EJPB 12357 No.

of Pages 13, Model 5G

24 November 2016

European Journal of Pharmaceutics and Biopharmaceutics xxx (2016) xxx–xxx

1

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics

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

2 Research paper
6
4 Pegylated oleic acid: A promising amphiphilic polymer
7
5 for nano-antibiotic delivery
8 Calvin A. Omolo a, Rahul S. Kalhapure a,⇑, Mahantesh Jadhav a, Sanjeev Rambharose a,
9 Chunderika Mocktar a, Valence M.K. Ndesendo b, Thirumala Govender a,⇑
10 a
Discipline of Pharmaceutical Sciences, School of Health Sciences, University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa
11 b
School of Pharmacy & Pharmaceutical Sciences, St. John’s University of Tanzania, P.O. Box 47, Dodoma, Tanzania

12
13
a r t i c l e i n f o a b s t r a c t
1
2 5
9
16 Article history: Vancomycin (VM), a last resort to control methicillin-resistant S. aureus (MRSA) infections, is on the verge 30
17 Received 7 October 2016 of becoming ineffective. Novel nano delivery systems of VM have the potential to combat MRSA. The 31
18 Revised 17 November 2016 search for novel materials for nanoantibiotic development is therefore an active research area. In this 32
19 Accepted in revised form 18 November 2016
study, oleic acid (OA) was coupled with monomethoxy polyethylene glycol (mPEG) to obtain a novel 33
20 Available online xxxx
bio-safe amphiphilic polymer, mPEG-OA. The critical micelle concentration of mPEG-OA, was found to 34
be 4.5
108 m/L. VM-loaded polymersomes were prepared from mPEG-OA and evaluated for size, poly- 35
21 Keywords:
dispersity index (PDI), zeta potential (ZP), surface morphology, drug release, in vitro and in vivo antibac- 36
22 Oleic acid
23 Polyethylene glycol
terial activity. The size, PDI and ZP of VM-loaded polymersomes were 142.9 ± 7.5 nm, 0.228 ± 0.03 and 37
24 Polymersomes 18.3 ± 3.55 mV respectively. Transmission electron microscopy images revealed the spherical shape 38
25 Vancomycin of polymersomes. The encapsulation efficiency was 53.64 ± 1.86%. The drug release from polymersomes 39
26 Antibiotic resistance was sustained and in vitro antibacterial activity was 42- and 5-fold more against S. aureus and MRSA, 40
27 Methicillin-resistant S. aureus compared with plain VM. An in vivo BALB/c mice, skin infection models revealed that treatment with 41
28
VM-loaded polymersomes significantly reduced the MRSA burden compared with plain VM and blank 42
polymersomes. There was a 183 and a 25-fold reduction in the MRSA colony finding units load in mice 43
skin treated with VM-loaded polymersomes compared to that treated with blank polymersomes and bare 44
VM respectively. In summary, the developed VM-loaded polymersomes from novel mPEG-OA polymer 45
were found to be a promising nanoantibiotic against MRSA. 46
Ó 2016 Elsevier B.V. All rights reserved. 47
48

49
50
51 1. Introduction MRSA has since become a global pandemic, with recent 65
research findings indicating incidences estimated to be 2.4% in Eur- 66
52 Misuse and overuse of antibiotics, as well as the limitations of ope, 4.8% in North America, 5.4% in South America, 2.5% in Asia and 67
53 conventional dosage forms, has resulted in high levels of resistance 3.1% in Africa [7] among patients undergoing microbial tests. There 68
54 to most of the first and second line antibiotics by common disease are dire consequences associated with MRSA disease burden due to 69
55 causing bacteria, such as Escherichia coli, Klebsiella pneumoniae and the additional costs for hospitals, individual, community and the 70
56 Staphylococcus aureus [1,2]. Among these bacteria, S. aureus, a government during treatment [8,9]. Vancomycin has been the 71
57 major human pathogen is the leading cause of hospital- most reliable antibiotic against infections caused by MRSA [10]. 72
58 associated infections [3]. Over time, S. aureus has experienced However, increased use of this glycopeptide to treat MRSA and 73
59 several waves of antibiotic resistance, which has included the other Gram-positive organisms has been associated with the emer- 74
60 entire b-lactam class of antibiotics, such as penicillins, cephalos- gence of insensitive strains of MRSA [11]. The limited number and 75
61 porins and carbapenems [4]. These b-lactam antibiotic resistant diversity of new antibiotic scaffolds is escalating this issue, with 76
62 strains of S. aureus are now referred to as methicillin-resistant S. the majority of new formulations having been reported until 77
63 aureus (MRSA) or superbugs, and were first identified in a London 2003 being from beta-lactam and fluoro-quinolones, and only a 78
64 hospital in 1961 [5,6]. handful having new mechanisms of action [12]. New ones will 79
eventually develop resistance if they are delivered with 80

⇑ Corresponding authors.
conventional dosage forms, as resistance is mainly the result of 81

E-mail addresses: rahul.kalhapure@rediffmail.com, kalhapure@ukzn.ac.za

bacterial mutations that are due to sub-optimal time of antibiotic 82

(R.S. Kalhapure), govenderth@ukzn.ac.za (T. Govender). exposure [13]. A recent review on the number of antibiotics in 83

http://dx.doi.org/10.1016/j.ejpb.2016.11.022
0939-6411/Ó 2016 Elsevier B.V. All rights reserved.

Please cite this article in press as: C.A. Omolo et al., Pegylated oleic acid: A promising amphiphilic polymer for nano-antibiotic delivery, Eur. J. Pharm. Bio-
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84 the development pipeline shows that in 2011 only 20 new scaf- (MHA) and Nutrient Broth were obtained from Biolab Inc., (South 147
85 folds were under clinical trials [14]. As very few antibiotics with Africa). Mueller–Hinton broth (MHB) was purchased from Oxoid 148
86 novel mechanism of action are likely to be available, new ones in Ltd. (England), and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetra 149
87 the existing classes are in danger of becoming ineffective due to zolium bromide (MTT) from Merck Chemicals (Germany). S. aureus 150
88 cross resistance [15,16]. (ATCC 25922) and S. aureus Rosenbach (ATCC BAA-1683TM) 151
89 In this regard, there is therefore a need to find new approaches (MRSA) strains were used. 152
90 for antimicrobial delivery for efficacy to be restored, to protect and
91 improve the activity of the currently used clinically effective 2.1. Synthesis and characterization of mPEG-OA (Scheme 1) 153
92 antibiotics. In the current scenario, nano-based approaches are
93 showing the potential to achieve these goals [17]. Nano structured The mPEG (10 g, 2.0 mmoL) was reacted with OA (16 g, 56.3 154
94 antibiotic systems have the potential to overcome resistance by mmoL) at 170 °C for five hours in a melted state. The reaction 155
95 improving the efficacy of the loaded antibiotic payloads with var- was performed under an inert nitrogen atmosphere to prevent oxi- 156
96 ious attributes. These include the possibility to control and manip- dation of OA during the esterification process (Scheme 1). Excess 157
97 ulate the structures at a molecular level. By doing so targeted OA was used to ensure complete esterification of free AOH in 158
98 delivery to infection sites and bacteria [18] is achieved, the serum mPEG. On completion of the reaction, the reaction mixture was 159
99 solubility of drugs is improved, prolonged systemic circulation life- washed with diethyl ether (3
100 mL) [29] to remove excess 160
100 time, release of drugs in a sustained and controlled manner, and OA. The isolated solid was dried in a vacuum desiccator for 24 h 161
101 preferentially deliver drugs to the tissues and cells of interest [19]. to obtain mPEG-OA conjugate off-white dry powder (9.23 g, 162
102 Nano systems, such as liposomes, solid lipid nano particles 87.36%). The conjugate was characterized using Fourier transform 163
103 (SLNs), micelles, polymeric nanoparticles, nano-emulsions, lipid- infra-red (FT-IR) spectroscopy and nuclear magnetic resonance 164
104 polymer hybrid nanoparticles [2] and recently lipid-dendrimer (NMR) imaging (1H and 13C). NMR spectra were recorded in deuter- 165
105 hybrid nanoparticles [20], have been widely used for antibiotic ated chloroform (CDCl3) on a Bruker Avance 400 MHz NMR (USA) 166
106 delivery. The ground breaking work done by Gibicki et al. in instrument, whereas a Bruker Alpha spectrophotometer (Ger- 167
107 1973 [21] led to understanding the formation of vesicles using nat- many) was used for FT-IR analysis. 168
108 ural fatty acid lipids and esters. Lipids have been used to formulate
109 drug delivery systems due to their attractive traits, which include 2.2. In vitro cytotoxicity 169
110 enhancing biological membrane penetration [22] and biocompati-
111 bility. Their inherent antimicrobial activity also makes them MTT assay was performed to assess the biological safety of the 170
112 attractive materials to formulate nanoantibiotics [23,24]. Oleic acid synthesized mPEG-OA using three cell lines i.e. human breast ade- 171
113 (OA), a free fatty acid from natural vegetable oils, is a widely used nocarcinoma (MCF 7), adenocarcinomic human alveolar basal 172
114 pharmaceutical excipient due to its non-toxicity, bio-compatibility, epithelial cells (A 549) and human liver hepatocellular carcinoma 173
115 bio-degradability, ready availability in the market and permeation (HepG 2). All cell lines were grown exponentially at 37 °C in a 174
116 enhancement efficacy [25], and displays some antibacterial activity humidified atmosphere of 5% CO2. The test material (mPEG-OA) 175
117 [26]. was dissolved in milli-Q water, and dilutions of concentrations of 176
118 Polyethylene glycol (PEG) has been widely used in pharmaceu- 20, 40, 60, 80 and 100 lg/mL were prepared [30]. Cell adherence 177
119 tical formulations to PEGylate nanoparticles, which results in a of the three cell lines was achieved by seeding the cell lines equiv- 178
120 reduction of the opsonization process of the nanoparticles by the alently (2.5
103) into 96-well plates and incubating them for 179
121 RES, thus increasing the circulation half-time, which translates to 24 h. Final treatment concentrations were attained by refilling 180
122 long circulation in the body [27]. It has been reported in the liter- the wells with fresh culture medium (100 lL per well) together 181
123 ature that conjugates of low molecular weight (MW 2000), mono- with the appropriate concentration of the test solutions. The posi- 182
124 methoxy polyethylene glycol (mPEG) and OA, form polymeric tive and negative control wells comprised of the culture medium 183
125 micelles and polymersomes [28]. The authors reported use of these only and culture medium without cells. After the 48 h incubation, 184
126 systems to efficiently delivering curcumin for anticancer therapy the culture medium and test materials were removed and replaced 185
127 [28]. No study to date has reported the use of mPEG and OA conju- with 100 lL of fresh culture medium and 100 lL of MTT solution 186
128 gate for antibiotic delivery, or its potential to enhance the activity (5 mg/mL in PBS) in each well. After four hours of incubation, the 187
129 of antibiotics. The present paper reports on the application of media and MTT solution were removed and solubilization of MTT 188
130 mPEG-OA for bacterial infections, via the synthesis of OA conju- formazan was achieved by adding 100 lL of dimethyl sulfoxide. 189
131 gated high molecular weight mPEG (MW 5000), in order to obtain The optical density of each well was measured on a microplate 190
132 a performance efficient biocompatible polymer for sustained spectrophotometer (spectrostar nano, Germany) at a wavelength 191
133 antibiotic delivery. of 540 nm (A540: absorbance at a wavelength of 540 nm [30]. All 192
134 This paper is the first report on the application of mPEG-OA for the experiments were performed with six replicates. The percent- 193
135 bacterial infections via the synthesis of high molecular weight age cell viability was calculated as follows. 194
195
136 mPEG-OA conjugate, and its use to develop nanoantibiotic to man-
137 age infections by S. aureus and MRSA. The promising in vitro and
138 in vivo results obtained through vancomycin (VM) encapsulation
139 into mPEG-OA polymersomes are reported in this paper.

140 2. Materials and methods

141 Vancomycin hydrochloride (VM) was purchased from Sino-

142 bright Import and Export Co., Ltd. (China), oleic acid, mono-
143 methoxy polyethylene glycol (mPEG) (MW 5000) and
144 tetrahydrofuran (THF) were purchased from Sigma–Aldrich Co.
145 Ltd. (USA). An Elix water purification system (Millipore Corp.,
146 USA) was used to obtain milli-Q water. Mueller Hinton Agar Scheme 1. Synthesis of mPEG-OA polymer.

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253
   
A540 nm treated cells Weight of VM in nanoparticlesx
% Cell viability ¼
100% %LC ¼
100%
197 A540 nm untreated cells Total weight of nanoparticles 255

198 2.3. Determining Critical Micelle Concentration (CMC) 2.7. Differential scanning calorimetry (DSC) 256

199 The CMC of mPEG-OA was determined using the Zetasizer Nano The thermal profiles of the VM, mPEG-OA, physical mixture and 257
200 ZS90 (Malvern Instruments Ltd., UK) equipped with a 633 nm laser lyophilized polymersomes were determined by DSC (Shimadzu 258
201 and 90° detection optics. A series of solutions, ranging from DSC-60, Japan). Briefly, samples (2 mg) were placed in an alu- 259
202 1
103 to 1.0
109 mol/L, were prepared from an aqueous stock minum pan and sealed using a crimper. The pan was heated to 260
203 solution of mPEG-OA (0.5 mol/L). The scattering intensity mea- 300 °C at a constant rate of 10 °C/min under a constant nitrogen 261
204 surements were performed in a polystyrene cuvette at 25 °C flow of 20 mL/min using an empty pan as a reference [38]. 262
205 (n = 3). Changes in intensity (kcps) were plotted against the corre-
206 sponding concentrations to determine CMC value [31]. 2.8. In vitro drug release 263

207 2.4. Formulating VM loaded mPEG-OA polymersomes The in vitro VM release studies were performed following a pre- 264
viously reported procedure [39]. Polymersomes and their respec- 265
208 The polymersomes were prepared using an o/w emulsion sol- tive blanks (2 mL) were loaded in dialysis bags (pore size: 8000– 266
209 vent evaporation method [32,33]. Several mPEG-OA:VM ratios 14,400 Da) and dialyzed against PBS of pH 7.4 (40 mL) at 37 °C in 267
210 and different water solvent organic solvents were tried before an incubator at 100 rpm. Samples (3 mL) were drawn from the 268
211 arriving at an optimized ratio of 10:1. To prepare the optimized receiver solution at specific time points, and an equal amount of 269
212 polymersomes, a solution of mPEG-OA (100 mg) in THF (5 mL) fresh PBS was added to keep a constant volume. The VM quantity 270
213 was added drop-wise to the aqueous solution of VM (10 mg) in in the sample was measured spectrophotometrically at 280 nm 271
214 milli-Q water (20 mL) under stirring. The formed emulsion was using blank polymersomes as a reference. The regression equation 272
215 stirred for 24 h at room temperature to ensure the complete evap- was y = 0.0039x  0.0055, with a linear regression coefficient (R2) 273
216 oration of the THF. The blank polymersomes were prepared using of 0.999. Sink Conditions were maintained throughout the release 274
217 the same procedure by replacing the aqueous VM solution with study and the experiments were performed in triplicate. In vitro 275
218 plain milli-Q water. drug release modeling and analysis was performed with DDSolver 276
[40,41], using data up to 60% cumulative release. The models used 277
219 2.5. Size, Polydispersity Index (PI), Zeta Potential (ZP) and morphology were zero order, first order, Higuchi, Weibull, Hixson-Crowell and 278
Korsmeyer–Peppas. The parameters calculated were correlation 279
220 The size, PI and ZP of mPEG-OA polymersomes were analyzed coefficient (R2), root mean square error (RMSE) and mean dissolu- 280
221 by dynamic light scattering using a Zetasizer Nano ZS90 (Malvern tion time (MDT) [41,42]. These were determined to obtain a best fit 281
222 Instruments Ltd., UK) equipped with a laser beam at 633 nm and a model for explaining the release kinetics and mechanism of drug 282
223 90° scattering angle. In a typical procedure, mPEG-OA polymer- release. 283
224 somes were diluted with milli-Q water in such a way that the scat-
225 tering intensity was within the instrument’s sensitivity range and 2.9. In vitro antibacterial activity 284
226 then analyzed for physical properties (size, PI and ZP). All measure-
227 ments were performed in triplicate on three different batches pre- The broth dilution method was used to determine minimum 285
228 pared separately. The morphological investigations were inhibitory concentration (MIC) values of VM-loaded mPEG-OA 286
229 performed using Jeol, JEM-1010 (Japan) transmission electron polymersomes [43]. Bacterial cultures of S. aureus and MRSA were 287
230 microscopy (TEM). The polymersomes solutions were diluted grown overnight in Nutrient Broth at 37 °C in a shaking incubator 288
231 appropriately and negatively stained with 1% uranyl acetate (UA) and diluted to match the turbidity of 0.5 McFarland using a DEN- 289
232 solution. The negative stain provided an electron dense layer that 1B suspension McFarland densitometer (Latvia) [44]. A 2-fold 290
233 resulted in reverse-contrast, negative-electron images. Samples serial dilution of these bacterial cultures was performed in order 291
234 mixed with UA were deposited on copper grid, with excess sample to obtain a final concentration of 5
105 colony forming units 292
235 being removed on the grid by blotting off with filter paper, after (CFU)/mL (39). Dilutions of bare VM (positive control), and VM- 293
236 which the grid was air-dried before measurement [34,35]. The loaded mPEG-OA polymersomes, were prepared in MHB and incu- 294
237 images were captured at an accelerating voltage of 100 kV. bated with the bacterial cultures at 37 °C in a shaking incubator 295
(100 rpm). At specified time, 10 lL was spotted on Mueller- 296
238 2.6. Entrapment efficiency (%EE) and drug loading capacity (LC) Hinton (MHA) plates and incubated for 37 °C to determine the 297
MIC values. The studies were performed in triplicate, and a blank 298
239 The ultrafiltration method [36], a convenient method to sepa- formulation of mPEG-OA polymersomes was used as negative 299
240 rate a free drug from entrapped drug [37], was used to determine control. 300
241 the %EE. polymersomes (2 mL) were placed in AmiconÒ Ultra-4
242 centrifugal filter tubes (Millipore Corp., USA) of 10 kDa pore size
2.10. Percentage cell viability 301
243 and centrifuged at 3000 rpm at 25 °C for 30 min. The amount of
244 free vancomycin in the filtrate was detected UV spectrophotomet-
Cell viability studies on S. aureus and MRSA cells were per- 302
245 rically (Shimadzu UV 1601, Japan) at 280 nm using suitable blanks.
formed following a MTT assay method using 96-well microplates 303
246 The regression equation used to obtain the unknown concentra-
[20]. The bacterial cultures were tested for percentage cell viability 304
247 tions from the absorbance values was y = 0.0039x  0.0055, with
after 18 h treatment with VM and VM loaded mPEG-OA polymer- 305
248 a linear regression coefficient (R2) of 0.999. The % EE and LC were
somes. The test material (100 lL) was added to each well contain- 306
249 calculated using the following equations [36].
250 ing 5
105 CFU/mL bacterial suspension in MHB (100 lL) and 307
 
Weight of VM in nanoparticles incubated at 37 °C for 18 h. The concentration of VM was double 308
%EE ¼
100%
252 Weight of VM added the MIC values to ensure that the final concentration in each well 309
equaled the MIC after dilution with bacterial suspension. The con- 310

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311 trol experiment was performed by diluting the bacterial suspen- and the solvents used usually affects the process [47]. In this study, 369
312 sion (100 lL) with MHB (100 lL) only. After eighteen hours of mPEG-OA, an amphiphilic polymer, was synthesized by a single 370
313 incubation, the plates were observed for the presence or absence step green reaction of heat esterification without the use of any 371
314 of visible growth, after which 10 lL of MTT (5 mg/mL) dye solution catalyst or solvent. The reaction occurs in a molten state and with 372
315 in dimethyl sulfoxide (DMSO) was added to each well and incu- a simpler purification process [29], and the mechanism in the for- 373
316 bated further for four hours. The plates were centrifuged at mation of the mPEG-OA polymer consisting of an esterification 374
317 4500 rpm for 20 min, after which the supernatant was discarded reaction between the mPEG and OA at a dehydrating temperature 375
318 and 100 lL of DMSO was added to the residue in each well. The of approximately 170 °C. The process involves removing 1 mol of 376
319 optical density (OD) of these wells was measured at 540 nm on a water of esterification per mole of acid reacted to form an ester 377
320 spectrophotometer (spectrostar nano, Germany). Data were [48], with the structure of the mPEG-OA being confirmed by FT- 378
321 obtained from six replicates, and the percentage cell viability was IR and NMR. 379
322 calculated using the following equation [20]: M.p. 56.45 °C; FT-IR: 2881.45, 1733.27, 1341.51, 1103.23, 958.58, 380
323
  842.23 cm1. 0.82 (t; 3H; ACH3), 1.19–1.23 (m; 22H; ACH2A), 1.55 381
A540 nm treated cells
% Cell viability ¼
100% (q; 2H; ACH2CH2COOA), 1.94 (m; 4H; ACH2ACH@CHACH2A), 2.25 382
325 A540 nm untreated cells
(t; 2H; ACH2COOA), 3.31 (s; 3H; AOCH3), 3.38–3.49 (m; 224H; 383
AOCH2ACH2OA), 3.75 (t; 2H; ACH2COOCH2CH2A) 4.15 (t; 2H; 384
326 2.11. In vivo antibacterial activity
ACH2COOCCH2A), 5.26 (m; 2H; ACH@CHA). 13C NMR (CDCl3) d 385
(ppm): 14.10, 22.65, 24.79, 24.87, 27.15, 29.09, 29.29, 31.84, 59.00, 386
327 A mouse skin infection model was used for in vivo antibacterial
57.12, 66.89, 70.53, 71.90, 129.73, 173.81. 387
328 activity [45,46]. The study protocol was approved by University of
329 KwaZulu-Natal’s Animal Research Ethics Committee (Approval
3.2. Critical Micelle Concentration (CMC) determination 388
330 number: AREC/104/015PD). BALB/c mice weighing 18–20 g were
331 procured from the Biomedical Research Unit, University of
To determine the CMC value, the intensity values recorded by 389
332 KwaZulu-Natal. One day prior to the experiment, the back hair of
scattering light were plotted verses different concentrations of 390
333 the mice was removed and the intact exposed skin was disinfected
mPEG-OA in solution, as illustrated in the Fig. 1. The CMC value 391
334 using 70% ethanol. On the next day, 50 lL of MRSA (1.5
108 CFU/
was found to be 4.5
108 mol/L from the intersection point of 392
335 mL) suspended in saline was injected intradermally. The mice were
the two straight lines drawn. This CMC value was almost 13-fold 393
336 then divided into three groups, these being treatment, positive
less than previously reported conjugate of OA with low molecular 394
337 control and negative control groups (n = 4). After 30 min of infec-
weight mPEG [28] and far less than CMC of sodium oleate 395
338 tion, 50 lL of VM-loaded MPEG-OA polymersomes, plain VM, and
(3.5
103 mol/L) [49]. This could be attributed to a balance 396
339 blank mPEG-OA were injected at the same site of infection to the
between the hydrophilic and hydrophobic chain lengths provided 397
340 treatment, positive control and negative control groups
by the high molecular weight mPEG. There should be a balance 398
341 respectively.
between the hydrophobic and hydrophilic chain lengths for the 399
342 The mice were kept under observation for 48 h with a normal
shape and stability of micelles as any kind of imbalance beyond a 400
343 12 h light and dark condition at 19–23 °C, and 55 ± 10% relative
certain point leads to increased CMC [50,51] micelles of less uni- 401
344 humidity with adequate ventilation. The mice were euthanized
form shape and non-spherical aggregates [52]. The CMC results 402
345 and the infected skin was homogenized in PBS of pH 7.4 (5 mL).
suggest the superiority of the synthesized mPEG-OA, with the par- 403
346 Tissue homogenates were serially diluted in PBS (pH 7.4) after
ticle size distribution being unimodal at and above the CMC value 404
347 which 20 lL were spotted on nutrient agar plates, incubated at
(Fig. 2). However, it was noticed that there was a multimodal size 405
348 37 °C for 24 h, and the number of colonies forming units (CFU)
distribution at all the concentrations below the CMC value (Fig. 2). 406
349 counted. For histological investigation, both the control and trea-
These observations were evidence of a stable micellar formation 407
350 ted skin samples were fixed in formalin for 7 days at room temper-
at the and above the CMC, as well as a disassembly below it 408
351 ature. The samples were dehydrated using an ethanol gradient
[53,54]. This phenomenon is because at CMC, the hydrophobic core 409
352 ranging from 50% up to 96% and embedded in paraffin wax. The
is completely shielded from interaction with water, resulting in 410
353 skin sections were collected on slides, dried and stained with
increased free energy and decreased entropy as a stable structure 411
354 hematoxylin and eosin (H&E). The sections were examined using
forms, due to rearrangements in the system [55]. mPEG-OA 412
355 a light microscope (Nikon 80i, Japan), and bright field images were
showed an amphipathic nature with very low CMC, which makes 413
356 digitally captured using NIS Elements D software and a camera
this material a promising polymer for nanoantibiotics. 414
357 (Nikon U2, Japan).

3.3. Preparation of mPEG-OA polymersomes 415

358 2.12. Statistical analysis
If the molecular weight of the hydrophilic block exceeds that of 416
359 One-way analysis of variance (ANOVA), followed by Bonfer- the hydrophobic block, an amphiphilic block copolymer self- 417
360 roni’s Multiple Comparison Test, was used for statistical analysis. assembles in aqueous media into bi-layered spherical vesicles 418
361 Individual groups were compared against each other using known as polymersomes. The self-assembly is driven by the need 419
362 unpaired t-test, with P values of <0.05 being considered statistically to minimize the contact between the hydrophobic block and water 420
363 significant. Values are represented as mean ± SD. [33]. In the mPEG-OA, the molecular weight of the hydrophilic 421
block was 5000 Da, which was almost 18-fold higher than the 422
364 3. Results and discussion hydrophobic OA block. This large difference in molecular weight 423
would result in an amphiphilic polymer that can easily self- 424
365 3.1. Synthesis and characterization of mPEG-OA assemble in water to form polymersomes. A solvent evaporation 425
method was employed, where mPEG-OA was dissolved in THF 426
366 Esterification of fatty acids usually involves multistep processes and then added drop by drop to the aqueous VM solution while 427
367 and needs catalysts, which necessitates further purification [28]. being continuously stirred. As the THF evaporated, the mPEG-OA 428
368 The transesterification process will normally have many impurities spontaneously self-assembled to form amphiphilic bi-layered 429

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Fig. 1. Plot of concentration (mol/L) against intensity (kcps) for mPEG-OA. (n = 3).

Fig. 2. Particle size distribution of mPEG-OA micelles above CMC value: (A) 1
107 mol/L; (B) 3
105 mol/L and (C) 8
105 mol/L. PDI; 0.117, 0.091, 0.230, respectively.
Particle size distribution below CMC value: (D) 8
109 mol/L; (E) 1
109 mol/L; and (F) 6
109 mol/L. PDI; 0.479, 0.557, 0.675 respectively.

430 structures by encapsulating VM in the aqueous filled core Polymersomes, tank-like systems with a liquid central core 433
431 [33,56,57]. Scheme 2 represents the mechanism involved in the enclosed in a thin polymer wall, can exhibit versatile transport 434
432 formation of the vancomycin-loaded mPEG-OA polymersomes. properties as both hydrophobic and hydrophilic drugs can be 435

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6 C.A. Omolo et al. / European Journal of Pharmaceutics and Biopharmaceutics xxx (2016) xxx–xxx

Scheme 2. The proposed mechanism involved in the formation of vancomycin-loaded mPEG-OA polymersomes.

Table 1
Effect of different ratios of VM and mPEG-OA (n = 3).

Ratio (VM:mPEG-OA) Size (nm) PDI ZP (mV) Solvent

2:5 61.23 ± 3.05 0.504 ± 0.015 0.0203 ± 0.037 Acetone
1:10 186.7 ± 25.7 0.346 ± 3.7 25.5 ± 7.3 Methanol
1: 5 72.9 ± 13.13 0.493 ± 0.018 0.089 ± 1.3 Acetone
1:10 142.9 ± 7.5 0.228 ± 0.03 18.3 ± 3.55 THF
3:10 84.7 ± 2.9 0.482 ± 0.01 2.05 ± 0.03 THF

436 encapsulated in them [58,59]. Hydrophobic drugs can be encapsu- 3.4. In vitro cytotoxicity 465
437 lated into the membrane of the carrier whereas hydrophilic drugs
438 can be encapsulated into the aqueous filled cavity [56,60]. Being a One of the most important requirements for new biological 466
439 hydrophilic drug, VM (hydrochloride salt) could have been application materials is their non-toxicity to mammalian cells 467
440 enclosed in the aqueous cavity of mPEG-OA similar to doxorubicin [66]. This is mainly determined by assessing the viability of cells 468
441 hydrochloride polymersomes reported in the literature [56,57]. after exposure to the test material, which is then quantified using 469
442 Several ratios for VM: mPEG-OA (w/w) were investigated in order a cytotoxicity assay such as MTT, this being based on its reduction 470
443 to optimize their formulation in terms of size, PDI, ZP and VM load- by viable cells into a crystalline formazan. The quantity of for- 471
444 ing (Table 1). mazan crystals formed in the cells are usually proportional to the 472
445 The ratio of 1:10 VM: mPEG-OA showed better results with number of viable cells [67]. Therefore, an in vitro cell culture sys- 473
446 regards to lower size, PDI and high ZP value. The assembled VM tem using the MTT assay was utilized to determine the biosafety 474
447 loaded mPEG-OA VM micellar structures were found to be in a of the synthesized mPEG-OA against MCF 7, A549 and Hep G2 cells. 475
448 nano size of 142.9 ± 7.5 nm, with PDI and ZP of 0.228 ± 0.028 and The test results showed a high percentage of cell viability ranging 476
449 18.3 ± 3.55 (mV) respectively. Entrapment efficiency and loading from 75.62 to 85.875% at all concentrations against all cell lines 477
450 capacity was found to be 53.64 ± 1.86% and 5.09 ± 0.17% w/w studied, which confirms the nontoxic nature of these materials. 478
451 respectively. These results were comparable to high hydrophilic The percentage cell viability obtained for mPEG-OA was between 479
452 drug encapsulated efficiencies [61,62] reported in the literature. 75.62 and 80.662% for the MCF 7 cells, 76.079–83.248% for the A 480
453 VM-loaded mPEG-OA polymersomes in aqueous solution were 549 cells, and 76.133–85.875% for the Hep G2 cells for all concen- 481
454 stable for a period of two months at 4 °C, and for one month at trations (Fig. 4). The mPEG-OA did not show any dose dependent 482
455 room temperature. This results were comparable to other polymer- toxicity towards all cell lines within the concentration range stud- 483
456 somes in the literature [63,64]. ied. The cell viabilities were above 75% can be considered to be bio- 484
457 The morphology of the polymersomes was determined using logically safe and nontoxic to mammalian cells [68,69]. These 485
458 TEM. The hollow structure of the polymeric vesicles can be findings demonstrate that mPEG-OA is a bio-safe polymer for 486
459 observed in TEM images (Fig. 3). The VM-loaded mPEG-OA poly- biomedical applications. 487
460 mersomes were spherical in shape, with most of the population
461 sizes being in the ranges that were comparable to the DLS size 3.5. Differential scanning calorimetry (DSC) 488
462 ranges. The TEM images of VM loaded mPEG-OA were identical
463 to previously reported polymersomes where vesicles can be clearly DSC studies were carried out to confirm the entrapment of VM 489
464 observed [65]. in the mPEG-OA polymersomes. The thermal behavior of VM, 490

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Fig. 3. TEM image of VM loaded mPEG-OA polymersomes.

491 physical mixture of VM and mPEG-OA, and the lyophilized formu- bly due to phase transition of drug-polymer system [71]. In addi- 512
492 lations was studied. Any sudden or forceful change in the thermal tion to phase transition of drug-polymer system, one of the 513
493 behavior of the drug or polymer may indicate a possible drug- reasons for appearance of the peak at 237.8 °C in lyophilized poly- 514
494 polymer interaction [70]. The thermal peak for bare VM was mersomes could be co-crystallization of formulation ingredients in 515
495 observed at about 111.53 °C, while the mPEG-OA thermal peak the presence of THF [74,75]. 516
496 was at 56.25 °C (Fig. 5). The physical mixture of VCM and mPEG-
497 OA showed nearly the same thermal behavior as the individual
498 components, indicating that there was no interaction between 3.6. In vitro drug release behavior 517
499 the drug and the polymer in the mixture. The thermogram of the
500 lyophilized mPEG-OA-vancomycin loaded polymersomes showed Fig. 6 represents the in vitro drug release profiles of the bare VM 518
501 a shift and broadening in the thermal peak of the mPEG-OA and and VM-loaded polymersomes in the PBS (pH 7.4). The cumulative 519
502 the disappearance of the VM peak. This disappearance can be VM release from the VM-loaded MPEG-OA polymersomes in the 520
503 attributed to its transformation into an amorphous form after initial three hours was less than 30%, indicating a sustained release 521
504 encapsulation in the mPEG-OA polymersomes [71]. The broaden- profile of the formulation. It took 48 h for the formulation to 522
505 ing of the mPEG-OA peak could be due to a combination of release 90% of VM, whereas almost 100% of the VM was released 523
506 endothermic contributions, such as the disruption of hydrogen from the bare VM solution in eight hours. In comparison with free 524
507 bonds [72], and exothermic factors, such as the break-up of VM, its much slower release rate from the VM-loaded Polymer- 525
508 hydrophobic interactions that bring about changes in the mem- somes can be attributed to the molecular structural characteristics 526
509 brane curvature restructuring [73] during entrapment of VM. The of the polymersomes. This delay of the VM release indicates the 527
510 appearance of peaks at 260.9 and 287.9 °C in the physical mixture potential applicability of the mPEG-OA as a sustained release drug 528
511 (Fig. 5C), and 237.8 °C in lyophilized polymersomes could be possi- carrier for nanoantibiotics. 529

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Fig. 4. Cytotoxicity evaluation of mPEG-OA against various concentrations of on MCF 7, A 549 and Hep G2 cells.

transport) [78,71]. This suggests more than one mechanism was 544
involved in the release of the drug [77]. Being an amphiphilic 545
copolymer of mPEG and fatty oleate, the release mechanism from 546
mPEG-OA might have been through enhanced diffusion due to 547
swelling and relaxation of the polymer system [79]. The mean dis- 548
solution time (MDT) was determined to enable the VM release rate 549
to be identified. The MDT value is inversely proportional to the 550
release rate, i.e. the lower the MDT the higher the release rate, 551
and vice versa [80]. The calculated MDT90% values for the VM 552
release from the mPEG-OA polymersomes and bare VM were 553
9.85 h and 3.83 h. The obtained MDT values suggest that the drug 554
release rate from the mPEG-OA was significantly slower than that 555
of the bare VM at a physiological pH (7.4). The in vitro release data 556
and calculated MDT values collectively suggest that the VM release 557
from the mPEG-OA polymersomes displayed sustained and con- 558
trolled release patterns. Such a system, which releases drug over 559
an extended period, could provide effective concentrations for ini- 560
tial activity and a slower release for prolonged activity. This will 561
lower the frequency of administration and the number of doses 562
in the treatment regime, thus improving treatment efficiency and 563
patients compliance. 564

Fig. 5. DSC thermogram of (A) VM; (B) mPEG-OA; (C) physical mixture of VM and
mPEG-OA and (D) lyophilized VM loaded mPEG-OA polymersomes. 3.7. In vitro antibacterial activity 565

The MIC values of bare VM against S. aureus and MRSA were 566
530 The kinetic analysis of the drug release was performed using 5.62 lg/mL and 31.25 lg/mL after 24 h respectively, whereas for 567
531 various models to understand the VM release profile (Table 2) from the VM-loaded mPEG-OA, the values were 0.37and 5.86 lg/mL 568
532 the VM-loaded mPEG-OA polymersomes. The maximum cumula- respectively. The MIC value of VM decreased by 42- and 5-fold 569
533 tive release percentage used for all the models was 60%, as linearity against S. aureus and MRSA after encapsulation into the mPEG- 570
534 of most of the equations can only be verified up to that percentage OA polymersomes (Table 3). It was also observed that the formula- 571
535 of released drug [76,77]. VM release from the prepared polymer- tion was effective up to 72 h, whereas the VM lost its activity after 572
536 somes was found to follow the Weibull model, as it had a higher day 1. Better activity of the formulation towards S. aureus than 573
537 correlation coefficient (R2 = 0.9942) and lower root mean square MRSA is attributed to the difference in peptidoglycan layers, the 574
538 error (RMSE = 1.750) values among all the models (Table 2). How- cell wall thickness being greater in the MRSA than the S. aureus 575
539 ever, to understand the drug release mechanism, the initial 60% [10]. The increased peptidoglycan layers in the MRSA cell wall lim- 576
540 drug release data was also fitted to the Korsmeyer-Peppas model its the VM molecules from crossing the membrane by affinity trap- 577
541 to obtain the ‘n’ exponent, which characterizes the different drug ping and clogging the VM in the peptidoglycan mesh [81]. Thus, a 578
542 release mechanisms. The ‘n’ value was found to be 0.614 which higher concentration of VM is needed to saturate all murein mono- 579
543 indicates the release mechanism to be non-Fickian (anomalous mers that are supplied at an increased rate [81] in the MRSA. Over 580

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Fig. 6. In vitro drug release profile of VM loaded MPEG-OA and bare VM.

Table 2
Release kinetics data from different models.

Model Equation R2 RMSE Release exponent (n)

Zero Order Q = k  t + Q0 0.8864 6.710 –
First Order Q = Q0  ekt 0.9738 3.228 –
Higuchi Q = k  t½ 0.9767 3.040 –
Korsmeyer-Peppas Q = k  tn 0.9898 2.148 0.614
Hixson-crowell Q⅓ = kt + Q⅓ 0 0.9546 4.245 –
Weibull Q = 1 exp [(t)a/b] 0.9942 1.750 –

R2 = linear regression coefficient, RMSE = Root mean square error.

Table 3
MICs of bare VM, blank and VM loaded mPEG-OA polymersomes, against S. aureus and MRSA.

Time (h) 24 48 72 24 48 72
S. aureus (MIC lg/mL) MRSA (MIC lg/mL)
Bare Vancomycin 15.62 NA NA 31.25 NA NA
VM loaded mPEG-OA polymersomes 0.37 0.37 0.37 5.86 5.86 5.86
Blank mPEG-OA polymersomes NA NA NA NA NA NA

NA = No activity. The values are expressed as mean ± SD, n = 3.

581 enhanced activity of the VM-loaded MPEG-OA can be attributed to between the cell membrane thicknesses. These results highlight a 595
582 an OA lipid part in the micellar structure. The OA could have facil- promising prospect of using VM-loaded mPEG-OA as a new thera- 596
583 itated the fusion of polymersomes with the MRSA membrane, peutic strategy against S. aureus and MRSA infections. 597
584 which further results in easy penetration of the drug into the bac-
585 terial cell [23]. This ensures proper drug concentration for a rela- 3.8. Percentage bacterial cell viability 598
586 tively long time at the desired site [2,82]. Therefore, narrow and
587 nano particle size, along with bacterial cell targeting, appear to Percentage bacterial cell decrease by VM and VM-loaded mPEG- 599
588 have enhanced the activity of the VM. The lower MICs associated OA polymersomes at their respective MIC values are represented in 600
589 with the VM-loaded mPEG-OA could result in a dose reduction of Table 4. The bare VM displayed a decrease in bacterial growth by 601
590 the VM. This reduced dose will assist in decreasing the 92 ± 1.81% and 90.5 ± 1.67% for S. aureus and MRSA respectively, 602
591 vancomycin-related nephrotoxicity that is associated with higher while VM-loaded mPEG-OA polymersomes had reduced growth 603
592 dose VM regimens [83] without compromising their therapeutic by 90.5 ± 1.43% and 86.8 ± 5.74% for S. aureus and MRSA respec- 604
593 efficacy. The possible reason for greater activity of the polymer- tively. The VM concentration in the polymersomes was 42.21 and 605
594 somes against S. aureus than MRSA could be the difference 5.33-fold lower than the concentration of bare VM for S. aureus and 606

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Table 4
Percentage cell viability and percentage decrease in bacterial cells growth after exposure to mPEG-OA-VM polymersomes and Bare vancomycin.

Formulation %Cell growth % Decrease bacteria growth

S. aureus MRSA S. aureus MRSA
Bare vancomycin 8. ± 1.81 9.5 ± 1.67 92 ± 1.81 90.5 ± 1.67
VM loaded MPEG-OA polymersomes 9.5 ± 1.43 13.2 ± 5.74 90.5 ± 1.43 86.8 ± 5.74

The values are expressed as mean ± SD, n = 6.

MRSA respectively. These results showed that the VM encapsu- 607

lated in the mPEG-OA polymersomes had similar activity as that 608
of bare VM, but at lower concentration (0.37 lg/mL for S. aureus 609
and 5.87 lg/mL for MRSA). This indicates the potential of the 610
VM-loaded MPEG-OA polymersomes as a nanoantibiotic to 611
increase the potency of VM, and its ability to reduce dose related 612
toxicities. These findings further confirm that VM-loaded mPEG- 613
OA at low concentrations displays greater activity against S. aureus 614
and MRSA compared to bare VM. 615

3.9. In vivo antibacterial activity 616

A mouse skin infection model was used to establish/test in vivo 617

antibacterial activity, where intradermal injections were delivered 618
into the dermis, or the skin layer underneath the epidermis. Intra- 619
dermal MRSA injections delivered the bacteria into the area 620
between the epidermal and the subcutaneous layer of the skin. 621
This caused short-term localization of the bacteria within this 622
layer, and prevented the direct entry of the bacteria into the sys- 623
temic circulation via the larger network of blood vessels found in 624
the subcutaneous layer [84]. The number of colony-forming units 625
(CFUs) were quantified for each treatment group and are repre- 626
sented as log10 (Fig. 7). There was a statistically significant 627
Fig. 7. MRSA burden after 48 h of treatment. Data represents mean ± SD (n = 3). ⁄⁄
(P < 0.0001) reduction in bacterial load of the skin samples treated 628
denotes significant difference when compared to blank mPEG-OA and bare VM. ⁄⁄⁄
denotes when compared to blank mPEG-OA compared to VM loaded mPEGO-OA with both VM-loaded mPEG-OA and bare VM, when compared to 629
and ⁄⁄⁄⁄ denotes significant difference between VM-loaded mPEG-OA and bare VM. the mPEG-OA control (Fig. 8). The mean bacterial load (log10 630
CFU) recovered from the blank mPEG-OA was 5.32 ± 0.14, which 631

Fig. 8. Photomicrographs of the control and the treated: (A) show the skin lesions at the site of injection for the control, (B) shows the injection site of the treated mice. The
selections for light microscopy (LM) stained with H&E; (4
), (C) bare VM treated, (D) VM-loaded mPEG-OA treated and (E) control/untreated.

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632 was 1.19-fold greater than that found in the bare VM treated sam- decrease in the MIC against susceptible and resistant S. aureus. It 695
633 ples (4.47 ± 0.05). These findings confirm that bare VM is able to was noticed that the developed nanoantibiotic was very effective 696
634 decrease the bacterial load significantly (P = 0.0007). In the VM- in controlling MRSA infections when tested using mice skin infec- 697
635 loaded mPEG-OA treated samples, the bacterial load was tion model. An in vivo evaluation of VM-loaded mPEG-OA in a mice 698
636 3.04 ± 0.23, which was a 1.75-fold decrease when compared to skin model further proved its superior antibiotic activity. The syn- 699
637 the blank mPEG-OA polymersomes (P = 0.0001). Interestingly there thesized novel mPEG-OA is a new non-toxic pharmaceutical poly- 700
638 was a 1.47-fold greater reduction in bacterial load after treatment mer to develop nanoantibiotics, and can therefore be exploited for 701
639 with VM-loaded mPEG-OA polymersomes compared with bare VM encapsulation of other antibiotic molecules to improve their effi- 702
640 (P = 0.0005). These in vivo investigations confirmed the superiority cacy against a range of bacteria to treat bacterial infections. 703
641 of VM-loaded mPEG-OA as an effective novel nanoantibiotic for
642 treating infections of MRSA origin. Disclosure 704
643 Histological analysis was performed after 48 h post the intra-
644 dermal infection study to assess skin integrity and histo- The authors report no conflicts of interest in this work. 705
645 morphological changes after the MRSA intradermal infection. The
646 skin from the injection site of the control group showed the forma- Acknowledgments 706
647 tion of fluid filled abscess, whereas the treated groups displayed no
648 abscess formation (Fig. 8A and B). The H&E images from the control The authors acknowledge the University of KwaZulu-Natal 707
649 group confirmed an inflammation and the formation of an abscess (UKZN), UKZN Nanotechnology Platform and the National Research 708
650 at the injection site (Fig. 8E). The control tissue image displayed Foundation of South Africa (Grant Nos. 87790 and 88453) for finan- 709
651 evidence of inflammation, as represented by the excessive swelling cial support. The Microscopy and Microanalysis Unit and Biomed- 710
652 of the dermal layer in the control image (Fig. 8E). While both the ical Resource Unit at UKZN are acknowledged for TEM and animal 711
653 bare VM and VM-loaded MPEG-OA treated samples did not display experiment facility respectively. We thank Ms Carrin Martin for 712
654 any definite abscess formation, there was evidence of minimal proof reading the manuscript. 713
655 inflammation in the dermal layer (Fig. 8D and E). In the control
656 group, there was evidence that a high number of cells were infil-
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