Hyperbranched poly (amine-ester)-poly (lactide-co-glycolide) copolymer and their nanoparticles as paclitaxel delivery system

Research Article

Received: 9 February 2010, Revised: 6 May 2010, Accepted: 6 May 2010, Published online in Wiley Online Library: 13 December 2010

(wileyonlinelibrary.com) DOI: 10.1002/pat.1765

Hyperbranched poly (amine-ester)-poly(lactide-co-glycolide) copolymer and theirnanoparticles as paclitaxel delivery system

Yan Wua*, Tie wei Wanga, Mingjun Lib* and Hongxia Gaob

A new hyperbranched poly (amine-ester)-poly (lactid

Polym. Adv

e-co-glycolide) copolymer (HPAE-co-PLGA) was synthesized byring-opening polymerization of D, L-lactide (DLLA) glycolid and branched poly (amine-ester) (HPAE-OHs) with Sn(Oct)2as catalyst. The chemical structures of copolymers were determined by FT-IR, 1H-NMR(13C NMR), TGA and theirmolecular weights were determined by gel permeation chromatography (GPC). Paclitaxel-loaded copolymer nano-particles were prepared by the nanoprecipitationmethod. Their physicochemical characteristics, e.g. morphology andnanoparticles size distribution were then evaluated by means of fluorescence spectroscopy, environmental scanningelectron microscopy (ESEM), and dynamic light scattering (DLS).Paclitaxel-loaded nanoparticles assumed a spherical shape and have unimodal size distribution. It was found that

the chemical composition of the nanoparticles was a key factor in controlling nanoparticles size, drug-loadingcontent, and drug release behavior. As themolar ratio of DL-lactide/glycolide to HPAE increased, the nanoparticles sizeand drug-loading content increased, and the drug release rate decreased. The antitumor activity of the paclitax-el-loaded HPAE-co-PLGA nanoparticles against human liver cancer H7402 cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The paclitaxel-loaded HPAE-co-PLGA nano-particles showed comparable anticancer efficacy with the free drug. Copyright � 2010 John Wiley & Sons, Ltd.

Keywords: antitumor activity; drug delivery; paclitaxel; hyperbranched poly (amine-ester); poly (lactide-co-glycolide);polymeric nanoparticles

* Correspondence to: M. Li, National Center for Nanoscience and Technology,No.11 Beiyitiao, Zhongguancun, Beijing, 100190, China.E-mail: [email protected]

a Y. Wu, T. w. Wang

National Center for Nanoscience and Technology, No.11 Beiyitiao, Zhong-

guancun, Beijing, 100190, China

b M. Li, H. Gao

The First Affiliated Hospital of Jiamusi University, Jiamusi 154002, China

Contract/grant sponsor: Chinese academy of sciences, Chinacontract/grant

number: KJCX2-YW-M02.

Contract/grant sponsor: State Key Development Program for Basic Research of

China (973); contract/grant number: 2009CB930200, 2010CB934004.

Contract/grant sponsor: State Key Development Program for Basic Research of

China (863); contract/grant number: 2007AA02Z150, 2006AA03Z321. 2

INTRODUCTION

There has been great interest in colloid microcarriers as effectivedrug delivery systems, for example, liposomes, and polymericspherical particles/micelles. It is well known that whenamphiphilic block copolymers are dissolved in a selective solvent,they usually form nanoscale core–shell structures in an aqueoussolution and can be used as a vehicle of drug delivery.[1–5]

Polymeric micelles have received special attention due to theirpotential application and academic interest in many interdisci-plinary field.[6–10] These core-shell type micelles may be used asdrug delivery vehicles for poorly water-soluble drugs, especiallywhen the micelles are made with suitable biodegradablepolymers.Recent trends in drug delivery technology have focused on

biodegradable polymers requiring no surgical removal once thedrug supply is depleted. PLGA is a well-known biodegradable andbiocompatible material with a hydrophobic character. In addition,it was reported that copolymers of DL-lactide and glycolide withother material such as PEG degraded much more rapidly thanPLGA homopolymer.[11–13]

The most widely studied amphiphilic polymers for drugdelivery include diblock copolymers and triblock copolymers.[14]

However, the conventional micelle drug delivery systems basedon the linear amphiphilic copolymers suffer from low encapsula-tion efficiency and fast initial release rate, which limit theirapplications in drug delivery.[15–16] Since the molecular archi-tecture of amphiphilic copolymers is the most important factor in

. Technol. 2011, 22 2325–2335 Copyright

determining micellar structure and property, research effortshave thus focused on the design of the molecular structures ofamphiphilic copolymers. Recently, it has been shown thatamphiphilic copolymers with nonlinear structure exhibitedimproved property as drug carriers.[17]

Over the past two decades, hyperbranched polymers havereceived extensive research attention as a new class of polymersof unique chain structure. Distinct from their linear analogs,hyperbranched polymers have structures and topologies similarto those of dendrimers, and possess some strikingly superiormaterial properties, such as low solution/melt viscosity, enhancedsolubility, abundance in terminal group, etc.[18–19] But, unlike

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dendrimers that often require tedious synthetic procedures,[20]

hyperbranched polymers are more easily produced on a largescale, which encourages their potential use in a variety ofimportant applications, including rheological additives, toughen-ing agents, drug delivery etc.[21–24] Hyperbranched polymerscontain numerous end-groups in their molecular structures andthe characteristics of these terminal groups have a greatinfluence on the properties of resulting hyperbranched polymers.Therefore, modification of the number and type of end-groups isa powerful tool to tailor the properties of hyperbranchedpolymers.[25] The present work is to associate the merits ofHPAE-OHs and PLGA and prepare HPAE-co-PLGA polymericnanoparticles as drug carriers. If PLGA can be grafted ontohydroxyl groups of HPAE-OHs, the amphiphilic copolymers wouldbecome affordable. Owing to the introduction of hydrophilicHPAE-OHs molecules, a drug can be encapsulated into theHPAE-co-PLGA polymeric nanoparticles without any deactivation.The new hyperbranched polymeric nanoparticles containabundant terminal groups, which enable binding drug withother target groups for drug delivery system. For this purpose, wesynthesize the HPAE-co-PLGA copolymers by opening polymeri-zation with hyperbranched HPAE, DL-lactide, and glycolide. Thechemical structure and physical properties of copolymers werecharacterized and the formation of polymeric nanoparticles wasinvestigated. Finally, Paclitaxel, extracted from the bark of anaturally occurring plant, Taxus brefolia has exhibited potentcytotoxic activity against a wide spectrum of cancers, especiallybreast and ovarian cancer.[26–27] However, it has difficulties inclinical formulation due to its extremely low aqueous solubility. Inthis paper, paclitaxel-loaded HPAE-co-PLGA nanoparticles usedas pharmaceutical carriers are prepared. The physicochemicalcharacteristics, drug release behavior, and anticancer activitiesof the paclitaxel-loaded HPAE-co-PLGA nanoparticles wereinvestigated.

MATERIALS AND METHODS

Materials

1, 1, 1-trimethylol propane of reagent grade, methyl acrylatepurified by vacuum distillation and diethanolamine waspurchased from National Medicines chemical Reagent Co. Ltd(China.). Titanium tetraisopropoxide (Ti(OiPr)4), benzoic anhy-dride, and imidazole were purchased from Beijing ReagentFactory,(China), DL-lactide and glycolide were purchased fromPURAC (The Netherlands) and used without further purification.Sn(Oct)2 was purchased from Sigma (America). Paclitaxelobtained from Hande Biotechnology Ltd., Yunnan, China. Allother reagents were analytical grade. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was purchasedfrom Sigma.

Synthesis of HPAE-OHs

HPAE-OHs were synthesized through a pseudo-one-step processby alcoholysis at 1208C using 1, 1, 1-trimethylol propane (as amolecular core) and N, N-diethylol-3-amine methylpropionate(as an AB2monomer) with Ti(OiPr)4 as the catalyst. The generationof HPAE-OHs was increased by repeatedly adding N,N-diethylol-3-amine methylpropionate monomer to the formergeneration product. N, N-diethylol-3-amine methylpropionatewas prepared by using methyl acrylate and diethanolamine, the

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feedmolar ratio is 1.5:1. After theMichael Reaction, excessmethylacrylate and methanol was removed by vacuum distillation. Thefourth-generation HPAE-OHs4 was obtained by repeating theprocess three times. The obtained product was dissolved in 10mlethanol, and the Ti(OiPr)4 produced the precipitate in ethanol.Then, the Ti(OiPr)4 was removed by filtration. Finally the ethanolwas evaporated and the product was dried at 408C in a vacuumoven and stored for further utilization.

Synthesis of HPAE-co-PLGA copolymer

HPAE-co-PLGA copolymer was synthesized utilizing aring-opening polymerization procedure. DLLA, glycolide andHPAE-OHs4 were dehydrated by using P2O5 under vacuum at458C for 24 hr and were used without further purification. A totalof 50 g of DL-lactide, glycolide plus HPAE was used for thepolymerization. Copolymerization in the bulk state was carriedout with various molar ratios of DL-lactide/glycolide (6/1, 10/1, 15/1) and the weight ratio of HPAE was adjusted to 30% (w/w).Stannous 2-ethylhexanoate (0.2%, w/w) was added into a driedpolymerization tube followed by the addition of DL-lactide,glycolide, and HPAE. Then the tube was sealed under vacuum.The sealed tube was immersed and kept in an oil baththermostated at 1508C. After 12 hr, the reaction product wascooled to ambient temperature. The obtained viscous materialwas dissolved with CH2Cl2 and then precipitated with ether/petroleum (1:1, v/v) to remove unreacted DL-lactide, glycolidemonomers. After ether and the petroleum were evaporated,the polymers were dissolved in a little of acetone and thenprecipitated in deionized water. The purified polymers were driedat 408C for 2 days in a vacuum oven.

Preparation of PLGA

PLGA was prepared at 1508C for 10 hr by the ring openingpolymerization of D, L-lactide and glycolide sealed in an ampoulein the presence of Sn(Oct)2. The product was purified by therepeat dissolution in chloroform and precipitation in coldmethanol and was finally dried for 48 hr at 408C in a vacuumoven. The molecular weights were measured by gel permeationchromatography (GPC).

Characterization of the chemical structure of HPAE-OHs andHPAE-co-PLGA copolymer

The hydroxyl values of HPAE-OHs were determined by thefollowing method:[28]HPAE-OHs was dissolved in excess benzoicanhydride with imidazole as a catalyst (in pyridine) at 808C ofwater bath for 3 hr to acetylate the hydroxyl groups in HPAE-OHs.By back-titrating the above mixture with NaOH solution (0.1mol/L, in water) at room temperature, the hydroxyl values ofHPAE-OHs were calculated. The hydroxyl values (I (OH))¼(V0�V1)�C�40/Ms; where V0 and V1 represent the volume (ml)of NaOH solution consumption as titrating sample and plain (thesample is not contained in it), respectively; C represents theconcentration of NaOH solution; Ms represents the weight ofsample. 40 represents the molecular weight of NaOH.IR spectra were recorded on Fourier-transform infrared (FTIR)

spectrometer (Spectrum One Perkin-Elmer, America). The spectraof HPAE-OHs were obtained by using film as a reference. TheHPAE-co-PLGA samples were mixed with KBr and then pressed toa plate for measurement.

0 John Wiley & Sons, Ltd. Polym. Adv. Technol. 2011, 22 2325–2335

NANOPARTICLES AS PACLITAXEL DELIVERY SYSTEM

2

The 1H NMR and 13C NMR spectra of HPAE-OHs andHPAE-co-PLGA were recorded on a (Bruker AVANCE 400) NMRspectrometer. 1H NMR and 13C NMR spectra of HPAE-OHs weredissolved in DMSO-d6. HPAE-co-PLGA copolymers were dissolvedin CDCl3.GPC was performed on a Waters 2410 GPC apparatus (USA).

Molecular weight and molecular weight distribution of thecopolymer were calculated using polystyrene as the standard.The thermal stability of HPAE-co-PLGA samples was measured

by TGA (Perkin-Elmer, America). The temperature range was25–9008C under nitrogen flow and the heating rate is 208C/min.The thermo-property of HPAE-co-PLGA samples was also

measured by DSC. Samples (3�5mg) were loaded into aluminumpans and the DSC thermo grams were recorded on a PyrisDiamond DSC apparatus (Perkin-Elmer, America). In order toobserve Tg, all of the DSC thermo grams were obtained from asecond heating procedure. Briefly, the heating rate was 208C/minin the range of 20–1008C by using nitrogen flowing, samples werestored at 1008C for 1min and then cooled to �508C, the coolingrate was 208C/min, then the samples were re-heated from0–1008C in the heating rate of 208C/min.

Biodegradation of HPAE-co-PLGA copolymer

A sample of the HPAE-co-PLGA copolymer in weight of 20mgwascompressed in a mold into a film on a Carver Laboratory Press(Fred S. Carver Inc., USA) at room temperature. Biodegradation(%)¼ 100(W1�Wd)/W1; where W1 and Wd represent the driedweight of the original film and that of incubating in phosphatebuffer solution (PBS; pH 7.4) at 378C for specific days, respectively.

Preparation of paclitaxel-loaded HPAE-co-PLGA polymericnanoparticles

Paclitaxel-loaded nanoparticles were prepared by the nanopre-cipitation method.[29] Briefly, 10–20mg paclitaxel and 100mgHPAE-PLGA were dissolved in 10ml acetonitrile. This organicsolution was drop-wise added to 20ml deionized water (or with0.25% w/v of Pluronic (F-68)) under magnetic stirring. Thenanoparticles were formed immediately and the solvent wasremoved through the overnight evaporation at room tempera-ture. The resulting suspension was filtered through 0.8mmmembrane filter (Whatman) and then centrifuged for 1 hr at11 000 rpm. The supernatant was discarded and the pellet wasresuspended in water. The final suspension volume was 10ml.Plain polymeric micelles of HPAE-co-PLGA copolymer wereprepared by same procedure described above with the exceptionof paclitaxel and Pluronic (F-68).

Characterization of HPAE-co-PLGA and paclitaxel-loadedHPAE-co-PLGA polymeric nanoparticles

Adrop of nanoparticles solution was deposited onto a silicon chipand air-dried before ESEM (environmental scanning electronmicroscope) observation with a Quauta 200FEG scanningelectron microscope (Quauta 200FEG, FEI). The size anddistribution of polymeric nanoparticles in aqueous solution weremeasured by dynamic light scattering (DLS) (Zetasizer Nanoseries ZEN 3600 Malvern Instruments Ltd., England). All DLSmeasurements were done with a wavelength of 532 nm at 258Cwith an angle detection of 908. All samples were prepared inaqueous solution at a concentration of 0.2mg/ml, and the datawere analyzed by Malvern Dispersion Technology Software 4.20.

Polym. Adv. Technol. 2011, 22 2325–2335 Copyright � 2010 John Wiley

Steady-state fluorescence spectra were recorded on a spectro-fluorophotometer (FL-920 England). A solution of hyperbranchedHPAE-co-PLGA copolymers which contain 6� 10�7M of pyrenewas placed in a square cell and the fluorescence spectrum wasobtained with a fluorometer (FL-920 England). The concen-trations of sample solution were varied from 1.0� 10�3 to0.5mg/ml. The excitation wavelength (lex) was 336 nm.

Nanoparticles yield, drug-loading content, and entrapmentefficiency

The obtained nanoparticles solutions were frozen and lyophilizedby freeze dryer system to obtain a dried nanoparticle product.The weight of nanoparticles was defined as the weight of theresultant dried product obtained by lyophilization. For evaluationof drug contents and loading efficiency, 5mg of paclitaxel loadedlyophilized polymeric nanoparticles were dissolved in 1mldicholoromethane (DCM) under vigorous vortexing. This solutionwas transferred to 10ml of the mixture of 50/50 (v/v) acetonitrileand water. The nitrogen was introduced to evaporate DCM and aclear solution was obtained for HPLC (Agilent LC 1100) analysis.The mobile phase of HPLC was composed of acetonitrile andwater of 50/50 (v/v).The nanoparticles yield, drug-loadingcontent, and drug entrapment efficiency were presented byeqn (1), (2), and (3), respectively:

Nano particles yieldð%Þ¼weight of nanoparticles=weight of polymer and drug feed initially

� 100

(1)

Drug� loading contentð%Þ¼weight of drug in nanoparticles=weight of nanoparticles

� 100

(2)

Entrapment efficiencyð%Þ¼weight of drug in nanoparticles=weight of drug feed initially

� 100

(3)

Each sample was assayed in triplicate.

Physical stability of paclitaxel -loaded nanoparticles

The drug-loaded nanoparticles (2mg/ml) were determined forphysical stability in PBS at 258C. Nanoparticle diameter changesas a function of time and scattering intensities were evaluated byDLS as mentioned above.

In vitro release experiment

One-milliliter (2mg/ml) particle suspension was put in acentrifuge tube containing 10ml PBS (pH 7.4). The tube wasplaced in an orbital shaker water bath at 378C. At particular timeintervals, the tube was taken out and centrifuged. Thesupernatant was poured out and extracted with 1ml DCM todetermine the content of the drug released. The pellet wasresuspended in 10ml fresh PBS for continuous release

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measurement. The analysis procedure was same as described inthe determination of the entrapment efficiency.

In vitro antitumor activity

Human liver cancer cells of H7402 were chosen as target cells toevaluate the antitumor activity of paclitaxel-loaded nanoparti-cles. The tumor cells were cultured in the Dulbecco’s Modificationof Eagle’s Medium (DMEM) growth medium (containing 10%fetal bovine serum, 100 IU/ml of penicillin, and 100mg/ml ofstreptomycin). The cell density was adjusted to 5� 104 cell/well.Aliquots (150ml) of this suspension were added to the wells in a96-well microplate and incubated for 24 hr in a humidifiedatmosphere containing 5% CO2 at 378C, and then the culturemedium was replaced with fresh DMEM. Predetermined amountsof the encapsulate nanoparticles and conjugate nanoparticleswere dissolved in dimethylsulfoxide (DMSO) so that the paclitaxelconcentrations after 100-fold dilution with PBS were 1, 10, 20, 30,40 ng/ml, and 0.1, 2, 4, 6, 8, 10mg/ml, respectively. Pure paclitaxeldissolved in DMSO and diluted with PBS was used as positivecontrol and HPAE-co-PLGA (6:1, 1.0mg/ml) was used as negativecontrol. DMSO, 100-fold-diluted with PBS, was used as the blankcontrol. Then the diluted solutions were added into the wells.After 56 hr incubation, 20ml of MTT solution (5mg/ml) was addedto each well of the plate. The incubation was continued foranother 4 hr. Then the MTT derivative was dissolved with DMSO(100ml) and the optical density of the solution was determinedby a microplate reader (Molecular Devices Co., Sunnyvale, CA) at560 nm. The relative cell viability was calculated using followequation:[30]

Cellviability %ð Þ ¼ Abstest cells=Abscontrol cells� 100

where Abstest cells and Abscontrol cells represent absorbances for thecells treated with paclitaxel or nanoparticles and the cells treatedwith the culture medium, respectively.

Statistical analysis

The results were reported as the mean� S.D. Between-groupcomparisons were carried out by the two-sample t-test usingSPSS11.5. p< 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Synthesis and characterizations of HPAE-co-PLGA copolymer

In our work, different generation HPAE-OHs4 with surfacehydroxyl groups were prepared according to the previouspaper[31] with few modifications. The synthesis route was shownin Scheme 1(A). In order to minimize the side reactions betweenthe AB2 monomer, the choice of effectively-available transester-ification catalysts which show the best compromise betweenchemioselecivity and activity has attracted growing attention inchemical reactions. It showed that titanates and zirconates werethe most interesting catalysts.[32] Because of above reasons,p-methylbenzene sulfonic acid (p-TsOH) was replaced byTi(OiPr)4. The final products of HPAE-OHs4 were soluble in water,ethanol and N, N-dimethylformamide (DMF).HPAE-co-PLGA copolymers were synthesized by a ring-opening

polymerization. Polymerization of DL-lactide and glycolide can beeffected by at least four different mechanisms categorized asanionic, cationic, coordination, and radical. Among them,


coordination polymerization is one of the most versatile methodsfor preparing PLGA and its copolymers, affording high molecularweights and conversions. In the present polymerization system,metal species are believed to function as a catalyst and thehydroxyl end-group of HPAE serves as an initiator.Stannous 2-ethylhexanoate has the advantage of having been

used to prepare polymers for which substantial toxicological dataare now available.[33] The active hydrogen atom at one end ofthe HPAE chains acted as an initiator and induced a selectiveacyl-oxygen cleavage of DL-lactide and glycolide. The polymeri-zation route was shown in Scheme 1(B). The solubility ofHPAE-co-PLGA was opposite to HPAE-OHs4, which could besolved in acetone, acetonitrile, dichloromethane, and tetrahy-drofuran (THF).The hydroxyl values of different generation HPAE-OHs were

measured and calculated by the method described in the previouspaper.[28] The results of different generation HPAE-OHs are shownin Table 1. It could be clearly seen that with the increase ofgeneration the hydroxyl values were close to the theoretical value,which indicated that the structure was apt to regulation.The different samples named HPAE-co-PLGA 6:1(feed ratio of

DL-lactide /glycolide molar ratio), HPAE-co-PLGA10:1, and HPAE-co-PLGA 15:1, respectively, were synthesized.The molecular weights and polydispersity indexes of the

HPAE-co-PLGA copolymers are shown in Table 2. The amount oflactide introduced to HPAE-OHs increased with the molar ratio ofDL-lactide /glycolide to HPAE-OHs. As the molar ratio of DL-lactide/glycolide to HPAE-OHs4 increased from 6:1 to 15:1, the molecularweights rose from 23 to 76 kDa. This indicated that the higherthe concentration of DL-lactide and glycolide, the higher theopportunity for the DL-lactide and glycolide to react withHPAE-OHs reactive centers. The reason of high polydispersityof the obtained copolymers may be due to the high polymermolecular weight, high viscosity of polymerization system in thelate reaction, so DL-lactide and glycolide are difficult to spreadevenly. Some of the active polymer end-groups do not haveaccess to DL-lactide and glycolide monomer, and the polymeri-zation was terminated prematurely.The hydrophobicity of the copolymer increases in the order

HPAE-co-PLGA (6/1), HPAE-co-PLGA (10/1), HPAE-co-PLGA (15/1)by increasing the molar ratio of DL-lactide/glycolide in the PLGAsegment because DL-lactide moiety is more hydrophobic thanglycolide.Figure 1(a,b) showed the infrared spectra of the HPAE-Ohs4

and HPAE-co-PLGA. It showed that absorption band ofHPAE-co-PLGA at 3455 cm�1 was assigned to terminal hydroxylgroups in the copolymer. The bands at 3010 and 2948 cm�1weredue to C–H stretch of CH3. A strong band at 1759 cm�1 wasassigned to C –– O stretch. The CH2 scissoring and waggingmodesat 1458, 1436, and 1384 cm�1 were different between the twographs. For the fourth generation of HPAE-Ohs4 (Fig. 1a), theintensity of the three absorption bands was weak and almostequivalent. While in HPAE-co-PLGA, the band located at 1458 and1384 cm�1 became much stronger than the other band. Theband at 1192 cm�1 was corresponding to the C–O stretchingvibration in HPAE-OHs4 alone, while in HPAE-co-PLGA theabsorption intensity became stronger than HPAE-OHs4 alone.All of these changes indicated the conformational change ofthe HPAE-OHs4 after the conjugation to the PLGA.The basic chemical structure of HPAE-OHs4 and HPAE-co-PLGA

were also confirmed by 1H NMR (Fig. 2a,b). Compared withHPAE-OHs4 (Fig. 2a), the 1H NMR spectra of the HPAE-co-PLGA


Scheme 1. The synthesis route of HPAE-OHs (A) and synthesis route of HPAE-co-PLGA copolymer (B).


2

copolymer (Fig. 2b) showed that the signals at �4.3 ppm wereassigned to the terminal methenyl protons of the branchedpolylactide. The signals at �1.4 and �1.5 ppm were attributed tothe methyl protons of the polylactied moiety located at theterminal groups and the repeat units of it in the chain.Overlapping doublets at �1.55 ppm were attributed to the


methyl groups of the D- and L-lactic acid repeat units. Themultiplets at �5.2 and �4.8 ppm corresponded to the lactic acidCH and the glycolic acid CH2, respectively, with the highcomplexity of the peaks resulting from different D-lactic, L-lactic,glycolic acid sequences in the polymer backbone (Structure ofthe HPAE-co-PLGA copolymer shown in Scheme 1B).


329

Table 1. The hydroxyl values of different generationHPAE-OHsa

Hydroxyl values of differentgeneration HPAE-OHs G2 G3 G4

Theoretical value (mgNaOH/g) 306 276 263Experimental value (mgNaOH/g) 322 250 273

a Represent the generation of HPAE-OHs.

Figure 1. IR spectra of HPAE–OHs4 (a) and HPAE-co-PLGA (6:1) (b).

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The basic chemical structure of HPAE-OHs4 and HPAE-co-PLGAwere further confirmed by 13CNMR (Fig. 2c, d). Compared withHPAE-OHs4 (Fig. 2c) the13CNMR spectra of the HPAE-co-PLGAcopolymer (Fig. 2d) showed that the peak at �170 ppm wasattributed to the C –– O group carbon peak of PLGA. The signalsat� 68 and�70 ppmwere assigned to –CH group carbon peak ofthe PLGA moiety located at the terminal groups and the repeatunits of it in the chain. The signals at �17 and �20 ppm wereattributed to the –CH3 group carbon peak of the PLGA moietylocated at the repeat units and the terminal groups. All theseresults evidenced that the hyperbranched copolymer containedside chains of polylactide and polyglycolide.The thermal properties of HPAE-co-PLGA copolymer were

examined by TGA measurement. Figure 3A showed the TGAthermo gram of the HPAE-OHs4, HPAE-co-PLGA, the mixture ofHPAE-OHs4 and PLGA (HPAE-mix-PLGA), and PLGA. There are twodecomposition events presented in TGA curves of the mixture ofHPAE-OHs4 and PLGA which may be caused by the HPAE-OHs4and PLGA segment. A fast process of weight loss appears in theTG curves response for the PLGA in thermal degradation ranges.Compared to HPAE-OHs4, HPAE-co-PLGA copolymer has lowerthermal degradation temperatures. A fast process of weight lossappeared in the TGA curves response for the HPAE-co-PLGAcopolymer in thermal degradation ranges. These results showedsome decrease of the thermal stability for HPAE-co-PLGAcopolymer relative to the original HPAE-OHs4.The DSC of the HPAE-OHs4, HPAE-co-PLGA, the mixture of

HPAE-OHs4 and PLGA (HPAE-mix-PLGA), and PLGA were alsomeasured and the representative DSC traces were shown in

Table 2. Composition and molecular weight distribution of HPAE

Sample Copolymer

Molecua

Mw (kDa)

1 HPAE-OHs2-co-PLGA(6/1) 4.52 HPAE-OHs2-co-PLGA(10/1) 8.63 HPAE-OHs2-co-PLGA(15/1) 134 HPAE-OHs3-co-PLGA(6/1) 155 HPAE-OHs3-co-PLGA(10/1) 316 HPAE-OHs3-co-PLGA(15/1) 587 HPAE-OHs4-co-PLGA(6/1) 238 HPAE-OHs4-co-PLGA(10/1) 419 HPAE-OHs4-co-PLGA(15/1) 76

aMeasured by GPC, HPAE-OHs2 (Mw¼ 1570Da); HPAE-OHs3 (Mw¼


Fig. 3B. A double peak presented in DSC of the mixture ofHPAE-OHs4 and PLGA may be caused by the HPAE-OHs4 andPLGA phase. The Tg of the PLGA segments was 528C. Only onesidestep presented in all of the copolymer samples showed that

-co-PLGA copolymersa

lr weight of copoly-mer Polydispersity indexes(Mw/Mn)

Mn (kDa)

4.0 1.137.2 1.1911 1.1813 1.1526 1.1947 1.2317 1.3526 1.5853 1.43

3450Da); HPAE-OHs4 (Mw¼ 7200Da).


Figure 2. The exhibited the typical 1H NMR spectrum of HPAE-OHs4 (a), HPAE-co-PLGA copolymer (b), 13CNMR spectrum of HPAE-OHs4 (c), and

HPAE-co-PLGA copolymer (d).


2

there were no existence of inhomogeneities.18.68C could be usedas the glass transition temperature of the HPAE-co-PLGA whichwas far low compared to pure PLGA. The reason could beexplained that HPAE-OHs4 with low Tg was elastic state at roomtemperature. The decrease in the Tg of PLGA after beingintroduced on HPAE indicated that the HPAE moiety couldplasticize the adjacent PLGA chain.

Biodegradation of HPAE-co-PLGA copolymer

The plots of weight loss versus time were demonstrated on Fig. 4.It was found that the hydrophilicity–hydrophobicity balanceplayed an important role in the biodegradation of theHPAE-co-PLGA copolymers. Since HPAE moieties were hydro-philic, water could be diffused into the HPAE-co-PLGA copolymermatrix so that the biodegradation could take place simul-taneously inside the copolymer film. In addition, the HPAE-co-PLGA copolymer with small molecular weight was biode-graded with the higher rate than those with large molecularweight. That was because the polymeric chains of smallmolecular weight possessed better mobility and more hydro-philicity so that the penetration of water molecules into thepolymer matrix became easy.


Characterization of HPAE-co-PLGA polymeric nanoparticles

Amphiphilic copolymer HPAE-co-PLGA can be dissolved inorganic solvents such as dichloromethane, acetone, acetonitrile,DMF, and THF, and it could self-assemble into micelles in selectivesolvents such as the aqueous medium. The main driving forcesof HPAE-co-PLGA micelles formation were known to be thesolvophobicity of PLGA segments in aqueous medium. Theinsolubility of PLGA segments in water causes the segment tocoagulate to form a core. The solvophilic segments, HPAE,stabilize the core as tethered chains from the core particle. Due tothe paclitaxel hydrophobicity, it could be incorporated into theHPAE-co-PLGA micelles during the progress of micelle formation.However, usually the encapsulation efficiency of the drugdepends on the structure, length, and molecular weight ofhydrophilic and hydrophobic fragments. Figure 5 gave theexcitation spectra of pyrene in its aqueous solutions with variousconcentrations and the change of I372/I383 with the concentration.The critical micelle concentration (cmc) was determined by theinterception of two straight lines. The cmc values of hyper-branched copolymer were listed in Table 3. From the table, it canbe seen that the cmc values of polymeric amphiphiles werelower than the cmc of low molecular weight surfactants,[34] and


331

Figure 3. TGA graphs of HPAE-OHs4, HPAE-co-PLGA (10:1), the mixture

of HPAE-OHs4 and PLGA (HPAE-mix-PLGA) and PLGA (Mw¼ 34 kDa) (A)

and DSC thermo grams of HPAE-OHs4, HPAE-co-PLGA (10:1), the mixture

of HPAE-OHs4 and PLGA (HPAE-mix-PLGA) and PLGA (Mw¼ 34 kDa) (B).

Figure 4. Weight Loss of the HPAE-co-PLGA copolymers in PBS (pH 7.4)

at 378C.

Figure 5. (A) Fluorescence emission spectra of pyrene in water in

the presence of the HPAE-co-PLGA copolymer at 208C (1: 0.5, 2: 0.25,3: 0.1, 4:0.01, 5: 0.005, 6:0.001mg/ml); (B) Change of the intensity ratio

(I372/I383) versus the concentration of the HPAE-co-PLGA copolymer

at 208C.


Y. WU ET AL.

2332

similar to other amphiphilic PLGA copolymer,[35] indicating thestability of micelles from hyperbranched copolymer at diluteconditions. The increase in hydrophobicity by introduction of alarge amount of hydrophobic groups further reduces the cmcvalues (Table 3).Figure 6(A, B) showed the ESEM image and the size distribution

of paclitaxel-loaded polymeric nanoparticles. It could beconfirmed that polymeric nanoparticles were regular sphericalin shape (Fig. 6A). Figure 6B showed a unimodal size distributionof paclitaxel-loaded polymeric nanoparticles.

Nanoparticles yield, drug-loading content, and entrapmentefficiency

The size and its size distribution of polymeric micelles andpaclitaxel-loaded polymeric nanoparticles (PaNP6, PaNP 10,PaNP15) were measured by DLS (Table 3). The size of theseplain polymeric micelles was 114–153 nm in water. The DLS datademonstrate that the micelle sizes increased with the increase ofDL-lactide/glycolide molar ratio. These results suggested that theelongation of hydrophobic PLGA side chain facilitates the growth


Table 3. Effect of different composition of copolymer on the properties of polymeric micelles and paclitaxel-loaded polymericnanoparticlesa

Sample Copolymer Mean diameter(nm)Polydispersity index of

mean diameter cmc� 102 (mg/ml)

1 HPAE-co-PLGA(6/1) 114 0.05 5.562 HPAE-co-PLGA(10/1) 136 0.08 2.013 HPAE-co-PLGA(15/1) 153 0.06 0.99PaNP6 HPAE-co-PLGA (6/1) 136 0.07 3.20PaNP10 HPAE-co-PLGA (10/1) 161 0.09 1.08PaNP15 HPAE-co-PLGA (15/1) 184 0.05 0.43

a Paclitaxel content 10% (w/w); HPAE-co-PLGA concentration 0.5mg/ml.


of the hydrophobic core of polymeric micelles. It can be seenfrom Table 3 that these micelles possess a narrow unimodaldistribution (polydispersity index). These results indicated thatthe micelle size was dependent on the ratio of hydrophobic PLGAsegment to hydrophilic HPAE segment in the chain.Table 4 summarized the nanoparticles yield, drug-loading

content, and entrapment efficiency of HPAE-co-PLGA copoly-meric nanoparticles. The nanoparticles yield and drug-loadingcontent depended mainly on the copolymer composition

Figure 6. The ESEM images (A) and the size distribution (B) of paclitax-el-loaded HPAE-co-PLGA copolymer nanoparticles in water (HPAE-

co-PLGA (10:1)).


ratio of HPAE to PLGA. The drug-loading content in nanopar-ticles increased from 7.5 to 12.1% with increase in the ratioof DL-lactide/glycolide to HPAE in copolymers. This resultcould be explained by the paclitaxel having a hydrophobiccharacter. Therefore, the higher the PLGA segment contentin copolymer, the more easily the drug was entrappedin nanoparticles. HPAE-co-PLGA (15:1) had the highestnanoparticles yield, 87.6%, within all of the samples. A largeamount of gelatinous coagulation was found during theacetonitrile evaporation process for preparation of HPAE-co-PLGA (6:1) and HPAE-co-PLGA (10:1) nanoparticles, whichwas the reason for the lower nanoparticles yield of the twosamples.

Physical stability of paclitaxel–loaded nanoparticles

In the clinical administration of the nanoparticle suspension,vessel occlusion due to the particle aggregation may occur. Thesteric stability of nanoparticles in the biological milieu is thusimportant aspect to be considered. An improved safety profileof amphiphilic copolymer nanoparticles was observed incomparison with the hydrophobic PLGA nanoparticles, whichwas attributed to the presence of hydrophilic segment onthe particles surface to prevent the coagulation cascade. In thepreparation of nanoparticles, the surfactant does not reallyattend the formation of nanoparticles. It acts as a stabilizer forkeeping the particles’ stability. HPAE-co-PLGA nanoparticleswere of an amphiphilic structure: the hydrophobic PLGAcompromised the pact core while the hydrophilic HPAEextended to the outer aqueous environment to form a shell.This structure may be believed to possess self-stabilizationfunction. In our work, the particles were suspended in PBSand the size was recorded (Fig. 7). Only a small size variationfrom 161 to 179 nm is observed in 14 days. This demonstratesthat HPAE-co-PLGA nanoparticles possess good steric stabilityin vitro.

2

In Vitro drug release studies

Figure 8 showed release profiles of paclitaxel from HPAE-co-PLGAnanoparticles with various DL-lactide/glycolide/HPAE ratios. Forall polymeric nanoparticles, paclitaxel release showed an initialburst release and after paclitaxel release profiles displayed asustained fashion. An initial burst release, a significant amount of


333

Table 4. Drug loading efficiency, drug entrapment efficiency, and nanoparticles yield of paclitaxel-loaded copolymeric nano-particlesa

Sample Copolymer Entrapment efficiency (%) Drug loading (%) Micelle yield (%)

1 HPAE-co-PLGA(6/1) 69.5 7.5 56.32 HPAE-co-PLGA(10/1) 74.2 8.9 71.23 HPAE-co-PLGA(15/1) 83.1 12.1 87.6

a The mass of paclitaxel used was 20% (w/w) in relation to polymer mass.

Y. WU ET AL.

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paclitaxel was released within 12 hr, 29% for HPAE-co-PLGA (6:1)copolymer nanoparticles. After the initial burst, HPAE-co-PLGArelease profiles displayed a sustained release. The amount ofcumulated HPAE-co-PLGA release over 14 days was 78.1% forHPAE-co-PLGA (6:1) polymeric nanoparticles (paclitaxel content20%). This sustained release could result from diffusion ofpaclitaxel into the polymer wall and the drug through polymerwall as well as the erosion of the polymers. The release of a drugfrom the polymer nanoparticles is rather complicated process. Itcan be affected by many factors such as the polymerdegradation, molecular weight, crystallinity, the binding affinitybetween the polymer and the drug, and so on.[36] In the study, thedrug release rate might be mainly determined by the diffusion ofthe drug through the polymer matrix. The initial burst might beattributed to the rapid release of drugs in the microchannelsprobably existing in nanoparticles. Paclitaxel, because of itslipophilic character, was physically entrapped in the hydrophobiccore of a nanoparticle. Accordingly, the in vitro release behaviorsof a lipophilic compound from these polymeric nanoparticlesystems were largely affected by its inner core with hydrophobicproperties.[36] Therefore, as the PLGA content of a copolymerincreased, the hydrophobic segments in a copolymer increased,resulting in the increase of the binding affinity between paclitaxeland PLGA. Consequently, in this nanoparticle system, the drugrelease rate was inversely proportional to the PLGA content ofa copolymer. Another reason for the fast release rate ofHPAE-co-PLGA was their small particle size with relatively highsurface area.

Figure 7. Steric stability of paclitaxel-loaded HPAE-co-PLGA (10:1) nano-

particles in PBS.


In vitro antitumor activity

The antitumor activity of the paclitaxel nanoparticles againsthuman liver cancer H7402 cells was evaluated by MTTmethod. Asshown in Fig. 9(a, b), the cancer cells were inhibited by paclitaxelnanoparticles after 56 hr incubation as a function of the paclitaxelamount (1 ng/ml–10mg/ml) used. It was seen that the antitumoractivities of the drug-loaded nanoparticles were comparable tothat of the pure paclitaxel and the concentration of 10mg/ml ofpaclitaxel is enough to kill about 75% of the cancer cells, itindicated that the paclitaxel could be released from paclitax-el-loaded nanoparticles without losing the anticancer activity.The IC50 value of free paclitaxel is 0.014mg/ml, and the IC50 valueof paclitaxel-loaded HPAE-co-PLGA (6:1, 1mg/ml) nanoparticles is0.017mg/ml. Paclitaxel can induce apoptosis of cancer cells. Ahigher concentration shows stronger antineoplastic activity andinhibition against tumor cells. It is noticed that the ability of thepaclitaxel-loaded HPAE-co-PLGA nanoparticles against livercancer is lower than that of pure paclitaxel after 56 hr incubation.The reason is that the paclitaxel-loaded HPAE-co-PLGA nano-particles display anticancer efficacy only when the paclitaxelmolecule is dissociated from the copolymer, and therefore thepaclitaxel-loaded HPAE-co-PLGA nanoparticles are releasedslower than the paclitaxel (data not shown) at the early stage.But since the paclitaxel-loaded HPAE-co-PLGA nanoparticlesshow a sustained release in PBS buffer in vitro, it is expected tohave a long-term anticancer activity. Animal trials are beingperformed to examine the long-term antitumor activity of the

Figure 8. Release profiles of paclitaxel from HPAE-co-PLGA polymeric

nanoparticles.


Figure 9. In vitro cytotoxicity of paclitaxel-loaded HPAE-co-PLGA nano-

particles against human liver cancer cell line H7402 (paclitaxel concen-

tration (a): 1–80 ng/ml; (b): 0.1–10mg/ml).


paclitaxel-loaded HPAE-co-PLGA nanoparticles. The results will bereported elsewhere.

CONCLUSION

In this study, HPAE-co-PLGA copolymer and paclitaxel-loadedHPAE-co-PLGA copolymer were amphiphilic and could self-assemble into nanoparticles in an aqueous media. Fluorescencespectrum, DLS, and ESEM analysis of the nanoparticles revealedtheir homogeneous spherical morphology and unimodal sizedistribution. The preliminary investigations for the novelnanoparticle system have shown that the composition of thecopolymer made a large influence on the nanoparticle size andsize distribution, and drug release behavior. Control of thenanoparticle size, drug-loading content, and drug releasebehavior could be achieved by optimizing the DL-lactide/glycolide to HPAE ratio of the copolymer. The particlessuspension exhibited good steric stability in vitro. In vitroantitumor activity of the paclitaxel-loaded polymeric nanopar-ticles against human liver cancer cell line H7402 was evaluatedby MTT method. The results showed that paclitaxel could be


released from the polymeric nanoparticles without losingcytotoxicity. Therefore, the HPAE-co-PLGA copolymer wasexpected to be used as a new formulation of paclitaxel andlipophilic drugs.

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Documents

Hyperbranched poly (amine-ester)-poly (lactide-co-glycolide) copolymer and their nanoparticles as paclitaxel delivery system