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Anti-Fas Antibody Conjugated Nanoparticles Enhancing the Antitumor Effect of Camptothecin by Activating the Fas−FasL Apoptotic Pathway Hongliang Yu,†,§,‡ Jian He,† Qian Lu,§ Da Huo,§ Shanmei Yuan,§ Zhengyang Zhou,*,† Peipei Xu,*,‡ and Yong Hu*,§ †
Department of Radiology and ‡Department of Hematology, Drum Tower Hospital, School of Medicine, Nanjing University, Nanjing, 210093, China § Institute of Materials Engineering and Collaborative Innovation Center of Chemistry for Life Sciences, College of Engineering and Applied Sciences, Nanjing University, Nanjing, 210093, China S Supporting Information *
ABSTRACT: Emerging evidence suggest that the introduction of Fas ligand (FasL) can enhance the Fas-dependent apoptosis and induce durable immune responses against tumor. However, selective triggering of apoptosis in tumor cells while sparing normal cells remains a great challenge for the application of FasL-based therapeutic strategies. Herein, smart nanoparticles (NPs) with a sandwich structure were fabricated. These NPs consist of a matrix metalloproteinase (MMP) cleavable PEG outer layer, an anti-Fas antibody middle layer, and a camptothecin (CPT)-loaded inner core. They could accumulate at a tumor site by the enhanced permeability and retention (EPR) effect. The removable PEG layer protects the cytotoxic anti-Fas antibody from premature contact with normal tissues, thus avoiding the unexpected lethal side effect before they reach the tumor site. Due to the high level of MMP expressed by tumor cells inside the tumor tissue, these NPs would shed their PEG layers, resulting in the exposure of anti-Fas antibody to bind the Fas receptor and triggering the apoptosis of tumor cells. Results of Western blot confirmed that these NPs could mimic the function of activated cytotoxic lymphocyte (CTL) to activate the Fas−FasL apoptosis pathway of tumor cells. With the aid of CPT payload, these anti-Fas antibody conjugated NPs achieved a high tumor inhibition in the B16 allograft tumor animal model. The design of these NPs provides a method for delivering cytotoxic ligand to targeting tissue, which may be valuable in cancer therapy. KEYWORDS: nanoparticles, anti-Fas antibody, polycaprolactone, camptothecin, matrix metalloproteinase
1. INTRODUCTION Fas and FasL belong to the family of tumor necrosis factor (TNF) receptors and TNF ligands, respectively. They can regulate apoptotic processes, including activation-induced cell death, T-cell induced cytotoxicity, immune privilege, and tumor surveillance.1,2 Generally, the Fas−FasL pathway was identified as a central route in the modulation of apoptosis against tumors within immune system.3 FasL is mostly expressed on the surface of activated lymphocytes to induce the apoptosis of tumor cells by contact with the Fas on the surface of tumor cell.4 However, most of tumor cells down regulate or completely lose the expression of Fas, and sometimes, they express invalid Fas on their surface, thus escaping from FasLmediated apoptosis.5,6 Simultaneously, a high level of FasL is expressed on the surface of tumor cells, which contrarily destroys infiltrated lymphocytes through the Fas−FasL pathway and contributes greatly to tumor immune evasion.7,8 Thus, appropriate population of Fas on the surface of tumor cells or © XXXX American Chemical Society
importing FasL into body will enhance the apoptosis of tumor cells.1,7 Treatments with anti-Fas antibody or FasL, which can activate Fas-mediated apoptosis, have been applied as immunotherapy against cancer.8,9 However, direct intravenous administration of soluble anti-Fas antibody induces serious side effects such as lethal liver hemorrhages and hepatocyte apoptosis due to its contact with normal cells before reaching the targeting site.10,11 Therefore, it is exigent to seek a suitable FasL delivery system, which can ensure the inertness of FasL in the circulatory system while activation occurs inside tumor tissue. Nanoparticles (NPs) have been widely used for cancer treatment and can efficiently deliver their payloads to tumor sites and minimize their systemic toxicity either by the EPR Received: August 4, 2016 Accepted: October 18, 2016 Published: October 18, 2016 A
DOI: 10.1021/acsami.6b09760 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Scheme 1. Schematic Illustration of the Structure of anti-Fas Antibody Conjugated NPs and the Therapy Strategy of NPs against Tumor Cellsa
a
These anti-Fas conjugated NPs are covered with MMP2/9 cleavable PEG layer on their surface, ensuring a long circulation time in blood. They will accumulate at the tumor site due to the EPR effect and shed the PEG layer, because of the high level of MMP2/9, to expose the anti-Fas antibody. Through contact with the Fas receptor on the surface of the tumor cells, these NPs can up-regulate the expression of casepase-8 and casepase-3, thus inducing tumor cell apoptosis through the Fas−FasL pathway.
effect12,13 or by a ligand-mediated active-targeting process.14 Moreover, NPs combining chemotherapy and immunotherapy to deliver membrane-associated proteins and antitumor molecules have shown a synergetic antitumor effect in vitro and in vivo.15,16 Conjugation of anti-Fas antibody onto the surface of camptothecin (CPT)-loaded NPs could definitely improve their antitumor effect against cancer cells by activating the Fas−FasL mediated apoptosis.17 However, the direct exposure of anti-Fas antibody on the surface of NPs indubitably brought the risk of autoimmunity when these NPs contacted with normal lymphocytes in the circulation system. So far, the delivery of anti-Fas antibody still remains a great challenge in the immunotherapy procedure, and there is little work related to the application of anti-Fas antibody in the treatment of cancer. Recently, on-demand drug-loaded NPs that could be programmable to realize their long circulation in blood and targeting and releasing property at tumor sites were achieved by precious surface engineering.18−20 With regards to this, we designed surface-engineered polymeric NPs, combining smallmolecule drug chemotherapy and anti-Fas antibody immunotherapy, which could expose anti-Fas antibody on demand. These NPs have a sandwich structure consisting of a cleavable PEG outer layer, anti-Fas antibody middle layer, and CPTloaded polycaprolactone (PCL) inner core. A matrix metalloproteinase (MMP) cleavable peptide was used as a linker between the PEG layer and the PCL inner core (Scheme 1). The unique structure imparts these NPs with an inactive state in circulation system (e.g., blood) that prevents nonspecific uptake by reticulo-endothelial system (RES) and accumulates at the tumor site because of the EPR effect. Due to the high level of MMP2/9 excreted by tumor cells,21 these NPs are activated by cleaving MMP-sensitive peptide linker and expose anti-Fas antibody to contact with Fas receptor on the surface of tumor cells, mimicking the function of CTL to induce the
apoptosis of tumor cells through the Fas−FasL pathway. Besides, some of the activated NPs can be engulfed by tumor cells, and the encapsulated CPT is released in intracellular compartments and performs the chemotherapy effect (Scheme 1). This design can screen the unexpected contact between anti-Fas antibody and normal cells, thus avoiding the autoimmunity before the arrival of these NPs at tumor site. The physicochemical properties, in vitro CPT delivery, cytotoxicity, uptake study, apoptosis degree, and in vivo antitumor efficacy of these combinational NPs were studied. We believe that the combined treatment with anti-Fas antibody and CPT will result in the sensitization of cancer cells to FasLmediated cytotoxicity, conferring synergistic antitumor effect.17,22
2. EXPERIMENTAL SECTION 2.1. Materials. The MMP2- and MMP9-cleavable peptide (PVGLIG) 23 was purchased from Shanghai HD Biosciences Company. mPEG-NHS (MW: 5000 Da) and NH2−PEG−COOH (MW: 2000 Da) were ordered from Beijing JenKem Technology Company. Anti-Fas antibody (anti-Fas mAb) was purchased from Abcam. ε-Caprolactone (ε-CL, Aldrich) and N,N-dimethylformamide (DMF) were purified as previously reported,20 All other chemicals were purchased from J & K Chemicals and used as received without further purification. 2.2. Synthesis of mPEG−Pep−PCL and PCL−PEG−COOH Polymers. mPEG−Pep−PCL was synthesized according to the previous report using mPEG−NHS (MW: 5000 Da) as the hydrophilic segment.19 PCL−PEG−COOH was synthesized using EDC/NHS coupling reagents. Briefly, PCL−COOH (0.002 mol), EDC (0.002 mol), and NHS (0.003 mol) were dissolved in DMF and kept at room temperature for 12 h with moderate stirring. Products were dialyzed and lyophilized, and PCL−NHS was obtained. PCL−NHS (0.001 mol) and NH2−PEG−COOH (0.0012 mol, MW: 2000 Da) were dissolved in DMF at 37 °C for 24 h to synthesize PCL−PEG−COOH. The molecular weight of these polymers was determined by Agilent GPC system. Chemical structure of these polymers was characterized B
DOI: 10.1021/acsami.6b09760 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces by Fourier transform infrared spectroscopy (FT-IR) and 1H NMR as described in the Supporting Information). 2.3. Preparation of NPs. CPT-loaded mPEG−Pep−PCL@PCL− PEG−COOH NPs (CPT-loaded NPs) were prepared according to our former work.20 Details can be found in the Supporting Information. As a control, CPT-loaded mPEG−PCL@PCL−PEG− COOH NPs without the MMP2/MMP9 cleavable peptide (CPTloaded con NPs) were also produced through the same procedure using mPEG−PCL instead of mPEG−Pep−PCL as the starting material. Empty NPs were prepared without the addition of CPT. After that, these NPs were purified through a 450 nm filter membrane to remove conglomeration and lyophilized for further use. 2.4. Conjugation of Anti-Fas mAb to NPs. NPs were first suspended at 5 mg/mL in MES buffer solution (pH = 5.0). Then, 200 μL of 0.1 M EDC and 600 μL of 0.1 M NHS were added into this suspension, and the mixture was kept at room temperature for 4 h with moderate stirring to activate NPs. Samples were centrifuged at 12 000 r/min for 10 min at 4 °C; NPs were collected, washed thrice with PBS (pH = 7.4) using suspension−spin cycles to remove unreacted reagents, and finally suspended in PBS. A total of 10 μg of anti-Fas mAb was added to 1 mL of suspension of 5 mg/mL activated NPs. Samples were then vortexed, incubated at 4 °C for 48 h, centrifuged at 15 000 r/min for 15 min at 4 °C, and washed twice with PBS to remove unconjugated antibodies. anti-Fas mAb conjugated mPEG− Pep−PCL@PCL−PEG−COOH NPs (anti-Fas NPs) and mPEG− PCL@PCL−PEG−COOH NPs (anti-Fas con NPs) were obtained. CPT-loaded anti-Fas NPs and CPT-loaded anti-Fas con NPs were prepared similarly. Their size was measured by dynamic light scattering (DLS, Malvern NanoZS instrument (Malvern, UK)) and transmission electron microscopy (TEM, JEOL-200, Japan) as we previously reported.20 The CPT-loading efficiency and in vitro drug releasing profiles were measured according to the Supporting Information. 2.5. Gelatinases-Triggered Destabilization of NPs. CPTloaded anti-Fas NPs were expected to be aggregated due to the shedding of PEG in the presence of gelatinases, which could cause changes in size and morphology. CPT-loaded anti-Fas con NPs and CPT-loaded anti-Fas NPs were incubated in phosphate buffer saline (PBS pH = 7.4) with or without gelatinases at 25 °C for 24 h. The changes in size were measured by DLS at 25 °C, and the morphology of CPT-loaded anti-Fas NPs was observed by TEM. 2.6. B16 Melanoma Cell Culture and In Vitro Cytotoxicity of NPs. B16 melanoma cells were cultured in DMEM, as we previously reported.20 In vitro cytotoxic effect of NPs against B16 melanoma cells was evaluated by the CCK-8 assay. The cells were plated in 96 well plates (4500 cells/well) and incubated at 37 °C for 24 h. The cells were added with 100 mL of NPs suspensions with different concentration and further incubated for different time. At the end, each well was added with 10 μL of WST-8 solution. After incubation for another 2 h in a humidified atmosphere at 37 °C in the dark, the absorbance in each individual well was determined at 490 nm with a microplate reader (Rayto, RT-6000). Cell viability was calculated according standard procedure. 2.7. Cellular Uptake of NPs. Laser confocal microscopy was used to evaluate the effect of MMPs-responsive property of NPs on the cellular uptake ability. First, coumarin-6-loaded NPs were prepared similarly as the synthesis of CPT-loaded NPs. Second, cells were seeded into a six-well Petri dish for confocal imaging. After 24 h, 500 μL of coumarin-6-loaded NPs, at a concentration of 2.5 mg/mL, were added to these cells for 2 or 4 h. Cells were then washed thrice with PBS. Finally, cells nuclei were stained by DAPI according to manufacturer’s instructions and fixed with 4% paraformaldehyde. After that, cells were washed with PBS to remove excess dye, and green fluorescence was observed using laser confocal microscopy. 2.8. Cell Apoptosis Analyses. The apoptosis-inducing ability of antibody conjugated NPs was determined using the annexin V-FITC− propidium iodide (PI) double staining method by flow cytometry. B16 cells were seeded in a six-well plate and allowed to grow overnight. Medium was changed and cells were incubated with 1 mL of different NPs suspensions for 24 h at 37 °C. The cells without any treatment were used as the control. Cells were harvested by trypsinization and
centrifugation at 1000 rpm for 5 min. After that, cells were washed with cold PBS repeatedly. An annexin V-FITC and PI apoptosis detection kit (BD Biosciences) was used according to manufacturer’s protocol to determine the cellular apoptosis. The test was performed on BD FACSVerse flow cytometer, and the data were analyzed using the Flowjo software. 2.9. Detection of Caspases Activation to Confirm Fas−FasL Apoptosis. The mechanism of apoptosis-like proapoptotic signaling or antiapoptotic signaling pathways, induced by CPT-loaded anti-Fas NPs, was further investigated by Western blotting. Cells were seeded in a culture flask (1 × 105/mL) for 24 h, added with 2 mg of different NPs, incubated for 6 h, and treated as following. Briefly, cells were washed with cold PBS and harvested by trypsinization. The retrieved samples were lysed in ice-cold lysis buffer containing proteinase inhibitor, phosphatase inhibitors, and PMSF. After the protein concentration was determined using a BCA protein assay kit, equal protein concentration from each sample was mixed with a Laemmli sample buffer, loaded, and separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) on a 12% (v/v) resolving gel. Proteins separated by SDS-PAGE were applied to an Immobilon-P membrane (Millipore Corp., Billerica, MA) and then probed with antibody for 1 h at room temperature. The membranes were incubated with AP-conjugated secondary antibody for 1 h at room temperature. The blots were obtained using an enhanced chemiluminescence detection system. 2.10. Animal Studies. All animal studies were in accord with the National Institute Guide for the Care and Use of Laboratory Animal. A B16 melanoma tumor bearing mouse model was set up according to our previous report.20 After the tumor reached ∼130 mm3 in volume 11 days after the transplantation, the animals were randomized into six groups (n = 6 each group). The mice were intravenously injected with 100 μL of antibody-conjugated NPs every 3 days at a dose of 20 mg/ kg, and mice administered with PBS were used as the control. Tumor volume and body weight were measured at determined interval. Mice were sacrificed on day 15 after the first treatment. The obtained tumor tissues were dissected and fixed in 10% neutral buffered formalin and then sectioned to slices with a thickness of 4 μm for hematoxylin− eosin (H & E) staining. Terminal deoxynucleoitidyltransferasemediated nick-end labeling (TUNEL) staining was conducted following manufacturer’s instructions for the In Situ Cell Death Detection Kit (Roche, Indianapolis, IN) to detect apoptotic cells. 2.11. Statistical Analysis. All data are the average value with standard deviation of triplicate measurements. A Student t test was used to determine if differences were statistically significant. A p value of
