Functionalized Amphiphilic Hyperbranched Polymers for Targeted

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Articles Functionalized Amphiphilic Hyperbranched Polymers for Targeted Drug Delivery Si Chen,† Xian-Zheng Zhang,† Si-Xue Cheng,*,† Ren-Xi Zhuo,† and Zhong-Wei Gu‡ Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China, and National Engineering Research Center in Biomaterials, Sichuan University, Chengdu 610064, People’s Republic of China Received April 7, 2008; Revised Manuscript Received June 22, 2008

Amphiphilic hyperbranched core-shell polymers with folate moieties as the targeting groups were synthesized and characterized. The core of the amphiphilic polymers was hyperbranched aliphatic polyester Boltorn H40. The inner part and the outer shell of the amphiphilic polymers were composed of hydrophobic poly(-caprolactone) segments and hydrophilic poly(ethylene glycol) (PEG) segments, respectively. To achieve tumor cell targeting property, folic acid was further incorporated to the surface of the amphiphilic polymers via a coupling reaction between the hydroxyl group of the PEG segment and the carboxyl group of folic acid. The polymers were characterized by 1H NMR, 13C NMR, and combined size-exclusion chromatography and multiangle laser light scattering analysis. The nanoparticles of the amphiphilic polymers prepared by dialysis method were characterized by transmission electron microscopy and particle size analysis. Two antineoplastic drugs, 5-fluorouracil and paclitaxel, were encapsulated into the nanoparticles. The drug release property and the targeting of the drugloaded nanoparticles to different cells were evaluated in vitro. The results showed the drug-loaded nanoparticles exhibited enhanced cell inhibition because folate targeting increased the cytotoxicity of drug-loaded nanoparticles against folate receptor expressing tumor cells.

Introduction Micelles formed by self-assembly of amphiphilic copolymers combining hydrophilic and hydrophobic segments have attracted significant attention in diverse biomedical fields such as drug and gene delivery.1,2 The advantages of micelle drug delivery systems include to improve the solubility of water-insoluble drugs, to stabilize and protect drugs that are sensitive to the surrounding environment, to reduce the nonspecific uptake by the reticuloendothelial system (RES), to prolong the circulation time in the blood, and thus to achieve the possible targeting delivery.1,2 However, the conventional micelle drug delivery systems based on the linear amphiphilic copolymers suffer from instability in vivo because the concentration of the copolymer is diluted in the bloodstream and micelles tend to disassemble once the concentration falls below the critical micelle concentration.3 To overcome the disadvantage of classical micelles, unimolecular micelles/nanoparticles based on amphiphilic copolymers with dendritic or hyperbranched structure have been developed. Because of the unique structure of the unimolecular micelles, the unimolecular micelles do not disassemble under dilution and are stable to environmental changes. Besides the good stability, the highly branched structure of the copolymers can provide many end groups for further functionalization.4-7 In this study, we synthesized amphiphilic hyperbranched polymers, H40-PCL-PEG-FA 1 and 2 with different molecular * To whom correspondence should be addressed. Tel./Fax: 86-2768754509. E-mail: [email protected]; [email protected]. † Wuhan University. ‡ Sichuan University.

weights, which have hyperbranched aliphatic polyester Boltorn H40 as the core, hydrophobic poly(-caprolactone) (PCL) segments as the inner part and hydrophilic poly(ethylene glycol) (PEG) segments as the outer shell. H40 is a commercial biodegradable hyperbranched aliphatic polyester,8 which has shown potential in biomedical applications.7 PCL is an important member of the aliphatic polyester family and of interest for biomedical applications owing to its good biocompatibility and low immunogenicity. For drug release applications, the advantages of PCL include its high permeability to drugs, and less acidic degradation products as compared to polylactide and polyglycolide.9 PEG is a commonly used hydrophilic polymer in biomedical fields.10 In the current study, PEG segments are also used as flexible spacers to connect with folate moieties. Folic acid (FA) is a targeting group which is extensively used to deliver therapeutic and imaging agents to folate receptor (FR) overexpressed cancer cells.5,10-13 Through incorporation of FA, the amphiphilic hyperbranched polymers are endowed with tumor cell targeting property. The in vitro study of the drugloaded nanoparticles indicates the great potential of H40-PCLPEG-FA nanoparticles as targeting drug carriers.

Experimental Section Materials. Boltorn H40 with 64 terminal hydroxyl groups (Perstorp, Sweden) was dried under vacuum at room temperature for 2 days. -Caprolactone (CL) (Aldrich) was dried over CaH2 for 2 days and distilled under reduced pressure. Stannous octoate (Sigma) was distilled under reduced pressure and then dissolved in dry toluene prior to use. PEG 600 and PEG 4000 were supplied by Shanghai Chemical Co. China and dried under vacuum. Folic acid (FA), dicyclohexylcarbo-

10.1021/bm800371n CCC: $40.75  2008 American Chemical Society Published on Web 07/30/2008

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Scheme 1. Structure of Amphiphilic Hyperbranched Polymer H40-PCL-PEG-FA

diimide (DCC), 4-dimethylaminopyridine (DMAP), succinic anhydride, and tin(II) chloride were of analytical grade and used as supplied by Shanghai Chemical Co., China. Dimethyl sulfoxide (DMSO) was purified by distillation over P2O5 and CaH2. 5-Fluorouracil (5-Fu; Amresco) and paclitaxel (Knowshine Pharmachemicals Inc., Shanghai, China) were used as received. Other reagents were of analytical grade and used without further purification. Polymer Synthesis. Boltorn H40 and CL, with a specific feed ratio of the hydroxyl group in H40 to monomer CL, were placed in a thoroughly dried silanized glass flask with a magnetic stirring bar. The flask was evacuated, purged with nitrogen three times, sealed under vacuum, and then immersed in an oil bath at 120 °C for 24 h. Then the reaction mixture was dissolved in dichloromethane, precipitated into cold methanol, filtrated, and dried under vacuum to obtain H40PCL (H40-PCL 1 with -OH/CL feed ratio of 1/10 and H40-PCL 2 with -OH/CL feed ratio of 1/20, respectively). H40-PCL with 2 mmol of -OH groups (the amount of H40-PCL was calculated based on that 1 mol H40-PCL contains 64 mol -OH groups), succinic anhydride (2.2 mmol) and tin(II) chloride (5 wt % to H40-PCL) were dissolved in dioxane (10 mL). The mixture was refluxed for 24 h to obtain H40-PCL-COOH (H40-PCL-COOH 1 from H40-PCL 1 and H40-PCL-COOH 2 from H40-PCL 2, respectively). H40-PCL-COOH with 1 mmol of -COOH groups (the amount of H40-PCL-COOH was calculated based on that 1 mol H40-PCL-COOH contains 64 mol -COOH groups), PEG (PEG 600 to obtain H40-PCLPEG 1 and PEG 4000 to obtain H40-PCL-PEG 2; 1 mmol), and DCC (1.1 mmol) were dissolved in anhydrous DMSO (20 mL) and stirred overnight at room temperature. Then DMAP (1 mmol) was added to the reaction mixture. The reaction mixture was stirred at room temperature for another 24 h. The product was dialyzed against DMSO for 3 h, then dialyzed against water for 24 h, during which the water was renewed every 3 h, and finally freeze-dried to obtain H40-PCLPEG (H40-PCL-PEG 1 from H40-PCL-COOH 1 and H40-PCL-PEG 2 from H40-PCL-COOH 2, respectively). Folic acid (0.1 mmol) and DCC (0.11 mmol) were dissolved in anhydrous DMSO (10 mL). The reaction mixture was stirred overnight at room temperature. Then H40-PCL-PEG with 1 mmol of -OH groups and DMAP (0.1 mmol) were added, and the reaction mixture was stirred at room temperature for another 24 h. The precipitated byproduct, dicyclohexylurea (DCU), was removed through centrifugation. The supernate was precipitated in cold methanol, filtrated, and dried under vacuum. The product was dissolved in DMSO, put in a dialysis tube (MWCO 8000-12000), dialyzed against DMSO for 3 h, then dialyzed

against water for 24 h, during which the water was renewed every 3 h, and finally freeze-dried to obtain H40-PCL-PEG-FA (H40-PCL-PEGFA 1 from H40-PCL-PEG 1 and H40-PCL-PEG-FA 2 from H40-PCLPEG 2, respectively). Polymer Characterizations. 1H NMR and 13C NMR spectra were recorded on a Varian Inova 600 MHz spectrometer. The solvent for H40-PCL-PEG-FA was DMSO-d6 and the solvent for other polymers was CDCl3. The molecular weights of the polymers were determined by combined size-exclusion chromatography and multiangle laser light scattering (SEC-MALLS) analysis. A dual detector system, consisting of a MALLS device (DAWN EOS, Wyatt Technology) and an interferometric refractometer (Optilab DSP, Wyatt Technology) was used. THF was used as the eluent at a flow rate of 0.3 mL/min. The MALLS detector was operated at a laser wavelength of 690.0 nm. Nanoparticle Formation and Characterizations. A total of 10 mg of functionalized H40-PCL-PEG-FA was dissolved in 3 mL of DMF. The solution was put into a dialysis tube (MWCO 8000-12000) and subjected to dialysis against 2000 mL of distilled water for 24 h to form nanoparticles. Transmission electron microscopy (TEM) observation was carried out on a JEM-100CXII transmission electron microscope. Before visualization, a droplet of nanoparticle suspension containing phosphotungstic acid was placed on a copper grid with Formvar film and dried. The size and size distribution of nanoparticles in an aqueous medium (2.5 mg/mL) were measured by a Zetasizer Nano ZS (Malvern Instruments). Drug Loading. The H40-PCL-PEG-FA 1 (10 mg) and 5-Fu (1 mg) were dissolved in 3 mL of DMF. The solution was put into a dialysis tube (MWCO 8000-12000) and subjected to dialysis against 2000 mL distilled water for 24 h, during which the water was renewed every 8 h. To determine the total drug loading, the drugloaded nanoparticle suspension was lyophilized and then dissolved in DMF. The UV absorbance at 270 nm was measured to determine the drug concentration. The H40-PCL-PEG-FA 2 (10 mg) and paclitaxel (1 mg) were dissolved in 3 mL of DMF. The solution was put into a dialysis tube (MWCO 8000-12000) and subjected to dialysis against 2000 mL distilled water for 24 h, during which the water was renewed every 8 h. To determine the total drug loading, the drug-loaded nanoparticle suspension was lyophilized and then dissolved in acetonitrile. The UV absorbance at 233 nm was measured to determine the drug concentration.

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Scheme 2. Synthesis of Amphiphilic Hyperbranched Polymer H40-PCL-PEG-FA

The drug loading content and entrapment efficiency were calculated as follows: drug loading content ) (drug recovered in nanoparticles/ nanoparticles recovered) × 100%; entrapment efficiency ) (drug recovered in nanoparticles/drug fed) × 100%. In Vitro Drug Release. After dialysis, the 5-Fu-loaded nanoparticle suspension in dialysis tube was immersed into 30 mL of phosphate buffer (PBS; 0.1 M, pH 7.4) and kept in a shaking water bath at 37 °C. Aliquots of 3 mL were taken out from the solution periodically. The volume of solution was kept constant by adding 3 mL of fresh PBS after each sampling. The drug concentration was determined by measuring the absorbance of 5-Fu at 270 nm in an ultraviolet-visible spectrophotometer (Perkin-Elmer Lambda Bio 40). The data are given as mean ( standard deviation (SD) based on three independent measurements.

In Vitro Evaluation of Cell Targeting Property. Human cervical carcinoma cell line Hela, human lung adenocarcinoma cell line A549 and mouse fibroblast cell line NIH 3T3 were obtained from China Center for Typical Culture Collection (Wuhan, China). The cells were seeded in a 96-well plate with a density of 5000 cells per well. The cells in each well were cultured in 200 µL of folate-free Dulbecco’s modified Eagle’s medium (DMEM; Sigma), supplemented with 10% fetal bovine serum, 2 mg/mL NaHCO3, and 100 unit/mL penicillin/ streptomycin. Cells were incubated at 37 °C in humidified air/5% CO2 for 24 h. Then the culture medium was replaced by 200 µL of fresh medium containing drug-loaded nanoparticles (H40-PCL-PEG-FA 1 nanoparticles containing 18 µg of 5-Fu or H40-PCL-PEG-FA 2 nanoparticles containing 20 µg of paclitaxel) or blank nanoparticles. For comparison, both folate-free medium and the medium containing


Figure 1.

13

C NMR spectra of (a) H40-PCL 1 and (b) H40-PCL 2.

1 µg/mL of folic acid were used. After incubation at 37 °C for 48 h, the culture medium was removed. Fresh medium (200 µL) and MTT (20 µL, 5 mg/mL) were added to each well, followed by 4 h of incubation at 37 °C. Then the supernatant was carefully removed, and 200 µL of DMSO was added in each well. The absorbance of the solution was measured using microplate reader (Bio-Rad 550) at 570 nm to determine the OD value. The cell viability was calculated as follows. Cell viability ) (ODtreated/ODcontrol) × 100%, where ODtreated was obtained for the cells treated by the nanoparticles, ODcontrol was obtained for the cells untreated by the nanoparticles, and the other culture conditions were the same. The data are given as mean ( standard deviation (SD) based on eight independent measurements.

Results and Discussion Synthesis and Characterizations of Amphiphilic Hyperbranched Polymer H40-PCL-PEG-FA. The schematic structure of amphiphilic hyperbranched polymer H40-PCL-PEG-FA is shown in Scheme 1. The core of the amphiphilic polymer is hyperbranched aliphatic polyester Boltorn H40. The reason we used Boltorn H40 as the core is due to its following advantages, including biodegradability, similarity to dendrimers, such as the existence of many functional groups that allow the modification for end use purposes and possibility to form unimolecular micelles with stability against dilution for drug delivery after incorporation of hydrophilic shell, and much lower cost as compared with commercial dendrimers. The hydrophobic PCL segments are connected with H40 core. Because both H40 core and PCL segments are hydrophobic, they could form the hydrophobic inner part of the nanoparticle when self-assembly occurs in an aqueous solution. The outer shell of the amphiphilic polymer is composed of hydrophilic PEG segments. To achieve tumor targeting property, folic acid moieties are further incorporated to some of the PEG segments. The detailed synthesis route is shown in Scheme 2. As we know, the ring-opening polymerization is an efficient method for producing aliphatic polyesters with high molecular weights. When initiators bearing hydroxyl groups are used, well-defined polymers with controlled architectures can be produced.14,15 In this study, using H40 with a theoretical molecular weight of 7316 g/mol and 64 terminal hydroxyl groups as a macroinitiator, H40-PCL was synthesized by the bulk ring-opening polymerization of CL. The polymerization was carried out under rigorously anhydrous condition to avoid the initiation by water and the formation of homopolymer PCL. In the current study,


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through adjusting the -OH/CL feed ratios at 1/10 and 1/20, two polymers, H40-PCL 1 and 2, with different molecular weights were synthesized. The 13C NMR spectra of H40-PCL 1 and 2 are shown in Figure 1. According to previous studies, the shift of the quaternary carbon in the initiator H40 is sensitive to the degree of substitution of the two hydroxyl groups adjacent to the quaternary carbon.14 The resonance of the quaternary carbon occurs at 50.6 if both hydroxyl groups are unreacted, at 48.8 if one hydroxyl group remains, and at 46.8 if both hydroxyl groups are reacted.14 As shown in Figure 1, the resonances at 48.8 and 46.8 indicate the existence of two reacted hydroxyl groups adjacent to some quaternary carbons, and one unreacted hydroxyl group and one reacted hydroxyl group adjacent to the other quaternary carbons for H40-PCL 1. The integral calculation shows that about 9 hydroxyl groups remain unreacted among the 64 hydroxyl groups in H40-PCL 1. For H40-PCL 2, only one peak at 46.8 ppm appears, showing that all hydroxyl groups reacted. According to the previous studies of other researchers, not all hydroxyl groups of H40 can initiate the ringopening polymerization of cyclic esters, especially when the degree of polymerization is low. Our result is in consistence with previous literatures.14,15 To achieve a high reactivity for further reactions, the next step was to convert hydroxyl groups of H40-PCL to carboxyl groups. This was achieved by reaction between H40-PCL and succinic anhydride in the presence of tin(II) in dioxane to obtain H40-PCL-COOH. After that, PEG segments were introduced to the hyperbrached polymer via the coupling reaction between -OH of PEG and -COOH of H40-PCL-COOH to form H40PCL-PEG. Corresponding to the different lengths of PCL segments in H40-PCL 1 and H40-PCL 2, two PEGs, PEG 600 and PEG 4000, with different molecular weights were used to achieve balanced hydrophobic/hydrophilic property of the resultant copolymers, H40-PCL-PEG 1 and H40-PCL-PEG 2, respectively. To achieve tumor targeting property, folic acid was further incorporated to the amphiphilic polymers via coupling reaction between the hydroxyl of the PEG segment and the carboxyl of folic acid to obtain the final product H40-PCL-PEG-FA.13 According to previous research, folic acid retains its receptor binding properties when derivatized via its γ-carboxyl.11 In the current study, the polymers exhibited FR mediated targeting property, indicating that the incorporation of folic acid was mainly through the reaction of γ-carboxyl because the affinity FR binding would not retain if the R-carboxyl group was reacted. As we know, folate moieties with pterin heterocycles can selfassemble in an aqueous solution by hydrogen bonds and stacking interactions.16 As a result, introducing too many FA moieties to the amphiphilic polymer would decrease the stability of the micelles formed by the amphiphilic polymer. Besides, the possible formation of dimers, trimers and even self-assembled tubular quartets16 at higher concentrations of folate molecules is definitely unfavorable for cell targeting since the FR can only bind one FA molecule and the self-assembled multimers of FA would be incapable of binding to the FR. So in the current study, about 10% of the -OH groups of H40-PCL-PEG was functionalized by FA. Figure 2 shows the 1H NMR spectrum of the final product synthesized. As shown in the spectrum of H40-PCL-PEG-FA 1, the typical signals of H40-PCL-PEG part can be observed at 1.15 (a), 1.29 (b), 1.53 (c), 2.26 (d), 3.09 (e), 3.51 (f), 4.05 (g), and 4.20 ppm (h). The typical signals from folate moiety can be detected at 8.68 (A), 8.17 (B), 7.68 (C), 6.98 (D), 6.67 (E),

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Figure 2. 1H NMR spectrum of H40-PCL-PEG-FA 1. Table 1. Synthesis and Molecular Weights of Different Polymers Determined by SEC-MALLS SEC-MALLS

polymer H40 H40-PCL 1 H40-PCL 2 H40-PCL-PEG 1 H40-PCL-PEG 2

feed ratio -OH/CL (mol/mol) 1/10 1/20 1/10 1/20

PEG used

Mw (g/mol)

Mw/Mn

PEG 600 PEG 4000

× × × × ×

1.08 1.20 1.18 1.07 1.22

6.55 6.46 2.53 1.07 4.79

3

10 104 105 105 105

4.51 (F), and 4.36 ppm (G). The appearance of these peaks confirms the successful conjugation. The molecular weights of polymers determined by SECMALLS analysis are listed in Table 1. The polymers have unimodal molecular weight distributions and reasonably narrow polydispersities. Because the ring-opening polymerization of CL was carried out under rigorously anhydrous conditions, the initiation by water and the formation of homopolymer PCL could be ignored. From SEC-MALLS, the unimodal molecular weight distributions of H40-PCL polymers further confirm that no homopolymer PCL could be detected. The absence of the molecular weight of H40-PCL-PEG-FA 1 and 2 is because of their poor solubility in THF due to the incorporation of folate moieties, which leads to the difficulty in SEC-MALLS analysis. Characterization of Polymeric Nanoparticles. The morphologies of H40-PCL-PEG-FA 1 and H40-PCL-PEG-FA 2 nanoparticles visualized by TEM are shown in Figure 3. From the TEM images, the mean size of the H40-PCL-PEG-FA 1

nanoparticles is less than 20 nm, and the mean size of the H40PCL-PEG-FA 2 nanoparticles is less than 100 nm. As determined by the size analyzer (Figure 4), both H40-PCL-PEG-FA 1 nanoparticles and H40-PCL-PEG-FA 2 nanoparticles exhibit unimodal size distributions with average diameters of 16 and 50 nm, respectively. The nanoparticle sizes determined by TEM are in accordance with the data measured by size analyzer. In Vitro Drug Release Property of H40-PCL-PEG-FA. The potential of using H40-PCL-PEG-FA as a delivery carrier was evaluated using 5-Fu and paclitaxel as model drugs. Since paclitaxel is highly hydrophobic, the entrapment efficiency is very low if it is loaded into H40-PCL-PEG-FA 1 nanoparticles since the hydrophobic inner part of H40-PCL-PEG-FA 1 is relatively small. So in the current study, H40-PCL-PEG-FA 1 nanoparticles were used to encapsulate 5-Fu, and H40-PCLPEG-FA 2 nanoparticles were used to load paclitaxel. The drug loaded nanoparticles were prepared by dialysis method. H40PCL-PEG-FA 1 nanoparticles have a drug loading content of 3.5 wt % with an entrapment efficiency of 41.3%, and H40PCL-PEG-FA 2 nanoparticles have a drug loading content of 13.3 wt % with an entrapment efficiency of 79.6%. The in vitro release profile of 5-Fu from H40-PCL-PEG-FA 1 nanoparticles is shown in Figure 5. A sustained release for about 200 h could be observed, and nearly all the loaded drug was released finally. For paclitaxel-loaded H40-PCL-PEG-FA 2 nanoparticles, because the solubility of paclitaxel in the aqueous solution is very low, the release of paclitaxel is controlled by the dissolution of paclitaxel when the volume of



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Figure 3. TEM images of (a) H40-PCL-PEG-FA 1 nanoparticles, and (b) H40-PCL-PEG-FA 2 nanoparticles.

Figure 5. In vitro drug release profile of 5-Fu from H40-PCL-PEGFA 1 nanoparticles.

Figure 4. Size distribution of (a) H40-PCL-PEG-FA 1 nanoparticles, and (b) H40-PCL-PEG-FA 2 nanoparticles.

the solution for release study is limited and a linear release curve could be observed as determined by HPLC (data not shown). In Vitro Targeting Property of H40-PCL-PEG-FA. To evaluate the folate mediated targeting of the H40-PCL-PEGFA nanoparticles, three different types of cells, Hela cells, A549 cells, and NIH 3T3 cells, were used for in vitro study. The cells were incubated with drug loaded nanoparticles in the medium

without or with 1 µg/mL of folic acid at 37 °C for 48 h. MTT assay was used to determine cell viabilities in the presence of the drug loaded nanoparticles, using the cells without the treatment by drug loaded nanoparticles or blank nanoparticles as control. As shown in Figure 6, the 5-Fu loaded H40-PCL-PEG-FA 1 nanoparticles show obvious cell inhibition for Hela cells, which are known as folate receptor positive tumor cells, in the absence of free folic acid in the cell culture medium. While the cell inhibition with the presence of 1 µg/mL of free folic acid is not so high because the targeting is suppressed due to the free folic acid binding. The data of the cell viabilities in free folic acid competition study indicate the cell inhibition is mainly caused by the cell internalization of the nanoparticles through folate-mediated targeting. For blank nanoparticles, cell viabilities are around 100% in both media without and with free folic acid, implying there is no cytotoxicity of H40-PCL-PEG-FA 1 for Hela cells. As for H40-PCL-PEG-FA 2 nanoparticles with larger inner hydrophobic parts, the highly hydrophobic anticancer drug, paclitaxel, which is widely used for lung, ovarian, and breast cancer treatments,17-19 could be effectively encapsulated inside

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Figure 6. Cell viability of Hela cells treated with 5-Fu-loaded H40PCL-PEG-FA 1 nanoparticles and blank H40-PCL-PEG-FA 1 nanoparticles: (a) in the absence of folic acid and (b) in the presence of 1 µg/mL of folic acid.

Figure 7. Cell viability of Hela cells treated with paclitaxel loaded H40PCL-PEG-FA 2 nanoparticles and blank H40-PCL-PEG-FA 2 nanoparticles: (a) in the absence of folic acid and (b) in the presence of 1 µg/mL of folic acid.

Figure 8. Cell viability of A549 cells treated with paclitaxel-loaded H40-PCL-PEG-FA 2 nanoparticles and blank H40-PCL-PEG-FA 2 nanoparticles: (a) in the absence of folic acid and (b) in the presence of 1 µg/mL of folic acid.

the nanoparticles. As depicted in Figure 7, the cell viability is about 4% after the treatment by paclitaxel loaded nanoparticles, and cell viability increases to 13% in the presence of free folic acid, further implying the cell inhibition is caused by the folatedmediated targeting of the nanoparticles. In the current study, the higher branching structure is favorable for targeting by the fact of presenting a larger number of folic acid moieties on the surfaces of nanoparticles. The effective cell inhibition of the drug loaded nanoparticles are particularly interesting for paclitaxel, since the poor hydrolytic stability of this drug constitutes a genuine barrier to its effective administration and it is impossible to use such a high concentration of free drug

Chen et al.

Figure 9. Cell viability of NIH 3T3 cells treated with paclitaxel-loaded H40-PCL-PEG-FA 2 nanoparticles and blank H40-PCL-PEG-FA 2 nanoparticles: (a) in the absence of folic acid and (b) in the presence of 1 µg/mL of folic acid.

for cancer treatment.20 For H40-PCL-PEG-FA 2 nanoparticles, the cell viabilities of blank nanoparticles are lower than 100%, indicating the cytotoxicity of H40-PCL-PEG-FA 2 to Hela cells. The in vitro study on Hela cells indicates that the drug loaded nanoparticles have enhanced cytotoxicity against FR expressing tumor cells due to the folate mediated targeting. Because paclitaxel-loaded H40-PCL-PEG-FA 2 nanoparticles have a strong inhibition effect on Hela cells, we also evaluated the cell inhibition effect on A549 cells having negative expression of folate receptor, and the result is shown in Figure 8. Similar to Hela cells, paclitaxel-loaded H40-PCL-PEG-FA 2 nanoparticles also exhibit strong cell inhibition activity on A549 cells in the medium without free folic acids. With the presence of folic acid in the culture medium, the cell viability after being treated by H40-PCL-PEG-FA 2 nanoparticles does not differ much. The strong inhibition effect of drug-loaded nanoparticles on A549 cells is mainly due to the high endocytic activity of A549 cells. A similar result has been reported in literature, that is, the difference in cytotoxicity between A549 and Hela cell lines after treated with drug-loaded micelles for 48 h was not so significant.21 According to previous studies, this is due to the fact that the cell uptake is mainly mediated by folic acid during short-time incubation. As the incubation proceeded, the cellular internalization of the micelles, which is dependent on micelle size, and structure gradually plays a main role.21 For comparison, in vitro study on nontumor cells, NIH 3T3 cells, was also carried out. As compared with Hela and A549 cells, the inhibition of drug-loaded nanoparticles on 3T3 cells is not so obvious, and the difference in cell viabilities between the 3T3 cells cultured in the media with and without free folic acid is small (Figure 9). The reason is that 3T3 cells do not have high endocytic activity. As a result, the cellular internalization of the nanoparticles is limited as compared with A549 cells.

Conclusions Amphiphilic hyperbranched polymers, H40-PCL-PEG-FA, with hyperbranched aliphatic polyester Boltorn H40 and PCL segments as the hydrophobic part, PEG segments as the hydrophilic outer shell, and folate moieties as the targeting groups were synthesized and characterized. The amphiphilic polymers can self-assemble to form nanoparticles with mean diameters less than 100 nm, and the size of nanoparticles increases with the increasing molecular weight of the polymer. Two antineoplastic drugs, 5-Fu and paclitaxel, were encapsulated


into the nanoparticles and the targeting properties of nanoparticles in the presence and in the absence of free folic acid were evaluated in vitro. The free folic acid competition study shows the drug-loaded nanoparticles exhibit strong inhibition on the folate receptor positive tumor cells because cell internalization could be significantly improved through the folate mediated targeting. Acknowledgment. Financial support from National Natural Science Foundation of China (20774070 and 50633020) and Ministry of Education of China (Cultivation Fund of Key Scientific and Technical Innovation Project 707043) are gratefully acknowledged. Special thanks are due to Ms Qing-Rong Wang for the SEC-MALLS measurement.

References and Notes (1) Torchilin, V. P. J. Controlled Release 2001, 73, 137. (2) Kataoka, K.; Harada, A.; Nagasaki, Y. AdV. Drug DeliVery ReV. 2001, 47, 113. (3) Wang, F.; Bronich, T. K.; Kabanov, A. V.; Rauh, R. D.; Roovers, J. Bioconjugate Chem. 2005, 16, 397. (4) Kreutzer, G.; Ternat, C.; Nguyen, T. Q.; Plummer, C. J. G.; Månson, J. A. E.; Castelletto, V.; Hamley, I. W.; Sun, F.; Sheiko, S. S.; Herrmann, A.; Ouali, L.; Sommer, H.; Fieber, W.; Velazco, M. I.; Klok, H. A. Macromolecules 2006, 39, 4507.


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(5) Majoros, I. J.; Myc, A.; Thomas, T.; Mehta, C. B.; Baker, J. R. Biomacromolecules 2006, 7, 572. (6) Zou, J.; Zhao, Y.; Shi, W. J. Phys. Chem. B 2006, 110, 2638. (7) Zou, J.; Shi, W.; Wang, J.; Bo, J. Macromol. Biosci. 2005, 5, 662. (8) Zˇagar, E.; Zˇigon, M. J. Chromatogr., A 2004, 1034, 77. (9) Sinha, V. R.; Bansal, K.; Kaushik, R.; Kumria, R.; Trehan, A. Int. J. Pharm. 2004, 278, 1. (10) Gabizon, A.; Shmeeda, H.; Horowitz, A. T.; Zalipsky, S. AdV. Drug DeliVery ReV. 2004, 56, 1177. (11) Sudimack, J.; Lee, R. J. AdV. Drug DeliVery ReV. 2000, 41, 147. (12) Leamon, C. P.; Low, P. S. Drug DiscoVery Today 2001, 6, 44. (13) Saul, J. M.; Annapragada, A.; Natarajan, J. V.; Bellamkonda, R. V. J. Controlled Release 2003, 92, 49. (14) Claesson, H.; Malmstro¨m, E.; Johansson, M.; Hult, A. Polymer 2002, 43, 3511. (15) Miao, Z. M.; Cheng, S. X.; Zhang, X. Z.; Wang, Q. R.; Zhuo, R. X. J. Biomed. Mater. Res. 2007, 81B, 40. (16) Ciuchi, F.; Nicola, G. D.; Franz, H.; Gottarelli, G.; Mariani, P.; Bossi, M. G. P.; Spada, G. P. J. Am. Chem. Soc. 1994, 116, 7064. (17) Park, E. K.; Kim, S. Y.; Lee, S. B.; Lee, Y. M. J. Controlled Release 2005, 109, 158. (18) Park, E. K.; Lee, S. B.; Lee, Y. M. Biomaterials 2005, 26, 1053. (19) Wang, Y.; Yu, L.; Han, L.; Sha, X.; Fang, X. Int. J. Pharm. 2007, 337, 63. (20) Licciardi, M.; Giammona, G.; Du, J.; Armes, S. P.; Tang, Y.; Lewis, A. L. Polymer 2006, 47, 2946. (21) You, J.; Li, X.; Cui, F. D.; Du, Y. Z.; Yuan, H.; Hu, F. Q. Nanotechnology 2008, 19, 045102.

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Functionalized Amphiphilic Hyperbranched Polymers for Targeted

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