ChemComm View Article Online
Published on 07 October 2014. Downloaded by University of Utah on 29/11/2014 04:20:16.
COMMUNICATION
Cite this: DOI: 10.1039/c4cc07563c Received 25th September 2014, Accepted 7th October 2014
View Journal
MMP-2 responsive polymeric micelles for cancer-targeted intracellular drug delivery† Wei-Hai Chen, Guo-Feng Luo, Qi Lei, Hui-Zhen Jia, Sheng Hong, Qing-Rong Wang,* Ren-Xi Zhuo and Xian-Zheng Zhang*
DOI: 10.1039/c4cc07563c www.rsc.org/chemcomm
Multifunctional
Biotin-PEG-b-PLL(Mal)-peptide-DOX
polymeric
micelles were prepared to selectively eliminate cancer cells. The micelles were able to enhance cancer cell uptake via the receptormediated endocytosis and respond to the stimulus of cancer cell excessive secreted protease MMP-2 to release the anticancer drug and induce apoptosis of cancer cells in a targeted manner.
In traditional cancer chemotherapy, anticancer drugs always lead to undesirable side effects and inevitable systemic toxicity.1 To overcome these limitations, great research effort has been made to design ideal drug carriers to improve the anticancer therapeutic efficacy with reduced side effects of anticancer drugs.2 Of particular significance, nanoscale polymeric micelles were demonstrated to show great potential as drug carriers due to their remarkable advantages, such as enhancing the solubility and bioavailability of drugs, improving pharmacokinetics and biodistribution profiles and increasing the accumulation at cancer sites via the passive enhanced permeability and retention (EPR) effects.3 However, the EPR effect could only enhance the aggregation of drug-loaded micelles in tumor tissues, while the insufficient cellular internalization as well as inefficient intracellular drug release always led to the available drug dosages to be below the therapeutic window and the therapeutic efficacy of cancer chemotherapy was much restricted.4 To address these challenges, it is desired to design the nanoscale micelles with the capability of recognizing particular cancer cells. In this regard, cancer targeting ligands were introduced to generate specific interaction between micelles and bio-markers overexpressed on cancer cells for enhancing the uptake of drug-loaded micelles by cancer cells.5 On the other hand, a stimuli-responsive strategy was also widely used and the intelligent drug-loaded micelles could respond to Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China. E-mail: [email protected], [email protected]; Fax: +86 27 6875 4509; Tel: +86 27 6875 5993 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4cc07563c
This journal is © The Royal Society of Chemistry 2014
intracellular or external stimuli, including pH, photo-irradiation or enzymes etc. with the controllable drug release behavior.6 Particularly, an enzyme-triggered reaction presents unique properties with high speed and specificity, and enzyme-responsive drug delivery systems exhibit on-demand release of the therapeutic agents. For instance, the ubiquitously overexpressed cathepsin B was used to cleave the peptide linkage to release the active drug and kill cancer cells in a targeted manner.7 Here, a new type of multifunctional polymeric micelles with the ability to release the anticancer drug in a targeted manner into cancer cells was designed. As shown in Scheme 1, the multifunctional micelles were constructed by using poly(ethylene glycol)blocked-poly(L-lysine) (PEG-b-PLL) as the polymer backbone, biotin
Scheme 1 Schematic illustration of the formation of nanoscale polymeric micelles and cancer-targeted intracellular drug delivery. (A) The amphiphilic Biotin-PEG-b-PLL(Mal)-peptide-DOX self-assembled into nanoscale polymeric micelles. (B) The drug loaded polymeric micelles were uptaken by cancer cell via the active targeting strategy and the anticancer drug was rapidly released for cancer therapy upon the triggering of overexpressed MMP protease in cancer cell.
Chem. Commun.
View Article Online
Published on 07 October 2014. Downloaded by University of Utah on 29/11/2014 04:20:16.
Communication
as an active targeting ligand, while the anticancer drug, doxorubicin (DOX) was conjugated to the PLL segment by the protease sensitive peptide linkage (peptide-DOX) via Michael addition. PEG as the hydrophilic shell was used to stabilize the micelles and prolong the blood circulation time. It is known that biotin can increase the selective uptake of micelles by cancer cells via receptor-mediated endocytosis.8 Importantly, peptide-DOX will act as the hydrophobic core to promote the formation of core–shell structural micelles. In addition, the release of DOX can be controlled upon the triggering of the matrix metalloproteinases (MMPs) excessively secreted by cancer cells.9 The cancer-targeted intracellular drug delivery from the multifunctional polymeric micelles was investigated to eliminate cancer cell selectively and reduce side effects. As illustrated in Scheme S1 (ESI†), Biotin-PEG-b-PLL(Z) was first prepared by the ring-opening polymerization of Z-Lys-NCA under the initiation of Biotin-PEG-NH2 (Mw = 3400 g mol 1).10 The molecular weight of Biotin-PEG-b-PLL(Z) was detected to be 22 090 g mol 1 using gel permeation chromatography (GPC) (PDI = 1.5), which was consistent with the data (Mw = 19 370 g mol 1) calculated from 1H NMR spectrometry (Fig. S3, ESI†). After treatment with HBr/HAc, the resonance signals from the protons of the carbobenzyloxy (Z) group in the 1H NMR spectrometry disappeared (Fig. S4, ESI†), suggesting the complete removal of Z groups in Biotin-PEG-bPLL(Z) and the successful synthesis of Biotin-PEG-b-PLL. For bonding with the peptide substrate, Biotin-PEG-b-PLL(Mal) was obtained through decoration of Biotin-PEG-b-PLL with 6-maleimidocaproic acid via an amidation reaction. Furthermore, BiotinPEG-b-PLL(Mal)-peptide-DOX was prepared by grafting the MMP-2 sensitive peptide conjugated doxorubicin (Ac-CPLGLAGG-DOX, peptide-DOX) onto Biotin-PEG-b-PLL(Mal) via Michael addition. The successful synthesis of Biotin-PEG-b-PLL(Mal)-peptide-DOX was confirmed using 1H NMR spectroanalysis (Fig. S5, ESI†). It was found that the drug loading in Biotin-PEG-b-PLL(Mal)-peptide-DOX was about 5.7 wt% as determined using fluorescence spectroscopy. In this study, Biotin-PEG-b-PLL(Mal)-peptide-DOX consists of a water-soluble polymeric backbone Biotin-PEG-b-PLL and a water-insoluble peptide-DOX. The inherent amphiphilicity property of Biotin-PEG-b-PLL(Mal)-peptide-DOX offers an opportunity to self-assemble into core–shell micelles in water.11 The critical micelle concentration (CMC) of Biotin-PEG-b-PLL(Mal)peptide-DOX micelles was estimated using fluorescence spectra and the CMC was detected to be 3.47 10 2 mg mL 1 (Fig. S6, ESI†). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to study the morphology and size distribution of the assemblies. As shown in Fig. 1A, the polymeric micelles self-assembled from Biotin-PEG-b-PLL(Mal)-peptide-DOX showed a regular spherical shape and were well dispersed as individual nanoparticles. The mean diameter measured using TEM was about 50 nm, which was smaller than the hydrodynamic diameter measured using DLS (around 75.4 nm, PDI = 0.245) (Fig. 1C) due to the shrinkage of nanoparticles in the dry state for TEM observations. To investigate the stimulus-responsive property of micelles, the assembled polymeric micelles were incubated with MMP-2 protease. As shown in Fig. 1B, the size of polymeric micelles increased significantly with aggregates emerging and some micelles got disassembled after treatment
Chem. Commun.
ChemComm
Fig. 1 Characterizations of the polymeric micelles by TEM and DLS. (A) TEM image and (C) size distribution of Biotin-PEG-b-PLL(Mal)peptide-DOX micelles. (B) TEM image and (D) size distribution of BiotinPEG-b-PLL(Mal)-peptide-DOX micelles treated with MMP-2 protease for 6 h.
with MMP-2. A similar result was also obtained by DLS measurements and the hydrodynamic diameter increased to about 110.3 nm with a large PDI of 0.495 (Fig. 1D). Additionally, the zeta potential of polymeric micelles increased from 16.5 0.5 to 6.9 0.4 mV, showing the dissociation of micelles. Moreover, DLS measurements demonstrated that the polymeric micelles were very stable in the normal physiological conditions (Fig. S7, ESI†). These results are easily understood since the MMP-2 protease can specifically cleave the peptide linkage, leading to the disassembly of micelles and rapid release of DOX from Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles. In order to evaluate the MMP-2 sensitivity of Biotin-PEG-bPLL(Mal)-peptide-DOX micelles, the in vitro drug release ability with or without the MMP inhibitor was studied and compared. As shown in Fig. 2, Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles exhibited apparent sensitivity to MMP-2 to cleave the peptide linkage and release the drug rapidly. Approximately
Fig. 2 Release profile of Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles under different conditions (red line: in the presence of MMP-2, blue line: in the presence MMP-2 and MMP inhibitor, and dark gray line: only in the presence of TCNB buffer).
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 07 October 2014. Downloaded by University of Utah on 29/11/2014 04:20:16.
ChemComm
46.2% of DOX was released after incubation with MMP-2 for 6 h. In contrast, almost no drug was released after treating with MMP-2 together with the protease inhibitor for 72 h. Similarly, very little drug was released when the micelles were incubated with TCNB buffer. These results indicate that Biotin-PEG-bPLL(Mal)-peptide-DOX micelles have a desirable MMP selectivity, which can be used to control the drug release in cancer cells using the up-regulated MMP-2 protease. Furthermore, confocal laser scanning microscopy (CLSM) was used to investigate the protease induced DOX release in cancer cells. The Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles were incubated with SCC-7 (squamous cell carcinoma) cancerous cells, which are well known for their high MMP expression, and COS7 (African green monkey kidney fibroblast cells) normal cells were used as a control. As shown in Fig. 3A1, significant red fluorescence was observed in SCC-7 cells when treated with
Fig. 3 Confocal laser scanning microscopy (CLSM) images of SCC-7 cancer cells incubated with Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles for 3 h (A–A2), 6 h (B–B2), and 6 h for SCC-7 cancer cells pre-treated with the excess of biotin in advance (C–C2). (A–C) blue fluorescence images of nuclei; (A1–C1) red fluorescence images of DOX; (A2–C2) the merge images of blue and red fluorescence. The scale bar is 14 mm. Quantitative flow cytometry analysis of the cellular DOX red fluorescence in SCC-7 cancer cells (D) and COS7 normal cells (E). The cells without treatment (blank line), treating with Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles for 3 h (red line), 6 h (green line) and 6 h (blue line) for cells pre-treated with the excess of biotin in advance, respectively.
This journal is © The Royal Society of Chemistry 2014
Communication
micelles for 3 h, indicating that polymeric micelles were quickly uptaken by cancer cells. This was attributed to the active targeting ability of biotin in micelles and increase the uptake via the receptor-mediated endocytosis. In general, many therapeutic agents need to be localized in particular subcellular compartments to elicit their pharmacological actions.12 For instance, DOX and cisplatin cannot exert their therapeutic actions unless they enter into the nuclei.13 After being uptaken by cells, the up-regulated MMP-2 protease secreted by SCC-7 cancer cells can specifically cleave the peptide substrate and result in the release of anticancer drug DOX rapidly. It was found that the red fluorescence intensity of DOX increased with a prolonged incubation time, and DOX entering into the targeted site of nuclei was also detected after SCC-7 cells were incubated with micelles for 6 h (Fig. 3B1). However, when SCC-7 cells were pre-incubated with the excess biotin prior to treatment with the polymeric micelles, the fluorescence intensity of DOX in the cancer cells was dramatically reduced due to the blocking of the receptormediated endocytosis of biotin in the micelles (Fig. 3C1). In contrast, a very weak fluorescence signal was observed in COS7 normal cells all the time (Fig. S8, ESI†) due to the shortage of the biotin-receptor in COS7 cells. Moreover, flow cytometry analysis was used to investigate the cellular red fluorescence of DOX quantitatively. It was found that the mean fluorescence intensity (MFI) values in SCC-7 cancer cells were 14.9- and 6.2-fold higher than the one of COS7 normal cells when incubated with micelles for 3 h or 6 h, respectively (Fig. 3D and E and Fig. S9, ESI†). When the cells were pre-treated with an excess of biotin in advance, the cellular fluorescence of DOX was decreased dramatically (Fig. S9a, ESI†) due to the fact that the free biotin occupies the receptor in cancer cells, leading to the disability of biotin in the polymeric micelles. These results indicate that Biotin-PEG-b-PLL(Mal)peptide-DOX micelles not only have the ability to increase the uptake by cancer cells via the receptor-mediated endocytosis but also respond to the triggering of cancer cell excessive secretion MMP protease to release the anticancer drug. To evaluate the anticancer effect of the Biotin-PEG-b-PLL(Mal)peptide-DOX micelles, the cell viability was determined using an MTT assay and the results are displayed in Fig. 4. The SCC-7 cell viability was more than 75% after incubation with the polymeric micelles in the presence of the MMP inhibitor. However, the cell viability decreased significantly to 15% in the absence of the MMP
Fig. 4 The cell viability of SCC-7 cancer cells (A) and COS7 normal cells (B) after incubated with Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles in the absence or in the presence of MMP-2 inhibitor.
Chem. Commun.
View Article Online
Published on 07 October 2014. Downloaded by University of Utah on 29/11/2014 04:20:16.
Communication
inhibitor. In the presence of the MMP inhibitor, the peptide linkage cannot be cleaved and the conjugated drug cannot be released from the micelles. In contrast, without the MMP inhibitor, MMP protease could hydrolyze the peptide linkage to induce drug release as well as the apoptosis of cancer cells. However, as shown in Fig. 4B, the viability of COS7 normal cells incubated with Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles was more than 85% with or without the MMP inhibitor. This high cell viability of COS7 cells was attributed to the low uptake of polymeric micelles and the lack of a specific MMP protease. In addition, the polymer backbone Biotin-PEG-b-PLL did not exhibit an obvious cytotoxicity in all cell lines (Fig. S10, ESI†). These results confirm that the selective therapy to cancer cells of the multifunctional micelles can be achieved due to the drug release upon applying the stimulus of cancer cells excessive secreted protease. In summary, the preparation of multifunctional Biotin-PEGb-PLL(Mal)-peptide-DOX polymeric micelles was demonstrated, which can specially respond to physiopathological signals of cancer cells. The biotin ligand could promote cancer cell uptake via the receptor-mediated endocytosis. Upon application of the stimulus of the overexpressed MMP protease in cancer cells, the anticancer drug DOX could be released effectively, leading to significant apoptosis of cancer cells. The targeted therapy to cancer cells could be achieved with much reduced side effects to normal cells, which will find great potential in cancer therapy. This work was supported by the National Natural Science Foundation of China (51125014 and 51233003), the Ministry of Science and Technology of China (2011CB606202), the Ministry of Education of China (20120141130003) and supported by the Fundamental Research Funds for the Central Universities (2014203020201 and 2014203020204).
Chem. Commun.
ChemComm
Notes and references 1 A. I. Minchinton and I. F. Tannock, Nat. Rev. Cancer, 2006, 6, 583. 2 (a) M. Ferrari, Nat. Rev. Cancer, 2005, 5, 161; (b) D. Peer, J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit and R. Langer, Nat. Nanotechnol., 2007, 2, 751; (c) A. Mukerjee, A. P. Ranjan and J. K. Vishwanatha, Curr. Med. Chem., 2012, 19, 3714. 3 (a) P. Sharma and S. Garg, Adv. Drug Delivery Rev., 2010, 62, 491; (b) H. Wei, R. X. Zhuo and X. Z. Zhang, Prog. Polym. Sci., 2013, 38, 503; (c) Z. Ge and S. Liu, Chem. Soc. Rev., 2013, 42, 7289; (d) J. Ding, L. Chen, C. Xiao, L. Chen, X. Zhuang and X. Chen, Chem. Commun., 2014, 50, 11274. 4 (a) J. Z. Du, X. J. Du, C. Q. Mao and J. Wang, J. Am. Chem. Soc., 2011, 133, 17560; (b) W. Xu, J. F. Burke, S. Pilla, H. Chen, R. Jaskula-Sztul and S. Gong, Nanoscale, 2013, 5, 9924. 5 (a) J. Nicolas, S. Mura, D. Brambilla, N. Mackiewicz and P. Couvreur, Chem. Soc. Rev., 2013, 42, 1147; (b) A. Mahmud, X. B. Xiong, H. M. Aliabadi and A. Lavasanifar, J. Drug Targeting, 2007, 15, 553; (c) Y. Zou, Y. Song, W. Yang, F. Meng, H. Liu and Z. Zhong, J. Controlled Release, 2014, DOI: 10.1016/j.jconrel.2014.05.016; (d) W. H. Chen, X. D. Xu, G. F. Luo, H. Z. Jia, Q. Lei, S. X. Cheng, R. X. Zhuo and X. Z. Zhang, Sci. Rep., 2013, 3, 3468. 6 (a) J. Dai, S. Lin, D. Cheng, S. Zou and X. Shuai, Angew. Chem., Int. Ed., 2011, 50, 9404; (b) Z. Chu, C. A. Dreiss and Y. Feng, Chem. Soc. Rev., 2013, 42, 7174; (c) M. Nakayama, J. Akimoto and T. Okano, J. Drug Targeting, 2014, 22, 584; (d) S. Mura, J. Nicolas and P. Couvreur, Nat. Mater., 2013, 12, 991. 7 X. Tian, K. H. Baek and I. Shin, Chem. Sci., 2013, 4, 947. 8 S. Bhuniya, S. Maiti, E. J. Kim, H. Lee, J. L. Sessler, K. S. Hong and J. S. Kim, Angew. Chem., Int. Ed., 2014, 53, 4469. ¨ha ¨ri, Curr. Cancer Drug 9 (a) P. Vihinen, R. Ala-aho and V. M. Ka Targets, 2005, 5, 203; (b) L. Aureli, M. Gioia, I. Cerbara, S. Monaco, G. F. Fasciglione, S. Marini, P. Ascenzi, A. Topai and M. Coletta, Curr. Med. Chem., 2008, 15, 2192. 10 (a) K. Wang, G. F. Luo, Y. Liu, C. Li, S. X. Cheng, R. X. Zhuo and X. Z. Zhang, Polym. Chem., 2012, 3, 1084; (b) B. Yang, Y. Lv, J. Y. Zhu, Y. T. Han, H. Z. Jia, W. H. Chen, J. Feng, X. Z. Zhang and R. X. Zhuo, Acta Biomater., 2014, 10, 3686. 11 P. Huang, D. Wang, Y. Su, W. Huang, Y. Zhou, D. Cui, X. Zhu and D. Yan, J. Am. Chem. Soc., 2014, 136, 11748. ¨lker and K. Simons, Nat. Rev. Drug Discovery, 12 L. Rajendran, H. J. Kno 2010, 9, 29. 13 L. Pan, Q. He, J. Liu, Y. Chen, M. Ma, L. Zhang and J. Shi, J. Am. Chem. Soc., 2012, 134, 5722.
This journal is © The Royal Society of Chemistry 2014
Published on 07 October 2014. Downloaded by University of Utah on 29/11/2014 04:20:16.
COMMUNICATION
Cite this: DOI: 10.1039/c4cc07563c Received 25th September 2014, Accepted 7th October 2014
View Journal
MMP-2 responsive polymeric micelles for cancer-targeted intracellular drug delivery† Wei-Hai Chen, Guo-Feng Luo, Qi Lei, Hui-Zhen Jia, Sheng Hong, Qing-Rong Wang,* Ren-Xi Zhuo and Xian-Zheng Zhang*
DOI: 10.1039/c4cc07563c www.rsc.org/chemcomm
Multifunctional
Biotin-PEG-b-PLL(Mal)-peptide-DOX
polymeric
micelles were prepared to selectively eliminate cancer cells. The micelles were able to enhance cancer cell uptake via the receptormediated endocytosis and respond to the stimulus of cancer cell excessive secreted protease MMP-2 to release the anticancer drug and induce apoptosis of cancer cells in a targeted manner.
In traditional cancer chemotherapy, anticancer drugs always lead to undesirable side effects and inevitable systemic toxicity.1 To overcome these limitations, great research effort has been made to design ideal drug carriers to improve the anticancer therapeutic efficacy with reduced side effects of anticancer drugs.2 Of particular significance, nanoscale polymeric micelles were demonstrated to show great potential as drug carriers due to their remarkable advantages, such as enhancing the solubility and bioavailability of drugs, improving pharmacokinetics and biodistribution profiles and increasing the accumulation at cancer sites via the passive enhanced permeability and retention (EPR) effects.3 However, the EPR effect could only enhance the aggregation of drug-loaded micelles in tumor tissues, while the insufficient cellular internalization as well as inefficient intracellular drug release always led to the available drug dosages to be below the therapeutic window and the therapeutic efficacy of cancer chemotherapy was much restricted.4 To address these challenges, it is desired to design the nanoscale micelles with the capability of recognizing particular cancer cells. In this regard, cancer targeting ligands were introduced to generate specific interaction between micelles and bio-markers overexpressed on cancer cells for enhancing the uptake of drug-loaded micelles by cancer cells.5 On the other hand, a stimuli-responsive strategy was also widely used and the intelligent drug-loaded micelles could respond to Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China. E-mail: [email protected], [email protected]; Fax: +86 27 6875 4509; Tel: +86 27 6875 5993 † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c4cc07563c
This journal is © The Royal Society of Chemistry 2014
intracellular or external stimuli, including pH, photo-irradiation or enzymes etc. with the controllable drug release behavior.6 Particularly, an enzyme-triggered reaction presents unique properties with high speed and specificity, and enzyme-responsive drug delivery systems exhibit on-demand release of the therapeutic agents. For instance, the ubiquitously overexpressed cathepsin B was used to cleave the peptide linkage to release the active drug and kill cancer cells in a targeted manner.7 Here, a new type of multifunctional polymeric micelles with the ability to release the anticancer drug in a targeted manner into cancer cells was designed. As shown in Scheme 1, the multifunctional micelles were constructed by using poly(ethylene glycol)blocked-poly(L-lysine) (PEG-b-PLL) as the polymer backbone, biotin
Scheme 1 Schematic illustration of the formation of nanoscale polymeric micelles and cancer-targeted intracellular drug delivery. (A) The amphiphilic Biotin-PEG-b-PLL(Mal)-peptide-DOX self-assembled into nanoscale polymeric micelles. (B) The drug loaded polymeric micelles were uptaken by cancer cell via the active targeting strategy and the anticancer drug was rapidly released for cancer therapy upon the triggering of overexpressed MMP protease in cancer cell.
Chem. Commun.
View Article Online
Published on 07 October 2014. Downloaded by University of Utah on 29/11/2014 04:20:16.
Communication
as an active targeting ligand, while the anticancer drug, doxorubicin (DOX) was conjugated to the PLL segment by the protease sensitive peptide linkage (peptide-DOX) via Michael addition. PEG as the hydrophilic shell was used to stabilize the micelles and prolong the blood circulation time. It is known that biotin can increase the selective uptake of micelles by cancer cells via receptor-mediated endocytosis.8 Importantly, peptide-DOX will act as the hydrophobic core to promote the formation of core–shell structural micelles. In addition, the release of DOX can be controlled upon the triggering of the matrix metalloproteinases (MMPs) excessively secreted by cancer cells.9 The cancer-targeted intracellular drug delivery from the multifunctional polymeric micelles was investigated to eliminate cancer cell selectively and reduce side effects. As illustrated in Scheme S1 (ESI†), Biotin-PEG-b-PLL(Z) was first prepared by the ring-opening polymerization of Z-Lys-NCA under the initiation of Biotin-PEG-NH2 (Mw = 3400 g mol 1).10 The molecular weight of Biotin-PEG-b-PLL(Z) was detected to be 22 090 g mol 1 using gel permeation chromatography (GPC) (PDI = 1.5), which was consistent with the data (Mw = 19 370 g mol 1) calculated from 1H NMR spectrometry (Fig. S3, ESI†). After treatment with HBr/HAc, the resonance signals from the protons of the carbobenzyloxy (Z) group in the 1H NMR spectrometry disappeared (Fig. S4, ESI†), suggesting the complete removal of Z groups in Biotin-PEG-bPLL(Z) and the successful synthesis of Biotin-PEG-b-PLL. For bonding with the peptide substrate, Biotin-PEG-b-PLL(Mal) was obtained through decoration of Biotin-PEG-b-PLL with 6-maleimidocaproic acid via an amidation reaction. Furthermore, BiotinPEG-b-PLL(Mal)-peptide-DOX was prepared by grafting the MMP-2 sensitive peptide conjugated doxorubicin (Ac-CPLGLAGG-DOX, peptide-DOX) onto Biotin-PEG-b-PLL(Mal) via Michael addition. The successful synthesis of Biotin-PEG-b-PLL(Mal)-peptide-DOX was confirmed using 1H NMR spectroanalysis (Fig. S5, ESI†). It was found that the drug loading in Biotin-PEG-b-PLL(Mal)-peptide-DOX was about 5.7 wt% as determined using fluorescence spectroscopy. In this study, Biotin-PEG-b-PLL(Mal)-peptide-DOX consists of a water-soluble polymeric backbone Biotin-PEG-b-PLL and a water-insoluble peptide-DOX. The inherent amphiphilicity property of Biotin-PEG-b-PLL(Mal)-peptide-DOX offers an opportunity to self-assemble into core–shell micelles in water.11 The critical micelle concentration (CMC) of Biotin-PEG-b-PLL(Mal)peptide-DOX micelles was estimated using fluorescence spectra and the CMC was detected to be 3.47 10 2 mg mL 1 (Fig. S6, ESI†). Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to study the morphology and size distribution of the assemblies. As shown in Fig. 1A, the polymeric micelles self-assembled from Biotin-PEG-b-PLL(Mal)-peptide-DOX showed a regular spherical shape and were well dispersed as individual nanoparticles. The mean diameter measured using TEM was about 50 nm, which was smaller than the hydrodynamic diameter measured using DLS (around 75.4 nm, PDI = 0.245) (Fig. 1C) due to the shrinkage of nanoparticles in the dry state for TEM observations. To investigate the stimulus-responsive property of micelles, the assembled polymeric micelles were incubated with MMP-2 protease. As shown in Fig. 1B, the size of polymeric micelles increased significantly with aggregates emerging and some micelles got disassembled after treatment
Chem. Commun.
ChemComm
Fig. 1 Characterizations of the polymeric micelles by TEM and DLS. (A) TEM image and (C) size distribution of Biotin-PEG-b-PLL(Mal)peptide-DOX micelles. (B) TEM image and (D) size distribution of BiotinPEG-b-PLL(Mal)-peptide-DOX micelles treated with MMP-2 protease for 6 h.
with MMP-2. A similar result was also obtained by DLS measurements and the hydrodynamic diameter increased to about 110.3 nm with a large PDI of 0.495 (Fig. 1D). Additionally, the zeta potential of polymeric micelles increased from 16.5 0.5 to 6.9 0.4 mV, showing the dissociation of micelles. Moreover, DLS measurements demonstrated that the polymeric micelles were very stable in the normal physiological conditions (Fig. S7, ESI†). These results are easily understood since the MMP-2 protease can specifically cleave the peptide linkage, leading to the disassembly of micelles and rapid release of DOX from Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles. In order to evaluate the MMP-2 sensitivity of Biotin-PEG-bPLL(Mal)-peptide-DOX micelles, the in vitro drug release ability with or without the MMP inhibitor was studied and compared. As shown in Fig. 2, Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles exhibited apparent sensitivity to MMP-2 to cleave the peptide linkage and release the drug rapidly. Approximately
Fig. 2 Release profile of Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles under different conditions (red line: in the presence of MMP-2, blue line: in the presence MMP-2 and MMP inhibitor, and dark gray line: only in the presence of TCNB buffer).
This journal is © The Royal Society of Chemistry 2014
View Article Online
Published on 07 October 2014. Downloaded by University of Utah on 29/11/2014 04:20:16.
ChemComm
46.2% of DOX was released after incubation with MMP-2 for 6 h. In contrast, almost no drug was released after treating with MMP-2 together with the protease inhibitor for 72 h. Similarly, very little drug was released when the micelles were incubated with TCNB buffer. These results indicate that Biotin-PEG-bPLL(Mal)-peptide-DOX micelles have a desirable MMP selectivity, which can be used to control the drug release in cancer cells using the up-regulated MMP-2 protease. Furthermore, confocal laser scanning microscopy (CLSM) was used to investigate the protease induced DOX release in cancer cells. The Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles were incubated with SCC-7 (squamous cell carcinoma) cancerous cells, which are well known for their high MMP expression, and COS7 (African green monkey kidney fibroblast cells) normal cells were used as a control. As shown in Fig. 3A1, significant red fluorescence was observed in SCC-7 cells when treated with
Fig. 3 Confocal laser scanning microscopy (CLSM) images of SCC-7 cancer cells incubated with Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles for 3 h (A–A2), 6 h (B–B2), and 6 h for SCC-7 cancer cells pre-treated with the excess of biotin in advance (C–C2). (A–C) blue fluorescence images of nuclei; (A1–C1) red fluorescence images of DOX; (A2–C2) the merge images of blue and red fluorescence. The scale bar is 14 mm. Quantitative flow cytometry analysis of the cellular DOX red fluorescence in SCC-7 cancer cells (D) and COS7 normal cells (E). The cells without treatment (blank line), treating with Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles for 3 h (red line), 6 h (green line) and 6 h (blue line) for cells pre-treated with the excess of biotin in advance, respectively.
This journal is © The Royal Society of Chemistry 2014
Communication
micelles for 3 h, indicating that polymeric micelles were quickly uptaken by cancer cells. This was attributed to the active targeting ability of biotin in micelles and increase the uptake via the receptor-mediated endocytosis. In general, many therapeutic agents need to be localized in particular subcellular compartments to elicit their pharmacological actions.12 For instance, DOX and cisplatin cannot exert their therapeutic actions unless they enter into the nuclei.13 After being uptaken by cells, the up-regulated MMP-2 protease secreted by SCC-7 cancer cells can specifically cleave the peptide substrate and result in the release of anticancer drug DOX rapidly. It was found that the red fluorescence intensity of DOX increased with a prolonged incubation time, and DOX entering into the targeted site of nuclei was also detected after SCC-7 cells were incubated with micelles for 6 h (Fig. 3B1). However, when SCC-7 cells were pre-incubated with the excess biotin prior to treatment with the polymeric micelles, the fluorescence intensity of DOX in the cancer cells was dramatically reduced due to the blocking of the receptormediated endocytosis of biotin in the micelles (Fig. 3C1). In contrast, a very weak fluorescence signal was observed in COS7 normal cells all the time (Fig. S8, ESI†) due to the shortage of the biotin-receptor in COS7 cells. Moreover, flow cytometry analysis was used to investigate the cellular red fluorescence of DOX quantitatively. It was found that the mean fluorescence intensity (MFI) values in SCC-7 cancer cells were 14.9- and 6.2-fold higher than the one of COS7 normal cells when incubated with micelles for 3 h or 6 h, respectively (Fig. 3D and E and Fig. S9, ESI†). When the cells were pre-treated with an excess of biotin in advance, the cellular fluorescence of DOX was decreased dramatically (Fig. S9a, ESI†) due to the fact that the free biotin occupies the receptor in cancer cells, leading to the disability of biotin in the polymeric micelles. These results indicate that Biotin-PEG-b-PLL(Mal)peptide-DOX micelles not only have the ability to increase the uptake by cancer cells via the receptor-mediated endocytosis but also respond to the triggering of cancer cell excessive secretion MMP protease to release the anticancer drug. To evaluate the anticancer effect of the Biotin-PEG-b-PLL(Mal)peptide-DOX micelles, the cell viability was determined using an MTT assay and the results are displayed in Fig. 4. The SCC-7 cell viability was more than 75% after incubation with the polymeric micelles in the presence of the MMP inhibitor. However, the cell viability decreased significantly to 15% in the absence of the MMP
Fig. 4 The cell viability of SCC-7 cancer cells (A) and COS7 normal cells (B) after incubated with Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles in the absence or in the presence of MMP-2 inhibitor.
Chem. Commun.
View Article Online
Published on 07 October 2014. Downloaded by University of Utah on 29/11/2014 04:20:16.
Communication
inhibitor. In the presence of the MMP inhibitor, the peptide linkage cannot be cleaved and the conjugated drug cannot be released from the micelles. In contrast, without the MMP inhibitor, MMP protease could hydrolyze the peptide linkage to induce drug release as well as the apoptosis of cancer cells. However, as shown in Fig. 4B, the viability of COS7 normal cells incubated with Biotin-PEG-b-PLL(Mal)-peptide-DOX micelles was more than 85% with or without the MMP inhibitor. This high cell viability of COS7 cells was attributed to the low uptake of polymeric micelles and the lack of a specific MMP protease. In addition, the polymer backbone Biotin-PEG-b-PLL did not exhibit an obvious cytotoxicity in all cell lines (Fig. S10, ESI†). These results confirm that the selective therapy to cancer cells of the multifunctional micelles can be achieved due to the drug release upon applying the stimulus of cancer cells excessive secreted protease. In summary, the preparation of multifunctional Biotin-PEGb-PLL(Mal)-peptide-DOX polymeric micelles was demonstrated, which can specially respond to physiopathological signals of cancer cells. The biotin ligand could promote cancer cell uptake via the receptor-mediated endocytosis. Upon application of the stimulus of the overexpressed MMP protease in cancer cells, the anticancer drug DOX could be released effectively, leading to significant apoptosis of cancer cells. The targeted therapy to cancer cells could be achieved with much reduced side effects to normal cells, which will find great potential in cancer therapy. This work was supported by the National Natural Science Foundation of China (51125014 and 51233003), the Ministry of Science and Technology of China (2011CB606202), the Ministry of Education of China (20120141130003) and supported by the Fundamental Research Funds for the Central Universities (2014203020201 and 2014203020204).
Chem. Commun.
ChemComm
Notes and references 1 A. I. Minchinton and I. F. Tannock, Nat. Rev. Cancer, 2006, 6, 583. 2 (a) M. Ferrari, Nat. Rev. Cancer, 2005, 5, 161; (b) D. Peer, J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit and R. Langer, Nat. Nanotechnol., 2007, 2, 751; (c) A. Mukerjee, A. P. Ranjan and J. K. Vishwanatha, Curr. Med. Chem., 2012, 19, 3714. 3 (a) P. Sharma and S. Garg, Adv. Drug Delivery Rev., 2010, 62, 491; (b) H. Wei, R. X. Zhuo and X. Z. Zhang, Prog. Polym. Sci., 2013, 38, 503; (c) Z. Ge and S. Liu, Chem. Soc. Rev., 2013, 42, 7289; (d) J. Ding, L. Chen, C. Xiao, L. Chen, X. Zhuang and X. Chen, Chem. Commun., 2014, 50, 11274. 4 (a) J. Z. Du, X. J. Du, C. Q. Mao and J. Wang, J. Am. Chem. Soc., 2011, 133, 17560; (b) W. Xu, J. F. Burke, S. Pilla, H. Chen, R. Jaskula-Sztul and S. Gong, Nanoscale, 2013, 5, 9924. 5 (a) J. Nicolas, S. Mura, D. Brambilla, N. Mackiewicz and P. Couvreur, Chem. Soc. Rev., 2013, 42, 1147; (b) A. Mahmud, X. B. Xiong, H. M. Aliabadi and A. Lavasanifar, J. Drug Targeting, 2007, 15, 553; (c) Y. Zou, Y. Song, W. Yang, F. Meng, H. Liu and Z. Zhong, J. Controlled Release, 2014, DOI: 10.1016/j.jconrel.2014.05.016; (d) W. H. Chen, X. D. Xu, G. F. Luo, H. Z. Jia, Q. Lei, S. X. Cheng, R. X. Zhuo and X. Z. Zhang, Sci. Rep., 2013, 3, 3468. 6 (a) J. Dai, S. Lin, D. Cheng, S. Zou and X. Shuai, Angew. Chem., Int. Ed., 2011, 50, 9404; (b) Z. Chu, C. A. Dreiss and Y. Feng, Chem. Soc. Rev., 2013, 42, 7174; (c) M. Nakayama, J. Akimoto and T. Okano, J. Drug Targeting, 2014, 22, 584; (d) S. Mura, J. Nicolas and P. Couvreur, Nat. Mater., 2013, 12, 991. 7 X. Tian, K. H. Baek and I. Shin, Chem. Sci., 2013, 4, 947. 8 S. Bhuniya, S. Maiti, E. J. Kim, H. Lee, J. L. Sessler, K. S. Hong and J. S. Kim, Angew. Chem., Int. Ed., 2014, 53, 4469. ¨ha ¨ri, Curr. Cancer Drug 9 (a) P. Vihinen, R. Ala-aho and V. M. Ka Targets, 2005, 5, 203; (b) L. Aureli, M. Gioia, I. Cerbara, S. Monaco, G. F. Fasciglione, S. Marini, P. Ascenzi, A. Topai and M. Coletta, Curr. Med. Chem., 2008, 15, 2192. 10 (a) K. Wang, G. F. Luo, Y. Liu, C. Li, S. X. Cheng, R. X. Zhuo and X. Z. Zhang, Polym. Chem., 2012, 3, 1084; (b) B. Yang, Y. Lv, J. Y. Zhu, Y. T. Han, H. Z. Jia, W. H. Chen, J. Feng, X. Z. Zhang and R. X. Zhuo, Acta Biomater., 2014, 10, 3686. 11 P. Huang, D. Wang, Y. Su, W. Huang, Y. Zhou, D. Cui, X. Zhu and D. Yan, J. Am. Chem. Soc., 2014, 136, 11748. ¨lker and K. Simons, Nat. Rev. Drug Discovery, 12 L. Rajendran, H. J. Kno 2010, 9, 29. 13 L. Pan, Q. He, J. Liu, Y. Chen, M. Ma, L. Zhang and J. Shi, J. Am. Chem. Soc., 2012, 134, 5722.
This journal is © The Royal Society of Chemistry 2014
