Targeted Delivery of Zoledronate to Tumor-Associated Macrophages

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Targeted Delivery of Zoledronate To Tumor Associated Macrophages For Cancer Immunotherapy Xinlong Zang, xiaoxu zhang, Haiyang Hu, Mingxi Qiao, Xiuli Zhao, yihui deng, and Dawei Chen Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.9b00261 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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Molecular Pharmaceutics

Targeted

Delivery

of

Zoledronate

to

Tumor

Associated Macrophages for Cancer Immunotherapy Xinlong Zang, Xiaoxu Zhang, Haiyang Hu, Mingxi Qiao, Xiuli Zhao, Yihui Deng* and Dawei Chen** School of Pharmacy, Shenyang Pharmaceutical University, Wenhua Road No. 103, Shenyang, PR China Key words: Tumor associated macrophages; Tumor immunotherapy; Nanoparticles; Zoledronate; pH sensitive

ABSTRACT

Tumor associated macrophages (TAMs) are recruited from circulatory monocytes by tumor derived factors, which differentiate into macrophages residing in the tumor microenvironment. TAMs play critical roles in promoting angiogenesis, invasion, metastasis, and immune escape, and the direct depletion of TAMs is a promising strategy for tumor immunotherapy. In this study, we developed lipid-coated calcium zoledronate nanoparticles (CaZol@pMNPs) containing conjugated mannose, which were sterically shielded with an extracellular pH-sensitive material. The NPs specifically targeted TAMs and induced their apoptosis in vitro and in vivo. In a S180 tumor-bearing mouse model, CaZol@pMNPs effectively depleted TAMs, markedly decreased angiogenesis, reduced immune suppression, and eventually restrained tumor growth without

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eliciting systemic effects. The collective data indicate the potential of the direct depletion of TAMs using CaZol@pMNPs as a cancer immunotherapy.

1. Introduction Tumor associated macrophages (TAMs) are a group of macrophages that reside in the tumor microenvironment. Emerging evidence has revealed the critical role of TAMs in tumor growth, progression, and chemotherapeutic resistance1-4. TAMs release vascular endothelial growth factor (VEGF) that has a dominant effect in neoangiogenesis. TAMs also enhance tumor invasion and metastasis into ectopic tissue through the paracrine loop consisting of tumor derived colonystimulating factor 1 (CSF-1) and epidermal growth factor as well as their receptors. More importantly, TAMs express a plethora of immunosuppressive factors, including interleukin (IL)10, transforming growth factor-beta (TGF-β), arginase 1, and others, which facilitate the escape of tumor cells from immune surveillance and elimination. The selective elimination of TAMs could effectively inhibit tumor growth and restore local immunosurveillance in the tumor microenvironment5. Additionally, TAMs do not have improved mutation rates that could inevitably produce drug resistance in tumor cells2. Therefore, the targeted depletion of TAMs is a potential strategy against tumors. Zoledronate (zoledronic acid, Zol) is a third-generation nitrogen-containing bisphosphonate that has selective cytotoxicity to TAMs and prolongs survival in cancer patients6. However, the short half-life (105 min) in circulation and maximum plasma concentration of only 1 μM hinders its therapeutic effects on TAMs. One strategy to enhance the biodistribution and concentration of Zol in tumors is to utilize lipid-coated calcium zoledronate nanoparticles (CaZol@NPs)7-9. Ligand conjugation is a feasible strategy to facilitate drug-targeted delivery. The conjugation can enhance cellular recognition and endocytosis mediated by specific cell receptors. TAMs are


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major, alternatively activated, M2-like macrophages that abundantly express macrophage mannose receptors (MMR, CD206)10. Mannosylated NPs can enhance cellular uptake in macrophages and so have been extensively explored concerning the targeting of the liver and spleen, since both these organs contain numerous macrophages11-12. To enable delivery to macrophages in the tumor microenvironment, mannose modified carriers should have a prolonged circulation time (achieved using a PEG shield to enable the enhanced permeation and retention effect) and stimulus responsive capabilities to take advantage of pathological characteristics at the target site (such as the exposure of target moieties by pH- or temperature-sensitive shielding). One of most commonly utilized stimuli to design a shell-detachable nanocarrier is the acidity (pH 6.26.9) induced by the Warburg effect in the tumor microenvironment, which exists in many different tumor types13-14. As an example, Wang et al. developed pH-sensitive NPs for tumor-targeted delivery of small interfering RNA or chemotherapeutics15-16. These carriers achieved both prolonged circulation due to PEG hindrance and enhanced cellular internalization following PEG detachment at tumor sites. Zhu et al developed a PEG-sheddable, mannose conjugated nanoparticles that could expose mannose to target TAMs after acid-sensitive PEG shedding in the acidic tumor microenvironment17. Notably, Bae et al. designed a series of pH-sensitive “pop-up” polymeric micelle systems that could present targeting ligands in response to marginal changes in pH18-20. In this study, we developed CaZol@pMNPs with conjugated mannose (Man-PEG1k-DOPE, sterically shielded with a pH-responsive aminomaleic acid linkage (2-propionic-3-methylmaleic anhydride, CDM) between PEG2000 and DOPE (PEG2K- CDM-DOPE). We hypothesized that the NPs would accumulate in tumors by EPR effect, followed by PEGs detachment in slightly acidic tumor microenvironment, exposing mannose to promote Zol delivery to TAMs via interactions


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with CD206. We investigated the efficiency of CaZol@pMNPs in the depletion of TAMs in vitro and in vivo, and the antitumor efficacy in a mouse tumor model. 2 Materials and methods 2.1 Materials Man-PEG1k-DOPE and PEG2k-CDM-DOPE were synthesized and characterized as described in the supplementary information (Figure S1-S5). Nile red and 1,1'-dioctadecyl-3,3,3',3'tetramethylindotricarbocyanine iodide (DiR) were obtained from Meilun Biotechnology Co., Ltd. (Dalian,

China).

1,2-dioleoyl-3-trimethylammonium-propane

(DOTAP)

and

Dioleoyl

Phosphoethanolamine (DOPE) were from AVT (Shanghai, China). 1,2-dioleoyl-sn-glycero-3phosphate (DOPA) was obtained from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Fluorescein isothiocyante (FITC)-labeled F4/80 antibody was from Biolegend (San Diego, CA, USA). Antimatrix metalloproteinase 9 (MMP9), anti-VEGF, and anti-IL-10 rabbit antibodies were purchased from Boster Biological Technology Co., Ltd. (Pleasanton, CA, USA). FITC-F4/80 antibody was from Proteintech (Wuhan, China). Zol, mannose, and other reagents were purchased from SigmaAldrich (St. Louis, MO, USA). 2.2 Cells isolation and culture RAW264.7 mouse macrophage cells and sarcoma S180 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). RAW264.7 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Zhejiang Tianhang Biotechnology, Huzhou, China). Male C57BL/6 and BALB/c mice were obtained from the Experimental Animal Center of Shenyang Pharmaceutical University (Shenyang, China). All mice were housed under SPF conditions and provided with adequate food and water. All animal experiments were conducted in


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accordance with the relevant laws and institutional guidelines for Care and Use of Research Animals established by Shenyang Pharmaceutical University Animal Studies Committee. The committee approved all animal experiments. S180 cells (5×106) were subcutaneously injected at the left armpits of male C57BL/6 mice to generate the heterotrophic tumor model. TAMs were isolated from S180 tumors as previously described21. Briefly, S180 tumors were excised, cut into 2mm×2mm pieces and digested with 0.3% collagenase Ⅳ and 0.05% DNase I at 37℃ for 40min. The cells were resuspended in RMPI 1640 medium with 10% FBS and allowed to adhere in 6-well plates overnight. The adhered cells were collected, stained with FITC-F4/80 antibody, and sorted for TAMs using fluorescence-activated cell sorting using a FACSAria device (BD, San Jose, CA, USA). Flow cytometry analysis suggested that the purity of TAMs was higher than 90%. 2.3 Preparation of CaZol@pMNPs Hydrophobic DOPA-coated CaZol@NPs were prepared using a previously described reverse microemulsion method7-8. Briefly, Zol solution (0.02 M, pH=9.0) was added dropwise to Igepal CO-520/cyclohexane (30/70, v/v) containing DOPA (0.9 mM) to form a Zol-based microemulsion. The CaCl2 reverse emulsion was prepared by mixing CaCl2 solution (0.15 M) with Igepal CO520/cyclohexane (30/70, v/v) in the absence of DOPA. The two microemulsions were combined to initiate the reaction between CaCl2 and Zol at room temperature for 20 min, followed by the addition of abundant anhydrous ethanol to disrupt the emulsion. The resulting suspension was centrifuged at 13,000 rpm for 20 min. The pelleted material was washed twice using ethanol. The final pellet was resuspended in chloroform and stored at -20℃ for further use. To prepare CaZol@pMNPs, DOTAP, cholesterol, mannose-lipid conjugate (Man-PEG1kDOPE), and pH-responsive PEG-lipid (PEG2k-CDM-DOPE) were dissolved (5/1/1/4, w/w/w/w) with CaZol@NPs in chloroform to obtain a transparent solution. The mixture was dried in a rotary


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evaporator under vacuum. The thin film was hydrated using 2 mL of distilled deionized water at 60℃ and sonicated for several minutes, followed by passage through a 0.22-μm filter and storage at 4℃. Similarly, preparation of Nile red or DiR labeled CaZol@pMNPs was performed using the same method described above. Non-targeting CaZol@pNPs were prepared using the same method in the absence of Man-PEG1k-DOPE. 2.4 Characterization of CaZol@pMNPs The sizes and zeta potential (ζ) were evaluated by dynamic light scattering (DLS) using a Zetasizer Nano ZS apparatus (Malvern Pananlytical, Malvern, UK). To observe morphology, the samples were prepared on copper mesh and then examined by transmission electronic microscopy (TEM; Hitachi, Tokyo, Japan). Zol loading efficiency was determined using ultraviolet spectroscopy22. Briefly, the NPs were digested overnight in 0.1 M HCl and then chloroform was added. After centrifugation to collect the water phase, the Zol concentration was measured by the absorbance at 215 nm. The release behavior of Zol from CaZol@pMNPs was evaluated by dialyzing the NPs against phosphate buffered saline (PBS) in a shaker incubator at 37℃ using a dialysis bag with a molecular weight cutoff of 10 kDa. At pre-determined times, 1 mL of the PBS was sampled and digested with 1 mL 0.1 M HCl. The Zol concentration was measured at 215 nm. All measurements were performed in triplicate. 2.5 In vitro uptake of Nile red-CaZol@pMNPs RAW264.7 cells and TAMs were seeded in 6-well plates at a density of 1×106 cells per well and cultured overnight in 2 mL RPMI 1640 medium at 37℃ in an atmosphere of 5% CO2. Media with different pH values containing Nile red-labeled CaZol@pMNPs and other formulations were then


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added to the wells. After incubating for 4 h, the medium was removed from each well and the cells were harvested for flow cytometry analysis. For confocal laser scanning microscopy (CLSM), 3×105 RAW264.7 cells and TAMs were seeded on a glass slide in each well of 6-well plates and cultured for 24 h to allow cell adhesion. Medium containing CaZol@pMNPs (pH 6.5 or 7.4) was added at a final Zol concentration of 0.5 μg·mL-1. Cell nuclei were stained with Hoechst 33258 (10 μg·mL-1). Images were taken with a confocal laser scanning microscope (Carl Zeiss, Jena, Germany). 2.6 In vitro cytotoxicity studies The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based assay was performed to investigate the cytotoxicity of CaZol@pMNPs on RAW264.7 cells and TAMs. Cells were seeded in 96-well plates (6×103 cells per well) and cultured overnight. After 4 h pretreatment at different pH conditions, CaZol@pMNPs or other formulations were added to designed wells. After 24 h, MTT was added and incubation was continued for another 4 h. The culture medium was replaced by dimethylsulfoxide to dissolve formazan crystals. The absorbance at 490 nm was determined using a microplate reader. The cytotoxicity of the nanoparticles against A549 cells was also investigate using the same method. 2.7 Apoptosis and necrosis assay RAW264.7 cells and TAMs were seeded in wells of 6-well plates at a density of 5×105 cells per well in 2 mL complete culture medium containing 10% FBS for 24 h. The medium containing CaZol@pNPs or CaZol@pMNPs (2 μg·mL-1) was then added to each well, followed by incubation for another 4 h. Cells without treatment served as the negative control. Cells were then harvested and washed twice with PBS, and then stained with FITC-Annexin V and PI according to the instructions of the manufacture (Beyotime, Shanghai, China) and analyzed using flow cytometry.


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2. 8 In vivo imaging The S180 heterotopic tumor-bearing male BALB/c mouse model was established as described in section 2.2 because fluorescence signal of DiR can be obtained from BALB/c. DiR labeled CaZol@pMNPs were administered to the S180 tumor-bearing mice via tail vein injection. At predetermined times, fluorescence images were obtained using the FX Pro In Vivo Imaging System (Carestream Molecular Imaging, Woodbridge, CT, USA). After sacrifice, tumors and major organs, including liver, spleen, lungs, kidneys, and heart, were obtained and imaged. 2.9 In vivo antitumor activity Male C57BL/6 mice bearing S180 tumors were randomly divided into four groups (n=6 per group). The mice were intravenously injected with saline, free Zol, CaZol@pNPs, or CaZol@pMNPs. The injection was performed once daily on days 7, 10, 13, 16, and 19 following the injection of S180 cells. The Zol dosage was 0.4 mg·kg-1 body weight. Body weight was determined every 3 days. Tumor length (L) and width (W) were measured, and tumor volume (TV) was calculated as TV (mm3) = L × W2/2. All mice were sacrificed on day 22. The tumors were weighted and then partly embedded in 4% paraformaldehyde for H&E and ICH experiments. The mRNA expression of VEGF, MMP9 and IL-10 in tumor were evaluated using RT-PCR. Major organs were also conserved for H&E staining. Hematoxylin and eosin (H&E) staining was performed to investigate the histopathological changes in tumors and major organs. Paraffin-embedded tumor sections (4-5μm thick) were stained with H&E and then observed using a light microscope (Nikon, Tokyo, Japan). The obtained tumor sections were deparaffinized in xylene and rehydrated in a graded series of alcohol and distilled water. The sections were treated with 3% hydrogen peroxide for 10 min, followed by a standard microwave heating technique for antigen retrieval. Primary antibodies


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against F4/80, VEGF, IL-10, and MMP9 were added and incubated overnight at 4℃. The sections were then incubated with secondary antibodies. The images were captured using an optical microscope (Nikon). Positive area of tumor sections was quantified using Image-Pro Plus software version 6.0 (Media Cybernetics, USA) and the quantification was performed at 3 separate sections per tumor (n=6). The total RNA was isolated from tumor tissues a kit purchased from Tiangen Biotech (Beijing, China). Real-time qPCR was performed using a commercial kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol. 2.10 Toxicity Studies To evaluate liver and renal functions, the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and blood urea nitrogen in the serum (BUN) were measured in tumor-free C57BL/6 mice. Mice were given a tail-vein injection with zoledronate and formulations at a dosage of 0.8mg·kg-1. Blood samples were collected after 4 days administration, and AST, ALT and BUN were tested according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, China). 2.11 Statistical analyses GraphPad Prism 7.0 software (GraphPad, La Jolla, CA, USA) was used for figure drawing and data analysis. Group comparisons were performed by Student’s t test or two-way ANOVA. P

Targeted Delivery of Zoledronate to Tumor-Associated Macrophages

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