PHARMACEUTICAL SCIENCE | RESEARCH ARTICLE

Preparation and characterization of PCL-PEG-PCL

polymersomes for delivery of clavulanic acid
Hossein Danafar

Cogent Medicine (2016), 3: 1235245

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Danafar, Cogent Medicine (2016), 3: 1235245
http://dx.doi.org/10.1080/2331205X.2016.1235245

PHARMACEUTICAL SCIENCE | RESEARCH ARTICLE

Preparation and characterization of PCL-PEG-PCL
polymersomes for delivery of clavulanic acid
Hossein Danafar1,2*

Received: 25 June 2016
Accepted: 07 September 2016
First Published: 16 September 2016
*Corresponding author: Hossein
Danafar, Zanjan Pharmaceutical
Nanotechnology Research Center,
Zanjan University of Medical Sciences,
Zanjan, Iran; Department of Medicinal
Chemistry, School of Pharmacy, Zanjan
University of Medical Sciences, Zanjan,
Iran
E-mail: danafar@zums.ac.ir
Reviewing editor:
Varun Khurana, Insys Therapeutics
Inc., USA
Additional information is available at
the end of the article
Abstract: The Clavulanic acid (CLV) is a suicide inhibitor of bacterial beta-lac-
tamase enzymes from Streptomyces clavuligerus. In the present study, a reli-
able drug delivery system using poly (ε-caprolactone)-poly (ethylene glycol)-poly
(ε-caprolactone) (PCL-PEG-PCL) was synthesized and the release profile of the CLV
from the drug-loaded polymersomes was evaluated. In this study, CLV was encap-
sulated within PCL-PEG-PCL nanoparticles by a double emulsion technique (w/o/w),
leading to creation of CLV-loaded PCL-PEG-PCL (CLV/PCL-PEG-PCL) polymersomes.
Characterization, stability of polymersomes, the particle size was determined by
DLS. The release profile of the CLV from the polymersomes which ready by the
drug-loaded copolymer, was evaluated. Our studies resulted in a successful estab-
lishment of uniformity and spherical CLV-loaded PCL-PEG-PCL polymersomes. The
loading efficiency of CLV was 16.00 ± 1.45%. The results of DLS show that the poly-
mersomes have spherical shapes with size of 113 nm. In vitro release of CLV from
CLV-entrapped polymersomes was remarkably sustained. The results indicate the
successful formulation of curcumin loaded PCL-PEG-PCL polymersomes.

Subjects: Allied Health; Health & Society; Health and Social Care; Midwifery

Keywords: PCL-PEG-PCL; polymersomes; clavulanic acid; drug delivery

ABOUT THE AUTHOR PUBLIC INTEREST STATEMENT

Hossein Danafar is Assistant Professor of
Medicinal Chemistry and Dean of Department of
Medicinal Chemistry at the School of Pharmacy,
Zanjan University of Medical Sciences, Zanjan,
Iran. He received his PhD (2013) degree in
Medicinal Chemistry from Tabriz University
of Medical Sciences, Tabriz, Iran. His current
research interests are the analytical chemistry,
pharmaceutical analysis, design, synthesis and
characterization of polymeric drug delivery
systems, including micelles, nanogels and
molecularly imprinted carriers, bioconjugates
and magnetic targeted drug delivery systems.
Hossein Danafar The research study titled is (Preparation and
characterization of PCL-PEG-PCL polymersomes
for delivery of clavulanic acid). The future study is
application of the other block copolymers in drug
delivery systems.
Clavulanic acid (CLV) and its salts and esters.
The acid is a suicide inhibitor of bacterial
beta-lactamase enzymes from Streptomyces
clavuligerus. In the present study, a reliable drug
delivery system using poly (ε-caprolactone)-poly
(ethylene glycol)-poly (ε-caprolactone) (PCL-
PEG-PCL) was established. In this study, CLV was
encapsulated within PCL-PEG-PCL nanoparticles
by a double emulsion technique (w/o/w), leading
to creation of CLV-loaded PCL-PEG-PCL (CLV/
PCL-PEG-PCL) polymersomes. Characterization,
stability of polymersomes, the particle size was
determined by DLS. Our studies resulted in a
successful establishment of uniformity and
spherical clavulanic acid-loaded PCL-PEG-PCL
polymersomes. The in vitro release of CLV from
CLV-entrapped polymersomes was remarkably
sustained. Our study showed that this nano-carrier
provided a suitable and appropriate system for
delivery of CLV. This developed nano-drug delivery
system can also be used as a promising base to
design further nanoparticle systems to improve the
therapeutic effect of CLV in the future.

© 2016 The Author(s). This open access article is distributed under a Creative Commons Attribution
(CC-BY) 4.0 license.

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1. Introduction
Polymersomes are hollow spheres, made of polymers that contain an aqueous solution in the core
surrounded by a bi-layer membrane. The bi-layer membrane is composed of hydrated hydrophilic
coronas both at the inside and outside of the hydrophobic middle part of the membrane separating
and protecting the fluidic core from the outside medium. In drug delivery context, the aqueous core
can be utilized for the encapsulation of hydrophilic therapeutic molecules such as small-molecule
drugs, enzymes, proteins, peptides, DNA and RNA fragments (Christian et al., 2009; Kim, Meeuwissen,
Nolte, & Hest, 2010; Lomas et al., 2007) while the membrane can incorporate hydrophobic drugs
within its hydrophobic core (Ahmed et al., 2006; Chen, Meng, Cheng, & Zhong, 2010; Kale & Torchilin,
2007; Khurana, Patel, Agrahari, Pal, & Mitra, 2015; Nie, 2010). Based on their multidrug loading capac-
ity, stability, long blood circulation times, membrane robustness and stealth properties, polymer-
somes are highly attractive for drug delivery of highly toxic therapeutics with tuned pharmacokinetics
in order to greatly increase therapeutic efficacy. Clavulanic acid (CLV) and its salts and esters. The acid
is a suicide inhibitor of bacterial beta-lactamase enzymes from Streptomyces clavuligerus.
Administered alone, it has only weak antibacterial activity against most organisms, but given in com-
bination with beta-lactam antibiotics prevents antibiotic inactivation by microbial lactamase. To
achieve drug controlled release, biodegradable polymeric nanoparticles are often used in advanced
anticancer drug delivery systems (Al-Musawi et al., 2014; Allen & Cullis, 2004; Danafar, Davaran,
Rostamizadeh, Valizadeh, & Hamidi, 2014, Danafar, Rostamizadeh, Davaran, & Hamidi, 2014, 2015;
Jackson & Singletary, 2004; Kumari, Yadav, & Yadav, 2010; Manjili et al., 2016). A number of drug de-
livery systems using recyclable polymer such as nanoparticles delivering antitumor agents are com-
mercially accessible. Poly (caprolactone)-poly (ethylene glycol) (PCL-PEG) copolymers are co-friendly,
easy to produce, amphiphilic, and have a sturdy probable function in drug delivery system (Danafar,
Davaran, et al., 2014; Danafar, 2016b; Khurana et al., 2015; Lin et al., 2012). Tian et al. evaluated al-
bumin based polymeric delivery system was successful and has the probable to enhance the thera-
peutic effect and anti-tumor activity of sulforaphane (Patel, Vaishya, Yang, Pal, & Mitra, 2014). For this
reason, it seems that encapsulating of CLV in PCL-PEG-PCL polymersomes may be a talented method
for developing an improved CLV formulation (Cheng et al., 2016; Danafar, 2016c; Danafar, Manjili, &
Najafi, 2016; Danafar, Sharafi, Kheiri, Manjili, & Andalib, 2016). In the current study, a polymersome
delivery system with PCL-PEG-PCL was prepared. In this study, an effective polymersomes delivery
system by PCL-PEG-PCL was obtained. We present an efficient nano-drug delivery system to improve
the combination and therapeutic level of CLV. It was presumed that the PCL-PEG-PCL could be a good
candidate for the delivery CLV. In this contribution, we are aimed to encapsulate CLV in PCL-PEG-PCL
polymersomes as a promising carrier with sustained release characteristics. In this way, a novel drug
delivery system with PCL-PEG-PCL was synthesized and the release profile of the CLV from the polym-
ersomes prepared using the drug-loaded copolymer was evaluated.

2. Materials and methods

2.1. Materials
All used chemicals were of analytical grade and purchased from commercial sources. Poly (ethylene
glycol) PEG (Mn = 6,000 Da) (Aldrich, St. Louis, USA, CAS.81323), ε-caprolactone (98% purity) (Acros,
New Jersy, USA, CAS.502443), clavulanate potassium (CLV) (Zahravi Company, Iran), stannous
2-ethyl-hexanoate (Sn (Oct)2) (Aldrich, St. Louis, USA, CAS. 301100), phosphate buffer (20 mM, pH
7.8), poly (vinyl alcohol), MW = 145,000 (Aldrich, St. Louis, USA) and corresponding salts which were
used throughout this research was obtained from Sigma-Aldrich or Merck.

2.2. Synthesis of PCL-PEG-PCL copolymer

The synthesis of PCL-PEG–PCL copolymers was prepared according to our previous report (Manjili
et al., 2016). That were synthesized by a ring opening polymerization of ε-caprolactone with PEG as
initial molecule and Sn (Oct) 2 as catalyst was performed. Briefly, ε-caprolactone (4 g), PEG (2 g), and
Sn (Oct)2 (0.01 mmol) were heated to 120°C to start polymerization. After 12 h, the resulting polymer
was cooled to room temperature (23°C), dissolved in chloroform, and precipitated in cold diethyl
ether. The copolymer was dried under vacuum at room temperature for 24 h.

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The obtained copolymers was characterized by Fourier Trans form Infrared spectroscopy (FT-IR)
(Bruker, Tensor 27) were used for characterization of the chemical structure of the PCL-PEG-PCL co-
polymer and Proton Nuclear Magnetic Resonance Spectroscopy (1H NMR) in CDCl3 at 400 MHz (Bruker,
Evans, 400). Thermal analysis of tri block copolymers was determined by differential scanning calo-
rimetry (DSC) (Mettler Toledo, model Star SW 9.30) and the data were recorded from 0 to 250°C.
Molecular weight and distribution of the PCL-PEG-PCL copolymers were calculated by gel permeation
chromatography (GPC) (Knaure, Berlin, Germany) composed of with an ultrastyragel column and dif-
ferential refract metric detector (4.6 × 30 mm) (Waters, Milford, USA, model HR 4E). Tetrahydrofuran
(THF) as a mobile phase was with a flow-rate of 1 ml/min and the injection volume was 100 μl of
stock solutions (0.1–0.5 w/v%). The standards of polystyrene mono-disperse in the range of 4,500-
29,500 DA (Varian Palo Alto, CA) obtained before measurements for use as the calibration curve for
determination of average molecular weight of the PCL-PEG-PCL copolymers(Manjili et al., 2016).

2.3. Preparation of CLV-loaded polymersomes

CLV loaded polymersomes were prepared by a double emulsion technique (w/o/w). In essence, 1 ml
aqueous solution of CLV with known concentration 6 mg/ml was first poured into the PCL-PEG-PCL
copolymer solution in 2 ml chloroform (25 mg/ml) to form a w/o emulsion. The resulting emulsion
was then injected drop-wise through a syringe (G = 22) into 20 ml of distillated water containing
0.4 wt% of poly (vinyl alcohol) under certain mixing rates. Then, the mixture stirring was continued
magnetically at room temperature until complete evaporation of the organic solvent. Subsequently,
evaporation of the organic solvent in aqueous environment leads the amphiphilic copolymers to
self-associate and form the nanoparticles. Finally, all of the samples were centrifuged at 20,000 g,
and freeze-dried (at pressure of 14 Pa and −78°C) to remove all of the residual solvents and to pro-
duce the final nanospowder form.

2.4. Characterization of the polymersomes

2.4.1. Determination of particle size

The particle size distribution of the prepared polymersomes was determined by dynamic light scat-
tering (DLS) using a nano/zetasizer (Malvern Instruments, Worcestershire, UK, model Nano ZS).

2.4.2. Stability of polymersomes

The physical stability of polymersomes was evaluated by monitoring the particle size distribution of
the polymersomes while suspended in phosphate-buffer saline(PBS, pH 7.4) and kept in in room
temperature at times 0, 15, and 30 days after preparation using the method described in 2.3.

2.4.3. Determination of loading efficiency

To determine the loading efficiency of the drugs in the polymersomes, two parameters including the
drug loading ratio and efficiency of entrapment were evaluated. Drug loading ratio was determined
as:

Weight of drug in polymersomes

DL% = × 100 (1)
Weight of polymersomes
where Wdrug in polymersomes and Wpolymersomes show weight of the encapsulated drug and the total weight of
the corresponding drug-encapsulated polymersomes, respectively. DL% is the drug loading ratio
(%). For determination of the drug loading ratio, 1 mg of the final freeze-dried Nano dispersion was
dissolved in 1  ml of chloroform, and the drug content was measured by high performance liquid
chromatography (HPLC) (Danafar, 2016a, 2016b; Danafar & Hamidi, 2013, 2016). The mobile phase
consisted of a mixture of buffer phosphate (pH 4.4) and methanol (90:10, v/v), and was delivered at
a flow rate of 1.0 ml/min using a double-reciprocation pump (Waters, model Breeze, USA). The sam-
ple was injected through a 20  μl sample loop. The column effluent at 1.52  min was detected at
220 nm with a variable wavelength detector (Waters, model Breeze, USA). A C8 analytical column
(150 × 4.6 mm, particle size 5 μm; Perfectsill, MZ-Analysen technik, Germany) equipped by a guard
column of the same packing was used.

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Entrapment efficiency was determined in reference to loading ratio and total dried nanodisper-
sion weight obtained using the following equation:

(Weight drug in polymersomes)

EE% = × 100 (2)
Weight of initial drug
where EE% is the efficiency of entrapment (%), weight drug in polymersomes and Wfeed drug stand for
the total mass of powders obtained after freeze-drying and the drug fed initially in the polymer-
somes preparation step, respectively.

2.5. Drug release study

This test was performed to evaluate the release behavior of CLV from polymersomed copolymer. The
solubility of CLV in PBS, pH 7.4 is approximately 10 mg/ml. Briefly, 5 mg of freeze-dried drug-loaded
carriers were dispersed in 2 ml phosphate-buffered saline (PBS) and the resulting suspension was
placed within a dialysis sac (Mw 12 kDa) and incubated at 37°C while immersed in 15 ml of PBS. Then,
at predetermined time intervals, 2 ml of the dialysate was taken out and replaced by 2 ml fresh PBS.
The concentration of CLV in the dialysate was measured at 220 nm using HPLC. All the release stud-
ies were carried out in triplicate. In order to study the pH-dependency of the drug release, the experi-
ments were performed using PBS at a pH of 5.5. As controls, the release of free CLV was studied in
PBS with pH 7.4 and pH 5.5.

3. Results and discussion

3.1. Synthesis and characterization of PCL-PEG-PCL copolymers

PCL-PEG-PCL tri-block copolymers was synthesized using the ring-opening polymerization of caprol-
actone in presence of PEG, whose hydroxyl end group initiated the ring opening and explain to pervi-
ous work (Manjili et al., 2016). The structure and composition of the synthesized PCL-PEG-PCL
tri-block copolymers was determined by H NMR spectroscopy in CDCl3 (Figure 1). The presence of
ethylene’s (CH2) in PCL was observed around 1.35, 1.69, 2.43 and 4.06  ppm, the methylene (CH2)
groups of PEG were around 3.72 ppm. Table 1 shows the characteristics of synthesized copolymers.
FT-IR spectrum of PCL-PEG-PCL copolymer show the sharp and intense bands at 1,725 and 1,111 cm−1
were awardable to the presence of carboxylic ester (C=O) and ether (C–O) groups, thereby indicating
that the formation of PCL-PEG-PCL copolymer has occurred successfully (Figure 2). To DSC thermo

Figure 1. H NMR spectrum

of PCL-PEG-PCL tri-block
copolymer in CDCl3.

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Table 1. Molecular characterics of the synthesized copolymers

Copolymer CL/EG feed Mn (Da)a Mw (Da)a PdIb Tm (°C)c DPPEG DPdPCL
PCL-PEG-PCL 2 13,343 8,310 1.6 44.01 136.36 73.09
a
Determined by GPC analysis using narrow molecular weight polystyrene standards.
b
Mw/Mn = Polydispersity index of the polymers (PdI) determined by GPC analysis.
c
Calculated from the first run of DSC as half of the extrapolated tangents.
d
DP: Degree of polymerization.

Figure 2. FTIR spectrum of PCL-

100

PEG-PCL tri-block copolymer.

80
60
Transmittance [%]
40
20
0

2891.50

1725.26

1111.81

962.29
841.93
731.76

526.11
452.86
4000 3500 3000 2500 2000 1500 1000 500

Wavenumber cm-1

Figure 3. DSC thermogram of

PCL-PEG-PCL copolymer.

grams copolymers the endotermic peak (44.01°C) including two merged peaks of PEG and PCL which
presented in Figure 3. GPC results showed that the weight- and number-based average molecular
weights of copolymer were 13.3 and 8.3 KDa, respectively (Figure 4).

3.2. Preparation and characterization of copolymeric polymersomes

The size of polymersomes was measured by dynamic light scattering technique (DLS). The Z-average
and Zeta potential of CLV loaded PCL-PEG-PCL polymersomes were found to be about 113 nm and
−7.42 mv, with their corresponding PDI being 0.096 (Figure 5). The loading ratio and encapsulation
efficiencies of CLV loaded to PCL-PEG-PCL polymersomes were determined by HPLC to be
16.00 ± 1.45% and 69.33 ± 1.12%, respectively. The chromatogram of CLV loaded to PCL-PEG-PCL
polymersomes was showed to Figure 6.

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Figure 4. GPC spectrum of PCL-

PEG-PCL tri-block copolymer.

3.3. In vitro release of CLV

3.3.1. The impact of different release media on drug release from polymersomes
In order to examine the influence of the chemical and biochemical factors on the release of CLV
from polymersomes, the release study was performed on drug-loaded polymersomes in neutral (pH
7.4) and acidified PBS solution (pH 5.5). As controls, the release of free CLV was studied to verify that
the diffusion of drug molecules across the dialysis membrane was not a rate-limiting step during the
release process. Free CLV was observed to be rapidly released and reached its peak of 85.15 and
86.03% of the total in the first 8 h at 7.4 and 5.5 pH respectively. Figure 7 shows the release profiles
of CLV from the drug-loaded polymersomes, at 7.4 and 5.5 pH. As expected, no considerable initial
burst CLV release was observed from the polymersomes. As shown in Figure 7, the percentage of CLV
released from the polymersomes increased as the pH value decreased from 7.4 to 5.5. For example,
after 96 h incubation, the amounts of CLV released in the media with pH values of 7.4 and 5.5 were
about 66.15, and 53.11%, respectively. This fact may be due to the physical loading of CLV in to the
polymersomes. The results revealed that the maximum drug releases were 68.34, and 62.87% re-
spectively for PBS pH 7.4 and pH 5.5 after a period of 120 h. The sustained release of CLV can be at-
tributed to the entrapment of CLV in core of polymersomes. Therefore, the obtained copolymeric
polymersomes can be regarded as highly attractive nano-carriers for time-controlled drug delivery
for hydrophobic drugs to achievement of different therapeutic objectives.

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Figure 5. Particle size

distribution and zeta
potential of CLV/PCL-PEG-PCL
nanoparticels (A) particle size
distribution (B) zeta potential.

3.4. Physical stability of polymersomes

In the clinical administration of nanoparticle dispersions, the stability of the sizes and volume of the
nano-carriers is of great importance both as a measure of the particle structure integrity and as an
indicator of the possible inter-particular associations (aggregation). For this purpose, the particle
size stability was monitored in this study over a 30-days course. The size of all polymersomes was
increased slightly throughout the measurement period. This observation cannot be a sign of aggre-
gation, which usually leads to several fold increases. Probably, some kind of copolymer swelling and/
or hydration (as a result of presence of the hydrophilic PEG portions in polymersomes surfaces) can
be responsible for this event.

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Figure 6. HPLC chromatogram

of (A) CLV loaded
polymersomes, (B) blank.

Figure 7. The release profiles 100

of CLV from CLV-PCL-PEG-PCL 90

polymersomes in different
80
release media (A) pH 7.4, (B)
pH 5.5. 70
Drug Release (%)

60
CLV-PCL-PEG-PCL
PH=5.5
50 CLV-PCLPEG-PCL
PH=7.4
40 CLV-PH=7.4"

CLV-PH= 5.5"
30

20

10

0
0 20 40 60 80 100 120 140 160 180
Time (hr)

4. Conclusion
A PCL-PEG-PCL copolymer was synthesized and characterized by H NMR, FTIR, DSC and GPC tech-
niques. The copolymer PCL-PEG-PCL was self-assembled into polymersomes in aqueous solution in
presence of CLV. The obtained polymersomes were characterized by DLS. The encapsulation effi-
ciency of CLV was 69.33 ± 1.12%. The results revealed that the polymersomes formed had spherical
structure with size of 113 nm. In vitro release of CLV from CLV-entrapped polymersomes was clearly
sustained in all the media tested for this purpose, with the apparent release plateau reached late at
about 120 h. Our study showed that this nano-carrier provided a suitable and appropriate system for
delivery of CLV. This developed nano-drug delivery system can also be used as a promising base to
design further nanoparticle systems to improve the therapeutic effect of CLV in the future.

Funding 1
 anjan Pharmaceutical Nanotechnology Research Center,
Z
The author received no direct funding for this research. Zanjan University of Medical Sciences, Zanjan, Iran.
2
Department of Medicinal Chemistry, School of Pharmacy,
Competing Interests Zanjan University of Medical Sciences, Zanjan, Iran.
The authors declare no competing interest.
Citation information
Author details Cite this article as: Preparation and characterization of
Hossein Danafar1,2 PCL-PEG-PCL polymersomes for delivery of clavulanic acid,
E-mail: danafar@zums.ac.ir Hossein Danafar, Cogent Medicine (2016), 3: 1235245.

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Cover image
Source: Author.
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Danafar, Cogent Medicine (2016), 3: 1235245
http://dx.doi.org/10.1080/2331205X.2016.1235245

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