as pH-responsiveness due to protonation-
deprotonation behaviour [12], there are very few reports on the use of AGSs for drug
delivery applications. For example, AGSs have been reported to enhance the
solubility of amphotericin B [13] and has been explored as a substitute for
phospholipids in liposome preparations [3].
In the current global antibiotic resistance crisis, responsive nano delivery systems for
antibiotics are emerging to combat resistant bacteria in a more efficient manner.
Responsive nano drug delivery systems reported for antibiotics include an enzyme
[14, 15] and a specific pH [16-20] as triggers to release an antibiotic at the bacterial
infection site. Among these two responses, pH seems to be an attractive trigger for
targeted antibiotic delivery, as some bacteria, such as Staphylococcus aureus,
methicillin-resistant S. aureus (MRSA) [19] and cariogenic bacteria [17] are known to
produce acids at the infection site due to combined actions of immune response and
bacterial metabolism. The polymers that have been used to prepare pH-responsive
nano antibiotic delivery systems include poly(b-amino ester) [16],
poly(dimethylaminoethyl methacrylate [17, 18], poly-l-histidine [19] and α-
norbornenyl-poly(ethylene oxide) [20].
Synthesizing pH-responsive polymers to prepare nano drug delivery systems for
antibiotics requires expensive materials and entails tedious procedures. An alternative
to these polymers could be the use of easy to synthesize and biocompatible AGSs
4

with pH-responsiveness. To the best of our knowledge, no study has reported the use
of AGS for pH-responsive antibiotic delivery systems. Therefore, the goals of the
proposed work were: 1) to synthesize a nontoxic pH-responsive AGS (twin-chain
anionic amphiphile) by facile chemical synthesis procedures using cheap and easily
available chemicals, and 2) to utilize the synthesized AGS to prepare a pH-responsive
nano delivery system for antibiotics to address the problem of bacterial resistance.
The synthesized AGS was explored to prepare vancomycin loaded pH-responsive
chitosan nanoparticles (CSNPs) capable of releasing their payload at an acidic pH to
treat S. aureus and MRSA infections.
2
. Materials and Methods
.1. Materials
Vancomycin hydrochloride was purchased from Sinobright Import & Export Co. Ltd.
China). Oleic acid (99% pure), chitosan (low mol. wt.; 75-85 % deacetylated;
2
(
viscosity 200-300 cp), 4-dimethylaminopyridine (DMAP), sodium bicarbonate and
sodium tripolyphosphate (TPP) were procured from Sigma-Aldrich (USA). Diethyl
ether and 2,2-dimethoxypropane were acquired from Sigma-Aldrich (Germany).
Pentaerythritol and succinic anhydride were procured from Sigma-Aldrich (Slovakia)
and Sigma-Aldrich (Switzerland) respectively. Triethylamine and MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were obtained from Merck
chemicals (Germany). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC.HCl) was procured from Carbosynth (UK). For thin layer
chromatography (TLC), Merck precoated silica gel 60F254 plates (Germany) was
used. Mueller Hinton Agar (MHA) and Mueller Hinton Broth (MHB) were acquired
from Biolab (South Africa). For drug release, dialysis tubing of MWCO 14000 Da
5

was obtained from Sigma-Aldrich (USA). Purified water, obtained from a Milli-Q
water purification system (Millipore corp., USA), was used throughout the
experiments.
2
.2.Instrumentation
FT-IR spectra were recorded on a Bruker Alpha-p spectrometer (Germany) with
1
13
diamond ATR. H NMR and C NMR spectra were recorded using Bruker 400/600
Ultrashield™ (United Kingdom) NMR spectrometer, while HRMS was performed on
a Waters Micromass LCT Premier TOF-MS (United Kingdom). Conductometric
experiments were performed using a Bante 901 benchtop conductivity meter (China).
2
.3.Synthesis of AGS 7 (Fig. 1)
2
.3.1. Synthesis of (2,2-dimethyl-1,3-dioxane-5,5-diyl)bis(methylene)
dioleate 4
To a stirred solution of (2,2-dimethyl-1,3-dioxane-5,5-diyl) dimethanol 3 (5.0
g; 0.0284 mol), synthesized by following a literature reported procedure [21]
(Supplementary Material), EDC.HCl (11.43 g; 0.0596 mol) and DMAP (1.74
g; 0.0142 mol) in dry DCM (30 ml), and oleic acid (16.84 g; 0.0596 mol) were
added, and the resulting reaction mixture stirred at room temperature for 24 h.
After completing the reaction, the solvent was evaporated under reduced
pressure and the obtained residue was purified by column chromatography
(silica gel #70-230 and 10-15% ethyl acetate in hexane as eluent) to obtain
1
compound 4 as a clear thick liquid (16.61 g, 83%). H NMR (CDCl
3
) δ (ppm):
), 1.53
-), 2.23 (t; 4H; -
0
. 81 (t; 6H; -CH
3
), 1.19-1.22 (m; 40H; -CH
2
-), 1.35 (s; 6H; -C(CH3)
2
(
q; 4H; -CH CH
2
2
COO-), 1.95 (m; 8H; -CH
2
CH=CHCH
2
6

CH
2
COO-), 3.67 (d; 4H; -CH
2
-), 4.03 (d; 4H; -CH
) δ (ppm): 14.10, 22.68, 23.57, 24.89, 27.17, 29.12 - 29.76,
1.15, 37.21, 62.34, 62.96, 98.55, 129.71, 130.00, 173.44.
2
-), 5.27 (m; 4H; -CH=CH-
1
3
)
. C NMR (CDCl
3
3
2
.3.2. Synthesis of 2,2-bis(hydroxymethyl)propane-1,3-diyl dioleate 5
To a solution of compound 4 (7.5 g; 0.0106) in THF (40 ml), 1M HCl (3 ml)
was added, and the reaction mixture was stirred at room temperature for 4 h.
After 4 h, TEA (1 ml) was added and the reaction mixture was stirred for 5
min, after which the solvent from the reaction mass was evaporated under
reduced pressure. The obtained residue was purified by column
chromatography (silica gel #70-230 and 25-35% ethyl acetate in hexane as
1
eluent) to obtain compound 5 as a thick liquid (5.52 g, 78 %). H NMR
(
CDCl
CH CH
.69 (d; 4H; -CH
CDCl
3
) δ (ppm): 0. 81 (t; 6H; -CH
COO-), 1.94 (m; 8H; -CH
-), 4.03 (d; 4H; -CH
3
), 1.19-1.23 (m; 40H; -CH -), 1.53 (q; 4H;
2
-
2
2
2
CH=CHCH
2
-), 2.24 (t; 4H; -CH
2
COO-),
1
3
3
2
2
-), 5.27 (m; 4H; -CH=CH-). C NMR
(
3
) δ (ppm): 14.13, 22.67, 23.59, 24.75, 27.23, 29.11 - 29.78, 31.11,
3
7.35 62.37, 63.15, 129.58, 130.51, 173.14.
2
.3.3. Synthesis of 4,4'-((2,2-bis((oleoyloxy)methyl)propane-1,3-
diyl)bis(oxy))bis(4-oxobutanoic acid) 6
To a solution of compound 5 (2.5 g; 0.0038mol) in dry THF (25 ml) was
added succinic anhydride (0.783 g; 0.0078 mol) and the mixture was refluxed
for 18 h, after which the solvent was evaporated. Excess unreacted succinic
anhydride was removed by washing the crude product with diethyl ether (3 x
1
2
5 ml) to obtain compound 6 as a viscous oil (2.63 g, 81%). H NMR (CDCl
3
)
7

δ (ppm): 0. 84 (t; 6H; -CH
CH CH COO-), 1.92 (m; 8H; -CH
.56 (t; 4H; -CH CH COOH), 2.59 (t; 4H; -CH
H; -CH -), 4.17 (d; 4H; -CH -), 5.27 (m; 4H; -CH=CH-). C NMR (CDCl
3
), 1.19-1.29 (m; 40H; -CH
CH=CHCH -), 2.23 (t; 4H; -CH
CH COOH), 3.37 - 3.67 (d;
2
-), 1.50 (q; 4H; -
2
2
2
2
2
COO-),
2
4
2
2
2
2
1
3
2
2
3
)
δ (ppm): 13.88, 14.10, 19.20, 22.67, 24.81, 27.17, 27.21, 28.77, 28.89, 28.94,
2
7
9.11 - 29.76, 31.62, 34.05, 41.94, 61.75, 61.89, 62.40, 63.97, 68.41, 71.12-
1.20, 98.55, 129.72, 129.99, 171.72, 172.29, 173.39, 173.87, 175.85, 176.52.
+
ESI-TOF MS m/z: [M] - calculated 864.596 (C49
84
H O12), found 864.573.
2
.3.4. Preparation of AGS 7
A solution of sodium bicarbonate (0.429 g; 0.0051 mol) in purified water (20
ml) was added to compound 6 (2.0 g; 0.0023 mol) and stirred for 3 h at room
temperature. The clear solution formed was filtered through a 0.2 µm filter
and the filtrate was freeze dried to obtain AGS 7 as a white fluffy solid (2.05
g, 98%).
2
.4. Determination of critical micelle concentration (CMC) and hydrophilic-lipophilic
balance (HLB) of AGS 7
An electrical conductivity method suitable for ionic surfactants, was used to
determine the CMC of AGS 7 [22]. In short, AGS 7 solution of highest concentration
(5 mM/L) was prepared and the conductivity measured using a step-by-step dilution-
extraction method reported in the literature [2, 23]. All measurements were performed
o
at 25 ± 2 C (n = 3), and the CMC was determined by plotting a graph of
concentration versus conductivity. The Davies and Rideal [24] method was followed
to calculate the HLB value of AGS 7 using the following formula.
8

HLB = ∑ (hydrophilic group numbers) - ∑ (lipophilic group numbers) + 7
2
.5. In vitro cytotoxicity
The biological safety of AGS 7 was assessed by performing an MTT assay on human
breast adenocarcinoma (MCF 7), adenocarcinomic human alveolar basal epithelial
cells (A 549) and human liver hepatocellular carcinoma (HepG 2) cell lines. A
literature reported procedure was followed to determine the cytotoxicity of AGS 7 at
2
0, 40, 60, 80 and 100 μg/ml concentration [25] using 5-Fluorouracil (5-FU) as a
positive control, with experiments being performed in six replicates. The percentage
cell viability was calculated as follows.
%
cell viability = (A540 nm treated cells/ A540 nm untreated cells) x 100
where A540 nm is absorbance at a wavelength of 540 nm, which was measured using a
microplate spectrophotometer (spectrostar nano, Germany).
2
.6.Preparation of chitosan nanoparticles (CSNPs)
A modified version of the ionic gelation technique [26, 27], based on the ionotropic
complexation of CS with TPP anions, was used to prepare blank and vancomycin
loaded CSNPs. A selective CS:TPP:AGS 7 ratio of 5:1:0.5 (w/w/w) was used to
prepare the CSNPs, with a solution of TPP (10 ml, 0.1 % w/v) and AGS 7 (5 ml, 0.1
%
w/v) being prepared (TPP to AGS 7 ratio = (1:0.5). Vancomycin (10 mg) was
added to the prepared TPP-AGS solution, and this formed premixed solution was
added drop wise to a stirred CS solution (50 ml, 0.1 % w/v) in acetic acid (1 % v/v).
The pH of the final solution was adjusted to 5.5 using 1 M NaOH [27, 28]. The
vancomycin free base loaded CSNPs (DL_CSSNPs) were formed spontaneously, and
the process of gelation was allowed to take place for 30 min under constant magnetic
9

stirring at 500 rpm. A similar technique was used to prepare blank CSNPs
B_CSSNPs) and control CSNPs without AGS 7 (blank, B_CSNPs and vancomycin
(
loaded, DL_CSNPs) by omitting vancomycin and AGS 7 respectively from the
procedure.
2
.7.Nanoparticle characterization
Dynamic light scattering (DLS) technique (Malvern Zetasizer Nano ZS90, Malvern
Instruments Ltd., UK) was used to determine the mean hydrodynamic diameter
o
(
MHD), polydispersity index (PDI) and zeta potential (ZP) of CSNPs at 25 C.
Transmission electron microscopy (TEM) (Jeol, JEM-1010, Japan) was used to
analyse the morphology and size of the CSNPs. A drop of CSNPs, dispersed in water,
was placed on a 3 mM forman (0.5% plastic powder in amyl acetate) coated copper
grid (300 mesh) to form a thin film. The sample was negatively stained using uranyl
acetate solution (2% w/v) and allowed to dry, and the images were obtained at an
accelerating voltage of 100 kV.
2
.8.Drug entrapment efficiency (%EE)
The CSNPs that were loaded with vancomycin were separated from the non-
o
entrapped drug by ultracentrifugation at 50,000 rpm for 50 min at 4 C [29], for which
a table top ultracentrifuge (OptimaTM MAX-XP, Beckman Coulter, USA) was used.
The amount of unentrapped vancomycin in the clear supernatant was measured using
a UV spectrophotometer (Schimadzu UV 1601, Japan) at 280 nm [30]. The
encapsulation efficiency (EE) was calculated using the equation [29]:
%EE = Total amount of drug-free amount of drug/Total amount of drug x 100
1
0

2
.9.In vitro release study
The CSNP formulations (1 ml) were added to PBS of pH 6.5 and 7.4 (1 ml) in a
dialysis tubing (molecular weight: 8000-14,400 Da) and dialyzed against respective
o
PBS solutions (10 ml) at 37 C in a shaking incubator at 100 rpm speed. The samples
(
3 ml) were withdrawn and replaced with an equal volume of fresh PBS at scheduled
time periods. The amount of vancomycin released was measured using UV
spectrophotometry (280 nm, Shimadzu 1601, Japan), with all experiments being
performed in triplicate. The release kinetics parameters were determined using
KinetDS by analyzing data up to 60% cumulative release [25].
2
.10. In vitro antibacterial activity
The in vitro antibacterial activity of DL_CSSNPs, bare vancomycin, B_CSSNPs and
®
AGS 7 was evaluated against S. aureus (ATCC 25922) and MRSA (ATCC BAA-
TM
1
683 ) at pH 6.5 and 7.4. Briefly, single bacterial culture colonies, grown in MHA,
8
were diluted in a saline solution and adjusted to 1.5 X 10 CFU/ml using a
densitometer (DEN-1B, Grant Instruments, China). Further dilutions were performed
5
in MHB (pH 6.5 and 7.4) to obtain 5.0 X 10 CFU/ml. The broth dilution method
(Supplementary Material) was used to determine the minimum inhibitory
concentrations (MICs) [25].
2
.11. In vivo antibacterial activity
In vivo antibacterial activity was evaluated against MRSA using BALB/c mice, the
protocol being approved by the Animal Research Ethics Committee of the University
of KwaZulu-Natal (Protocol No. AREC/104/015PD). A literature reported procedure
[31], with some modifications, was followed for this purpose (Supplementary
1
1

Material). Histological evaluations were performed on freshly harvested excised skin
from the injection site (Supplementary Material).
2
.12. Stability study
Three batches of DL_CSSNPs were prepared under optimum conditions and their
MHD, PDI and ZP were analyzed. Thereafter, the samples were stored in borosilicate
o
glass vials at room temperature (RT) and 4 C, and their stability assessed using DLS
analyses for three months. The parameters monitored to assess the stability were
physical appearance, MHD, PDI and ZP.
2
.13. Statistical analysis
The data is expressed as mean ± standard deviation (SD), and was statistically
analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni’s
multiple comparison test or t-test followed by non-parametric Mann-Whitney tests,
with a p value <0.05 being considered statistically significant. GraphPad Prism®
(
Graph Pad Software Inc., Version 5, San Diego, CA) was used.
3
. Results and Discussion
.1.In vitro cytotoxicity
3
An MTT assay was performed to evaluate the biosafety of AGS 7 using an in vitro
cell culture system. The results showed that AGS 7 was non-toxic against all cell lines
tested (Fig. S1). The percentage cell viability ranged between 77.05 ± 5.94 to 81.86 ±
1
1
0.35 for MCF 7 cells, 81.16 ± 10.79 to 91.18 ± 8.16 for A549 cells and 77.34 ±
0.62 to 89.35 ± 9.36 for Hep G2 cells (Fig S1) whereas 5-FU was found to be toxic
with 0% cell viability at 100 µg/ml concentration. Although a slight dose dependant
1
2

trend was observed in the percentage cell viability across all cell lines, the lowest cell
viabilities obtained can be considered biologically safe, as they were all greater than
7
5 %, which is considered non-toxic [32]. These results therefore suggest that the
application of AGS in biological studies would be safe.
3
.2.Determination of CMC and HLB of AGS 7
A conductometric method was used to determine the CMC of the AGS 7, as it is
considered the most suitable method for ionic surfactants [2, 23]. A plot of
conductance versus concentration was plotted. The CMC, intersection point of two
straight lines drawn, of AGS 7 was found to be 1.3 mM/L (Fig. 2). The CMC value
of this oleic acid based AGS 7 was 2.7-fold less than sodium oleate with a CMC of
3
.5 mM/L [2]. Surfactants with low CMC values have higher surface activity and a
tendency to form micelles at low concentrations [2]. The calculated HLB of AGS 7
was 15.45, which revealed that it can form a clear solution in water [33] and be a
good solubilizer [2].
3
.3.Nanoparticle characterization
Blank CSNPs without AGS 7 (B_CSNPs), vancomycin free base loaded CSNPs
without AGS 7 (DL_CSNPs), blank CSNPs with AGS 7 (B_CSSNPs), and
vancomycin free base loaded CSNPs with AGS 7 (DL_CSSNPs) were prepared using
the ionic gelation technique to analyze the effect of AGS 7 and vancomycin free base
on MHD, PDI and ZP of CSNPs. The DLS results indicated that there were no
significant differences (p > 0.05) in MHD, PDI and ZP of all the formulated CSNPs.
The MHD, PDI and ZP for all the CSNPs were in the range of 202.40 ± 3.73 to
2
20.57 ± 5.90 nm, 0.266 ± 0.01 to 0.301 ± 0.004 and 20.1 ± 3.50 to 22.00 ± 0.90 mV
1
3

respectively (Table 1). These results show that AGS 7 and vancomycin free base had
no significant effect on the DLS parameters. The PDI values below 0.35 and ZP
values > 19.00 mV indicated that all formulations were relatively homogenous in size
with high steric stability.
The TEM analysis performed on DL_CSSNPs showed that the nanoparticles were
homogenous and spherical in shape, and that the size range was 55-90 nm, which is
much smaller (Fig. 3a) than the MHD determined by DLS. This could be due to the
fact that, unlike DLS, TEM determines particles’ diameter alone in a dry state [34,
3
5].
3
.4.Drug entrapment efficiency
Vancomycin free base was used to prepare the DL_CSSNPs as the aim was to
encapsulate higher quantity of drug inside the nanoparticles. The %EE for
vancomycin free base loaded DL_CSSNPs was 59.89 ± 2.33%, which can be
attributed to the hydrophobicity of vancomycin free base compared to its
hydrochloride salt. This hypothesis was confirmed by preparing vancomycin
hydrochloride loaded DL_CSSNPs. It was observed that the %EE for these
nanoparticles was 31.74 ± 6.66% which was almost 2-fold lower than the vancomycin
free base loaded DL_CSSNPs. In addition, the particle size for vancomycin
hydrochloride loaded CSSNPs was 276.83 ± 37.69 which was higher than the
DL_CSSNPs. The excess unencapsulated hydrochloride salt of vancomycin could
have ionically interacted with -COONa and free -NH function of AGS 7 and chitosan
2
respectively resulting in aggregation of nanoparticles to some extent and subsequent
increase in particle size. The lower amount of encapsulated drug could have also
1
4

affected the geometry of the particle and particle formation mechanism thereby
altering particle size.
3
.5.In vitro release study
The cumulative amount of vancomycin free base released from the DL_CSSNPs at
pH 6.5 and 7.4 is shown in Fig. 3b. A control experiment using DL_CSNPs was also
performed under similar conditions to confirm the pH-responsive drug release from
DL_CSSNPs. The cumulative amount released from the DL_CSSNPs after the initial
3
1
6
0 min was almost 3-fold greater at pH 6.5 (12.61 ± 3.55 %) than at pH 7.4 (4.33 ±
.40 %). At each time point, the drug release from the DL_CSSNPs was greater at pH
.5 compared to at pH 7.4. The drug release from DL_CSSNPs at the end of 8 and 24
h time period at pH 6.5 was 77.58 ± 3.36 % and 94.29 ± 5.30 % respectively, while at
pH 7.4, it was 53.92 ± 6.89 % and 57.30 ± 6.47 % respectively. The increase in drug
release at pH 6.5 could be attributed to the pH dependent activity of AGS 7. The
mechanism involved in greater drug release at pH 6.5 could be due to the decrease in
ionization of AGS 7 under mildly acidic conditions, resulting in a destabilization of
the nanoparticles’ structure. A similar kind of triggered release of methotrexate under
acidic conditions was observed for CSNPs that was prepared using an anionic
surfactant 77 KL [27]. The drug release from the DL_CSNPs did not follow any
notable pH dependent trend. The drug release after 8 and 24 h from DL_CSNPs at pH
6
5
.5 was 46.87 ± 4.44 % and 65.34 ± 11.38 % respectively, whereas at pH 7.4 it was
4.38 ± 5.29 % and 67.42 ± 5.71 %. These results confirm that the pH dependent
cumulative release from the DL_CSSNPs was due to the AGS 7.
The vancomycin release at both pH values followed zero order kinetics as indicated
2
by R value obtained from KinetDS software. This suggests that the drug release
1
5

process took place at a constant rate that was independent of the concentration
involved in the process. At both pH values, the drug release from all formulations
2
followed the Weibull model (R closer to 1 and low RMSE values), a model which is
useful for comparing the release profile of matrix type drug delivery systems. The n
value of the Korsemeyer-peppas model at pH 6.5 and 7.4 for all the formulations was
>
1, which is an indication of a super case II transport [36]. The mean dissolution time
(MDT) values for DL_CSSNPs at pH 6.5 and 7.4 were 2.12 and 4.18 h respectively.
This ~ 2-fold decrease in MDT for DL_CSSNPs at acidic pH clearly suggests that the
drug release was pH dependent. On the contrary, there was no significant difference
in the MDT value of the DL_CSNPs at pH 6.5 (MDT = 1.08) and 7.4 (MDT = 1.09).
The results from this study confirm that the pH-dependent release from the
DL_CSSNPs was exclusively due to AGS 7.
3
.6.In vitro antibacterial activity
The MIC for bare vancomycin free base at both the pH values was constant whereas
the MIC for the DL_CSSNPs was lower (enhanced activity) at the acidic pH
compared to the physiological pH. The MICs of the DL_CSSNPs against S. aureus at
pH 6.5 and 7.4 were 7.81 and 31.25 µg/ml respectively, whilst against MRSA, they
were 7.81 and 62.5 µg/ml at pH 6.5 and 7.4 respectively (Table 2). This 4- and 8-fold
enhanced activity of the DL_CSSNPs against S. aureus and MRSA at pH 6.5 could be
due to a greater release of the vancomycin free base at acidic pH compared to
physiological pH as observed during the in vitro release study. These results indicated
that the DL_CSSNPs were more active at acidic pH than at neutral pH and confirm
the potential of this nanosystem for targeted release at bacterial infection sites.
1
6

3
.7.In vivo antibacterial activity
Emergence of community associated MRSA has recently contributed to an increased
number of skin infections. Therefore, having confirmed the in vitro potential of
DL_CSSNPs to inhibit MRSA growth, its in vivo efficacy was subsequently
evaluated using a mice skin infection model. The MRSA burden was highest in blank
CSSNPs treated mice (3- and 26-fold more than bare vancomycin and DL_CSSNPs
respectively). There was a significant difference (p = 0.0001) in the bacterial colony
forming units per ml (CFU/ml) remaining in the skin of mice treated with bare
vancomycin and those treated with DL_CSSNPs. The MRSA burden in the
DL_CSSNPs treated mice was almost 8-fold less compared to the bare vancomycin
(Fig. 4). This enhanced activity of the DL_CSSNPs could be ascribed to a sequence of
steps: i) a decrease in the ionization of AGS 7 due to the acidic pH at the infection
site, ii) the attachment of protonated amino groups to the negatively charged bacterial
cell wall, and iii) vancomycin release due to a degradation of the nanoparticle
structure. In addition, some chitosan nanoformulation can escape the efflux pump at
cell membrane level and enter into the bacterial cell [37]. It has been reported that: i)
there is a decrease in the ionization of an anionic surfactant in a nanoformulation due
to mild acidic pH which results in nanoparticle destabilization [27] and ii) at low
environmental pH chitosan binds to the bacterial cell wall [38].
In this study, intradermal injections delivered MRSA between the epidermal and
subcutaneous layers of the skin. B_CSSNPs (control) and bare vancomycin groups
showed fluid filled abscesses at the injection site, while there were no visible signs of
abscess formation in the DL_CSSNP injected group. Tissue from the injection site
were harvested and processed for further histological evaluation to determine the
morphological changes after intradermal MRSA infection. The H&E stained samples
1
7

provided evidence of abscess formation in the B_CSSNPs and bare vancomycin
groups (Fig 5A and 5C), with no evident signs in the DL_CSSNP treated group (Fig.
5
E). Furthermore, images from B_CSSNPs and bare vancomycin groups showed
signs of tissue inflammation at the injection site, as presented by swelling of the
dermal layer (Fig. 5A and 5C). In contrast, the images from the DL_CSSNP treated
group displayed minimal signs of tissue inflammation (Fig. 5E). In the B_CSSNPs
and bare vancomycin groups, the large area of the abscess indicated that a high
number of skin cells were infiltrated by MRSA (Fig. 5A and 5C). As MRSA is
recognized as foreign to the body, its introduction into skin would result in an
immune response (inflammation) that is proportional to the quantity of bacteria
(bacterial load) present at the infection site. Images from the treated group (Fig. 5F)
showed a significant lower number of immune cells present at infection site as
compared to control group (Figure 5B and 5D). This suggests that a greater immune
response was evoked by the B_CSSNPs and bare vancomycin groups due to the larger
bacterial load present in the skin. The histomorphological evaluations directly
correlated with findings of the in vivo antibacterial study, where the B_CSSNPs and
bare vancomycin groups displayed a significantly higher bacterial load compared to
the DL_CSSNP injected treated group (Fig. 4). These histomorphological evaluations
further confirmed the antimicrobial superiority of the DL_CSSNPs.
3
.8.Stability study
Stability studies at a developmental stage of formulation development help in
obtaining important information to determine storage conditions, shelf-life and
container closure system of a proposed new product. This study provides preliminary
data for confirming the potential of the product to eventually meet full stability
1
8

requirements as per regulatory guidelines [36]. The DL_CSSNPs were stable for the
o
period of three months at both 4 C and RT as no change in physical appearance of
formulation was observed, and there were no statistically significant differences in
MHD, PDI and ZP at different time points within the samples kept at specific storage
conditions (p > 0.05). In addition, no statistical differences were observed for these
o
parameters between samples stored at 4 C versus their RT counterparts at all the time
points (p > 0.05) (Table 3).
4
. Conclusion
A gemini surfactant, AGS 7, based on oleic acid tails (hydrophobic portion) and
anionic sodium carboxylate head groups (hydrophilic portion) was successfully
synthesized and its biosafety confirmed by MTT assay. The CMC value of 1.3 mM/L
revealed that this surfactant could be a good solubilizer and stabilizer for colloidal
formulations. Vancomycin loaded pH responsive CSNPs, DL_CSSNPs, were
formulated using AGS 7. Although the drug release from DL_CSSNPs was sustained
at both acidic and normal physiological pH, it was higher at acidic pH. The in vitro
antibacterial activity of DL_CSSNPs was significantly greater at acid pH than at pH
7
.4 against both S. aureus and MRSA. Furthermore, in vivo anti-MRSA activity
confirmed that the prepared DL_CSSNPs had a greater potential to inhibit the growth
of MRSA at acidic infection site than bare vancomycin. In conclusion, this study has
introduced: i) AGS 7, a novel safe ionic gemini surfactant, for pharmaceutical and
biological applications, and ii) a pH-responsive nanoantibiotic from AGS 7 for
effectively managing sensitive and resistant S. aureus infections. The DL_CSSNPs
can be of major significance in improving the therapeutic efficacy of antibacterial
drugs, thereby addressing drug resistance challenges by resistant bacteria.
1
9

Acknowledgements
The authors thank College of Health Sciences (CHS), University of KwaZulu-Natal (UKZN),
UKZN Nanotechnology Platform and the National Research Foundation (NRF) of South
Africa (NRF Grant No. 87790 and 88453) for financial support and Microscopy and
Microanalysis Unit (MMU), UKZN for technical assistance. Ms. Carrin Martin is
acknowledged for proof-reading the manuscript.
2
0

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3

Figure Legends
Fig. 1. Scheme for synthesis of AGS 7.
Fig. 2. Plot of concentration versus conductance for AGS 7 solution. Data are expressed as
mean ± S.D (n = 3).
Fig. 3. (a) TEM image of DL_CSSNP and (b) in vitro release pattern of DL_CSNPs and
DL_CSSNPs at pH 6.5 and 7.4 (n = 3).
Fig. 4. MRSA burden post 48 h treatment. Data represents mean ± SD (n = 3). *denotes
significant for Blank CSSNPs versus bare vancomycin free base. **denotes significant
difference between blank CSSNPs and DL_CSSNPs and ***denotes significant difference
between bare vancomycin free base and DL_CSSNPs.
Fig. 5. Photomicrographs of the blank CSSNPs, bare vancomycin and the DL_CSSNP treated
skin selections for light microscopy (LM) stained with H&E (4X and 40X); blank CSSNPs
(A and B), bare vancomycin (C and D) and DL_CSSNP (E and F).
2
4