Multifunctionalized Micelles Facilitate Intracellular Doxorubicin

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Multi-functionalized micelles facilitate intracellular doxorubicin delivery for reversing multidrug resistance of breast cancer Aichen Cao, Panqin Ma, Tong Yang, Yang Lan, Shuangyu Yu, Lu Liu, Yue Sun, and Yanhua Liu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00094 • Publication Date (Web): 18 Apr 2019 Downloaded from http://pubs.acs.org on April 19, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

OH CH3

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Molecular Pharmaceutics

H N O

O

O

N

NH

Blood vessel

1 2 O 3 O C CH2OH 4 CH2OH COOH O O O O O 5 OH DOX O O OH O O HA-DOCA-His 6 OH OH OH NHCOCH 3 NHCOCH 3 OH 7 n 8 9 Self-assembly O H 10 HO DOX/HA-DOCA-His-PF 11 O O PF 127 12 micelles 106 106 70 13 CH3 14 15 16 CD44 receptor 17 18 bly 19 m e s s 20 Disa 21 pH 6.5-7.2 22 23 Endosomal 24 25 pH 5.0-6.0 26 27 28 pH-triggered 29 release 30 Endocytosis pH 7.4 Nucleus 31 32 33 34 35 MDR reversion 36 37 38 ACS Paragon Plus Environment 39 EPR effect P-gp protein 40 41

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Article

Title:

Multi-functionalized micelles facilitate intracellular doxorubicin delivery for reversing multidrug resistance of breast cancer

Author: Aichen Cao †, ¶, Panqin Ma §, ¶, Tong Yang †, ¶, Yang Lan †, Shuangyu Yu †, Lu Liu †, Yue Sun †, Yanhua Liu †, ‡, *

Affiliation: †: 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 §: Kangya of Ningxia Pharmaceuticals Corporation Limited, Yinchuan, 750002, China

*Corresponding Author: Associate Prof. Yanhua Liu, Ph D Tel/Fax: +86-951-6880693 E-mail: [email protected]

ACS Paragon Plus Environment

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ABSTRACT Intracellular doxorubicin (DOX) pumping out of cells through P-glycoprotein (P-gp) transporter lead to the reduction of intracellular DOX levels and induce multidrug resistance (MDR). A hyaluronic acid-deoxycholic acid-histidine and Pluronic F127 (PF127) mixed micellar system, named HA-DOCA-His-PF micelles, functionalized with active targeted endocytosis mediated via CD44 receptor, intracellular triggered DOX release under endosome-pH, and combined with PF127 mediated P-gp efflux inhibition was developed for sufficiently intracellular DOX delivery and MDR reversion. The DOX/HA-DOCA-His-PF drug-loaded micelles displayed endosomal pH-mediated self-assembly/disassembly characteristics, triggered DOX release under an endosomal (pH 5.5) environment, and demonstrated enhanced cytotoxicity and superior MDR reversion performance against drug-resistant MCF-7/Adr tumor cells. Importantly, superior antitumor activity of DOX/HA-DOCA-His-PF micelles was presented on the growth inhibition of MCF-7/Adr bearing tumor, by further inhibiting the P-gp activity on intracellular DOX efflux through depleting intracellular ATP content. This multifunctional micellar system could be facilitated intracellular DOX delivery for reversing MDR of breast cancer.

KEYWORDS: DOX; HA-DOCA-His-PF micelles; CD44 receptor targeted endocytosis;

endosomal pH triggered drug release; P-gp mediated efflux inhibition; MDR reversion



1. INTRODUCTION Doxorubicin (DOX) is widely used in the breast cancer chemotherapy [1]. However, its therapeutic activity was limited by the multidrug resistance (MDR) emerged in the entire therapeutic treatment course [2-5]. It is generally believed that when DOX are delivered into cytoplasm, P-glycoprotein (P-gp) transporter will pump the drugs out of MDR cells, which causes the lower DOX accumulation in cytoplasm before reaching its action site of nucleus and further leads to the limited therapy efficacy on MDR tumors [6]. With the development of nanotechnology, polymeric micellar systems provide an alternative strategy to overcome MDR through providing an approach to physical encapsulation or chemical conjugation of therapeutic agents [7-14]. High intracellular endocytosis and triggering drug release into cytoplasm is a primary factor for polymeric micelles designing on MDR reversion [15]. A CD44 receptor- and endosome pH sensitive- dual targeting hyaluronic acid-deoxycholic acid-histidine micellar delivery system, named HA-DOCA-His, was designed for intracellular delivery of therapeutic agents in our previous study [16]. Although this micellar system offered an efficient strategy on cytosolic delivery of PTX, there are also showed limitations on intracellular DOX delivery in multidrug resistant cells due to the lack of inhibition actions on P-gp transporter mediated drug efflux. We have previously found HA-DOCA-His micelles could only effective delivered and released sufficiently DOX into the cytoplasm thoroughly and rapidly, afterwards the intracellular released DOX still would be pumped out from the multidrug resistant cells by P-gp transporters, before being delivered to the drug action site of nucleus [15, 17-19]. Thus, in general, an ideal nano drug delivery system facilitating intracellular DOX delivery for MDR reversion based on HA-DOCA-His carrier is further required to inhibit the P-gp mediated drug pumping


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action on cytosolic released DOX, and effective delivers DOX to its site action, then plays its antitumor action on cancer cells killing [15, 20-23]. Due to the energy dependent property of P-gp transporter, “cutoff” the energy supplement for inhibiting the P-gp function is an efficient approach for P-gp efflux reduction [24-26]. There are important discoveries revealed that Pluronic F127 (PF127) inhibits the pumping action of P-gp through the depletion of ATP in drug-resistant tumor cells [27-29]. It has been utilized as a promising MDR modulator in nano drug delivery system [30-32]. Currently, Pluronic-based mixed micelles with PF127 and Pluronic L61 are under phase III clinical trials for DOX delivery [33-34]. Therefore, combining the MDR tumor-sensitizing features of PF127 with the developed multifunctionalized HA-DOCA-His micelles might be an efficient strategy for intracellular DOX delivery on MDR reversion of breast cancer treatment. Herein, a micellar delivery system (HA-DOCA-His-PF) formed with HA-DOCAHis and PF127 polymers was designed for efficient DOX delivery on MDR reversion achievement. The established micelles could be accumulated and then penetrated into tumors by the enhanced permeability and retention (EPR) effect. Afterwards, the micelles were then endocytosed into MCF-7/Adr tumor cells through active targeted internalization mediated by CD44 receptor, followed by the triggering release of DOX release into cytoplasm under endosomal-pH microenvironment. Afterwards, DOX/HADOCA-His-PF micelles further inhibited P-gp mediated efflux on the cytosolic released DOX, then facilitated the efficient delivery of DOX into nucleus, thereby achieving improved antitumor efficacy on MDR tumor models (Fig. 1). The most important advantage of the DOX/HA-DOCA-His-PF micellar system developed in this study is the integration of active targeting strategy, endosome-pH sensitive targeting and P-gp efflux inhibition approaches in one drug delivery system



provides a promising strategy of MDR reversion on MDR tumor models.

Fig. 1. The DOX loading and micellar-assembly of HA-DOCA-His-PF nano drug delivery system, and the DOX delivery into MCF-7/Adr tumor tissues and cells of HADOCA-His-PF micelles.

2. EXPERIMENTAL SECTION 2.1 Materials HA-DOCA-His polymer was synthesized by our laboratory [16]. PF127 was obtained from Shanghai Chineway Pharmaceutical Tech. Co., Ltd. Doxorubicin·HCl was obtained from Beijing Hvsf Chemical Materials. Hoechst 33342, penicillinstreptomycin solution and LysoTracker Green DND-26 were purchased by SigmaAldrich Co.


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2.2 Cell culture MCF-7 and MCF-7/Adr are DOX-sensitive and DOX-resistant human breast cancer cell lines, respectively, which over-expressed CD44 receptors [35-36]. These two type cell lines are incubated in DMEM medium with FBS (10%, v/v), streptomycin (100 μg/mL) and penicillin (100 IU/mL) in air atmosphere of 5% CO2-95% air atmosphere at 37 °C.

2.3 DOX loaded HA-DOCA-His-PF micelles preparation The doxorubicin base (DOX), encapsulated in micellar system, was synthesized according to the following procedure: Doxorubicin·HCl (1 equiv) was reacted with triethylamine (1.5 equiv) for 24 h in DMSO [37]. HA-DOCA-His (10 mg) and PF127 (2 mg) carriers were dissolved in DMSO. The prepared DOX solution described above at designated drug/carrier weight ratios of 1/5 and 1/10 was mixed with carrier/DMSO solution. The carrier/DOX DMSO solution was then loaded into dialysis bag with a MWCO of 3500 Da, and then dialyzed in PBS (pH 7.4, 10 mM) for removing the organic solvent and free DOX. The DOX loaded HA-DOCA-His micelles were prepared by the same procedure without PF127 added. The preparation of corresponding blank micelles was according to the same procedure described above without DOX added.

2.4 Endosome pH sensitivity The endosome pH-mediated self-assembly/disassembly characteristics was investigated via measuring the pH-dependent particle size changes and the structure integrity changes of DOX/HA-DOCA-His-PF micelles. The size distributions at different pHs (7.4, 6.5 and 5.5) were measured by Malvern Nanosizer through dynamic



light scattering (DLS). The fluorescence intensity of nile red in the micelles under various pHs was measured to estimate the critical micellar concentration (CMC) changes of DOX/HA-DOCA-His-PF micelles under physiological, extracellular and intracellular pH environments [16, 38].

2.5 Characterization of DOX/HA-DOCA-His-PF micelles The size distribution, zeta potential and morphologies of blank carrier and DOXloaded micelles were studied by DLS and transmission electron microscopy (TEM), respectively. Drug loading capacity (DLC) and encapsulation efficiency (DEE) was evaluated by measuring the DOX concentration in micellar formulations, which was determined by micelle-breaking method through HPLC method with fluorescence detector. Following equations are utilized for the DEE and DLC calculation. DEE (%)=

the drug amount incorporated in the micelles ×100 the input drug amount

DLC (%)=

the drug amount incorporated in the micelles ×100 the input drug and carrier amounts

Endosomal pH-triggered release manners of DOX from HA-DOCA-His-PF micelles were measured through dynamic dialysis method with DOX solution as a comparison. Briefly, DOX/HA-DOCA-His-PF micelles were loaded into dialysis bag with MWCO of 3500 Da, and then immersed into PBS (10mM) of various pHs, under 37 ℃ with 100 rpm stirring. The release sample were withdrawn at designated time intervals and analyzed by fluorescence spectrometry.

2.6 Intracellular endosomal pH-triggered DOX release MCF-7/Adr cells were seeded in a 6-well plate with coverslips. Following 24 h cultured, DOX/HA-DOCA-His-PF micelles at a DOX concentration of 20 μg/mL were


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added into cells. After treatment, The DAPI and LysoTracker Green DND-26 were added into the cells for 20 and 30 min, respectively, following washed with PBS. The intracellular DOX triggering release in endosome compartment of HA-DOCA-His-PF micelles was observed under a confocal laser scanning microscope (CLSM) [39].

2.7 Intracellular uptake efficiency MCF-7/Adr and MCF-7 cells were seeded in a 6-well plate with coverslips and cultured 24 h. Then DOX formulations at a DOX concentration of 20 μg/mL were treated into the cells and incubated for 4 h. Afterwards, DAPI was added into the cells, followed by PBS washing. The cellular DOX internalization efficiency of different DOX preparations was investigated by CLSM. To verify the active targeted endocytosis function mediated by CD44 receptors on intracellular internalization facilitating of HA-DOCA-His-PF micelles, HA solution (10 mg/mL) was pre-treated into MCF-7/Adr cells for 2 h before DOX/HA-DOCAHis-PF micelles treatment. The following procedure was carried out as described above.

2.8 In vitro cytotoxicity The MDR reversion was evaluated by studying the in vitro cytotoxicity of DOX/HA-DOCA-His micelles against sensitive and DOX-resistant cells. MCF-7 and MCF-7/Adr cells were seeded in 96-well plates (2 × 103 cells/well) and incubated for 24 h. Then gradient concentrations of various DOX formulations were treated into the cells. After 72 h incubation, the cell proliferation inhibition rate was assayed by MTT method as described before [16]. The parameters of resistant index (RI) and reversal factor (RF) were utilized for evaluation the MDR reversion efficacy, which was calculated by IC50 values obtained



from the in vitro cytotoxicity studies. The RI and RF calculated equations are listed as follows [40-42]. RI = IC50 (DOX formulation)-MCF-7/Adr /IC50 (DOX formulation)-MCF-7 RF = IC50 (DOX solution)-MCF-7/Adr /IC50 (DOX micelles)-MCF-7/Adr The cancer cells with stronger resistance to drugs was indicated a higher RI value. The higher RF values indicate the stronger MDR-reversing efficiency.

2.9 MDR reversion mechanism 2.9.1 Rhodamine 123 accumulation Fluorescent dye rhodamine 123 (Rh123), has been widely utilized as a P-gp substrate in DOX-resistant tumor cells for evaluation of P-gp activity. The effect of the developed micellar systems on Rh123 accumulation in MCF-7/Adr tumor cells was studied. MCF-7/Adr cells were seeded into 6-well plates and incubated for 24 h. Then free Rh123 solution, Rh123 loaded HA micelles were incubated into the cells. After 4 h incubation, 0.1% of Triton X-100 was treated into the cells after PBS washing, and then centrifuged. Rh123 content in supernatants was assayed by fluorescence microplate reader, and the intracellular Rh123 accumulation was corrected by determination of protein amount in supernatants.

2. 9.2 Intracellular ATP level assaying MCF-7/Adr cells were seeded in 12-well plates and incubated 24 h. PF127 empty HA-DOCA-His and HA-DOCA-His-PF micelles were treated into the cells. PF127 unimers and FBS-free culture medium were investigated as controls. After 4 h incubation, 0.1% Triton X-100 was added into the cells after PBS washing, and then the intracellular ATP content was measured by ATP assay kit and corrected by


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determination of protein amount in supernatants.

2.10 In vivo therapeutic efficacy Female nude mice (aged 4-6 weeks, Ningxia Medical University) were s.c. inoculated with MCF-7/Adr cells. The mice were randomly divided into 4 groups (n = 6) as the tumor sizes grew around 50-100 mm3. Saline and various DOX formulations were i.v. administered at 5 mg/kg DOX dosage on 1, 4 and 7 days. The tumor volumes were calculated according to the previous reports. The equation of (L × W2)/2 was utilized for tumor volume calculation. W and L in above equation is the shortest and longest diameter (mm) in tumor, respectively. The relative tumor volume was calculated by the following equations. Body weights changes of mice with different formulation treatments were investigated in the experiment. Relative tumor volume =

the tumor volume at a given time point the tumor volume prior to first treatment

After the anti-tumor activity study completed, the tumors were excised, fixed with formaldehyde (10% in PBS), and then embedded with paraffin. The tumor samples embedded in paraffin were cut into slices, followed by hematoxylin and eosin (H&E) staining for histopathological observation. The survival rate of the mice bearing MCF-7/Adr tumor was also investigated. The survival end points were considered as the day of the tumor volume grew around 1000 mm3 or the animal death. Kaplane Meier curves was plotted for the survival rate description and the mean survival day was figured up. All animal studies were executed following the guidelines for ethical conduct in the use of animals in the research approved by Ningxia Medical University.

3. RESULTS AND DISCUSSION ACS Paragon Plus Environment


3.1 DOX/HA-DOCA-His-PF micelles characterization The blank carrier and DOX-loaded micelles were prepared by a dynamic dialysis method. The HA-DOCA-His-PF micelles showed a CMC of ~18 µg/mL, which would present superior structure stability during blood circulation after i.v. administration. The micelles formed by HA-DOCA-His and HA-DOCA-His-PF carriers showed average sizes of 160.3 nm and 189.4 nm in DLS, respectively. The DOX-loaded micelles presented average sizes in the range of 189.4-234.0 nm at various weight ratios of carrier/DOX (Table 1), and presented homogeneous size distributions in pH 7.4 PBS (Fig. 2A & B), which was appropriate for EPR effect mediated tumor accumulation and penetration. The increased sizes of micelles after DOX encapsulation might be due to the fact that the HA-DOCA-His-PF formed the micellar structure with a hydrophobic core packed tightly, and further DOX encapsulation resulted in the micellar core volume increased. The DOX-loaded micelles had nearly spherical morphologies and well-dispersed in TEM observation (Fig. 2C & D). The DOX encapsulation performance and colloidal stability of the DOX/HADOCA-His-PF micelles were then examined and compared. HA-DOCA-His-PF micelles exhibited high DLC of 16.0% and DEE of 96.1% for DOX at carrier/drug input weight ratios ranging from 5/1 to 10/1. The blank and DOX-loaded micelles could be stable for at least 5 days under 4°C, with no precipitation appeared or obvious sizes changes (Table 1). In addition, the DEE and colloidal stability were remarkedly improved with the increased of the carrier amount.


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Fig. 2. The measurements of size distributions and morphologies of DOX/HA-DOCAHis (A & C) and DOX/HA-DOCA-His-PF (B & D) micelles at designated carrier/DOX input ratio (w/w) of 5/1.

3.2 Endosome pH sensitivity As observed in Fig. 3, particle size distribution and CMC data of DOX/HADOCA-His-PF micelles remained almost unchanged at pH 7.4 and 6.5. As pH goes down to pH 5.5 (endosome pH microenvironment), the mean particle size obviously increased to 1160.8 nm and a significant size distribution was observed. Moreover, there was also a significant increase in CMC with a value of ~86 µg/mL, which further verified the hydrophobic core integrity of micelles was disrupted under endosomal pH microenvironment. Above significant changes in CMC values and size distribution results suggested DOX/HA-DOCA-His-PF micelles would disassemble into more loose and irregular particles under pH 5.5, which confirmed the pH response of micelles in the endosomal microenvironment.



Fig. 3. (A) The particle size distribution of DOX/HA-DOCA-His-PF micellar solution (B) Plots of nile red fluorescence intensity against DOX/HA-DOCA-His-PF micellar concentration at different pHs (7.4, 6.5 and 5.5).

3.3 pH-dependent DOX release A burst release profiles was observed for DOX solution, with almost 100% of DOX released after 12 h incubation, and no obvious differences were found on the DOX release manner as solution at physiological, extracellular and intracellular pH environments. A slow DOX release rate was observed of DOX/HA-DOCA-His-PF micelles at physiological pH of 7.4 and extracellular pH of 6.5, with only 29.7% and 30.5% of DOX released at 12 h, respectively. Notably, the cumulative amount of DOX released from DOX/HA-DOCA-His-PF micelles at pH 5.5 increased to 74.9 % at 12 h incubation (Fig. 4). The pH-dependent assembly/disassembly characteristics and DOX release performance of DOX/HA-DOCA-His-PF micelles provided strong evidence that the protonation of the imidazole rings in His groups of HA-DOCA-His-PF carrier promoted the micellar structure deformation, then triggering DOX release under endosomal pH microenvironment. Overall, it is evident suggested the DOX/HA-DOCA-His-PF micelles with endosomal pH-responsive characteristics can be facilitated to maintain structure


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stability in blood circulation for further tumor accumulation and in tumor extracellular environment, and then triggered the loaded-DOX release under the endosome microenvironment after intracellular endocytosis, which would be definite beneficial for effective intracellular drug delivery on MDR tumor treatments.

Fig. 4. DOX release profiles from DOX/HA-DOCA-His-PF micelles with DOX solution as a comparison in PBS at various pH conditions.

3.4 Intracellular endosomal pH-triggered DOX release Fig. 5 presents the endosomal drug release and intracellular distribution behavior of DOX/HA-DOCA-His-PF micelles after internalization by CLSM. As observed for 2 h treatment, DOX fluorescence signals (red) were mainly distributed in perinuclear region of cells and mainly co-localized with endo/lysosomes fluorescence (green), which afforded a yellow fluorescence in merged image. It is indicated that micelles were distributed in endosomes firstly followed by being uptaken into cells. After the treatment prolonged to 4 h, more DOX signals were appeared in the nuclei while the yellow fluorescence afforded by co-localization was disappeared gradually. This indicated DOX/HA-DOCA-His-PF could be disassembled under endosomal microenvironment and triggered a burst DOX released into intracellular compartment,



thus overlapped the perinuclear region of MCF-7/Adr cells. However, the cytosolic released DOX might be pumped out of cells through the P-gp efflux action. This would limit further efficient distribution of DOX into nucleus region and lower the treatment efficacy on MDR tumors.

Fig. 5. CLSM images of MCF-7/Adr tumor cells treated with DOX/HA-DOCA-HisPF micelles for (A) 2 h and (B) 4 h.

3.5 Internalization observation The potential MDR overcoming efficacy was further evaluated by qualitatively observing the intracellular internalization of DOX/HA-DOCA-His-PF micelles in DOX-resistant MCF-7/Adr and DOX-sensitive MCF-7 cells. As shown in Fig. 6, the fluorescence signals of free DOX in DOX-resistant cells were significantly weaker than those in sensitive cells after 4 h treatment, thus implying the notable DOX pumped out by the P-gp transporter in DOX-resistant MCF-7/Adr cells. On contrary, DOX endocytosis efficacy in MCF-7/Adr cells was increased by the delivery of HA-DOCAHis micelles, whereas still weaker in comparison to that in MCF-7 cells, thereby indicating a partly reversion effect on DOX efflux by integration of active-targeted internalization and intracellular DOX triggering-release. Furthermore, comparable red


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fluorescence signals of DOX were found in sensitive and drug-resistant tumor cells treated with DOX/HA-DOCA-His-PF micelles, thus implying that the further integration with PF127 mediated P-gp efflux inhibition function in micellar system showed efficient synergistic action on promoting the DOX accumulation, which would further facilitate in the enhancement of cell proliferation inhibition in drug-resistant cancer cells. Furthermore, it was observed that the competitive interaction of CD44 receptors in MCF-7/Adr cells with free HA contributed to an internalization reduction of HADOCA-His-PF micelles (Fig. 6B). This clearly support that HA-dependent CD44 receptor-specific internalization pathway take a key role in intracellular delivery of HADOCA-His-PF micelles.



Fig. 6. CLSM images of MCF-7 (A) and MCF-7/Adr (B) cells for 4 h treatment with different DOX formulations, and MCF-7/Adr cells with 10 mg/mL of free HA pre-


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treatment (B).

3.6 In vitro cytotoxicity The proliferation inhibition activity of DOX encapsulated in HA-DOCA-His-PF micelles was studied to demonstrate the MDR reversion combinational effect of activetargeted internalization, intracellular drug triggering-release and PF127 mediated P-gp efflux inhibition. As shown in Fig. 7, DOX solution showed a cell viability of 4.7% against MCF-7 cells at 5 μg/mL of DOX, whereas 43.9% of cell viability against MCF7/Adr cells at 50 μg/mL of DOX treatment. This further confirmed that free DOX would be pump out from the MCF-7/Adr cells through the efflux activity mediated by P-gp. Moreover, DOX loaded in HA-DOCA-His micelles showed less sensitivity on proliferation inhibition with cell viability of 20.9% at 50 μg/mL of DOX against MCF7/Adr than those against MCF-7 cells with cell viability of 5.7% at 5 μg/mL of DOX. However, a proliferation inhibition enhancement of DOX/HA-DOCA-His-PF micelles treatment against MCF-7/Adr cells was observed with cell viabilities of 17.8% at 5 μg/mL of DOX, which showed slightly lower cytotoxicity than those of 3.4% against MCF-7 cells.

Fig. 7. MTT cytotoxicities of different DOX formulations treatment for 72 h in MCF7 (A) and MCF-7/Adr (B) cells.



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3.7 MDR reversion evaluation The RI and RF values are listed in Table 2, based on the IC50 values calculated from in vitro cytotoxicity studies. The calculated RI of DOX against MCF-7/Adr cells was 45.0, which indicated typical DOX resistance against MCF-7/Adr cells. Notably, HA-DOCA-His micelles showed moderate RI and RF values of 9.7 and 4.6, respectively. The RI value of HA-DOCA-His-PF micellar system remarkably reduced to 1.9 and the RF was calculated to be 23.7, suggesting the enhancement of the DOX sensitivity and MDR reversion activity against MCF-7/Adr cells through loading and delivery by HA-DOCA-His-PF micelles. Overall, taking into accounts of intracellular uptake and endosomal pH triggering DOX release results, it is clear indicated the internalized HA-DOCA-His-PF micelles were endocytosed mediated by CD44 receptor, triggered intracellular DOX burst release in endosomal microenvironment, then further inhibited drug pumping action mediated by P-gp, which would be beneficial for a higher intracellular DOX accumulation in MCF-7/Adr cells. Furthermore, considering the MDR reversion and in vitro cytotoxicity results, it is implied the superior MDR reversion and efficient tumor cell growth inhibition activity might result from a synergistic action on multifunctionalization of HA-DOCA-His-PF micelles.

Table 2 The RI and RF values of different DOX formulations for 72 h treatment against MCF-7 and MCF-7/Adr cells. IC50 (μg/mL) DOX Formulations

Free DOX

MCF-7

MCF-7/Adr

1.0±0.1

46.6±0.2


RI

RF

45.0

/

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DOX/HA-DOCA-His micelles

1.4±0.1

13.2±0.1

9.7

4.6

DOX/HA-DOCA-His-PF micelles

1.3±0.2

2.6±0.1

1.9

23.7

3.8 MDR reversion mechanism The probably mechanism of HA-DOCA-His-PF micelles on MDR reversion was related to the P-gp activity inhibition. An increased Rh123 internalization was observed with HA-DOCA-His-PF micelles treatment than those treated with HA-DOCA-His micelles and Rh123 solution in MCF-7/Adr cells, suggesting that the inserted PF127 take an important part in P-gp action inhibition and further enhanced Rh123 accumulation (Fig. 8A). As shown in Fig. 8B, there was no significant decrease in intracellular ATP level for HA-DOCA-His treatment, which remained the same level as control. In contrast, PF127 unimers and HA-DOCA-His-PF micelles led to a remarkedly reduction in the intracellular ATP levels, which would contribute to the Pgp efflux action inhibition and DOX internalization enhancement in MDR cells. Furthermore, it is noticed that there was no significant difference on intracellular ATP content between HA-DOCA-His micelles and Rh123 solution groups, whereas HA-DOCA-His micelles showed moderate higher Rh123 accumulation than Rh123 solution, which suggested the achieved MDR reversion efficacy and moderate cytotoxicity was mainly resulted from higher drug accumulation in cytoplasm instead of P-gp efflux inhibition for HA-DOCA-His micelles. Rh123 accumulation and P-gp level determination data of HA-DOCA-His micelles in the MDR tumor cells indicate the combination of CD44 receptor mediated internalization and intracellular triggering drug release showed limitation on MDR reversion efficacy. The further integration with P-gp activity inhibition of HA-DOCA-



His-PF micelles would contribute to a synergistic effect on reversion activity in MDR tumors.

Fig. 8. (A) Rh123 accumulation of HA-DOCA-His-PF micelles with free Rh 123 and HA-DOCA-His micelles as controls. (B) Intracellular ATP content in MCF-7/Adr cells treated with HA-DOCA-His-PF micelles with PF127 and HA-DOCA-His micelles as controls.

3.9 In vivo therapeutic efficacy In vivo anticancer activity of DOX/HA-DOCA-His-PF micelles was examined, with DOX solution and DOX/HA-DOCA-His-treated groups as comparisons, in MCF7/Adr tumor bearing nude mice. It was presented in Fig. 9A that the mice against salinetreatment showed a fast tumor growth rate during therapeutic treatment, DOX solution showed a moderate tumor growth inhibition effect, whereas a moderate inhibition activity was achieved for the DOX solution treatment group. It was also obvious that encapsulation of DOX into HA-DOCA-His micellar carriers led to a further improved therapeutic activity. The best tumor growth inhibition efficacy was achieved for DOXloaded HA-DOCA-His-PF micellar treated group. As observed in Fig. 9B, no obvious changes were found in body weight of mice with DOX formulation-treated groups.


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The survival rate profiles of the mice treated with different formulations were further studied. A short mean survival time of 19 days was monitored for mice with saline-treated. The mean survival time of mice in DOX solution and DOX/HA-DOCAHis micelles groups were 34 days and 37 days, respectively. The mice treated with DOX/HA-DOCA-His-PF micelles exhibited substantial survival advantage with prolonged median survival time of 43 days (Fig. 9C). Above mice survival data further approved DOX/HA-DOCA-His-PF micelles achieved a superiority on therapeutic efficacy with minimal toxicity on MDR tumor treatment. Fig. 9D further presents histological analysis of mice against different DOX preparation treatments. The tumor in saline-treated group presented a typical morphology with large nuclei, which resulted from the fast proliferation of tumor cells. Contrary, the tumors of DOX formulation-treatment groups exhibited morphology changes to some extent with declined tumor cells density and nuclei shrunken. The mice under DOX/HA-DOCA-His-PF micelles treatment showed the maximum area of cell necrosis in morphology. The improved therapeutic efficacy of MDR tumor bearing mice treated with DOX/HA-DOCA-His-PF micelles was obtained based on the following proposed mechanisms:

firstly,

DOX/HA-DOCA-His-PF

micelles

facilitated

efficiently

accumulating and penetrating in the poorly vascularized tumors via EPR effect. Secondly, DOX/HA-DOCA-His-PF micelles promoted internalization into cancer cells through active-targeted endocytosis mediated with CD44-receptors. Thirdly, the endocytosed DOX/HA-DOCA-His-PF micelles triggered intracellular DOX release into cytoplasm under endosome pH microenvironment. Lastly, PF127 inserted in HADOCA-His-PF micelles further supressed the pumping action to the cytosolic DOX, mediated by P-gp transporter. Overall, HA-DOCA-His-PF micellar carrier with above



mechanisms facilitated in the therapeutic efficacy improvement on MDR tumor treatments.

Fig. 9. (A) Changes of relative tumor volume profiles of mice treated with DOX formulations (mean ± SEM, n = 6), *P < 0.05; **P < 0.01. (B) Body weight of mice treated with various formulations. (C) Survival profiles of MCF-7/Adr tumor bearing mice following different treatments. (D) H&E staining images of tumors under various formulations treatment at day 19.

4. CONCLUSION HA-DOCA-His-PF nanodrug delivery micellar system integrated with active


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targeting, endosome pH-sensitive targeting and drug-pumping inhibition functions was developed for MDR overcoming of DOX. The HA-DOCA-His nanocarrier formed the mixed micelles increased the intracellular uptake via HA-dependent active-targeted endocytosis and His facilitated intracellular triggered DOX release. PF127 inserted in this micelle could further increase internalization of DOX into cytoplasm and delivery into nucleus by the P-gp mediated drug efflux inhibition, with the reduction of ATP level and P-gp activity in the cells. It could be concluded that the HA-DOCA-His-PF micellar delivery system achieved superior intracellular DOX delivery and excellent therapeutic activity in drug-resistant cancer treatment.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Phone: +86-951-6880693. Fax: +86-951-6880693.

Author Contributions ¶ A. C., P. M. and T. Y. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by the Nature Science Foundation of Ningxia (NO. NZ17057).

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

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Table 1. Physicochemical characterization of blank, DOX-loaded HA-DOCA-His and HA-DOCA-His-PF micelles. carrier / DOX

Size

Zeta Potential

DLC

DEE

Stability

(w/w)

(nm) a

(mV) a

(%) b

(%) c

(days) d

HA-DOCA-His micelles

--

160.3

-23.1

--

--

11

HA-DOCA-His-PF micelles

--

189.4

-27.1

--

--

9

10:1

191.4

-20.4

8.4

93.4

6

5:1

234.0

-24.3

14.5

87.2

5

10:1

200.8

-23.7

8.5

93.2

7

5:1

218.7

-19.6

16.0

96.1

5

Micellar formulations

DOX/HA-DOCA-His micelles

DOX/HA-DOCA-His-PF micelles a: Measured by DLS particle sizer. b: DLC = DOX loading capacity. c: DEE = DOX encapsulation efficiency. d: The stability study was tested the formulation at 4 °C. The samples were followed for either noticeable precipitates or significant changes in sizes as determined by DLS.


Multifunctionalized Micelles Facilitate Intracellular Doxorubicin

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