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Tumor-Targeting Peptide Conjugated pH-Responsive Micelles as a Potential Drug Carrier for Cancer Therapy Xiang Lan Wu,†,‡ Jong Ho Kim,†,| Heebeom Koo,§ Sang Mun Bae,| Hyeri Shin,| Min Sang Kim,‡ Byung-Heon Lee,| Rang-Woon Park,| In-San Kim,| Kuiwon Choi,§ Ick Chan Kwon,§ Kwangmeyung Kim,*,§ and Doo Sung Lee*,‡ Department of Polymer Science and Engineering, Sungkyunkwan University, Suwon, 440-746, Republic of Korea, Biomedical Research Center, Korea Institute of Science and Technology, Seongbuk-Gu, Seoul, 136-791, South Korea, and Advanced Medical Technology Cluster for Diagnosis & Prediction, Kyungpook National University, Daegu 700-422, South Korea. Received December 2, 2009; Revised Manuscript Received January 4, 2010

Herein, we prepared tumor-targeting peptide (AP peptide; CRKRLDRN) conjugated pH-responsive polymeric micelles (pH-PMs) in cancer therapy by active and pH-responsive tumor targeting delivery systems, simultaneously. The active tumor targeting and tumoral pH-responsive polymeric micelles were prepared by mixing AP peptide conjugated PEG-poly(D,L-lactic acid) block copolymer (AP-PEG-PLA) into the pH-responsive micelles of methyl ether poly(ethylene glycol) (MPEG)-poly(β-amino ester) (PAE) block copolymer (MPEG-PAE). These mixed amphiphilic block copolymers were self-assembled to form stable AP peptide-conjugated and pH-responsive APPEG-PLA/MPEG-PAE micelles (AP-pH-PMs) with an average size of 150 nm. The AP-pH-PMs containing 10 wt % of AP-PEG-PLA showed a sharp pH-dependent micellization/demicellization transition at the tumoral acid pH. Also, they presented the pH-dependent drug release profile at the acidic pH of 6.4. The fluorescence dye, TRITC, encapsulated AP-pH-PMs (TRITC-AP-pH-PMs) presented the higher tumor-specific targeting ability in vitro cancer cell culture system and in vivo tumor-bearing mice, compared to control pH-responsive micelles of MPEG-PAE. For the cancer therapy, the anticancer drug, doxorubicin (DOX), was efficiently encapsulated into the AP-pH-PMs (DOX-AP-pH-PMs) with a higher loading efficiency. DOX-AP-pH-PMs efficiently deliver anticancer drugs in MDA-MB231 human breast tumor-bearing mice, resulted in excellent anticancer therapeutic efficacy, compared to free DOX and DOX encapsulated MEG-PAE micelles, indicating the excellent tumor targeting ability of AP-pH-PMs. Therefore, these tumor-targeting peptide-conjugated and pH-responsive polymeric micelles have great potential application in cancer therapy.

INTRODUCTION Polymeric micelles formed by amphiphilic block copolymers in aqueous conditions have been investigated as a tumortargeting drug delivery system in cancer therapy, because they can form a stable nanosized micellar structure, resulting in accumulation and extravasation into the target tumor tissue, which is called an enhanced permeation and retention effect (EPR effect) (1, 2). Importantly, pH-responsive polymeric micelles (pH-PMs) are more attractive for their sharp pHdependent structural disruption in tumoral acidic pH, which results in rapid drug release at target tumor tissue (3-5). This is because tumor tissues have a more acidic environment, due to the lactic acid produced by acidic intracellular organelles and hypoxia. Due to the tumoral acidic pH microenvironments, various amphiphilic copolymers containing pH-responsive histidine and sulfonamide moieties have been designed to present a sharp pH-dependent micellization/demicellization transition, resulting in the demicellization leading to rapid drug release at target tumor tissues (3, 6). Therefore, pH-PMs not only successfully targeted at tumor tissue, but also enabled rapid drug release into the target tumor tissue (7-9). * To whom correspondence should be addressed.Tel: +82-2-9585912; Fax: +82-2-958-5909; E-mail: [email protected] (K. Kim). Tel: +82-31-290-7282; Fax: +82-31-292-8790; E-mail: [email protected] (D. S. Lee). † Both authors contributed equally. ‡ Sungkyunkwan University. § Korea Institute of Science and Technology. | Kyungpook National University.

We also developed pH-PMs composed of poly(ethylene glycol) methyl ether-poly(β-amino ester) (MPEG-PAE) (10), which presented a sharp pH-responsive micellization/demicellization transition at the tumoral acidic pH condition (pH 6.4-6.8), due to its tertiary amine with a pKb of about 6.5 (11, 12). However, the pH-responsive MPEG-PAE micelles at the acidic pH buffer might take a few hours to present the sharp pH-dependent responsiveness at the tumoral weakly acidic pH microenvironment. It is also reported that most pHresponsive polymeric micelles at the acidic buffer condition required a prolonged reaction time to present a pH-responsiveness. On the basis of the time-delayed pH-responsiveness of polymeric micelles, the in vivo pH responsiveness of various polymeric micelles at the target tumor tissue might be less effective than that in the in vitro pH buffer condition, due to the very small tumor volume and the lower tumor targeting ability. Therefore, the pH-responsive polymeric micelles should be specifically targeted and remained for a long time at the target tumors, wherein the pH-responsive polymeric micelles might present a sharp pH-dependent micellization/demicellization transition. Herein, to enhance the specificity and retention time of pHPMs at the target tumor, we prepared tumor-specific peptide (AP peptide; CRKRLDRN)-conjugated pH-responsive MPEGPAE micelles (AP-pH-PMs). It was reported that AP peptide had very specific binding affinity to IL-4 receptors of atherosclerotic plaques and breast tumor tissues (13-15). Therefore, we anticipated that AP-pH-PMs may increase the pH-responsiveness at the target tumors, due to the enhanced tumor

10.1021/bc9005283  2010 American Chemical Society Published on Web 01/14/2010

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Figure 1. Design strategy of peptide-conjugated pH-responsive micelle: (A) Chemical structure of tumor-targeting AP peptide (CRKRLDRN) coupled PEG-PLA block copolymer (AP-PEG-PLA) and (B) pH-responsive MPEG-PAE block copolymer (MPEG-PAE). (C) Schematic diagram depicting tumor-targeting and pH-responsive polymeric micelles of AP-PEG-PLA/MPEG-PAE (AP-pH-PM), which triggered doxorubicin (DOX) release by the sharp pH-dependent micellization/demicellizaton transition at the tumoral acidic pH.

targeting ability in vivo. In order to prepare AP peptide-modified polymeric micelles, AP peptide grafted PEG-poly(D,L-lactic acid) block copolymer (AP-PEG-PLA) was prepared and mixed with pH-responsive MPEG-PAE block copolymer in aqueous condition. These mixed amphiphilic block copolymers were selfassembled to form stable AP-pH-PMs (Figure 1). The AP-pHPMs containing 10 wt % of AP-PEG-PLA showed a sharp pHdependent micellization/demicellization transition at the tumoral acid pH (pH 6.8-6.9). To assay the tumor targeting ability of AP-pH-PMs in MDA-MB231 human breast tumor-bearing mice, fluorescence dye, TRITC, encapsulated AP-pH-PMs were noninvasively monitored using optical fluorescence imaging technology. Finally, doxorubicin (DOX) was encapsulated into the AP-pH-PMs (DOX-AP-pH-PMs), and its enhanced therapeutic efficacy was studied in MDA-MB231 human breast tumor-bearing mice, compared to control free DOX and DOX encapsulated pH-responsive MEPG-PAE micelles (DOX-pHPMs), respetively (16).

RESULTS AND DISCUSSION In order to enhance the targeting specificity and retention time of pH-PMs at the target tumor, first, human breast tumor-specific AP peptide (CRKRLDRN) chemically conjugated to the PEGPLA block copolymer (AP-PEG-PLA) was prepared by chemical coupling thiolated AP peptide to maleimide-terminated PEGPLA block copolymer in acetonitrile/water (Figure 1A). After the reaction, the solution was lyophililized and reprecipitated in cold diethyl ether for twice the time, followed by drying in a vacuum oven for 12 h (yield: 85%), resulting in AP-PEGPLA (Mw ) 19.6 kDa). Second, pH-responsive block copolymer was prepared by a Michael-type step polymerization between hydrophilic methyl ether poly(ethylene glycol) (MPEG) and pHresponsive/biodegradable poly(β-amino ester) (PAE) (Figure 1B), wherein the MPEG-PAE (Mw ) 17.4 kDa) has 2.6 mol equiv of PAE per 1 M of PEG (see the Supporting Information), as previously reported (10). The AP peptide-modified pHresponsive micelles (AP-pH-PMs) were simply prepared by a solvent evaporation method (Figure 1C). The polymer mixture containing 10 wt % of AP-PEG-PLA and 90 wt % of MPEGPAE in organic solvent was evaporated to give the polymer thin film, and the film was incubated in distilled water or PBS

at 37 °C for 30 min. After the incubation, the dried polymer film was dispersed by signification for 2 min. This amphiphilic AP-PEG-PLA/MPEG-PAE mixture was spontaneously selfassembled to form stable polymeric micelles with an average diameter of 156 nm at normal pH of 7.4. Also, the AP-pHPMs showed a sharp micellization/demicellization transition at tumoral acidic pH values (pH 6.8-6.9). It is deduced that amphiphilic MEPEG-PAEs are ionized and completely dissolved at the weakly acidic condition. For the assay, the tumor targeting ability and anticancer therapeutic efficacy of AP-pH-PMs, 10 wt % of fluorescence dye, TRITC, or 10 wt % of anticancer drug, doxorubicin (DOX), was encapsulated into the AP-pH-PMs by the solvent evaporation method, respectively (see Supporting Information) (17). Hydrophobic TRITC or DOX was well-encapsulated into the hydrophobic inner cores of the polymeric micelles with a higher loading efficiency of about 90%. The TRITC or DOX encapsulated polymeric micelles (TRITC-AP-pH-PMs or DOX-APpH-PMs) showed a slightly increased average size of about 180 nm, measured by dynamic light scattering (Figure 2A), wherein the polydisersity of each polymeric micelle was fairly low (0.021 mg/mL), indicating narrow size distribution of each polymeric micelle. The TEM images also showed the spherical particle morphologies of DOX-AP-pH-PMs. As control, TRITC or DOX was also encapsulated into the pH-PMs without AP peptide (TRITC-pH-PMs and DOX-pH-PMs) using the same solvent casting method. The overall characteristics of each polymeric micelle, including loading efficiency, average size, and pH transition, are summarized in Table 1. As expected, DOX-AP-pH-PMs began to be rapidly demicellized with decreasing pH values because the tertiary amines of PEA polymer were completely ionized at the lower pH value (6.8-6.9) (Figure 2B). This pH-dependent micellization/demicellization transition of DOX-AP-pH-PMs was very similar to that of MPEG-PEA micelles at the pH of 6.4-6.8, which may specifically respond to the tumoral weakly acidic extracellular pH (5). DOX release profile from the AP-pH-PMs was examined using a dialysis method. It was obvious from Figure 2C that DOX release was highly influenced by pH value, indicating a pH-dependent drug release profile. At pH 7.4, the release rate of DOX was very low with less than 20% of the DOX released

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Figure 3. Confocal laser scanning microscopy of TRITC encapsulated pH-PMs (TRITC-pH-PMs) and AP-pH-PMs (TRITC-AP-pH-PMs) in MDA-MB-231 cancer cell culture system. The scale bar is 20 µm in the image. Figure 2. Characteristics of DOX-encapsulated AP-pH-PMs (DOXAP-pH-PMs): (A) Size distribution of DOX-AP-pH-PMs (1 mg/mL in PBS at pH 7.4). The inset image presents TEM image of DOX-APpH-PMs (1 mg/mL in distilled water). (B) Average size change of DOXAP-pH-PMs as a function of pH. (C) In vitro release profiles of DOX from DOX-AP-pH-PMs at different pH values (6.4 and 7.4) at 37 °C measured by UV-vis spectroscopy. Table 1. Characterization of pH-Responsive Polymeric Micelles sample

feed amount (wt %)

loading efficiency (%)

size (nm)a

µ2/Γ2b

pH-PM AP-pH-PM TRITC-pH-PM TRITC-AP-pH-PM DOX-pH-PM DOX-AP-pH-PM

10 10 10 10

86 ( 2.5 90 ( 2.8 82 ( 2.8 86 ( 2.8

62 150 132 182 183 181

0.014 0.018 0.021 0.019 0.023 0.024

b

a The average size was measured with dynamic light scattering. Polydispersity factor for polymeric micelles.

in 20 h, while a noticeably increased release rate of DOX was observed at pH 6.4 with more than 65% of the DOX released within 4 h. It is deduced that, at pH 7.4, most AP-pH-PMs maintained their micellar structure and kept DOX in the micellar core, while they were rapidly demicellized in a short time at pH 6.4, followed by releasing DOX. This drug release profile of AP-pH-PMs indicated that AP peptides on the micelle’s surface did not have any effect on the pH-dependent drug release profile of pH-responsive MPEG-PEA micelles (18). The tumor-specific targeting ability of fluorescence dye, TRITC, encapsulated polymeric micelles (TRITC-AP-pH-PMs) in the MDA-MB-231 human breast cancer cell culture system was evaluated using confocal laser microscopy, whereas, as control, TRITC-pH-PMs without AP peptide moieties at the micelle’s surface were used. Figure 3 shows the enhancement of cellular uptake with AP-pH-PMs over pH-PMs, suggesting that AP peptide modified micelles were more rapidly taken into the cells by the specific binding affinity to IL-4 receptors of breast cancer cells (15). The TRITC’s fluorescence intensity of

the TRITC-AP-pH-PM treated cells was always higher than that of TRITC-pH-PM treated cells at any moment. In particular, only after 1 min postincubation, TRITC-AP-pH-PMs-treated cells presented substantial fluorescence intensity at the cytoplasm part, indicating rapid receptor-medicated endocytosis (red arrows; see NIR fluorescence histogram in Supporting Information Figure S1) (19). As incubation time increased, the fluorescence intensity increased up to 1 h and most cells presented strong fluorescence in the cytoplasm and perinuclear compartments. However, TRITC-pH-PMs did not present rapid cellular uptake in the cell culture system, due to the bioinert PEG surface of polymeric micelles. It suggested that the surface modification of pH-PMs with tumor-targeting AP peptides greatly enhanced the cellular uptake profile of pH-responsive polymeric micelles by rapid receptor-medicated cellular adherence and uptake. The tumor targeting ability of the TRITC-AP-pH-PMs was also measured using noninvasive and live optical imaging system. When the tumor volume grew to 150-200 mm3, TRITC-pH-PMs and TRITC-AP-pH-PMs containing 1 mg/kg of TRITC were administrated intravenously into the MDAMB231 human breast tumor-bearing mice. After the scheduled time points, the fluorescence signals of TRITC-pH-PMs and TRITC-AP-pH-PMs were noninvasively quantified by measuring total photon counts at the target tumor tissues (Figure 4A) (20). At 1 h postinjection of TRITC-AP-pH-PMs, the inoculated tumor was clearly delineated from the body, indicating rapid tumor accumulation of TRITC-AP-pH-PMs. Moreover, the fluorescence signals gradually increased up to 24 h postinjection. However, TRITC-pH-PM-treated mice showed minimum fluorescence intensity at the target tumors up to 12 h. It means that pH-PMs without AP peptides might be less effective in targeting at the tumor tissues, due to the bioinert PEG surface of pHPMs, compared to AP-pH-PMs. After 24 h postinjection, the delayed tumor targeting ability of pH-PMs was observed, indicating that the pH-responsiveness of MPEG-PAE micelles is not very effective in the real tumor model in vivo. The fluorescent total photon counts per gram of TRITC-AP-pH-PMtreated tumors were 2-fold higher than those of the TRITC-

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Figure 4. In vivo and ex vivo biodistribution of peptide conjugated pH-responsive micelles: (A) In vivo fluorescence images of MDA-MB-231 tumor xenografted athymic nude mice injected with TRITC-pH-PM and TRITC-AP-pH-PM, respectively. (B) Quantification of in vivo tumor targeting ability of TRITC-pH-PMs and TRITC-AP-pH-PMs were recorded as total photon counts per centimeter squared per steradian (p/s/cm2/sr) per each tumor at 1 day postinjection (n ) 3). The data represent mean ( s.e. (C) Ex vivo fluorescence image of dissected organs after 24 h postinjection of TRITC-pH-PMs and TRITC-AP-pH-PMs.

Figure 5. In vivo anti-tumor therapy of doxorubicin encapsulated micelles: (A) In vivo anti-tumor therapeutic efficacy of saline (0), pH-PM (O), AP-pH-PM (4), free DOX (2 mg/kg) (9), DOX- pH-PM with 2 mg/kg of DOX (•), and DOX-AP-pH-PM 2 mg/kg of DOX (∆) (n ) 5). Values represent the mean ( SD. (B) Tumor weights of MDA-MB-231 tumor-bearing mice 15 days post-treatment. (C) Immunohistological detection of apoptotic cells (TUNNEL assay) in MDA-MB-231 tumor-bearing mice 15 days post-treatment.

pH-PM-treated tumors over any time period (Figure 4B). These results indicated that the AP-pH-PMs could be specifically targeted in a short time period and localized for a long time at the target tumors. Interestingly, ex vivo fluorescence images of excised organs showed substantial evidence of the tumortargeting ability of TRITC-AP-pH-PMs (Figure 4C). After 24 h

postinjection, mice were sacrificed, and major organs such as liver, lung, spleen, kidney, and heart, as well as the tumors, were isolated to evaluate the in vivo biodistribution of TRITCAP-pH-PMs and TRITC-pH-PMs. Strong fluorescence signals were observed in the tumor tissues, indicating the tumor targeting ability of both pH-PMs and AP-pH-PMs, while other

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tissues showed negligible NIRF signals, except for the liver, in which biodegradable polymers were metabolized. Interestingly, TRITC-AP-pH-PMs presented the stronger fluorescence signal in the target tumor, compared to TRITC-pH-PM. The tumor targeting ability of AP peptide-modified polymeric micelles could increase the tumor targeting specificity and retention time at the target tumor. The in vivo anti-tumor efficacy of the DOX-encapsulated APpH-PMs (DOX-AP-pH-PMs) was also evaluated in MDA-MB231 human breast tumor-bearing mice (21). Figure 5A shows the growth curves of the tumors after intravenous injection of saline, polymeric micelles alone (pH-PMs, AP-pH-PMs), free DOX (2 mg/kg), or DOX-loaded polymeric micelles (DOXpH-PMs, DOX-AP-pH-PMs) with 2 mg/kg of free DOX. The tumor growth rate of pH-PMs and AP-PMs-treated mice were similar to that of mice treated with saline, indicating that polymeric micelles had no therapeutic effect on the tumor growth. However, free DOX, DOX-pH-PMs, and DOX-APpH-PMs-treated mice showed significant anti-tumor therapeutic efficacy, respectively. At 15 day postinjection, the average tumor volume in free DOX-treated mice was suppressed and reached only 544 ( 98 mm3, compared with 911 ( 137 mm3 attained in saline-treated mice. DOX-pH-PM-treated mice also showed more efficient anti-tumor efficacy than free DOX, with the average tumor volume of 304 ( 37 mm3. In particular, DOXAP-pH-PM-treated mice decreased substantially the tumor volumes by 183 ( 37 mm3, indicating that AP-pH-PMs could successfully deliver the encapsulated DOX at the target tumor. It suggested that AP peptide-modified polymeric micelles could specifically deliver encapsulated drugs to the target tumor by the tumor-targeting ability of AP peptides on their micelle’s surface, as previously confirmed by in cell culture system and in vivo imaging data. At 15 day postinjection, DOX-AP-pHPMs greatly suppressed the tumor weight to 18.6%, compared to the saline-treated mice, whereas free DOX and DOX-pHPMs suppressed the tumor weight to 60% and 32%, respectively. The anti-tumor efficacy of pH-PM-DOX was also evaluated by the TUNEL assay, which detects apoptosis at the tumor tissues (Figure 4C) (22). No significant apoptotic cells were seen in tumors from saline and polymeric micelle-treated micelles. However, tumor tissues treated with DOX-AP-pH-PMs showed the highest TUNEL signals compared to tumors treated with free DOX and DOX-pH-PMs, indicating the excellent anticancer therapeutic efficacy of DOX-AP-pH-PMs. Collective results from both in vitro and in vivo studies strongly supported the anticancer therapeutic efficacy of APpH-PMs as a new nanosized drug carrier with higher tumortargeting ability. DOX molecules were efficiently loaded into the AP-pH-PMs and rapidly released under acidic conditions with higher tumor-targeting ability. The increased tumortargeting characteristics of AP peptide-modified and pHresponsive polymeric micelles were evaluated in a cell culture system and tumor-bearing mice using a fluorescence imaging technique, which showed that AP-pH-PMs efficiently deliver anticancer drugs, resulting in excellent anticancer therapeutic efficacy. Taken together, the tumor-homing and pH-responsive AP-pH-PMs have great potential application in cancer therapy.

ACKNOWLEDGMENT This work was financially supported by the Real-Time Molecular Imaging Project, 2009K 001594, and GRL Program of MEST and by grant to the Intramural Research Program (Theragnosis) of KIST, by a grant of by BK21 BNT Scientist Renovating for the Drug Development Coping with Aged Society, and Advanced Medical Technology Cluster for Diagnosis and Prediction at Kyungpook National University from MOCIE.

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Supporting Information Available: Experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org.

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