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Article Cite This: ACS Appl. Bio Mater. 2019, 2, 2481−2489
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Enhanced Antitumor Immunity Using a Tumor Cell LysateEncapsulated CO2‑Generating Liposomal Carrier System and Photothermal Irradiation Ji Eun Won,† Yeongseon Byeon,† Tae In Wi,† Jae Myeong Lee,† Tae Heung Kang,† Jeong Won Lee,‡ Byung Cheol Shin,§ Hee Dong Han,*,†,∥ and Yeong-Min Park*,†,∥
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Department of Immunology, School of Medicine, Konkuk University, 268 Chungwondaero, Chungju-Si, Chungcheongbuk-Do 380-701, South Korea ‡ Department of Obstertrics and Gynecology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 135-710, South Korea § Bio/Drug Discovery Division, Korea Research Institute of Chemical Technology, Daejeon 305-600, South Korea S Supporting Information *
ABSTRACT: Dendritic cell (DC)-based cancer immunotherapies have been studied extensively. In cancer immunotherapy, the initial key step is the delivery of tumor-specific antigens, leading to the maturation and activation of DCs. To promote effective antigen delivery, liposome-based delivery systems for tumor-specific antigens have been investigated, and although promising, a triggered release of the antigen from the liposome is required to attain an optimum immune response. In this study, we developed CO2-bubble-generating thermosensitive liposomes (BG-TSLs) that encapsulate whole tumor cell lysates (TCLs). The release of the lysate from BG-TSLs can be triggered using near-infrared (NIR) irradiation. We also developed BG-TSLs able to encapsulate doxorubicin (DOX) for combination therapy. The DOX-BG-TSLs and TCL-BGTSLs have a mean particle size of 114.17 ± 8.28 nm and 123.8 ± 10.2 nm and a surface charge of −22.56 ± 1.3 mV and −28.9 ± 0.8 mV, respectively. CO2 bubble generation within TCL-BG-TSLs and DOX-BG-TSLs by NIR irradiation led to the burst release of TCL or DOX. TCL release from TCL-BG-TSLs promoted dendritic cell maturation and activation, leading to the emergence of antigen-specific cytotoxic CD8+ T cells. The combination of TCL-BG-TSLs with DOX-BG-TSLs showed a significantly greater antitumor efficacy in B16F10 tumor-bearing mice compared to that seen in the control mice (P < 0.001). Taken together, our liposomal delivery system, combined with NIR irradiation, could enhance the therapeutic efficacy of cancer immunotherapies. KEYWORDS: immune response, liposome, CO2 generation, photothermal irradiation, cancer immunotherapy
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INTRODUCTION
effective delivery of tumor-specific antigens to the DCs, a process that is commonly inadequate, leading to insufficient maturation of the DCs. In addition, a single antigen cannot represent the entire antigen diversity required for tumor cell specificity in such systems.11 Recently, instead of using a single antigen, whole tumor cell lysates (TCLs), potentially representing the complete repertoire of cancer cell target antigens, have been reported to improve the cytotoxic CD8+ T cell immune response.12,13 Whole tumor antigens represent an attractive alternative source of antigens for DC-based immunotherapy compared to tumor-derived peptides and full-length recombinant tumor proteins. Unlike defined tumor-derived peptides and proteins,
Immunotherapy is a highly promising therapeutic strategy for the treatment of cancer.1−3 Cancer immunotherapies increase the ability of the patient’s own immune system to recognize tumor-associated antigens, thus eliciting an immune response directed toward the cancer. To increase the immune response in cancer immunotherapy, a key objective is to increase the antigen presenting cell (APC)-dependent activation of cytotoxic CD8+ T cells through the major histocompatibility complex (MHC) class I pathway.4,5 Dendritic cells (DCs), the most effective APCs, have been extensively studied for the development of a variety of DCbased cancer immunotherapeutic strategies to improve tumorspecific antigen presentation in the immune response.6,7 Although several promising studies have been reported, the development of successful DC-based vaccines remains a challenge.8−10 One obstacle of cancer immunotherapy is the © 2019 American Chemical Society
Received: March 4, 2019 Accepted: May 21, 2019 Published: May 22, 2019 2481
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Scheme 1. Schematic Illustration of the Combination Therapy Platform Using TCL-BG-TSLs and DOX-BG-TSLs Designed To Enhance the Antigen-Specific Immune Responsea
a
(A) TCL-BG-TSLs and DOX-BG-TSLs. (B) Overall scheme used for the combination therapy.
results from heating these to temperatures above the transition temperature of lipid composition.26 In this study, we developed CO 2-bubble-generating thermosensitive liposomes (BG-TSLs) containing TCL or doxorubicin (DOX) (Scheme 1A). Following NIR irradiation, TCL or DOX could be rapidly released from TCL-BG-TSLs or DOX-BG-TSLs, respectively. Therapeutically, DOX released from DOX-BG-TSLs caused a slight stimulation of tumor cell apoptosis combined with simultaneous NIR irradiation. TCL, as a tumor-specific antigen, was released from the TCL-BGTSLs, leading to DC maturation and enhancement of the ̈ T cells immune response through increased activation of naive (Scheme 1B). Our system provides a novel immunotherapeutic method for the controlled release of drugs and antigens using external stimulation at a designated time at a particular site. In addition, DOX-BG-TSLs can induce antigen release through immunogenic cell death, and TCL-BG-TSLs can increase the antitumor immune response by TCL release. Taken together, our results indicate that the combination of TCL-BG-TSLs and DOX-BG-TSLs showed synergistic cooperation with respect to DC maturation and the induction of antigen-specific cytotoxic CD8+ T cells. Therefore, this study provides a novel combination therapeutic strategy using both chemotherapeutics and immune response and has potential for the use in cancer immunotherapies.
whole tumor lysate therapy is applicable to all patients regardless of their HLA type.14 Several clinical trials have used whole TCLs as a tumor-specific antigen for DC maturation and for priming their activation.15 Therefore, the use of whole TCLs is a potential approach that can be used to increase the effectiveness of immunotherapy for the treatment of cancer. Chemotherapy induces immunogenic cell death against tumor cells. Nevertheless, chemotherapy has been studied for the development of chemotherapy-induced cancer vaccines in the field of cancer immunotherapy due to inefficient DC maturation.16 Thus, for effective immunization, a TCLencapsulated vaccine was used in combination with chemotherapy. To effectively deliver tumor-specific antigens to the DCs, nanotechnology-based platforms using chitosan nanoparticles (NPs),17 poly(lactic-co-glycolic acid) (PLGA)-NPs,18−20 goldNPs,21 or liposomes22,23 have been developed to increase the efficiency of antigen delivery.24 Among these nanoparticles, liposomes are capable of transferring hydrophilic drugs and can be triggered to release the drug upon external stimulation.25 Thermosensitive liposomes (TSLs) have been developed to increase drug release from liposomes at a hyperthermal temperature.26 The release of drugs from TSLs depends entirely on an increase in lipid bilayer permeability, which 2482
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Figure 1. Physical properties of TSLs and BG-TSLs. (A) Size and (B) ζ potential of TSLs or BG-TSLs were measured using laser light scattering with a particle size analyzer. (C) The loading efficiency of DOX into DOX-TSLs or DOX-BG-TSLs was measured using UV−vis spectrophotometry at 490 nm, and (D) the loading efficiency of TCL into TCL-TSLs or TCL-BG-TSLs was determined using the BCA protein assay. (E) Morphologies of BG-TSLs and DOX-BG-TSLs were monitored using atomic force microscopy. Scale bar: 100 nm. (F) Morphologies of BG-TSLs and DOX-BG-TSLs were monitored by cryoTEM. The arrow denotes DOX encapsulation. Scale bar: 100 nm. (G) TCL encapsulation into TCL-BG-TSLs was assessed using silver staining using the SDS-PAGE gel retardation method. Error bars represent SEM.
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RESULTS AND DISCUSSION Characteristics of the Various TSLs. To promote an effective anticancer immunotherapy efficacy, the delivery strategies of tumor-specific antigens into the tumor microenvironment have been studied extensively, in particular with regard to the triggered release of tumor-specific antigens at the tumor site. Therefore, in this study, we developed a potential carrier system of CO2-bubble-generating thermosensitive liposomes (BG-TSLs) containing TCL or DOX stimulated with NIR. Initially, we analyzed the physicochemical characteristics of TSLs, DOX-TSLs, TCL-TSLs, BG-TSLs, DOX-BGTSLs, and TCL-BG-TSLs. The mean size of the TSLs was 92.16 ± 1.43 nm, whereas the mean sizes of the DOX-TSLs, TCL-TSLs, BG-TSLs, DOX-BG-TSLs, and TCL-BG-TSLs were slightly increased, at 115.56 ± 7.59 nm, 128.8 ± 7.98 nm, 94.16 ± 2.14 nm, 114.17 ± 8.28 nm, and 123.8 ± 10.21 nm, respectively (Figure 1A). The surface charges of BG-TSLs, DOX-BG-TSLs, and TCL-BG-TSLs were −17.8 ± 2.86 mV, −22.57 ± 1.3 mV, and −28.9 ± 0.8 mV, respectively (Figure 1B). Furthermore, the loading efficiency of DOX into DOXBG-TSLs was 38.6% (DOX concentration in DOX-BG-TSLs was 800 μg/mL, Figure 1C) and that of TCL in TCL-BGTSLs was 43.58% (435.8 μg/mL, Figure 1D). The morphology of BG-TSLs and DOX-BG-TSLs was confirmed using atomic force microscopy (AFM) and cryogenic transmission electron microscopy (cryoTEM). BG-TSLs and DOX-BG-TSLs were observed to be spherical with a size of approximately 100 nm (Figure 1E,F). Notably, we identified a phospholipid bilayer structure and encapsulation of DOX into BG-TSL as a black bar at the center.27 Next, we separated TCL proteins using sodium dodecyl sulfate gel electrophoresis (SDS-PAGE)28 and
used the silver staining to verify the loading of TCL into TCLBG-TSLs. The proteins in the TCL were clearly visible in lane 1 (TCL alone) and lane 2 (TCL after dissolution of TCL-BGTSLs); however, bands from the TCL could not be seen in lane 3 (TCL-BG-TSLs), which indicated that the TCL was completely encapsulated in the TCL-BG-TSLs (Figure 1G). Release Behavior of the Payload from DOX-BG-TSLs or TCL-BG-TSLs. The amount of DOX or TCL released from DOX-BG-TSLs and TCL-BG-TSLs was measured at different temperatures and incubation times. DOX release from DOXBG-TSLs significantly increased compared to that from DOXTSLs at 41 °C (Figure 2A). Drug release from DOX-TSL depended on the transition temperature of the composition of lipids, such as DPPC. Here, the temperature sensitivity of TSL is caused by the phase transition temperature of DPPC in response to heat.29 Moreover, the transition temperature of TSL was observed by differential scanning calorimetry (DSC) (Figure S2).29,30 This result indicated that the increased DOX release may be due to CO2 bubble generation. The release of TCL from TCL-BG-TSLs also increased at 41 °C (Figure 2B), again indicating that CO2 bubble generation triggered TCL release as a result of destabilization of the liposomal membrane. Therefore, we selected 41 °C as the optimum temperature for CO2 bubble generation for subsequent experiments. Next, we observed DOX and TCL release from DOX-BG-TSLs or TCL-BG-TSLs using NIR irradiation (2 W/ cm2, 808 nm) as an external heat source to increase local temperatures in the tumor tissue.31 Following NIR irradiation, 70% of DOX was released from the DOX-BG-TSLs, which was significantly higher than the release observed in the absence of NIR irradiation (Figure 2C). The release of TCL from TCLBG-TSLs following NIR irradiation was also significantly 2483
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Figure 2. Release behavior of different payloads from liposomes. Release of (A) DOX from DOX-TSLs or DOX-BG-TSLs at various temperatures and (B) TCL release from TCL-TSLs or TCL-BGTSLs. (C) Release of DOX from DOX-TSLs or DOX-BG-TSLs with or without NIR irradiation (2 W/cm2) and (D) TCL release from TCL-TSLs or TCL-BG-TSLs with or without NIR irradiation (2 W/ cm2). Error bars represent SEM.
Figure 3. Intracellular uptake of DOX-TSLs or DOX-BG-TSLs by B16F10 cells. Intracellular uptake was assessed using (A) flow cytometry and (B) confocal microscopy. Sytox green represents nuclei and red represents DOX. Scale bar: 20 μm. (C) Cell viability of DOXTSLs or DOX-BG-TSLs with NIR irradiation (2 W/cm2). Error bars represent SEM *P < 0.005.
higher than that observed in the absence of NIR irradiation (Figure 2D). These results clearly indicate that CO2 bubble generation can effectively contribute to drug and antigen release from different BG-TSLs. Intracellular Uptake of DOX-TSLs and DOX-BG-TSLs. Prior to determining the effect of DOX-TSLs and DOX-BGTSLs on cell viability, we determined the intracellular uptake of DOX-BG-TSLs by B16F10 cells using flow cytometry (FACS) and confocal microscopy (Figure 3A,B). Both DOXTSLs and DOX-BG-TSLs showed an intracellular uptake of up to 97% compared to that of controls (Figure 3A). Based on the data shown in Figure 3A, both DOX-TSLs and DOX-BG-TSLs had a similar intracellular localization in B16F10 cells (Figure 3B). Next, we assessed the viability of B16F10 cells treated with DOX-TSLs or DOX-BG-TSLs at different temperatures using the MTT assay (Figure 3C). Cell viability upon treatment with DOX-BG-TSLs at 41 °C decreased compared to that of cells treated with DOX-BG-TSLs at 37 °C. This result clearly indicates that CO2 bubble generation within DOX-BG-TSLs at 41 °C may trigger the release of DOX, leading to a decrease in cell viability owing to the cytotoxic effect of DOX. DC Maturation upon Treatment with TCL-BG-TSLs: Intracellular Uptake of BG-TSLs in DCs. Prior to determining DC maturation or activation, we first assessed delivery efficiency using ovalbumin (OVA) as a model antigen by FACS. The intracellular delivery efficiency of the OVAencapsulating liposomal system was significantly increased compared to OVA alone (Figure S3). To determine the effect
of TCL-BG-TSLs, with or without NIR irradiation, on DC maturation, we used flow cytometry to measure the expression of activation markers in DCs (Figure 4). DCs were isolated from the bone marrow of C57/BL6 mice and incubated with BG-TSLs, TCL (20 μg), and TCL-BG-TSLs (20 μg of TCL), with or without NIR irradiation. DCs treated with TCL-BGTSLs with NIR irradiation showed a significantly higher expression of surface maturation markers, such as CD40, CD80, and MHC class I, compared to those observed in the untreated DCs used as a control, DCs treated with BG-TSLs, and DCs treated with TCL (Figure 4A and Figure S4). Notably, DCs treated with TCL-BG-TSLs with NIR irradiation showed significantly increased levels of activated cell markers compared to DCs treated with TCL-BG-TSLs without NIR irradiation. The intracellular delivery of tumor-specific antigens, such as TCL, is the first step for maturation and activation of DCs. However, TCL alone showed significantly low maturation compared to TCL-BG-TSLs. Generally, intracellular delivery of antigen alone showed a low efficiency and was rapidly degraded in an in vivo environment.32 Therefore, antigen alone showed weak DC maturation compared to the liposomal system. To overcome this limitation, we used a liposomal delivery system to increase the intracellular antigen delivery efficiency.33 These results indicate that NIR irradiation-mediated CO2 bubble generation promotes the release of TCL from TCL-BG-TSLs, leading to the acceleration of DC maturation. In addition, DCs treated 2484
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Figure 5. (A) CO2 bubble generation from BG-TSLs after uptake of liposomes by B16F10 cells and (B) CO2 bubble generation from BGTSLs after i.v. or i.t. injection of BG-TSLs into tumor-bearing mice for 1 h. CO2 bubble generation was monitored using an ultrasound imaging system.
Figure 4. Maturation and activation of DCs induced by TCL-BGTSLs. (A) Expression of surface maturation markers (CD40, CD80, and MHC class I) was verified using flow cytometry. (B) Quantification of pro-inflammatory cytokines and DC activation factors from the culture supernatants of DCs treated with TCL-BGTSLs for 24 h was performed using ELISA. Error bars represent SEM *P < 0.05.
CO2 bubble generation at the tumor site in the control group (TSL) not subjected to NIR irradiation, whereas there was significant CO2 bubble generation at the tumor site after NIR irradiation (1.00 W/cm2, 1 min, Figure 5B). From the above data, we concluded that CO2 bubble generation from BG-TSLs occurred as a result of the photothermal effect caused by NIR irradiation. These results indicate that CO2 bubble generation from BG-TSLs in combination with NIR irradiation could leads to drug release at the tumor site. Antitumor Efficacy. To determine the therapeutic efficacy of the combination of DOX-BG-TSLs and TCL-BG-TSLs, we assessed their antitumor effects using the B16F10 melanoma tumor model. B16F10 tumor cells (1 × 106 cell/mouse) were inoculated into C57/BL6 mice using subcutaneous (s.c.) injection, and the mice were randomly allocated to one of the following treatment groups (n = 5 mice per group): (1) control, (2) TCL-BG-TSLs without NIR irradiation, (3) TCLBG-TSLs with NIR irradiation (1.00 W/cm2, 1 min), (4) combination of TCL-BG-TSLs and DOX-BG-TSLs without NIR irradiation, and (5) combination of TCL-BG-TSLs and DOX-BG-TSLs with NIR irradiation (1.00 W/cm2, 1 min). The groups were administered two intravenous (i.v.) injections of the appropriate TSLs at weekly intervals (Figure 6A). The therapeutic efficacy of TCL-BG-TSLs, either with or without NIR irradiation, did not show any significant inhibition of tumor growth. However, the combination treatment (TCLBG-TSLs + DOX-BG-TSLs) with NIR irradiation showed the highest inhibition of tumor growth compared to the control group (*P < 0.001), TCL-BG-TSLs with NIR irradiation (*P < 0.003), and upon combination treatment (TCL-BG-TSLs + DOX-BG-TSLs) without NIR irradiation (*P < 0.004, Figure 6B). Consistently, the weight of tumors from the mice in the combination plus NIR irradiation group significantly decreased on day 20 compared to the mice from the control group (*P < 0.001), TCL-BG-TSLs with NIR irradiation (*P < 0.003), and combination (TCL-BG-TSLs + DOX-BG-TSLs) without NIR irradiation (*P < 0.04, Figure 6C). There were no significant differences in the body weights of the mice (Figure 6D), their feeding habits, or behavior among all groups, suggesting that there were no overt toxicities related to the therapy. Next, we examined the tumors for markers of cell proliferation (antiKi67), micro vessel density (MVD, anti-CD31), apoptosis
with TCL-BG-TSLs with NIR irradiation showed a significant increase in the expression of pro-inflammatory cytokines IL-6, IL-12, and TNF-α during DC maturation, compared to DCs treated with TCL-BG-TSLs without NIR irradiation, and upon administering other treatments to DCs (Figure 4B). These data clearly indicate that treatment with TCL-BG-TSLs combined with NIR irradiation induces DC maturation and activation. Observation of CO2 Bubble Generation in TumorBearing Mice. Prior to evaluating the therapeutic efficacy of BG-TSLs, we examined CO2 bubble generation after injection of BG-TSLs into B16F10 tumor-bearing mice followed by NIR irradiation, using an ultrasound imaging system. We first verified CO2 bubble generation from BG-TSLs after their uptake by B16F10 cells. BG-TSLs showed no evidence of CO2 bubble generation at 37 °C; however, they clearly showed CO2 bubble generation at 41 °C (Figure 5A). Although NH4HCO3 is decomposed at 35−60 °C, we speculated that a partial amount of NH4HCO3 may be encapsulated into BG-TSL. To confirm CO2 generation, we prepared another BG-TSL-1 using the same hydration method at 34 °C. The physical properties of BG-TSL-1 (34 °C hydration method) and BG-TSL (43 °C hydration) showed similar characteristics (Figure S1A,B). Additionally, we observed CO2 generation in BG-TSL-1 (34 °C hydration) at 37 and 41 °C (Figure S1C). In this study, we focused on CO2 bubble generation in liposomes by external temperature control using NIR irradiation. BG-TSL-1 was unable to control CO2 generation at body temperature (37 °C), which indicated that the anticancer drug (DOX) is released from BG-TSL after being injected into the patient. In this case, we cannot control DOX release from BG-TSL, leading to side effects or nontargeted delivery of DOX. However, the hydration method at 43 °C showed CO2 bubble generation at 41 °C rather than 37 °C (Figure 5A). This result could allow for the safe circulation or maintenance of BG-TSL without DOX release after injection. Next, we monitored CO2 bubble generation in tumorbearing mice after injection of BG-TSLs. We did not observe 2485
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Figure 6. Therapeutic efficacy of the combination of TCL-BG-TSLs and DOX-BG-TSLs plus NIR irradiation in the B16F10 tumor model. Treatment with TCL-BG-TSLs and DOX-BG-TSLs was initiated 1 week after subcutaneous injection of B16F10 tumor cells into C57/BL6 mice. The TCL-BG-TSLs and DOX-BG-TSLs were injected i.v. once a week at doses of 5 mg/kg TCL and 5 mg/kg DOX based on body weight. One hour after injection of BG-TSLs, the tumor site was irradiated by NIR (1.00 W/cm2) for 1 min. (A) Experimental schedule for the treatments. (B) Tumor volume. (C) Tumor weight and tumor tissues. (D) Body weights of mice. (E) Immunohistochemistry results for markers of cell proliferation (Ki67), microvessel density (MVD, CD31), and the TUNEL assay were performed using B16F10 tumor tissues following treatment with TCL-BG-TSLs and DOX-BG-TSLs (scale bar: 50 μm). IHC analysis for CD8+ T cell localization in tumor tissues (anti-CD8 staining, scale bar: 50 μm) was performed. The bar graph indicates the percentage of CD8+ T cells in the same tissue area. Cytotoxic CD8+ T cells (positive for anti-CD8 and IFN-γ immunostaining) were identified using flow cytometry from mice splenocytes from the treatment group. The bar graph indicates the percentage of CD8+ and IFN-γ+ cells in the same tissue area. Scale bar: 50 μm. All analyses were performed using five random fields recorded for each slide. Error bars represent SEM *P < 0.002 and **P < 0.001.
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(TUNEL assay), and CD8+ T cell population (anti-CD8) using immunohistochemistry. The combination plus NIR irradiation group showed significant inhibition of cell proliferation (*P < 0.001), a significant decrease in MVD (*P < 0.001), and a significant increase in the number of apoptotic cells (*P < 0.001, Figure 6E). To assess the immune response of the TCL, we examined the CD8+ T cell population (anti-CD8) in the tumor tissue as well as activation of IFN-γ secreting cytotoxic CD8+ T cells in splenocytes. The combination plus NIR irradiation group showed a significant increase in the number of CD8+ T cells in the tumor tissue (*P < 0.001, Figure 6E), and a significant increase in the number of IFN-γ+ secreting CD8+ T cells (*P < 0.001, Figure 6E). These data suggest that the combination of TCL-BGTSLs and DOX-BG-TSLs plus NIR irradiation was efficacious, providing a therapeutic option that involves a combination of chemotherapy and immunotherapy.
CONCLUSIONS
This study demonstrated that combining TCL-BG-TSLs and DOX-BG-TSLs, potential carriers for the delivery of chemotherapeutic agents, with a tumor-specific antigen delivery system triggered payload release by NIR irradiation, leading to an increased antitumor efficacy. Herein, we demonstrated that BG-TSLs are capable of triggering payload release using external stimulation with NIR irradiation. This system provides a nanotechnology platform that uses a combination of chemotherapy and immunotherapy and demonstrate the potential use of an antitumor chemo-immunotherapeutic agent for the treatment of cancer. Our combination strategy using TCL-BG-TSLs and DOX-BG-TSLs has a broad applicability as a delivery platform for combination therapies and can be used or modified to suit other therapeutic and experimental approaches. 2486
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The amount of DOX was determined using UV spectroscopy at 490 nm and that of TCL was determined using the BCA protein assay kit. The percent release of DOX or TCL from DOX-BG-TSLs or TCL-BG-TSLs was calculated using the following formula:40
MATERIALS AND METHODS
Materials. Hydrogenated soy phosphatidylcholine (HSPC), dipalmitoylphosphatidyl-choline (DPPC), monostearoylphosphatidylcholine (MSPC), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG-2000) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Doxorubicin hydrochloride (DOX), ammonium bicarbonate (NH4HCO3), and Sephadex G-50 columns were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Welgene (Gyeongsan, Korea). All other materials were of analytical grade and used without further purification. Preparation of TSLs, BG-TSLs, DOX-BG-TSLs, and TCL-BGTSLs. TSLs were prepared using the thin lipid film hydration method.31,34,35 TSLs were composed of DPPC/MSPC/DSPE-mPEG2000 at a molar ratio of 79.5:6.5:14. The lipids were dissolved in 3 mL of a chloroform−methanol mixture (8:2, v/v), and the solvent was evaporated using a rotary evaporator under a vacuum to obtain a thin lipid film. The lipid film was then hydrated with 6 mL of 300 mM citrate acid solution (pH 4) at 43 °C. The TSLs were isolated using a Sephadex G-50 column (GE Healthcare, Buckinghamshire, U.K.). BG-TSLs were prepared using a slight modification of the TSL preparation method.31,34,36 BG-TSLs were composed of DPPC/ MSPC/DSPE-mPEG-2000 in the same molar ratio as the TSLs. The lipids were dissolved in 3 mL of a chloroform−methanol mixture (8:2, v/v), and the solvent was evaporated using a rotary evaporator under a vacuum to obtain the thin lipid film. The lipid film was hydrated with 6 mL of 253 mM NH4HCO3 solution (pH 4) at 43 °C, after which the BG-TSLs were isolated using a Sephadex G-50 column. Here, we selected 43 °C for the hydration temperature during liposome preparation, despite NH4HCO3 decomposition, which induced DOX release at 41 °C compared to 37 °C. DOX-BG-TSLs were prepared using a slight modification of the BG-TSL preparation method. DOX was encapsulated into BG-TSLs using the pH gradient loading method.37 DOX (2 mg/mL) was mixed with BG-TSLs (2:1 w/v), after which the mixture was incubated at 29 °C overnight. The DOX-BG-TSLs were isolated using a Sephadex G50 column at 4 °C to remove free DOX. TCL-BG-TSLs were prepared using a slight modification of the BG-TCL preparation method. Briefly, to prepare the TCL-BG-TSLs, the lipid film was hydrated with a 1 mg/mL TCL solution containing 253 mM of NH4HCO3 at 43 °C. The TCL-BG-TSLs were isolated using a Sephadex G-50 column at 4 °C to separate unencapsulated TCL and NH4HCO3. The TSLs, BG-TSLs, DOX-BG-TSLs, and TCL-BG-TSLs were stored at 4 °C until further use. The encapsulation efficiency of DOX into the DOX-BG-TSLs was measured using UV−vis spectrophotometry (UV-mini, Shimadzu, Japan) at 490 nm after dissolution of the liposomal membrane with a chloroform−methanol mixture (4:1 v/v).38 The encapsulation efficiency of TCL into the TCL-BG-TSLs was determined using a BCA protein assay after dissolution of the liposomal membrane using a chloroform and methanol mixture (4:1 v/v). The size and surface charge of the BG-TSLs, DOX-BG-TSLs, and TCL-BG-TSLs were measured by laser light scattering using a particle size analyzer (SZ100, Horiba, Japan).39 The morphology of the BG-TSLs and DOXBG-TSLs was examined using atomic force microscopy (AFM, XE100, Park Systems, Korea). In addition, the DOX-BG-TSL morphologies were monitored by cryogenic transmission electron microscopy (cryoTEM, Tecnai T12 Bio-TWIN, FEI Company, Hillsboro, OR, USA). TCL encapsulation into the TCL-BG-TSLs was determined using silver staining to visualize protein bands from the lysate using an EZ-silver staining kit.20 Release of the Payload from DOX-BG-TSLs or TCL-BG-TSLs at Different Temperatures. The temperature-sensitive release of DOX or TCL from the DOX-BG-TSLs or TCL-BG-TSLs was measured. Briefly, DOX-BG-TSLs or TCL-BG-TSLs were added to a glass tube, which was then placed in a water bath for a predetermined time. After incubation, the DOX-BG-TSLs or TCL-BG-TSLs were centrifuged (15 700g for 30 min) and the supernatants were collected to measure the release of DOX or TCL.
release (%) = (Ft − F0)/(Fmax − F0) × 100 where Ft is the payload intensity of the DOX-BG-TSLs or TCL-BGTSLs, F0 is the initial background payload intensity of the DOX-BGTSLs or TCL-BG-TSLs, and Fmax is the intensity of payload in the DOX-BG-TSLs or TCL-BG-TSLs in an organic solvent mixture consisting of chloroform and methanol (4:1 v/v). Release Behavior of the Payload from DOX-BG-TSLs or TCLBG-TSLs with NIR Irradiation. The triggered release of DOX or TCL from the DOX-BG-TSLs or TCL-BG-TSLs, with or without NIR irradiation, was determined by measuring the amount of DOX or TCL released using UV spectroscopy or the BCA protein assay kit, respectively. The DOX-BG-TSLs or TCL-BG-TSLs were added to a glass tube, and the tube was then placed in a water bath at 37 °C followed by near-infrared (NIR) laser (2 W/cm2, 808 nm) irradiation as a heat source for hyperthermia. After irradiation, the liposomal solution was centrifuged at 15 700g for 30 min and the supernatant was collected to measure the levels of the DOX or TCL released from the DOX-BG-TSLs or TCL-BG-TSLs using the equation provided above. Intracellular Uptake of DOX-BG-TSLs. The melanoma cell line, B16F10, was maintained and propagated in DMEM supplemented with 10% fetal bovine serum (FBS) and 0.1% gentamicin sulfate at 37 °C in a 5% CO2 incubator. The intracellular uptake of DOX-BG-TSLs was assessed using flow cytometry (FACSCallibur with CELLQuest Software, BD Biosciences, Franklin Lakes, NJ, USA). B16F10 cells (2 × 105 cells/well) were seeded into a 6-well plate. After 24 h incubation at 37 °C in a CO2 incubator, the medium was replaced with fresh DMEM containing DOX-BG-TSLs. The cells were then further incubated for 20 min. Thereafter, the medium was removed, and the cells were collected. The intracellular uptake of DOX-BGTSLs in B16F10 cells was monitored using flow cytometry. Additionally, the morphology of the tumor cells containing DOXBG-TSLs was observed using confocal microscopy (Carl Zeiss, LSM 710, Germany). Viability of B16F10 Cells Subjected to DOX-BG-TSLs at Different Temperatures. The viability of the cells subjected to DOX-BG-TSLs at different temperatures was determined using the MTT assay.41 After the intracellular uptake of DOX-BG-TSLs by the B16F10 cells, the cells were incubated at 37 or 41 °C for 5 min. The cells were then further incubated for 24 h in a CO2 incubator, and the MTT assay was performed to assess cell viability. The absorbance was measured at 570 nm using a microplate reader (EL808, Bio-Tek, Winooski, VT, USA). DC Maturation and Activation. Bone-marrow-derived dendritic cells (BMDCs) were harvested from the bone marrow of C57/BL6 mice.42 Briefly, bone marrow was collected from the tibiae and femora of the mice. Red blood cells were depleted using RBC-lysis buffer (Sigma-Aldrich, St Louis, MO, USA), and bone marrow cells (2 × 106 cells/well) were collected and cultured in a 6-well plate containing 4 mL of RPMI 1640 supplemented with 10% FBS, 0.1% gentamycin, and 20 ng/mL mouse recombinant GM-CSF at 37 °C in a 5% CO2 incubator. To verify DC maturation, DCs were cultured in 6-well plates at a density of 2 × 106 cells per well. DCs alone as a control, BG-TSLs, TCL (50 μg), and TCL-BG-TSLs (50 μg of TCL) were then incubated with the cells for 30 min. After incubation, DCs were irradiated with NIR (2 W/cm2) and cultured for an additional 24 h, after which DC maturation was assessed using flow cytometry.42 DCs were stained with FITC-conjugated anti-CD11c, PE-conjugated antiCD40, anti-CD80, and anti-MHC class I antibodies. In addition, cytokines (IL-1β, IL-6, IL-12p70, and TNF-α) secreted from DCs during maturation were analyzed using cytokine-specific ELISA kits (eBioscience, San Diego, USA).42 CO2 Generation in BG-TSLs in Tumor-Bearing Mice. Following NIR irradiation, CO2 generation by the B16F10 cells containing BG-TSLs was monitored using an ultrasound imaging 2487
DOI: 10.1021/acsabm.9b00183 ACS Appl. Bio Mater. 2019, 2, 2481−2489
ACS Applied Bio Materials
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system with a 7 MHz transducer (Telemed, Vilnius, Lithuania).43 B16F10 cells were incubated with BG-TSLs for 30 min at 37 °C. Then, the cells were collected in a round-bottom tube, which was heated to produce temperatures of 37 or 41 °C. CO2 bubble generation was automatically observed using the B-mode of the Echo Blaster 128 CEXT.43 In addition, we verified CO2 bubble generation following NIR irradiation in tumor-bearing mice after intratumoral injection of BG-TSLs. To produce tumors, mice were administered B16F10 (1 × 106 cells in 50 μL of HBSS) cells via subcutaneous (s.c.) injection. BG-TSLs (50 μL) were injected into tumors via intratumoral (i.t.) and intravenous (i.v.) injection. One hour after the injection of the BG-TSLs, the tumor site was irradiated with NIR (1 W/cm2). CO2 bubble generation was monitored using the B-mode of the Echo Blaster 128 CEXT. Antitumor Efficacy. Female C57/BL6 mice (6−7 weeks old) were purchased from Orient Co. (Seoul, South Korea). All mouse studies were approved by the Konkuk University Institutional Animal Care and Use Committee (ref. no. KU18025-1). To produce tumors, B16F10 cells (1 × 106 cells in 50 μL of HBSS) were subcutaneously injected into mice. After the tumor volumes reached 60 mm3, the mice were randomly allocated to five groups, as follows (n = 5/ group): (1) control, (2) TCL-BG-TSLs without NIR, (3) TCL-BGTSLs with NIR, (4) TCL-BG-TSLs + DOX-BG-TSLs without NIR, and (5) TCL-BG-TSLs + DOX-BG-TSLs with NIR. Different BGTSLs were injected once a week via i.v. injection using TCL (5 mg/ kg) or DOX (5 mg/kg). One hour after the injection of the different BG-TSLs, a NIR laser (1 W/cm2) was used as a heat source to irradiate the tumor site for 1 min. Drug treatment was continued until the mice became moribund. The tumor volume, tumor weight, and body weight of the mice were then recorded. The individuals who performed the necropsies, tumor collections, and tissue processing were blinded to the assigned treatments. Tissue specimens were fixed using either formalin or OCT (optimum cutting temperature; Miles, Inc., Elkhart, IN, USA). Immunohistochemical Staining. Immunohistochemical analysis of cell proliferation (Ki67), angiogenesis (CD31), apoptosis (TUNEL), and the CD8+ T cell population (anti-CD8) were performed using tumor tissues isolated from the mice.33,44 All analyses were recorded in five random fields of each slide at ×400 magnification. All staining was scored by two blinded investigators. CD8+ T Cell Activation after Treatment of Tumor-Bearing Mice. The activation of cytotoxic CD8+ T cells from splenocytes was assessed using tissues from groups of treated mice. After dissection of the mice, splenocytes (1 × 107) were collected from the spleens, resuspended in 1 mL of RPMI 1640 supplemented with 10% FBS, 0.1% gentamycin, and 0.5% β-mercaptoethanol, and incubated for 16 h with GolgiPlug (BD Biosciences, San Diego, CA, USA). Cells were washed and stained with a PE-conjugated anti-CD8a antibody and a FITC-conjugated anti-IFN-γ antibody to verify activation of IFN-γ secreting CD8+ T cells. Statistical Analysis. Differences in continuous variables were analyzed using the Student’s t-test to compare two groups, and ANOVA was performed to compare differences between multiple groups. For values that were not normally distributed, the Mann− Whitney rank sum test was used. The statistical package for the Social Sciences (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. A P value of
