Article pubs.acs.org/molecularpharmaceutics
Inducing Optimal Antitumor Immune Response through Coadministering iRGD with Pirarubicin Loaded Nanostructured Lipid Carriers for Breast Cancer Therapy Caifeng Deng,† Mengdi Jia,† Guangfei Wei,† Tiantian Tan,† Yao Fu,† Huile Gao,† Xun Sun,† Quan Zhang,*,†,‡ Tao Gong,*,† and Zhirong Zhang† †
Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu 610041, China ‡ School of Pharmacy, Chengdu Medical College, Chengdu 610083, China ABSTRACT: Chemotherapeutic agents trigger antitumor immune response through inducing immunogenic tumor cell death. However, severe toxicity to immune system and insufficient immunogenic cell death hinder chemotherapy from arousing efficient antitumor immunity in vivo. In this study, the cytotoxic drug, pirarubicin (THP), was entrapped into nanostructured lipid carriers (NLC); THP-NLC significantly reduced the toxicity of THP to immune system and improved immune status of breast cancer bearing mice. When THP-NLC was coinjected with iRGD (a tumor-penetrating peptide), drug accumulation in tumors was greatly elevated, which led to significant control of tumor growth and increase of immunogenic tumor cell death. Subsequently, the cytotoxic T lymphocytes (CD3+ and CD8+ cells) infiltration and cytokine (IFN-γ and INF-α) secretion in tumors were heavily increased. The efficient T-cell dependent control of tumors in the late stage and the lower side effects contributed to the longest whole survival of THP-NLC + iRGD treated mice. Therefore, the coadministration of THP-NLC with iRGD resulted in increased tumor cell direct-killing death and enhanced antitumor immune response. Our results illustrated that THP could serve as an immunogenic cell death inducer and the proposed drug delivery strategy might impact cancer immunotherapy by arousing increased immunogenic tumor cell death. KEYWORDS: immunogenic tumor cell death, antitumor immune response, targeted chemotherapy, iRGD, nanostructured lipid carriers, anthracyclines cancer cells.13,14 Tumor cell apoptosis is generally assumed nonimmunogenic. However, studies demonstrated that apoptotic cancer cells induced by anthracyclines could elicit immune response by emitting “eat me” and “danger” signals, inducing molecular chaperones and releasing high mobility group box 1 (HMGB1). Those molecular events of apoptotic cancer cells were the features of immunogenic cell death due to their ability to promote the maturation and antigen presentation of dendritic cells (DCs).15−17 The antigens derived from apoptotic tumor cells were presented by DCs to T cells, which led to a tumor antigen specific proliferation of T cells.9,18,19 Furthermore, the increase of tumor infiltrating lymphocytes in tumor microenvironment caused by some chemotherapeutic agents could slow the growth of residual tumor cells and prolong the whole survival.20,21 Those facts provide us the potential to optimize the antitumor efficacy of
1. INTRODUCTION Immune cells in tumors are important for regulating tumor progression. The anticancer immune response could be beneficial to eliminate residual cancer cells, which fail to be directly killed by antitumor drugs or maintain micrometastases in a stage of dormancy after conventional chemotherapy.1,2 Therefore, triggering an effective antitumor immune reaction would be a promising strategy in cancer treatment. Chemotherapy has been believed to play antitumor effect by directly killing tumor cell without inducing innate or adaptive immune responses.3,4 However, growing evidence have recently manifested that complex interactions occur between the immune response and cytotoxic drugs.5,6 Some of chemotherapeutic drugs could trigger antitumor immunity response by causing immunogenic cell death.7−9 Immunogenic cell death represents the “first-hit” of some chemotherapeutic agents to tumor cells. The tumor-specific immune response induced by immunogenic cell death is the “second-hit” to the residual cancer tissues, which can lead to long-term tumor treatment benefit indirectly from the initial drug cytotoxicity.10−12 Immunogenic cell death is characterized by the immunostimulatory damage-associated molecular patterns of apoptotic © 2016 American Chemical Society
Received: Revised: Accepted: Published: 296
October 17, 2016 November 13, 2016 November 28, 2016 November 28, 2016 DOI: 10.1021/acs.molpharmaceut.6b00932 Mol. Pharmaceutics 2017, 14, 296−309
Article
Molecular Pharmaceutics
Trypsine-EDTA (0.25%), and Annexvin V-FITC Apoptosis/ propidium iodide Dectection kit were purchased from Nanjing Keygen Biotch. Co., Ltd. (Nanjing, China). Mouse antibodies including PerCP/Cy4 anti-CD4, FITC anti-CD8a, and PE antiCD3e were obtained from Affymetrix eBioscience Co., Ltd. (San Diego, USA). 2.2. Cells and Animals. 4T1 mouse breast cancer cells and DC 2.4 cells were purchased from Chinese Academy of Science Cell Bank for Type Culture Collection (Shanghai, China). Male SD rats (200 ± 20 g) and female BaLB/c mice (20 ± 2 g) were provided by Chengdu Dossy Biological Technology Co., Ltd. (Chengdu, China). All animals were maintained at 25 ± 1 °C with free access to food and water. All the animal experiments were carried out meeting the requirements of the National Act on the use of experimental animals (China) and in accordance with guidelines evaluated and approved by the Animal Ethics Committee of Sichuan University. 2.3. Preparation and Characterization of THP-NLC. THP-NLC was prepared by the thin-film hydration method as described elsewhere.35 Briefly, THP, oleic acid, E80, HS15, αtocopherol, and structured triglyceride were dissolved into dichloromethane. Dichloromethane was removed by rotary evaporation at 30 °C, and the thin film was hydrated with the distilled water. Finally, the gained coarse emulsion was passed through a high pressure homogenizer (EmulsiFlex-C5, Avestin, Canada) at an operating pressure of 10 000 psi for ten cycles to obtain THP loaded nanostructured lipid carriers (THP-NLC). The particle size and zeta potential of THP-NLC were determined by dynamic light scattering (DLS) using Zetasizer Nano ZS90 instrument (Malvern, UK). The morphology of THP-NLC was performed by transmission electron microscopy (TEM, H-600, Hitachi, Japan). The encapsulation efficiency (EE %) was determined by ultrafiltration method with an ultrafree MC unit (Filter Ultrafree MC, 10 000 mw, Millipore, Bedford, USA). The drug loading efficiency (DLE) was calculated by using drug encapsulated in NLC versus the amount of equivalent dried NLC according to our previous report.38 2.4. In Vitro Evaluation. 2.4.1. Cellular Uptake. 4T1 mouse breast cancer cells and DC 2.4 cells were seeded in 6well plates at a density of 1 × 105 cells per well, respectively. Cells were treated with 10 μg/mL THP solution or THP-NLC for 1 and 3 h. Thereafter, cells were collected and centrifuged at 2000 rpm for 3 min, and the intensity of drug fluorescence was determined through flow cytometry analysis. 2.4.2. Mechanism of Cellular Uptake. Endocytosis inhibition experiments were performed to study the endocytic pathways of THP-NLC against 4T1 cells and DC 2.4 cells. Briefly, 4T1 cells and DC 2.4 cells were seeded in 6-well plates at a density of 1 × 105 cells per well, respectively, and cultured for 24 h. Cells were treated with different endocytosis inhibitors for 1 h. Chlorpromazine (10 μg/mL), amiloride (30 μg/mL), mycostatin (25 μg/mL), and polylysine (PLL, 200 μg/mL) were used as clathrin-mediated endocytosis inhibitor, macropinocytosis inhibitor, caveolae-mediated endodytosis inhibitor, and cell adhesion inhibitor, respectively.36,37 After being incubated with THP-NLC (10 μg/mL) for another 1 h, cells were collected for flow cytometry analysis. 2.4.3. Cell Viability. To evaluate cell viability, 4T1 cells and DC 2.4 cells were seeded in 96-well plates and treated with various predetermined concentrations of THP solution and THP-NLC formulation for 24 h. MTT (1 mg/mL) and DMSO
chemotherapy by activating enhanced host antitumor immune response. Chemotherapy inducing antitumor immunity represents a challenging task for most chemotherapeutic drugs are immunosuppressive.22,23 As is well-known, lacking of selective cytotoxicity and nonspecific biodistribution, chemotherapeutic agents could cause unexpected toxicity to immune system like spleen and bone marrow, which has severely hindered the occurrence of antitumor immunity.22,24 What’s more, the toxicity of chemotherapy to normal cells and healthy tissues has negative influence on the quality of life.23,25 Nanostructured lipid carriers (NLC), as one type of novel nanocarriers,26 were proved to possess good qualities of simple production, excellent biocompatibility, and good tolerability.27 They also have played an important role in reducing the unexpected side effects of antitumor drugs.28 Although, NLC were well developed to deliver drugs to tumors, they never have been investigated to increase the antitumor immune response of chemotherapy. Here, we hypothesized that NLC might serve as a prominent candidate to overcome the immunosuppression of chemotherapy. Arousing efficient host antitumor immune response also requires sufficient immunogenic tumor cell death. High interstitial fluid pressure and multiple layers of tumor cells hinder antitumor drugs loaded nanocarriers from penetrating into tumor tissues.29,30 The low level of chemotherapeutic drug distribution in tumor would not effectively kill tumor cells and could not cause sufficient immunogenic tumor cell death. Therefore, to elicit an efficient antitumor immune response, tumor cells were always treated by chemotherapeutic drugs in vitro and then injected into animals.5−7 Recently, iRGD (CRGDKGPDC) has been reported as a safe tumorpenetrating peptide for having the ability of vascular extravasation and tumor tissues penetration.31,32 Studies also found that coadministering iRGD with drug loaded nanocarriers could improve the tumor penetration of antitumor drugs and that coadministration was more effective than conjugation.33,34 We speculated that the coadministration of iRGD with antitumor drugs loaded NLC would facilitate drugs penetrating from tumor vessels and cause a higher level of immunogenic cell death, thus contributing to a better antitumor immune response. In sum, we developed a rational drug delivery strategy that could optimize the antitumor immune response of chemotherapy. Briefly, pirarubicin (THP), a commonly used anthracycline, was loaded into NLC to reduce its side-effect especially for immune system, thus laying base for triggering host antitumor immune response. iRGD was coadministered with THP loaded NLC to increase the drug distribution in tumors, which would result in enhanced cytotoxicity and antitumor immune response of THP.
2. EXPERIMENTAL SECTION 2.1. Materials. Pirarubicin (THP) was provided by Sichuan Baili Pharmaceutical CO., Ltd. (Chengdu, China). Egg yolk lecithin 80 (E80) and oleic acid (OA) were purchased from Lipoid Co., Ltd. (Ludwigshafen, Germany). Solutol HS 15 was a gift from BASF (Ludwigshafen, Germany). iRGD (CRGDKGPDC) was custom-synthesized by GL Biochem (Shanghai) Ltd. (Shanghai, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and 4,6diamidino-2-phenylindole (DAPI) were obtained from SigmaAldrich Co., LLC. (Saint Louis, USA). Wright’s Giemsa Dye, 297
DOI: 10.1021/acs.molpharmaceut.6b00932 Mol. Pharmaceutics 2017, 14, 296−309
Article
Molecular Pharmaceutics
intestine) and tumors was also quantitatively analyzed by LC− MS/MS (Agilent 6410B, USA). For pharmacokinetic studies, male SD rats were intravenously injected 7 mg/kg of THP solution and THP-LNC with or without 4 mg/kg of iRGD. Blood was withdrawn at preset time intervals and centrifuged at 5000 rpm for 5 min. The gained plasma was stored at −80 °C for further analysis. 2.7. Flow Cytometry Analysis of Immune Cells in Vivo. Female BaLB/c mice (20 ± 2 g) bearing 4T1 breast cancers were established as indicated above. At day 7, 10, 13, 16, and 19 after 4T1 breast cancer cells implantation, four formulations including THP solution, THP-NLC, THP solution + iRGD, and THP-NLC + iRGD were given into mice via tail vein, respectively, at the dose of THP of 3.5 mg/kg and iRGD of 4 mg/kg, and the saline group was used as control. Blood samples, spleens, and tumors were collected 7 days after the last treatment. For the preparation of peripheral blood cell suspension, blood samples were centrifuged at 5000 rpm for 5 min, and then red blood cells were lysed using lysis buffer (BD Biosciences). The remaining cells were washed twice with PBS for further analysis. For the preparation of spleen cells suspension, spleens were weighted, excised, and forced through a 70 μm cell strainer. After that, the prepared spleen cells suspension was centrifuged and red blood cells were lysed, then the gained cells were washed and suspended in PBS to prepare single cell suspension. Tumor tissues were dissected into approximately 3 mm fragments, followed by agitation with collagenase (175 U/mL) for 7 h at 37 °C. Tumor cell suspension was obtained by filtration through 70 μm nylon mesh and washed twice with PBS. The single cell suspensions from blood, spleen, and tumor were incubated with antibodies of PerCP/Cy4 anti-CD4, FITC anti-CD8a, and PE anti-CD3e at 4 °C for 1 h, respectively. After that, cell suspensions were washed and analyzed using a flow cytometer (FCM, CytomicsTM FC 500, USA). 2.8. Cytokine Assay. Tumors were harvested 7 days following treatment and were homogenized on ice with 200 μL of RIPA solution containing protease per mg of tissue. The cell debris was removed by centrifugation. IFN-γ and INF-α levels in the supernatants was detected using ELISA according to the manufacturer’s instructions (Affymetrix eBioscience). 2.9. Histological and Immunohistochemical Analysis. Female BaLB/c mice bearing 4T1 breast cancers were treated as indicated in flow cytometry analysis of immune cells in vivo study. The mice were sacrificed 28 days after tumor cells implantation, then tumors were harvested for H&E staining, TUNEL staining, and immunostaining. H&E staining was used for histological analysis. Ki-67 antigen staining and TUNEL staining were used to assess antitumor efficacy on tumor cell proliferation and apoptosis according to the manufacturer’s protocol. CD3+ and CD8+ staining was used to determine the antitumor immune response. 2.10. Antitumor Efficacy. To evaluate the antitumor efficacy, female BaLB/c mice (20 ± 2 g) bearing 4T1 breast cancers was established as indicated above. At day 7, 10, 13, 16, and 19 after 4T1 breast cancer cell implantation, four formulations including THP solution, THP- NLC, THP solution + iRGD, and THP-NLC + iRGD were given into mice via tail vein, respectively, at the dose of THP of 3.5 mg/kg and iRGD of 4 mg/kg, and the saline group was used as control. Tumor size of each mouse was measured every 2 days after first administration. The survival of animals was recorded,
were added, and the absorbance at 490 nm was measured by Varioskan flash multimode plate reader (Thermo, NH, USA). 2.4.4. Cell Cycle Arrest and Cell Apoptosis Assay. 4T1 cells were seeded in 6-well plates and cultured for 24 h. The cell cycle arrest assay was carried out according to our previous study,38 except cells were treated with 100 ng/mL of THP formulations. The apoptosis induced by THP formulations was determined by using an Annexin V-FITC Apoptosis Detection kit according to the manufacturer’s instructions. 2.4.5. High Mobility Group Box 1 (HMGB1) Assay. 4T1 cells were plated in 1 mL of cell culture medium at a density of 1 × 105 cells per well and cultured for 12 h. After that, the culture medium was replaced by 1 mL of 2.5 mg/kg of THP solution or THP-NLC formulation. Cells were incubated for 24 h. Supernatants were collected and dying tumor cells were removed by centrifugation. Quantification of HMGB1 in the supernatants was analyzed by ELISA according to the manufacturer’s instructions (Affymetrix eBioscience). 2.5. Safety Evaluation. To assess the bone marrow toxicity induced by THP solution and THP-NLC, healthy male SD rats (200 ± 20 g) were intravenously administered with 7 mg/kg of THP solution, THP-NLC, and an equal volume of saline, respectively. Blood samples were withdrawn in tubes containing EDTA-2K salt at the day before administration and every other day after administration. The amount of white blood cells was counted by MEK-6318K Automated Hematology Analyzer (Nihonkohden, Shinjuku-ku, Japan). At day 9 after drug administration, bone marrows from thighbone were obtained and incubated with Wright’s Giemsa Dye. The stained mature erythrocytes and nucleated cells were captured by a light microscope (Axiovert 40CFL, Germany). For major organ histopathologic assay, rats were randomly injected 2 times (at day 1 and day 4) with 4 mg/kg dose of THP solution and THP-NLC via tail vein, and saline was used as control. At day 7, blood samples and major organs including heart, liver, spleen, lung, kidney, pancreas, stomach, duodenum, jejunum, ileum, and colon were collected. The gained blood samples were centrifuged to collect serum. The levels of creatine kinase (CK), lactate dehydrogenase (LDH), creatine kinase MB (CK-MB), serum aspartate transaminase (AST), alanine transaminase (ALT), alkalinephosphatase (ALP), total protein (TP), urea nitrogen (BUN), and creatinine (CREA) in serum were detected by Hitachi 7020 automatic biochemical analyzer (Hitachi 7020, Japan). The above organs were stained with hematoxylin and eosin (H&E) and observed by a light microscope. 2.6. In Vivo Distribution and Pharmacokinetic Studies. Female BaLB/c mice (20 ± 2 g) bearing 4T1 breast cancers were used for in vivo tumor penetration study. Briefly, 5 × 106 cells were subcutaneously injected into the right flank of female BaLB/c mice. Once the volume of tumors reached 300 mm3, the tumor-bearing mice were intravenously administrated with THP solution, THP-NLC, THP solution + iRGD, and THPNLC + iRGD, respectively, at the dose of 7 mg/kg of THP and 4 mg/kg of iRGD. Mice were sacrificed at 1 h after treatment to obtain the tumors. The gained tumors were fixed in 4% paraformaldehyde to prepare frozen section (10 μm thickness). The tumor slices were stained with mouse anti-CD31 antibody, Cy3-labled donkey antimouse secondary antibody, and DAPI. The fluorescent distribution was captured by confocal microscope (LSM710, Carl Zeiss, Germany). Besides, the drug distribution in major organs (heat, liver, spleen, lung, kidney, 298
DOI: 10.1021/acs.molpharmaceut.6b00932 Mol. Pharmaceutics 2017, 14, 296−309
Article
Molecular Pharmaceutics Table 1. Characterization of the THP-NLC nanoparticles
size (nm)
PDI
zeta potential (mV)
EE (%)
DLE (wt %)
THP-NLC
112 ± 2.8
0.18 ± 0.02
−17.5 ± 1.3
90.01 ± 3.03
2.78 ± 0.04
Figure 1. (A) Size distribution of THP-NLC measured by dynamic light scattering (DLS). (B) Morphology of THP-NLC observed by transmission electron microscopy (TEM). (C) Pharmacokinetics behaviors of THP solution and THP-NLC in rats after intravenous injection of different THP formulations with or without iRGD at an equivalent dose of 7 mg/kg of THP and 4 mg/kg of iRGD (n = 5).
(Figure 2D). Those results illustrated that the THP-NLC endocytosis was mainly mediated by caveolae for DC 2.4 cells and by clathrin, caveolae, and macropinocytosis for 4T1 cells. 3.2.3. Cell Viability. From MTT assay, it was shown that 4T1 cells treated with THP-NLC displayed lower cell viabilities compared with THP solution group. On the contrary, DC 2.4 cells incubated with THP-NLC had higher cell viabilities than that of THP solution group (Figure 2E,F). 3.2.4. Cell Cycle Arrest and Cell Apoptosis Assay. We also analyzed the apoptosis caused by different THP formulations. Compared with control group and THP solution treated group, accumulation of cells in G2/M phase after incubation with THP-NLC was increased from 30.91% to 46.3% (Figure 3B). What’s more, the percentage of late apoptosis and necrosis of cells treated with THP solution was 6.32% and 3.01%, respectively. While those treated with THP-NLC was significantly increased to 11.18% and 11.21% (Figure 3A). 3.2.5. High Mobility Group Box 1 (HMGB1) Assay. Immunogenic cell death occurs when apoptotic tumor cell elicits specific molecular events.14 HMGB1 is a marker of immunogenic cell death.14 We analyzed the release of HMGB1 in the supernatants of THP formulation treated cells. THPNLC significantly increased the release of HMGB1 compared with THP solution (Figure 3C). 3.3. Safety Evaluation. The myelosuppression is one of the serious toxicities induced by THP treatment to the immune system. Here, the white blood cell (WBC) counts in serum considered as useful index for assessing myelosuppression was recorded in our experiment.39 The normal value of WBC counts for healthy rats is 5−25 (×109/L). As shown in Figure 4A, similar to THP solution, THP-NLC could cause a decrease of WBC, but WBC counts of THP-NLC treated rats could go back to normal value within 9 days, and no rat death was found (Figure 4B,C). What’s more, THP solution induced a significant reduction of proliferation of bone marrow cells with reduced granulocytes, whereas the granulocyte cells counts of THP-NLC treated group were much higher (Figure 4D).
presented by Kaplane−Meier plots, and analyzed with log-rank test. 2.11. Statistical Analysis. All quantitative parameters in this study are expressed as mean with standard deviation (SD). Statistical analysis was performed by the ANOVA, and survival analysis was presented by Kaplane−Meier plots and compared by the log-rank test using the SPSS software. P values of
