Materials Science and Engineering C 44 (2014) 386–390
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Polymerizable disulfide paclitaxel prodrug for controlled drug delivery Yi Ding a, Wulian Chen a, Jianhua Hu a,c, Ming Du b,⁎, Dong Yang a,⁎⁎ a b c
State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai 200433, China Medical Center for Diagnostics & Treat of Cervical Disease, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA (Fudan University), Shanghai 201203, China
a r t i c l e
i n f o
Article history: Received 26 April 2014 Received in revised form 15 July 2014 Accepted 8 August 2014 Available online 27 August 2014 Keywords: Polymerizable prodrug Disulfide bond Paclitaxel Controlled drug delivery
a b s t r a c t A polymerizable disulfide paclitaxel (PTX) prodrug was synthesized by the consequential esterification reactions of 3,3′-dithiodipropionic acid (DTPA), a disulfide compound containing two active carboxyl groups, with 2-hydroxyethyl methacrylate (HEMA) and PTX. The structure of the prodrug was confirmed by 1H NMR characterization. Then, the polymerizable prodrug was copolymerized with poly(ethylene glycol) methyl ether methacrylate (PEGMEA) to obtain a copolymer with hydrophilic PEG side chains and PTX covalently linked onto the backbone via disulfide bonds. The loading content of PTX was 23%. In aqueous solution, this copolymer prodrug could self-assemble into micelles, with hydrophobic PTX as the cores and hydrophilic PEG-segment as the shells. In vitro cell assay demonstrated that this copolymer prodrug showed more apparent cytotoxicity to cancer cells than to human normal cells. After incubation for 48 h, the cell viability of HEK-293 cells (human embryo kidney cells) at 0.1 μg/mL PTX still remained more than 90%, however, that of HeLa cells (human cervical cancer cells) decreased to 52%. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Chemotherapy, as an indispensable and important strategy in the comprehensive treatment of cancer, has drawn increasing attentions [1–4]. Paclitaxel (PTX) has been proved to be a potent drug for treating a variety of cancers, such as metastatic breast cancer and ovarian cancer [5]. However, the poor water-solubility and non-distinctive cytotoxicity to cancer cells and normal cells greatly hindered its administration dosage and application scope [6–9]. Thus, it's highly required to develop a convenient and safe controlled drug delivery system (CDDS) to maximize the therapeutic efficacy at tumor sites and minimize the side effects of PTX [10–13]. Up to date, a variety of PTX loaded CDDSs, based on pH, thermal, light, or ion strength responsive release mechanisms, have been reported [14]. Many materials, such as liposomes [15], mesoporous silica nanoparticles (MSNs) [16], block copolymer [17], and dendrimers [18], have been utilized as the drug carriers. However, most of these CDDSs loaded PTX by non-covalent interactions. The general routine entraps the drug with the hydrophobic core and function the vehicles with the hydrophilic shell and targeting moieties ensuring the prodrug to release the drug cancer cells. But the burst release of the loaded PTX was inevitable once in the medium [19], due to the weak interaction between the drug and the carrier. Furthermore, the differences of pH,
⁎ Corresponding author. Tel.: +86 21 55665280; fax: +86 21 64650293. ⁎⁎ Corresponding author. Tel.: +86 21 65643575; fax: +86 21 65640293. E-mail addresses: [email protected] (M. Du), [email protected] (D. Yang).
http://dx.doi.org/10.1016/j.msec.2014.08.046 0928-4931/© 2014 Elsevier B.V. All rights reserved.
temperature, or light between the lesion sites and the normal tissues were tiny, for example, the pH and temperature of the tumor tissues were approximately 0.5 lower and 1.0 °C higher than those of the normal tissues, respectively. Thus, external complementary assistants were often requisite to enlarge the circumstance differences between the lesion locations and the normal tissues. It's still a challenging work to explore a more efficient and sensitive CDDS for PTX. Recently, a disulfide bond has attracted growing attentions due to its highly sensitive responsibility [20,21]. A disulfide bond could be readily broken in response to thiol compounds through the thiol–disulfide exchange reaction [22,23]. Only 2 to 3 orders of higher level of glutathione (GSH) tripeptide (approximately 2–10 mM) than the extracellular fluids (approximately 2–20 μM) is enough to induce the disassembly and release of drug in the cytosol [24]. It has been found that the concentration of GSH, is 7-fold higher in human tumor cells than that in normal cells [25]. Thus, a disulfide bond would be stable in the blood circulation and normal tissues, and be broken in tumor tissues [26,27]. The S–S linkage has been widely explored in the design of smart drug vehicles for its redox-responsive property [23]. For example, Ojima et al. have covalently loaded taxoid onto single-walled carbon nanotubes via a disulfide linker, and found that this CDDS showed specific cytotoxicity to L1210FR leukemia cell line [28]. Previously, our group has covalently loaded PTX onto copolymer backbone, and found that this CDDS showed apparent cytotoxicity to OS-RC-2 kidney tumor cells and low cytotoxicity to macrophage human normal cells [29]. However, to our knowledge, all the CDDSs covalently loaded drug via disulfide bonds were constructed by functionalizing carriers firstly, and then linking drug to the carriers.
Y. Ding et al. / Materials Science and Engineering C 44 (2014) 386–390
Herein, a new strategy to construct a CDDS covalently loaded PTX via disulfide bonds was reported. Firstly, a polymerizable PTX prodrug containing a disulfide bond was synthesized by two sequent esterification reactions of 3,3′-dithiodipropionic acid (DTPA) with 2-hydroxyethyl methacrylate (HEMA) and PTX. Then, the polymerizable PTX prodrug was copolymerized with poly(ethylene glycol) methyl ether methacrylate (PEGMEA) to obtain a copolymer with hydrophilic PEG side chains and PTX covalently linked onto the backbone via disulfide bonds (see Fig. 1). The loading content of PTX could reach up to 23%. In aqueous solution, this copolymer could self-assemble into micelles, with hydrophobic PTX as the cores and hydrophilic PEG-segment as the shells. The mean diameter of the micelles evaluated by dynamic light scattering (DLS) was approximately 210 nm. In vitro cell assays were performed to evaluate the cytotoxicity of the CDDS to human embryo kidney cells (HEK-293) and HeLa human cervical carcinoma cells. 2. Experimental section 2.1. Materials Paclitaxel (PTX) was purchased from Beijing Huafeng United Technology Co., Ltd. Poly(ethylene glycol) methyl ether methacrylate (PEGMEA, Mn = 475) and 2-hydroxyethyl methacrylate (HEMA, 98%) were purchased from Aladdin and purified by passing through a neutral alumina column to remove the inhibitor. 2,2′-Azobis(isobutyronitrile) (AIBN) was purchased from Tianjin Guangfu Fine Chemical Research
387
Institute, and purified by dissolving in chloroform and precipitating in methanol. 3,3′-Dithiodipropionic acid (DTPA, 99%), N,N′-diisopropylcarbodiimide (DIC, 98%) and dimethylaminopyridine (DMAP, 99%) were purchased from Aldrich. Tetrahydrofuran (THF), hexane, ethyl acetate, methylbenzene, dichloromethane, and diethyl ether were purchased from Shanghai Medpep Co., Ltd. Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. 2.2. Synthesis of polymerizable PTX prodrug 2 Compound 1 (DTPA-HEMA) was synthesized according to the former report [30]. Typically, DTPA (4.4 g, 20 mmol), HEMA (0.6 mL, 5 mmol) and DMAP (0.05 g, 0.4 mmol) were dissolved in THF (50 mL). Then, DIC (0.5 mL, 3.2 mmol) was added to the mixture, and the solution was stirred at room temperature overnight. After reaction, the mixture was concentrated and purified by flash column chromatography on silica using hexane/ethyl acetate (1:1, v/v) as the eluent, to give the desired product 1 (1.17 g, yield: 72%). Then, PTX (1.0 g, 2.4 mmol), DMAP (0.024 g, 0.2 mmol) and DTPA–HEMA (0.78 g, 2.4 mmol) were dissolved in dichloromethane (70 mL). After adding DIC (23 μL, 0.16 mmol), the solution was stirred at room temperature overnight. After reaction, the mixture was concentrated and purified by flash column chromatography on silica using hexane/ethyl acetate (1:1, v/v) as the eluent, to give the desired polymerizable PTX prodrug 2 (PTX–DTPA–HEMA, 1.328 g, yield: 48%).
Fig. 1. Schematic illustration of the synthesis of the copolymer.
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2.3. Free radical polymerization to synthesize copolymer 3 Typically, PTX–DTPA–HEMA (75.8 mg, 0.065 mmol), PEGMEA (0.3 mL, 0.5 mmol) and AIBN (7.5 mg, 0.04 mmol) were dissolved in methylbenzene (3 mL). Then, the solution was purged with nitrogen and heated at 68 °C for 2 h. After reaction, the solution was precipitated into cold diethyl ether. The crude product was dried in vacuo at room temperature overnight, to give a colorless colloidal copolymer 3 (P(PTX–DTPA–HEMA)-co-PPEGMEA, 121 mg, yield: 44%). 2.4. In vitro cell assay The in vitro cytotoxicity of P(PTX–DTPA–HEMA)-co-PPEGMEA toward HeLa and HEK-293 cells was evaluated by CCK-8 kit assay. The cells were seeded in 96-well plates with a density of 1 × 104 cell per well and incubated at 37 °C in an atmosphere containing 5% CO2 for 24 h to allow cell attachment. Then, the medium was replaced with a fresh medium containing the indicated concentration of P(PTX–DTPA–HEMA)-co-PPEGMEA. After incubation for 24 and 48 h, the medium was aspirated and replaced by 100 mL of fresh medium containing 10 mL of CCK-8. The cells were incubated for another 2 h at 37 °C in dark. Afterward, the absorption at a wavelength of 450 nm of each well was measured using a microplate reader.
spectrum of PTX–DTPA–HEMA, peaks from 7.40 to 8.14 ppm were attributed to the protons from the phenyls of PTX. All the 1H NMR results indicated that the PTX–DTPA–HEMA was successfully synthesized. Because the reactivity of HEMA and PEGMEA are similar, they could be easily copolymerized to get P(PTX–DTPA–HEMA)-co-PPEGMEA by free radical copolymerization. The number-average molecular weight (Mn) and the PDI of P(PTX–DTPA–HEMA)-co-PPEGMEA determined by GPC were 10,000 and 1.3, respectively (Fig. 3A). The chemical structure was characterized by 1H NMR. As seen in Fig. 3B, P(PTX– DTPA–HEMA)-co-PPEGMEA showed two strong peaks at 3.35 ppm (a) (OCH3) and 3.50 ppm (b) (OCH2CH2), characteristic of the protons of PEG. The four peaks from 7.93 to 8.11 ppm originating from the phenyl groups of PTX were also clearly observed. The loading content of PTX
2.5. Characterization 1 H NMR spectra were recorded on a Bruker Avance 500 spectrometer using CDCl3 as solvent, trimethylsilane was used as internal standard. Fourier transform infrared (FT-IR) measurements were performed on a Nicolet 6700 spectrometer. The molecular weight and molecular weight distribution were determined by gel permeation chromatography (GPC), which performed in THF at 35 °C with an elution rate of 1.0 mL/min on Aginent 1100 equipped with a G1310A pump, a G1362A refractive index detector, and a G1314A variable wavelength detector. The system was calibrated with linear polystyrene standards. The size distribution of the micelles was measured by dynamic light scattering (DLS) using a Malvern autosizer 4700 instrument. Morphological characteristics of self-assembled micelles were investigated by a Hitachi H-600 transmission electron microscope (TEM) at 75 kV. UV–visible (Vis) absorption spectra were measured with a Perkin-Elmer Lambda 35 spectrophotometer.
3. Results and discussion DTPA was chosen as the initial disulfide reagent, because DTPA contains two active carboxyl groups, that one could react with HEMA to introduce polymerizable double bonds, and the other could covalently load drug. Firstly, DTPA was esterified with HEMA to yield DTPA–HEMA. To insure that only one of the carboxyl groups of DTPA was reacted with HEMA, the concentration of DTPA was four times than that of HEMA. The 1H NMR spectra of DTPA and DTPA– HEMA (Fig. 2A and B) confirmed this assumption. Due to the loss of the hydrogen bonds originating from the double carboxy groups of every molecular after esterification, the chemical shifts of DTPA protons undergo significantly shifts from 2.01 (b) and 1.69 (a) to 2.91 (f) and 2.79 (e), respectively. The integration ratio of the peaks at 6.14 (g) and 5.61 (h) ppm, originating from the double bond of HEMA (\CH = CH 2 ), to the peaks at 2.91 and 2.79 ppm, corresponding to the methylene group of DTPA (\CH2SSCH2− and \O(O)CCH2CH2SS\) is 1:4, indicating that the molar ratio of DTPA and HEMA was 1:1, i.e. only one of the carboxyl groups in DTPA was reacted with HEMA. And the resonance of the proton of PTX attached to the ester bond undergoes slight shift from 4.78 (j) to 4.93 (j′). The 2′-hydroxyl group of PTX is significantly reactive, it would be facilely esterified with the remaining carboxyl group of DTPA in the presence of DIC/DMAP catalyst, to give PTX–DTPA–HEMA. Fig. 2D showed the 1H NMR
Fig. 2. 1H NMR spectra of (A) DTPA (B) DTPA–HEMA, (C) PTX, (D) PTX–DTPA–HEMA.
Y. Ding et al. / Materials Science and Engineering C 44 (2014) 386–390
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Fig. 3. (A) GPC trace and (B) 1H NMR spectrum of P(PTX–DTPA–HEMA)-co-PPEGMEA.
Fig. 4. (A) FT-IR spectra and (B) UV–vis spectra of P(DTPA–HEMA)-co-PPEGMEA, PTX and P(PTX–DTPA-HEMA)-co-PPEGMEA.
on the copolymer was 23 wt.%, determined from the integration area ratio of the peak at 5.50 ppm, assigned to tert-butyl groups of PTX, to peak a. As it could be seen from the FT-IR spectra of Fig. 4A. Compared with P(DTPA–HEMA)-co-PPEGMEA, which was prepared as the control copolymer with the similar synthetic process of P(PTX–DTPA–HEMA)co-PPEGMEA, P(PTX–DTPA–HEMA)-co-PPEGMEA showed a new peak at 710 cm−1, ascribed to PTX. In addition, the UV–vis spectra (Fig. 4B) also confirmed the presence of PTX in P(PTX–DTPA–HEMA)-coPPEGMEA, due to the absorption at 227 nm. All these results suggested that P(PTX–DTPA–HEMA)-co-PPEGMEA was successfully synthesized. P(PTX–DTPA–HEMA)-co-PPEGMEA is amphiphilic, and could selfassemble into micelles in aqueous solution, with hydrophobic PTX as
the core and hydrophilic PEG side chains as the shell. The size and morphology of P(PTX–DTPA–HEMA)-co-PPEGMEA micelles were examined by DLS and TEM. As seen from the TEM image of Fig. 5A, all the micelles were spherical nanoparticles, with gray periphery and dark inner regions, which might correspond to PEG-enriched and PTXL-enriched domains, respectively. The average diameter of the micelles was determined by DLS measurement (Fig. 5B), which was approximately 135 nm, with a PDI of 0.12. To evaluate the controlled drug release behavior of the P(PTX– DTPA–HEMA)-co-PPEGMEA prodrug, its in vitro cytotoxicity against HEK-293 cells (human embryo kidney cells) and HeLa (human cervical cancer cells) cells were investigated by CCK-8 assay. Fig. 6 showed the cell viability of HEK-293 cells and HeLa cells incubated with different
Fig. 5. (A) TEM image and (B) DLS curve of the self-assembled micelles.
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Fig. 6. Cell viability of HEK-293 cells and HeLa cells incubated with different concentrations of P(PTX–DTPA–HEMA)-co-PPEGMEA for (A) 24 h and (B) 48 h.
concentrations of P(PTX–DTPA–HEMA)-co-PPEGMEA for 24 and 48 h. Both of the cell viability of HEK-293 cells and HeLa cells decreased with the increase of the concentration of P(PTX–DTPA–HEMA)-coPPEGMEA. However, the cell viability of HeLa decreased more notably than that of HEK-293 cells. For example, after incubation for 48 h, the cell viability of HEK-293 cells at 0.1 μg/mL PTX still remained more than 90%, that of HeLa cells decreased to 52%. 4. Conclusion In summary, we reported a novel strategy to construct controlled drug delivery ststem based on disulfide bond covalently loading drug. Firstly, a polymerizable disulfide paclitaxel (PTX) prodrug was synthesized by the consequential esterification reactions of 3,3′dithiodipropionic acid (DTPA), a disulfide compound containing two active carboxyl groups, with 2-hydroxyethyl methacrylate and PTX. The structure of the prodrug was confirmed by 1H NMR characterization. Then, the polymerizable prodrug was copolymerized with poly(ethylene glycol) methyl ether methacrylate (PEGMEA) to obtain a copolymer with hydrophilic PEG side chains and PTX covalently linked onto the backbone via disulfide bonds. The loading content of PTX was 23%. In aqueous solution, this copolymer prodrug could self-assemble into micelles, with hydrophobic PTX as the cores and hydrophilic PEG-segment as the shells. In vitro cell assay demonstrated that after incubation for 48 h, the cell viability of HEK-293 cells (human embryo kidney cells) at 0.1 μg/mL PTX still remained more than 90%, however, that of HeLa cells (human cervical cancer cells) decreased to 52%. Acknowledgments The authors thank the financial support from the National Natural Science Foundation of China (51073042, 51103026, 51373035, 51373040 and 81372796), the Shanghai Natural Science Funds (11ZR1403100), the Shanghai Scientific and Technological Innovation Project (11JC1400600, 10431903000 and 124119a2400), the Shanghai Rising Star Program (12QB1402900), the Fund of Shanghai Bureau of Health (20124097), the Specialized Research Fund for the Doctoral Program of Higher Education (20110071120006), and the School of Pharmacy, Fudan University & The Open Project Program of Key Lab of Smart Drug Delivery (Fudan University), Ministry of Education & PLA, China.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Polymerizable disulfide paclitaxel prodrug for controlled drug delivery Yi Ding a, Wulian Chen a, Jianhua Hu a,c, Ming Du b,⁎, Dong Yang a,⁎⁎ a b c
State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai 200433, China Medical Center for Diagnostics & Treat of Cervical Disease, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China Key Laboratory of Smart Drug Delivery, Ministry of Education & PLA (Fudan University), Shanghai 201203, China
a r t i c l e
i n f o
Article history: Received 26 April 2014 Received in revised form 15 July 2014 Accepted 8 August 2014 Available online 27 August 2014 Keywords: Polymerizable prodrug Disulfide bond Paclitaxel Controlled drug delivery
a b s t r a c t A polymerizable disulfide paclitaxel (PTX) prodrug was synthesized by the consequential esterification reactions of 3,3′-dithiodipropionic acid (DTPA), a disulfide compound containing two active carboxyl groups, with 2-hydroxyethyl methacrylate (HEMA) and PTX. The structure of the prodrug was confirmed by 1H NMR characterization. Then, the polymerizable prodrug was copolymerized with poly(ethylene glycol) methyl ether methacrylate (PEGMEA) to obtain a copolymer with hydrophilic PEG side chains and PTX covalently linked onto the backbone via disulfide bonds. The loading content of PTX was 23%. In aqueous solution, this copolymer prodrug could self-assemble into micelles, with hydrophobic PTX as the cores and hydrophilic PEG-segment as the shells. In vitro cell assay demonstrated that this copolymer prodrug showed more apparent cytotoxicity to cancer cells than to human normal cells. After incubation for 48 h, the cell viability of HEK-293 cells (human embryo kidney cells) at 0.1 μg/mL PTX still remained more than 90%, however, that of HeLa cells (human cervical cancer cells) decreased to 52%. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Chemotherapy, as an indispensable and important strategy in the comprehensive treatment of cancer, has drawn increasing attentions [1–4]. Paclitaxel (PTX) has been proved to be a potent drug for treating a variety of cancers, such as metastatic breast cancer and ovarian cancer [5]. However, the poor water-solubility and non-distinctive cytotoxicity to cancer cells and normal cells greatly hindered its administration dosage and application scope [6–9]. Thus, it's highly required to develop a convenient and safe controlled drug delivery system (CDDS) to maximize the therapeutic efficacy at tumor sites and minimize the side effects of PTX [10–13]. Up to date, a variety of PTX loaded CDDSs, based on pH, thermal, light, or ion strength responsive release mechanisms, have been reported [14]. Many materials, such as liposomes [15], mesoporous silica nanoparticles (MSNs) [16], block copolymer [17], and dendrimers [18], have been utilized as the drug carriers. However, most of these CDDSs loaded PTX by non-covalent interactions. The general routine entraps the drug with the hydrophobic core and function the vehicles with the hydrophilic shell and targeting moieties ensuring the prodrug to release the drug cancer cells. But the burst release of the loaded PTX was inevitable once in the medium [19], due to the weak interaction between the drug and the carrier. Furthermore, the differences of pH,
⁎ Corresponding author. Tel.: +86 21 55665280; fax: +86 21 64650293. ⁎⁎ Corresponding author. Tel.: +86 21 65643575; fax: +86 21 65640293. E-mail addresses: [email protected] (M. Du), [email protected] (D. Yang).
http://dx.doi.org/10.1016/j.msec.2014.08.046 0928-4931/© 2014 Elsevier B.V. All rights reserved.
temperature, or light between the lesion sites and the normal tissues were tiny, for example, the pH and temperature of the tumor tissues were approximately 0.5 lower and 1.0 °C higher than those of the normal tissues, respectively. Thus, external complementary assistants were often requisite to enlarge the circumstance differences between the lesion locations and the normal tissues. It's still a challenging work to explore a more efficient and sensitive CDDS for PTX. Recently, a disulfide bond has attracted growing attentions due to its highly sensitive responsibility [20,21]. A disulfide bond could be readily broken in response to thiol compounds through the thiol–disulfide exchange reaction [22,23]. Only 2 to 3 orders of higher level of glutathione (GSH) tripeptide (approximately 2–10 mM) than the extracellular fluids (approximately 2–20 μM) is enough to induce the disassembly and release of drug in the cytosol [24]. It has been found that the concentration of GSH, is 7-fold higher in human tumor cells than that in normal cells [25]. Thus, a disulfide bond would be stable in the blood circulation and normal tissues, and be broken in tumor tissues [26,27]. The S–S linkage has been widely explored in the design of smart drug vehicles for its redox-responsive property [23]. For example, Ojima et al. have covalently loaded taxoid onto single-walled carbon nanotubes via a disulfide linker, and found that this CDDS showed specific cytotoxicity to L1210FR leukemia cell line [28]. Previously, our group has covalently loaded PTX onto copolymer backbone, and found that this CDDS showed apparent cytotoxicity to OS-RC-2 kidney tumor cells and low cytotoxicity to macrophage human normal cells [29]. However, to our knowledge, all the CDDSs covalently loaded drug via disulfide bonds were constructed by functionalizing carriers firstly, and then linking drug to the carriers.
Y. Ding et al. / Materials Science and Engineering C 44 (2014) 386–390
Herein, a new strategy to construct a CDDS covalently loaded PTX via disulfide bonds was reported. Firstly, a polymerizable PTX prodrug containing a disulfide bond was synthesized by two sequent esterification reactions of 3,3′-dithiodipropionic acid (DTPA) with 2-hydroxyethyl methacrylate (HEMA) and PTX. Then, the polymerizable PTX prodrug was copolymerized with poly(ethylene glycol) methyl ether methacrylate (PEGMEA) to obtain a copolymer with hydrophilic PEG side chains and PTX covalently linked onto the backbone via disulfide bonds (see Fig. 1). The loading content of PTX could reach up to 23%. In aqueous solution, this copolymer could self-assemble into micelles, with hydrophobic PTX as the cores and hydrophilic PEG-segment as the shells. The mean diameter of the micelles evaluated by dynamic light scattering (DLS) was approximately 210 nm. In vitro cell assays were performed to evaluate the cytotoxicity of the CDDS to human embryo kidney cells (HEK-293) and HeLa human cervical carcinoma cells. 2. Experimental section 2.1. Materials Paclitaxel (PTX) was purchased from Beijing Huafeng United Technology Co., Ltd. Poly(ethylene glycol) methyl ether methacrylate (PEGMEA, Mn = 475) and 2-hydroxyethyl methacrylate (HEMA, 98%) were purchased from Aladdin and purified by passing through a neutral alumina column to remove the inhibitor. 2,2′-Azobis(isobutyronitrile) (AIBN) was purchased from Tianjin Guangfu Fine Chemical Research
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Institute, and purified by dissolving in chloroform and precipitating in methanol. 3,3′-Dithiodipropionic acid (DTPA, 99%), N,N′-diisopropylcarbodiimide (DIC, 98%) and dimethylaminopyridine (DMAP, 99%) were purchased from Aldrich. Tetrahydrofuran (THF), hexane, ethyl acetate, methylbenzene, dichloromethane, and diethyl ether were purchased from Shanghai Medpep Co., Ltd. Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies, Inc. 2.2. Synthesis of polymerizable PTX prodrug 2 Compound 1 (DTPA-HEMA) was synthesized according to the former report [30]. Typically, DTPA (4.4 g, 20 mmol), HEMA (0.6 mL, 5 mmol) and DMAP (0.05 g, 0.4 mmol) were dissolved in THF (50 mL). Then, DIC (0.5 mL, 3.2 mmol) was added to the mixture, and the solution was stirred at room temperature overnight. After reaction, the mixture was concentrated and purified by flash column chromatography on silica using hexane/ethyl acetate (1:1, v/v) as the eluent, to give the desired product 1 (1.17 g, yield: 72%). Then, PTX (1.0 g, 2.4 mmol), DMAP (0.024 g, 0.2 mmol) and DTPA–HEMA (0.78 g, 2.4 mmol) were dissolved in dichloromethane (70 mL). After adding DIC (23 μL, 0.16 mmol), the solution was stirred at room temperature overnight. After reaction, the mixture was concentrated and purified by flash column chromatography on silica using hexane/ethyl acetate (1:1, v/v) as the eluent, to give the desired polymerizable PTX prodrug 2 (PTX–DTPA–HEMA, 1.328 g, yield: 48%).
Fig. 1. Schematic illustration of the synthesis of the copolymer.
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2.3. Free radical polymerization to synthesize copolymer 3 Typically, PTX–DTPA–HEMA (75.8 mg, 0.065 mmol), PEGMEA (0.3 mL, 0.5 mmol) and AIBN (7.5 mg, 0.04 mmol) were dissolved in methylbenzene (3 mL). Then, the solution was purged with nitrogen and heated at 68 °C for 2 h. After reaction, the solution was precipitated into cold diethyl ether. The crude product was dried in vacuo at room temperature overnight, to give a colorless colloidal copolymer 3 (P(PTX–DTPA–HEMA)-co-PPEGMEA, 121 mg, yield: 44%). 2.4. In vitro cell assay The in vitro cytotoxicity of P(PTX–DTPA–HEMA)-co-PPEGMEA toward HeLa and HEK-293 cells was evaluated by CCK-8 kit assay. The cells were seeded in 96-well plates with a density of 1 × 104 cell per well and incubated at 37 °C in an atmosphere containing 5% CO2 for 24 h to allow cell attachment. Then, the medium was replaced with a fresh medium containing the indicated concentration of P(PTX–DTPA–HEMA)-co-PPEGMEA. After incubation for 24 and 48 h, the medium was aspirated and replaced by 100 mL of fresh medium containing 10 mL of CCK-8. The cells were incubated for another 2 h at 37 °C in dark. Afterward, the absorption at a wavelength of 450 nm of each well was measured using a microplate reader.
spectrum of PTX–DTPA–HEMA, peaks from 7.40 to 8.14 ppm were attributed to the protons from the phenyls of PTX. All the 1H NMR results indicated that the PTX–DTPA–HEMA was successfully synthesized. Because the reactivity of HEMA and PEGMEA are similar, they could be easily copolymerized to get P(PTX–DTPA–HEMA)-co-PPEGMEA by free radical copolymerization. The number-average molecular weight (Mn) and the PDI of P(PTX–DTPA–HEMA)-co-PPEGMEA determined by GPC were 10,000 and 1.3, respectively (Fig. 3A). The chemical structure was characterized by 1H NMR. As seen in Fig. 3B, P(PTX– DTPA–HEMA)-co-PPEGMEA showed two strong peaks at 3.35 ppm (a) (OCH3) and 3.50 ppm (b) (OCH2CH2), characteristic of the protons of PEG. The four peaks from 7.93 to 8.11 ppm originating from the phenyl groups of PTX were also clearly observed. The loading content of PTX
2.5. Characterization 1 H NMR spectra were recorded on a Bruker Avance 500 spectrometer using CDCl3 as solvent, trimethylsilane was used as internal standard. Fourier transform infrared (FT-IR) measurements were performed on a Nicolet 6700 spectrometer. The molecular weight and molecular weight distribution were determined by gel permeation chromatography (GPC), which performed in THF at 35 °C with an elution rate of 1.0 mL/min on Aginent 1100 equipped with a G1310A pump, a G1362A refractive index detector, and a G1314A variable wavelength detector. The system was calibrated with linear polystyrene standards. The size distribution of the micelles was measured by dynamic light scattering (DLS) using a Malvern autosizer 4700 instrument. Morphological characteristics of self-assembled micelles were investigated by a Hitachi H-600 transmission electron microscope (TEM) at 75 kV. UV–visible (Vis) absorption spectra were measured with a Perkin-Elmer Lambda 35 spectrophotometer.
3. Results and discussion DTPA was chosen as the initial disulfide reagent, because DTPA contains two active carboxyl groups, that one could react with HEMA to introduce polymerizable double bonds, and the other could covalently load drug. Firstly, DTPA was esterified with HEMA to yield DTPA–HEMA. To insure that only one of the carboxyl groups of DTPA was reacted with HEMA, the concentration of DTPA was four times than that of HEMA. The 1H NMR spectra of DTPA and DTPA– HEMA (Fig. 2A and B) confirmed this assumption. Due to the loss of the hydrogen bonds originating from the double carboxy groups of every molecular after esterification, the chemical shifts of DTPA protons undergo significantly shifts from 2.01 (b) and 1.69 (a) to 2.91 (f) and 2.79 (e), respectively. The integration ratio of the peaks at 6.14 (g) and 5.61 (h) ppm, originating from the double bond of HEMA (\CH = CH 2 ), to the peaks at 2.91 and 2.79 ppm, corresponding to the methylene group of DTPA (\CH2SSCH2− and \O(O)CCH2CH2SS\) is 1:4, indicating that the molar ratio of DTPA and HEMA was 1:1, i.e. only one of the carboxyl groups in DTPA was reacted with HEMA. And the resonance of the proton of PTX attached to the ester bond undergoes slight shift from 4.78 (j) to 4.93 (j′). The 2′-hydroxyl group of PTX is significantly reactive, it would be facilely esterified with the remaining carboxyl group of DTPA in the presence of DIC/DMAP catalyst, to give PTX–DTPA–HEMA. Fig. 2D showed the 1H NMR
Fig. 2. 1H NMR spectra of (A) DTPA (B) DTPA–HEMA, (C) PTX, (D) PTX–DTPA–HEMA.
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Fig. 3. (A) GPC trace and (B) 1H NMR spectrum of P(PTX–DTPA–HEMA)-co-PPEGMEA.
Fig. 4. (A) FT-IR spectra and (B) UV–vis spectra of P(DTPA–HEMA)-co-PPEGMEA, PTX and P(PTX–DTPA-HEMA)-co-PPEGMEA.
on the copolymer was 23 wt.%, determined from the integration area ratio of the peak at 5.50 ppm, assigned to tert-butyl groups of PTX, to peak a. As it could be seen from the FT-IR spectra of Fig. 4A. Compared with P(DTPA–HEMA)-co-PPEGMEA, which was prepared as the control copolymer with the similar synthetic process of P(PTX–DTPA–HEMA)co-PPEGMEA, P(PTX–DTPA–HEMA)-co-PPEGMEA showed a new peak at 710 cm−1, ascribed to PTX. In addition, the UV–vis spectra (Fig. 4B) also confirmed the presence of PTX in P(PTX–DTPA–HEMA)-coPPEGMEA, due to the absorption at 227 nm. All these results suggested that P(PTX–DTPA–HEMA)-co-PPEGMEA was successfully synthesized. P(PTX–DTPA–HEMA)-co-PPEGMEA is amphiphilic, and could selfassemble into micelles in aqueous solution, with hydrophobic PTX as
the core and hydrophilic PEG side chains as the shell. The size and morphology of P(PTX–DTPA–HEMA)-co-PPEGMEA micelles were examined by DLS and TEM. As seen from the TEM image of Fig. 5A, all the micelles were spherical nanoparticles, with gray periphery and dark inner regions, which might correspond to PEG-enriched and PTXL-enriched domains, respectively. The average diameter of the micelles was determined by DLS measurement (Fig. 5B), which was approximately 135 nm, with a PDI of 0.12. To evaluate the controlled drug release behavior of the P(PTX– DTPA–HEMA)-co-PPEGMEA prodrug, its in vitro cytotoxicity against HEK-293 cells (human embryo kidney cells) and HeLa (human cervical cancer cells) cells were investigated by CCK-8 assay. Fig. 6 showed the cell viability of HEK-293 cells and HeLa cells incubated with different
Fig. 5. (A) TEM image and (B) DLS curve of the self-assembled micelles.
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Fig. 6. Cell viability of HEK-293 cells and HeLa cells incubated with different concentrations of P(PTX–DTPA–HEMA)-co-PPEGMEA for (A) 24 h and (B) 48 h.
concentrations of P(PTX–DTPA–HEMA)-co-PPEGMEA for 24 and 48 h. Both of the cell viability of HEK-293 cells and HeLa cells decreased with the increase of the concentration of P(PTX–DTPA–HEMA)-coPPEGMEA. However, the cell viability of HeLa decreased more notably than that of HEK-293 cells. For example, after incubation for 48 h, the cell viability of HEK-293 cells at 0.1 μg/mL PTX still remained more than 90%, that of HeLa cells decreased to 52%. 4. Conclusion In summary, we reported a novel strategy to construct controlled drug delivery ststem based on disulfide bond covalently loading drug. Firstly, a polymerizable disulfide paclitaxel (PTX) prodrug was synthesized by the consequential esterification reactions of 3,3′dithiodipropionic acid (DTPA), a disulfide compound containing two active carboxyl groups, with 2-hydroxyethyl methacrylate and PTX. The structure of the prodrug was confirmed by 1H NMR characterization. Then, the polymerizable prodrug was copolymerized with poly(ethylene glycol) methyl ether methacrylate (PEGMEA) to obtain a copolymer with hydrophilic PEG side chains and PTX covalently linked onto the backbone via disulfide bonds. The loading content of PTX was 23%. In aqueous solution, this copolymer prodrug could self-assemble into micelles, with hydrophobic PTX as the cores and hydrophilic PEG-segment as the shells. In vitro cell assay demonstrated that after incubation for 48 h, the cell viability of HEK-293 cells (human embryo kidney cells) at 0.1 μg/mL PTX still remained more than 90%, however, that of HeLa cells (human cervical cancer cells) decreased to 52%. Acknowledgments The authors thank the financial support from the National Natural Science Foundation of China (51073042, 51103026, 51373035, 51373040 and 81372796), the Shanghai Natural Science Funds (11ZR1403100), the Shanghai Scientific and Technological Innovation Project (11JC1400600, 10431903000 and 124119a2400), the Shanghai Rising Star Program (12QB1402900), the Fund of Shanghai Bureau of Health (20124097), the Specialized Research Fund for the Doctoral Program of Higher Education (20110071120006), and the School of Pharmacy, Fudan University & The Open Project Program of Key Lab of Smart Drug Delivery (Fudan University), Ministry of Education & PLA, China.
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