Communication
A Smart Drug Delivery System from Charge-Conversion Polymer–Drug Conjugate for Enhancing Tumor Therapy and Tunable Drug Releasea Hailong Huang, Yapeng Li,* Zongpeng Sa, Yuan Sun, Yuzhen Wang, Jingyuan Wang
A smart drug delivery system is prepared by citraconylated polyaspartic acid (PASP) derivate–drug conjugate. The conjugate contains two pH-sensitive groups: citraconic amide and hydrazone linker. Citraconic amide group can enhance tumor therapy efficiency by the extracellular pH-sensitive charge-conversion property. Hydrazone linker between polymer and drug can cleave efficiently in the intracellular pH environment. The resulting conjugate shows dual-pH sensitive properties: extracellular pH-triggered enhanced tumor targeting and intracellular pH-triggered drug release. The results of physicochemical properties, intracellular location, and cytotoxicity of conjugate micelles demonstrate that this novel smart drug delivery system can enhance intracellular delivery of drug at a low pH and then release drug rapidly.
1. Introduction In recent years, smart amphiphilic polymers have been developed and have attracted much attention because of their environment–responsive properties including pH, temperature, ionic strength, light, photochemical process.[1–4] Under appropriate environment conditions, the
Dr. H. Huang, Prof. Y. Li, Dr. Z. Sa, Y. Sun, Y. Wang, Prof. J. Wang Alan G. MacDiarmid Institute of Jilin University, 2699 Qianjin Street, Changchun 130012, China E-mail: [email protected] a Supporting Information is available online from the Wiley Online Library or from the author.
Macromol. Biosci. 2014, 14, 485–490 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
novel polymer micelles based on amphiphilic environment–responsive polymers can enhance the delivery efficiency of anticancer drugs, genetic drugs, and imaging agents.[5–8] Among the environment–responsive polymers, the smart pH-sensitive polymers, sensitive to intracellur environment of tumor cells, are especially attactive in the drug delivery field which requires selective controlled release.[9,10] pH-Sensitive polymers are so well developed that it can incorporate hydrazone,[11] ortho ester,[12] and acetal[13] into it as its acid labile linkage, with which drug release can be achieved in the endosomal component (pH 4–6). In particular, among those linkages above, the hydrazone linker are interested by researchers because of the high breaking effciency.[14] Although a selective degradation has
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been showed in the endosome, the process of drug release was relatively passive and slow,[6] because without uptake selection, inspite of EPR effect,[15] the drugs-loaded micelles would be endocytosed by both normal cells and cancer cells during the circulation before it was released in the endosomal of cancer cells. Therefore, in order to achieve better diagnosis and therapy, targeting ligands, such as peptides, antibodies, and aptamers, which can specifically bind to the receptors overexpressed only in cancer cells, have been popularly tethered on the surface of the smart delivery system to deliver the drugs to the tumor sites.[16–18] However, there also exist certain disadvantages for targeting ligands, including high cost, short storage time, and low conjugation efficiency. As one of the alternative approaches, chargeconversional polymeric micelles in response to a small pH drop have been developed for anti-cancer drug, genetic Scheme 1. Schematic illustration of the process of enhanced intracellular delivery by drugs, imaging agents.[19,20] The enhanced sensing extracellular pH and drug release by sensing intracellular pH. intracellular delivery shows a great potendelivery systems obtain better therapeutic effect because tial for cancer therapy and tumor imaging. of the drug release efficiency. Therefore, the dual-responCitraconic anhydride is an a-methyl derivative of maleic sive drug release system has a wide application. In this anhydride, which can be used to enhance tumor targeting study, as is shown in Scheme 1, an amphiphilic polymer– therapy efficiency.[6,20] As shown in Scheme S1, amide drug conjugate was synthesized via hydrazone linker with bonds are formed from the reaction between primary combtype side groups of the citraconic amide and their amines and citraconic anhydride. The resulting amides physicochemical properties, including the degree of degradhave negative charges owing to the carboxylated groups at ability and the charge conversion were characterized. The the end.[6] The citraconic amide is stable at both neutral smart drug delivery system combined two distinct advanand basic pH, but at acidic pH it becomes unstable and tages of citraconic amide and hydrazone linker, extracellular promptly degrades back into the cationic primary amine. pH-triggered tumor targeting of citraconic amide, and So when in the acidic environment, the polymer could intracellular pH-triggered drug release of hydrazone linker. achieve charge reversal from negative to positive by With efficient tumor targeting and controlled drug release, it cleavage of citraconic amide linkage. It is well known is obvious that this system would be of great potential for that human and rodent solid tumor tissues exhibit a more cancer therapy. acidic microenvironment than normal tissues, and their extracellular pH ranges from 5.8 to 7.4,[21,22] where the polymer would be changed from negative to positve. With positive charges, the cationic system could be combined 2. Results and Discussion with the anionic tumor cell membrane. Thus, this chargeconversion in response to low pH drug delivery system 2.1. Citraconylation Reaction of Polymer Drug could increase the extent of cellular uptake only at the Conjugate [6,21] acidic tumor region. The sythesis of the conjugate p(aspartate)-graft-p(ethylene In recent years, researchers have provided dualglycol)-dodecylamine-(hydrazone-doxorubicin)-(ethyleneresponsive drug delivery system based on charge-converdiamine-citraconic amide) PASP-g-PEG-DDA-(Hyd-DOX)sion polymers and disulfide group. The methods included: (EDA-Cit) is illustrated in Scheme S1. The details of synthesis i) charge-conversion polymer drug conjugate based on were described in the Supporting Information. After disulfide group;[23] ii) drug-loaded dual-responsive crosscitraconylation reaction for 2 h at various molar ratios of linked polymer micelles.[24] The dual-responsive drug
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A Smart Drug Delivery System from Charge-Conversion Polymer–Drug Conjugate . . . www.mbs-journal.de
citraconic anhydride/amine, the amount of remaining aminegroup in the PASP conjugate was measured by fluorescamine assay. As shown in Figure 1A, 60% of total amine groups in the PASP conjugate was modified at the citraconic anhydride/amine molar ratio of 0.7, suggesting that the charge of conjugate under that condition was slightly negative. The negative micelles may prevent nonspecific interactions with negatively charged serum components from forming thrombi in the capillary.[20] Therefore, the conjugate micelles were very steady in the long body circulation. 2.2. Degradation of Citraconic Amide in the Conjugate To show the charge regeneration of primary amine groups in the conjugate at different pH conditions, the amount of primary amine groups was quantitatively analyzed by fluorescamine assay after incubating at different pH conditions (Figure 1B). When the citraconylated conjugate was incubated at pH 6.6 and 7.4. The relative amounts of exposed amine groups were 72 and 42%, respectively, after 5 h incubation. Compared with that at pH 7.4, the extent of regenerated amine group was significantly increased at pH
6.6 in a time-dependent manner, indicating that only in the acidic conditions was the net charge in the conjugate recovered from negative to positive. The zeta-potential data also proved the presence of charge-conversion (Figure 1C). As is known, the micelles can be enriched in the tumor region by EPR effect. After enriched in the tumor region whose extracellular pH is almost ranged from 5.8 to 6.8, the charge of micelles would change from negative to positive by the charge-conversion property in response to extracellular environment of tumor cells. The intracellular delivery of micelles would be enhanced by the electrostatic interaction between micelles and cell membrane. This targeting endocytosis process could achieve better diagnosis and therapy. 2.3. Drug Release Analysis of the Conjugate Drug release curve in Figure 1D showed that the drug release content was obviously high at the tumor intracellur pH, while the drug release content was very low at both the tumor extracellur pH and neutral pH. Therefore, the conjugate micelles were very steady in the long body circulation suggesting no initial burst release and relatively low side effect. After the endocytosis
Figure 1. A) The relative percent of primary amine after reaction by citraconic anhydride at various citraconic anhydride/amine molar ratios. B) The relative percent of remaining amine group in the PASP-g-PEG-DDA-(Hyd-DOX)-(EDA-Cit) after incubation at pH 6.6, and pH 7.4 at 37 8C as a function of incubation time. The error bars mean standard deviation. C) The z-potential of PASP-g-PEG-DDA-(Hyd-DOX)-(EDA-Cit) after incubation at pH 6.6, and pH 7.4 at 37 8C as a function of incubation time. The error bars mean standard deviation. The error bars mean standard deviation. D) Cumulative DOX release from the micelles to a series of buffer solutions (pH 7.4–5.0). E) AFM imaging of the conjugate micelles. F) The hydrodynamic diameters as a function of storage time at 37 8C for the conjugate micelles in PBS buffer (pH 7.4 and 6.6).
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process, the micelles were trapped in the endocytic vesicles, under which condition (pH 4–6) DOX could be released efficiently.
reduction in the repulsive forces due to the partial charge neutralization. 2.6. Confocal Laser Scanningmicroscopy Analysis
2.4. Characterization of the Conjugate-Drug Micelles The process of forming micelles by the citraconylated conjugate was shown supporting information. Dynamic light scattering (DLS) measurement showed that the micelles had a hydrodynamic size of 60 nm. AFM imaging showed the morphology of the nanoparticles was uniformly spherical with a length size of 52 nm in Figure 1E. 2.5. Stability of the Conjugate-Drug Micelles As shown in Figure 1F, the conjugate micelles maintained a diameter of around 60 nm at pH 7.4, but there was an increase in its size at pH 6.6. After 3 h, the diameter was up to 110 nm. The reason for the aggregation is probably the
After incubating for 3 h in buffer solutions of pH values 6.6 and pH 7.4, the micelles were treated to HepG2 cells to show intracellular uptake extent of the DOX. After 3 h incubation, cellular images visualized by confocal microscopy revealed that the micelles incubated at pH 6.6 (Figure 2C) was more significantly taken up by the cells than those at pH 7.4 (Figure 2A). It is obvious that the charge of the surface-anchored citraconylated conjugate was reversed from negative to positive, thereby facilitating nonspecific endocytic uptake of micelles by increasing their charge interactions with the cell membrane. After the fresh medium replaced the medium with conjugate, the cells continued to be cultured until the accumulation time reached 24 h. The significant increase in fluorescence intensity in Figure 2D proved that DOX could be released
Figure 2. Confocal microscopy images of intracellular micelles with citraconic amide (dosage: 10 mg DOX equiv. mL 1). For each panel, the images from left to right show cell nuclei stained by DAPI (blue), DOX fluorescence in cells (red), and overlays of the two images. The scale bars correspond to 20 mm in all the images. A) 3 h incubation, pH 7.4; B) 24 h incubation, pH 7.4; C) 3 h incubation, pH 6.6; D) 24 h incubation, pH 6.6.
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Figure 3. Cytotoxicity assay results on HepG2 with different DOX concentrations and different incubation conditions (pH 7.4 and 6.6). DOX concentration of medium range from 0.01–100 mg mL 1.
from the micelles when the conjugate respond to the intracellular pH. 2.7. Analysis of the Cellular Uptake of the Conjugate In order to confirm our hypothesis that the conjugate micelles can be taken up by cancer cells and release DOX, flow cytometric (FCM) analysis was carried out by changing the exposure conditions. The increasing fluorescence of the cells was monitored. As shown in Figure 3A, the fluorescence intensity of the conjugate incubated at pH 6.6 was much higher than that of at pH 7.4. After 24 h incubation, the fluorescence intensity increased further. This phenomenon could prove that DOX released from the micelles due to the disappearance of the characteristic fluorescence quenching effects of the micelles. 2.8. Cytotoxicity In Vitro We also measured the cytotoxicity in vitro of conjugate micelles, which were incubated for 3 h in buffer solutions of pH values 6.6 and pH 7.4. MTT results are shown in Figure 3B. After 24 h incubation, the cell viability of polymer–drug micelles incubated in buffer solutions of pH values 6.6 decreased much more significantly than that incubated in buffer solutions of pH values 7.4 and was close to free DOX. As time goes on, it is noted that the cell viability of conjugate incubated in buffer solution of pH values 6.6 decreased significantly. This phenomenon proved that the intracellular released DOX play a leading role in the antitumor process. As described above, the micelles were designed to enhance tumor targeting therapy and tunable drug release, and therefore the difference in cytotoxicity indicates that
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a charge-conversion effect should be the reason for the enhanced efficacy of the extracellular environmentsensitive polymeric micelles. After intracellular delivery of conjugate micelles, the drug released from the micelles rapidly because that the intracellular pH-sensitive hydrazone bonds would broken.
3. Conclusion A novel smart drug delivery system based on amphiphilic polymer–drug conjugate via hydrazone linker with combtype side groups of the citraconic amide was demonstrated, exhibiting surface charge reversal at tumor extracellular pH and high drug release efficiency at tumor extracellular pH. Therefore, it was able to avoid the initial burst drug release and drug loss in the blood circulation, and at the same time drug can be delivered into tumor cells directly. This novel extracellular and intracellular pH-sensitive delivery system can be expected to deliver other therapeutic agents to solid tumors. The conjugate micelles can also deliver imaging agents to the tumor sites for cancer diagnosis because of the special amphiphilic strcuture of the polymers.
Acknowledgements: This work is financially supported by the Natural Science Foundation of China (No. 20080119), the Science foundation for youths of Jilin Committee of Science and Technology (No. 20904014), the Fund for Doctoral Program of Chinese Ministry of Education (No. 20090061110017) and the Fund for youths of Chinese Ministry of Education (No. 2070310).
Received: July 19, 2013; Revised: October 8, 2013; Published online: DOI: 10.1002/mabi.201300337
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Keywords: amphiphiles; biomaterials; charge transfer; conjugated polymers; drug delivery systems
[1] S. Deka, A. Quarta, R. Corato, A. Riedinger, R. Cingolani, T. Pellegrino, Nanoscale 2011, 3, 619. [2] Y. Pang, Q. Zhu, D. Zhou, J. Liu, Y. Chen, Y. Su, J Polym. Sci., Part A: Polym. Chem. 2011, 49, 966. [3] C. Chuang, T. Don, W. Chiu, J. Polym. Sci., Part A: Polym. Chem. 2010, 48, 2377. € ller, Macromol. Rapid Commun. [4] X. Andr e, M. Zhang, A. Mu 2005, 26, 558. [5] K. T. Oh, H. Yin, E. S. Lee, Y. H. Bae, J. Mater. Chem. 2007, 17, 3987. [6] Y. Lee, S. Fukushima, Y. Bae, S. Hiki, T. Ishii, K. Kataoka, J. Am. Chem. Soc. 2007, 129, 5362. [7] A. S. Mikhail, C. Allen, Biomacromolecules 2010, 11, 1273. [8] K. Shirashi, K. Kawano, T. Minowa, Y. Maitani, M. Yokoyama, J. Controlled Release 2009, 136, 14. [9] Y. Bae, S. Fukushima, A. Harada, K. Kataoka, Angew. Chem. Int. Ed. 2003, 42, 4640. [10] M. D. Howard, A. Ponta, A. Echman, M. Jay, Y. Bae, Pharm. Res. 2011, 28, 2435. [11] Y. Bae, N. Nishiyama, S. Fukushima, H. Koyama, M. Yasuhiro, K. Kataoka, Bioconjug. Chem. 2005, 16, 122.
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[12] H. Sun, B. Guo, X. Li, R. Cheng, F. Meng, H. Liu, Biomacromolecules 2010, 11, 848. [13] Y. Chan, V. Bulmus, M. Hadi Zareie, F. Byrne, L. Barner, M. Kavallaris, J. Controlled Release 2006, 115, 848. [14] Y. Bae, W. Jang, N. Nishiyama, S. Fukushima, K. Kataoka, Mol. Biosyst. 2005, 1, 242. [15] H. Maeda, Y. Matsumura, Crit. Rev. Ther. Drug Carrier Syst. 1989, 6, 193. [16] G. Kong, R. D. Braun, M. W. Dewhirst, Cancer Res. 2000, 60, 4440. [17] F. Krotz, C. Wit, H. Y. Sohn, S. Zahler, T. Gloe, U. Pohl, C. Plank, Mol. Ther. 2003, 7, 700. [18] D. Schmaljohann, Adv. Drug Delivery Rev. 2006, 7, 700. [19] K. Zhang, W. Yang, D. Wang, C. Liu, L. Qi, Y. Wang, J. Controlled Release 2011, 152, 95. [20] Y. Lee, K. Miyata, M. Oba, T. Ishii, S. Fukushima, M. Han, H. Koyama, N. Nishiyama, K. Kataoka, Angew. Chem. 2008, 120, 5241. [21] H. Mok, J. W. Park, T. G. Park, Bioconjug. Chem. 2008, 19, 797. [22] I. F. Tannock, D. Rotin, Cancer Res. 1989, 49, 4373. [23] Z. Zhou, Y. Shen, J. Tang, M. Fan, Adv. Funct. Mater. 2009, 19, 3580. [24] L. Wu, Y. Zou, C. Deng, R. Cheng, F. Meng, Z. Zhong, Biomaterials 2013, 34, 5262.
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A Smart Drug Delivery System from Charge-Conversion Polymer–Drug Conjugate for Enhancing Tumor Therapy and Tunable Drug Releasea Hailong Huang, Yapeng Li,* Zongpeng Sa, Yuan Sun, Yuzhen Wang, Jingyuan Wang
A smart drug delivery system is prepared by citraconylated polyaspartic acid (PASP) derivate–drug conjugate. The conjugate contains two pH-sensitive groups: citraconic amide and hydrazone linker. Citraconic amide group can enhance tumor therapy efficiency by the extracellular pH-sensitive charge-conversion property. Hydrazone linker between polymer and drug can cleave efficiently in the intracellular pH environment. The resulting conjugate shows dual-pH sensitive properties: extracellular pH-triggered enhanced tumor targeting and intracellular pH-triggered drug release. The results of physicochemical properties, intracellular location, and cytotoxicity of conjugate micelles demonstrate that this novel smart drug delivery system can enhance intracellular delivery of drug at a low pH and then release drug rapidly.
1. Introduction In recent years, smart amphiphilic polymers have been developed and have attracted much attention because of their environment–responsive properties including pH, temperature, ionic strength, light, photochemical process.[1–4] Under appropriate environment conditions, the
Dr. H. Huang, Prof. Y. Li, Dr. Z. Sa, Y. Sun, Y. Wang, Prof. J. Wang Alan G. MacDiarmid Institute of Jilin University, 2699 Qianjin Street, Changchun 130012, China E-mail: [email protected] a Supporting Information is available online from the Wiley Online Library or from the author.
Macromol. Biosci. 2014, 14, 485–490 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
novel polymer micelles based on amphiphilic environment–responsive polymers can enhance the delivery efficiency of anticancer drugs, genetic drugs, and imaging agents.[5–8] Among the environment–responsive polymers, the smart pH-sensitive polymers, sensitive to intracellur environment of tumor cells, are especially attactive in the drug delivery field which requires selective controlled release.[9,10] pH-Sensitive polymers are so well developed that it can incorporate hydrazone,[11] ortho ester,[12] and acetal[13] into it as its acid labile linkage, with which drug release can be achieved in the endosomal component (pH 4–6). In particular, among those linkages above, the hydrazone linker are interested by researchers because of the high breaking effciency.[14] Although a selective degradation has
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been showed in the endosome, the process of drug release was relatively passive and slow,[6] because without uptake selection, inspite of EPR effect,[15] the drugs-loaded micelles would be endocytosed by both normal cells and cancer cells during the circulation before it was released in the endosomal of cancer cells. Therefore, in order to achieve better diagnosis and therapy, targeting ligands, such as peptides, antibodies, and aptamers, which can specifically bind to the receptors overexpressed only in cancer cells, have been popularly tethered on the surface of the smart delivery system to deliver the drugs to the tumor sites.[16–18] However, there also exist certain disadvantages for targeting ligands, including high cost, short storage time, and low conjugation efficiency. As one of the alternative approaches, chargeconversional polymeric micelles in response to a small pH drop have been developed for anti-cancer drug, genetic Scheme 1. Schematic illustration of the process of enhanced intracellular delivery by drugs, imaging agents.[19,20] The enhanced sensing extracellular pH and drug release by sensing intracellular pH. intracellular delivery shows a great potendelivery systems obtain better therapeutic effect because tial for cancer therapy and tumor imaging. of the drug release efficiency. Therefore, the dual-responCitraconic anhydride is an a-methyl derivative of maleic sive drug release system has a wide application. In this anhydride, which can be used to enhance tumor targeting study, as is shown in Scheme 1, an amphiphilic polymer– therapy efficiency.[6,20] As shown in Scheme S1, amide drug conjugate was synthesized via hydrazone linker with bonds are formed from the reaction between primary combtype side groups of the citraconic amide and their amines and citraconic anhydride. The resulting amides physicochemical properties, including the degree of degradhave negative charges owing to the carboxylated groups at ability and the charge conversion were characterized. The the end.[6] The citraconic amide is stable at both neutral smart drug delivery system combined two distinct advanand basic pH, but at acidic pH it becomes unstable and tages of citraconic amide and hydrazone linker, extracellular promptly degrades back into the cationic primary amine. pH-triggered tumor targeting of citraconic amide, and So when in the acidic environment, the polymer could intracellular pH-triggered drug release of hydrazone linker. achieve charge reversal from negative to positive by With efficient tumor targeting and controlled drug release, it cleavage of citraconic amide linkage. It is well known is obvious that this system would be of great potential for that human and rodent solid tumor tissues exhibit a more cancer therapy. acidic microenvironment than normal tissues, and their extracellular pH ranges from 5.8 to 7.4,[21,22] where the polymer would be changed from negative to positve. With positive charges, the cationic system could be combined 2. Results and Discussion with the anionic tumor cell membrane. Thus, this chargeconversion in response to low pH drug delivery system 2.1. Citraconylation Reaction of Polymer Drug could increase the extent of cellular uptake only at the Conjugate [6,21] acidic tumor region. The sythesis of the conjugate p(aspartate)-graft-p(ethylene In recent years, researchers have provided dualglycol)-dodecylamine-(hydrazone-doxorubicin)-(ethyleneresponsive drug delivery system based on charge-converdiamine-citraconic amide) PASP-g-PEG-DDA-(Hyd-DOX)sion polymers and disulfide group. The methods included: (EDA-Cit) is illustrated in Scheme S1. The details of synthesis i) charge-conversion polymer drug conjugate based on were described in the Supporting Information. After disulfide group;[23] ii) drug-loaded dual-responsive crosscitraconylation reaction for 2 h at various molar ratios of linked polymer micelles.[24] The dual-responsive drug
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citraconic anhydride/amine, the amount of remaining aminegroup in the PASP conjugate was measured by fluorescamine assay. As shown in Figure 1A, 60% of total amine groups in the PASP conjugate was modified at the citraconic anhydride/amine molar ratio of 0.7, suggesting that the charge of conjugate under that condition was slightly negative. The negative micelles may prevent nonspecific interactions with negatively charged serum components from forming thrombi in the capillary.[20] Therefore, the conjugate micelles were very steady in the long body circulation. 2.2. Degradation of Citraconic Amide in the Conjugate To show the charge regeneration of primary amine groups in the conjugate at different pH conditions, the amount of primary amine groups was quantitatively analyzed by fluorescamine assay after incubating at different pH conditions (Figure 1B). When the citraconylated conjugate was incubated at pH 6.6 and 7.4. The relative amounts of exposed amine groups were 72 and 42%, respectively, after 5 h incubation. Compared with that at pH 7.4, the extent of regenerated amine group was significantly increased at pH
6.6 in a time-dependent manner, indicating that only in the acidic conditions was the net charge in the conjugate recovered from negative to positive. The zeta-potential data also proved the presence of charge-conversion (Figure 1C). As is known, the micelles can be enriched in the tumor region by EPR effect. After enriched in the tumor region whose extracellular pH is almost ranged from 5.8 to 6.8, the charge of micelles would change from negative to positive by the charge-conversion property in response to extracellular environment of tumor cells. The intracellular delivery of micelles would be enhanced by the electrostatic interaction between micelles and cell membrane. This targeting endocytosis process could achieve better diagnosis and therapy. 2.3. Drug Release Analysis of the Conjugate Drug release curve in Figure 1D showed that the drug release content was obviously high at the tumor intracellur pH, while the drug release content was very low at both the tumor extracellur pH and neutral pH. Therefore, the conjugate micelles were very steady in the long body circulation suggesting no initial burst release and relatively low side effect. After the endocytosis
Figure 1. A) The relative percent of primary amine after reaction by citraconic anhydride at various citraconic anhydride/amine molar ratios. B) The relative percent of remaining amine group in the PASP-g-PEG-DDA-(Hyd-DOX)-(EDA-Cit) after incubation at pH 6.6, and pH 7.4 at 37 8C as a function of incubation time. The error bars mean standard deviation. C) The z-potential of PASP-g-PEG-DDA-(Hyd-DOX)-(EDA-Cit) after incubation at pH 6.6, and pH 7.4 at 37 8C as a function of incubation time. The error bars mean standard deviation. The error bars mean standard deviation. D) Cumulative DOX release from the micelles to a series of buffer solutions (pH 7.4–5.0). E) AFM imaging of the conjugate micelles. F) The hydrodynamic diameters as a function of storage time at 37 8C for the conjugate micelles in PBS buffer (pH 7.4 and 6.6).
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process, the micelles were trapped in the endocytic vesicles, under which condition (pH 4–6) DOX could be released efficiently.
reduction in the repulsive forces due to the partial charge neutralization. 2.6. Confocal Laser Scanningmicroscopy Analysis
2.4. Characterization of the Conjugate-Drug Micelles The process of forming micelles by the citraconylated conjugate was shown supporting information. Dynamic light scattering (DLS) measurement showed that the micelles had a hydrodynamic size of 60 nm. AFM imaging showed the morphology of the nanoparticles was uniformly spherical with a length size of 52 nm in Figure 1E. 2.5. Stability of the Conjugate-Drug Micelles As shown in Figure 1F, the conjugate micelles maintained a diameter of around 60 nm at pH 7.4, but there was an increase in its size at pH 6.6. After 3 h, the diameter was up to 110 nm. The reason for the aggregation is probably the
After incubating for 3 h in buffer solutions of pH values 6.6 and pH 7.4, the micelles were treated to HepG2 cells to show intracellular uptake extent of the DOX. After 3 h incubation, cellular images visualized by confocal microscopy revealed that the micelles incubated at pH 6.6 (Figure 2C) was more significantly taken up by the cells than those at pH 7.4 (Figure 2A). It is obvious that the charge of the surface-anchored citraconylated conjugate was reversed from negative to positive, thereby facilitating nonspecific endocytic uptake of micelles by increasing their charge interactions with the cell membrane. After the fresh medium replaced the medium with conjugate, the cells continued to be cultured until the accumulation time reached 24 h. The significant increase in fluorescence intensity in Figure 2D proved that DOX could be released
Figure 2. Confocal microscopy images of intracellular micelles with citraconic amide (dosage: 10 mg DOX equiv. mL 1). For each panel, the images from left to right show cell nuclei stained by DAPI (blue), DOX fluorescence in cells (red), and overlays of the two images. The scale bars correspond to 20 mm in all the images. A) 3 h incubation, pH 7.4; B) 24 h incubation, pH 7.4; C) 3 h incubation, pH 6.6; D) 24 h incubation, pH 6.6.
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Figure 3. Cytotoxicity assay results on HepG2 with different DOX concentrations and different incubation conditions (pH 7.4 and 6.6). DOX concentration of medium range from 0.01–100 mg mL 1.
from the micelles when the conjugate respond to the intracellular pH. 2.7. Analysis of the Cellular Uptake of the Conjugate In order to confirm our hypothesis that the conjugate micelles can be taken up by cancer cells and release DOX, flow cytometric (FCM) analysis was carried out by changing the exposure conditions. The increasing fluorescence of the cells was monitored. As shown in Figure 3A, the fluorescence intensity of the conjugate incubated at pH 6.6 was much higher than that of at pH 7.4. After 24 h incubation, the fluorescence intensity increased further. This phenomenon could prove that DOX released from the micelles due to the disappearance of the characteristic fluorescence quenching effects of the micelles. 2.8. Cytotoxicity In Vitro We also measured the cytotoxicity in vitro of conjugate micelles, which were incubated for 3 h in buffer solutions of pH values 6.6 and pH 7.4. MTT results are shown in Figure 3B. After 24 h incubation, the cell viability of polymer–drug micelles incubated in buffer solutions of pH values 6.6 decreased much more significantly than that incubated in buffer solutions of pH values 7.4 and was close to free DOX. As time goes on, it is noted that the cell viability of conjugate incubated in buffer solution of pH values 6.6 decreased significantly. This phenomenon proved that the intracellular released DOX play a leading role in the antitumor process. As described above, the micelles were designed to enhance tumor targeting therapy and tunable drug release, and therefore the difference in cytotoxicity indicates that
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a charge-conversion effect should be the reason for the enhanced efficacy of the extracellular environmentsensitive polymeric micelles. After intracellular delivery of conjugate micelles, the drug released from the micelles rapidly because that the intracellular pH-sensitive hydrazone bonds would broken.
3. Conclusion A novel smart drug delivery system based on amphiphilic polymer–drug conjugate via hydrazone linker with combtype side groups of the citraconic amide was demonstrated, exhibiting surface charge reversal at tumor extracellular pH and high drug release efficiency at tumor extracellular pH. Therefore, it was able to avoid the initial burst drug release and drug loss in the blood circulation, and at the same time drug can be delivered into tumor cells directly. This novel extracellular and intracellular pH-sensitive delivery system can be expected to deliver other therapeutic agents to solid tumors. The conjugate micelles can also deliver imaging agents to the tumor sites for cancer diagnosis because of the special amphiphilic strcuture of the polymers.
Acknowledgements: This work is financially supported by the Natural Science Foundation of China (No. 20080119), the Science foundation for youths of Jilin Committee of Science and Technology (No. 20904014), the Fund for Doctoral Program of Chinese Ministry of Education (No. 20090061110017) and the Fund for youths of Chinese Ministry of Education (No. 2070310).
Received: July 19, 2013; Revised: October 8, 2013; Published online: DOI: 10.1002/mabi.201300337
Macromol. Biosci. 2014, 14, 485–490 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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H. Huang, Y. Li, Z. Sa, Y. Sun, Y. Wang, J. Wang www.mbs-journal.de
Keywords: amphiphiles; biomaterials; charge transfer; conjugated polymers; drug delivery systems
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Macromol. Biosci. 2014, 14, 485–490 ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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