Charge-Reversible Multifunctional HPMA Copolymers for

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Charge-reversible Multi-functional HPMA Copolymers for Mitochondrial Targeting Fengling Wang, Wei Sun, Lian Li, Lijia Li, Yuanyuan Liu, Zhirong Zhang, and Yuan Huang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09693 • Publication Date (Web): 01 Aug 2017 Downloaded from http://pubs.acs.org on August 2, 2017

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ACS Applied Materials & Interfaces

Charge-reversible

Multi-functional

HPMA

Copolymers

for

Mitochondrial Targeting Fengling Wang1, Wei Sun1, Lian Li, Lijia Li, Yuanyuan Liu, Zhi-rong Zhang, Yuan Huang*

†

Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China

School of Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, China

KEYWORDS. HPMA Copolymers, Charge-reversible, Tumor extracellular acidity, Guanidine, Mitochondrial Targeting,

ABSTRACT Mitochondrial-oriented delivery of anticancer drugs has been considered as a promising strategy to improve the anti-tumor efficiency of chemotherapeutics. However, the physiological and biological barriers from the injection site to the final mitochondrial action site remain to be great challenges. Herein, a novel mitochondrial-targeted multifunctional nano-complex based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers (MPC) is designed to enhance drug accumulation in mitochondria. The MPC possess various functions such as extracellular pH response, superior cellular uptake, lysosome escape and mitochondrial targeting. In detail, the MPC was formed by two oppositely charged HPMA copolymers, i.e., a positively charged mitochondrial targeting guanidine group modified copolymers (P-GPMA-KLA) and a charge reversible 2,3-dimethylmaleicanhydride (DMA) modified copolymers (P-DMA). The MPC could remain stable in the circulation (pH7.4) but be cleaved to expose the positive charge of guanidine group immediately in response to the mild acidity of tumor tissues (pH 6.5). The gradually exposure of positive charged guanidine will simultaneously facilitate the ACS Paragon Plus Environment


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endocytosis, endo/lysosome escape and mitochondrial targeting. The in vitro experiments showed that compared with copolymers without guanidine modification, the cellular uptake and mitochondrial targeting ability of MPC in simulated tumor environment ([email protected]) separately increased 4.3 folds and 23.8 folds. The in vivo experiments were processed on B16F10 tumor-bearing C57 mice and the MPC showed the highest accumulation in the tumor site and a peak tumor inhibition rate of 82.9%. In conclusion, multi-functional mitochondrial targeting HPMA copolymers provide a novel and versatile approach for cancer therapy.

1. INTRODUCTION The mitochondria-mediated apoptosis pathway is an effective alternative for cancer therapy due to the presence of cell-suicide weapons. 1-3 During last several decades, mitochondrial-oriented delivery of anticancer drugs has been considered as a promising strategy to improve the anti-tumor efficiency of chemotherapeutics.4 However, to achieve mitochondrial targeting, the physiological and biological barriers remain great challenges. Commonly, there exist two strategies to achieve mitochondrial targeting.5 One option is based on the specific interaction between the targeting ligands/molecules and the receptor expressed in the mitochondrial. But the different targeting efficiency due to the heterogeneous receptor expression in the tumor tissue impeded its application.6 Another option is based on the negative potential of -180mV~-200mV on the matrix face of the inner mitochondrial membrane.7-9 Charged cationic molecules were utilized to modify drug carriers to achieve mitochondrial targeting by electrostatic interaction.10-11 The mitochondrial-targeted carrier should be able to integrate these features: a) negative or neutral charge to avoid the non-specific protein absorption and rapid elimination by the reticuloendothelial system (RES); b) increased cellular uptake; c) rupture the endosomes to facilitate endosomal escape; and d) with high positive charge to bind to mitochondria.12 Despite extensive ACS Paragon Plus Environment

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research in the field has been performed, the majority of previous reports have only investigated one or two issues above all. It is still very difficult to design a drug delivery system that can overcome successive physiological and biological barriers ranging from blood circulation to mitochondria in the next millennium. Guanidine group, an unsaturated amino group with strong positive charge, has drawn a broader attention due to its function of promoting cell penetration. The most widely used example is in the arginine residues of the cell-penetrating peptides (CPPs).13 Furthermore, the guanidine group is similar with polyether imide (PEI) with unsaturated amino functional parts which can combine with the proton in the lysosome, produce "proton sponge effect", and then escape from the lysosome.14-16 Mitochondrial co-localization was observed by attachment of guanidine to porphyrin derivatives or sorbitol scaffold derivatives but the quantitation determination and in vivo investigation were absent.17-18 Furthermore, whether the guanidine can be used in anticancer drug carrier to improve the accumulation of drug in the tumor cell mitochondrial or not, is still kept unknown. Therefore, in the present study, we developed a guanidine containing HPMA copolymers based mitochondrial targeting delivery system to deeply investigate the in vitro and in vivo mitochondria targeting efficiency of guanidine modification. However, the positive nature of guanidine would restrict its in vivo application due to the non-specific protein absorption and rapid elimination by RES.19-21 Therefore, a rational strategy is required to mask the positive charge in the circulation (pH7.4). Previous studies in our laboratory have proved that the anionic 2,3-dimethylmaleicanhydride (DMA) modified N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers (P-DMA) could shield the positive charge of the cationic mesoporous silica nanoparticles, significantly prolong the blood circulation. Meanwhile, DMA can be hydrolyzed to trigger the charge reversal and expose the positive charge of mesoporous silica nanoparticles immediately in response to the mild acidity of tumor tissues (pH 6.5),22 demonstrating P-DMA is a good candidate to mask the positive charge of guanidine group. ACS Paragon Plus Environment


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Herein, we firstly synthesized the positively charged guanidine containing HPMA copolymers with D[KLAKLAK]2

(KLA) peptide as the model drug (P-GPMA-KLA). HPMA copolymers was used as

drug carrier for its advantage of non-immunogenicity, non-toxicity, biocompatibility and multi-functionality. It was reported that KLA could disrupt mitochondrial membrane and thus induce mitochondria-dependent cell-free apoptosis, which would be a good candidate for mitochondria targeting research.23-24 Then the positively changed P-GPMA-KLA was complexed with negatively charged P-DMA to get the charge shielding multi-functional HPMA copolymers nano-complex (MPC), as shown in the Fig 1. We hypothesize it could remain stable in circulation (pH7.4) but be disassembly to expose the positively charged P-GPMA-KLA in the mild acidity of tumor tissues (pH 6.5). The gradually exposure of positive charged guanidine will simultaneously facilitate the cellular endocytosis, endo/lysosome escape and mitochondrial targeting.

Fig 1. Schematic illustration of the composition and transportation pathway of charge-reversible mitochondrial targeting HPMA copolymers nano-complex (MPC). 1. Charge shielding of cationic ACS Paragon Plus Environment

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P-GPMA-KLA in circulation by complexing with charge-reversible negatively charged P-DMA to avoid being quickly cleared. 2. The cleavage of DMA in the acidic tumor extracellular microenvironment induces charge reversal of P-DMA, causing the disassembly of MPC, thus exposing the positive charged P-GPMA-KLA. 3. Increased uptake by tumor cells. 4. Entering into endo/lysosome. 5. The endo/lysosome was destroyed by proton sponge effect. 6. Escaping from lysosome to target to mitochondrial.

2. EXPERIMENTAL SECTION

2.1 materials. N-3-aminopropylmethacrylamide hydrochloride (APMA), 3-(4,5-dimethyl-2-tetrazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT), 2-ethyl-thiopseudourea hydrobromide, fluorescein isothiocyanate (FITC) and 4, 6-diamidino-2-phenylindole (DAPI) were all obtained from Sigma-Aldrich (St. Louis, MO). The azide-modified N3-D(KLAKLAK)2 peptide was synthesized by Chinese Peptide Co. Ltd. (Hangzhou, China). Cyanine 5 NHS ester (Cy5-NHS) was purchased from Lumiprobe (FL, USA). 2,2'-[azobis(1-methylethylidene)]bis[4,5-dihydro-1H-imidazole dihydrochloride (VA-044) and 4-cyanopentanoic acid dithiobenzoate (CTA) were purchased from Sigma-Aldrich (St Louis, MO). Lysosome red fluorescence probe was purchased from Invitrogen (Carlsbad, CA). Mito Tracker Red was obtained from Invitrogen (Eugene, OR, USA). The detection kits of ROS level and membrane potential were both bought from Beyotime Institute of Biotechnology (Haimen, China). The rest of the reagents are commercially available analytical reagent.

2.2. Synthesis and characterization of HPMA copolymers. 2.2.1. Synthesis of monomers. The synthetic process of N-3-guanidinopropyl methacrylamide (GPMA)25-27 was as follows. 5.0 g of N-3-aminopropylmethacrylamide hydrochloride (APMA) was dissolved in 20 ml of deionized water. 50 wt% sodium hydroxide (NaOH) solution was used to regulate the pH to 11. Then the APMA was extracted by dichloromethane (CH2Cl2) and yellow oily product was ACS Paragon Plus Environment


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collected after removing CH2Cl2. The yellow oily product was mixed with 4.56 g of 2-ethyl-thiopseudourea hydrobromide, 2.49 g of trimethylamine and 20 ml of acetonitrile in the round bottom flask and stirred at room temperature for 16 h. Solvent was removed under vacuum using a rotavapor. The crude product was purified by column chromatography (silica gel 60 Å, 100–200 mesh, ethanol:ethyl acetate = 1:1). And the other monomers, such as N-methacryloyl-glycylglycyl-propargyl (MA-GG-C≡CH)

28

and N-(2-hydroxypropyl) methacrylamide (HPMA)

29

were synthesized according

to previous reports.

2.2.2. The synthesis of copolymers precursor. P-GPMA-GG-C≡CH was prepared through radical solution polymerization. HPMA, GPMA and MA-GG-C≡CH（87.5:5:7.5mol%）were dissolved in methanol with AIBN (2 wt %) as the initiator, and stirred at 50℃ for 24 h in a sealed ampoules filled with nitrogen. Then the reaction mixture was precipitated into diethyl ether to obtain the copolymers. The crude product was purified by dialysis in the distilled water and freeze-dried. The synthesis of P-GG-C≡CH and P-APMA was similar as mentioned above with the monomers ratio adjusted to HPMA: MA-GG-C≡CH (92.5:7.5 mol %) and HPMA: APMA (80:20 mol %), separately.

2.2.3 Synthesis of P-GPMA-KLA, P-KLA and P-DMA. KLA peptide was attached by specific click reactions dazide-alkyne cycloaddition. Briefly, P-GPMA-GG-C≡CH or P-GG-C≡CH copolymers precursor was mixed with azide modified KLA peptide at the molar ratio of alkynyl groups: azide group of 1:1 and dissolved in the mixed solution of tertiary butanol and deionized water (1:1 v/v). Cupric sulfate penthydrate (CuSO4·5 H2O) and vitamin C sodium were added as catalyst and reductant, stirred at room temperature for 24 h. Then crude product was purified by dialysis in 4 ℃ deionized water for 48 h, and freeze-dried to get spongy solid product. Negatively charged P-DMA was prepared by conjugating DMA to P-APMA through amidation reaction. Briefly, P-APMA was dissolved in 0.1% of sodium bicarbonate (pH 8.5), and 3 equivalents (to APMA monomer) of 2, 3-dimethylmaleic anhydride (DMA) was slowly added under stirring. The whole procedure was maintained at pH 8.0-8.5 under the ACS Paragon Plus Environment

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regulation of 0.2N NaOH. Then the solution was stirred for 4 h at room temperature and dialyzed against sodium bicarbonate buffer (0.1 M, pH 8.5) for 2 days. Finally P-DMA copolymers were obtained after freeze-drying the resultant solution. The HPMA copolymers containing fluorescently labeled sub-units (P-GPMA-KLA-FITC/Cy5, P-KLA-FITC/Cy5, and P-DMA-Rho) were synthesized according to the same method as described above. The FITC/Cy5 /Rho were conjugated to APMA containing HPMA copolymers through amide interaction.

2.2.4 Preparation of charge shielding multi-functional HPMA copolymers nano-complex (MPC). P-GPMA-KLA and P-DMA were dissolved into deionized water separately at a concentration of 2.0 mg/mL. Then, P-DMA was added drop wise to P-GPMA-KLA solution under stirring. MPC was obtained after stirring the mixture at 500 r/min for another 10 minutes.

2.2.5 The synthesis of P-KLA copolymers with slightly higher Mw. For P-KLA-2, firstly, copolymers precursor P-GG-C≡CH (7.5 mol%) with higher Mw was synthesized through RAFT copolymerization. Briefly, HPMA (1.0 g, 7 mmol), MA-GG-C≡CH (117.92 mg, 0.5 mmol) and CTA (3.14 mg, 7.69 µmol) were dissolved in 5ml cold water containing initiator VA044 (1.04 mg, 3.12 µmol). After bubbling the solution with nitrogen (N2) for 20 min, The polymerization was proceed in oil bath at 50 °C for 6 h. Afterward, the resultant polymer solution was precipitated in acetone/diethyl ether (1:1, v/v, 200 ml) and dried under vacuum at room temperature. Secondly, KLA peptide was attached to the precursor by specific click reactions (dazide-alkyne cycloaddition) as described previously. P-KLA-3 was synthesized using the same method as described above, only changed the VA044 (0.64 mg, 1.92 µmol) and CTA (1.96 mg, 4.8 µmol). Also, Cy5 labeled HPMA copolymers were synthesized using the same method.

2.2.6 Characterization of HPMA copolymers. To measure the loading efficiency of FITC, the P-KLA-FITC or P-GPMA-KLA-FITC were dissolved in sodium tetra borate buffer and determined by ACS Paragon Plus Environment


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UV-Vis at 490 nm. The amount of Cy5 in the P-KLA-Cy5 and P-GPMA-KLA-Cy5 (excitation at 675 nm and emission at 697 nm) was measured by Varioskan Flash (Thermo Fisher Scientific, MA, USA). The drug loading efficiency of KLA peptide was measured by amino analysis (Commonwealth Biotech, VA). The molecular weight and polydispersity index (PDI) of different copolymers were measured by size exclusion chromatography on a Superose 200 10/300 GL analytical column (Amersham Biosciences, NJ). The size and zeta potential of various copolymers and MPC in the presence of 50% fetal bovine serum were estimated by Malvern Zetasize NanoZS90 (Malvern Instruments Ltd, Malvern, UK). The morphologies of MPC at pH 7.4 or 6.5 for 4 h were observed via transmission electron microscopy (TEM) (Tecnai G2 F20).

2.2.7 pH responsible self-assembly and disassembly of MPC. To verify the pH responsible self-assembly and disassembly behavior of MPC, FRET assay was conducted. P-GPMA-KLA was labelled with FITC (P-GPMA-KLA-FITC) and P-DMA copolymers were modified with rhodamine (P-DMA-Rho), which formed a FRET pair. The emission images of P-GPMA-KLA-FITC, P-DMA-Rho, [email protected] and [email protected] at the excitation of 440 nm

32

were obtained, respectively. To further identify the pH

responsible, the charge reversible ability of P-DMA was investigated by monitoring the variation of zeta potential in pH 7.4 or 6.5 at predetermined time points (0, 0.25, 0.5, 1, 2, 4, 6, 8, 10h).30-31 Briefly, P-DMA was dissolved with phosphate buffer solution (PBS) at pH 7.4 or 6.5. Then, the solution was shaked at a rate of 100 r/min at 37℃. And the zeta potential was measured by Malvern Zetasize NanoZS90 (Malvern Instruments Ltd, Malvern, UK) at predetermined time intervals.

2.3 Cell experiments 2.3.1 Cell culture. Mice melanoma cell line B16F10 was purchased from the Chinese Academy of Science Cell Bank for Type Culture Collection (Shanghai, China), cells were cultured in 1640 medium containing 10% FBS and 1% penicillin-streptomycin (both 100 IU/mL) at 37℃ in a 5% CO2/95% air atmosphere. ACS Paragon Plus Environment

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2.3.2 Cellular uptake study. For quantitative analysis, B16F10 were seeded into 12-well plates (1× 105 cells/well) and cultured for 24h. And then the cells were treated with P-KLA-FITC, P-GPMA-KLA-FITC, [email protected] (MPC dissolved in pH6.5 medium) and [email protected] (MPC dissolved in pH6.5 medium) (equivalent FITC concentration: 5µg/ml) for 2 h or 4h. After incubation, the cells were washed with PBS for three times, then trypsinized, centrifuged (1200 rpm, 3 min), and resuspended in cold PBS. Finally, the fluorescence intensity of the cells was measured by flow cytometry. For qualitative analysis, after washing the cells with PBS for three times, 4% paraformaldehyde was used to fix the cells for 20 min. then 4, 6-diamidino-2- phenylindole (DAPI, 5mg/mL) was used to stain the nuclei for 5 min. Finally, cell fluorescent images were recorded by confocal laser scanning microscopy (CLSM, Zeiss Ism 510 due, BD).

2.3.3 Cellular uptake mechanisms assays in B16F10 cells. Chlorpromazine (10µg/mL), Lovastatin (10 µg/mL), Amiloride (0.2mg/mL), Sodium azide（1mg/mL）were used as clathrin endocytosis inhibitor, caveolae endocytosis inhibitor, macropinocytosis inhibitor and active transport inhibitor, respectively. In brief, the B16F10 cells were incubated with various inhibitors for 1 h, and then incubated with P-KLA, P-GPMA-KLA, [email protected], [email protected] for another 2h. The cellular uptake was measured as described above.

2.3.4 Lysosome escape. In order to investigate whether GPMA modified HPMA copolymers possessed lysosome escape ability, Lyso Tracker Red was used to trace the lysosome.33 B16F10 were seeded into 12-well plates at a density of 3× 104 cells/well and cultured in an environment of 37 ℃ with 5% CO2 for 24h. And then the cells were treated with P-KLA-FITC, P-GPMA-KLA-FITC, [email protected] and [email protected] (equivalent FITC concentration: 5µg/ml) for 4h. After incubation, the cells were washed with PBS for three times, and incubated with Lyso Tracker Red (50 nmol/mL) for 30min at 37℃. After washing the cells with PBS for three times again, 4% paraformaldehyde was used to fix the cells for 20 ACS Paragon Plus Environment


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min, then DAPI (5 mg/mL) was used to stain the nuclei for 5 min. Finally, cell fluorescent images were obtained by CLSM.

2.3.5 Mitochondrial targeting. Accumulation of various HPMA copolymers in mitochondrial fractions was quantitatively analyzed by flow cytometer.34 B16F10 cells were seeded in 6-well plates (5×105 cells/well) for 24 h, and then the cells were treated with P-KLA-FITC, P-GPMA-KLA-FITC, [email protected] and [email protected] (equivalent FITC concentration: 5ug/ml) respectively for 4h. After incubation, the cells were washed with PBS for three times followed by collection. Mitochondrial isolation was performed by a cell mitochondria isolation kit (Beyotime Institute of Biotechnology, China).35 Finally, the fluorescence of different copolymers in the mitochondria (1×104 cells) was measured with flow cytometry. Simultaneously, the qualitative experiment was performed. After incubation with various HPMA copolymers, the cells were incubated with Mitotracker Red (50 nmol/mL) for 30min at 37℃. Then the cells were washed with PBS for three times and stained with DAPI for 5min. Finally the images were obtained by CLSM.

2.3.6 Mitochondrial damage detect. The reactive oxygen species (ROS) and mitochondrial membrane potential

(∆ψm)

are

often

regarded

as

important

index

of

mitochondrial.36

Therefore,

2′,7′-dichlorofluorescein diacetate (DCFH-DA) fluorescent dyes and JC-1 were applied respectively to investigate the ROS and membrane potential of B16F10 cells. Briefly, after incubated with HPMA copolymers for 4h, B16F10 cells were incubated with10 µM of DCFH-DA or 5 µg/mL of JC-1 for 30 min in the dark. Next, the cells were washed with PBS for three times, then trypsinized, centrifuged (1200 rpm, 3 min), and resuspended in cold PBS. Finally, flow cytometer was used to measure the fluorescence intensity. For ROS detection, the DCFH-DA was transformed into DCF oxidized by the ROS in the damaged cells with detectable green fluorescence intensity, while there is no fluorescence in the normal cells. So, the green fluorescent was used to represent the level of ROS. For mitochondrial ACS Paragon Plus Environment

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membrane potential (∆ψm) detection, JC-1 aggregates in normal mitochondria with a red color, and disperses into a single state accompanied with the color changing from red to green when membrane potential collapses. So, the ratio of green fluorescence value of JC-1 monomer and red fluorescence value of JC–1 polymer could represent mitochondrial membrane potential (∆ψm).

2.3.7 In vitro antitumor activity. MTT assay was used to investigate the cytotoxicity of free KLA or HPMA copolymers against B16F10 cells. Briefly, B16F10 cells were seeded onto 96 well plates (3000 cells/well). After 24 h, cells were treated with free KLA, P-KLA, P-GPMA-KLA, [email protected] or [email protected] for 24 h at 37, followed by incubation with MTT (5 mg/mL, 20 µL per well) for another 4 h. Finally Varioskan Flash were used to measure the formazane of MTT dissolved by dimethyl sulfoxide (150 µL per well).

2.4. In vivo imaging. B16F10 cells (4×105 cells/C57 mouse) were subcutaneously injected to induce tumors. Experiments were performed when the tumors reached 1000 mm3 after implanting for 10 days. The mice were intravenously administrated with P-KLA-Cy5, P-GPMA-KLA-Cy5 or Cy5 labelled MPC at a dose of 1.5 mg/kg, and were observed by a near-infrared reflection (NIR) fluorescence imaging system at predetermined time points (24, 48, and 72 h). Finally, the tumors were harvested at 72h to be imaged either.

2.5 In vivo antitumor efficacy. B16F10 melanoma tumor-bearing C57 mice were used to evaluate the in vivo antitumor efficacy of different copolymers or free KLA. 4 days after tumor implantation, mice were randomly divided into 5 groups (7 mice per group), and intravenously administrated with saline, free KLA, P-KLA, P-GPMA-KLA or MPC (10 mg/kg KLA equivalents) at 4, 5, 6, 7 day. Meanwhile, the tumor size and body weight of the mice were measured every day. The mice were sacrificed to obtain the tumor in the end of the experiment. Tumor volumes and tumor inhibition rate were calculated according to the previous reports.

2.6. Histological analysis. For histology, the obtained organs including heart, liver, spleen, lung, kidneys, ACS Paragon Plus Environment


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and tumors after sacrificing the drug-treated mice at day 14 were fixed in 10% paraformaldehyde and embedded in paraffin blocks. Then each section was cut into 4 mm slices and stained with hematoxylin and eosin (H&E), and visualized with an optical microscope (XD30A-RFL, Ningbo Sunny, Zhejiang, China).

2.7. Statistical analysis. Data were presented as mean±SD. Statistical analysis was performed using the Statistical Program for Social Sciences (SPSS) software 17.0 (SPSS Inc., Chicago, IL). The threshold of significance was defined as P< 0.05 and highly significance was defined as P< 0.01.

3.

RESULTS AND DISCUSSIONS

3.1 Synthesis and characterization of different HPMA copolymers A series of HPMA copolymers (P-GPMA-KLA, P-KLA and P-DMA) were synthesized as shown in Fig S1 (supporting information). Firstly, GPMA monomers with guanidine group were synthesized. Next, copolymers precursor with alkynyl was synthesized by the radical solution reaction. Subsequently, the drug (KLA peptide) was attached to copolymers by specific click reactions dazide-alkyne cycloaddition to get P-GPMA-KLA. As control to validate the special function of guanidino, P-KLA without GPMA group, was prepared. P-DMA was synthesized by conjugating DMA to APMA containing HPMA copolymers through amide interaction. Also, FITC/Cy5/Rho labeled HPMA copolymers were synthesized using the same method. The characteristics of different copolymers including molecular weight (Mw), poly dispersity index (PDI), size and zeta potential, drug/FITC/Cy5.5 contents were shown in table S1. The molecular weights of all copolymers were in the range of 26-82.5 kDa with polydispersity indices (Mw/Mn) around 1.28-1.89. Zeta potential indicated that guanidine modified HPMA copolymers have a strong positive charge +19.4 mv while the undecorated was +4.68 mv. The KLA content was among 16-19 wt%. ACS Paragon Plus Environment

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Then negatively charged P-DMA was complexed with positively charged P-GPMA-KLA to form nano-complex (MPC) by electrostatic interaction. The feeding ratio of both HPMA copolymers was screened according to size and zeta potential (Table S2 supporting information). Optimal size (17.26 nm) and nearly neutral zeta potential (+2.94 mv) were achieved with feeding ratio of 1:1 (w/w) (Fig 2A), indicating successful charge shielding. The drug loading efficiency in MPC was 9.67%. Although MPC self-assembled from two kinds of liner HPMA copolymers, there is a negligible increase of the size. A plausible reason should be the compacting force from the electrostatic interaction between the two kinds of polymers. A highly compacted structure formed once cross-linking of the two oppositely charged HPMA copolymers happened in the solution. So the final size of the MPC showed a negligible increase compared with the loosely dispersed starting polymers. A similar results were observed when hydrophobic groups (cholesteryl or β-sitosterol) containing HPMA copolymers self-assemble into micelle. The final size of the micelles were less than 20 nm with negligible size increase.37-38 Moreover, the hardly changed particle size and zeta potential of MPC over 24 h in the presence of 50% fetal bovine serum, indicated a strengthened structure compactness of MPC, which could remain relatively stable in plasma (Fig S2, Supporting Information). What more, it may minimize the aggregation with charged plasma protein due to the nearly neutralized surface charge of MPC.

3.2 pH responsible self-assembly and disassembly of MPC

An ideal charge shielding approach must not only be stable in the circulation (pH7.4), but also fulfill charge-reverse to regenerate positive charge immediately to facilitate cellular uptake in response to the mild acidity of tumor tissues (pH6.5).39 To verify the pH responsible self-assembly and disassembly behavior of MPC, the spatial interaction between P-GPMA-KLA-FITC and rhodamine labelled P-DMA (P-DMA-Rho) at pH7.4 or 6.5 was evaluated by FRET assay. Theoretically, FRET signal will occur by the energy transfer between excited donor chromophore (FITC) and the acceptor chromophore ACS Paragon Plus Environment


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(rhodamine) when they are within 10 nm giving rise to a peak around 585 nm.40 As shown in Fig. 2B, there was a FRET signal when MPC was incubated at pH 7.4, clearly indicating an energy transfer will happen due to the compacted structure of MPC at pH 7.4. However, followed by the disassembly of MPC at pH 6.5, this FRET signal disappeared, as FITC and Rho were separated and could not transfer energy. Further insight into the stability of MPC at pH 7.4 and disassembly at pH 6.5 was obtained by analyzing the variation of zeta potential at pH7.4 or 6.5. As shown in Fig 2C, the zeta potential of P-DMA was negatively charged at pH 7.4 even after 10h but underwent a sharp charge reverse from -16 mv to +0.34 mv within 1 h at pH 6.5 and increased to +13.7 mv after 6h. Therefore, we could speculate the positive charge of GPMA could be masked by P-DMA at pH 7.4, and undergo charge reversal to expose the positive charge of GPMA at pH 6.5. The morphology of MPC at different pH values evaluated by transmission electron microscopy (Fig 2 D and E) could directly verify the pH responsible self-assembly and disassembly. At pH 7.4, MPC exhibited a compact and homogeneous nanometer-sized shape with the mean diameter of 16.5 nm, which agreed well with the size (17.92 nm) measured by DLS (Fig 2F). By comparison, MPC appeared to be loose and irregular, and showed inhomogeneous size at pH 6.5, size measured by DLS also appeared multimodal (Fig 2G). All these results validated that MPC could be relatively stable in the circulation, and a positive charge intended for efficient uptake by tumor cells could be attained with the aid of tumor acid environment.

ACS Paragon Plus Environment

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Fig 2. (A) Size and zeta potential of P-DMA, P-GPMA-KLA copolymers and MPC. (B) The emission images of P-GPMA-KLA-FITC, P-DMA-Rho, [email protected] and [email protected] at the excitation of 440 nm (C) Variation of zeta potential of P-DMA at pH 7.4 or 6.5. TEM images of MPC after incubating at pH 7.4 (D) or 6.5 (E). Size distribution of MPC after incubating at pH 7.4 (F) or 6.5 (G) determined by DLS.

3.2. Effect of guanidine group and pH responsive charge shielding on the cellular uptake of HPMA copolymers

After the copolymers were trapped around the tumor tissues, a quick and massive cellular uptake is considered a prerequisite for restoring the anticancer activity and subsequent cytotoxicity.41 So we evaluated the cellular uptake of P-KLA, P-GPMA-KLA, [email protected] (MPC incubated with pH 6.5 medium) and [email protected] (MPC incubated with pH 7.4 medium) on B16F10 cells. As the results shown in Fig 3, GPMA modified HPMA copolymers showed a tremendous increased cellular uptake compared with P-KLA. This observation is quite expectable, as the strong positive charge of P-GPMA-KLA could firstly adhere to the cell surface by electrostatic attraction. Meanwhile, the function of promoting cell penetration by GPMA forming a bidentate hydrogen bond with the ACS Paragon Plus Environment


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phosphates, carboxylates, and/or sulfates of the cell surface by also leads to an enhanced cellular uptake. MPC exhibited a pH responsible uptake with significantly higher fluorescence intensity at pH 6.5 than pH 7.4, indicating that MPC could minimize the cellular uptake in the normal tissues by masking the positive charge, as cell membranes are generally negatively charged. When trapped around the tumor tissues (pH 6.5), enhanced cellular internalization took place after the stimulated hydrolysis of positive charge-shielded groups (Fig 3A). Coincidence with the qualitative analysis, the quantitative investigation by flow cytometer presented a similar tendency (Fig 3B). All of the copolymers displayed time-dependent cellular uptake especially for P-GPMA-KLA, which showed 5.05- and 4.27-fold higher (p

Charge-Reversible Multifunctional HPMA Copolymers for

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