AB2-type amphiphilic block copolymer containing a pH-cleavable hydrazone linkage for targeted antibiotic delivery

Article abstract of DOI:10.1016/j.ijpharm.2019.118948

A novel AB2-type amphiphilic block copolymer [OA-C[dbnd]N-NH-(PEG)₂] with hydrazone linkage was synthesized and explored for pH-triggered antibiotic delivery. Vancomycin (VCM) loaded micelles of the polymer [OA-C[dbnd]N-NH-(PEG)₂-VCM] were spherical in shape with size, polydispersity index, zeta potential and entrapment efficiency of 130.33 ± 7.36 nm, 0.163 ± 0.009, ?4.33 ± 0.55 mV and 39.61 ± 4.01% respectively. The dilution stability study exhibited no significant change in the size distribution of OA-C[dbnd]N-NH-(PEG)₂-VCM micelles up to 320-fold dilution. An in vitro drug release assay confirmed greater release of VCM from OA-C[dbnd]N-NH-(PEG)₂-VCM at pH 6, compared to pH 7.4. An in vitro antibacterial activity evaluation of OA-C[dbnd]N-NH-(PEG)₂-VCM showed 2-fold enhancement in antibacterial activity of VCM after 54 h of incubation against Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) at acidic pH compared to physiological pH. An in vivo antibacterial activity of OA-C[dbnd]N-NH-(PEG)₂-VCM further proved the enhanced activity of OA-C[dbnd]N-NH-(PEG)₂-VCM against MRSA. In conclusion, micelles from pH-responsive OA-C[dbnd]N-NH-(PEG)₂ could be utilized for site-specific delivery of VCM at the infection site.

Full text of DOI:10.1016/j.ijpharm.2019.118948

International Journal of Pharmaceutics 575 (2020) 118948
Contents lists available at ScienceDirect
International Journal of Pharmaceutics
journal homepage: www.elsevier.com/locate/ijpharm
AB2-type amphiphilic block copolymer containing a pH-cleavable
hydrazone linkage for targeted antibiotic delivery
a
a,b,⁎
a
a,c
Sandeep J. Sonawane , Rahul S. Kalhapure
, Mahantesh Jadhav , Sanjeev Rambharose ,
a
a,⁎
Chunderika Mocktar , Thirumala Govender
a
Discipline of Pharmaceutical Sciences, School of Health Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa
Odin Pharmaceuticals, 300 Franklin Square Drive, Somerset, NJ 08873, USA
Division of Emergency Medicine, Department of Surgery, University of Cape Town, Cape Town, South Africa
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
2
A novel AB2-type amphiphilic block copolymer [OA-C]N-NH-(PEG) ] with hydrazone linkage was synthesized
Hydrazone linkage
Amphiphilic polymer
pH cleavable
Vancomycin
MRSA
and explored for pH-triggered antibiotic delivery. Vancomycin (VCM) loaded micelles of the polymer [OA-C]N-
NH-(PEG) -VCM] were spherical in shape with size, polydispersity index, zeta potential and entrapment effi-
ciency of 130.33 ± 7.36 nm, 0.163 ± 0.009, −4.33 ± 0.55 mV and 39.61 ± 4.01% respectively. The
dilution stability study exhibited no significant change in the size distribution of OA-C]N-NH-(PEG) -VCM
micelles up to 320-fold dilution. An in vitro drug release assay confirmed greater release of VCM from OA-C]N-
NH-(PEG) -VCM at pH 6, compared to pH 7.4. An in vitro antibacterial activity evaluation of OA-C]N-NH-
PEG) -VCM showed 2-fold enhancement in antibacterial activity of VCM after 54 h of incubation against
Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA) at acidic pH compared to physio-
logical pH. An in vivo antibacterial activity of OA-C]N-NH-(PEG) -VCM further proved the enhanced activity of
OA-C]N-NH-(PEG) -VCM against MRSA. In conclusion, micelles from pH-responsive OA-C]N-NH-(PEG)
could be utilized for site-specific delivery of VCM at the infection site.
2
2
Site-specific delivery
2
(
2
2
2
2
1
. Introduction
Fernandes and Martens, 2017; Sharma et al., 2012). Therefore, the
design and development of novel strategies for efficient delivery of
currently existing antibiotics to improve their efficacy so that they can
combat bacterial resistance more effectively is necessary (Kalhapure
et al., 2014).
Nanotechnology has proved to be an effective strategy to address
the problems related to antibiotic resistance (Pelgrift and Friedman,
2013). Conventional dosage forms of antibiotics have various dis-
advantages including occurrence of bacterial resistance with time, non-
targeted delivery to the infections site, low therapeutic index, undesired
side effects and enhanced frequency of administration. These limita-
tions can be overcome using various nanoparticulate drug delivery
systems (Bawa et al., 2009; Sharma et al., 2012). Various
Most bacterial species have developed clinically significant re-
sistance towards available antibiotics due to frequent and suboptimal
use of antibiotics in treating bacterial infections (Sharma et al., 2012).
Misuse and overuse of antibiotics especially in developing countries
have contributed to evolution of resistant bacteria such as methicillin-
resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphy-
lococcus aureus (VRSA) and vancomycin-resistant Enterococcus (VRE)
(
Fernandes and Martens, 2017; Klevens et al., 2006; Rice, 2009). The
development of new antibiotics is in a stagnant phase and the available
pool of antibiotics is not sufficient enough to address the risk of rapidly
growing antibiotic resistance crisis (Allahverdiyev et al., 2011;
Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; VRSA, vancomycin-resistant Staphylococcus aureus; VRE, vancomycin-resistant Enterococcus; NEs,
nanoemulsions; PNPs, polymeric nanoparticles; SLNs, solid lipid nanoparticles; LPHNs, lipid polymer hybrid nanoparticles; LDHNs, lipid dendrimer hybrid nano-
particles; MHA, Mueller Hinton Agar; MHB, Mueller-Hinton broth; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; FT-IR, Fourier-transform
1
13
infrared; H NMR, proton nuclear magnetic resonance; C NMR, carbon nuclear magnetic resonance; HRMS, high resolution mass spectrometry; RBF, round bottom
flask; NIBS, non-invasive back scatter; LCMS, liquid chromatography-mass spectrometry; DCM, dichloromethane; VCM, vancomycin; TEM, transmission electron
microscope; PI, polydispersity index; ZP, zeta potential; RT, room temperature; SD, standard deviation; CFU, colony forming units; ANOVA, analysis of variance;
CMC, critical micelle concentration; TLC, thin layer chromatography; RF, retention factor; MIC, minimum inhibitory concentration
⁎
Corresponding authors at: Private Bag X54001, Durban 4000, KwaZulu-Natal, South Africa.
E-mail addresses: kalhapure@ukzn.ac.za (R.S. Kalhapure), govenderth@ukzn.ac.za (T. Govender).
https://doi.org/10.1016/j.ijpharm.2019.118948
Received 1 August 2019; Received in revised form 1 December 2019; Accepted 7 December 2019
Available online 16 December 2019
0378-5173/ © 2019 Elsevier B.V. All rights reserved.

S.J. Sonawane, et al.
International Journal of Pharmaceutics 575 (2020) 118948
nanoparticulate drug delivery systems have been reported in the lit-
erature for efficient delivery of antibiotics. These nano systems include;
nanoemulsions (NEs), polymeric nanoparticles (PNPs), solid lipid na-
noparticles (SLNs), liposomes, dendrimers, lipid polymer hybrid nano-
particles (LPHNs), lipid dendrimer hybrid nanoparticles (LDHNs)
systemic circulation will certainly solve the problem of superbugs by
improving antibiotic efficiency. Among various responsive materials,
use of pH-responsive materials could be an effective approach for de-
livery of sufficient amount of antibiotics specifically at infection site, as
bacterial infection sites are known for acidic conditions (Kalhapure
et al., 2017b; Radovic-Moreno et al., 2012). Various researchers have
shown promise of developing new polymers with pH-responsiveness to
antibiotic delivery. However, the reported polymers require difficult
synthesis and purification steps, the use of hazardous chemicals for
their synthesis and purification, and have low production yields.
(
Sonawane et al., 2016), and nanostructures composed of pure carbon
and nanohybrids (Kalhapure et al., 2015).
Although these nanoantibiotics are therapeutically effective com-
pared to conventional dosage forms (Kalhapure et al., 2015), they are
not smart enough to respond to the changes in metabolic states of the
body (Bawa et al., 2009). Ideally, the release of a drug from a delivery
system should be in accordance with the physiological need of the body
2
Thus, a new AB type amphiphilic block copolymer containing a pH-
cleavable hydrazone linkage was designed, synthesized by conjugation
of aldehyde functional group of PEG aldehyde with carbohydrazide
function of G1 oleodendrimer (Kalhapure and Akamanchi, 2013), and
utilized for pH-responsive delivery of vancomycin (VCM). This polymer
was successfully used in the formulation development of VCM loaded
polymeric micelles, which showed pH-dependent enhanced drug re-
lease and antibiotic activity against both susceptible and resistant S.
aureus strains.
The goal of the research undertaken in this manuscript was pri-
marily to enhance antibacterial potency of VCM against susceptible and
resistant bacteria through a pH-responsive micellar delivery system to
specifically target the infection site. The promising in vitro and in vivo
results obtained through this study are presented in this manuscript.
(
Gupta et al., 2002). Therefore, application of stimuli-responsive drug
delivery systems using different triggers for drug targeting to the site of
action has become a major focus area (Ganta et al., 2008; Kalhapure
and Renukuntla, 2018). Amongst different stimuli, pH has been studied
widely for site specific drug delivery (Liu et al., 2016). The materials
employed for preparing pH-responsive drug delivery systems contain a
particular chemical functional group in their structure that can show
response to various pH gradients existing in both normal and disease
conditions in the body (Gillies et al., 2004). Various chemical functional
groups, such as, ortho ester (Tang et al., 2011; Tang et al., 2010), acetal
(
Chen et al., 2010; Kim et al., 2010), vinyl ether (Shin et al., 2003; Xu
et al., 2008), amine (Lee et al., 2003; Radovic-Moreno et al., 2012) and
hydrazone (Bae et al., 2003; Etrych et al., 2010) have been utilized to
fabricate materials that are efficient to release their payload at the
lower pH conditions, specifically in the management of various can-
cerous tumours. To date only three to four papers have reported use of
pH-responsive materials for antibiotic delivery, including a pH-re-
sponsive surfactant (Kalhapure et al., 2017a) and lipids (Jadhav et al.,
2. Materials and methods
2.1. Materials
Benzyl amine, methyl acrylate, thionyl chloride and trimethylamine
were purchased from Merck Chemicals (Germany). Pd/C (10%), oleic
acid, hydrazine hydrate solution (50–60%) and poly (ethylene glycol)
methyl ether (average Mn = 5000) were procured from Sigma-Aldrich
Co., Ltd. (USA). All the solvents utilized were of analytical grade and
procured from Merck Chemicals (Germany). VCM was obtained from
Sinobright Import and Export Co., Ltd. (China). Nutrient Broth and
Mueller Hinton Agar (MHA) were procured from Biolab Inc., (South
Africa). Mueller-Hinton broth (MHB) was obtained from Oxoid Ltd.
(England), and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) from Merck Chemicals (Germany). All other chemicals
used in this research work were obtained from Sigma-Aldrich Co., Ltd.
(USA). Distilled water used was obtained in the laboratory with a Milli-
Q purification system (Millipore corp., USA). For antibacterial studies,
S. aureus (ATCC 25922) and S. aureus Rosenbach (ATCC®BAA-1683TM)
(MRSA) were used. Fourier-transform infrared (FT-IR) spectra of all the
synthesized molecules were produced on a Bruker Alpha-p spectro-
meter with diamond ATR (Germany), as per standard procedures.
2018; Kalhapure et al., 2017b; Mhule et al., 2018).
The hydrazone linkages have been commonly used for conjugating
drugs to polymer backbones to design pH-responsive delivery systems
due to their rapid hydrolysis at acidic pH compared to neutral phy-
siological pH, the purpose being to minimize systemic toxicity by tar-
geting drug to the site of action in the body (Yoshida et al., 2013).
Hydrazone linkages have been proven to be an effective strategy in
designing several pH-responsive drug delivery systems, such as linear
polymers (Lu et al., 2009), star shaped polymers (Etrych et al., 2011),
dendrimers (Kono et al., 2008), micelles (Aryal et al., 2009) and PE-
Gylated systems (Lai et al., 2010) for the site specific delivery of an-
ticancer drugs. The site specific delivery of anticancer drugs reported in
most of the articles has been achieved through hydrazone conjugates
formed by conjugating hydrazide functional group of carrier polymers
or inorganic materials or dendrimers with an aldehyde or ketone
functional group of anticancer drugs (Sonawane et al., 2017). Although
the hydrazone conjugates have been reported to be effective systems for
the site specific delivery of anticancer drugs, their limitations must be
taken into consideration for design of new pH-responsive systems for
other classes of drugs containing no ketone or aldehyde functional
group in their structures. In the present available pool of various drug
classes, very few contain aldehyde or ketone functional groups in their
structures. For example, only doxorubicin and pirarubicin from the
anticancer class and streptomycin antibiotic fulfill the criterion of
possessing aldehyde/ketone function in their structures for the forma-
tion of pH-responsive hydrazone bond with hydrazide functional group
of various carriers (Sonawane et al., 2017). Considering the limitations
associated with hydrazone conjugates and since there are very few re-
ports on a polymer itself containing hydrazone linkage, there is an
urgent need to design and develop a novel polymer containing hy-
drazone linkage for encapsulation and site specific delivery of any class
of drugs. An inadequate concentration of antibiotics at the bacterial
infection site is one of the reasons for the evolution of resistant bacterial
species by supporting gene mutations (Kalhapure et al., 2017b). Design
and development of novel responsive materials for site specific anti-
biotic delivery to maximize its utilization and reduce elimination via
1
13
Proton and carbon nuclear magnetic resonance ( H NMR and C NMR)
spectra were determined using a Bruker 400/600 Ultrashield™ (U.K.)
NMR spectrometer. High resolution mass spectrometry (HRMS) spectra
were obtained on a Waters Micromass LCT Premier TOF-MS (U.K.).
2.2. Methods
2
2.2.1. Synthesis of OA-C]N-NH-(PEG) (Fig. 1)
2.2.1.1. Synthesis of compounds (i) to (iv). A procedure reported in the
previous literature was followed for the synthesis of compounds (i) to
(iv) (Kalhapure and Akamanchi, 2013).
2.2.1.2. Synthesis of compound (v). Compound (iv) (0.5 g, 1.102 mmol)
in ethanol (20 ml) was added drop wise to an ethanolic solution (30 ml)
of hydrazine hydrate (50–60%) (quantity equivalent to approx. 1.41 g,
44.08 mmol of pure hydrazine) in 100 ml of round bottom flask (RBF)
over a period of 30 min. After complete addition of hydrazine hydrate,
the resulting mixture was heated under reflux for 12 h. The ethanol and
residual amount of hydrazine hydrate were removed, using a rotary
2

S.J. Sonawane, et al.
International Journal of Pharmaceutics 575 (2020) 118948
evaporator and the obtained product was further freeze dried to
completely remove entrapped water from product. Complete removal
of water yielded a light yellow semisolid (0.455 g, 91.07%).
5 mg/ml MTT solution in PBS (100 µl) and incubated for 4 h.
Thereafter, MTT containing solution was discarded, and DMSO (100 µl)
was introduced into to the wells to solubilize the MTT formazan.
Absorbance was measured at 540 nm wavelength. A microplate spec-
trophotometer (Mindray MR-96A) was used for this purpose (Sonawane
et al., 2015). The equation used to calculate the percentage cell viabi-
lity was as follows:
2.2.1.3. Synthesis of mPEG-aldehyde (mPEG-CHO). mPEG-CHO was
synthesized from poly (ethylene glycol) methyl ether (average
Mn = 5000) according to a previously reported method (Sugimoto
et al., 1998).
%
cell survival
=
[A540 nm treated cells]/[A540 nm untreated cells] × 100
2
.2.1.4. Synthesis of compound (vi) [OA-C]N-NH-(PEG)
2
]. Compound
(
v) (0.435 g, 0.958 mmol) and mPEG-CHO (12 g, 2.4 mmol) were
dissolved in chloroform (200 ml) in 500 ml of RBF and heated under
reflux for 24 h. The progress of reaction was monitored using FT-IR
spectrophotometer. The product was purified by dialyzing against
5
1
2
.2.6. Preparation of VCM loaded micelles [OA-C]N-NH-(PEG)
2
-VCM]
OA-C]N-NH-(PEG)
cation method. Briefly, 100 mg of OA-C]N-NH-(PEG)
ml of dichloromethane (DCM) and added drop by drop through the
sidewall of a beaker containing 10 mg of VCM dissolved into 10 ml of
pH 7.4 PBS. The resulting emulsion was allowed to stir overnight for the
complete removal of DCM. A similar method was followed to prepare
VCM free (blank) micelles.
2
-VCM micelles were prepared using emulsifi-
2
was dissolved in
00 ml of methanol for 48 h (fresh methanol was replaced after each
2 h for 2 days) using 7000 MWCO dialysis tubing (Thermo Scientific,
1
U.S.A.), followed by removal of methanol using a rotary evaporator to
get a light yellowish-white solid (4.5 g, 45%).
2.2.2. Characterization of compounds
Structural characterization of compound (v) and (vi) was performed
2
.2.7. Determination of particle size, polydispersity index (PI) and zeta
potential (ZP)
The measurement of particle size, PI and ZP of OA-C]N-NH-
PEG) -VCM was performed using the dynamic light scattering method
after diluting OA-C]N-NH-(PEG) -VCM micelles solution (200 µl) with
using FT-IR spectroscopy (Bruker Alpha-p spectrometer with diamond
1
13
ATR, Germany), H NMR and C NMR spectroscopy (Bruker 400/600
Ultrashield™ NMR spectrometer, U.K.) and HRMS spectrometry on a
Waters Micromass LCT Premier TOF-MS (U.K.).
(
2
2
milli-Q water (1 ml). The PI value represented size distribution, and ZP
value showed the total charge present on the surface of micelles
2
.2.3. Critical micelle concentration (CMC)
The CMC of the synthesized OA-C]N-NH-(PEG) was determined
2
(
Sonawane et al., 2015).
according to a previously reported dynamic light scattering method
Topel et al., 2013). Zetasizer Nano ZS (Malvern Instruments Ltd., UK)
was utilized to determine the scattered intensity from each solution of
different concentration of OA-C]N-NH-(PEG) . The scattered intensity
(
2.2.8. Surface morphology
2
Surface morphology of OA-C]N-NH-(PEG) -VCM micelles was in-
2
vestigated using transmission electron microscope (TEM). A drop of
suitably diluted micelles was allowed to dry on a copper grid (300
mesh) coated with a 3 mM forman (0.5% plastic powder in amyl
acetate), and uranyl acetate stain (2% w/v) was further added for 30
sec, dried. A 100 kV of accelerating voltage was used to visualize mi-
celles using a mega view III camera (Jeol, JEM-1010, Tokyo, Japan).
measurements were carried out at an angle of 175° in order to maintain
the non-invasive back scatter (NIBS) optic arrangement, which in-
creases the detection of scattered light while retaining signal quality.
The solutions of different concentrations ranging from 1 to 30 µg/ml
were prepared using stock solution (1 mg/ml) of OA-C]N-NH-(PEG)
2
in water. All the measurements were carried out in triplicate using a
polystyrene cell at 25 °C.
2.2.9. Entrapment efficiency (% EE) and drug loading capacity (LC)
The total amount of VCM entrapped in OA-C]N-NH-(PEG) -VCM
2
2.2.4. pH dependent breakdown
micelles was determined using an ultrafiltration method (Sonawane
et al., 2016). Briefly, 2 ml of micelle solution was filled into Amicon®
Ultra-4 centrifugal filter tube (Millipore Corp., USA) having molecular
weight cut-off of 10 kDa, and allowed to centrifuge at 300g at 25 °C for
In vitro hydrolytic breakdown of hydrazone linkage present in the
OA-C]N-NH-(PEG)
2
was assessed by incubating 20 mg of polymer in
1
0 ml of PBS at pH 6 and 7.4 at 37 °C. After a period of 4, 8, 16 and 24 h
of incubation, aliquots of polymeric solution in PBS were collected and
analyzed using liquid chromatography-mass spectrometry (LCMS)
analysis. LCMS analysis was performed using Shimadzu mass spectro-
meter (Japan) equipped with an Xbridge C18 (4.6 × 100 mm, 3.5 mm)
column. The mobile phases prepared for analysis were 0.1% formic acid
in acetonitrile (mobile phase A), and 0.1% formic acid in water (mobile
phase B). A linear gradient, from 5 to 95% of B (water, 0.1% formic
acid) into A (acetonitrile, 0.1% formic acid) at a flow rate of 1 ml/min,
was used for the analysis.
3
0 min. The filtrate containing unentrapped amount of VCM was ana-
lyzed using UV spectrophotometer (Schimadzu UV 1601, Japan) at
80.5 nm, with appropriate blanks. The regression equation and line-
2
2
arity (r ) obtained were y = 0.004x + 0.0082 and 0.9991, respectively.
The method was specific, as blank micelles showed no interference at
the specified wavelength. The following equations were used to calcu-
late the % EE and LC of OA-C]N-NH-(PEG)2-VCM micelles (Sonawane
et al., 2016; Wang et al., 2012):
%
EE = (Weight of VCM in micelles/Weight of VCM added)
2
.2.5. In vitro cytotoxicity study
In vitro cytotoxicity of the OA-C]N-NH-(PEG)
×
100%
2
against adeno-
carcinomic human alveolar basal epithelial cells (A549), breast cancer
cell line (MCF7) and human liver hepatocellular carcinoma (HepG2)
cell lines was tested using an MTT assay. A 96-well plate was employed
LC = (Weight of VCM in micelles/Weight of micelles) × 100%
3
to equivalently seed the cell lines (2.5 × 10 ) and further allowed to
2.2.10. Dilution stability of the OA-C]N-NH-(PEG)
2
-VCM micelles
adhere by incubating for 24 h. Following adherence, the fresh culture
The dilution stability of the OA-C]N-NH-(PEG) -VCM micelles was
2
medium was placed in the wells and aqueous solutions of OA-C]N-NH-
studied by slight modifications in previously reported method (Song
(
PEG)
2
was added to attain final concentrations of 20–100 µg/ml. The
et al., 2014). In brief, the micelles sample (containing 10 mg/ml of OA-
controls were prepared by adding culture medium only, whilst wells
with media only were used as blanks. The plates were further incubated
2
C]N-NH-(PEG) ) was diluted using PBS (pH 7.4) from 1.25 to 320-fold
at room temperature (RT), and changes in particle size were determined
using Zetasizer Nano ZS (Malvern Instruments Ltd., UK).
(
48 h), the wells were replenished with the fresh media (100 µl) and
3

S.J. Sonawane, et al.
International Journal of Pharmaceutics 575 (2020) 118948
2
.2.11. In vitro drug release
further allowed to stain with hematoxylin and eosin (H&E). A Leica
Microscope DM 500, fitted with a Leica ICC50 HD camera (Leica
Biosystems, Germany) was used to examine the sections and capture the
images.
OA-C]N-NH-(PEG) -VCM micelles, blank micelles and bare VCM
2
solution (1 ml) (donor solutions) were added in dialysis tubes (mole-
cular weight: 8000–14,400 Da), and dialysis was performed against
40 ml of PBS at pH 6 and 7.4 (receiver solutions), respectively, in 50 ml
bottles at 37 °C in an incubator at 100 rpm. At fixed time intervals,
samples (3 ml) from the receiver solutions were withdrawn for analysis
and replaced with an equal volume of fresh PBS (pH 6 and 7.4) to
maintain sink conditions. The amount of VCM released after each time
interval was determined spectrophotometrically at 280.5 nm and
2.2.14. Stability studies
2
The physical stability assessment of OA-C]N-NH-(PEG) -VCM mi-
celles was performed for three months both at room temperature and at
4 °C. Various parameters such as particle size, PI, ZP and appearance
were considered for the physical stability assessment.
2
80.4 nm at pH 7.4 and 6, respectively with blank micelles as a re-
2
ference. The regression equation and linearity (r ) obtained at pH 7.4
were y = 0.004x + 0.0082 and 0.9991, respectively, whereas at pH 6
they were y = 0.0043x + 0.0004 and 1, respectively. This experiment
was performed in three replicates.
2.2.15. Statistical analysis
The data generated is stated as mean ± standard deviation (SD),
and bio-statistical analysis was performed using one-way analysis of
variance (ANOVA), followed by non-parametric Kruskal-Wallis and
paired t-test were used. GraphPad Prism (Graph Pad Software Inc.,
Version 5, San Diego, CA) software was used for statistical analysis and
a p value < 0.05 was considered statistically significant.
2
.2.12. In vitro antibacterial activity
A broth dilution method was used to determine the minimum in-
hibitory concentration (MIC) values for bare VCM and OA-C]N-NH-
PEG) -VCM micelles against S. aureus and MRSA at 18, 36 and 54 h at
3. Results and discussion
(
2
pH 7.4 and 6, respectively. Briefly, a densitometer (DEN-1B densit-
ometer, Shanghai, China) was used to adjust overnight grown cultures
of S. aureus and MRSA to 0.5 McFarland, and subsequently diluted
3
.1. Synthesis and characterization of newly synthesized compound (v) and
(vi)
(
1:150) using distilled water to obtain 1 × 106 CFU/ml. The serial
Compound (v) containing hydrazide functional groups was synthe-
dilutions of the test samples at pH 7.4 and 6 were performed by adding
35 µl of bare VCM solution and OA-C]N-NH-(PEG) -VCM micelles to
sized by refluxing hydrazine hydrate with an ethanolic solution of
compound (iv) containing ester functional groups. Then, OA-C]N-NH-
1
2
each well of a 96-well plate containing 135 µl of MHB of pH 7.4 and 6,
respectively. To the serially diluted samples, bacterial cultures (15 µl)
were added and further allowed to incubate in a shaking incubator at
(
2
PEG) (compound-vi) polymer containing hydrazone linkage was
synthesized by conjugating m-PEG-CHO to the free hydrazide func-
tional groups of the compound (v).
37 °C, for 18 h at 100 rpm. The MIC values at pH 7.4 and 6 were de-
termined by spotting the respective dilutions (5 µl) on MHA plates and
allowing to incubate for 18, 36 and 54 h. Blank micelles were used as
the control. All the experiments were performed in three replicates.
3
.1.1. Compound (v)
Light yellow semi-solid, FT-IR: 3317.52, 3288.93, 2956.70,
2
1
1
4
871.33,
161.86 cm
1628.60,
1533.97,
1471.77,
) δ (ppm): 0.81 (t; 3H; eCH
CH CONe), 1.94 (m;
CONe), 2.41 (t; 4H; eN
CH CONHe) ), 3.97 (bs;
), 5.26 (m; 2H; eCH]CHe). C NMR (CDCl ) δ (ppm):
1378.26,
1260.62,
−
1
1
.
H NMR (CDCl
3
3
),
2.2.13. In vivo antibacterial activity
.19–1.23 (m; 20H; eCH
H; eCH CH]CHCH e), 2.29 (t; 2H; eCH
CH CONHe) ), 3.45–3.57 (t; 4H; eN(CH
H; eNHNH
2
e), 1.49 (q; 2H; eCH
2
2
The in vivo antibacterial activity of plain VCM and OA-C]N-NH-
2
2
2
(
2
PEG) -VCM micelles was performed against MRSA using BALB/c mice.
(
CH
2
2
2
2
3
2
2
The protocol (Protocol No. AREC/104/015PD) followed for this study
was approved by Animal Research Ethics Committee (AREC) of the
University of KwaZulu-Natal (UKZN). The guidelines developed by the
AREC of UKZN and South African National Standard (SANS
1
4
1
4
2
3
4.10, 22.67, 25.44, 27.24, 29.28–29.77, 31.90, 31.92, 33.22, 33.84,
3.35, 45.20, 129.69, 130.02, 170.85, 172.14, 174.52. ESI-TOF MS m/
+
+
z: [M+Na]
4
47 5 3
– calculated 476.3577 (C24H N O +Na ), found
10386:2008) were followed for the human care of the animals used in
76.3581.
this study. A day prior to the experiments, male BALB/c mice (n = 4 for
each group) were shaved to remove the back hairs, and the shaved skin
was disinfected with ethanol. The following day, 50 µl of 0.5 McFarland
of MRSA suspension in sterile saline was injected intradermally at the
shaved area at the back of all the animals. After 20 min, plain VCM and
3.1.2. Compound (vi)
Light yellowish-white solid, melting point = 64 °C, FT-IR: 2881.23,
739.36, 1683.11, 1642.22, 1466.24, 1358.74, 1340.52, 1279.25,
2
1
1
4
−
1 1
145.79, 1101.86 cm . H NMR (CDCl
.19–1.23 (m; 20H; eCH e), 1.52 (q; 2H; eCH
H; eCH CH]CHCH e), 2.29 (t; 2H; eCH eCONe), 2.41 (t; 4H; -N
), 3.30 (s; 3H; eCH ), 3.45–3.47 (t; 4H; eN
), 3.57–3.69 [m; CH of PEG (eOCH CH Oe
e), 4.61 (t; 4H; eN]CHCH e), 5.26
m; 2H; eCH]CHe), 8.02 (t; 1H; eN]CHCH
e). 13C NMR (CDCl3) δ
ppm): 13.81, 14.16, 20.94, 22.66, 27.22, 29.28 – 29.76, 31.88, 58.99,
3
) δ (ppm): 0.81 (t; 3H; eCH
3
),
2
OA-C]N-NH-(PEG) -VCM micelles (50 µl; equivalent to 25 µg/ml of
2
2
CH CONe), 1.93 (m;
2
VCM) were injected at the same place as the MRSA infection. The mice
were observed over 48 h for lesion development. Thereafter, the mice
were then euthanized, and the infected skin was surgically removed and
homogenized in sterile PBS (8 ml). The achieved suspensions were di-
2
2
2
(
(
CH
CH
2
CH
CH
2
CONHe)
CONHe)
2
2
3
2
2
2
2
2
1
4
chain)], 4.14 (t; 4H; -N]CHCH
(
(
2
2
luted 10 to 10 , and 20 µl of each diluted sample was spotted on a
nutrient agar plate and incubated overnight at 37 °C, to quantify the
number of colony forming units (CFU) of MRSA in the skin tissues. The
following equation was used to calculate the CFU/ml:
2
6
1
1.61, 62.99, 63.41, 63.58, 66.29, 67.08–70.60, 71.97, 72.68, 74.92,
29.84, 160.99, 164.66, 170.87. ESI-TOF MS m/z: [M-10413.6700], [M
+
6/6] - calculated 1736.6116, found 1736.8472.
.2. Critical micelle concentration (CMC)
The CMC of OA-C]N-NH-(PEG) was determined at 25 °C, using
CFU/ml
=
(number of colonies × dilution factor)/volume of culture plate
3
Morphological evaluations were made on freshly harvested samples
from the injection site. After harvesting, the skin was moved from saline
to 10% buffered formalin. After 7 days, skin samples were embedded in
paraffin wax following dehydration in an ethanol gradient. A micro-
tome (Leica RM2235, Leica Biosystems, Germany) was used to section
the tissue wax blocks. Sections were collected and dried on slides and
2
dynamic light scattering method to determine micelle formation cap-
ability and stability of micelles for site specific delivery of the en-
capsulated drug. To obtain the CMC value of OA-C]N-NH-(PEG)
polymer, the intensity values of scattered light obtained from each
2
4

S.J. Sonawane, et al.
International Journal of Pharmaceutics 575 (2020) 118948
Fig. 1. Synthesis of AB
2
type amphiphilic block copolymer [OA-C]N-NH-(PEG)
2
] containing hydrazone linkage.
(
PEG)
2
would remain stable even upon severe dilution in the blood-
stream following intravenous injection. This further indicates the suit-
ability of an amphiphilic polymer [OA-C]N-NH-(PEG)
carrier for in vivo applications.
2
] as a drug
3.3. pH dependent breakdown
Hydrolytic breakdown (degradation) of polymers is considered as
an important step in achieving controlled and site specific delivery of
drugs to the target sites (Liechty et al., 2010). Therefore pH dependent
degradation behaviour of the newly synthesized polymeric assemblies
containing hydrazone linkages at various physiological pH is essential.
2
The degradation of OA-C]N-NH-(PEG) polymer was performed in PBS
at pH 6 and 7.4, respectively at 37 °C for 24 h. Initially, a thin layer
chromatography (TLC) method was tried for monitoring progress of the
degradation process, but similar retention factor (RF) values of de-
Fig. 2. Determination of the critical micelle concentration of OA-C]N-NH-
PEG) polymer using dynamic light scattering measurements.
(
2
gradation product (compound-v) and OA-C]N-NH-(PEG)
2
polymer
(
compound-vi) complicated the interpretation of results. Therefore,
solution of different concentration of OA-C]N-NH-(PEG)
were plotted against different concentrations of OA-C]N-NH-(PEG)
polymer. This plot of intensity versus concentration yielded two
straight lines intersecting at data points corresponding to 6 µg/ml. This
2
value indicates the CMC value of OA-C]N-NH-(PEG) polymer (Fig. 2),
which was found to be much lower compared to previously reported
CMC value (0.87 mg/L) of tri-block copolymer (PEG-DiHyd-PLA-18K)
containing hydrazone linkages (Qi et al., 2018).
2
polymer
aliquots of polymeric solution in PBS (pH 6 and 7.4) collected after a
period of 24 h of incubation were analyzed using LCMS method. LCMS
spectra of OA-C]N-NH-(PEG) sample incubated in PBS (pH 6) in-
2
dicated the presence of fragment peak at 455 (M + 1), corresponding to
the molecular weight of compound-v (Mol. Wt. = 454) (Fig. 3B). On
2
the other hand, LCMS spectra of OA-C]N-NH-(PEG) sample incubated
in PBS (pH 7.4) did not show any fragment peak at 455 (Fig. 3A).
The polymer sample at acidic pH showed the presence of mass peak
at 455, which indicates the hydrolytic cleavable ability of hydrazone
2
The scattered intensity values obtained for OA-C]N-NH-(PEG)
2
concentrations below CMC were almost constant, corresponding to that
of deionized water, as reported previously (Topel et al., 2013).
Whereas, there was linear increase in the intensity with concentration
at the CMC because of increase in number of micelles in the solution.
The CMC values are important to decide the stability of micelles for in
vivo applications. A low CMC provides stability to polymeric micelles by
allowing great resistance to dilution (Sahoo and Labhasetwar, 2003;
Song et al., 2014). Therefore, the low CMC (6 µg/ml) obtained in this
study indicates that the micelle structure formed by OA-C]N-NH-
linkage of OA-C]N-NH-(PEG)
the absence of mass peak at 455 of polymer sample incubated in PBS
pH 7.4), indicates stability of hydrazone linkage at neutral physiolo-
gical pH. These results therefore indicate the suitability of OA-C]N-
NH-(PEG) polymer for pH-responsive delivery of VCM to the bacterial
infection sites.
2
polymer at acidic conditions, whereas
(
2
5

S.J. Sonawane, et al.
International Journal of Pharmaceutics 575 (2020) 118948
2
Fig. 3. LCMS spectra of OA-C]N-NH-(PEG) polymer samples incubated in PBS of pH 7.4 (A) and 6 (B).
3.4. In vitro cytotoxicity study
than 75% cell viability and showed acceptable toxicity against all cell
lines tested, thereby suggesting that synthesized polymer OA-C]N-NH-
The viability of cells upon their exposure to newly synthesized/
2
(PEG) was safe for biological application.
formulated nanomaterials is essential to establish dosages that are safe
and suitable for bio-applications (Sonawane et al., 2015). Biological
efficacy of OA-C]N-NH-(PEG) polymer was assessed using an in vitro
2
cell culture system. Cytotoxicity studies were performed against A549,
MCF7 and HepG2 cells using the MTT assay. The basic principle in-
volved in the MTT assay is the biochemical reduction of MTT dye by
viable cells via enzymatic dehydrogenase activity (Rambharose et al.,
3
.5. Preparation and characterization of OA-C]N-NH-(PEG)
2
-VCM
micelles
After the confirmation of micelles forming ability, pH-responsive
degradation and non-toxicity of OA-C]N-NH-(PEG) polymer, the
study then proceeded to the preparation of OA-C]N-NH-(PEG) -VCM
micelles. The VCM loaded micelles were formulated using emulsifica-
tion technique, in which the polymer dissolved in a water immiscible
organic solvent (DCM) was added into an aqueous solution (PBS, pH
2
2
2015). The cell viability within the concentration range studied
(
20–100 µg/ml) was between 79.17 ± 12.67–78.81 ± 6.65% for A
549 cells, 86.71
±
11.16–89.25
±
6.99% for MCF 7 cells and
76.10 ± 9.00–76.27 ± 6.304% for Hep G2 cells (Fig. 4).
7
.4) containing VCM under vigorous stirring. The PBS solution (pH 7.4)
These results exhibited a dose independent effect of OA-C]N-NH-
was selected as an aqueous solvent for micelles preparation in order to
provide long term storage stability to the pH-sensitive micelles (Fang
et al., 2016).
(
PEG)
2
on cell viability, across all cell lines. The MTT assay results re-
showed more
vealed that the synthesized polymer OA-C]N-NH-(PEG)
2
2
Fig. 4. Cytotoxicity assay presenting percentage cell viability after exposure of cells (A549, MCF7 and HepG2) to OA-C]N-NH-(PEG) polymer (n = 6).
6

S.J. Sonawane, et al.
International Journal of Pharmaceutics 575 (2020) 118948
After overnight stirring for complete removal of DCM, micelles with
particle size, PI and ZP of 130.33 ± 7.36 nm, 0.163 ± 0.009 and
drug loading efficiency of polymeric micelles is decided by miscibility
between polymers and drugs (Xiong et al., 2011). Practically, the
loading efficiency of hydrophobic drugs in polymeric micelles is mainly
affected by the length of the hydrophobic block and the type of sub-
stituent present on it (Jette et al., 2004; Shuai et al., 2004). For ex-
ample, modification in the core of poly(ethylene oxide)-poly(L-aspartic
acid) (PEO-P (Asp)) micelles by conjugating fatty acid side chains on p
(Asp), resulted in 13 times higher amphotericin B encapsulation as
compared to the PEO-P(Asp) micelles containing benzyl core structures
(Lavasanifar et al., 2002). Therefore, it is postulated that for OA-C]N-
−
4.33 ± 0.55 mV respectively were obtained. The particle size ob-
tained for the OA-C]N-NH-(PEG) -VCM micelles was well below
00 nm, which is considered as ideal in order to achieve prolonged
blood circulation by avoiding their scavenging by the liver and spleen
Aliabadi and Lavasanifar, 2006; Qiu et al., 2007). The size of micelles
2
2
(
obtained in this study was in line with recently reported work, where
micelles prepared using triblock copolymer containing hydrazone lin-
kages exhibited particle size ranging from 70 to 130 nm (Qi et al.,
2018). The PI of 0.163 ± 0.009 was an indication that the formulated
NH-(PEG)
2
-VCM micelles, the high % EE (39.61 ± 4.01%) could be
micelles were monodisperse. The ZP plays an important role in the
stability of a colloidal system. The ZP of −4.33 ± 0.55 mV indicated
that micelles had a negative charge on their surfaces, which was in line
with previously reported micelles formulations (Song et al., 2014; Su
et al., 2016). This low ZP was due to the presence of the neutral charge
due to use of oleic acid (fatty acid) chain as a hydrophobic component
in the development of amphiphilic OA-C]N-NH-(PEG) polymer con-
2
taining hydrazone linkage.
3.8. Dilution stability of the OA-C]N-NH-(PEG) -VCM micelles
2
on the structure of OA-C]N-NH-(PEG)
charged structure of OA-C]N-NH-(PEG)
electrostatic repulsion, the presence of PEG chains in the micellar
structure could make them sterically stable. Therefore, these para-
2
polymer. Although this un-
yielded micelles with no
2
Dilution stability of polymeric micelles loaded with drugs using
physical methods is an important property for in vivo applications. This
is because upon intravenous injection they are severely diluted in the
bloodstream and become thermodynamically unstable, which can lead
to the disaggregation of micelles and burst-release of entrapped drugs
before reaching the site of action (Su et al., 2016). It has been reported
in the literature that premature release of drug from polymeric micelles
could significantly decrease the drug concentration at tumour sites
(Deng et al., 2012; Su et al., 2016).
2
meters in general suggest that the OA-C]N-NH-(PEG) -VCM micelles
formulated using an emulsification technique could be a superior al-
ternative for pH- responsive delivery of VCM to the bacterial infection
sites.
3.6. Surface morphology
2
Therefore, to illustrate the dilution stability of OA-C]N-NH-(PEG) -
The morphological studies using TEM showed that micelles were
distinct and spherical in shape without any signs of aggregation. The
size of spherical micelles obtained using TEM was in the range of
VCM micelles, samples were diluted with PBS (pH 7.4) from 1.25 to
320-fold and changes in the particle size was measured using Zetasizer
Nano ZS. It is interesting that there was no significant change observed
1
31–150 nm, which correlated well with the micelles size obtained
in the sizes of OA-C]N-NH-(PEG)
tion (Fig. 6). The reason for this is that the concentration of OA-C]N-
NH-(PEG) polymer was 31.25 µg/ml when diluted to 320-fold, which
was still above the determined CMC of the OA-C]N-NH-(PEG)
polymer. Therefore, these results indicate the suitability of the OA-C]
N-NH-(PEG) -VCM micelles for in vivo applications due to structural
integrity at high dilution values.
2
-VCM micelles up to 320-fold dilu-
using a zetasizer (Fig. 5A). Fig. 5B is an image captured using a blank
grid to prove that the spherical shape white spots appeared in Fig. 5A
were part of the grid background only and not a part of micelles. The
TEM image of spherical shaped micelles therefore confirms the ability
2
2
of OA-C]N-NH-(PEG)
2
polymer to assemble into a nanoformulation.
2
3.7. Entrapment efficiency (% EE) and drug loading capacity (LC)
3.9. In vitro drug release
The % EE and LC of VCM in the OA-C]N-NH-(PEG)
were found to be 39.61 ± 4.01% and 3.6 ± 0.36 (% w/w), respec-
tively. % EE obtained using OA-C]N-NH-(PEG) was found to be much
higher, compared to previously reported amphiphilic macromolecular
carriers containing hydrazone linkages, where micelles prepared using
pH-responsive macromolecular carriers AM-1 and AM-2 showed % EE
of 24.3 ± 0.8 and 15.8 ± 1.1, respectively for doxorubicin (Gu et al.,
2
-VCM micelles
2
In vitro release profiles of bare VCM and OA-C]N-NH-(PEG) -VCM
2
micelles at pH 7.4 and 6 are depicted in Fig. 7. The % drug release from
bare VCM and OA-C]N-NH-(PEG) -VCM micelles at pH 7.4 for the
2
initial 30 min period was 10.46 ± 2.51 and 2.13 ± 0.57, respec-
tively, whereas at pH 6, it was 11.10 ± 1.07 and 4.27 ± 1.61, re-
spectively. Afterwards, 100% release was observed for bare VCM after
2
017). Micellar systems have been shown to be beneficial in enhancing
8 h at pH 7.4 and 6, respectively, whereas 51.85
57.99 ± 3.40% of VCM was released from OA-C]N-NH-(PEG)
±
1.54 and
-VCM
the solubility and entrapment efficiency of hydrophobic drugs. The
2
Fig. 5. Morphology of OA-C]N-NH-(PEG)
2
-VCM micelles by TEM analysis (A) and TEM image showing blank grid background (B).
7

S.J. Sonawane, et al.
International Journal of Pharmaceutics 575 (2020) 118948
0
0
.97 µg/ml, respectively, whereas, at pH 6 it was 0.97 µg/ml and
.97 µg/ml respectively, which was similar to bare VCM against both
the bacterial strains. This similar antibacterial activity of OA-C]N-NH-
PEG) -VCM micelles to bare VCM can be a result of quick availability
of unentrapped VCM from OA-C]N-NH-(PEG) -VCM micelles for-
mulation to interact with bacteria after 18 h period of time (Fig. 7).
After 36 h, the MIC of OA-C]N-NH-(PEG) -VCM micelles decreased by
-folds against S. aureus at pH 7.4 and 6 respectively (MIC = 1.95 µg/
(
2
2
2
2
ml), whereas it retained the same MIC against MRSA at both the pH
values (MIC = 0.97 µg/ml) (Table 1). It is interesting to note that after
2
54 h, OA-C]N-NH-(PEG) -VCM micelles showed a 2-fold increase in
antibacterial activity against S. aureus and MRSA at pH 6 compared to
pH 7.4 (Table 1). This enhanced antibacterial activity of VCM at pH 6
can be a result of hydrolytic breakdown of the hydrazone linkage of OA-
2
C]N-NH-(PEG) polymer in acidic conditions, which led to sustained
Fig. 6. Dilution stability study: size changes of OA-C]N-NH-(PEG)2-VCM mi-
celles against different dilutions in PBS (pH 7.4) (n = 3).
release of entrapped VCM from micelles. The lower antibacterial ac-
tivity of VCM at pH 7.4 suggests the stability of hydrazone linkage at
neutral physiological pH to hold the entrapped drug inside the micelle
core. Therefore, the results of this study confirmed the potential of OA-
C]N-NH-(PEG)
system to enhance the performance of VCM against susceptible and
resistant bacterial strains, indicating the OA-C]N-NH-(PEG) -VCM
2
-VCM micelles as a pH-responsive drug delivery
2
micelles could be used as an alternative for pH-responsive delivery of
antibiotics.
3.11. In vivo antibacterial activity
2
An in vivo antibacterial activity of OA-C]N-NH-(PEG) -VCM mi-
celles was established using a mouse skin infection model, where
samples were injected intradermally into the dermis layer of the skin.
Delivery of MRSA into the area between the epidermal and the sub-
cutaneous layer was achieved by intradermal injections into the shaved
skin of mice. The calculated colony-forming units (CFUs) values of each
treatment group were expressed as log10 (Fig. 8). It was interesting to
note that, there was a statistically significant (P = 0.0388) depletion in
bacterial concentration of the skin samples treated with OA-C]N-NH-
2
Fig. 7. In vitro release profiles of bare VCM and OA-C]N-NH-(PEG) -VCM
micelles (n = 3).
(
2
PEG) -VCM micelles, compared to the untreated control. In the OA-C]
micelles at pH 7.4 and 6 after 8 h, respectively. Furthermore, the re-
lease of VCM from micelles was 85.64 ± 1.64% at pH 7.4, whereas
N-NH-(PEG) -VCM micelles treated samples, the log10 CFU/ml value
obtained was 3.13 ± 0.20, which was found to be a 1.62-fold lower
2
100% VCM was released at pH 6 after 48 h incubation, respectively. As
(P = 0.0388), compared to the untreated control. The log10 CFU/ml
value obtained for the untreated control was 5.09 ± 0.15, which was
1.06-fold higher compared to the bare VCM treated samples
(4.76 ± 0.12). These values therefore indicate that there was no sta-
tistically significant depletion (P = 0.0566) in the bacterial load of the
skin sample treated with bare VCM after 48 h, compared to the un-
treated control. However, interestingly, there was a 1.52-fold reduction
expected, the in vitro drug release profile of the OA-C]N-NH-(PEG)
2
-
VCM micelles showed faster drug release at pH 6 (corresponding to
bacterial infection site), compared to pH 7.4 (neutral physiological pH).
As it is well known that the lower pH of the medium enhances the
hydrolysis rate of hydrazone linkages (Li et al., 2015), this faster drug
release was because of the hydrolytic breakdown of hydrazone linkage
of OA-C]N-NH-(PEG)
2
polymer at acidic conditions which resulted in
in MRSA load after 48 h of treatment with OA-C]N-NH-(PEG) -VCM
2
decreased micelles stability. Therefore, the results of the in vitro release
study confirmed stability of the formulated micelles at neutral physio-
logical pH (pH 7.4) and their potential to release the VCM at bacterial
micelles, compared to bare VCM (P = 0.0260).
The MRSA was introduced to the intradermal layer of the skin via
injections with the bacteria being contained between the epidermis and
subcutaneous regions. After 48 h, the presence of fluid filled abscesses
were noted in the control group (Untreated). Following tissue har-
vesting, histomorphological analysis was performed to assess the skin
integrity after the MRSA intradermal infection. Tissue sections from the
untreated group showed signs of inflammation evidenced by excessive
swelling of the dermal layer (Fig. 9A). The images also confirmed the
formation of an abscess at the infection site, as evidenced by the abscess
wall/capsule in Fig. 9A. The large area of the abscess in Fig. 9A is in-
dicative of the extent of tissue infiltration and destruction by the 48 h
MRSA infection. Due to the foreign nature of the bacteria, their in-
troduction would have stimulated an immune response that triggered
tissue inflammation and abscess formation at the infection site in an
attempt to contain and destroy the invading microbes. The extent of
this response is directly relative to the bacterial load in the tissue.
infection site (pH 6), indicating the OA-C]N-NH-(PEG)
2
-VCM micelles
could be a good approach for pH-responsive delivery of antibiotics.
3.10. In vitro antibacterial activity
The MIC value obtained for free VCM against S. aureus and MRSA at
pH 7.4 was 0.97 µg/ml and 0.97 µg/ml, respectively, whereas, at pH 6
it was 1.95 µg/ml and 0.97 µg/ml, respectively (Table 1). Blank mi-
celles were not active against either of the bacterial strains at both pH
during the entire study period, whereas VCM loaded micelles exhibited
activity up to 54 h against both the bacteria. After 36 h of incubation,
only OA-C]N-NH-(PEG)
activity, which can be attributed to the controlled release of VCM from
the OA-C]N-NH-(PEG) -VCM micelles over 48 h (Fig. 7). At the initial
time period of 18 h, the MIC value for OA-C]N-NH-(PEG) -VCM mi-
celles against S. aureus and MRSA at pH 7.4 was 0.97 µg/ml and
2
-VCM micelles showed sustained antibacterial
2
2
Conversely, the OA-C]N-NH-(PEG)
2
-VCM samples showed no definite
signs of abscess formation although there was minimal inflammation
8

S.J. Sonawane, et al.
International Journal of Pharmaceutics 575 (2020) 118948
Table 1
2
Sustained in vitro antibacterial activity results for VCM, blank micelles and VCM loaded micelles [OA-C]N-NH-(PEG) -VCM] (n = 3).
Formulation
Bacteria
pH
MIC (µg/ml)
S. aureus
7.4
MRSA
7.4
6
6
Time (h)
18
36
54
18
36
54
18
36
54
18
36
54
Vancomycin
Blank micelles [OA-C]N-NH-(PEG)
0.97
NA
NA
NA
NA
NA
1.95
NA
NA
NA
NA
NA
0.97
NA
NA
NA
NA
NA
0.97
NA
NA
NA
NA
NA
2
]
Vancomycin loaded micelles [OA-C]N-NH-(PEG)
2
-VCM]
0.97
1.95
3.90
0.97
1.95
1.95
0.97
0.97
1.95
0.97
0.97
0.97
NA = No activity
evident in the dermal region (Fig. 9B). Interestingly, these histological
assessments are in agreement with what was observed in the in-vivo
antibacterial study. The OA-C]N-NH-(PEG) -VCM samples only
2
showed minimal signs of inflammation as it had the lowest isolated
bacterial load (Fig. 8). However, the untreated control group displayed
more evidence of inflammation and abscess formation as it had a sta-
tistically significant larger quantity of isolated bacteria (Fig. 8). These
results provide further evidence of OA-C]N-NH-(PEG)
2
-VCM micelles
effectiveness as a novel nanoantibiotic for treating MRSA infections.
3.12. Stability studies
The stability studies were performed to evaluate the potential of the
prepared micelles to withstand different storage conditions specified by
regulatory authorities. The physical appearance, particle size, PI and ZP
of the micelles stored at 4 °C and RT were evaluated to assess their
storage stability. There was no change in the physical appearance in-
cluding color change and aggregation of micelles throughout the sto-
rage period at 4 °C and RT for three months. The statistical analysis
performed using one-way analysis of variance (ANOVA), followed by
non-parametric Kruskal-Wallis and paired t-test showed no significant
changes (p > 0.05) in the particle size, PI and ZP values of micelles
(
Table 2) stored at 4 °C. Statistical analyses performed on data obtained
at RT indicated no significant changes (p > 0.05) in the particle size
and ZP values, however there was a statistically significant change
observed (p = 0.0415) for PI values of micelles. These results therefore
confirm the storage stability of VCM loaded micelles at both 4 °C and
RT.
Fig. 8. MRSA load after 48 h of treatment. Data represents mean
±
SD
(n = 3). # denotes significant difference when compared to untreated (control)
2
and OA-C]N-NH-(PEG) -VCM micelles. @ denotes when compared to un-
4. Conclusion
treated (control) and bare VCM and * denotes significant difference between
bare VCM and OA-C]N-NH-(PEG) -VCM micelles.
2
2
The novel amphiphilic OA-C]N-NH-(PEG) polymer containing the
Fig. 9. Photomicrographs of the control and the treated skin selections for light microscopy (LM) stained with H&E; (X40) (A) Control (MRSA injected, untreated) (B)
Treated [OA-C]N-NH-(PEG) -VCM].
2
9

S.J. Sonawane, et al.
International Journal of Pharmaceutics 575 (2020) 118948
Table 2
Effect of storage condition and time on particle size, PI and ZP of VCM loaded micelles [OA-C]N-NH-(PEG)
2
-VCM] (n = 3).
Storage condition
Particle size (nm)
4 °C
PI
ZP
Time (days)
0
RT
4 °C
RT
4 °C
RT
130.3 ± 7.36
135.6 ± 7.00
137.1 ± 6.57
137.2 ± 6.74
130.3 ± 7.36
138.1 ± 8.51
144.5 ± 3.04
142.2 ± 3.55
0.163 ± 0.009
0.172 ± 0.029
0.169 ± 0.035
0.179 ± 0.012
0.163 ± 0.009
0.207 ± 0.011
0.239 ± 0.030
0.255 ± 0.048
−4.16 ± 0.32
−4.13 ± 0.53
−4.53 ± 0.07
−4.27 ± 0.27
−4.16 ± 0.32
−5.94 ± 2.38
−6.03 ± 0.60
−5.05 ± 0.35
30
60
90
Particle size, PI and ZP expressed as mean ± SD.
hydrazone linkage was synthesized and characterized successfully. The
micelles prepared using OA-C]N-NH-(PEG) polymer used to en-
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2
capsulate VCM, showed pH dependent sustained drug release properties
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S. aureus and MRSA at acidic pH, compared to neutral physiological pH.
An anti-dilution assay performed on drug loaded micelles confirmed
their circulation stability for in vivo antibacterial experiments. The re-
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superior in treating resistant S. aureus infections as compared to bare
VCM. In a field of strategic solutions to an antibiotic resistance, this
biocompatible, noncomplex nano system engineered using a novel pH-
responsive amphiphilic polymer would be a significant addition for the
site specific delivery of VCM to treat resistant bacterial infections. In
addition to antibiotic delivery, this pH-responsive micellar delivery
system could be adopted for encapsulation of other classes of drugs for
effective management of diseases such as cancer that are characterized
by lower pH conditions.
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CRediT authorship contribution statement
Gupta, P., Vermani, K., Garg, S., 2002. Hydrogels: from controlled release to pH-re-
sponsive drug delivery. Drug Discovery Today 7, 569–579.
Sandeep J. Sonawane: Methodology, Formal analysis,
Jadhav, M., Kalhapure, R.S., Rambharose, S., Mocktar, C., Singh, S., Kodama, T.,
Govender, T., 2018. Novel lipids with three C18-fatty acid chains and an amino acid
head group for pH-responsive and sustained antibiotic delivery. Chem. Phys. Lipids
212, 12–25.
Investigation, Data curation, Writing
- original draft. Rahul S.
Kalhapure: Conceptualization, Validation, Writing - review & editing,
Funding acquisition. Mahantesh Jadhav: Project administration,
Validation. Sanjeev Rambharose: Methodology, Investigation.
Chunderika Mocktar: Validation, Resources. Thirumala Govender:
Supervision, Writing - review & editing, Funding acquisition.
Jette, K.K., Law, D., Schmitt, E.A., Kwon, G.S., 2004. Preparation and drug loading of poly
(ethylene glycol)-block-poly (ε-caprolactone) micelles through the evaporation of a
cosolvent azeotrope. Pharm. Res. 21, 1184–1191.
Kalhapure, R.S., Akamanchi, K.G., 2013. Oleodendrimers: A novel class of multicephalous
heterolipids as chemical penetration enhancers for transdermal drug delivery. Int. J.
Pharm. 454, 158–166.
Kalhapure, R.S., Jadhav, M., Rambharose, S., Mocktar, C., Singh, S., Renukuntla, J.,
Govender, T., 2017a. pH-responsive chitosan nanoparticles from a novel twin-chain
anionic amphiphile for controlled and targeted delivery of vancomycin. Colloids
Surf., B 158, 650–657.
Kalhapure, R.S., Mocktar, C., Sikwal, D.R., Sonawane, S.J., Kathiravan, M.K., Skelton, A.,
Govender, T., 2014. Ion pairing with linoleic acid simultaneously enhances en-
capsulation efficiency and antibacterial activity of vancomycin in solid lipid nano-
particles. Colloids Surf., B 117, 303–311.
Kalhapure, R.S., Renukuntla, J., 2018. Thermo-and pH dual responsive polymeric mi-
celles and nanoparticles. Chem.-Biol. Interact. 295, 20–37.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgement
Kalhapure, R.S., Sikwal, D.R., Rambharose, S., Mocktar, C., Singh, S., Bester, L., Oh, J.K.,
Renukuntla, J., Govender, T., 2017b. Enhancing targeted antibiotic therapy via pH
responsive solid lipid nanoparticles from an acid cleavable lipid. Nanomedicine 13,
2067–2077.
Kalhapure, R.S., Suleman, N., Mocktar, C., Seedat, N., Govender, T., 2015.
Nanoengineered drug delivery systems for enhancing antibiotic therapy. J. Pharm.
Sci. 104, 872–905.
Kim, J.K., Garripelli, V.K., Jeong, U.H., Park, J.S., Repka, M.A., Jo, S., 2010. Novel pH-
sensitive polyacetal-based block copolymers for controlled drug delivery. Int. J.
Pharm. 401, 79–86.
Klevens, R.M., Edwards, J.R., Tenover, F.C., McDonald, L.C., Horan, T., Gaynes, R.,
System, N.N.I.S., 2006. Changes in the epidemiology of methicillin-resistant
Staphylococcus aureus in intensive care units in US hospitals, 1992–2003. Clin.
Infect. Dis. 42, 389–391.
The authors acknowledge the University of KwaZulu-Natal (UKZN),
the National Research Foundation (NRF) of South Africa for financial
support (Grant# 87790 and 88453) and South African Medical
Research Council (MRC), The Biomedical Resource Unit (BRU) (UKZN)
and Microscopy and Microanalysis Unit (MMU) UKZN for technical
assistance, and Charlotte Ramadhin for proof reading.
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