Nanovaccine Incorporated with Hydroxychloroquine Enhances

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Biological and Medical Applications of Materials and Interfaces

Nanovaccine Incorporated with Hydroxychloroquine Enhances Antigen Cross-presentation and Promotes anti-Tumor Immune Responses Jiale Liu, Xiaoxuan Liu, Yanfeng Han, Jing Zhang, Dan Liu, Guilei Ma, Chen Li, Lanxia Liu, and Deling Kong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09348 • Publication Date (Web): 23 Aug 2018 Downloaded from http://pubs.acs.org on August 24, 2018

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ACS Applied Materials & Interfaces

Nanovaccine Incorporated with Hydroxychloroquine Enhances Antigen Cross-presentation and Promotes Anti-Tumor Immune Responses Jiale Liu1, Xiaoxuan Liu1, Yanfeng Han2,3, Jing Zhang1, Dan Liu1, Guilei Ma1, Chen Li1, Lanxia Liu1*, Deling Kong1,4* 1.The Tianjin Key Laboratory of Biomaterials, Institute of Biomedical Engineering, Peking Union Medical College & Chinese Academy of Medical Sciences, Tianjin 300192, China 2.Institute of Biomedical & Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006, China 3. School of Biomedical Sciences, University of Queensland, St Lucia QLD 4072, Australia 4. College of Life Science, Nankai University, Tianjin300071, China

*

Corresponding author

Corresponding to: Deling Kong, Ph.D. Professor Institute of Biomedical Engineering, Peking Union Medical College & Chinese Academy of Medical Sciences, College of Life Science, Nankai University. Phone & Fax: +86 (22) 87458056 E-mail: [email protected]

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Abstract Induction of effective antigen-specific CD8+ T-cell responses is critical for cancer immunotherapy success. Hydroxychloroquine (HCQ) is a widely used classical antimalarial and antirheumatic drug. HCQ is also an endosomal membrane disrupting agent that can lead to vesicular swelling and membrane permeabilization, which likely facilitates the release of therapeutic agents from lysosomes into the cytoplasm. Here, we

develop

a

minimalistic

nanovaccine,

which

is

comprised

of

poly

(lactide-co-glycolide) acid (PLGA) nanoparticles encapsulating a physical mixture of ovalbumin (OVA, a model antigen) and HCQ (HCQ-OVA-PLGA NPs). We tested whether HCQ could spatiotemporally control the cytosolic delivery of antigens, enhance antigen processing and presentation via the MHC-I pathway, and thus generate a sufficient anti-tumor cytotoxic T-cell response. The results of in vitro experiments showed that HCQ-OVA-PLGA NPs significantly enhanced OVA escape from lysosomes into the cytoplasm within bone marrow-derived dendritic cells (BMDCs). We also observed that HCQ-OVA-PLGA NPs enhanced the expression level of MHC-I on dendritic cells (DCs) and improved cross-presentation of antigen, compared to Free OVA or OVA-PLGA NPs. Results of in vivo experiments confirmed that HCQ initiated Th1-type responses and strong CD8+ T-cell responses that induced tumor cell apoptosis. Moreover, vaccination of mice with HCQ-OVA-PLGA NPs effectively generated memory immune responses in vivo and prevented tumor progression. We conclude that co-encapsulation of HCQ with antigens in nanovaccines can boost antigen-specific antitumor immune responses, particularly through CD8+ T-cells, serving as a simple and effective platform for the treatment of tumors and infectious diseases. Keywords: Vaccine; nanovaccine; Hydroxychloroquine; Immune response; PLGA nanoparticles

2


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1. Introduction On the basis of the cancer immunoediting hypothesis, the immune system plays a pivotal role in cancer occurrence and progression. The immune system can not only recognize and subsequently destroy cancer cells, but also has the capacity to shape cancer immunogenicity and promote cancer progression1. Therefore, in recent years immunotherapy that guides the immune system to kill cancer cells has been pursued. Consequently, innovative therapies, such as engineered chimeric antigen receptor T cells (CAR-T), immune checkpoint inhibitors and many other immunotherapeutic techniques2-6, have been invented. With the development of nanotechnology, nanovaccines have emerged as a promising approach in the cancer immunotherapy toolbox. Compared to traditional vaccines, the nanovaccine delivery system has multifaceted and unique advantages, for example constant release of antigens from nanoparticles at the injected site for a prolonged period7, protection of antigens from enzymatic degradation, co-delivery of antigens and adjuvants8, facilitating the access of antigens to antigen-presenting cells, and enhancing cytosolic delivery of antigens9-10. Cytosolic

antigen

processing

and

MHC-I

presentation

(known

as

cross-presentation) is essential for the initiation of CD8+ T-cell responses (CTLs), which are critically important for effectiveness of cancer immunotherapy11-12. However, vaccination with soluble protein/peptide antigens usually results in poorly primed cell-mediated immune responses due to exogenous antigens processed through the endosomal pathway, leading to the presentation of peptides on major histocompatibility complex (MHC) class II molecules (MHC-II), rather than processing via the cytosolic pathway and presentation on MHC class I (MHC-I) machinery. Compared to conventional peptides/adjuvant complexes, nanovaccines that are made through the encapsulation of antigens within nanoparticles, are more likely to enhance functional CTLs, as they feature increased efficiency of MHC-I antigen presentation. To achieve an optimal exogenous antigen MHC-I presentation and trigger antigen-specific CD8+ T-cell responses, researchers have made efforts in 3



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several directions, such as the modification of nanovaccine carriers with ligands/molecules targeted to dendritic cells (DCs) or draining lymph nodes13-14, and co-delivery

of

antigens

and

Toll-like

receptor

(TLR)

agonists

or

other

immunopotentiators using spherical nucleic acids or polymer nanocarriers15-17. All strategies have achieved success with varying degrees. Numerous studies have demonstrated that the pivotal feature important in achieving cross-presentation by cancer nanovaccine, is antigen spatiotemporal orchestration and antigen escape from lysosomes in antigen-presenting cells18-19. Recently, Gao J. et al reported that a minimalistic nanovaccine, made by physically mixing antigens with synthetic polymeric

nanoparticles,

achieved

efficient

cytosolic

antigen

delivery

in

antigen-presenting cells and generated a potent cytotoxic T-cell response19. Hydroxychloroquine (HCQ), an analog of Chloroquine (CQ), is a widely used antimalarial and antirheumatic drug with low cytotoxicity effects to normal cells20-21. HCQ has also been shown to exert anti-tumor effects by suppressing autophagy, which is a self-protective mechanism of tumor cells in response to stress, thereby sensitizing the cells to apoptosis by chemotherapy or other cancer treatments22-24. HCQ is an endosomal membrane disrupting agent, upon entering acidic endosomes, HCQ promotes vesicular destabilization and endosomal membrane permeabilization, facilitating the release of therapeutic agents into cytoplasm. Kataoka et al, conjugated HCQ to siRNA, and formed functionalized HCQ-siRNA gold nanoparticles (GNPs). Delivery of HCQ-siRNA-GNPs facilitated siRNA endosomal escape into the cytoplasm, enhanced siRNA loading of the RNA-Induced Silencing Complex (RISC), and improved the efficiency of knock-down25. However, little is known whether HCQ could spatiotemporally control the cytosolic delivery of antigens, to generate a strong cytotoxic T-cell response. Poly (lactide-co-glycolide) acid (PLGA) is a biodegradable copolymer and has been approved by the FDA for numerous clinical applications. In addition, previous studies have demonstrated that PLGA nanoparticles exhibit significant potential as nanovaccine carriers and immune adjuvants12,

26-27

. In our previous work, we

formulated various OVA-nanoparticles based on PLGA, using a double emulsion 4


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(W/O/W)/solvent evaporation method, and evaluated the effect of these nanoparticles on antigen-specific immune responses28. To promote MHC-I restricted CD8+ T-cell responses, we successfully prepared hyaluronic acid (HA) decorated cationic lipid-PLGA hybrid nanoparticles as nanovaccines29. In the present study, to address whether HCQ could achieve antigen spatiotemporal orchestration and generate sufficient anti-tumor cytotoxic T-cell responses, we developed nanovaccines by physically mixing OVA (a model antigen) and HCQ, and then encapsulating the mixture into PLGA NPs. Our objectives were to investigate whether the prepared nanovaccines (HCQ-OVA PLGA NPs) could enhance the cytosolic delivery of antigens, improve cross-presentation of antigens and subsequently boost MHC-I restricted cytotoxic CD8+ T-cell responses.

2. Materials and methods 2.1 Encapsulation of PLGA NPs with antigens PLGA NPs loaded with antigens or with antigens and HCQ were prepared using a two-stage emulsification method30. Briefly, 1 mg of OVA (Sigma-Aldrich Co. MO, USA) or 1 mg of OVA and 5 mg of hydroxychloroquine sulfate (Huamaike Biotechnology Co. Beijing, China) was dissolved in distilled water and then each of the solutions was added to 1 mL of poly(D,L-lactide-co-glycolide) (lactide:glycolide 75:25, Mw 4,000-15,000, Sigma-Aldrich Co. MO, USA) dichloromethane solution to make OVA-PLGA NPs or HCQ-OVA-PLGA NPs. The primary water-in-oil (W/O) emulsion was ultrasonicated at 30 kW of power output for 1 min, and subsequently transferred into 4 mL of 2% w/v polyvinyl alcohol (PVA, Sigma-Aldrich Co. MO, USA) in de-ionized water with additional sonication for 3 min, to generate a secondary (Water-Oil-Water, W/O/W) emulsion. This emulsion was magnetically stirred in fume hood overnight for organic solvent volatilization. The produced OVA-PLGA NPs and HCQ-OVA-PLGA NPs were collected by centrifugation (Beckman Coulter, Avanti J-26S XP, USA) at 20,000 rpm for 20 min. The NPs were washed with distilled water 3 times to remove residual PVA. 5



2.2 Characterization of antigen-loaded PLGA NPs The size of HCQ-OVA-PLGA NPs and OVA-PLGA NPs were measured using dynamic light scattering (DLS). Zeta potential was detected using Zetasizer Nano ZP (Malvern Instruments Ltd., UK) by laser Doppler electrophoresis. The particle morphology was analyzed by transmission electron microscopy (TEM) (Tecnai-F20, FEI, The Netherlands). The loading capacity of OVA in PLGA NPs was quantified by measuring OVA concentration using Enhanced BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai,China) after OVA was released from NPs. Briefly, a certain amount of NPs were suspended in a solution containing 0.1 M NaCl and 1% w/v SDS at room temperature overnight. The pellucid liquid was mixed with BCA working solution and incubated at 60℃ for 30 min. Absorption was detected at 562 nm by Thermo Scientific Microplate Reader (Varioskan LUX, Finland). The in vitro release behavior of antigen was evaluated using following method. Approximately 10 mg of lyophilized NPs were suspended in 1 mL of phosphate buffer saline (PBS) with pH value of 7.4. The suspension was gently shaken at 200 rpm in an orbital shaker at 37℃. At established intervals, the supernatant was withdrawn after centrifugation and an equal volume of fresh PBS was added. The OVA concentrations in collected supernatant were detected with BCA assay kits. Data is displayed as the mean ± SD.

2.3. In vitro Immunization Studies 2.3.1 Intracellular trafficking of PLGA NPs in bone-marrow-derived dendritic cells The bone marrow of 6-8 weeks old C57BL/6 mice (Beijing HFK Bioscience Co., Beijing, China) was used to harvest bone marrow-derived dendritic cells (BMDCs), according to previous protocol31-32. BMDCs were suspended in 10 mL complete RPMI 1640 medium (10% fetal bovine serum (FBS), 10 ng/ml IL-4 and 20 ng/ml GM-CSF), and cultured in 5% CO2 incubator at 37 °C for 6 days (RPMI 1640 6


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medium containing 10% fetal bovine serum (FBS), IL-4 (10 ng/ml) and GM-CSF (20 ng/ml)) before they were used for in vitro experiments. To observe whether HCQ encapsulated in HCQ-OVA-PLGA NPs facilitates OVA lysosomal escape into cytoplasm of BMDCs, fluorescein isothiocyanate (FITC) labeled OVA (Beijing Solarbio Science & Technology Co., Beijing, China) was used to prepare nanoparticles. After BMDCs co-incubated with Free FITC-OVA (20µg/mL), OVA(FITC)-PLGA NPs or HCQ-OVA(FITC)-PLGA NPs (equivalent OVA concentration: 20µg/mL) for 6 h, respectively, cells were washed 3 times with PBS to deplete Free OVA and NPs. The lysosomes and nuclei of cells were visualized with Lyso-Tracker Red and DAPI (Beyotime Biotechnology, Shanghai, China), respectively. Finally, cells were fixed and imaged with laser scanning confocal microscope (Zeiss LSM 800, Germany). To further analyze the fluorescence intensity of OVA in lysosomes and OVA that had escaped into the cytoplasm of BMDCs, the cells were imaged and analyzed using ImageJ software. For assessment of fluorescence intensity of OVA in lysosomes, images were converted to 8-bit grey scale images, threshold was applied so that only 2.5% of the brightest pixels remained, and the remaining fluorescence was counted as lysosomal vesicles. To analyze the quantity of OVA escaped into cytoplasm, 8-bits images had the threshold set so that only pixels with a grey value above 5 remained bright. The remaining pixels within the cell were denoted as OVA signals. This was followed by deduction of OVA signals within the nuclei and lysosomes from the total intensity. The ratio of OVA fluorescence intensity in the cytosol to that in total (lysosome plus cytosol) was calculated.

2.3.2 Activation of BMDCs and Cross-presentation of antigens. To test whether HCQ-OVA-PLGA NPs could activate and help BMDCs mature, immature BMDCs were stimulated with Free OVA, OVA-PLGA NPs and HCQ-OVA-PLGA NPs for 6 hours. Cells were then incubated in complete RPMI 1640 medium overnight. BMDCs were collected and stained with a mixture of antibodies labeled with different fluorescent dyes against CD11c, MHC-I and CD86. 7



Membrane MHC-I and CD86 on CD11c+ DCs were analyzed using a BD Accuri™ C6 flow cytometer (BD Biosciences, CA, USA). Results were analyzed and plotted using BD Accuri™ C6 software. Concentrations of IL-12 (p70) and tumor necrosis factor-ɑ (TNF-ɑ) in culture supernatants were quantified with ELISA kits according to manufacturer protocol (Tianjin Anoric Bio-technology CO., Tianjin, China).

2.4 In vivo Immunization Studies 2.4.1 Tumor models and immunization All experiments incurring the use of animals were conducted in accordance with the guidelines of the local Institutional Animal Care and Use Committee. Female C57BL/6 mice were purchased from the Beijing Laboratory Animal Center and raised in flow chambers. E.G7-OVA cell line purchased from American Type Culture Collection (ATCC) was used to generate mouse tumor model. To produce xenograft mouse tumor model, 50 µl of E.G7-OVA cells (2×107 cells/ml) at logarithmic growth phase were subcutaneously injected into shaved right flanks of mice. When tumors grew to diameters between 4-5 mm, mice were immunized with 100 µL suspension of Free OVA, OVA-PLGA NPs or HCQ-OVA-PLGA NPs (each containing 20 µg of OVA) by subcutaneous injecting in the right inguinal region. The immunization was performed three times (at week 0, 2 and 4). Mice were sacrificed at week 5. The spleen and tumors were collected for immunological response analysis.

2.4.2 Antibody responses in blood Before mice were sacrificed, whole blood was collected from the retrobulbar vascular plexus. Serum was prepared by centrifugation whole blood at 3,000 rpm for 10 min. OVA-specific IgG, IgG1 and IgG2a were quantified using ELISA kits (Tianjin Anoric Bio-technology CO., Tianjin, China) following the manufacturer's instructions.

2.4.3 Intracellular IFN-γ staining To determine the proportion of IFN-γ-producing CD8+ T cells, after re-stimulation 8


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with OVA for 4 hours, splenocytes were treated, fixed, permeabilized and stained using BD Cytofix/Cytoperm™ Plus Fixation/Permeabilization Kit as described before29. Re-stimulated splenocytes were also analyzed using a BD Accuri C6 flow cytometer.

2.4.4 Histological Assay of tumor tissue Following sacrifice, tumor tissues were collected from mice. The tissues were fixed in 4 % paraformaldehyde solution and embedded in paraffin for sectioning (5 µm per section). The histology characteristics of tumor issue was examined by hematoxylin and eosin (H&E) stain method.

2.4.5 Analysis of T cells memory immune responses To observe memory T cell responses, splenocytes (2×106 cells), harvested from immunized mice in different groups, were re-stimulated with OVA (50 µg/ml) for 72 h and subsequently stained with anti-mouse CD4-FITC, CD8-FITC, CD44-APC and CD62L-PE. Subsequently, Accuri C6 flow cytometer was used to analyze percentage of central memory T cells (CD44Hi CD62LHi) and effector memory T cells (CD44Hi CD62LLo) in splenocytes.

2.4.6 Tumor prevention experiments To further evaluate the tumor prevention effectiveness of HCQ-OVA-PLGA NPs, mice were vaccinated with Free OVA, OVA-PLGA NPs or HCQ-OVA-PLGA NPs, (OVA 40 mg/mouse) on day 0, 7 and 14 days, respectively before tumor cells EG.7-OVA implanted. Tumor length(A) and width(B) were measured using a digital caliper. Tumor volume (mm3) was calculated using the formula (A × B2)/2. All mice bearing tumors larger than 2000 mm3 were sacrificed.

2.5 Statistical Analysis Data were displayed in the form of mean ± standard deviation (SD). Student's t-test or ANOVA followed by Tukey's multiple comparison was used to detect statistic 9



differences between groups. p values of 0.05 or less were considered to be statistically significant.

3.Results 3.1 Characterization of OVA-PLGA NPs and HCQ-OVA-PLGA NPs The TEM images showed that OVA-PLGA NPs and HCQ-OVA-PLGA NPs were uniformly spherical particles (Figure 1A and 1B). The size of HCQ-OVA-PLGA NPs was determined by DLS. As shown in Table 1, the Z-average diameter of the particles in water was 207.53±2.84 nm with a polydispersity index (PDI) of 0.12±0.01. As a control, OVA-PLGA NPs were prepared under the same condition and the hydrodynamic diameter is 225.90±2.65 nm with the PDI of 0.16±0.02. The surface charge of the two particles was almost the same (-20 mV). To monitor the releasing kinetics of OVA from HCQ-OVA-PLGA NPs and OVA-PLGA NPs, protein content in collected supernatant was measured. About 20% of OVA in the two particles (OVA-PLGA NPs 20.49 ± 1.56, HCQ-OVA-PLGA NPs 24.06 ± 1.73) was released after only 2 days, and subsequent release was relatively slower, reaching about 60-70% (OVA-PLGA NPs 65.55 ± 2.54, HCQ-OVA-PLGA NPs 70.62 ± 2.05) after 22 days (Figure 1C).

Figure 1. Typical TEM images of OVA-PLGA NPs (A) and HCQ-OVA-PLGA NPs(B), and in vitro OVA release profile from nanoparticles (C). Data is presented as the mean ± SD (n = 3).

Table1 Size and Zeta potential of OVA-PLGA NPs and HCQ-OVA-PLGA NPs 10


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Nanoparticles

Diameter(nm)

Polydispersity

Zeta potential(mV)

OVA-PLGA NPs

225.90±2.65

0.16±0.02

-20.93±0.78

HCQ-OVA-PLGA NPs

207.53±2.84

0.12±0.01

-20.77±0.38

3.2 In vitro Immunization Studies 3.2.1 Intracellular trafficking of PLGA NPs in bone-marrow-derived dendritic cells To investigate the subcellular localization of OVA in BMDCs, after 6 h incubation of BMDCs with Free FITC-OVA, OVA-PLGA NPs or HCQ-OVA-PLGA NPs, images were taken with confocal fluorescence microscopy. Images (Figure 2A) show that the majority of OVA (green) of the OVA-PLGA NPs group was co-localized with lysosomes (red), indicating that little of the protein antigen escaped from the lysosomes. However, much more significant OVA signal from the HCQ-OVA-PLGA NPs group appeared in the cytoplasm of BMDCs, compared to

OVA-PLGA NPs

and Free OVA, indicating that HCQ encapsulated in HCQ-OVA-PLGA NPs could facilitate OVA lysosomal escape into the cytoplasm of BMDCs. However, we observed that some OVA signal from the HCQ-OVA-PLGA NPs group remained co-localized with lysosomes. The results support the conclusion that HCQ encapsulated in HCQ-OVA-PLGA NPs facilitated OVA escape from the lysosomes into the cytoplasm of BMDCs, suggesting that protein antigens delivered by HCQ-OVA-PLGA NPs may be processed and presented through both the MHC-I and MHC-II pathways. To further demonstrate that OVA escape from lysosomes into cytoplasm was mainly a result of HCQ co-encapsulation in PLGA NPs, we analyzed the fluorescence intensity of OVA within lysosomes and OVA that had escaped into the cytoplasm of BMDCs. As shown in Figure 2B and 2C, the fluorescence intensity of OVA within lysosomes was significantly stronger when delivered by NPs, compared to Free OVA (OVA-PLGA NPs vs Free OVA P=0.0056, HCQ-OVA-PLGA NPs vs Free OVA P=0.0088). However, there was no statistically significant difference in the intensity 11



of OVA signal within lysosomes, between the two NP groups (P=0.66). Similarly, the quantity of OVA escaped into the cytoplasm of BMDCs treated by NPs was significantly increased compared to Free OVA. The capability of OVA escape induced by HCQ-OVA-PLGA NPs was markedly elevated, compared to Free OVA (P

Nanovaccine Incorporated with Hydroxychloroquine Enhances

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