Enhanced Cellular Internalization and On-Demand Intracellular

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Enhanced cellular internalization and on-demand intracellular release of doxorubicin by stepwise pH/reduction-responsive nanoparticles Fang Li, Weiliang Chen, Bengang You, Yang Liu, Shudi Yang, Zhiqiang Yuan, Wenjing Zhu, Jizhao Li, Chenxi Qu, Yejuan Zhou, Xiaofeng Zhou, Chun Liu, and Xue-Nong Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b09604 • Publication Date (Web): 02 Nov 2016 Downloaded from http://pubs.acs.org on November 6, 2016

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ACS Applied Materials & Interfaces

Enhanced cellular internalization and on-demand intracellular release of doxorubicin by stepwise pH/reduction-responsive nanoparticles

Fang Lia+, Wei-liang Chen a+, Ben-gang Youa, Yang Liua, Shu-di Yanga, Zhi-qiang Yuana, Wen-jing Zhua, Ji-zhao Lia,Chen-xi Qua,Ye-juan Zhoua, Xiao-feng Zhoub, Chun Liuc, Xue-nong Zhanga∗

a. Department of Pharmaceutics, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123, People’s Republic of China b. Changshu Hospital of Traditional Chinese Medicine, Changshu 215500, People’s Republic of China c. The hospital of Suzhou People’s Hospital affiliated to Nanjing Medical University， Suzhou, 215000，People’s Republic of China

Keywords: charge reversion; dually pH/reduction-responsive; smart NPs; efficient delivery.

+ The two authors contributed equally to the paper.

Correspondence should be addressed to the following: The Department of Pharmaceutics, College of Pharmaceutical Sciences, Soochow University, DuShuHu High Education Zone, Su Zhou, Jiang Su Province, People’s Republic of China, 215123; Tel/Fax: +86 (0512) 65882087; E-mail: [email protected] 1

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Abstract: The efficient delivery of anti-tumor agents to tumor sites faces numerous obstacles, such as poor cellular uptake and slow intracellular drug release. In this regard, smart NPs that respond to the unique microenvironment of tumor tissues have been widely used for drug delivery. In this study, novel charge-reversal and reduction-responsive histidine-grafted chitosan-lipoic acid NPs (HCSL-NPs) were selected for efficient therapy of breast cancer by enhancing cell internalization and intracellular pH- and reduction-triggered doxorubicin (DOX) release. The surface charge of HCSL-NPs presented as negative at physiological pH and reversed to positive at the extracellular and intracellular pH of the tumor. In vitro release investigation revealed that DOX/HCSL-NPs demonstrated a sustained drug release under the physiological condition, whereas rapid DOX release was triggered by both endo-lysosome pH and high-concentration reducing glutathione (GSH). These NPs exhibited enhanced internalization at extracellular pH, rapid intracellular drug release and improved cytotoxicity against 4T1 cells in vitro. Excellent tumor penetrating efficacy was also found in 4T1 tumor spheroids and solid tumor slices. In vivo experiments demonstrated that HCSL-NPs exhibited excellent tumor targeting ability in tumor tissues as well as excellent anti-tumor efficacy and low systemic toxicity in breast tumor-bearing BALB/c mice. These results indicated that the novel charge-reversal and reduction-responsive HCSL-NPs have great potential for targeted and efficient delivery of chemotherapeutic drugs in cancer treatments. Keywords: charge reversion; dually pH/reduction-responsive; smart NPs; efficient delivery.

Abbreviations DOX: doxorubicin CSO: chitosan oligosaccharide EPR: enhanced permeability and retention GSH: Glutathione CSL: lipoic acid grafted chitosan oligosaccharide 2


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HCSL: histidine-grafted chitosan oligosaccharide-lipoic acid DOX/CSL-NPs: DOX loaded CSL NPs DOX/HCSL-NPs: DOX loaded HCSL NPs DOX•HCl: doxorubicin hydrochloride NPs: NPs EDC·HCl: 1-(3-Dimethylaminopropyl)-3-ethyl carbon carbodiimide hydrochloride, NHS: N-hydroxysuccinimide DS: degree of substitution DLS: dynamic light scattering TEM: transmission electron microscopy LC%: Drug loading content EE%: encapsulation efficiency MTT: 5-diphenyl-2H-tetrazolium bromide CMC: critical micelle concentration ROI: region of interest

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1. Introduction Nanoparticles (NPs) have been extensively investigated in cancer therapy because of their ability to improve the solubility of poorly soluble chemotherapeutic agents, prolong circulation time in vivo, and enhance accumulation in tumor sites through size-dependent enhanced permeability and retention (EPR) effect1-4. However, the EPR effect just contributes to the enhanced accumulation of NPs in tumor sites5. The poor cell internalization as well as slow and incomplete intracellular drug release remains to be great challenges for efficient treatment; these phenomena diametrically result in low intracellular drug concentration below the required therapeutic level and unsatisfactory therapy efficacy6. Hence, smart NPs based on the special microenvironment of tumor tissues have gained increased attention7-9. The key factors for internalization of NPs by tumor cells mainly include particle size, zeta potential, and other properties. Among them, zeta potential plays an important role10. Generally, positively charged NPs tend to be quickly and abundantly internalized by negatively charged tumor cells because of electrostatic interaction11. However, NPs with a positive charge will interact with the negative-charged components in blood circulation and meanwhile are quickly captured by the reticuloendothelial system (RES) and are further eliminated by the liver, leading to the loss of pharmacological activity and less accumulation in tumor tissues12. To address this contradiction, the charge reversal concept has obtained great attention in effectively enhancing cell internalization13, 14. The pH gradients between the normal tissue (blood physiological pH 7.4) and tumor site (tumor extracellular pH 6.5) are used to design charge-conversional NPs; these particles are expected to remain as negative charge at physiological pH in favor of prolonged circulation while being reversed to positive charge responding to tumor extracellular pH, thereby facilitating cellular uptake by negatively charged tumor cells15. The charge-conversion property can be achieved through cleavage of amide linkages or protonation of amino groups and imidazole groups16,

17

. Common materials including poly (β-amino ester),

polyetherimide (PEI), and polyhistidine have been developed to prepare charge-conversional NPs for efficient drug delivery. For example, He et al. designed a novel pH-sensitive charge conversion nanocarrier (M-HHG2C18-L) that can reverse 4


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surface zeta potential from negative to positive at tumor extracellular pH based on the protonation of imidazole group; these particles facilitate cancer cell internalization, showing considerable tumor accumulation and potent suppression of tumor growth in Lewis lung carcinoma tumor therapy18. Another unignorable challenge for drug delivery is the slow and insufficient intracellular drug release after internalization of drug-loading NPs by tumor cells. Hence, smart NPs in response to special internal signals in tumor cells have been investigated to develop nanocarriers for the rapid and complete intracellular release of payloads19, 20. Of these signals, low pH in endo-lysosomes21 as well as GSH (1–10 mM in tumor cell cytoplasm and 2-20 µM in blood fluids)22 have been extensively investigated. Numerous studies have utilized reduction-sensitive nanocarriers containing disulfide bonds, such as dithiodipropionic acid and lipoic acid, to promote sharp and sufficient drug release, wherein the disulfide bonds would break up in response to internal high concentration of GSH and further cause destabilization and cleavage of drug-loading nanocarriers23,

24

. For example, Zhong et al. reported

reduction-sensitive reversibly crosslinked nanoparticle based on lipoic acid conjugates (HA-Lys-LA) to achieve high extracellular stability and fast intracellular drug release. In addition, dually pH/reduction-sensitive NPs can take advantage of both endo-lysosome pH and high concentration of GSH in the cytoplasm, leading to more efficient intracellular drug delivery25. Considerable progress has been obtained in improving poor cell internalization or uncontrolled intracellular release of payloads26, 27. Thus far, few types of NPs have been reported to simultaneously enhance cell internalization and improve intracellular drug release. In the present study, novel stepwise pH/reduction-responsive NPs based on the assembly of histidine-grafted chitosan-lipoic acid (HCSL) were developed for efficient delivery and enhanced anti-tumor activity of the chemotherapeutic agent DOX (Scheme 1). Chitosan oligosaccharide (CSO), a natural cationic oligosaccharide with good biocompatibility, biodegradability, and water solubility, was used as the backbone of the amphiphilic polymer28. Histidine containing the imidazole group with pKa of approximately 6.5 was chosen as the pH-sensitive block, which would be rapidly protonated in slight acidic surroundings, such as extracellular pH, leading to 5



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charge conversion of NPs29, 30. Lipoic acid (LA) containing a disulfide bond was used as the reduction section and hydrophobic core, in which the chemotherapeutic drug DOX was loaded31, 32. HCSL NPs with a negative charge in physiological conditions were assumed to be stable in blood and could accumulate in tumor tissues via the EPR effect. The surface charge of HCSL NPs reversed from negative to positive in response to the mildly acidic environment of tumor tissues, leading to facilitated internalization. Further pH sensitivity occurred in endosomes, resulting in swelling of HCSL NPs and rapid release of partial DOX. Moreover, complete release of DOX was achieved in response to the high concentration of GSH in the cytoplasm after the NPs escaped from the endosomes via the proton sponge effect. These stepwise pH/reduction-responsive HCSL NPs could achieve enhanced cell internalization and efficient intracellular drug release, leading to enhanced cytotoxicity. In this study, the stepwise pH/reduction-responsive nanocarrier (HCSL) copolymer was successfully synthesized, and DOX-loaded NPs were prepared and characterized. Cell internalization, intracellular drug release, cytotoxicity in vitro, tumor targeting ability, and penetrating efficiency, as well as anti-tumor activity in vivo of DOX/HCSL-NPs, were comprehensively evaluated.

2. Experimental section 2.1 Materials Chitosan oligosaccharide with molecular weight (MW) of 3-5 kDa and deacetylation degree of 95% was obtained from Qingdao Yunzhou Biochemistry Co., Ltd

(Qingdao,

China).

3-(3-dimethylamin-opropyl)-1-ethylcarbodiimide

hydrochloride (EDC·HCl), N-Hydroxysuccinimide (NHS) and R-Alpha-Lipoic acid (CAS No.52-46-4) were purchased from Adamas-beta-Reagent Co., Ltd. L-Histidine (CAS No.71-00-1) was from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Doxorubicin hydrochloride of over 98% purity was obtained from Melone Pharmaceutical Co., Ltd (Dalian, China). Methyl thiazolyl tetrazolium bromide (MTT) was from Sigma-Aldrich (St. Louis, MO, USA). Hoechst 33258 was from Invitrogen (Eugene, OR, USA). RPMI-1640 was purchased from Hyclone Thermo-Fisher Biochemical Products Co., Ltd (Beijing, China). Fetal bovine serum (FBS) was 6


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obtained from Zhejiang Tianhang Biotechnology Co., Ltd (Hangzhou, China). Trypsin EDTA solution and penicillin-streptomycin solution were from Beyotime Institution of Biotechnology (Shanghai, China). All other chemicals were of analytical grade and used without further purification. Murine breast cancer cell lines 4T1 from Jiangsu Province Key laboratory of Biotechnology and Immunology (Suzhou, China) were cultured in RPMI-1640 with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin solution under 37 ℃ and 5% CO2 condition. Female BALB/c mice (age, 3-5 w) were provided by the Experimental Animal Center of Soochow University (Suzhou, China). All mice were raised in the environment complying with the guidelines of the National Institute of Health for the care and use of laboratory animals. All animal procedures were performed following the protocols approved by the Institutional Animal Care and Use Committee. 2.2 Synthesis and characterization 2.2.1 Synthesis of the CSO-LA (CSL) copolymer The CSL copolymer was synthesized by an amidation reaction. Briefly, CSO (1.0 g) was dissolved in 50 mL of distilled water. LA (0.5 g) was dissolved in 50 mL of ethanol. Then, EDC·HCl (0.25 g) and NHS (0.25 g) were added and stirred at 45 °C for 15 min to activate the carboxyl group of LA. Later on, the CSO solution was added dropwise and stirred at the same temperature for another 12 h. The mixture was then dialyzed against distilled water (MWCO = 1000) for 24 h, followed by freeze-drying, and then the CSO-LA (CSL) copolymer was obtained.

2.2.2 Synthesis of the His-CSO-LA (HCSL) copolymer The His-CSO-LA (HCSL) copolymer was synthesized as follows: histidine (0.193 g) was dissolved in 20 mL of distilled water and then activated for 15 min by adding EDC·HCl (0.239 g) and NHS (0.143 g) under stirring at 45 °C. The reaction was conducted for another 12 h at the same temperature after the dropwise addition of 20 mL of CSL solution (10 mg/mL). Then, the HCSL copolymer was obtained by dialysis against distilled water for 48 h (MWCO = 1000), followed by freeze-drying.

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2.2.3 Characterization of copolymer The structures of the CSO, CSL, and HCSL copolymer were characterized by 1

H-NMR (Varian, Palo Alto, CA, USA) at 400 MHz using deuterium water (D2O) as

the solvent.

2.3 Surface charge-conversion and pH/reduction-responsiveness of HCSL-NPs The changes in particle size and surface zeta potential of HCSL-NPs responding to the trigger of GSH (10 mM) or pH from 7.4 to 5.3 were observed by dynamic light scattering (DLS) method at 25 °C using a ZEH 3600 (Malvern Instruments, Malvern, UK). Briefly, the blank HCSL-NPs solution (2 mg/mL) was prepared with the probe ultrasonic method. 1 mL of sample was incubated with 5 mL of phosphate buffer solutions (PBS) with or without GSH (10 mM) at different pH values for a predesigned time and particle size was determined. The zeta potential of the blank HCSL-NPs was also monitored with PBS at different pH values after incubation for 1 h.

2.4 Preparation and characterization of DOX-loaded NPs DOX-loaded NPs were prepared by the dialysis method. 5 mL of blank HCSL or CSL NPs (2 mg/mL) was prepared with the above mentioned method, followed by the addition of 0.5 mL DOX·HCl solution (10 mg/mL) and 0.5 mL trimethylamine dropwise. The mixture was dialyzed against fresh distilled water for 8 h to remove the free DOX. Then, DOX-loaded NPs were obtained after filtering through 0.22 µm filter membranes. The micelle morphology of DOX-loaded NPs was observed by a transmission electron microscope (TEM) instrument (JEOL Ltd., Japan). In addition, the size distribution and zeta potential of DOX-loaded NPs was determined. The amounts of DOX encapsulated in NPs were measured using a full wavelength microplate reader (Infinite M1000 PRO, TECAN, Switzerland) with an excitation and emission wavelength of 484 and 598 nm, respectively33. The drug encapsulation efficiency (EE%) and the drug loading capacity (LC%) of the DOX-loaded NPs were calculated by formula 1,2: w

EE%= w0 ×100%

(1)

1

8


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LC%=

w0 w

×100%

(2)

where w0 and w1 are the amounts of loaded DOX and the total weight of DOX in the solution respectively, and w is the total weight of DOX-loaded NPs.

2.5 In vitro release of DOX from DOX-loaded NPs The in vitro release features of DOX from DOX-loaded NPs were investigated with the dialysis membranes method. Briefly, a 5.0 mL DOX-loaded NPs solution was placed in a dialysis bag (MWCO = 3500). The dialysis bag was then transferred into a bottle containing 100 mL of release medium with or without 10 mM GSH at different pH values and was stirred at 37 ± 0.5 °C with 100 rpm. 1 mL of release medium was taken out at predestinate time points and replenished with equal volumes of the fresh medium. The amount of DOX released from DOX-loaded NPs was determined using a full wavelength microplate reader.

2.6 In vitro cellular uptake and distribution of DOX-loaded NPs 2.6. 1 Cellular uptake by confocal laser scanning microscopy 4T1 cells were seeded on microscope slides in a 6-well plate with an intensity of 5×103 cell per well and were incubated for 24 h. The culture medium was replaced with DOX/HCSL-NPs in PBS (pH 7.4 or pH 6.5) with the final DOX concentration of 5 µg/mL in comparison with DOX·HCl and DOX/CSL-NPs. After incubation for 2 h or 8 h respectively, the cells were rinsed with cold PBS (pH 7.4) and were then fixed with 4% paraformaldehyde for 10 min. The cell nuclei were stained with Hoechst 33258 (10 µg/mL) for 15 min. The intracellular fluorescence images were taken by a Confocal Laser Scanning Microscope (CLSM, ZEISS710, Germany). To identify the intracellular delivery efficiency of the drug delivery system, the intracellular distribution, and trafficking of NPs labeled by fluorescein isothiocyanate (FITC) were also investigated by CLSM. Lysosomes were stained with Lyso-tracker Red and the cell nuclei were stained as described above.

2.6.2 In vitro quantitative uptake detected by flow cytometry To quantitatively investigate the in vitro cellular uptake of DOX in different 9



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formulations by tumor cells, 4T1 cells were seeded on a 6-well plate at an intensity of 5×103 cell per well followed by a 24-h incubation. The cells were then treated with DOX-loaded NPs (DOX concentration, 5 µg/mL) under pH 7.4 or pH 6.5 incubation conditions. After incubation for 2 h, the fluorescence intensity of DOX was determined using a flow cytometry analysis (FC500, Beckman Coulter, USA).

2.7 Cytotoxicity study of DOX-loaded NPs The in vitro cytotoxicity of the blank and DOX-loaded NPs against 4T1 cells were studied using the MTT assay. Briefly, 4T1 cells were cultivated in the 96-well plates (5000 cells/well) and were incubated for 24 h. Then, the incubation medium was replaced with fresh medium (pH 7.4 or pH 6.5) containing DOX·HCl, DOX/CSL-NPs, DOX/HCSL-NPs, or blank NPs at different concentrations. After further incubation for 24 h, 20 µL/well of MTT solution (5 mg/mL) was added and the cells were incubated for another 4 h, followed by the replacement of 150 µL DMSO per well. The absorption values at 570 nm were measured using a microplate reader (ELx808, Bio-Tek, USA). The cytotoxicity of different formulations against 4T1 cells were calculated by formula 3: Cell viability ሺ%ሻ=

A570(treated) -A0 A570(non-treated) -A0

×100%

(3)

where A570(treated) and A570(non-treated) represent the absorbance of cells treated with free DOX, blank, or DOX-loaded NPs at 570 nm, whereas A0 represents the medium-only samples.

2.8 In vivo tumor targeting imaging The in vivo tumor targeting ability of polymeric micelles was evaluated with Dir as a fluorescent dye. Briefly, a subcutaneous breast tumor model was established by the subcutaneous injection of 2×107 4T1 cells. Then, the mice were intravenously administrated with Dir-loaded NPs at a dose of 300 µg/kg body weight via the tail vein when the tumor volume reached 100 ± 30 mm3. The fluorescence distribution imaging was obtained at predesigned time points using IVIS Spectrum (Caliper life science) with an excitation wavelength of 710 nm and an emission wavelength of 745 10


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nm. 48 h later, the mice were sacrificed and the major organs and tumors were excised, followed by immediate imaging under the condition described above.

2.10 In vivo tissue distribution To analyze the tissue distribution of DOX in vivo, breast tumor-bearing BALB/c mice were injected with DOX in different formulations (5 mg/kg) via the tail vein. 24 h later, the mice were sacrificed and the major organs were excised, followed by homogenization. DOX in different tissues was extracted with the organic solvent (ratio of chloroform to methanol 3:1, v/v). DOX concentration was measured using a multifunctional microplate reader with an excitation wavelength of 484 nm and an emission wavelength of 598 nm.

2.11 DOX distribution in tumor spheroids and solid tumors To prepare the three-dimensional tumor spheroids, 4T1 cells were seeded on 96-well plates coated with 80 µL of 2% low-melting-temperature agarose at a density of 600 cells/200 µL. After incubation for approximately four days, the tumor spheroids were transferred to a glass bottom petri-dish and treated with DOX in different formulations at a DOX concentration of 5 µg/mL. 2 h later, the fluorescent intensity was monitored by a CLSM. The frozen slices of tumor tissues were obtained 24 h after the administration with DOX-loaded NPs or DOX·HCl to investigate DOX distribution in solid tumor tissues. The cell nuclei were stained with Hoechst 33258 and then the solid tumor tissues slices were prepared for observation with a CLSM.

2.12 In vivo anti-tumor growth efficiency study BALB/c mice subcutaneous 4T1 tumor model was produced as described in section 2.9. The mice were randomly divided into the following four groups with six mice in each group: physiological saline, DOX·HCl, DOX/CSL-NPs, and DOX/HSCL-NPs. The mice in each group were given the intravenous administration of DOX·HCl or DOX-loaded NPs at a dose of 5 mg/kg through the tail vein every four days since the tumor volume reached approximately 80 mm3. The day before the first injection was set as day 0. The body weight and the tumor volume of mice were 11



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measured every two days. The tumor volume was calculated as the formula of V = 0.5 LS2, where L and S represented the longest (mm) and the shortest (mm) diameters, respectively, which were measured by an electronic Vernier caliper (ASONE, Japan). The animals were sacrificed five days after the last injection and the tumor tissues were excised and weighed.

2.13 Statistical analysis All the experiments were conducted for at least three times. All data were presented as mean±standard deviation (SD). Results were analyzed by ANOVA and Students’ T test was used for pair-wise comparisons. Statistical significance was showed as ***p

Enhanced Cellular Internalization and On-Demand Intracellular

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