CD44 Receptor Targeting and Endosomal pH-Sensitive Dual

Article pubs.acs.org/molecularpharmaceutics

CD44 Receptor Targeting and Endosomal pH-Sensitive Dual Functional Hyaluronic Acid Micelles for Intracellular Paclitaxel Delivery Yanhua Liu,*,†,‡ Chengming Zhou,§ Wenping Wang,†,‡ Jianhong Yang,†,‡ Hao Wang,†,‡ Wei Hong,∥ and Yu Huang†,‡ †

Department of Pharmaceutics, School of Pharmacy, Ningxia Medical University, No. 1160, Shengli Street, Yinchuan 750004, China Key Laboratory of Hui Ethnic Medicine Modernization, Ministry of Education, Ningxia Medical University, Yinchuan 750004, China § Department of Pharmacy, Tumor Hospital of General Hospital, Ningxia Medical University, Yinchuan 750004, China ∥ School of Chemistry and Chemical Engineering, Beifang University of Nationalities, Yinchuan 750021, China ‡

ABSTRACT: A novel CD44 receptor targeting and endosome pH-sensitive dual functional hyaluronic acid−deoxycholic acid−histidine (HA-DOCA-His) micellar system was designed for intracellular paclitaxel (PTX) delivery. The HADOCA-His micelles exhibited desirable endosome pH (5.0− 6.0)-induced aggregation and deformation behavior verified by size distribution, critical micellar concentration, and zeta potential changes. The HA-DOCA-His micelles presented excellent encapsulation efficiency and loading capacity of 90.0% and 18.9% for PTX, respectively. The PTX release from HA-DOCA-His micelles was pH-dependent, with more rapid PTX release at pH 6.0 and 5.0 than those at pH 7.4 and 6.5. The cellular uptake performance of HA-DOCA-His micelles was enhanced comparing with pH-insensitive HA-DOCA micelles by qualitative and quantitative measurements. HA-DOCA-His micelles could be taken up via CD44-receptor mediated endocytosis, transported into endosomes, and triggered drug release to cytoplasm. In vitro cytotoxicity study exhibited PTX-loaded HA-DOCA-His micelles were more active in tumor cell growth inhibition in MCF-7 cells at pH 5.8 than those at pH 6.8 and pH 7.4. A superior antitumor efficacy was demonstrated with HADOCA-His micelles in a MCF-7 breast tumor model. These indicated that the dual functional HA-DOCA-His micelles combined targeted intracellular delivery and endosomal release strategies could be developed as a promising nanocarrier for anticancer efficacy improvement of PTX. KEYWORDS: HA-DOCA-His micelles, CD44 receptor targeting, endosomal pH-sensitivity, cytosolic PTX delivery

1. INTRODUCTION Efficient tumor tissue accumulation and intracellular delivery of therapeutic agents are key factors for cancer chemotherapy. Nowadays, nanodrug delivery systems have presented as a promising approach for cancer treatment.1,2 Hydrophobized polysaccharide micelles, as a potent nanocarrier, have been utilized for targeting chemotherapeutic agents delivery.3,4 It can be beneficial for passive tumor targeting via enhanced permeability and retention (EPR) effect.5 However, the poor intracellular uptake and inefficient intracellular drug release limit the cancer treatment based on nanodrug delivery system. Therefore, it is urgent to develop multifunctional nanodrug delivery systems for efficient anticancer therapy.6 Conjugation of hydrophobized polysaccharide with active targeting ligands or antibodies can facilitate the efficient intracellular uptake via highly efficient receptor-mediated endocytosis. Hyaluronic acid (HA), an active targeting polysaccharide that possesses specific binding characteristic to tumor cells overexpressing CD44 receptors, has been © XXXX American Chemical Society

developed as promising nanocarriers for targeting delivery of therapeutic agents.7−12 Paclitaxel (PTX), a first-line anticancer agent, has been reported to conjugate or encapsulate to HA-based micelles, such as HA-grafted-dendritic oligoglycerol,13 HA-graftedglycyrrhetinic acid,14 HA-conjugated-PTX,15 and HA-amino acid-conjugated-PTX,16 which have emerged as novel targeting carriers for PTX delivery. Although these HA-based PTX micelles are benefits to accumulate in tumor tissue via EPR effect mediated passive targeting and internalize into cells by CD44 receptor-mediated active targeting endocytosis, these systems are limited in controlling intracellular release of encapsulated PTX in tumor cells, which lower PTX’s antitumor efficacy. To overcome this limitation, new HA micellar delivery Received: September 27, 2016 Revised: October 16, 2016 Accepted: October 19, 2016

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DOI: 10.1021/acs.molpharmaceut.6b00870 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics

Figure 1. Schematic illustration of the self-assembly, tumor tissue accumulation, tumor cells endocytosis, and pH-responsive intracellular PTX release of HA-DOCA-His micelles. (a) PTX-encapsulated micelles based on HA-DOCA-His polymers were formed in aqueous solution. (b) After intravenous injection, micelles accumulated in tumor tissue via EPR effect. (c) By CD44 receptor-mediated endocytosis, micelles were selectively endocytosed into MCF-7 cells and delivered to the endosomes, triggered PTX release into the cytoplasm, increased intracellular PTX concentration, and improved antitumor efficacy.

system combining smart functions with targeting controlled micellar dissociation and triggered drug release mechanism in efficient intracellular PTX delivery is of high demand in cancer treatment. To date, the combination of active targeting and intracellular pH sensitive targeting strategies for micellar systems in cancer therapy helps trigger the activation of nanosystems to improve tumor targeting and intracellular drug delivery. This novel approach provides a promising opportunity for therapeutic efficacy improvement.17−19 The pH gradient of tumor tissue to normal tissue and extracellular compartment to intracellular compartment has provided a effective strategy for targeting delivery of therapeutic agents in tumor tissue, extracellular, and/or intracellular compartment. The pH of blood and normal tissues is 7.4, while the extracellular environment of tumors has a pH of 6.5− 7.2.20 In addition, after endocytosis into cells, the micelles would encounter a lower pH in endosomes of 5.0−6.0 and in lysosomes of 4.0−5.0, respectively.13,21 Based on pH differences between the tumor tissues and the normal tissues, as well as the endosomal and lysosomal acidic microenvironments, endosomal-pH sensitive targeting strategy has been emerging as an effective pathway for intracellular release of therapeutic agents after the micelle has been taken up by cells, which is competitive and favorable for chemotherapy.22−24 Histidine (His) has been utilized as a potent pH-sensitive group in micellar systems for tumor extracellular pH or more acidic endosomal pH-triggered drug releasing. His exhibited pH-sensitive property by protonation−deprotonation of the unsaturated nitrogen in imidazole ring. In addition, the proton sponge effect of His can promote the disassembly of micelles after endocytosed into cells and delivered into endosomes, then release encapsulated drugs into cytoplasm by the disruption of endosomal membrane, facilitating the therapeutic efficacy improvement.25−27

Triggering micelles disassemble and drug release in tumor intracellular compartment while maintaining minimal drug leaking during blood circulation and in tumor extracellular compartment is essential for cancer chemotherapy. However, the pKa of His is 6.5, which is too sensitive to tumor extracellular pH. It could be expected drug might be released from such pH sensitive micelles before endocytosed into tumor cells, resulting in the drug’s efficacy decrease. Conjugating a pH-insensitive polymer to the pH-sensitive His polymer may provide an effective approach to achieve endosomal-pH (5.0− 6.0) sensitivity. In addition, more stable His-micelles would be formed by conjugation with hydrophobic pH-insensitive polymers.28−30 Here, in our study, HA-deoxycholic acid-His (HA-DOCAHis) micelles with CD44 targeting and endosomal pHresponsive features was developed for active targeting and intracellular delivery of PTX, which can be internalized via CD44 receptor-mediated endocytosis pathway to enhance cellular uptake and respond to endosome environment to promote intracellular PTX release. HA-deoxycholic acid (HADOCA) micelles with CD44 receptor targeting, lacking pHsensitive property, was also developed for comparison. As illustrated in Figure 1, PTX could be loaded in micelles by the self-assembly of HA-DOCA-His polymer in aqueous solution. After intravenous injection, the micelles accumulated at tumor site via EPR effect. The micelles then could be endocytosed into tumor cells by CD44 receptor-mediated endocytosis and delivered to endosomes, followed by endosomal-pH triggered micellar disassembly and endosomal membrane disruption, showing effective delivery of PTX into cytoplasm. The most important advantage of this novel design is that the HA-based micelle combined dual functions of active targeting and endosomal pH-triggered drug release provides an effective approach for targeting intracellular delivery and endosomal B


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Molecular Pharmaceutics Scheme 1. Synthesis Scheme of HA-DOCA-His Polymer

2.2.2. Synthesis of HA-DOCA-His Polymer. HA-DOCA-His polymer was synthesized by a three-step reaction, as shown in Scheme 1. First, DOCA was amidated with H-His(Trt)-OMe· HCl. Briefly, DOCA (500 mg), NHS (288 mg), and EDC (1030 mg) were added in 5 mL of anhydrous DMF and reacted at room temperature for 24 h. Afterward, H-His(Trt)-OMe· HCl dissolved in anhydrous DMF with triethylamine was added to above reaction mixture slowly and stirred for 24 h at room temperature. The resultant solution was precipitated in Na2CO3−NaHCO3 (pH > 8) buffer solution. DOCA-His (Trt) was obtained after filtration. The second step was the ester bond formation between −COOH of HA and −OH of DOCA-His (Trt). HA (100 mg), EDC (96 mg), and NHS (58 mg) were dissolved in formamide and stirred in ice bath for 2 h. Then, DOCA-His (Trt) dissolved in anhydrous DMF was added to HA solution and stirred at 50 °C for 6 h and at room temperature for further 48 h. The resultant solution was dialyzed against ethanol/water (1:1−1:3, v/v) and distilled water for 2 days, respectively. Following filtration, the solution was lyophilized and HADOCA-His (Trt) powder was obtained. One hundred milligrams of HA-DOCA-His (Trt) was dissolved in 1 mL of formamide, and 1 mL of trifluoroacetic acid and 25 μL of thioanisole were added. The mixture was stirred for 3 h to deprotect the Trt group. The reaction solution was dialyzed against Na2CO3−NaHCO3 buffer solution (pH 9−10) and distilled water for 2 days, respectively, then lyophilized. 2.2.3. Nuclear Magnetic Resonance Spectroscopy. The chemical structure of HA and the obtained HA-DOCA and

drug release, resulting in significant increases in antitumor therapeutic efficacy.

2. EXPERIMENTAL SECTION 2.1. Materials. Sodium hyaluronate (Mw = 11 kDa) was obtained from Freda Biopharm Co., Ltd. (Shandong, China). Deoxycholic acid (DOCA) was supplied by An Hui Tian Qi Chemical Engineering Co., Ltd. (Anhui, China). 1-(Triphenylmethyl)-L-histidine methyl ester monohydrochloride (HHis(Trt)-OMe·HCl) was obtained from GL Biochem Co. Ltd. (Shanghai, China). PTX was obtained from Xian Helin Pharm Co., Ltd. (Xian, China). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5diphenyl-tetrazolium bromide (MTT), coumarin-6, pyrene, diamidino-phenylindole (DAPI), and propidium iodide (PI) were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). DMEM were supplied by Gibco (BRL, MD, USA). Lysotracker Red DND-99 was supplied by Shanghai qcbio Science and Technologies Co., Ltd. (Shanghai, China). Other reagents were chromatographic or analytical grade. Human breast carcinoma MCF-7 cell line, CD44 receptor overexpressed,31,32 was cultured as described before.10 2.2. Polymer Synthesis and Characterization. 2.2.1. Synthesis of HA-DOCA Polymer. HA (100 mg), NHS (58 mg), and EDC (96 mg) were dissolved in formamide under stirring for 2 h. DOCA (196 mg) in N,N-dimethylformamide (DMF) was added into HA solution, and the reaction mixture was stirred at 50 °C for 12 h and at room temperature for further 24 h. The resultant solution was dialyzed against ethanol/water (1:1−1:3, v/v) and distilled water for 2 days, respectively, then filtered and lyophilized.10 C


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Molecular Pharmaceutics HA-DOCA-His polymers were confirmed by nuclear magnetic resonance spectrometery (NMR, 400 Hz, Bruker, Switzerland). HA was measured with D2O as solvent; HA-DOCA and HADOCA-His were dissolved in D2O/CD3OH (1/3, v/v), respectively. 2.3. pH-Dependent Assembly and Disassembly of HADOCA-His Micelles. The pH-dependent assemble and disassemble performance of HA-DOCA-His micelles was examined by monitoring the changes of size distribution, zeta potential, transmittance values, and critical micellar concentration (CMC) at pHs ranging from 7.4 to 5.0. The zeta potential and size distribution changes of HA-DOCA-His micelles at different pH were examined by dynamic light scattering (DLS) instrument (Nicomp-380/ZLS, USA). The CMC value of micelles at various pHs (7.4, 6.5, 6.0, 5.0) was determined to investigate the core integrity change of HADOCA-His micelles by pH. The changes in transmittance value of HA-DOCA-His micelles were assayed with UV spectrometry. 2.4. Preparation of PTX-Loaded Micelles. Polymeric micelles loading with PTX were prepared using our previous reported probe-type ultrasonication method.10 HA-DOCA or HA-DOCA-His dispersed in phosphate buffered saline (PBS, pH 7.4). PTX (30% mass weight of polymer) dissolved in anhydrous ethanol was added into HA-DOCA or HA-DOCAHis solution under stirring. The micelle solution was ultrasonicated at 200 W for 5 min in ice bath. The PTXloaded micelles was filtered through 0.45 μm microfiltration membrane. 2.5. Characterizations of HA-DOCA and HA-DOCA-His Micelles. 2.5.1. 1H NMR Identification of Micellar Structure. The micellar forming characteristic of HA-DOCA-His with core−shell structure in aqueous environment was confirmed by analyzing HA and HA-DOCA-His micelles on a H 1NMR spectroscopy (400 Hz, Bruker, Switzerland) in D2O and HADOCA-His micelles in D2O/CD3OH (1:3, v/v). 2.5.2. CMC Measurement. The micellar forming characteristics of HA-DOCA and HA-DOCA-His were validated by quantitatively assaying the CMC with pyrene as fluorescence probe.10 In brief, HA-DOCA and HA-DOCA-His micelles at concentrations ranging from 1.0 × 10−4 to 5 × 10−1 mg/mL were added into pyrene, which was controlled at a final concentration of 6.0 × 10−7 M. The fluorescence spectra of micelles was performed with a excitation at 336 nm and emission recorded from 360 to 450 nm. The CMC was calculated by the intensity ratio of the peak at 373 nm to the peak at 384 nm plotted versus the logarithm of micellar concentration. 2.5.3. Physicochemical Characterization of Micelles. The size distribution and zeta potential of micelles were determined by DLS technique. The morphology of HA micelles loading with PTX was evaluated by transmission electron microscopy (TEM, Tecnai 275 G220, FEI, USA). Drug loading capacity (DLC) and drug encapsulation efficiency (DEE) were determined to evaluate the PTX entrapment performance of micellar system. PTX concentration in micellar solution was assayed by high performance liquid chromatography (HPLC) method, which was described in our previous report.10 DEE and DLC were calculated by the following equations: DEE(%) =

WPTX incorporated in micelle Wtotal PTX added in micelle

DLC(%) =

WPTX incorporated in micelle Wtotal micelle

× 100

The existence form of PTX in micellar system was characterized by studying the physical properties of PTX, blank micelles, physical mixture of PTX and blank micelles, and PTX-loaded micelles by X-ray diffraction (XRD). 2.5.4. In Vitro PTX Release. In vitro pH-dependent PTX release pattern was investigated by our previous reported dynamic dialysis method.10 Typically, 1 mg of PTX-loaded HADOCA-His micelles were transferred into dialysis bag (molecular weight cutoff = 7000) and immersed in 80 mL of PBS with 2% Cremophor EL at pHs of 7.4, 6.5, 6.0, and 5.0, under stirring at 100 rpm and 37 °C. PTX release from HADOCA-His micelles was assayed at predetermined time by HPLC method. The PTX release from commercial Taxol formulation and PTX-loaded HA-DOCA micelles were also investigated for comparison. 2.6. Intracellular Trafficking of HA-DOCA-His Micelles. Coumarin-6 was loaded in HA micellar system as fluorescence probe to investigate the intracellular trafficking and uptake of drug-loaded micelles. MCF-7 cells were seeded in a 6-well plate (1 × 105 cells/well) with coverslips and incubated for 24 h. Following incubation with HA-DOCA-His micelles loading with coumarin-6 (10 μg/mL) at 37 °C for 1 and 3 h, the cells were washed with cold PBS, stained with Lysotracker Red DND-99, fixed by 70% ethanol, then stained the nuclei with DAPI. After washing the cells with PBS and mounting on a slide, the intracellular distribution and endosomal triggered drug release behavior of HA-DOCA-His micelles were observed by confocal laser scanning microscopy (CLSM).29 2.7. Intracellular Uptake. MCF-7 cells were seeded in a 6well plate (1 × 105 cells/well) with coverslips and incubated for 24 h. The coumarin-6 formulations dispersed in serum-free culture medium were added and incubated for 1 and 3 h. Cells were washed by cold PBS, fixed by 70% ethanol, further stained the nuclei by PI. After washed with PBS, the cell monolayer was mounted on a slide. Afterward, the intracellular uptake and distribution of micelles was visualized using a CLSM. Cellular uptake efficiency of micellar systems was quantitatively assayed by flow cytometry. MCF-7 cells treated with different coumarin-6 formulations were performed as described above. After treated for 1 and 3 h, respectively, cells were washed and resuspended by PBS, then assayed by flow cytometry. To confirm the receptor mediated endocytosis function of HA micelles interacted with CD44 receptors for uptake improvement, coumarin-6 loaded micellar formulations were added into MCF-7 cells after preincubated with HA (10 mg/ mL) polymer for 2 h. The following procedure were performed as described in CLSM. 2.8. pH-Dependent Cytotoxicity Assays. To testify the effect of CD44 receptor-targeting and endosomal pH-triggered drug release of HA micelles on tumor cell growth inhibition, the MTT assay was carried out as a function of pH in MCF-7 cells.27 Briefly, MCF-7 cells were seeded in a 96-well plate (3 × 103 cells/well) and incubated for 24 h. Blank micelles, PTX solution, and PTX-loaded micelles at a gradient concentration in culture medium of different pH (7.4, 6.8, and 5.8) were added into cells and incubated for 48 h. MTT assay was carried out as described in our previous report.10 The relative cell viability was calculated by the following equation:

× 100 D


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Molecular Pharmaceutics cell viability(%) =

A sample − A blank Acontrol − A blank

× 100

Asample and Acontrol are the absorbance in the presence and absence of sample treatment, respectively. Ablank is the absorbance of culture medium. 2.9. In Vivo Therapeutic Efficacy. MCF-7 cells were inoculated into female nude mice at a density of 1× 107 cells/ mouse. When the tumor volume reached 50−100 mm3, mice were divided into four groups. Saline, taxol, PTX-loaded HADOCA micelles, and PTX-HA-DOCA-His micelles were intravenously injected into mice, respectively, on days 0, 3, and 6 with PTX dose of 10 mg/kg. The tumor sizes and body weights of all mice were measured every 3 days. Tumor volumes were calculated by the following equation: (longest tumor diameter × widest tumor diameter2)/2. Mice were sacrificed after 21 days, and tumors were harvested. 2.10. Statistical Analysis. All measured data are expressed as mean ± standard deviation (SD). Two-tailed Student’s t test between two groups is performed for statistical analysis. In all statistical analyses, p < 0.05 was considered statistically significant, and p < 0.01 was considered highly statistically significant.

3. RESULTS AND DISCUSSION 3.1. Polymer Synthesis and Characterization. Multifunctional micelles combined active targeting with intracellular pH-sensitivity strategies facilitated the chemotherapy efficacy improvement. In our design, HA was used as a tumor targeting moiety for hydrophobized modification due to its CD44 receptor targeting property. His was conjugated to make HA polymer with pH responsive property. The further attachment with DOCA shifted the pH-sensitivity of His to exhibit an endosome pH. This novel approach might help trigger the activation of nanosystems to improve tumor targeting and intracellular drug delivery. The chemical structure of the synthesized polymers was verified using 1H NMR (Figure 2). As shown in the spectrum of HA, the peaks appeared at 1.95 ppm (−NHCOCH3), 3.0− 4.0 ppm (glucosidic H), and 4.35−4.45 ppm (anomeric H) could be identified as the characteristic signals of HA. In the spectrum of HA-DOCA-His, the characteristic signal of methyl protons (c, NHCO−CH3) in HA presented at 2.0 ppm. The peak at 7.11 and 8.44 ppm showed the methylene protons (a, −N−CHC−; b, −NCH−) located in imidazole group of His. The signals appeared at 0.6−2.5 ppm presented the protons (−CH2−, −CH3) of DOCA. Above results proved the presence of DOCA-His in the HA-DOCA-His conjugate. The characteristic signals presented at 0.67−1.60 ppm confirmed the grafting of DOCA to HA. 1H NMR results indicated HADOCA and HA-DOCA-His were successfully synthesized. The degree of substitution (DS) of DOCA to HA was determined from intensity ratio of −CH3 in DOCA and −NHCOCH3 in HA, and DS of DOCA-His to HA was calculated by the intensity ratio of the methylene protons (a, −N−CHC−; b, −NCH−) located in imidazole of His and −NHCOCH3 (c) in HA. The DS of DOCA and DOCAHis in HA-DOCA and HA-DOCA-His polymers calculated from 1H NMR spectrum was 19.2% and 16.7%, respectively. 3.2. pH-Responsive Disassemble Behavior of HADOCA-His Micelles. The pH-dependent structural transformation behavior of HA-DOCA-His micelles was measured in terms of the change of size distribution, zeta potential, CMC,

Figure 2. 1H NMR spectrum of HA in D2O (A), HA-DOCA (B), and HA-DOCA-His (C) polymers in D2O/CD3OH (1/3, v/v), and HADOCA-His micelles in D2O (D).

and transmittance as a function of pH, which is presented in Figure 3. The transmittance of micelles was 97.7% at pH 7.4, while the transmittance decreased gradually as pH reduced below 6.0, indicating the transmittance transition point of E


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Figure 3. pH-dependent aggregation and deformation of HA-DOCA-His micelles. (A) Plots of the transmittance and zeta potential; (B) particle size distribution; (C) plots of the particle size and PI; (D) changes in the intensity ratio I373/I384 from pyrene emission spectra of HA-DOCA-His micelles at different pHs.

Figure 4. (A) Plots of I373/I384 against the logarithrm of HA-DOCA and HA-DOCA-His concentration. (B) TEM image of PTX-loaded HA-DOCA micelles. (C) TEM image of PTX-loaded HA-DOCA-His micelles. (D) XRD profiles of PTX, blank micelles, physical mixture of PTX and blank micelles, and PTX-loaded HA-DOCA-His micelles.

micelle occurred at pH 6.0 (Figure 3A). Meanwhile, the size distribution and zeta potential of micelles remained almost unchanged at pH > 6.5. As pH goes down between 6.0 and 5.0 (endosomal pH value), the size distribution, polydispersity index (PI), and zeta potential increased obviously (Figure 3B,C).

In addition, the pH-induced deformation of HA-DOCA-His micelles was investigated by monitoring the CMC value changes at different pHs. The CMC value began to increase gradually below pH 6.0, suggesting pyrene molecules entrapped in micellar core were exposed to aqueous solution, verifying the disassembly of the core−shell structural micelles (Figure 3D). Above obvious change in transmittance, size distribution, zeta F


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Molecular Pharmaceutics Table 1. Physicochemical Characterization of Blank and PTX-Loaded Micelles (Mean ± SD) formulation HA-DOCA PTX-HA-DOCA HA-DOCA-His PTX-HA-DOCA-His

PTX/carrier (w/w, %) 30 30

size (nm) 234.6 197.3 237.4 184.9

± ± ± ±

zeta (mV) −25.0 −18.3 −23.9 −17.9

1.2 3.5 13.1 7.4

± ± ± ±

0.7 1.2 0.3 1.9

DEE (%)

DLC (%)

stability (d)

90.9 ± 0.9

18.6 ± 1.3

7

91.0 ± 0.8

18.9 ± 3.9

6

Figure 5. Cumulative release of PTX from taxol solution: PTX-loaded HA-DOCA micelles at pH 7.4 (A) and PTX-loaded HA-DOCA-His micelles at different pHs (B).

blank and PTX-loaded micelles ranged from −17.9 to −25.0 mV. DEE and DLC were calculated to evaluate the PTX entrapment efficacy of HA-DOCA and HA-DOCA-His micelles, which are summarized in Table 1. HA-DOCA-His micelles showed excellent PTX encapsulation performance, with DLC of 18.9% and DEE of 91.0%. The morphology of HA micelles were characterized by TEM (Figure 4B,C). The HA-DOCA-His and HA-DOCA micelles loading with PTX had nearly spherical morphology and were well dispersed. Compared with the size determination by dynamic light scattering, the smaller size was probably due to the micellar shrinkage during TEM sample preparation. As shown in Figure 4D, in XRD spectrum of PTX, three sharp diffraction peaks at 2θ of 5.581°, 8.904°, and 12.387° and numerous small diffraction peaks between 2θ of 15° to 30° were shown. The micelles loading with PTX had no diffraction peak of PTX, and presented similar XRD pattern to blank micelles, but physical mixture of blank micelle and PTX showed similar diffraction peaks of PTX. It was indicated PTX could be physical encapsulated in HA-DOCA-His micelles as molecular or amorphous state. With the purpose of endosomal-pH sensitivity, the HADOCA-His micelles for targeting drug delivery to tumors and releasing PTX inside tumor cells were stable at physiological pH of 7.4 and tumor extracellular pH of 6.5−7.2 but disassemble under endosomal pH of 5.0−6.0, facilitating drug release inside tumor cells.13,33 With this in mind, the PTX release patterns from HA-DOCA-His micelles were tested at four different pH (7.4, 6.5, 6.0, and 5.0) values at 37 °C (Figure 5). PTX release from HA-DOCA-His micelles was pH-dependent. HA-DOCA-His micelles showed a slow PTX release at pH 7.4 and 6.5, with only 14.5% and 19.6% release after 6 h. However, the PTX release was accelerated at pH 6.0 and 5.0, with 41.8% and 58.6% of PTX being released in 6 h, respectively. This phenomenon could be explained by the endosomal pH-induced deformation of the HA-DOCA-His micelles, contributing to more rapid PTX release from micelles. For comparison, Taxol showed an essentially complete release

potential, and CMC results indicated that HA-DOCA-His micelles disassembled into irregular and loose particles below pH 6.0, demonstrating the endosomal-pH sensitive property of HA-DOCA-His micelles. As a comparison, the size of insensitive HA-DOCA micelle did not change at pH ranging from 5.0 to 7.4. It was showed strong evidence that the endosomal-pH triggered disassembly of HA-DOCA-His micelles was contributed to the protonation of imidazole ring in His. Taken together, it was indicated HA-DOCA-His micelles would be stable at physiological condition (pH 7.4) and tumor extracellular compartment (pH > 6.5), and be endocytosed as the micelle entity into tumor cells, then would disintegrate and trigger PTX quick release at endosomal microenvironment (pH 5.0−6.0). 3.3. Preparation and Characterization of Micelles. PTX was physically incorporated in HA micelles using ultrasonic method. Figure 2 presented the core−shell structural confirmation results of HA-DOCA-His micelles by 1H NMR. HA-DOCA-His dissolved in D2O/CD3OH presented all proton signals, owing to its monomer state existed in organic solvent (Figure 2B). However, the characteristic peaks of DOCA and His disappeared from HA-DOCA-His micelles dissolved in D2O, showing similar spectrum of HA in D2O (Figure 2D). These data strongly suggested that the micellar structure could be formed by the self-assembly of HA-DOCAHis in aqueous solution, with HA served as hydrophilic shell and DOCA-His embedded as interior core. Figure 4A shows the I373/I384 value changes against the logarithm of HA-DOCA and HA-DOCA-His concentration. HA-DOCA and HA-DOCA-His comb-type polymers had CMC value of 26.2 and 19.8 μg/mL, respectively. The lower CMC value would ensure the micelle keep good stability during blood circulation.4 Table 1 summarized the physicochemical characteristics of blank micelles and PTX-loaded micelles. PTX-loaded HADOCA-His and HA-DOCA micelles presented mean particle size of 184 and 197 nm, respectively, which were favorable for passive tumor targeting by EPR effect. The zeta potentials of G


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Figure 6. Intracellular trafficking of coumarin-6 loaded in HA-DOCA-His micelles incubated in MCF-7 cells after 1 and 3 h.

Figure 7. CLSM images of coumarin-6 solution, coumarin-6 loaded HA-DOCA, and HA-DOCA-His micelles incubated in MCF-7 cells for 1 h (A) and 3 h (B), and cells pretreated with free HA polymer (B). H


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Figure 8. Flow cytometry histograms of coumarin-6 uptake in MCF-7 cells treated with coumarin-6 solution, HA-DOCA, and HA-DOCA-His micelles loaded with coumarin-6 for 1 h (A) and 3 h (B).

Figure 9. In vitro cytotoxicity of blank micelles (A); PTX solution and PTX-loaded HA micelles (B) against MCF-7 cells after incubation in culture medium of pH 7.4, 6.8, and 5.8 for 48 h.

extracellular compartments, but also with the burst release of encapsulated PTX in endosome within cells. This would ensure effective intracellular drug delivery and be favorable for cancer therapy. 3.4. Intracellular Trafficking of HA-DOCA-His Micelles. The CD44 receptor-mediated endocytosis pathway facilitates

of PTX in 16 h, and PTX loaded in HA-DOCA micelles exhibited a slow release manner in 48 h under physiological condition (pH 7.4). From this point of view, it was clear that the endosomal pHresponsive ability of HA-DOCA-His micelles was not only associated with superior stability during circulation and tumor I


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Molecular Pharmaceutics delivery of the endocytosed HA-DOCA-His micelles to endosomes. To illustrate the feasibility of HA-DOCA-His micelles for PTX cytosolic delivery, the internalization and endosomal drug release behavior of micelles was evaluated (Figure 6). After an incubation time of 1 h, green coumarin-6 fluorescence signal colocalized with red fluorescence signal of endosomes, affording a yellow fluorescence in merged images. This suggested micelles were first delivered in endosomes after being endocytosed into cells. After a prolonged incubation time of 3 h, strong coumarin-6 fluorescence signal was mainly distributed in perinuclei region, while yellow fluorescence gradually disappeared. This suggested the HA-DOCA-His micelles was able to disassemble rapidly in endosomal microenvironment, promoted the release of coumarin-6, and covered all intracellular regions of cells. The results indicated the protonation of His segments favored the micellar disassembly in the acidic endosomal compartments after endocytosis, followed by the incorporated drug release and escaped from endosome via His induced endosomal membrane disruption, which facilitated the therapeutic efficacy improvement of the loaded drugs. 3.5. Cellular Uptake. The intracellular distribution and uptake efficiency of HA-DOCA and HA-DOCA-His micelles were observed by CLSM in MCF-7 cells (Figure 7). A weak fluorescence signal of coumarin-6 solution marked was mainly localized in cytoplasm after incubated for 1 h. HA-DOCA and HA-DOCA-His micelles showed stronger coumarin-6 fluorescence signal than that of coumarin-6 solution. Maybe the CD44 receptor-mediated endocytosis pathway could facilitate and promote the efficient internalization of HA micelles into CD44 receptor-overexpressed cells. Additionally, HA-DOCAHis micelles show stronger fluorescent signals than those of HA-DOCA micelles. The coumarin-6 loaded in HA-DOCA-His micelles could be effectively delivered into cytoplasm by combined CD44 receptor active targeting mediated endocytosis with endosomal pH-responsive triggered burst drug release targeting strategy, resulting in more efficient cytosolic drug delivery. The coumarin-6 fluorescence signal was markedly increased, and a large amount of coumarin-6 was distributed in perinuclei region for all the groups after 3 h incubation, which indicated the cellular uptake of HA micelles was time dependent. The uptake efficiency of micellar system, with free drug form as comparison, was further quantitatively assayed by flow cytometry. As shown in Figure 8, cells treated with HA-DOCAHis micelles showed the highest level of coumarin-6 fluorescence signal. All intracellular uptake studies confirmed the hypothesis that the combination of CD44 receptor mediated active targeting and endosomal-pH sensitivity strategies could facilitate the intracellular uptake and release of HA-DOCA-His micelles. As presented in Figure 7B, it was found that the cells pretreated with free HA showed a decreased intracellular uptake of HA-DOCA and HA-DOCA-His micelles. The results verified the specific binding of HA to CD44 receptors and suggested the important role of HA-dependent specific CD44 receptor-mediated endocytosis pathway in efficient internalization of HA-DOCA and HA-DOCA-His micelles. 3.6. pH-Dependent in Vitro Cytotoxicity. The MTT assays of PTX-loaded HA-DOCA-His micelles characterized in normal tissue, tumor extracellular compartment, and endosomal compartment were performed in the culture medium of pH 7.4, 6.8 and 5.8, respectively.28

As shown in Figure 9A, blank micelles showed no significant cytotoxicity at pH 7.4, 6.8, and 5.8, even with a polymer concentration of 1 mg/mL. This indicated that HA-DOCA and HA-DOCA-His polymers could be employed as nontoxic nanocarriers for chemotherapeutic drugs. To evaluate the feasibility of active targetability and endosomal pH-sensitivity dual functional combination of micelles for chemotherapy, the cytotoxicity of PTX solution, CD44 receptor targeting single functional HA-DOCA micelles, and CD44 receptor targeting combined with endosomal pHsensitive dual functional HA-DOCA-His micelles at different pHs was studied (Figure 9B). Compared with free PTX passive diffused through cell membrane, PTX-encapsulated in HA-DOCA micelles were uptaken into MCF-7 cells by CD44 receptor-mediated endocytosis pathway, resulting in an increased cellular uptake efficiency and higher cytotoxicity. However, there was no significant difference among the cell viability of HA-DOCA micelles in pH 7.4, 6.8, and 5.8, owing to the slow PTX release at these pHs. At pH 7.4 and 6.8, PTX-loaded HA-DOCA-His micelles demonstrated similar cytotoxicity with HA-DOCA micelles, and no obvious difference occurred between these pH values. More important, the cytotoxicity of PTX-loaded HA-DOCAHis micelles exhibited a pH-dependent manner and especially exhibited much higher cytotoxicity at pH 5.8. On entry into MCF-7 cells by CD44 receptor mediated endocytosis, the endosomal pH-triggered micellar disassembly and burst release of PTX into cytoplasm contributed to the cytotoxicity enhancement of PTX loaded in HA-DOCA-His micelles. The results were consistent with the pH-dependent PTX release profile, intracellular distribution, and endosomal drug release behavior of HA-DOCA-His micelles. Above results suggested the dual targeting combination of efficient active internalization and endosomal-pH triggered drug release strategies were facilitated to efficient cancer cells growth inhibition. The dual function of targeting and intracellular pHresponsive strategies could generate a combination effect. After endocytosed, endosomal pH-sensitivity property and fusogenic activity of HA-DOCA-His micelles could promote to trigger PTX release from endosomal compartment to cytoplasm. Figure 10 demonstrates such combined effect. The targeting and endosomal pH-sensitive PTX-loaded HA-DOCAHis micelles showed an enhanced cell growth inhibition, with 6% cell viability at PTX concentration of 25 μg/mL, but the targeting and pH-insensitive HA-DOCA micelles showed 27% cell viability at equal PTX concentration. The difference in cytotoxicity might be due to the triggered PTX release and efficient endosomal escape of HA-DOCA-His micelles. In conclusion, the results demonstrated the enhanced intracellular accumulation of HA-DOCA-His micelle in MCF7 cells via receptor mediated endocytosis, endosomal-pH triggered PTX release, and endosomal escape could provide a more efficient way to cell proliferation inhibition and anticancer therapeutic efficacy enhancement. 3.7. In Vivo Therapeutic Efficacy. Antitumor activity of PTX-HA-DOCA-His micelles was studied in nude mice bearing MCF-7 tumor and compared to Taxol and PTX-HA-DOCA micelles. As shown in Figure 11A, the tumor size increased rapidly for the mice treated with saline. The tumor growth inhibition rates were calculated to be 43.2%, 60.0%, and 76.9%, respectively, for the treated groups of taxol, PTX-loaded HADOCA, and HA-DOCA-His micelles. As shown in Figure 11B, J


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Body weight loss is one of the indicators for evaluation of formulation toxicity. The toxicity of all the formulations was evaluated by following their effects on body weight. As shown in Figure 11C, the body weight of mice increased slightly for all treatment groups, indicating our new HA-DOCA-His micellar system could achieved effective antitumor activity with minimal toxicity. The superior antitumor efficacy and excellent safety of PTXloaded HA-DOCA-His micelles could be attributed to their minimal drug leaking during blood circulation and in tumor extracellular compartment, enhanced tumor-targeting ability via the particle size-mediated EPR effect, increased internalization in tumor cells via CD44-receptor active targeting endocytosis, and intracellular triggered PTX release induced by His protonation in endosomal pH.

4. CONCLUSION A novel HA-DOCA-His micelle integrating CD44 receptor active targeting and endosomal pH-sensitivity properties was successfully developed for effective cytosolic delivery of PTX. The HA-DOCA-His micelles showed excellent endosomal pHresponsive ability and triggered PTX release performance. The dual functional HA-DOCA-His micelles could keep stable during blood circulation and in tumor extracellular pH, then endocytosed into cells as intact micelle form by receptor-

Figure 10. In vitro cytotoxicity of PTX solution, PTX-loaded HADOCA, and HA-DOCA-His micelles against MCF-7 cells after incubation in pH 5.8 culture medium for 72 h (mean ± SD, n = 3).

the isolated tumors for the HA-DOCA-His micelles group showed much smaller sizes, which further confirmed the superiority in therapeutic efficacy improvement of HA-DOCAHis micelles.

Figure 11. In vivo antitumor activity of taxol, PTX-loaded HA-DOCA, and HA-DOCA-His micelles in MCF-7 tumor-bearing nude mice. (A) The plots of relative tumor volume of mice treated with different PTX formulations against different days postinoculation (mean ± SEM, n = 6), *P < 0.05; **P < 0.01. (B) Images of excised tumor between different treatment groups on 21 days post-first treatment. (C) Body weight changes of mice monitored in different treatment groups (mean ± SD, n = 6). K


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activatable paclitaxel prodrug micelles effectively target and treat CD44-overexpressing human breast tumor xenografts in vivo. Biomaterials 2016, 84, 250−261. (14) Zhang, L.; Zhou, J. P.; Yao, J. Improved anti-tumor activity and safety profile of a paclitaxel-loaded glycyrrhetinic acid-graft-hyaluronic acid conjugate as a synergistically targeted drug delivery system. Chin. J. Nat. Med. 2015, 13 (12), 915−924. (15) Xu, C. R.; Lv, Y. P.; Qin, C.; Shen, L. J.; Yin, L. F. Selfassembled nanoparticles from hyaluronic acid-paclitaxel prodrugs for direct cytosolic delivery and enhanced antitumor activity. Int. J. Pharm. 2015, 493 (1−2), 172−181. (16) Xin, D. C.; Wang, Y.; Xiang, J. N. The use of amino acid linkers in the conjugation of paclitaxel with hyaluronic acid as drug delivery system: synthesis, self-assembled property, drug release, and in vitro efficiency. Pharm. Res. 2010, 27 (2), 380−389. (17) Torchilin, V. P. Multifunctional and stimuli-sensitive pharmaceutical nanocarriers. Eur. J. Pharm. Biopharm. 2009, 71 (3), 431−444. (18) Sun, J.; Luo, C.; Wang, Y. J.; He, Z. G. The holistic 3M modality of drug delivery nanosystems for cancer therapy. Nanoscale 2013, 5 (3), 845−859. (19) Zhao, H. J.; Li, Q.; Hong, Z. H. Paclitaxel-loaded mixed micelles enhance ovarian cancer therapy through extracellular pH-triggered PEG detachment and endosomal Escape. Mol. Pharmaceutics 2016, 13, 2411. (20) Tian, L.; Bae, Y. H. Cancer nanomedicines targeting tumor extracellular pH. Colloids Surf., B 2012, 99, 116−126. (21) Danhier, F.; Feron, O.; Preat, V. To exploit the tumor microenvironment: passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J. Controlled Release 2010, 148 (2), 135− 146. (22) Wu, Y. L.; Chen, W.; Meng, F. H.; Wang, Z. J.; Cheng, R.; Deng, C.; Liu, H. Y.; Zhong, Z. Y. Core-crosslinked pH-sensitive degradable micelles: a promising approach to resolve the extracellular stability versus intracellular drug release dilemma. J. Controlled Release 2012, 164 (3), 338−345. (23) Quadir, M. A.; Morton, S. W.; Deng, Z. J.; Shopspwz, K. E.; Murphy, R. P.; Epps, T. H.; Hammond, P. T. PEG-polypeptide block copolymers as pH-responsive endosome-solubilizing drug nanocarriers. Mol. Pharmaceutics 2014, 11 (7), 2420−2430. (24) Jin, Y. H.; Hu, H. Y.; Qiao, M. X.; Zhu, J.; Qi, J. W.; Hu, C. J.; Zhang, Q.; Chen, D. W. pH-sensitive chitosan-derived nanoparticles as doxorubicin carriers for effective anti-tumor activity: preparation and in vitro evaluation. Colloids Surf., B 2012, 94, 84−191. (25) Hu, W.; Qiu, L. P.; Cheng, L.; Hu, Q.; Liu, Y.; Hu, Z. Y.; Chen, D. W.; Cheng, L. F. Redox and pH dual responsive poly(amidoamine) dendrimer-poly(ethylene glycol) conjugates for intracellular delivery of doxorubicin. Acta Biomater. 2016, 36, 241−253. (26) Sun, Y.; Li, Y. P.; Nan, S. L.; Zhang, L. Z.; Huang, H. L.; Wang, J. Y. Synthesis and characterization of pH-sensitive poly(itaconic acid)poly(ethylene glycol)-folate-poly(L-histidine) micelles for enhancing tumor therapy and tunable drug release. J. Colloid Interface Sci. 2015, 458, 119−129. (27) Zhang, X. J.; Chen, D. W.; Ba, S.; Zhu, J.; Zhang, J.; Hong, W.; Zhao, X. L.; Hu, H. Y.; Qiao, M. X. Poly(l-histidine) Based Triblock Copolymers: pH Induced Reassembly of Copolymer Micelles and Mechanism Underlying Endolysosomal Escape for Intracellular Delivery. Biomacromolecules 2014, 15 (11), 4032−4045. (28) Wu, H.; Zhu, L.; Torchilin, V. P. pH-sensitive poly (histidine)PEG/DSPE-PEG co-polymer micelles for cytosolic drug delivery. Biomaterials 2013, 34 (4), 1213−1222. (29) Kim, D.; Lee, E. S.; Oh, K. T.; Gao, Z. G.; Bae, Y. H. Doxorubicin-loaded polymeric micelle overcomes multidrug resistance of cancer by double-targeting folate receptor and early endosomal pH. Small 2008, 4 (11), 2043−2050. (30) Li, Z.; Qiu, L. P.; Chen, Q.; Hao, T. N.; Qiao, M. X.; Zhao, H. X.; Zhang, J.; Hu, H. Y.; Zhao, X. L.; Chen, D. W.; Mei, L. pHsensitive nanoparticles of poly(L-histidine)-poly(lactide-co-glycolide)tocopheryl polyethylene glycol succinate for anti-tumor drug delivery. Acta Biomater. 2015, 11, 137−150.

mediated endocytosis, followed by endosomal-pH triggered drug release into the cytoplasm, induced the increased cellular uptake, and pronounced highest cytotoxicity of PTX at lowered pH values. In vivo, PTX-loaded HA-DOCA-His micelles showed superior efficacy in tumor growth inhibition compared with PTX-loaded HA-DOCA micelles as well as taxol and an excellent safety profile. All the results suggested that the dual functional HA-DOCA-His micelles held promising approach for intracellular delivery of hydrophobic therapeutic agents, which was favorable of antitumor efficacy improvement.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86-951-6880693. Fax: +86-951-6880693. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Nature Science Foundation of China (No. 81360483 and No. 81660590). REFERENCES

(1) Misra, R.; Acharya, S.; Sahoo, S. K. Cancer nanotechnology: application of nanotechnology in cancer therapy. Drug Discovery Today 2010, 15 (19−20), 842−850. (2) Davis, M. E. Nanoparticle therapeutics: an emerging treatment modality for cancer. Nat. Rev. Drug Discovery 2008, 7 (9), 771−782. (3) Yokoyama, M. Polymeric micelles as a new drug carrier system and their required considerations for clinical trials. Expert Opin. Drug Delivery 2010, 7 (2), 145−158. (4) Liu, Y. H.; Sun, J.; Zhang, P.; He, Z. G. Amphiphilic polysaccharide-hydrophobicized graft polymeric micelles for drug delivery nanosystems. Curr. Med. Chem. 2011, 18 (17), 2638−2648. (5) Torchilin, V. P. Tumor delivery of macromolecular drugs based on the EPR effect. Adv. Drug Delivery Rev. 2011, 63 (3), 131−135. (6) Nicolas, J.; Mura, S.; Brambilla, D.; Mackiewicz, N.; Couvreur, P. Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery. Chem. Soc. Rev. 2013, 42 (3), 1147−1235. (7) Negi, L. M.; Talegaonkar, S.; Jaggi, M.; Ahmad, F. J.; Iqbal, Z.; Khar, R. K. J. Role of CD44 in tumor progression and strategies for targeting. Drug Target. 2012, 20 (7), 561−573. (8) Li, J.; Huo, M. R.; Wang, J.; Zhou, J. P.; Mohammad, J. M.; Zhang, Y. L.; Zhu, Q.; Waddad, A. Y.; Zhang, Q. Redox-sensitive micelles self-assembled from amphiphilic hyaluronic aciddeoxycholic acid conjugates for targeted intracellular delivery of paclitaxel. Biomaterials 2012, 33 (7), 2310−2320. (9) Choi, K. Y.; Yoon, H. Y.; Kim, J. H.; Bae, S. M.; Park, R. W.; Kang, Y. M.; Kim, I. S.; Kwon, I. C.; Choi, K.; Jeong, S. Y.; Kim, K.; Park, J. H. Smart nanocarrier based on PEGylated hyaluronic acid for cancer therapy. ACS Nano 2011, 5 (11), 8591−8599. (10) Liu, Y. H.; Sun, J.; Cao, W.; Yang, J. H.; Lian, H.; Li, X.; Sun, Y. H.; Wang, Y. J.; Wang, S. L.; He, Z. G. Dual targeting folateconjugated hyaluronic acid polymeric micelles for paclitaxel delivery. Int. J. Pharm. 2011, 421 (1), 160−169. (11) Prestwich, G. D. Hyaluronic acid-based clinical biomaterials derived for cell and molecule delivery in regenerative medicine. J. Controlled Release 2011, 155 (2), 193−199. (12) Zhao, X. B.; Jia, X.; Liu, L.; Zeng, J.; Tian, K.; Zhou, T. T.; Liu, P. Double-cross-linked hyaluronic acid nanoparticles with pH/ reduction dual-responsive triggered release and pH-modulated fluorescence for folate-receptor-mediated targeting visualized chemotherapy. Biomacromolecules 2016, 17 (4), 1496−1505. (13) Zhong, Y. N.; Goltsche, K.; Cheng, L.; Xie, F.; Meng, F. H.; Deng, C.; Zhong, A. Y.; Haag, R. Hyaluronic acid-shelled acidL


Article

Molecular Pharmaceutics (31) Lee, H.; Lee, K.; Park, T. G. Hyaluronic acid-paclitaxel conjugate micelles: synthesis, characterization, and antitumor activity. Bioconjugate Chem. 2008, 19, 1319−1325. (32) Lee, E. S.; Na, K.; Bae, Y. H. Polymeric micelles for tumor pH and folate-mediated targeting. J. Controlled Release 2003, 91, 103−113. (33) Yin, H.; Lee, E. S.; Oh, K. T.; Kim, D. I.; Bae, Y. H. Physicochemical properties of pH sensitive poly(L-histidine)-bpoly(ethylene glycol)/poly(L-lactide)-b-poly(ethylene glycol) mixed micelles. J. Controlled Release 2008, 126, 130−138.

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CD44 Receptor Targeting and Endosomal pH-Sensitive Dual

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