Nanostructured Lipid Carriers Delivering Sorafenib to Enhance Immunotherapy Induced by Doxorubicin for Effective Esophagus Cancer Therapy.

The tumor microenvironment (TME) plays a significant role in weakening the effect of cancer immunotherapy, which calls for the remodeling of TME. Herein, we fabricated a nanostructured lipid carrier (NLC) to codeliver doxorubicin (Dox) and sorafenib (Sfn) as a drug delivery system (NLC/D-S). The Sfn was expected to regulate the TME of esophagus cancer. As a result, the immune response induced by Dox-related immunogenicity cell death could be fully realized. Our results demonstrated that Sfn was able to remodel the TME through downregulation of regulatory T cells (Treg), activation of effector T cells, and relieving of PD-1 expression, which achieved synergistic effect on the inhibition of primary tumor but also subsequent strong immune response on the regeneration of distant tumor.

Introduction

Cancer immunotherapy is a novel promising approach for cancer therapy, which is getting more and more attention in recent years.[1,2] Different from conventional chemotherapy or radiotherapy, which employed additional drugs or assistance to kill cancer cells, immunotherapy is designed to utilize the endogenous human immune system to combat cancer.[3] With the advances of cancer therapy, recent studies have found several drawbacks in single cancer immunotherapy. In particular, the tumor microenvironment (TME) is demonstrated to significantly downregulate the activation of immune response in tumor tissue, which finally resulted in impaired cancer immunotherapy.[4] For example, TME can reduce the infiltration of dendritic cell (DC) and suppress the activation of effector T cells to finally silence the immune system to current therapies. As a result, it was generally recognized that the remodeling of TME using other approaches, such as chemotherapy, in combination with immunotherapy might be a promising approach for effective cancer therapy. However, the delivering of proper drugs using the suitable drug delivery system (DDS) to satisfy both TME regulation and cancer immunotherapy is challenging.[5,6]

Recently, the phenomenon that some chemotherapeutics, such as anthracyclines and oxaliplatin, induce immunogenicity cell death (ICD) along with apoptosis of cancer cells is getting more and more attention.[7,8] It was reported that the ICD is capable of effectively presenting the cancer-specific antigen to the surface of the cells with the following activation of related immune cells, such as CD4 and regulatory T cells (Treg). As a result, the chemotherapy-induced ICD has been employed by previous studies to stimulate the immune response, which showed promising benefits in cancer therapy.[9,10]

Sorafenib (Sfn) is a widely applied small molecule of multikinase inhibitor, which is adopted in various cancer treatments through cell cytotoxicity.[11] Surprisingly, recent discoveries also revealed its regulation potential on TME by inhibiting the immuno-suppressive Treg cells[8] and enhancing the function of effector T cells.[12] As a result, we suggested that the combination of Sfn with anthracyclines, such as doxorubicin (Dox), might be a suitable choice for cancer therapy. It was suggested that Dox can induce strong ICD for immune response while the negative effects of TME can be neutralized by Sfn.[13] However, the codelivery of both drugs in one DDS, which integrates decent drug-loading capacity and promising tumor targetability, is a critical issue that requires careful selection of suitable carrier.[14,15]

In recent years, nanoparticle-derived DDSs have shown many advantages, including enhanced drug bioavailability, increased tumor-homing, and reduced cytotoxicity.[16−18] The DDSs adopted in cancer therapy were widely studied by previous reports with promising outcomes.[19−21] In particular, nanostructured lipid carrier (NLC) is a widely adopted organic platform for drug delivery,[22] which composed of biocompatible lipids at solid or liquid state and was suitable for the delivery of hydrophobic drugs.[22,23] As a result, the application of NLC for the safe and effective delivery of drugs for cancer therapy has been successfully developed by many previous researchers.[24,25]

Herein, we employed the mice drug-resistant esophagus carcinoma cells (AKR/Dox) as the model cell line. Moreover, NLC was selected as the carrier for the loading of Sfn and Dox (NLC/D-S) and was surface modified with folic acid (FA) as the targeting moiety to increase the tumor-homing of the DDS. It was suggested that the NLC/D-S with nanoscale size can specifically homing to tumor tissue via enhanced penetration and retention effect and deliver both drugs to target cells. Upon drug release in cells, the Dox induced ICD to express the cancer-specific antigen while Sfn regulated the TME. Both effects were believed to facilitate the immune response of the subject for effective treatment of AKR/Dox cancer.

Results and Discussion

The NLC is prepared by a solvent diffusion method in a one-pot route. The drugs can be encapsulated within the hydrophobic region of the NLC to afford decent drug loading and safe delivery. Here, in our study, under the given condition, using dynamic light scattering, it was shown that the size of the acquired NLC/D-S was around 100 nm (Figure A) with well dispersion, suggesting the successful preparation of uniform nanosized NLC using this method. By adjusting the charge ratio, the drug loading of NLC/D-S was 5.96% for Dox and 6.11% for Sfn, which nearly 1:1 in weight ratio.

Figure 1

The size and stability of NLC/D-S. (A) The size distribution of NLC/D-S. (B) Colloidal stability of NLC/D-S in PBS (pH 7.4) and mouse plasma at 37 °C for up to 48 h. Data were expressed as mean standard deviation with three parallel experiments.

Previous studies have shown that colloidal stability plays a significant role in the in vivo fate of DDS, therefore, the colloidal stability of NLC/D-S was evaluated using two physiological media (PBS 7.4 and mouse plasma). Because the distribution of DDS to the target side is a time-dependent process, the DDS should maintain stability long enough without leakage for tumor targeting.[26] Therefore, the changes in particle size of NLC/D-S were monitored for 48 h. As displayed in Figure B, NLC/D-S showed almost no changes in size in both media, which suggested that NLC/D-S might be able to maintain stability upon in vivo applications, which was a primary requirement for drug delivery in cancer therapy.[19,27]

Then, the biocompatibility of the DDS as another important parameter was also studied. The hemolysis of NLC/D-S was studied using 2% red blood cell (RBC) from New Zealand rabbit. It was suggested that hemolysis suggested the irritation of DDS on RBC, which was critical for safe drug delivery upon in vivo applications.[28] As shown in Figure A, NLC/D-S showed almost no hemolysis on RBC (1.21% h at the concentration of 1 mg/mL) under all given condition, which might be even lower because of the blood dilution upon in vivo applications. Therefore, the NLC/D-S was suggested to be a highly biocompatible DDS without hemolysis upon in vivo applications.[29]

Figure 2

The biocompatibility of NLC/D-S. (A) Hemolysis of NLC/D-S on 2% RBC under different concentrations at 37 °C for 1 h. (B) Cytotoxicity of various concentrations of NLC after 48 h of incubation with AKR/Dox cells. Data were expressed as mean standard deviation with three parallel experiments.

The biocompatibility of the DDS was further investigated by investigating the cytotoxicity of drug-free carrier on AKR/Dox cells. The cell viability of AKR/Dox cells recorded after cells was exposed to various concentrations of NLC for 48 h. As depicted in Figure B, the viability of AKR/Dox cells at the end of the test remained over 90% at the concentration of 200 μg/mL, which further suggested the high biocompatibility of NLC.[30]

Afterward, the cellular uptake of Dox in AKR/Dox cells was investigated to understand the role of FA modification in the cellular uptake of the DDS. With the aim to find the true result of NLC-mediated drug uptake in comparison to free drug, the NLC/Dox was employed instead of NLC/D-S. As illustrated in Figure A, in line with previous reports, the strong drug-resistance nature of AKR/Dox cells resulted in weak cellular accumulation of free Dox while this phenomenon significantly overcame by introduction of NLC/Dox. It was reported that DDS can facilitate drug retention in cells through endocytosis, especially receptor-mediated pathways to partially reverse drug resistance of cancer cells.[3,31] In order to identify this merit, the AKR/Dox cells were pretreated with excess free FA to further study the drug accumulation changes between free Dox and NLC/Dox. As expected, the cellular uptake of free Dox was not affected by FA, suggesting that the accumulation of free Dox was not related to FA. However, compare with the untreated group, FA pretreatment resulted in significant drop in intracellular accumulation of Dox, and this phenomenon was observed in all tested time intervals, which showed a half drop (53.6%) of drug accumulation at 6 h postincubation. These observation was in line with previous reports that FA modified in the NLC mediated the cellular uptake of the DDS through the corresponding receptor, which was beneficial for cancer therapy.[32,33]

Figure 3

The tumor targetability of NLC/D-S. (A) Quantitative analysis of intracellular time-dependent uptake of NLC/Dox in AKR/Dox cells in comparison with free Dox and pretreated with/without FA. (B) Total fluorescence intensity of dissected tumors and major organs of mice treated with NLC/D-S at 4 and 8 h post-injection. Data were expressed as mean standard deviation with three parallel experiments.

Next, the in vivo targetability of NLC was further explored. The indocyanine green as a probe was loaded into NLC to indicate the location of DDS. At predetermined time interval (4 and 8 h), the mice were sacrificed, and the tumor and major organs were harvested to study the distribution of DDS. As displayed in Figure B, NLC/D-S showed preferable accumulation in tumor at 4 h and further time extension resulted in more elevated DDS accumulation. In addition, it was noted that the distribution of NLC/D-S in major organs, especially the liver and spleen, was less than that in the tumor, which further suggested the promising tumor targetability of this DDS.

The in vitro anticancer ability of this DDS was evaluated using MTT assay. As demonstrated in Figure A, the strong drug resistance of AKR/Dox cells resulted in impaired cytotoxicity of Dox with cell viability over 63.6% at the Dox dosage of 50 μM in the NLC/Dox group. Because of cytotoxicity of Sfn, the NLC/Sfn also exerted moderate cytotoxicity on AKR/Dox cells. Most importantly, the combination of Dox and Sfn demonstrated significant drop in cell viability, which reached 32.7% at the same Dox concentration of 50 μM as compared to NLC/Dox. The combination index of the drugs was 0.47, which indicated the strong synergistic suppression effect on AKR/Dox cells.[5]

Figure 4

The in vitro anticancer assay of NLC/D-S. (A) Cell viability of AKR/Dox cells treated with different formulations at different Dox concentrations for 48 h. (B) Western blot assays of the expression of caspase-3, cytochrome C and Bcl-2 proteins after different treatments (Dox concentration: 20 μM). Data were expressed as mean standard deviation with three parallel experiments.

The western blot assay was conducted to assess the apoptosis level of cells after different treatments and as another proof to reveal the in vitro anticancer effects of NLC/D-S. As shown in Figure B, the bcl-2 level in NLC/D-S-treated cells was the lowest among all groups while the caspase-3 and cytochrome-3 levels were higher than other groups. These results provided decisive evidence to show that severe apoptosis was occurred upon NLC/D-S treatment, which explained the best cell inhibition effect of NLC/D-S.[34]

The multicellular tumor spheroid (MCTS), which simulates the in vivo solid tumor, was further employed to assess the in vivo anticancer effects of different treatments. As shown in Figure A,B, the growth of MCTS in the free Dox group was uncontrollable, which was almost 3-fold of original volume, suggesting the neutralization profile of AKR/Dox on the cytotoxicity effect of Dox. Moreover, the inhibition effect of single delivery systems (NLC/Dox and NLC/Sfn) is only moderate without reversing the growth of MCTS. In contrast, NLC/D-S significantly reversed MCTS growth, which was merely 0.72-fold of the original volume at day 5, which suggested the promising synergetic effects of both drugs on the inhibition of drug resistance cells.[35]

Figure 5

The inhibition effect on MCTS. (A) The volume changes of MCTS after different treatments (Dox concentration: 20 μM) for 5 days. (B) The representative optical image of MCTS at day 0 (left) and day 5 (right) after different treatments. Scale bar: 200 μm. Data were expressed as mean standard deviation with three parallel experiments.

Afterward, the core design of our study, verifying the role of NLC/D-S in activating immune responses, was evaluated in vivo using the AKR/Dox tumor-bearing model. Mice were randomly divided and treated with different formulations in parallel. The tumor volume of the subjects was recorded before every administration. After 15 days of treatment, the mice were challenged with tumor cells on the other side of the primary tumor, and the growth in distant tumor was monitored for another 15 days without any treatment to assess the immune responses upon different treatments. As displayed in Figure A, the growth of primary tumors was significantly suppressed in NLC/D-S group (286 mm3) as compared with other groups. In contrast, the NLC/Dox and NLC/Sfn groups showed faster tumor growth during the whole period (final tumor volume of 416 and 553 mm3, respectively). In particular, the further tumor challenging using the same tumor cells also revealed the promising effects of NLC/D-S. As displayed in Figure B, the distant tumor in the control group persistently increases to the inferior acquired immunity. In contrast, both NLC/Dox and NLC/Sfn treatments triggered elevated immune response as the tumorigenicity of AKR/Dox cells was suppressed to some extent. It was noted that the performance of NLC/Dox was better than the NLC/Sfn group, suggesting the critical role of ICD in the immune response. In particular, the NLC/D-S group showed the lowest tumorigenicity of cancer cells with a final distant tumor volume of 101 mm3, which was in line with results, as obtained in Figure A, and our suggestions.

Figure 6

In vivo antitumor efficacy of different formulations for AKR/Dox tumor-bearing Balb/c mice. Tumor volume changes of primary (A) and distant tumors (B) after different treatments as a function of time were recorded. Data were expressed as mean standard deviation with six parallel experiments.

In order to further confirm this conclusion and understand the underlying mechanisms responsible for this phenomenon, the cytokine (IL-6) was selected as a signal for DC maturation, and its plasma concentration after different treatments was measured by the ELISA kit. As demonstrated in Figure A, the plasma levels of IL-6 increased upon the administration of different treatments, which further increased as a function of time. These results suggested the DC maturation as a result of immunotherapy. As expected, the NLC/D-S group showed higher elevation on IL-6 levels than other groups, which further confirmed the preferable activation effect of NLC/D-S on the immune system.[36] In order to understand the role of ICD in DC activation, the ICD in tumor tissues was further assessed. In line with results, as obtained in Figure A, compared to the inferior ICD level in Sfn and control groups, Dox could induce strong ICD while the aid of Sfn in the NLC/D-S group further enhanced the ICD level.

Figure 7

The (A) cytokine IL-6 level in peripheral blood serum (indicating in vivo DC stimulation) after different time intervals of treatment. Data were expressed as mean standard deviation with three parallel experiments. (B) The ICD of tumor tissues at the end of the test after different treatments. Scale bar: 100 μm.

In order to identify the role of Sfn, the strong immune response effect of NLC/D-S, the Treg, and effector T cell regulation effects after Sfn treatment were explored. As displayed in Figure A, the percentage and number of CD4+CD25+ Treg in tumor-infiltrating lymphocytes were significantly dropped after Sfn treatment in a concentration-dependent manner. Moreover, as confirmed in Figure B, the proliferation of Treg was also negatively regulated by Sfn in a concentration-dependent manner. As CD4+CD25+ Treg was responsible for the immune suppression of TME, the negative regulation effect of Sfn on these cells was supposed to exert beneficial effects on the restoration of the function of effector T cells and DC cells to fully induce immune response.[37]

Figure 8

(A) Number of CD4+CD25+ Tregs cells among the CD4+ T cell population in the tumors tissue after different dosage treatment of Sfn. (B) Effect of different concentrations of Sfn on Treg proliferation in vitro. Data were expressed as mean standard deviation with three parallel experiments.

Cytotoxic (CD8+) T lymphocytes (CTLs) are important cells in cancer killing through the release of effector molecules and/or effector cytokines. Its activation was critical to the final performance of immunotherapy. As a result, the inhibition or regulation effect of Sfn on CTLs is also our concern. As demonstrated in Figure A, the cell number of CTL among CD8+ cells was increased by Sfn treatment and was positively related to drug concentration, which suggested the beneficial effect of Sfn on CTL activation. Considering that PD-1 signaling of cancer cells can mediate potential immune escape, we next aim to explore if Sfn could further facilitate the recognition of CTL on cancer cells. As a result, the PD-1-positive CD8+ T cells in TME were also examined before and after Sfn treatments. As depicted in Figure B, the percentage of PD-1+ CD8+ T cells in the TME decreased drastically with the increase of Sfn dosing, suggesting that Sfn can regulate the PD-1 expression in the effector T cells to block the PD-1/PD-L1 pathways in the major cases of tumor immune escape. In all, we concluded that Sfn was able to remodel TME on the AKR/Dox tumors through elevation of CTL percentage and decrease of proportion/proliferation of PD-1+ CD8+ effector T cells and Treg cells, which finally sensitized the subject to ICD-induced immune responses with promising inhibition on tumors.[38]

Figure 9

Sfn treatment augmented effector function of tumor-specific T cells and downregulated the PD-1 expression of CD8+ T cells in TME. (A) The mean percentage of CD25 (activation marker) expressing cells among tumor-infiltrating CD8+ T cells and (B) corresponding flow cytometry graphs. (C) Percentage of PD-1-expressing CD81 T cells in tumor draining lymph nodes of tumor bearing mice. Data were expressed as mean standard deviation with three parallel experiments.

Conclusions

In summary, we successfully fabricated dual drug-loaded NLC as an effective DDS for TME remodeling and ICD-based cancer immunotherapy (NLC/D-S). Our results demonstrated that nanosized NLC/D-S was highly stable and biocompatible with promising tumor targetability. The synergistic effect of Dox and Sfn on NLC/D-S showed the best in vitro and in vivo anticancer benefits as compared to single delivery systems (NLC/Dox and NLC/Sfn). Most importantly, the NLC/D-S with the combination of Dox-induced ICD and Sfn-mediated TME remodeling showed strong immune response after treatment. Our results further revealed that the TME remodeling effect of Sfn was through the combination of Treg inhibition/effector T cell activation/PD-1 relieving. In all, the NLC/D-S might be promising DDSs for effective cancer immunotherapy.

Experimental Section

Detailed information about Materials and Method can be found in the Supporting Information.