Article pubs.acs.org/Biomac
Biodegradable Hyperbranched Polyglycerol with Ester Linkages for Drug Delivery Mei Hu,†,‡,∥ Mingsheng Chen,§,∥ Guolin Li,§ Yan Pang,§ Dali Wang,§ Jieli Wu,§ Feng Qiu,§ Xinyuan Zhu,*,§ and Jian Sun*,† †
Department of Oral and Maxillofacial Surgery, Ninth People’s Hospital, School of Stomatology, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 200011, People’s Republic of China ‡ Department of Oral and Maxillofacial Surgery, First Teaching Hospital of Xinjiang Medical University, 137 Liyushannan Road, Urumqi 830054, People’s Republic of China § School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China S Supporting Information *
ABSTRACT: Biodegradable hyperbranched polyglycerols (dHPGs) were synthesized through oxyanionic initiating hybrid polymerization of glycerol and glycidyl methacrylate. Due to the introduction of ester linkages into the hyperbranched polyglycerol backbone, dHPGs showed good biodegradability and low cytotoxicity. Benefiting from the existence of terminal hydroxyls and methacryloyl groups, both the anticancer drug methotrexate (MTX) and fluorescent probe Rhodamine-123 could be conjugated onto the surface of dHPGs easily. The resultant MTX-conjugated polymers (dHPG-MTXs) exhibited an amphiphilic character, resulting in the formation of micelles in an aqueous solution. The release of MTX from micelles was significantly faster at mildly acidic pH of 5.0 compared to physiological pH of 7.4. dHPG-MTX micelles could be efficiently internalized by cancer cells. MTT assay against cancer cells showed dHPG-MTXs micelles had high anticancer efficacy. On the basis of their good biodegradability and low cytotoxicity, dHPGs provide an opportunity to design excellent drug delivery systems.
■
INTRODUCTION Dendritic polymers including dendrimers and hyperbranched polymers have gained widespread attention in biomedical fields due to their unique physical and chemical properties.1−5 Among these dendritic polymers, hyperbranched polyglycerol (HPG) exhibits good hydrophilicity, excellent biocompatibility, low/absent immunogenicity, and high chemical stability.6−11 Therefore, HPG and its derivatives are widely regarded as promising biomaterials for various bioapplications, especially in the construction of drug delivery systems. Considering that the maximum size for renal clearance is about 4−6 nm, it is necessary for polymers to degrade into low-molecular-weight oligomers or monomers after completion of a controlled drug release.12,13 However, HPG is a highly branched polymer with a flexible aliphatic polyether backbone, which is not degradable in aqueous solution. It has been well-known that a lot of intracellular esterases exist in living cells, which enhances the cleavage rate of ester bond greatly.14,15 It can be imaged that if the ester linkages are introduced into the polymeric backbone of HPG, a novel type of biodegradable hyperbranched polyglycerol (dHPG) with excellent biocompatibility and biodegradability can be obtained. Generally speaking, HPG is synthesized by cationic and anionic ring-opening polymerizations of glycidol.6−11 It is not © 2012 American Chemical Society
easy to prepare dHPG by introduction of ester bonds into a highly branched polymer backbone. Here, we notice that glycidyl methacrylate (GMA) has a glycidyl group with an ester bond. Very recently, we developed the oxyanionic initiating hybrid polymerization of GMA and various oligo(ethylene glycol)s to prepare degradable hyperbranched poly(esterether)s.16−20 Provided that the initiators of various oligo(ethylene glycol)s are replaced by glycerol, then the oxyanionic initiating hybrid polymerization of glycerol and GMA will result in the formation of dHPG with ester linkages in the polymer backbone. On the basis of this concept, dHPG has been successfully prepared in the present work. Thanks to the combination of good biodegradability and low cytotoxicity, dHPG can be used as a promising carrier for drug delivery.
■
EXPERIMENTAL SECTION
Materials. Potassium hydride (KH, 30 wt % dispersion in mineral oil, Acros), N-hydroxysuccinimide (NHS, Acros), N,N-dicyclohexylcarbodiimide (DCC, Aldrich), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Aldrich), and 4-dimethylaminopyridine (DMAP, Aldrich) were used as received. GMA (Acros) and Received: June 24, 2012 Revised: September 12, 2012 Published: September 24, 2012 3552
dx.doi.org/10.1021/bm300966d | Biomacromolecules 2012, 13, 3552−3561
Biomacromolecules
Article
dimethyl sulfoxide (DMSO) were dried over calcium hydride (CaH2) and distilled under reduced pressure. Tetrahydrofuran (THF) was dried by refluxing over fresh sodium-benzophenone complex (a deep purple color indicating an oxygen- and moisture-free solvent) and distilled just before polymerization. Methotrexate (MTX) was purchased from Sangon Biotech (Shanghai) Co., Ltd. Glycerol (Alfa) was dried by azeotropic distillation with methylbenzene and then distilled under reduced pressure. Cell culture medium was from Shanghai Key Laboratory of Stomatology. Clear polystyrene tissueculture-treated 6-well, 12-well, and 96-well plates were obtained from Corning Costar. Other reagents were purchased from Shanghai Chemical Reagent Co. and used as received. Characterization. 1H and 13C NMR spectra of the polymers were recorded using a Bruker Avance III 400 spectrometer with dimethyl sulfoxide-d6 (DMSO-d6) as a solvent. Quantitative 13C NMR spectra were measured by the method of inverse gated 1H decoupling (100 MHz). The number of scans was 2048, the acquisition time was 1.363 s, and the relaxation delay was 15.0 s. In DEPT experiments, the 1H tip angle θ was set to 135° to determine carbon multiplicities with CH, CH3 up and CH2 down. 1H,1H−COSY and 13C,1H-HSQC spectra were recorded using the standard pulse sequence provided by Bruker. Fourier transform infrared (FTIR) measurements were carried out on a Bruker Equinox-55 FTIR spectrometer with a disk of KBr. The molecular weights and molecular weight distributions were determined by gel permeation chromatography (GPC). GPC was performed on a Perkin-Elmer series 200 system (10 μm PL gel 300 × 7.5 mm mixed-B and mixed-C column, poly(ethylene glycol) calibration) equipped with a refractive index (RI) detector. Dimethylformamide (DMF) containing 0.01 mol/L lithium bromide was used as the mobile phase at a flow rate of 1 mL/min at 70 °C. Transmission electron microscopy (TEM) was performed on a JEOL 2010 instrument operating at a voltage of 200 kV. Solution sample was dropped onto carbon-coated copper grids and then immersed in liquid nitrogen. After freeze-drying, the sample was used for TEM observation. Dynamic light scattering (DLS) experiments were carried out with a Malvern Zetasizer Nano S apparatus equipped with a 4.0 mW laser operating at λ = 633 nm. The scattering angle was set to 173°, and the data were the average of three tests. Synthesis of dHPGs. dHPGs were prepared by oxyanionic initiating hybrid polymerization. A typical polymerization procedure is as follows: A suspension of KH in mineral oil (30% in weight) was introduced in a dry preweighted 100 mL Schlenk flask under nitrogen. The mineral oil was removed by three extractions with THF, and the remaining THF was removed by vacuum. When KH was completely dried, the flask was weighted again to determine the amount of KH (0.8019 g, 20.0 mmol). Then, DMSO (30 mL) and glycerol (3.7256 g, 40.5 mmol) were introduced to the flask. The solution was stirred for 30 min to form the potassium alcoholate. Subsequently, GMA (5.7500 g, 40.5 mmol) was added by syringe, and the polymerization was conducted at 80 °C for 48 h. Upon completion of the polymerization, the mixture was precipitated in acetone/diethyl ether (1 L, v/v = 1/4). The product was redissolved in methanol and neutralized by filtration over cation-exchange resin. The obtained polymer was precipitated twice from methanol solution in cold diethyl ether and then dried in vacuo at 50 °C for 24 h. Similarly, polymers with different dendritic structures were synthesized by changing KH/glycerol or glycerol/ GMA molar ratios (see Table 1). The resultant purified products were highly viscous. 1H NMR (400 MHz, DMSO-d6): δ = 1.04 (−CH3), 1.86 (−CH2C(CH3)− or −CH2CH(CH2)O−) 3.15−3.77 (−CHO−, −CH2O−), 4.1−4.7 (−OH), 5.66, 6.04 (−CCH2) ppm; IR (KBr): ν = 3404, 2925, 2879, 1722, 1457, 1311, 1255, 1128, 943, 802, 659, 586 cm−1. Synthesis of dHPG-MTX. A typical example for the preparation of dHPG-MTXs is described as follows: According to the abovementioned procedure, dHPG was prepared by polymerization in DMSO for 48 h. After that, DCC (1.2361 g, 6.0 mmol), NHS (0.6899 g, 6.0 mmol), DMAP (0.7320 g, 6.0 mmol) were added into polymer solution. The mixture was stirred at room temperature. Subsequently, MTX (0.6780 g, 1.5 mmol) was added to the reaction system. The solution was stirred for another 72 h at 50 °C under a nitrogen
Table 1. Reaction Conditions and Results of Oxyanionic Initiating Hybrid Polymerization of Glycerol and GMA entrya
KH/ Gb
G/ GMAc
Mwd ( × 103)
Mnd ( × 103)
PDId
DBe
yield (%)
P1 P2 P3 P4 P5
1:2 1:2 1:3 1:3 1:3
1:1 1:1.2 1:1 1:1.2 1:1.5
3.9 5.0 4.7 4.3 4.7
2.3 2.3 2.8 2.4 2.5
1.69 2.17 1.68 1.79 1.88
0.50 0.47 0.46 0.45 0.43
55 60 51 60 58
a Polymer obtained from the corresponding condition listed. bMolar ratio of KH to glycerol (G). cMolar ratio of glycerol (G) to GMA. d Molecular weights and polydispersity (PDI) were determined by GPC. eDegree of branching (DB) was calculated from quantitative 13C NMR analysis.
atmosphere. Then, the mixture was precipitated in acetone/diethyl ether (1 L, v/v = 1/4). The product was redissolved in distilled water and dialyzed against deionized water using a dialysis tube (MWCO = 1000 Da) until the absence of MTX signals in dialysate by UV measurements. The solution was centrifuged at 1000 rpm for 5 min in order to remove the insoluble materials. After being dried in vacuo at 50 °C for 24 h, dHPG-MTX was obtained. Moreover, due to the existence of some methacryloyl groups in the surface of dHPGs, the fluorescence-labeled dHPG-MTXs could be readily obtained by the same procedure, except for adding small amount of Rhodamine-123 into the system during the polymerization. The Michael addition of methacryloyl groups in dHPGs and amino groups in Rhodamine-123 gave the fluorescence-labeled dHPG-MTXs. Degradation of Polymers. First, the degradation behavior of polymers was estimated by 1H NMR measurements. The polymer dHPGs (P1) were dissolved in the different phosphate buffered saline (PBS) D2O solutions (pH 7.4 and 5.0), respectively. The polymer concentrations were set at 20 mg/mL. Then the dHPGs solutions were injected into NMR tubes respectively and placed in a shaking incubator (37 °C, 100 rpm). At the predetermined intervals (2, 4, 6, and 10 days), 1H NMR spectra were measured in tubes directly and the corresponding curves were analyzed. Next, the extent of polymer degradation was estimated by measuring the decrease in molecular weight of dHPGs. Similarly, polymers were dissolved in different PBS solutions (pH 7.4 and 5.0, respectively) and were placed in centrifuge tubes. The polymer concentrations were set at 20 mg/mL. Then they were incubated in a shaking incubator (37 °C, 100 rpm). At the predetermined intervals, 1 mL of PBS solution of degradation product was taken out, then kept frozen, and finally subjected to GPC for molecular weight determination or tested by MTT assay. Preparation of dHPG-MTX Micelles. Typically, a total of 10 mg of dHPG-MTX polymer was dissolved in 2 mL of DMF and stirred at room temperature for 4 h. Then 8 mL of deionized water was slowly added into the polymer solution under stirring for 2 h, and the solution was transferred into dialysis tubing (MWCO = 3.5 kDa) and dialyzed against the deionized water for 24 h. During the process, the water was renewed at appropriate intervals. In Vitro Release Study. In vitro release studies were performed in a glass apparatus at 37 °C in two different buffers (pH 7.4 and 5.0). First, 50 mg of dHPG-MTX micelles was dispersed in 5 mL of buffer solution and placed in a dialysis bag (MWCO = 1000 Da). The dialysis bag was then immersed in 95 mL of the release medium and kept in a shaking water bath at 37 °C to acquire sink conditions. At predetermined time intervals, 2 mL of the external buffer was withdrawn and replenished with an equal volume of fresh media. The amount of released MTX was analyzed with ultraviolet−visible (UV− vis) measurements on a Perkin-Elmer Lambda 20/2.0 UV−vis spectrometer, and the UV absorbance calibration curve of MTX was used to determine the drug content with a correlation coefficient of >99.9%. The release experiments were conducted in triplicate, and the results were the average data. 3553
dx.doi.org/10.1021/bm300966d | Biomacromolecules 2012, 13, 3552−3561
Biomacromolecules
■
Cell Culture. NIH/3T3 cells (a mouse embryonic fibroblast cell line), CAL27 cells (an oral adenosquamous carcinoma cell line), and Hela cells (a human cervical carcinoma cell line) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplied with 10% fetal bovine serum (FBS) and antibiotics (50 units/mL penicillin and 50 units/mL streptomycin) at 37 °C in a humidified atmosphere containing 5% CO2. Cytotoxicity Measurements. For cytotoxicity, the polymer samples were dissolved in PBS, respectively. NIH/3T3 cells were seeded in 96-well plates at an initial seeding density of 8.0 × 103 cells/ well in 200 μL medium. After 24 h of incubation, 20 μL of PBS containing serial dilutions of polymers was added to each well. The final polymer concentrations in the culture medium were from 0.001 to 10 mg/mL. The cells were grown for another 48 h. Then, 20 μL of 5 mg/mL MTT assays stock solution in PBS was added to each well. After incubating the cells for 4 h, the medium containing unreacted dye was removed carefully. The obtained blue formazan crystals were dissolved in 200 μL per well DMSO, and the absorbance was measured in a BioTek Elx800 at a wavelength of 490 nm. Cytotoxicity of the degradation products was also evaluated by the MTT assay with the same method mentioned above. Cell Internalization. Cell internalization was characterized by flow cytometry and confocal laser scanning microscopy (CLSM). Flow cytometry was used to provide statistics on the adhesion of dHPGMTXs into CAL27 cells. CAL27 cells (5.0 × 105 cells per well) were seeded in a 6-well tissue culture plate. After 24 h of culture, dHPGMTXs dissolved in DMEM culture medium with a polymer concentration of 10 mg/mL were added to different wells, and the cells were incubated at 37 °C for predetermined time intervals. After incubation, samples were prepared for flow cytometry analysis by removing the cell growth media, rinsing with PBS buffer, and treating with trypsin. Data for 1.0 × 104 gated events were collected, and analysis was performed by means of a BD FACScalibur flow cytometer and CELLQuest software. For CLSM, CAL27 cells (2.0 × 105 cells per well) were seeded on coverslips in a 12-well tissue culture plate. After 24 h of culture, dHPG-MTXs dissolved in DMEM culture medium with a polymer concentration of 10 mg/mL were added to different wells, and the cells were incubated at 37 °C for predetermined time intervals. After being washed with PBS, the cells were fixed with 4% formaldehyde for 30 min at room temperature, and the slides were rinsed with PBS three times. Finally, the cells were stained with 2-(4-amidinophenyl)-6indolecarbamidine dihydrochloride (DAPI) for 10 min, and the slides were rinsed with PBS three times. The slides were mounted and observed with a LSM510 META. Activity Analyses. The inhibition of dHPG-MTX micelles against CAL27 cells and Hela cells was evaluated in vitro by MTT assay. Here, free MTX with the same concentration was used as the control. Both CAL27 cells and Hela cells were seeded in 96-well plates at an initial seeding density of 8.0 × 103 cells/well in 200 μL medium. After 24 h of incubation, the culture medium was removed and replaced with 200 μL of medium containing serial concentrations of MTX-conjugated polymers. The MTX concentrations were varied as follows: 0.01, 0.1, 1, 5, 10, 50, 100, 200, and 500 μg/mL. The tumor cell proliferation was evaluated from 24 to 72 h by MTT assay. Then, 20 μL of 5 mg/ mL MTT assays stock solution in PBS was added to each well. After incubating the cells for 4 h, the medium containing unreacted MTT was removed carefully. The obtained blue formazan crystals were dissolved in 200 μL per well DMSO, and the absorbance was measured in a VarioskanFlash at a wavelength of 490 nm. Statistics. All experiments were repeated at least three times. Data are presented as means ± standard deviation. Statistical significance (p < 0.05) was evaluated by using Student’s t test when only two groups were compared. If more than two groups were compared, evaluation of significance was performed using one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc test. In all tests, statistical significance was set at p < 0.05.
Article
RESULTS AND DISCUSSION
Synthesis and Characterization. It has been well reported that HPG is a kind of hyperbranched polymers with excellent biocompatibility, but it is not biodegradable. To endow the degradability into biocompatible HPG, the oxyanionic initiating hybrid polymerization of commercially available glycerol and GMA with a glycidyl group and an ester bond was performed in the presence of KH catalysis. GMA is a typical monomer for hybrid polymerization because of the existence of two different reactive groups, namely, a methacryloyl group and an epoxy group. Both the epoxy group and the double bond of GMA could be polymerized through oxyanionic initiation. As shown in Scheme 1, glycerol 1 is easily initiated by KH, producing the initiators 2 and 3. Because GMA Scheme 1. The Preparation of dHPGs through Oxyanionic Initiating Hybrid Polymerization
3554
dx.doi.org/10.1021/bm300966d | Biomacromolecules 2012, 13, 3552−3561
Biomacromolecules
Article
has a methacryloyl group and an epoxy group, there are two possibilities for the reaction of GMA with 2 or 3, respectively. Both 2 and 3 can separately initiate the epoxy group of GMA, leading to 4 and 8. Meanwhile, 2 and 3 can react with the methacryloyl group of GMA, producing 11 and 14, respectively. It has been well recognized that the active anion center of the secondary alkoxide or carbonanion can transfer immediately to the more stable primary alkoxide through proton-transfer, thus producing 5, 6, 7, 9, 10, 12, 13, and 15. Further polymerization gives rise to the degradable hyperbranched polyglycerol with ester linkages 16 (dHPG). After the polymerization was carried out in DMSO at 80 °C for 48 h, highly viscous product was obtained. The reaction proceeded homogeneously, and no gelation took place throughout the polymerization. By changing the KH/glycerol or glycerol/GMA molar ratios, various dHPGs with different dendritic structures were prepared. Table 1 summarizes the experimental conditions and corresponding characterization data. FTIR results provide the chemical structure information of dHPGs. As shown in Figure S1 in the Supporting Information, the strong absorption peak at 1128 cm−1 is attributed to the C− O−C stretching vibration. The band at 1457 cm−1 results from the asymmetric deformation vibration of the −CH2− group. A strong ester carbonyl band at 1722 cm−1 confirms the presence of ester bonds. The bands around 2879 and 2925 cm−1 correspond to the symmetric −CH3 stretching vibration and asymmetric −CH2− stretching vibration, while the broad O−H stretching vibration around 3404 cm−1 indicates the appearance of many hydroxyl groups. The structure of the resulting polymers was also analyzed by various NMR techniques, including 1H NMR, 13C NMR, DEPT-135 NMR, 1H,1H-COSY, and 13C,1H-HSQC spectra. Careful examination of the dHPG’s structure reveals that four terminal subunits, six linear subunits, and five dendritic subunits might be present, as shown in Figure 1. Figure 2 gives the 1H NMR spectra of various dHPGs. The peak at 1.04 ppm is ascribed to the methyl group of GMA units, and the peaks at 1.86 and 2.50 ppm are assigned to the methylene of GMA units in the spectrum of dHPGs. The signals at 3.15−3.77 ppm can be ascribed to the methylene and methine protons adjacent to oxygens in both the ether and alcohol moieties, while the wide peak from 4.0 to 4.6 ppm can be assigned as the hydroxyl protons. The small characteristic peaks at 5.66 and 6.04 ppm come from the vinyl protons of the GMA unit. Figure 3 gives the quantitative 13C NMR and DEPT-135 spectra of a representative dHPG (P1), in which methyl, methylene, or methine carbon atoms can be distinguished. To further verify the branched topology of the polymerized products, the two-dimensional NMR (2D-NMR) technique was used.21−24 Figure 4 presents the 1H,1H-COSY, and 13C,1HHSQC spectra, and the detailed assignment is given in Figures 1, 3, and 4. Correspondingly, the degree of branching (DB) for dHPGs can be calculated from the following equation:25
Figure 1. Various structural units of dHPGs.
Figure 2. 1H NMR spectra of dHPGs (P1−P5) in DMSO-d6.
polymers are around 4.5 × 103, with PDI about 1.8. The real molecular weights of dHPGs may be even larger than the values estimated by GPC analysis using linear poly(ethylene glycol) as the calibration, because dendritic polymers generally have smaller sizes than linear polymers with the same molecular weight and can hardly be expanded in solution. Comparing to its linear counterpart, the active anion center of hyperbranched polymer is readily deactivated by the side reaction such as the chain transfer and cyclization of the active center. Correspondingly, the molecular weights of dHPGs are low, and some methacryloyl moieties exist in the final products. Nevertheless, it facilitates further modification for biomedical applications. In Vitro Degradation. The biodegradability of polymeric materials is of great importance for biomedical applications. As is well known, the polymer degradation reduces the cytotoxicity and makes it easy to eliminate through the excretion pathway in
DB = (D + T )/(D + T + L)
where D, T, and L represent the fractions of the dendritic, terminal, and linear units, respectively. As shown in Table 1, the DBs of dHPGs are at 0.43−0.50, suggesting the formation of hyperbranched products. In addition, the molecular weights and polydispersity index (PDI) values of these polymers were measured by GPC, and the data are summarized in Table 1. GPC measurements suggest that the weight average molecular weights of the 3555
dx.doi.org/10.1021/bm300966d | Biomacromolecules 2012, 13, 3552−3561
Biomacromolecules
Article
analyses. First, the in vitro degradation behavior of dHPG (P1) was evaluated in PBS at 37 °C under neutral (pH 7.4) or acidic (pH 5.0) conditions by in situ measurements of degradation in a NMR tube. The 1H NMR spectra at pH 7.4 are almost unchanged in 10 days (Figure S2 of the Supporting Information), indicating the high stability of dHPG in neutral conditions. On the contrary, Figure 5 shows in acidic
Figure 5. 1H NMR spectra of dHPG (P1) in D2O at different degradation times at 37 °C under acidic (pH 5.0) conditions.
conditions (pH 5.0) a sharp triplet methyl signal at 1.0 ppm (−CH3), and several new peaks at 3.3−3.5 ppm (−CH2OH) related to degradation products appear, and their intensities enhance quickly with the prolongation of degradation time. It has been well recognized that the ester bond is readily hydrolyzed at a low pH. Therefore, the NMR variation can be attributed to the hydrolysis of ester linkages into hydroxyl and carboxylic groups. To further confirm the degradation of dHPG, the molecular weights of degraded products at pH 7.4 and 5.0 were measured by GPC technique. At a neutral pH of 7.4, the molecular weights are almost unchanged in 10 days. Figure 6 and Figure S3 give the degradation profile of dHPG as a function of time at pH 5.0. It can be found that the molecular weights decrease quickly with degradation time, suggesting that the ester bonds in the backbone of dHPG are susceptible to
Figure 3. Quantitative 13C NMR and DEPT-135 NMR spectra of dHPG (P1) in DMSO-d6. (A and B are the same 13C DEPT-135 NMR spectrum with different regions from 200 to 0 ppm and 90 to 10 ppm, respectively.)
Figure 4. 2D-NMR spectra of dHPG (P1): (A) 1H,1H−COSY spectrum, (B) the enlargement of A, (C) 13C,1H-HSQC spectrum, and (D) the enlargement of C.
vivo.26,27 Comparing to the conventional biocompatible HPG, a prime advantage of dHPG is its biodegradability due to the introduction of ester linkages into the polymer backbone. The polymer degradation was monitored by NMR and GPC
Figure 6. Molecular weights of dHPG (P1) as a function of degradation time (pH = 5.0). 3556
dx.doi.org/10.1021/bm300966d | Biomacromolecules 2012, 13, 3552−3561
Biomacromolecules
Article
hydrolysis at acidic conditions. Both NMR and GPC results corroborate the fast degradation of dHPG in an acidic situation, which originates from the cleavage of ester bonds. In Vitro Cytotoxicity of dHPGs. For drug delivery, it is necessary to lower the potential toxicity of polymeric carriers. To evaluate the cytotoxicity of dHPGs, in vitro MTT assay against NIH/3T3 normal cells (a mouse embryonic fibroblast cell line) was determined with the PEI and HPG as positive and negative controls, respectively. The MTT assay is based on the ability of a mitochondrial dehydrogenation enzyme in viable cells to cleave the tetrazolium rings of the pale yellow MTT and form formazan crystals with a dark blue color. Therefore, the number of surviving cells is directly proportional to the level of formed formazan.28,29 NIH/3T3 cells were cultured for 24 h to permit cell attachment. After this time, the culture medium was removed and replaced with culture medium containing serial dilutions of polymers. Figure 7 gives the cell viability after 48 h
Figure 8. Cytotoxicity of the degradation products of dHPGs (P1 and P2) against NIH/3T3 cells for 48 h. *P < 0.05 versus the cell.
similar with HPG. These results confirm that the degradation products of dHPG have low cytotoxicity to NIH/3T3 cells. Synthesis and Characterization of dHPG-MTXs. The low cytotoxicity of dHPGs encouraged us to further evaluate their potential as a polymeric drug carrier. MTX, a folic acid antagonist, is a widely used agent for the treatment of certain human cancers. The MTX-conjugated polymers (dHPGMTXs) were prepared by coupling the end hydroxyl groups of dHPG with the carboxylic groups of MTX in the presence of N,N-dicyclohexyl-carbodiimide (DCC) and DMAP in dry DMSO. The resulting dHPG-MTXs were then purified by being repeatedly dissolved in methanol and precipitated in diethyl ether. The product was redissolved in distilled water and dialyzed against deionized water using a dialysis tube until the absence of MTX signals in dialysate by UV measurements, followed by centrifugation at 1000 rpm for 5 min to remove the insoluble materials. Figure 9A gives the FTIR spectra of dHPG (P1), MTX, and dHPG-MTX. As above-mentioned, the 1722 and 1128 cm−1 bands of dHPG originate from the ester carbonyl and ether groups, respectively. For free MTX, the characteristic absorptions at 1644 and 1603 cm−1 come from the amide groups. After the conjugation of dHPG and MTX, all of these peaks appear in the spectrum of dHPG-MTX. Moreover, 1H NMR analysis was performed to evaluate the drug content of final obtained conjugates, and the result is given in Figure 9B. For MTX, the signals at 7.71 and 6.81 ppm can be ascribed to the protons from the phenyl group. On the other hand, the methyl peak of dHPG from the GMA unit is located at 1.0 ppm. For dHPG-MTX conjugates, both MTX and dHPG characteristic signals can be observed, suggesting the successful conjugation between MTX and dHPG. Based on the relative intensity of 1H NMR peaks at 7.71/6.81 and 1.0 ppm, the MTX content is determined to be 6.66 wt %. In addition, the dHPGMTX was further measured by UV−vis spectra in DMF (Figure S4 in the Supporting Information). The UV−vis result indicates that MTX was successfully conjugated to dHPG, which is consistent with the results of FTIR and NMR techniques. Micellization of dHPG-MTXs. Under a physiological pH value (pH 7.4), dHPGs could be dissolved in water very well, but MTX was insoluble. Benefiting from their amphiphilicity, dHPG-MTXs self-assembled into polymeric micelles. Both TEM and DLS techniques were employed to investigate the morphology and size distribution of MTX-conjugated polymeric micelles. The TEM image in Figure 10A shows the
Figure 7. Cytotoxicity of dHPGs against NIH/3T3 cells for 48 h. PEI and HPG as positive and negative controls. *P < 0.05 versus the cell.
incubation with polymers at different concentrations from 0.001 to 10 mg/mL. Cells cannot tolerate the treatment with PEI at only 0.01 mg/mL. In contrast, the viability of NIH/3T3 cells cultured with HPG and dHPGs after 48 h remains above 90% compared with the untreated cells, even when the polymer concentration is up to 10 mg/mL. The MTT assay of dHPGs against NIH/3T3 cells for 24 h gives a similar result (Figure S5). These results suggest the low cytotoxicity of the obtained dHPGs, which might be related to the excellent biocompatibility of glycerol units and the presence of biodegradable ester bonds in the polymer backbone. The cytotoxicity of the degradation products is a very important parameter to evaluate the biological safety of biomaterials. For example, poly(lactide) and poly(lactide-coglycolide) have the satisfactory biocompatibility, but their high concentrated degradation products show a toxic influence according to the previous report. Here, the cytotoxicity of the degradation products of dHPGs, which were hydrolyzed under acidic condition (pH 5.0) over 20 days, was determined by MTT viability assay against NIH/3T3 cells. The cell viability after 24 and 48 h of incubation with the degradation products (P1 and P2) at different concentration is shown in Figure S6 and Figure 8. Compared with the untreated cells, the cell viability is higher than 90% after 24 and 48 h of incubation with the concentration of degradation products up to 10 mg/mL, 3557
dx.doi.org/10.1021/bm300966d | Biomacromolecules 2012, 13, 3552−3561
Biomacromolecules
Article
TEM is reasonable, because DLS and TEM show a different morphology in the swollen and solid states, respectively. In Vitro Drug Release. The drug therapeutic effect is closely related to the release behavior of the drug delivery system in the body. The normal pH of blood for sustaining human life is about 7.4, while the endosomes and lysosomes of cells have a more acidic environment. Therefore, a drug release study of dHPG-MTX micelles was carried out in pH 7.4 PBS and pH 5.0 acetate buffer solution, respectively. On the basis of the standard curves of the UV−vis absorbance of free MTX in 7.4 and 5.0 buffer solutions, the release profiles of dHPG-MTX micelles at pH 7.4 and 5.0 are calculated. Figure 11 shows that the release amount of MTX (including MTX modified with low molecular weight dHPG residues) is less than 20% at pH 7.4 in 80 h. By comparison, a noticeably increased release of MTX is observed at pH 5.0 with 50%. Furthermore, the release rate of MTX from the dHPGMTX micelles at pH 5.0 is much faster than that at pH 7.4. The fast release of MTX in mild acidic environment could be attributed to the accelerated hydrolysis of ester linkages at low pH values. Therefore, it can be inferred that the MTX delivery system may exhibit relative stability during the drug transportation in the blood. When the delivery system is internalized into a mild acidic pH environment such as the endosomes and lysosomes, a large amount of MTX could be released to inhibit the proliferation of cancer cells in vivo. Furthermore, the existence of many intracellular esterases is also favorable for the drug release. Cell Internalization. To study the cellular uptake of dHPG-MTX micelles, Rhodamine-123 was used as a fluorescence probe and conjugated to dHPG-MTXs. On the basis of the standard curves of the UV−vis absorbance of free Rhodamine-123, the Rhodamine-123 content in dHPG-MTXsRhodamine was determined to be 1.0 wt %. The synthesis details have been given in the experimental part. With the help of flow cytometry technique, the cellular adhesion of dHPGMTX micelles could be determined by measuring the intracellular fluorescence intensity. Figure 12 gives the histograms of cell-associated Rhodamine-123 fluorescence intensity for CAL27 cells (an oral adenosquamous carcinoma cell line) incubated with dHPG-MTX micelles at the predetermined time intervals, 5 and 60 min. Here, CAL27 cells without any treatment were used as a blank control. The relative geometrical mean fluorescence intensities (GMFIs) of polymer pretreated cells for 5 min are about 410-fold greater than those of of nonpretreated cells. After incubation for 1 h, the relative GMFIs of polymer pretreated cells are about 742-
Figure 9. (A) FTIR and (B) 1H NMR spectra of MTX, dHPG (P1), and dHPG-MTX.
appearance of spherical nanoparticles with an average diameter of 150 nm, illustrating the formation of multimolecular micelles. The small nanoparticles less than 200 nm facilitate the endocytosis. Furthermore, it is observed that these nanoparticles are well-separated from each other, which indicates that the hydrophobic MTX is effectively stabilized by polymer micelles. To confirm the TEM results, the DLS measurements of MTX-conjugated polymeric micelles were performed. The DLS curve in Figure 10B gives a monomodal size distribution with a hydrodynamic diameter of 160 nm. The larger size observed by DLS as compared to that determined by
Figure 10. (A) TEM image of dHPG-MTX micelles. (B) DLS measurements of the dHPG-MTX micelles in aqueous solution. 3558
dx.doi.org/10.1021/bm300966d | Biomacromolecules 2012, 13, 3552−3561
Biomacromolecules
Article
Figure 11. In vitro release profiles of MTX from dHPG-MTX micelles at different pH values.
the Rhodamine-123 fluorescence is still localized in the cytosol, suggesting the delayed release of drugs due to the covalent linkage between MTX and dHPG, which is in agreement with the flow cytometry observation. CLSM results confirm the efficient cell internalization of dHPG-MTX micelles by living cells. Proliferation Inhibition of Tumor Cells. To demonstrate the therapeutic potential of the obtained dHPG-MTX micelles, in vitro evaluation was performed by MTT assay against CAL27 cells and Hela cells (a human cervical carcinoma cell line). To compare the antitumor activity of the dHPG-MTX micelles with that of the free drug, MTX in free form was used as a control. The cellular growth inhibition was calculated from the following formula:
Figure 12. The cellular adhesion of the dHPG-MTX micelles by CAL27 cells versus the incubation time by flow cytometry analysis.
inhibition% = 100 × (Ablank − A test )/Ablank
where, Ablank = absorbance of the blank, and Atest = absorbance in the presence of the samples. All analysis was done in triplicate, and results were expressed as ± mean SD. It was observed that dHPG-MTX exhibited highly efficient abilities of proliferation inhibition in CAL27 and Hela cancer cells. Meanwhile, dHPG-MTX inhibited the proliferation of cancer cells in a dose- and time-dependent fashion (Figure 14). When the incubation time is prolonged, the cellular growth inhibition is improved against cancer cells. These results indicate that the dHPG-MTX micelles are able to enter the cells and produce the desired pharmacological action. With the increase of incubation time, the proliferation inhibition of tumor cells becomes more efficient.
fold greater than those of nonpretreated cells. The fast enhancement of fluorescence signals indicates high cellular adhesion of dHPG-MTX micelles by CAL27 cells. Because of their partially hydrophobic nature with a small size, dHPGMTX micelles might be readily adhered to the cell surface through hydrophobic interactions, which facilitates the occurrence of cellular uptake. Confocal laser scanning microscopy (CLSM) was exploited to investigate and compare the cell uptake behavior and intracellular distribution of dHPG-MTX micelles by CAL27 cells. Both the yellow fluorescence from Rhodamine-123 and blue fluorescence from DAPI were used to study the localization of dHPG-MTX micelles within the cells. First, CAL27 cells were incubated with dHPG-MTX micelles for different time intervals. Subsequently, the cell nucleus was stained by DAPI, and then the treated cells were fixed with formaldehyde. As shown in Figure 13A, yellow fluorescence was predominantly localized in the cytosol compared with the blue nucleus after cell incubation for 5 min. When prolonging the incubation time to 60 and 180 min, Figure 13B,C shows that
■
CONCLUSIONS Through oxyanionic initiating hybrid polymerization of glycerol and GMA, the ester linkages were successfully introduced into the backbone of hyperbranched polyglycerols, resulting in the formation of dHPGs. The structure and property of dHPGs were well characterized by FTIR, NMR, and GPC. The highly branched architecture of polymerized products was confirmed 3559
dx.doi.org/10.1021/bm300966d | Biomacromolecules 2012, 13, 3552−3561
Biomacromolecules
Article
Figure 13. CLSM photos of CAL27 cells incubated with the Rhodamine-123-labeled dHPG-MTX micelles for different intervals. The pictures from left to right are differential interference contrast (DIC), Rhodamine-123 fluorescence, DAPI fluorescence, and the merge image, respectively. (Cell nucleuses were stained with DAPI). Rhodamine-123-labeled dHPG-MTX treated cells for (A) 5 min, (B) 60 min, (C) 180 min. The excitation wavelengths of DAPI and Rhodamine-123 were 340 and 507 nm, respectively.
Figure 14. Proliferation inhibition of free MTX and dHPG-MTX micelles against CAL27 (A: 48 h, B: 72 h) and Hela (C: 48 h, D: 72 h) cells.
evaluation confirmed the low cytotoxicity of dHPGs, which was closely related to the excellent biocompatibility of glycerol units and the presence of biodegradable ester bonds in polymer backbone. Benefiting from the existence of plentiful terminal hydroxyls, the hydrophobic anticancer drug MTX could be
by 2D-NMR techniques. By changing the molar ratios of glycerol/GMA or KH/GMA, the degree of branching could be adjusted between 0.43 and 0.50. The NMR and GPC analyses showed that the hydrolytic degradation of dHPGs was accelerated greatly in an acidic environment. The MTT 3560
dx.doi.org/10.1021/bm300966d | Biomacromolecules 2012, 13, 3552−3561
Biomacromolecules
Article
(14) Zhang, W.; Zhang, Y.; Lobler, M.; Schmitz, K. P.; Ahmad, A.; Pyykko, I.; Zou, J. Int. J. Nanomed. 2011, 6, 535−546. (15) Steinhilber, D.; Sisson, A. L.; Mangoldt, D.; Welker, P.; Licha, K.; Haag, R. Adv. Funct. Mater. 2010, 20, 4133−4138. (16) Jia, Z. F.; Li, G. L.; Zhu, Q.; Yan, D. Y.; Zhu, X. Y.; Chen, H.; Wu, J. L.; Tu, C. L.; Sun, J. Chem. −Eur. J. 2009, 15, 7593−7600. (17) Pang, Y.; Zhu, Q.; Liu, J. Y.; Wu, J. L.; Wang, R. B.; Chen, S. Y.; Zhu, X. Y.; Yan, D. Y.; Huang, W.; Zhu, B. S. Biomacromolecules 2010, 11, 575−582. (18) Pang, Y.; Liu, J. Y.; Wu, J. L.; Li, G. L.; Wang, R. B.; Su, Y.; He, P.; Zhu, X. Y.; Yan, D. Y.; Zhu, B. S. Bioconjugate Chem. 2010, 21, 2093−2102. (19) Li, G. L.; Liu, J. Y.; Pang, Y.; Wang, R. B.; Mao, L. M.; Yan, D. Y.; Zhu, X. Y.; Sun, J. Biomacromolecules 2011, 12, 2016−2026. (20) Pang, Y.; Liu, J. Y.; Su, Y.; Wu, J. L.; Zhu, L. J.; Zhu, X. Y.; Yan, D. Y.; Zhu, B. S. Polym. Chem. 2011, 2, 1661−1670. (21) Chen, L.; Zhu, X. Y.; Yan, D. Y.; Chen, Y.; Chen, Q.; Yao, Y. F. Angew. Chem., Int. Ed. 2006, 45, 87−90. (22) Jia, Z. F.; Chen, H.; Zhu, X. Y.; Yan, D. Y. J. Am. Chem. Soc. 2006, 128, 8144−8145. (23) Zhu, X. Y.; Chen, L.; Chen, Y.; Yan, D. Y. Sci. China, Ser. B 2008, 51, 1057−1065. (24) Wan, H. S.; Chen, Y.; Chen, L.; Zhu, X. Y.; Yan, D. Y.; Li, B.; Liu, T.; Zhao, L.; Jiang, X. L.; Zhang, G. Z. Macromolecules 2008, 41, 465−470. (25) Hawker, C. J.; Lee, R.; Fréchet, J. M. J. J. Am. Chem. Soc. 1991, 113, 4583−4588. (26) Prabaharan, M.; Grailer, J. J.; Pilla, S.; Steeber, D. A.; Gong, S. Biomaterials 2009, 30, 6065−6075. (27) Prabaharan, M.; Grailer, J. J.; Pilla, S.; Steeber, D. A.; Gong, S. Biomaterials 2009, 30, 3009−3019. (28) Aryal, S.; Prabaharan, M.; Pilla, S.; Gong, S. Int. J. Biol. Macromol. 2009, 44, 346−352. (29) Kolhe, P.; Misra, E.; Kannan, R. M.; Kannan, S.; Lieh-Lai, M. Int. J. Pharm. 2003, 259, 143−160.
conjugated onto the surface of dHPGs. Due to their amphiphilicity, the MTX-conjugated dHPGs (dHPG-MTXs) self-assembled into micelles with a diameter of about 160 nm, and the conjugated MTX could be released quickly at a mild acidic condition. Flow cytometry and CLSM analysis suggested that the dHPG-MTX micelles could be easily internalized by cancer cells and preferentially accumulated in the cytoplasm. The ability of dHPG-MTX micelles to inhibit proliferation of CAL27 and Hela cancer cells was investigated in vitro. It was found that dHPG-MTX micelles exhibited high anticancer efficacy. All of these results indicate that biodegradable hyperbranched polyglycerols could be used to construct safe and promising drug delivery systems.
■
ASSOCIATED CONTENT
S Supporting Information *
FTIR spectra of dHPGs, 1H NMR spectra of dHPG at different degradation times, GPC chromatograms at different degradation times, and cytotoxicity of dHPGs and their degraded products against NIH/3T3 cells. This material is available free of charge via the Internet at http://pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*Tel: +86-21-34205699. Fax: +86-21-34205722. E-mail:
[email protected] (X.Z.);
[email protected] (J.S.). Author Contributions ∥
These authors are joint first authors.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (20974062), the National Basic Research Program (2009CB930400, 2012CB821500), and China National Funds for Distinguished Young Scientists (21025417).
■
REFERENCES
(1) Fréchet, J. M. J.; Tomalia, D. A. In Dendrimers and Other Dendritic Polymers; Wiley: New York, 2001. (2) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem. Rev. 1999, 99, 1665−1688. (3) Stiriba, S. E.; Frey, H.; Haag, R. Angew. Chem., Int. Ed. Engl. 2002, 41, 1329−1334. (4) Voit, B. I.; Lederer, A. Chem. Rev. 2009, 109, 5924−5973. (5) Zhou, Y. F.; Huang, W.; Liu, J. Y.; Zhu, X. Y.; Yan, D. Y. Adv. Mater. 2010, 22, 4567−4590. (6) Sunder, A.; Hanselmann, R.; Frey, H.; Mülhaupt, R. Macromolecules 1999, 32, 4240−4246. (7) Sunder, A.; Quincy, M. F.; Mülhaupt, R.; Frey, H. Angew. Chem., Int. Ed. Engl. 1999, 38, 2928−2930. (8) Haag, R. Angew. Chem., Int. Ed. 2004, 43, 278−282. (9) Kainthan, R. K.; Hester, S. R.; Levin, E.; Devine, D. V.; Brooks, D. E. Biomaterials 2007, 28, 4581−4590. (10) Calderón, M.; Quadir, M. A.; Sharma, S. K.; Haag, R. Adv. Mater. 2010, 22, 190−218. (11) Wilms, D.; Stiriba, S. E.; Frey, H. Acc. Chem. Res. 2010, 43, 129− 141. (12) Afrimzon, E.; Deutsch, A.; Shafran, Y.; Zurgil, N.; Sandbank, J.; Pappo, I.; Deutsch, M. Clin. Exp. Metastasis 2008, 25, 213−224. (13) Zimra, Y.; Nudelman, A.; Zhuk, R.; Rabizadeh, E.; Shaklai, M.; Aviram, A.; Rephaeli, A. J. Cancer Res. Clin. Oncol. 2000, 126, 693−698. 3561
dx.doi.org/10.1021/bm300966d | Biomacromolecules 2012, 13, 3552−3561
