In recent year, there has been an increasing interest in biodegradable star-shaped
polyesters of poly(L-lactide) (Wang and Dong, 2006;
Zhang and Zheng, 2007), poly(D,L-lactide) (Srisa-ard
and Baimark, 2010) and poly(ε-caprolactone) (Xie
and Gan, 2009) which are branched polymers distinguished by a structure
containing three or more linear arms radiating from a center. The star-shaped
polyesters are expected to display peculiar viscosity, thermal and mechanical
properties and degradation profiles compared with linear polyesters (Odelius
and Ann-Christine, 2008). The polyesters with different arm numbers have
been synthesized using initiators containing different hydroxyl end-groups.
The chemical structures in each arm were the same. Influences of arm number
and arm length on crystallinity, melting temperature and thermal degradation
of these star-shaped polyesters have been reported.
Methoxy poly(ethylene glycol) (MPEG) blocks have been attached to polyester
blocks as biodegradable amphiphilic diblock copolymers for increasing its hydrophilicity
and flexibility. These amphiphilic diblock copolymers have been prepared as
nanoparticle, film and tube forms for biomedical applications (Baimark
et al., 2008; Srisuwan et al., 2008;
Baimark and Phromsopha, 2009; Khamhan
and Yodthong, 2009; Kotseang et al., 2009;
Phromsopha and Baimark, 2009). Biodegradable amphiphilic
MPEG-b-polyester diblock copolymers have been prepared as the surfactant-free
nanoparticles for drug controlled release (Aliabadi et
al., 2005; Baimark, 2009). These nanoparticles
are core-shell structure with hydrophobic polyester core and hydrophilic MPEG
shell. The MPEG blocks enhanced retention time of diblock copolymeric nanoparticles
in the human blood system.
The star-shaped MPEG-b-polyester block copolymers have been synthesized
and reported by many researchers (Cai et al., 2006;
Lemmouchi et al., 2007; Wang
et al., 2008; Lin and Zhang, 2010). However,
the drug-loaded nanoparticle characteristics and drug release behaviors of the
star-shaped MPEG-b-polyester block copolymers have been scarcely published
(Quaglia et al., 2006).
In this study, the influence of arm number of poly(L-lactide)-b-MPEG (PLL-b-MPEG) on characteristics and drug release behaviors of drug-loaded PLL-b-MPEG nanoparticles was investigated. The PLL with arm numbers of 1, 4 and 6 were synthesized before coupling with carboxylic acid end-group MPEG.
MATERIALS AND METHODS
This study was conducted on November 2010-October 2011 at Mahasarakham University, Mahasarakham, Thailand.
Materials: L-Lactide (LL) monomer was synthesized by well established procedure from L-lactic acid (88% Purac, Thailand). LL was purified by repeated recrystallization from distilled ethyl acetate for 4 times and dried in a vacuum oven at 45°C for 48 h before use. 1-dodeccanol (98%, Fluka, Switzerland) was purified by distillation under reduced pressure before being stored over molecular sieves. Pentaerythritol (99%, Aldrich, USA) and dipentaerythritol (99%, Aldrich, USA) were dried in a vacuum oven at 100°C for 24 h before use. Methoxy poly(ethylene glycol) (MPEG, Fluka, USA) with molecular weight of 5,000 g mol-1 was dried at 120°C in a vacuum oven for 4 h before use. Stannous octoate (Sn(Oct)2, 95% Sigma, USA), Dicyclohexylcarbodiimide (DCC, 99% Fluka, USA), 4-dimethylaminopyridine (DMAP, 99% Fluka, USA), succinic anhydride (99% Acros Organic, USA), triethylamine (99% Acros Organic) and indomethacin (99%, Sigma, USA) were used without further purification. All solvents in analytical grade were used.
Synthesis of poly(L-lactide): The poly(L-lactide)s (PLL) with arm numbers of 1, 4 and 6 were polymerized in bulk at 140°C for 24 h under nitrogen atmosphere. The 1-dodecanol, pentaerythritol and dipentaerythritol were used as hydroxyl end-group initiators to prepare linear, 4-armed and 6-arm PLLs, respectively. LL/initiator ratio of 208/1 by mole was used. The theoretical molecular weight of PLLs calculated from feed ratio was approximately 30,000 g mol-1. Hydroxyl end-group compound and Sn(Oct)2 were used as the initiating system. Sn(Oct)2 concentration was kept constant at 0.02 mol%. The as-polymerized PLL was purified by being dissolved in chloroform before precipitating in cool n-hexane. The PLL was dried to constant weight in a vacuum oven at room temperature.
Synthesis of carboxylic acid end-group methoxy poly(ethylene glycol):
Carboxylic acid end-group methoxy poly(ethylene glycol) (MPEG-COOH) was synthesized
by changing a hydroxyl end-group of MPEG to a carboxylic acid end-group. For
this purpose, MPEG (10 g, 2.0 mmol), succinic anhydride (304.8 mg, 3.0 mmol),
DMAP (246.4 mg, 2.0 mmol) and triethylamine (203.6 mg, 2.0 mmol) were dissolved
in dioxane (13 mL) and vigorous stirring for 48 h at room temperature. The solvent
was then completely evaporated. The obtained residue was dissolved in methylene
chloride before precipitating in ether. The precipitated MPEG-COOH was then
dried in a vacuum oven at 40°C overnight.
Synthesis of PLL-b-MPEG: The linear and star-shaped PLL-b-MPEG diblock copolymers were prepared using the coupling reaction between the hydroxyl end-group of PLL and the carboxylic acid end-group of MPEG-COOH. The coupling reaction was illustrated in Fig. 1. For this purpose, PLL, MPEG-COOH, DCC and DMAP were dissolved in anhydrous methylene chloride (4.0 mL) and stirring for 48 h under nitrogen atmosphere at room temperature. The amounts of substances were summarized in Table 1. The acetone was added to precipitate dicyclohexylcarbodiurea by-product and residue DCC before filtering off. The filtered solution was dried by solvent evaporation. The dried solid was dissolved in chloroform before extraction with a 0.5 wt% HCl solution, followed by water. The obtained PLL-b-MPEG was dried over anhydrous Na2SO4 before evaporation.
Characterization of PLL and PLL-b-MPEG: The intrinsic viscosity, [η], of PLL and PLL-b-MPEG were determined from flow-time measurements on a diluted series of solutions in chloroform as solvent at 30°C using viscometrically. Molecular weight characteristics of the samples were characterized by Gel Permeation Chromatography (GPC) using a Waters 717 plus Autosampler GPC equipped with an Ultrastyragel® column operating at 40°C and employing universal calibration. Chloroform was used as the solvent at a flow rate of 1 mL min-1. Thermal transition properties of the samples were carried out by means of Differential Scanning Calorimetry (DSC) using a Perkin-Elmer DSC Pyris Diamond. For DSC, the sample (3-5 mg) was sealed in aluminum pan before heated at 10°C/min under a helium atmosphere.
Preparation of drug-loaded nanoparticles: Surfactant-free drug-loaded
PLL-b-MPEG nanoparticles were prepared via a nanoprecipitation method.
Briefly, 5 mg of indomethacin and 80 mg of copolymer were dissolved in 8 mL
of acetone/chloroform (7/1 v/v) mixture. The organic solution was added drop-wise
into 80 mL of distilled water under magnetic stirring.
|| Synthesis of PLL-b-MPEG by coupling reaction
|aThe theoretical number-average molecular weight,
as calculated from feed LL/initiator mole ratio
|| Coupling reaction of PLL with MPEG-COOH
The nanoparticles were immediately formed after solvent diffusion. The organic
solvents were then evaporated at room temperature for 6 h in a fume hood. The
resultant nanoparticle suspension was centrifuged at 15,000 rpm 4°C for
2 h. The supernatant was carefully discarded and the precipitated nanoparticles
were re-suspended in a phosphate buffer solution media (0.1 M, pH 7.4). The
dried drug-loaded nanoparticles were obtained by freeze-drying the precipitated
Characterization of drug-loaded nanoparticles: Morphology of the drug-loaded nanoparticles was determined by Transmission Electron Microscopy (TEM) using a JEOL JEM 1230 TEM. For TEM analysis, a drop of nanoparticle suspension was placed on a formvar film coated on the copper grid. The specimen on the copper grid was not stained. Average particle size of the drug-loaded nanoparticles was measured from the nanoparticle suspension by Light Scattering (LS) analysis using a Coulter LS230 particle size analyzer at 25°C. Thermal transition properties of the freeze-dried nanoparticles were carried out by mean of DSC as described above.
Theoretical drug loading content (DLCtheoretical), actual drug loading
content (DLCactual) and Drug Loading Efficiency (DLE) were calculated
from Eq. 1-3, respectively. The DLCactual
is an average value from three measurements. For DLCactual measurement,
the freeze-dried sample of drug-loaded nanoparticles was dissolved in dichloromethane.
The weight of actual drug entrapped in the drug-loaded nanoparticles was determined
by UV-vis spectrophotometry using a Perkin-Elmer Lambda 25 UV-vis spectrophotometer
at 319 nm compared to standard curve of indomethacin:
In vitro drug release test: In vitro drug release from
nanoparticles was performed by dialysis bag diffusion technique. Ten mL of drug-loaded
nanoparticle re-suspension was placed in a dialysis bag, tied and immersed into
100 mL phosphate buffer solution (0.1 M, pH 7.4). The entire system was kept
at 37°C with horizontal shaking at about 150 rpm.
At predetermined time intervals, 5 mL of aliquots of the release medium were withdrawn from the release medium and the same volume of fresh buffer solution was added for continuing the drug release test. The concentration of indomethacin released was monitored using an UV-vis spectrophotometer at 319 nm. According to a predetermined indomethacin concentration-UV absorbance standard curve, indomethacin concentration of the release medium was obtained. Percentage of indomethacin release was calculated based on ratio of drug release in each release time and initial drug content within nanoparticles. The average % release was calculated from the three measurements.
Synthesis and characterization of PLL and PLL-b-MPEG: The PLLs with different arms were synthesized through ring-opening polymerization of LL monomer by using different hydroxyl end-group compounds. The % yields of resultant linear (1-arm) and star-shaped (4-and 6-arm) PLLs after purification were higher than 95%. The intrinsic viscosity ([η]), number-average molecular weight (Mn) and Molecular Weight Distribution (MWD) of the PLLs are summarized in Table 2. It was found that the [η] of PLLs slightly decreased as the arm number increased, whereas the Mn and MWD of the PLLs are in range of 25, 100-26, 500 g mol-1 and 1.2-1.4, respectively. The PLL-b-MPEG was prepared by coupling hydroxyl end group of PLL with MPEG-COOH. The % yields of PLL-b-MPEG were higher than 90%. The [η], Mn and MWD of PLL-b-MPEG are also reported in Table 2. The [η] and Mn of PLL increased but MWD did not after coupling with MPEG-COOH. It should be noted that the increasing of [η] and Mn of PLL after coupling MPEG-COOH are in order 6-arm>4-arm >1-arm PLL.
Thermal transition properties such as a melting temperature (Tm) and a heat of melting (ΔHm) of the PLL and PLL-b-MPEG were determined from DSC analysis. The DSC results are reported in Table 3. The Tm and ΔHm of PLLs decreased significantly when the arm number was increased. Degree of crystallinity (χc) of the PLLs directly related to its ΔHm is also summarized in Table 3 that decreased as the arm number increased. DSC thermograms of the PLL-b-MPEG are shown in Fig. 2. The Tm, ΔHm and χc of the PLL-b-MPEG were slightly lower than the starting PLL. The higher arm number PLL-b-MPEG showed lower Tm and ΔHm. In addition, the crystallinity of MPEG block was suppressed after coupling with PLL.
||Intrinsic viscosity and molecular weight characteristics of
PLL and PLL-b-MPEG
|aIntrinsic viscosity ([η]) was determined
at 30°C using chloroform as the solvent. bNumber-average
molecular weight (Mn) and Molecular Weight Distribution (MWD)
were measured from GPC analysis
|| Thermal transition properties of PLL and PLL-b-MPEG
|aMelting temperature (Tm) and heat of
melting (ΔHm) were determined from DSC analysis. bDegree
of crystallinity (χc) was calculated from χc
||DSC thermograms of (a) 1-arm PLL-b-MPEG, (b) 4-arm
PLL-b-MPEG and 6-arm PLL-b-MPEG
Preparation and characterization of drug-loaded nanoparticles: The surfactant-free
PLL-b-MPEG nanoparticles containing indomethacin were prepared by the
nanoprecipitation. The morphology of drug-loaded nanoparticles was determined
from TEM micrographs as shown in Fig. 3. It can be seen that
they were nearly spherical in shape. The average size of nanoparticles was measured
by light-scattering analysis, as example of which is shown in Fig.
4 for the drug-loaded nanoparticles of linear PLL-b-MPEG. The particle
sizes were less than 200 nm with narrow size distribution. The average sizes
of drug-loaded nanoparticles of PLL-b-MPEG are reported in Table
4. They were in range of 118-128 nm. This indicates that the arm number
of PLL-b-MPEG did not affect average particle size of the drug-loaded
nanoparticles. The Tm and ΔHm of nanoparticles determined
from DSC thermograms in Fig. 5 and reported in Table
4 were slightly lower than its PLL-b-MPEG (Table 3).
||TEM images of drug-loaded nanoparticles of (a) 1-arm PLL-b-MPEG,
(b) 4-arm PLL-b-MPEG and (c) 6-arm PLL-b-MPEG. All bars =
|| Particle size graph of drug-loaded 1-arm PLL-b-MPEG
||DSC thermograms of drug-loaded nanoparticles of (a) 1-arm
PLL-b-MPEG, (b) 4-arm PLL-b-MPEG and (c) 6-arm PLL-b-MPEG
||Drug release profiles from nanoparticles of 1-arm PLL-b-MPEG,
4-arm PLL-b-MPEG and 6-arm PLL-b-MPEG
|| Average particle size and drug content of drug-loaded nanoparticles
|aMelting temperature (Tm) and heat of
melting (ΔHm) were determined from DSC analysis. bAverage
particle size was measured from light-scattering analysis. cActual
drug loading content (DLCactual) and Drug Loading Efficiency
(DLE) were calculated from Eq. 2 and 3,
The theoretical drug loading content (DLCtheoretical) of the drug-loaded nanoparticles calculated from Eq. 1 is 5.88%. The actual drug loading content (DLCactual) and Drug Loading Efficiency (DLE) of the drug-loaded nanoparticles were calculated from Eq. 2 and 3, respectively that are also summarized in Table 4. The DLCactual and DLE of the drug-loaded nanoparticles are similar that in range of 2.11-2.48 and 36-42%, respectively.
In vitro drug release: Figure 6 shows in vitro drug release patterns from PLL-b-MPEG nanoparticles with different arm numbers in phosphate buffer solution pH 7.4 at 37°C for 21 days. It can be clearly observed that the drug release profiles exhibit biphasic containing rapid initial burst release within the first day of release time followed with sustained release. The % drug releases at 21 days of release time are 41, 72 and 82% for 1-, 4-and 6-arm PLL-b-MPEG nannoparticles, respectively. The drug release significantly increased as the arm number increased. The results suggested that the drug release pattern strongly depended upon the arm number of PLL-b-MPEG.
Synthesis and characterization of PLL and PLL-b-MPEG: The hydroxyl
end-group compound/Sn(Oct)2 system has been widely used to polymerize
the LL monomer (Aliabadi et al., 2005; Wang
and Dong, 2006). The shape or arm number of PLL depended upon the hydroxyl
group of co-initiator. The 1-dodecanol, pentaerythritol and dipentaerythritol
containing 1, 4 and 6 hydroxyl end-groups have used to prepare 1-, 4-and 6-arm
PLLs, respectively (Srisa-ard and Baimark, 2010). The
high % yields (>95%) of PLLs in this work suggested that the polymerization
condition (140°C for 24 h) was appropriate. Table 2 reports
the [η], Mn and MWD of the PLLs and PLL-b-MPEG. The [η]
of PLL decreased steadily as the arm number increased. The [η] of PLL solution
directly related to hydrodynamic volume of PLL molecule in solution state. The
higher arm number polyester exhibited smaller hydrodynamic volume (Wang
and Dong, 2006; Srisa-ard and Baimark, 2010). The
hydrodynamic volume of PLL molecules decreased as the arm number increased for
the similar molecular weight. Thus, the higher arm number PLL induced lower
the [η] value. However, the Mn and MWD of these PLLs from GPC
For PLL-b-MPEG preparation, the hydroxyl end-group of PLL was reacted
with carboxylic acid end-group of MPEG-COOH, as shown in Fig.
1. The [η] and Mn of PLL-b-MPEG in Table
2 were higher than the starting PLL for the same arm number. The results
supported that the PLL reacted with MPEG to form as the block copolymer. The
coupling reaction did not affect the MWD. The 6-arm PLL-b-MPEG showed
the highest increasing of [η] and Mn. This may be due to the
6-arm PLL contained 6 hydroxyl end-groups that connected to 6 molecules of MPEG-COOH
according to the literature (Wang et al., 2008).
From DSC results in Table 3, it can be seen that the 1-arm
PLL exhibited the highest Tm, ΔHm and χC
values. These values of the PLL decreased as the arm number increased according
to the literatures (Wang and Dong, 2006; Zhang
and Zheng, 2007). The arm length of 1-arm PLL was longer than the 4-and
6-arm PLLs. The longer arm length PLL induced higher Tm, Δhm
and χC. For PLL-b-PEG, these thermal properties slightly
decreased when the PLL block was attached to MPEG block. The MPEG block may
inhibit crystallizing of PLL block. Moreover, the crystallinity of MPEG block
within PLL-b-MPEG disappeared. This confirmed the attachment between
PLL and MPEG blocks. The rigid PLL blocks prevented crystallization of flexible
MPEG blocks. Suppression of MPEG crystallizability by copolymerizing poly(D,L-lactide)
block has been reported in our previous works (Kotseang
et al., 2009; Baimark and Phromsopha, 2009).
Preparation and characterization of drug-loaded nanoparticles: The emulsification-diffusion
method was used to prepare the surfactant-free nanoparticles of amphiphilic
diblock copolymers using acetone/chloroform mixture as the organic solvent.
The MPEG blocks can act as stabilizer to prevent nanoparticle aggregation (Baimark
et al., 2008). The core-shell nanoparticles with nearly spherical
shape were formed. From Table 4, the drug-loaded nanoparticles
with 118-128 nm in size were prepared with narrow size distribution. The results
suggested that the MPEG blocks of PLL-b-MPEG with different arm numbers
showed similar effective to stabilize the nanoparticle formation.
The Tm and ΔHm of the PLL-b-MPEG (Table 3) were slightly decreased in the nanoparticle matrix form (Table 4). This may be due to the drug molecules distributed into the nanoparticles that inhibited crystallizing of PLL-b-MPEG. However, the arm number of PLL-b-MPEG did not affect the DLE of nanoparticles.
In vitro drug release: The effect of arm numbers of PLL-b-MPEG on in vitro drug release behavior from PLL-b-MPEG nanoparticles are illustrated in Fig. 6. The rapid initial burst release within the first day from the nanoparticles was probably due to the releasing of drug that entrapped on the nanoparticle surfaces. After that the sustain release may be due to drug diffusion mechanism through nanoparticle matrix. The drug release contents are in order 6-arm>4-arm>1-arm PLL-b-MPEG. This may be explained that the drug can easier diffuse through the amorphous phase than the crystalline phase. From Table 3, the crystallinity values of PLL-b-MPEG are in order 1-arm>4-arm>6-arm PLL-b-MPEG. Thus the amorphous values of PLL-b-MPEG are in order 6-arm>4-arm>1-arm PLL-b-MPEG.
In the present study, the nanoparticles of linear and star-shaped PLL-b-MPEG
were prepared by the emulsification-diffusion method for controlled-release
of a poorly water-soluble model drug, indomethacin. The drug-loaded nanoparticles
with less than 200 nm in size had nearly spherical in shape. The different arm
numbers of PLL-b-MPEG did not affect the average size, shape and DLE
of the drug-loaded nanoparticles. However, the drug release contents from PLL-b-MPEG
nanoparticles were in order 6-arm>4-arm>1-arm PLL-b-MPEG. Therefore,
the drug release rate can be tailored by varying the arm number of PLL-b-MPEG
nanoparticle matrix. These nanoparticles may have potential to provide other
poorly water-soluble drugs for use as controlled-release drug delivery systems.
This study was supported by the Mahasarakham University and the Center of Excellence for Innovation in Chemistry (PERCH-CIC), Commission on Higher Education, Ministry of Education, Thailand.