INTRODUCTION
Poly (∈-caprolactone) (PCL) is a biodegradable and biocompatible polymer that
is widely used in medicine and pharmaceutical applications. Diblock copolymers
composed of methoxy poly (ethylene glycol) (MPEG) and PCL have been synthesized
to attain versatile biodegradable polymers having more water-absorbing capacity
because of the inclusion of hydrophilic MPEG segments within the relative hydrophobic
PCL segments (He et al., 2004; Shuai
et al., 2004; Aliabadi et al., 2005).
These diblock copolymers have been used for the preparation of drug-loaded nanoparticles
(Kim and Lee, 2001; Shuai et al.,
2004; Aliabadi et al., 2005; Zhang
and Zhuo, 2005). The nanoparticles have shown potential as drug delivery
systems because of their small sizes, improving circulation times in the body
and creates more available routes of administration than do microparticles,
which are rapidly cleared by the reticulo-endothelial tissues (Kumar,
2000).
The modified-spontaneous emulsification-solvent diffusion method (modified-SESD
method) for the preparing surfactant-free nanoparticles of hydrophilic-hydrophobic
diblock copolymer was first proposed as previously described by Baimark
et al. (2007). MPEG-b-poly (D, L-lactide) was dissolved in
volatile water-miscible organic solvents with lower toxicity, acetone and ethanol.
Higher energy apparatus, such as a homogenizer or a sonicator (usually applied
in larger scale preparation of polymer nanoparticles), was not used for this
technique. However, preparation of drug-loaded nanoparticles of MPEG-b-PCL
by the modified-SESD method has not been reported.
The aims of present study were to prepare surfactant-free drug-loaded
MPEG-b-PCL nanospheres by the modified-SESD method and to investigate
the influences of PCL block lengths and drug loading content on the nanosphere
characteristics and drug release behaviours. Ibuprofen was used as a poorly-water
soluble model drug. Interactions between ibuprofen and nanosphere matrices
were also determined.
MATERIALS AND METHODS
Materials: Methoxy poly (ethylene glycol) (MPEG) with a molecular
weight of 5,000 g mol-1 (Fluka, Germany) was dried at 120°C
under vacuum for 4 h before use. The ∈-caprolactone (CL) monomer
(99%, Acro, USA) was purified by drying with CaH2 followed
by distillation under reduced pressure before storage over molecular sieves
in a refrigerator. Stannous octoate (Sn(Oct)2, 95%, Sigma,
USA) was used as received. Acetone (Merck, Germany) and ethanol (Merck,
Germany) in analytical grade were used.
Methods
Synthesis of MPEG-b-PCL: MPEG-b-PCL diblock copolymers
with different molecular weights of PCL block were synthesized by ring-openning
polymerization of CL monomer in bulk at 130°C for 48 h under nitrogen
atmosphere. MPEG:CL feed mole ratios of 1:350 and 1:700 were used. Sn(Oct)2
and MPEG were used as the initiating system. Sn(Oct)2 concentration
was kept constant at 0.04 mol%. The MPEG-b-PCL products were purified
by dissolving them in chloroform before precipitation in cool n-hexane
and then drying to a constant weight under reduced pressure at room temperature.
The both purified MPEG-b-PCLs were obtained with approximately
95% yields.
Characterization of MPEG-b-PCL: Chemical compositions
of the MPEG-b-PCL were determined by 1H-nuclear magnetic
resonance (NMR) spectrometry using a Bruker Advanced DPX 300 1H-NMR
spectrometer. CDCl3 was used as a solvent at room temperature
and tetramethysilane was used as the internal standard. The number-average
molecular weight (Mn) and Molecular Weight Distribution (MWD)
were determined by Gel Permeation Chromatography (GPC) using a Waters
717 plus Autosampler GPC equipped with a Ultrastyragel®
column operating at 30°C. A refractive index detector was employed.
Tetrahydrofuran was used as the solvent at a flow rate of 1 mL min-1.
The thermal properties of the MPEG-b-PCL were characterized by
non-isothermal Differential Scanning Calorimetry (DSC) using a Perkin-Elmer
Pyris Diamond DSC. For DSC analysis, approximately 10 mg of the sample
was placed in a seal aluminium pan and heated at the rate of 10°C
min-1 under helium flow to measure the melting temperature
(Tm) and the heat of melting (ΔHm).
Preparation of ibuprofen-loaded nanospheres: Ibuprofen-loaded nanospheres
of the MPEG-b-PCL were prepared according to the modified-SESD method
without surfactant used (Baimark et al., 2008).
Briefly, 0.2 g of ibuprofen/MPEG-b-PCL mixtures with different ratios
were dissolved in 20 mL of the 3/3 (v/v) acetone/ethanol mixture solvent. The
ibuprofen with 1.25, 2.5 and 5.0% (w/w) were used. These solutions were added
drop-wise into 160 mL distilled water in a 250 mL beaker with stirring at 600
rpm. After evaporation of organic solvents at room temperature for 6 h in a
fume hood, the nanosphere colloid was centrifuged at 12,000 rpm for 1 h at 4°C
before freeze-drying for 48 h. Then dried nanospheres were obtained. The ibuprofen-free
nanospheres of both MPEG-b-PCL were also prepared by the same method
as controls.
Characterization of ibuprofen-loaded nanospheres: Functional
groups of ibuprofen and MPEG-b-PCL of the nanospheres and interactions
between them were studied by FTIR spectroscopy using a Perkin-Elmer Spectrum
GX FTIR spectrophotometer with air as the reference. The resolution of
4 cm-1 and 32 scans were used. FTIR spectra were obtained from
KBr disk of dried nanospheres. Particle sizes and size distributions of
the nanospheres with and without ibuprofen loading were directly determined
from their nanosphere colloids by light-scattering analysis using a Coulter
LS230 light scattering particle size analyzer at 25°C. Morphology
of the nanospheres was investigated by Scanning Electron Microscopy (SEM)
using a JEOL JSM-6460LV SEM. Before SEM measurement, the dried nanospheres
were sputter coated with gold for enhancing the surface conductivity.
Thermal properties of the dried nanospheres were measured by DSC as described
earlier.
Ibuprofen loading content and encapsulation efficiency: Ibuprofen
loaded in nanospheres was determined by UV-Vis spectroscopy using a Perkin-Elmer
Lambda 25 UV-Vis spectrophotometer at 264 nm (Borovac et
al., 2006). The ibuprofen-loaded nanospheres were dissolved in
dichloromethane for this purpose. Ibuprofen loading content and encapsulation
efficiency were calculated from Eq. 1 and 2,
respectively. The measurement was performed in triplicate.
In vitro ibuprofen release: An exact amount (about 10 mg) of
ibuprofen-loaded nanospheres was disclosed into a dialysis bag (molecular weight
cut off was 8,000 to 12,000) and immersed into a flask containing 150 mL of
Phosphate Buffer Solution (PBS) at pH 7.4. The sample flasks were incubated
at 37°C under shaking at the rate of 150 rpm. At predetermined time intervals,
10 mL samples were withdrawn and 10 mL of fresh PBS was added into the flask
for continuing the release test. The released ibuprofen was measured by UV-Vis
spectrophotometer at 220 nm (Borovac et al., 2006).
According to a predetermined ibuprofen concentration-UV absorbance standard
curve, ibuprofen concentration of the release medium was obtained and ibuprofen
(%) released was calculated. The average value was calculated from the three
measurements.
RESULTS
Characterization of MPEG-b-PCL: The chemical compositions
of MPEG-b-PCLs were determined from the 1H-NMR spectra
by calculating the ratio of the integral peak areas corresponding to the
Ethylene Oxide (EO, repeating units of MPEG) methylene protons at δ
= 3.6- 3.7 ppm and the CL ∈-methylene protons at δ = 4.0-4.2
ppm. From the peak area integrations of the peaks a and b in Fig.
1a and b, the copolymer compositions can be determined
as EO:CL = 24:76 and 14:86 (mol%) corresponding to the MPEG:CL mole ratios
of 1:361 and 1:701 for the MPEG-b-PCL40000 and the MPEG-b-PCL80000,
respectively.
The Mns of MPEG-b-PCLs obtained from GPC curves were
44,000 and 82,500 g mol-1 for the MPEG-b-PCL40000 and
the MPEG-b-PCL80000, respectively. The DSC curve of MPEG in Fig.
2a showed the melting temperature (Tm) and the heat of
melting (ΔHm) and they were found to be 61°C and 177.4
J g-1, respectively. The DSC curves of MPEG-b-PCL revealed
a semi-crystalline morphology with a single-Tm as shown in
Fig. 2b, c. The Tms of
MPEG-b-PCL were 58 and 60°C, whereas the ΔHms
were 92.8 and 101.2 J g-1 for the MPEG-b-PCL40000 and
the MPEG-b-PCL80000, respectively.
|
|
| Fig. 1: |
1H-NMR spectra of (a) MPEG-b-PCL40000
and (b) MPEG-b-PCL80000 |
Characterization of nanospheres: In this study, the drug-loaded
nanospheres of MPEG-b-PCL were firstly prepared by the modified-SESD
method without any surfactants for used as controlled release drug delivery
systems.
Functional groups and interactions of the MPEG-b-PCL nanosphere
matrices and the loaded ibuprofen were determined from FTIR spectra, as
example of which is shown in Fig. 3a-e
for the MPEG-b-PCL80000 nanosphere (No. 5-8) and the ibuprofen.
The FTIR spectra of the pure MPEG-b-PCL nanospheres (Fig.
3a) and the ibuprofen (Fig. 3e) showed carbonyl
absorption bands at 1761 and 1721 cm-1, respectively. The FTIR
spectra of ibuprofen-loaded nanospheres of MPEG-b-PCL40000 with
different drug loading contents showed similar evidence (Fig.
3a-e).
The surfactant-free nanosphere colloids with and without ibuprofen loading
were clear aqueous suspensions. The sizes of colloidal nanospheres were
investigated by the light-scattering analysis.
|
| Fig. 2: |
DSC thermograms of (a) MPEG, (b) MPEG-b-PCL40000 and 8 MPEG-b-PCL80000 |
|
| Fig. 3: |
FTIR spectra of nanosphere No. (a) 5, (b) 6, 8 7 and (d) 8 and (e)
ibuprofen |
|
| Fig. 4: |
Particle size graphs of nanosphere No. (a) 5, (b) 6, 8 7 and (d)
8 |
| Table 1: |
Particle sizes and thermal properties of ibuprofen-loaded
MPEG-b-PCL nanospheres |
 |
| aNanospheres of MPEG-b-PCL40000, bNanospheres
of MPEG-b-PCL80000, cObtained from light-scattering
analysis, dObtained from DSC thermograms |
Their sizes were found
in the range of 82 to 97 nm. These data are shown in Fig. 4a-d and
summarized in Table 1. The average particle size of surfactant-free
nanosphere colloids of the MPEG-b-PCL40000 was slightly larger than the
MPEG-b-PCL80000. However, the different drug loading contents did not
significantly effect to their average particle sizes.
The morphologies of nanospheres were determined from SEM micrographs
as examples are shown in Fig. 5a and b
for the nanosphere No. 4 and 8. It was found that the colloidal nanospheres
have a spherical shape in the nanometer size range and smooth surfaces.
The particle sizes significantly decreased as the PCL block length increased
according to the light-scattering analysis. However, the nanospheres observed
from SEM micrographs were similar in size.
The Tm and the ΔHm of ibuprofen measured from
DSC curve were 74°C and 116.4 J g-1, respectively, as shown
in Fig. 6.
|
|
| Fig. 5: |
SEM micrographs of nanosphere No. (a) 4 and (b) 8 (bar = 1 μm) |
|
| Fig. 6: |
DSC thermogram of ibuprofen |
The ibuprofen crystallites entrapped in the nanospheres were not detected
as example of which is shown in Fig. 7a-d
for the MPEG-b-PCL80000 nanospheres. The thermal properties of
nanospheres are also shown in Table 1. In addition,
the both Tm and ΔHm of MPEG-b-PCL nanosphere
matrices significantly decreased as the drug loading content increased.
|
| Fig. 7: |
DSC
thermograms of nanosphere No. (a) 5, (b) 6, 8
7 and (d) 8 |
|
|
| Fig. 8: |
(a, b) Ibuprofen release profiles of nanosphere No. |
Ibuprofen loading content and encapsulation efficiency: The calculated
results of ibuprofen loading content and encapsulation efficiency are
also shown in Table 1. It was found that the ibuprofen-loaded
nanospheres with different ibuprofen loading contents can be prepared
by using different ibuprofen feed ratios. The ibuprofen encapsulation
efficiencies were in the range of 48-64%. These values of the MPEG-b-PCL80000
were slightly lower than the MPEG-b-PCL40000.
In vitro release of ibuprofen: The ibuprofen release profiles
from the nanospheres of MPEG-b-PCL40000 and MPEG-b-PCL80000
investigated in PBS pH 7.4 at 37°C are shown in Fig.
8a and b. The release profiles of ibuprofen-loaded
nanospheres were biphasic containing rapid initial burst release and sustaining
release. The initial burst releases from the MPEG-b-PCL80000 nanospheres
were approximately 60% at the first 3 h of release time (Fig.
8a, b). Then, release profiles
were followed by a constant slow release until to 98, 95 and 85% ibuprofen
released within 312 h with ibuprofen loading contents of 0.8, 1.6 and
3.0%, respectively for the MPEG-b-PCL40000 nanospheres.
The drug release behaviors from the MPEG-b-PCL80000 nanospheres
also showed similar phenomenon. Moreover, Fig. 8a and
b showed that the drug loading content was an important
factor of the drug release rate. The nanospheres with lower drug loading
content showed the higher drug release rate. Finally, the drug release
from the nanospheres of MPEG-b-PCL40000 was faster than that of
MPEG-b-PCL80000.
DISCUSSION
Characterization of MPEG-b-PCL: As could be expected, the copolymer
compositions obtained from the 1H-NMR were very similar to the MPEG:CL
feed mole ratios (1:350 and 1:700 for MPEG-b-PCL40000 and MPEG-b-PCL80000,
respectively). Therefore, the synthesized reaction was taken to near-quantitative
conversion. The Mns obtained from GPC (44000 and 82500 g mol-1
for MPEG-b-PCL40000 and MPEG-b-PCL80000, respectively) were closely
similar to that obtained from the feed ratios (45000 and 85000 g mol-1
for MPEG-b-PCL40000 and MPEG-b-PCL80000, respectively). The lower
molecular weights obtained from GPC curves may be due to the degradation-side
reactions such as hydrolysis and thermal degradation reactions. From DSC results
suggested that the both MPEG and MPEG-b-PCL contained semi-crystalline
morphology. The MPEG crystallinity was disappeared when the MPEG block was connected
with the poly (D, L-lactide) (PDLL) block as previously described by Baimark
et al. (2007). Then the obtained MPEG-b-PDLL showed completely
amorphous structure. Thus, the crystallinity of MPEG was also suppressed when
connected to the PCL block. It can be concluded that the crystallinity of MPEG-b-PCL
synthesized in this study can be attributed to PCL crystallites.
Characterization of nanospheres: The carbonyl absorption band at 1721
cm-1 attributed to the crystalline form of ibuprofen (Kazarian
and Martirosyan, 2002) which shifting to higher wave number for the ibuprofen
loaded in nanospheres as shown in Fig. 3b-d
suggested that the ibuprofen-ibuprofen interactions in its crystalline fraction
were destroyed when the ibuprofen was entrapped in the MPEG-b-PCL nanosphere
matrices. This may be indicated that the ibuprofen molecules were well distributed
throughout the nanosphere matrices.
The broader carbonyl absorption bands in the region of 1785-1735 cm-1
were assigned to shift of the carbonyl bands of the both MPEG-b-PCL and
ibuprofen indicated the existence of interactions between the MPEG-b-PCL
nanosphere matrices and the loaded ibuprofen. These intermolecular interactions
were expected as hydrogen bonding between carbonyl groups of MPEG-b-PCL
and hydroxyl groups of ibuprofen corresponding to the hydrogen bonding between
carbonyl groups of poly (vinylpyrrolidone) matrices and loaded ibuprofen as
previously reported by Kazarian and Martirosyan (2002).
The intermolecular bonding between MPEG-b-PCL40000 nanosphere matrices
and loaded ibuprofen can be also detected from their FTIR spectra. In addition,
intensities of the absorption band at 1560 cm-1 assigned to ibuprofen
characteristics increased as the increasing ibuprofen content for the all ibuprofen-loaded
nanospheres supported that the ibuprofen contents were strongly depended upon
the ibuprofen feed ratio.
The sizes of nanospheres with and without ibuprofen loading measured
from the both light-scattering and SEM analyses were in the nanometer
size ranges. The nanosphere sizes obtained from the SEM looked larger
than those obtained from the light-scattering analysis. It was due to
the colloidal nanospheres, which were slightly flatten during the centrifugation
and drying processes before SEM measurement. The nanospheres of MPEG-b-PCL80000
were slightly smaller than the MPEG-b-PCL40000. The MPEG-b-PCL
with higher ΔHm usually leads to higher crystallinity
that was larger self-condensed than the lower one during nanosphere solidification.
The MPEG-b-PCL80000 contained higher crystallinity than the MPEG-b-PCL40000.
Therefore, the nanosphere sizes decreased when the molecular weight of
PCL block was increased.
The DSC results indicated that the MPEG-b-PCL nanospheres and
the ibuprofen contained crystalline structures. It is significant to note
that the ibuprofen crystallizability in the all of drug-loaded nanospheres
was suppressed after loaded into the MPEG-b-PCL nanospheres supported
that the ibuprofen loaded in the nanospheres had a completely amorphous
state corresponding to the FTIR results as described earlier. Meanwhile,
the increasing drug content can also inhibited the PCL crystallization
due to the ibuprofen interpenetrated between the MPEG-b-PCL chains.
It concluded that the ibuprofen was well distributed into the MPEG-b-PCL
nanosphere matrices.
Ibuprofen loading content and encapsulation efficiency: The drug loading
content of the ibuprofen-loaded MPEG-b-PCL nanospheres was strongly depended
upon the drug feed ratio. The obtained ibuprofen loading contents as shown in
Table 1 were lower than the ibuprofen feed ratios of 1.25,
2.5 and 5.0% (w/w) for the nanosphere No. 2, 6, 3, 7 and 4, 8, respectively.
This can be indicated that the some drug has released out during the nanosphere
formation. The changes of ibuprofen encapsulation efficiency directly related
to the ibuprofen loading content (Eq. 1, 2).
The both ibuprofen loading content and encapsulation efficiency of the drug-loaded
nanospheres decreased when the PCL block length increased for the same ibuprofen
feed ratios. This could be due to higher PCL crystallinity which could decrease
the drug loading content, the drug molecules were squeezed out because crystallization
of PCL blocks, as only the amorphous PCL phase is likely to accommodate drug
molecules (Shuai et al., 2004).
In vitro release of ibuprofen: The rapid initial burst
releases of drug from the nanospheres were probably due to the releasing
of drug that was entrapped or adsorbed near to the nanosphere surfaces.
After that the slow release may be due to diffusion and matrix erosion
mechanisms. The drug release rates increased when the drug loading content
increased. This may be due to higher ibuprofen loading content and encapsulation
efficiency. Finally, a faster rate of ibuprofen releasing was obtained
when a less hydrophobic matrix (MPEG-b-PCL40000) due to the shorter
PCL block length was used.
CONCLUSION
The MPEG-b-PCL diblock copolymers with different PCL block lengths
were successfully synthesized by ring-opening polymerization of the CL
monomer using Sn(Oct)2 and MPEG as the initiating system. They
were semi-crystalline diblock copolymers. The surfactant-free nanospheres
of the MPEG-b-PCL with and without ibuprofen, a hydrophobic model
drug, loading were successfully prepared by the modified-SESD method using
an acetone/ethanol mixture as the organic solvent. The average sizes of
nanospheres were less than 100 nm. The prepared nanospheres looked spherical,
had a smooth surface and a narrow size distribution under SEM. The nanosphere
size and drug encapsulation efficiency were found mainly affected by the
drug loading and the PCL block length. The drug release rate can be optimally
controlled by adjusting these parameters.
ACKNOWLEDGMENTS
The author would like to acknowledge the Research Development and Support
Unit, Mahasarakham University and the Center of Excellence for Innovation
in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of
Education, Thailand for financial supports.
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