Star-Shaped Polycaprolactone-Polyethyleneglycol Copolymer Micelle-Like Nanoparticles for Picropodophyllin Delivery

Jing Zhao, Yajing Wang, and Libiao Luan∗

Abstract

The purpose of this work was to develop a novel picropodophyllin-loaded micelle-like nanoparticle with a biodegradable amphiphilic star-shaped polycaprolactone-polyethyleneglycol copolymer (S-PCL-PEG). S-PCL-PEG was synthesized using star-shaped polycaprolactone (S-PCL) as a hydrophobic block and monomethoxy polyethyleneglycol (PEG) as a hydrophilic block and characterized by 1H-NMR. It was confirmed by the pyrene fluorescence probe method that the obtained S-PCL-PEG could form micelles through self-assembly in aqueous media. In addition, picropodophyllin (PPP), a hydrophobic anticancer drug, could be entrapped in the hydrophobic inner core of the micelles using the thin film hydration method, forming PPP-loaded micelle-like nanoparticles (PPP-NPs). PPP-NPs had a high encapsulation efficiency of greater than 90%, an average size of 90–110 nm with a symmetrical monodisperse distribution and a zeta potential of −18 mV. Additionally, in vitro release tests showed that approximately 70% of the drug was released from PPP-NPs intoDelivered by Publishing Technology to: Rice University PBS (pH 7.4) containing 0.2% Tween 80 at 37IP: 117.245.76.159 On: Thu, 07 Jan 2016 04:06:57C for 96 h, and the drug release data fit well to the Higuchi equation. Furthermore, an in vitro tumor cell growth inhibition assay showed that the ICCopyright: American Scientific Publishers50 values of the PPP solution and PPP-NPs against SMMC7721 liver cancer cell lines were 0.4 g/ml and 0.2 g/ml respectively, which indicated that the cytotoxicity of PPP-NPs against tumor cells was greater than that of the PPP solution. In conclusion, S-PCL-PEG micelle-like nanoparticles loaded with PPP have a promising future for administration by injection.

KEYWORDS: Picropodophyllin, Star-Shaped Polycaprolactone-Polyethyleneglycol, Nanoparticle, Thin Film Hydration, In Vitro Release, Cytotoxicity.

INTRODUCTION

Many anti-cancer drugs usually show poor water solubility, thus presenting one of the most frequent and greatest challenges for drug development. Recently, nanotechnology has been employed to improve the poor water solubility of hydrophobic drugs.1–10 The use of nanotechnology enables these hydrophobic drugs to be entrapped in the form of nanoscale particles. Such nanoparticles can be well-dispersed in aqueous solutions to produce a stable and homogenous suspension, thus meeting the requirements of clinical parenteral administration. Additionally, nanoparticles may be able to deliver more anticancer drugs to specific tumor tissues or cells than to normal tissues because of their enhanced permeability and retention (EPR) effect. Therefore, various biomaterials like polymeric micelles,1–10 nanoparticles,1112 liposomes13 and polymer-drug conjugates14 have been investigated as potential carriers for hydrophobic anticancer drug delivery.
Synthetic amphiphilic copolymers are able to selfassemble into micelle-like polymeric nanoparticles in aqueous solutions with a hydrophobic inner core and a hydrophilic outer shell.15–17 The hydrophobic core, serving as the sustained release reservoir of insoluble molecules or unstable agents, is mainly composed of aliphatic polyesters, typically poly(-caprolactone) (PCL), poly(lactide) (PLA) or their copolymers. The hydrophilic shell commonly consists of polyethyleneglycol and works as a hydrated steric barrier to avoid being recognized and sequestered by the body’s reticuloendothelial system (RES), thus increasing the blood circulation time of micelle-like nanoparticles.
Star-shaped amphiphilic polymers are branched, multiarmed amphiphilic polymeric materials in which the branches radiate from a central core. Owing to their particular architecture, star-shaped amphiphilic block copolymers are expected to exhibit useful rheological, mechanical and biomedical properties that are inaccessible to linear polymers.18 For example, star-shaped amphiphilic block copolymers are able to self-assemble into unimolecular micelles which have significant advantages in terms of stability over micelles of linear copolymers.19–21 Additionally, star-shaped amphiphilic block copolymers are superior in host–guest interactions with small organic molecules compared to their linear counterparts. Moreover, compared with linear copolymers, star-shaped amphiphilic block copolymer micelles tend to show better thermal stability and kinetic stability due to their lower critical micelle concentration (CMC) values.
Picropodophyllin (PPP) is a specific small molecule inhibitor of the insulin-like growth factor 1 receptor (IGF1R) with potential antineoplastic activity.2223 IGF1R, a tyrosine kinase receptor, is overexpressed in a variety of human cancers and plays a critical role in the growth and survival of many types of cancer cells. Picropodophyllin can specifically downregulate the cellular expression of IGF1R without interfering with other growth factor receptors, such as receptors for insulin, epidermal growth factor, platelet-derived growth factor, fibroblast growth factor and mast/stem cell growth factor. PPP displays potent activity

EXPERIMENTAL DETAILS

Materials

Monomethoxy polyethylene glycol (PEG, Mn = 2000, Sigma-Aldrich Chemie GmbH, Riedstr) was dried under vacuum before use. Pentaerythritol (AR, Aladdin) and -caprolactone (-CL) (AR, Aladdin) were sublimed under vacuum at 200 C. Methylene chloride (AR, Sinopharm Chemical Reagent Co., Ltd.) was dried over calcium hydride and distilled under an argon atmosphere. Stannous octoate (Sn(Oct)2, 4-dimethylamiopyridine (DMAP) and N,N -dicyclohexyl carbodiimide (DCC) were purchased from Sinopharm Chemical Reagent Co. Ltd., China.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were from Sigma, USA, while Dulbecco’s modified Eagle’s medium (DMEM) was from GIBCO, USA. Linear polycaprolactone- polyethyleneglycol copolymer (L-PCLPEG) was synthesized in our laboratory through ringopening polymerization of -CL using monomethoxy polyethylene glycol as an initiator. Picropodophyllin (PPP, 99%) was prepared by chemical isomerization of podophyllotoxin (Xian senmu technical Co. Ltd., China) in our laboratory. All other chemicals and solvents were of analytical reagent (AR) grade.

Synthesis of S-PCL-PEG

In this study, we developed a novel picropodophyllinloaded micelle-like nanoparticle based on a star-shaped polycaprolactone-polyethyleneglycol copolymer (S-PCLPEG). First, we synthesized and characterized a biodegradable S-PCL-PEG block copolymer consisting of star-shaped polycaprolactone (S-PCL) and monomethoxy polyethylene glycol (PEG) and studied its self-assembly behavior. Then, PPP-loaded S-PCL-PEG nanoparticles (PPP-NPs) were prepared and optimized by the thin film hydration method in order to increase the solubility of PPP in water. The entrapment efficiency of PPP-NPs was investigated using the ultrafiltration method. Additionally, the size and zeta potential of PPP-NPs were determined with a ZetaPlus instrument while the morphology of PPP-NPs was examined by means of atomic force microscopy (AFM). The drug release profile in vitro was also investigated by the dialysis method. Finally, the cytotoxicity of PPP-NPs against SMMC7721 liver cancer cell lines was evaluated with PPP solution as the control.

Synthesis of 4-Arm Star-Shaped Polycaprolactone S-PCL

S-PCL was synthesized through ring-opening polymerization of -caprolactone (-CL) using pentaerythritol as the initiator and Sn(Oct)2 as the catalyst. Typically, a certain amount of pentaerythritol (e.g., 0.0298 g, 0.22 mmol), -CL (e.g., 2 ml, 17.5 mmol) and Sn(Oct)2 were reacted in a three-neck round-bottom flask at 140 C for 12 h under a nitrogen atmosphere. The resulting product was dissolved in methylene chloride and then excess methanol was poured in to form a precipitate. Finally, the precipitate was collected and dried under vacuum at room temperature to obtain star-shaped PCL. The yield was about 70%.

Synthesis of Carboxyl-Terminated PEG PEG-COOH

PEG-COOH was synthesized by introducing a carboxylic acid group to the chain end of monomethoxy polyethylene glycol (PEG). Briefly, a certain amount of monomethoxy polyethylene glycol (e.g., 2.4 g, 1.2 mmol) and succinic anhydride (e.g., 0.6 g, 6 mmol) was reacted at 50 C for 5 h in the presence of anhydrous pyridine. At the end of the reaction, the mixture was poured into excess ice-cold ether to precipitate. The precipitate was then dissolved in isopropanol and was filtered to remove unreacted succinic anhydride. The filtrate was precipitated with excess diethyl ether. The precipitate was filtered and dried in a vacuum to obtain carboxyl-terminated PEG (PEG-COOH). The yield was greater than 59%.

Coupling Reaction of S-PCL and PEG-COOH

S-PCL-PEG was synthesized through the coupling reaction of star-shaped PCL with carboxyl-terminated PEG (PEGCOOH). Briefly, calculated masses of S-PCL (e.g., 0.46 g, 0.05 mmol), PEG-COOH (e.g., 0.63 g, 0.3 mmol), DCC, DMAP and dichloromethane were added into a threenecked round-bottom flask. The reaction was carried out at room temperature for 24 h under stirring. The reaction byproduct, dicyclohexylcarbodiurea, was removed by filtration, and then the filtered solution was evaporated to dryness. Subsequently, the residue was dissolved in dichloromethane and purified by precipitating in diethyl ether. After being filtered and dried under vacuum for 24 h, S-PCL-PEG copolymer was obtained. The yield was more than 60%.

Characterization of S-PCL-PEG

Differential scanning calorimetry (DSC) curves were recorded on a differential scanning calorimeter (NETASCH, DSC2004) with a heating rate of 10 C/min. Proton nuclear magnetic resonance (1H-NMR) spectra were obtained on a Bruker AV-500 spectrometer (Bruker, a dry polymer-drug film. Afterwards, the film was reconstituted with distilled water at 60 C for 10 min under vigorous stirring in the presence of glass beads and was further sonicated for 20 min, allowing the polymer to self-assemble into PPP-loaded micellar-like nanoparticles. Finally, the nanoparticle solution was filtered through a 0.45 m filter.
In addition, PPP-loaded L-PCL-PEG micelle-like nanoparticles were also prepared by a similar thin film hydration method.

Characterization of Drug-Loaded Nanoparticles

Drug Loading and Entrapment Efficiency of Nanoparticles Switzerland) at frequencies of 300 Hz or 500 Hz usingDelivered by Publishing Technology to: Rice UniversityTo determine the entrapment efficiency, free PPP was CDCl3 as the solvent. IP: 117.245.76.159 On: Thu, 07 Jan 2016 04:06:57separated from the PPP-loaded nanoparticle solution by Copyright: American Scientific Publishersultrafiltration through Ultra-4 filters (Amicon, 30 kDa
In order to determine the PPP content loaded into the nanoparticles, the nanoparticles were dissolved in methanol–water (5:1, v/v) and a clear solution was obtained for HPLC analysis using a LC-10A Shimadzu high-pressure liquid chromatograph. A Hypersil BDS, 5 m, 250×4.6 mm column was used. The mobile phase consisted of methanol–water (55:45) and the flow rate was 1 ml/min. The ultraviolet detector was set at 290 nm. PPP concentrations were calculated based on a calibration curve previously created by a plot of peak areas (A) against PPP concentrations (C) in the range of 1.0–100 g/ml. The linear regression equation of the calibration curve was A = 24751C +11087, R2 = 09998.

Determination of Critical Micelle Concentrations

The critical micelle concentrations (CMCs) of S-PCL-PEG were determined by fluorescence spectrometry (Shimadzu RF5301, Japan) using pyrene as a hydrophobic fluorescence probe, as previously described.16 A predetermined amount of pyrene in acetone was added to a series of volumetric flasks and then the acetone was evaporated completely. Subsequently, a predetermined volume of the copolymer micelle solution and ultrapure water were added to the volumetric flasks consecutively to obtain solutions of different micelle concentrations ranging from 0.1 mg/ml to 40 mg/ml, while the concentration of pyrene in each flask was fixed at 2.95×10−7 mol/l. The excitation spectra (333–338 nm) were recorded with an emission wavelength of 390 nm. The excitation and emission bandwidths were set at 5 nm. The fluorescence intensity ratios of pyrene at I338 and I333 were plotted as a function of the logarithm of polymer concentration. The inflection points of the curves were defined as the CMC value.

Preparation of PPP-Loaded S-PCL-PEG Nanoparticles

PPP-loaded S-PCL-PEG nanoparticles (PPP-NPs) were prepared by the thin film hydration method. In brief, S-PCL-PCL and PPP were both fully dissolved in dichloromethane and then evaporated in a rotavapor to obtain molecular weight cutoff) at 4000 rpm for 4 min. The drug entrapment efficiency of the nanoparticles was calculated from the following equation: Entrapment Efficiency EE% where MT is the weight of PPP in the nanoparticle solution and MF is the weight of PPP in the ultrafiltrate.

Particle Size and Morphology Measurements

Measurements of the particle size and zeta potential of PPP-NPs were carried out using a ZetaPlus instrument (Brookhaven). The surface morphology of PPP-NPs was observed by atomic force microscopy (AFM-Veeco/ Bruker) after the PPP-NP solution was dropped onto coverslips and air dried.

In Vitro PPP Release from Nanoparticles

The release profiles of PPP from S-PCL-PEG nanoparticles were investigated in vitro. In brief, the PPP-NP solution (2 ml) was introduced into dialysis membrane tubing (MWCO 3000) and the dialysis bag was placed in a 200 ml bottle which contained 100 ml of pH 7.4 phosphate buffer solution with 0.2% Tween 80 (PBST). The release test was performed in a shaking incubator at 37 C with a shaking speed of 50 rpm. At predetermined intervals, aliquots of PBST were taken from the release medium and an equal volume of fresh PBST was added back. The concentrations of the released PPP were measured by HPLC as described above.
Similarly, the in vitro drug release profiles of PPPloaded L-PCL-PEG nanoparticles and free PPP solution were also evaluated.

In Vitro Tumor Cell Growth Inhibition Assay

In this test, the SMMC7721 liver cancer cell line, maintained in DMEM (containing 10% fetal bovine serum) and incubated at 5% CO2 at 37 C, was used to evaluate the cytotoxicity of PPP-incorporated nanoparticles (PPP-NPs). The cells were seeded into 96-well plates at a density of 3,000 cells per well and then incubated overnight. Next, the cells were exposed to a series of PPPNP solutions and free PPP DMSO solutions for 72 h. Growth inhibition of tumor cells was measured in triplicate by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based cell proliferation assay.

RESULTS AND DISCUSSION

Firstly, star-shaped PCL was synthesized by ring-opening polymerization of -CL using pentaerythritol as the initiator. Secondly, the monomethoxy polyethylene glycol (PEG) was activated using succinic anhydride to form carboxyl-terminated PEG (PEGCOOH). Thirdly, S-PCL was conjugated to PEG-COOH with DMAP as the catalyst.
The DSC curve of S-PCL-PEG is shown in Figure 2. The melting temperature of S-PCL-PEG was 52.3 C. A single melting transition peak suggests that the product was highly pure.
The 1H-NMR spectra of S-PCL-PEG and its intermediates, i.e., S-PCL and carboxyl-terminated PEG (PEGCOOH), are shown in Figures 3(A)–(C), respectively. According to Figure 3(A), the peak at 4.10 ppm belongs to the methylene protons of the pentaerythritol moiety. The peaks at 4.06 ppm and 2.31 ppm correspond to the methylene protons of –CH2OCO– and –OCCH2– in the PCL units, respectively. The peaks at 1.66 ppm and 1.35 ppm correspond to the methylene protons of –(CH23– in the PCL units. These proton peaks from the hydrophobic block of S-PCL-PEG resemble those of S-PCL in Figure 3(B). In Figure 3(B), the weak peak at 3.65 ppm corresponds to the methylene protons of –CH2OH in the PCL units. Additionally, in Figure 3(A), the sharp peak at 3.64 ppm is due to the methylene protons of –CH2CH2O– in the PEG units of the block copolymer.

Synthesis of S-PCL-PEG Delivered by Publishing Technology to: Rice UniversityThe peak at 4.23 ppm is due to the methylene protons IP: 117.245.76.159 On: Thu, 07 Jan 2016 04:06:57

S-PCL-PEG copolymers were prepared following a three-Copyright: American Scientific Publishersof –OCOCH2– in the PEG units, which result from the step synthetic procedure using the “core-first” approach, succinylation of monomethoxy polyethylene glycol with succinic anhydride. The very weak peak at 3.38 ppm is due to the methyl protons of –OCH3 in the PEG units, whereas the peak at 2.65 ppm corresponds to the proton resonance of ethylene group between the two ester groups, indicating that carboxyl-terminated PEG was successfully coupled to S-PCL. The above proton peaks from the hydrophilic block of S-PCL-PEG resemble those of carboxyl-terminated PEG (PEG-COOH) in Figure 3(C). Therefore, the 1H-NMR results show the successful synthesis of S-PCL-PEG block copolymers. Additionally, the molecular weight of S-PCL-PEG was calculated from the ratio of the peak area at 2.31 ppm to the peak area at

2.65 ppm in Figure 3(A); the results are listed in Table I.

Delivered by Publishing Technology to: Rice University As shown in Table I, the molecular weight of S-PCL-PEGIP: 117.245.76.159 On: Thu, 07 Jan 2016 04:06:57 calculated from the 1H-NMR data was about 17,004 DaCopyright: American Scientific Publishers and the mean yield was 62.67±1.92%. Our experimental study revealed that the yield of S-PCL-PEG synthesis is mainly related to the feeding ratio of S-PCL to PEGCOOH. As the feeding ratio of PEG-COOH to S-PCL increased, the yield of S-PCL-PEG improved correspondingly; however, the yield did not increase after the feeding ratio of PEG-COOH to S-PCL reached 6 to 1 (mol/mol). At the optimized feeding ratio, the yields of three batches of S-PCL-PEG were all greater than 60%, indicating that the process had good reproducibility.

CMC of S-PCL-PEG Copolymer Micelles

Figure 4 shows the fluorescence excitation spectra of pyrene in various concentrations of S-PCL-PEG nanoparticle micelles. Obviously, a red shift of the absorption band from 333 nm to 338 nm was observed when the concentration of the copolymer increased. This red shift resulted from the formation of micelles in the system since the pyrene probe molecules which entered the micelle phase exhibited much stronger fluorescence than those in the water phase. In addition, the CMC value was 2.88× 10−3 mg/ml (i.e., 1.69 × 10−7 mol/l), which was determined from the intersection of the two straight lines (the horizontal line of I338 nm/I333 nm with nearly constant values and the diagonal line with an increase in the I338 nm/ I333 nm values) on a plot of the I338 nm/I333 nm ratio versus the log polymer concentration (Fig. 4). The amphiphilic block copolymers exhibited a much lower CMC compared to the low molecular weight surfactants. Typically, the CMC of low molecular weight surfactants is on the order of 10−3 to 10−4 mol/l. Due to the low CMC, amphiphilic polymeric micelles remained stable at very low polymer concentrations, which makes them relatively insensitive to dilution, resulting in an enhanced circulation time.

Preparation and Characterizations of PPP-Loaded S-PCL-PEG Micelle-Like Nanoparticles

PPP was loaded into the hydrophobic core of S-PCL-PEG micelle-like nanoparticles using the thin film hydration method. The entrapment efficiency of PPP-loaded S-PCLPEG micelle-like nanoparticles was over 96%, which was higher than the efficiency using their linear counterparts listed in Table III. The average diameters of the micelles were in the range of 90–110 nm, which indicated that the star-shaped copolymer had self-assembled into micellelike nanoparticles. The copolymer nanoparticles were negatively charged at their surface with a zeta potential of around −18 mV, likely attributed to the polarization effect (Table III). (around 85%), as shown in Table II. One of the major rolesDelivered by Publishing Technology to: Rice Universitywith the size distribution result presented in Table III. of S-PCL-PEG micelle-like nanoparticles as a deliveryIP: 117.245.76.159 On: Thu, 07 Jan 2016 04:06:57 vehicle is to enhance the solubility of highly hydrophobicCopyright: American Scientific PublishersPPP Release from Nanoparticles
Atomic force microscopy (AFM) is ideally suited for characterizing nanoparticles. AFM offers topographic measurements of surfaces with accuracy in the nanometric scale. Unlike transmission electron microscopy and scanning electron microscopy, which provide a twodimensional image of a sample, AFM provides a threedimensional surface image. Therefore, AFM was utilized to investigate the surface morphology and the size of S-PCLPEG micelle-like nanoparticles. As shown in Figure 5, the S-PCL-PEG copolymer formed spherical nanoparticles with a diameter of close to 100 nm, which was consistent drugs in an aqueous medium. It was found in our experiment that the maximum amount of PPP loaded into 1.0 ml of micelle-like nanoparticle solution with 12.5 mg/ml of S-PCL-PGE was 100 g, while the solubility of PPP in a physiological solution (pH 7.4) is only 1.9 g/ml at 25 C. The S-PCL-PEG micelle-like nanoparticles had enhanced the solubility of PPP in water by a factor of more than 50. The particle size and zeta potential of the PPP-NPs were measured using a ZetaPlus instrument; the results are
The drug release profiles from S-PCL-PEG micelle-like nanoparticles, L-PCL-PEG micelle-like nanoparticles and the free PPP solution are shown in Figure 6. The cumulative release rates of PPP from S-PCL-PEG nanoparticles and L-PCL-PEG nanoparticles were both much slower than that from PPP solution. It was found that only 15% of PPP was released from S-PCL-PEG nanoparticles within 12 h, whereas L-PCL-PEG nanoparticles released approximately 50% of PPP and free PPP solution released as much as 84% of the drug into the media within 12 h.
In addition, the release profiles of S-PCL-PEG nanoparticles and L-PCL-PEG nanoparticles were both typical twophase release profiles, involving an initial rapid release phase which was followed by another phase of relatively slow release (plateau phase).26 The burst release of PPP may be due to the dissolution and diffusion of the drug that was poorly entrapped in the polymer matrix, while the slower and continuous release may be attributed to the diffusion of the drug entrapped in the PCL core of the nanoparticles. The in vitro release profiles demonstrated that S-PCL-PEG nanoparticles did not reach plateau phase until 48 h, while L-PCL-PEG nanoparticles entered the plateau phase at 12 h. These interesting results indicated that the PPP release from S-PCL-PEG nanoparticles was slower than from L-PCL-PEG nanoparticles. The much slower release rate of PPP from S-PCL-PEG nanoparticles can be attributed to the molecular structural characteristics of star-shaped polymeric micelles. This showed that the

Delivered by Publishing Technology to: Rice University0.08 g/ml, 0.16 g/ml and 0.32 g/ml), the cell growth

S-PCL-PEG PPP-NPs were more suitable as a drug carrier compared to L-PCL-PEG. Copyright: American Scientific Publishersinhibition induced by PPP-NPs was much greater than using MTT assay. Figure 7 shows the growth inhibition of cancer cells treated with PPP-NPs of different concentrations for 72 h, compared with those treated with PPP in a DMSO solution. The results demonstrated that S-PCL-PEG PPP-NPs and the PPP solution inhibited cancer cell growth in a PPP concentration-dependent manner. Moreover, at low PPP concentrations (0.008 g/ml, Since the solubility of PPP in aqueous media is very poor, the release media should be composed of PBS (pH 7.4) containing 20% Tween 80; this makes PPP soluble in the release media to satisfy the sink conditions required for this assay.

In Vitro Tumor Cell Growth Inhibition Assay

The growth inhibition of SMMC7721 liver cancer cell lines exposed with S-PCL-PEG PPP-NPs was evaluated that by PPP solution (P < 005), while at high concentrations (1.6 g/ml and 3.2 g/ml), there was no significant difference between PPP-NPs and the PPP solution (P > 005). The IC50 values of PPP solution and PPPNPs against SMMC7721 liver cancer cells were 0.4 g/ml and 0.2 g/ml respectively, indicating that PPP-NPs had greater growth inhibition activity on tumor cells than the PPP solution. The improved anticancer activity of S-PCLPEG PPP-NPs may be attributed to the non-specific internalization of nanoparticles by cells via endocytosis or phagocytosis.2728 However, the detailed mechanisms of this process require further study.

CONCLUSIONS

A star-shaped amphiphilic block copolymer, S-PCL-PEG, was synthesized using pentaerythritol, -caprolactone (-CL) and monomethoxy polyethylene glycol (PEG) as raw materials, and was characterized with 1H-NMR. Fluorescence measurements confirmed that S-PCL-PEG copolymers could self-assemble into micelle-like nanoparticles in aqueous solutions and contained a hydrophobic S-PCL core and a hydrophilic PEG shell. The S-PCLPEG copolymers could be used to successfully entrap PPP and to develop a novel PPP-loaded micelle-like nanoparticle (PPP-NP). PPP-NPs had a drug encapsulation efficiency of greater than 90% and a size of about 100 nm.
In vitro release tests showed that PPP-NPs possessed good sustained release behavior. Finally, cytotoxicity tests indicated that the growth inhibition activity of PPP-NPs on tumor cells was greater than that of the PPP solution. In conclusion, a new PPP-loaded micelle-like nanoparticle using the amphiphilic block copolymer S-PCL-PEG as a carrier was successfully developed. PPP-NPs showed higher anti-cancer activity than PPP solution, and thus warrant further investigation.

REFERENCES

A. Richter, C. Olbricha, M. Krause, and T. Kissel, Solubilization of sagopilone, a poorly water-soluble anticancer drug, using polymeric micelles for parenteral delivery. Int. J. Pharm. 389, 244 (2010).
K. Letchford, R. Liggins, and H. Burt, Solubilization of hydrophobic drugs by methoxy poly(ethylene glycol)-block-polycaprolactone diblock copolymer micelles: Theoretical and experimental data and correlations. J. Pharm. Sci. 7, 1179 (2008).
P. Prabhu and V. Patravale, The upcoming field of theranostic nanomedicine: An overview. J. Biomed. Nanotechnol. 8, 859 (2012).
P. Dong, X. Wang, Y. Gu, Y. Wang, Y. Wang, C. Gong, F. Luo, G. Guo, X. Zhao, Y. Wei, and Z. Qian, Self-assembled biodegradable micelles based on star-shaped PCL-b-PEG copolymers for chemotherapeutic drug delivery. Colloids Surf. A: Physicochem. Eng. Aspects 358, 128 (2010).
Z. Liu, D. Liu, L. Wang, J. Zhang, and N. Zhang, Docetaxel-loaded pluronic P123 polymeric micelles: In vitro and in vivo evaluation. Int. J. Mol. Sci. 12, 1684 (2011).
T. L. Lapenda, W. A. Morais, F. J. Almeida, M. S. Ferraz, M. C. Lira, N. P. Santos, M. A. Maciel, and N. S. Santos-Magalhães, Encapsulation of trans-dehydrocrotonin in liposomes: An enhancement of the antitumor activity. J. Biomed. Nanotechnol. 9, 499 (2013).
H. S. Yoo, K. H. Lee, J. E. Oh, and T. G. Park, In vitro and in vivo anti-tumor activities of nanoparticles based on doxorubicin-PLGA conjugates. J. Control. Rel. 68, 419 (2000).
J. A. Yáñez, M. L. Forrest, Y. Ohgami, G. S. Kwon, and N. M. Davies, Pharmacometrics and delivery of novel nanoformulated PEG-b-poly(-caprolactone) micelles of rapamycin. Cancer Chemother. Pharmacol. 61, 133 (2008).
K. Letchford, J. Zastre, R. Liggins, and H. Burt, Synthesis and micellar characterization of short block length methoxy poly(ethylene glycol)-block-poly(caprolactone) diblock copolymers. Colloids Surf. B: Biointerfaces 35, 81 (2004).
H. Ding, B. D. Sumer, C. W. Kessinger, Y. Dong, G. Huang, D. A. Boothman, and J. Gao, Nanoscopic micelle delivery improves the photophysical properties and efficacy of photodynamic therapy of protoporphyrin IX. J. Control Rel. 151, 271 (2011).
D. J. Cameron and M. P. Shaver, Aliphatic polyester polymer stars: Synthesis, properties and applications in biomedicine and nanotechnology. Chem. Soc. Rev. 40, 1761 (2011).
R. K. Kainthan, C. Mugabe, H. M. Burt, and D. E. Brooks, Unimolecular micelles based on hydrophobically derivatized hyperbranched polyglycerols: Ligand binding properties. Biomacromolecules 9, 886 (2008).
J. Wang, L. S. del Rosario, B. Demirdirek, A. Bae, and K. E. Uhrich, Comparison of PEG chain length and density on amphiphilic macromolecular nanocarriers: Self-assembled and unimolecular micelles. Acta Biomater. 5, 883 (2009).ration of core–shell type nanoparticle for encapsulation of anticancerDelivered by Publishing Technology to: Rice University(2010).
N. V. Cuong, J. L. Jiang, Y. L. Li, J. R. Chen, S. C. Jwo, and M. F.IP: 117.245.76.159 On: Thu, 07 Jan 2016 04:06:57(2010).Copyright: American Scientific Publishers22. M. Doghman, M. Axelson, and E. Lalli, Potent inhibitory effect
X. Yang, J. J. Grailer, S. Pilla, D. A. Steeber, and S. Gong, Tumortargeting, pH-responsive, and stable unimolecular micelles as drugHsieh, Doxorubicin-loaded PEG-PCL-PEG micelle using xenograft model of nude mice: Effect of multiple administration of micelle on the suppression of human breast cancer. Cancers 3, 61 (2011).
M. Ukawala, T. Rajyaguru, K. Chaudhari, A. S. Manjappa, S. Pimple, A. K. Babbar, R. Mathur, A. K. Mishra, and R. S.Murthy, Investigation on design of stable etoposide-loaded PEG-PCL micelles: Effect of molecular weight of PEG-PCL diblock copolymer on the in vitro and in vivo performance of micelles. Drug Delivery19, 155 (2012).
H. Cho, G. L. Indig, J. Weichert, H. C. Shin, and G. S. Kwon, In vivo cancer imaging by poly(ethylene glycol)-b-poly(-caprolactone) micelles containing a near-infrared probe. Nanomedicine 8, 228 (2012). 10. K. Rajagopal, A. Mahmud, D. A. Christian, J. D. Pajerowski, A. E. Brown, S. M. Loverde, and D. E. Discher, Curvature-coupled hydration of semicrystalline polymer amphiphiles yields flexible worm micelles but favors rigid vesicles: Polycaprolactone-based block copolymers. Macromolecules 43, 9736 (2010).
P. Lee, R. Zhang, V. Li, X. Liu, R. W. Sun, C. M. Che, and K. K. Wong, Enhancement of anticancer efficacy using modified lipophilic nanoparticle drug encapsulation. Int. J. Nanomedicine 7, 731(2012).
F. Figueiró, A. Bernardi, R. L. Frozza, T. Terroso, A. Zanotto-Filho, E. H. Jandrey, J. C. Moreira, C. G. Salbego, M. I. Edelweiss, A. R. Pohlmann, S. S. Guterres, and A. M. Battastini, Resveratrol-loaded lipid-core nanocapsules treatment reduces in vitro and in vivo glioma growth. J. Biomed. Nanotechnol. 9, 516 (2013). of the cyclolignan picropodophyllin (PPP) on human adrenocortical carcinoma cells proliferation. Am. J. Cancer Res. 1, 356 (2011).
A. Girnita, C. All-Ericsson, M. A. Economou, K. Astrom, M. Axelson, S. Seregard, O. Larsson, and L. Girnita, The insulinlike growth factor-I receptor inhibitor picropodophyllin causes tumor regression and attenuates mechanisms involved in invasion of uveal melanoma cells. Clin. Cancer Res. 12, 1383 (2006).
S. Ekman, J. E. Frödin, J. Harmenberg, A. Bergm. A. Hedlund, P. Dahg, C. Alvfors, B. Ståhl, S. Bergström, and M. Bergqvist, Clinical phase I study with an insulin-like growth factor-1 receptor inhibitor: Experiences in patients with squamous non-small cell lung carcinoma. Acta Oncol. 50, 441 (2011).
K. Letchford, J. Zastre, R. Liggins, and H. Burt, Synthesis and micellar characterization of short block length methoxy poly(ethylene glycol)-block-poly(caprolactone) diblock copolymers. Colloids Surf. B: Biointerfaces 35, 81 (2004).
F. Danhier, N. Lecouturier, B. Vroman, C. Jérôme, J. MarchandBrynaert, O. Feron, and V. Préat, Paclitaxel-loaded PEGylated PLGA-based nanoparticles: In vitro and in vivo evaluation. J. Control. Rel. 133, 11 (2009).
C. Allen, Y. Yu, A. Eisenberg, and D. Maysinger, Cellular internalization of PCL(20)-b-PEO(44) block copolymer micelles. Biochim. Biophy. Acta 1421, 32 (1999).
A. des Rieux, V. Fievez, M. Garinot, Y.-J. Schneider, and V. Préat, Nanoparticles as potential oral delivery systems of proteins and vaccines: A mechanistic approach. J. Control. Rel. 116, 1 (2006).