Mesoporous Silica Nanoparticles Doped with Gold Nanoparticles for

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Mesoporous Silica Nanoparticles Doped with Gold Nanoparticles for Combined Cancer Immunotherapy and Photothermal Therapy Chunwei Ong, Bong Geun Cha, and Jaeyun Kim ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00483 • Publication Date (Web): 12 Jul 2019 Downloaded from pubs.acs.org on July 18, 2019

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ACS Applied Bio Materials

Mesoporous Silica Nanoparticles Doped with Gold Nanoparticles for Combined Cancer Immunotherapy and Photothermal Therapy Chunwei Ong1, Bong Geun Cha1, and Jaeyun Kim1,2,3*

1School

of Chemical Engineering, Sungkyunkwan University (SKKU), Suwon 16419,

Republic of Korea

2Department

of Health Sciences and Technology Samsung Advanced Institute for

Health Sciences & Technology (SAIHST), Sungkyunkwan University (SKKU), Suwon 16419, Republic of Korea

3Biomedical

Institute for Convergence at SKKU (BICS), Sungkyunkwan University

(SKKU) Suwon 16419, Republic of Korea

* Correspondence should be addressed to J.K. (e-mail: [email protected])

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Abstract Cancer immunotherapy is a treatment that utilizes the host immune system to fight against cancer. Inducing a cancer antigen-specific adaptive immune response is key in cancer immunotherapy. Although diverse immune cells including dendritic cells (DCs) and T cells infiltrate a tumor, the activation of such immune cells is inhibited owing to an immunosuppressive tumor microenvironment. In this study, we propose mesoporous silica nanoparticles (XL-MSNs) decorated with gold nanoparticles (Au@XL-MSNs) for the delivery of a high amount of CpG-ODNs to the tumor site to activate DCs infiltrated within the tumor for the induction of antigen-specific adaptive immune response. During an in vitro test, CpG-ODNs delivered using Au@XL-MSNs were shown to be more effectively internalized by bone marrow-derived dendritic cells (BMDCs), resulting in an enhanced expression of co-stimulatory molecules and an increased secretion of proinflammatory cytokines compared to soluble CpG-ODNs. Furthermore, an in vivo test demonstrated a more significant tumor growth inhibition and an enhanced survival rate result from the intratumoral injection of Au@XL-MSN-CpG compared to that treated using soluble CpG-ODNs. Furthermore, through the induction of a photothermal effect


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based on the assembled AuNPs on XL-MSNs, an enhancement of the cancer immunotherapy was achieved by generating a cancer antigen at the tumor site, which can be processed by tumor-infiltrated DCs. These findings suggest that our approach can be applied as a synergistic platform for an efficient cancer immunotherapy enabling a delivery of immunostimulating signals as well as in-situ antigen generation through a photothermal effect.

Keywords: mesoporous silica nanoparticles, gold nanoparticles, CpG-ODNs, cancer immunotherapy, photothermal therapy



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Introduction Cancer immunotherapy is a treatment that utilizes the host immune system to fight against cancer.1-2 Inducing a cancer antigen-specific adaptive immune response is a key in cancer immunotherapy.3-4 Although diverse immune cells including dendritic cells (DCs) and T cells infiltrate a tumor, the activation of such antigen-specific immune responses

is

usually

inhibited

owing

to

an

immunosuppressive

tumor

microenvironment.5-7 As cancer antigens are released from the cancer cells in a tumor site, an activation of tumor-infiltrated immune cells is a possible way to induce a cancer antigen-specific immune response for cancer treatment.8-9 DCs are potent professional antigen-presenting cells (APCs) that can prime naïve CD8+ cells to induce the antigenspecific cytotoxic T cells. The antigen presenting function of DCs for T cells is essential for effective cancer immunotherapy. To enhance the priming of naïve T cells by DCs, immune adjuvants, typically pathogen-associated molecular patterns (PAMPs), are used to activate the DCs.10-12 CpG-ODNs, a type of PAMPs, are short synthetic single-stranded DNA molecules that contain unmethylated CpG motifs that can be used as an adjuvant in


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cancer vaccines. CpG-ODNs have potent immunostimulatory effects and can therefore enhance the anti-cancer activity of a variety of cancer treatments.13-15 These CpG motifs are specifically recognized by toll-like receptor 9 (TLR9), a transmembrane pattern recognition receptor (PRR) located in many immune cells, particularly DCs.16-17 Once CpG motifs are recognized by TLR9 in DCs, the adapter protein MyD88 will be recruited and then triggering the activation of the transcription factor nuclear factor kappa B (NFB).18-20 This initiates the expression of pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-12 (IL-12), and tumor necrotic factor α (TNF-α), thus enhancing the innate and adaptive immune responses. For instance, the secretion of IL-6 induces B cells to become apoptosis resistant, prolonging its function in humoral immunity. IL-12 secreted by DCs induces type-1 helper (Th1) immunity against tumors through the differentiation of naïve CD4+ T cells into Th1 cells secreting IFN-γ. TNF-α is a potent pro-inflammatory cytokine involved in a cellular immune response by enhancing the T cell proliferation and activation. At the same time, DCs are promoted to express higher CD40, CD54, CD80, and CD86 and therefore have more potential to activate both CD4 and CD8 T cells.21-25 Despite their potent immunostimulatory function, the use of CpG-



ODNs in cancer vaccines is limited owing to the rapid degradation in physiological condition by nuclease and poor cellular uptake attributed to the negative charge in the oligonucleotide backbone.26 Therefore, a high amount of soluble CpG-ODNs is usually required for effective cancer immunotherapy. To address these challenges, current approaches include a chemical modification of the CpG-ODNs or their incorporation into the nanocarriers. Nanocarriers, specifically, have the potential to improve the pharmacokinetics of CpG-ODNs, as well as their cellular uptake, thereby optimizing the therapeutic effect of CpG-ODNs in cancer vaccines.27-32 The main types of cancer immunotherapy approved are monoclonal antibodies, immune checkpoint inhibitors, cancer vaccines, and chimeric antigen receptor T (CAR-T) cells.33-35 Recently, there has been intensive studies on the use of nanomaterials as a delivery platform for enhanced cancer immunotherapy. For examples, diverse nanoparticles were used for the efficient delivery of cancer antigen and adjuvants in cancer vaccination.36-38 Besides, different drugs or reagents can be delivered via single nanoparticles delivery platform for combined cancer therapy. For instance, hollow mesoporous silica nanocages and surfactant-polymer hybrid nanoparticles were used to


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deliver chemotherapeutic drug and photosensitizer for combined cancer chemotherapy and photodynamic therapy.39-40 Furthermore, carbon nanotubes and chitosan-coated hollow copper sulfide nanoparticles can act as photothermal converting agent as well as immune checkpoint inhibitor delivery platform.41-42 In this study, we designed a CpG-ODN delivery system based on extra-large pore mesoporous silica nanoparticles (XL-MSNs) decorated with small gold nanoparticles (AuNPs) for a synergistic cancer immunotherapy with a high CpG delivery and photothermal therapy (Figure 1a). XL-MSNs have been used extensively in drug delivery owing to several characteristics that make them a good candidate for drug delivery, including a good thermal stability and biocompatibility, easy synthesis and functionalization, and high surface area and porosity.43-45 XL-MSNs with extra-large pores were used to load high amounts of AuNPs (1-2 nm) that were subsequently conjugated with thiol-modified CpG-ODNs (SH-CpG-ODNs) and SH-PEG (Figure 1b). The resulting Au@XL-MSN-CpG/PEG showed a more efficient immunoactivating property than free CpG-ODNs in in vitro DC experiments. An in vivo tumor therapeutic study revealed that mice treated with our nanocarriers loaded with CpG-ODNs as a



vaccine adjuvant have achieve a good tumor suppression effect and longer survival rate; and these effects were further significantly enhanced through additional photothermal therapy using laser irradiation. These findings suggest that the use of Au@XL-MSNs is an effective platform for synergistic cancer immunotherapy and photothermal therapy.

Experimental Section Materials Sodium oleate was purchased from Tokyo Chemical Industry (Seoul, Korea). Chloroform, ethyl acetate, methanol, and ethanol were purchased from Samchun Chemicals (Seoul, Korea). Sodium hydroxide, hexadecyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate (TEOS), tetrakis(hydroxymethyl)phosphonium chloride (THPC), chloroauric acid (HAuCl4.3H2O), (3-aminopropyl) trimethoxysilane (APTMS), ammonium hydroxide, iron chloride (FeCl3.6H2O (III)), hexane, oleic acid, 1-octadecene, and Tween-20 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric acid (HCl) and sulfuric acid were purchased from Daejung Chemicals (Seoul, Korea). Thiol-modified CpG-ODNs (SH-CpG-ODNs) were purchased from Genotech (Daejeon,


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Korea). Methoxy polyethylene glycol sulfhydryl (SH-mPEG, 2,000 M.W) was purchased from SunBio (Anyang, Korea). AlamarBlue was purchased from Invitrogen (Frederick, MD, USA). Fetal bovine serum (FBS) was purchased from Millipore (Billerica, MA, USA). Recombinant murine GM-CSF was purchased from Peprotech (Rocky Hill, NJ, USA). Antibodies against the following proteins were used: CD11c, MHC class II, and CD86. These antibodies and FcR blocking reagent were purchased from Miltenyi Biotech Korea (Seoul, Korea). IL-12 and TNF-α ELISA kits were purchased from BD Biosciences (San Diego, CA, USA). Synthesis of Extra-Large Pore Mesoporous Silica Nanoparticles (XL-MSNs). XL-MSNs with extra-large pores were prepared using iron oxide nanoparticles as a seed material following the previous report with some modifications.46-47 Iron oxide nanoparticles stabilized with oleic acid were first synthesized based on the heating-up method48 using an iron-oleate complex as a precursor and were dispersed in chloroform (5.8 mg/mL). The resulting iron oxide nanoparticles in chloroform (0.5 mL) were mixed with an aqueous CTAB solution (0.055 M, 5 mL), and the mixture was vigorously stirred for 30 min to form oil-in-water microemulsions. The mixture was then heated to 60 oC and



stirred for 10 min to evaporate the chloroform, resulting in CTAB-stabilized iron oxide nanoparticles. The resulting solution was added to a mixture of DI water (95 mL), methanol (5 mL), ammonium hydroxide (3 mL), CTAB solution (0.055 M, 5 mL), and ethyl acetate (25 mL). Then, TEOS (0.5 mL) was added to the reaction solution and stirred for 8 h at room temperature for a silica sol-gel reaction. The as-synthesized XLMSNs were washed with excess ethanol three times and dispersed in ethanol (15 mL). For the extraction of the CTAB, HCl solution was added to XL-MSNs in ethanol, and the pH was adjusted to 1.5. The resulting solution was stirred for 3 h at 60 oC. The extracted XL-MSNs were dispersed in ethanol (20 mL) after washing with excess ethanol three times. To introduce an amine functional group, APTMS (0.224 mM, 40 µL) was added to the as-synthesized XL-MSNs and stirred for 12 h in ethanol (15 mL). Then, the amine group-functionalized XL-MSNs were washed three times with excess ethanol and dispersed in 15 mL of ethanol. Attachment of AuNPs on XL-MSNs. AuNPs were synthesized according to the previous report with several modifications with reactants concentrations.49-50 THPC from an 80% aqueous solution (0.12 mL) was diluted in DI water (10 mL). Sodium hydroxide solution


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(0.2 M, 1.5 mL) and the diluted THPC solution (1 mL) were then added to DI water (45 mL). The mixture solution was stirred for 2 min, followed by the addition of chloroauric acid solution (29 mM, 2 mL). The color of the mixture changed to a brown color, indicating that the AuNPs were synthesized. Amine-modified XL-MSNs (1 mg) were washed three times with DI water. DI water (100 µL) and an aqueous AuNP solution (900 µL) were added to the XL-MSNs, and the resulting solution was rotated using an auto-rotator for 5 min. The mixture solution was then washed, and the supernatant was discarded. The attachment steps were repeated seven times. During the last attachment step, the Au@XL-MSNs were washed three times with DI water. SH-CpG-ODN Loading and subsequent PEGylation on Au@XL-MSNs. Au@XL-MSNs (100 µg) were washed three times and re-suspended in DI water (100 µL). The stock solution of SH-CpG-ODNs (1 µg/µL, 20 µL) was mixed with an Au@XL-MSN solution, and the mixture was vigorously vortexed for 30 min. The solution was then washed three times with DI water to remove unbounded SH-CpG-ODNs. The supernatant collected during the washing steps was analyzed for its absorbance at 260 nm using a UV-VIS spectrophotometer to calculate the loading amount of SH-CpG-ODNs on the



Au@XL-MSNs. For further surface modification with poly(ethylene glycol) (PEG), SHmPEG (5 mg, 2000 M.W) was dissolved in DI water (1 mL) and mixed with an Au@XLMSN-CpG solution (1 mg/mL, 100 µL) with stirring for 30 min. The resulting Au@XLMSN-CpG/PEG was centrifuged and washed three times using DI water. To check the release of the SH-CpG-ODNs, Au@XL-MSN-CpG (100 µg) was dispersed in DI water (120 µL) in a 1 mL microcentrifuge tube. After the predetermined time interval, the Au@XL-MSN-CpG in DI water was centrifuged (10,000 rpm, 5 min) and the supernatant was collected for a quantitative analysis of the released SH-CpG-ODNs using a UV-VIS spectrophotometer at an absorbance wavelength of 260 nm. The same amount of DI water was refilled into the microcentrifuge tube for a further study on the release. BMDCs Culture. To prepare bone marrow-derived dendritic cells (BMDCs), bone marrow cells collected from the tibias and femurs of mice were cultured in a growth medium (RPMI 1640 supplemented with 10% of heat-inactivated fetal bovine serum (FBS), β-mercaptoethanol, granulocyte-macrophage colony-stimulating factor (GMCSF), and penicillin/streptomycin) for seven days in 37oC, 5 % CO2. On day 3, a fresh cell culture medium and GM-CSF were added to the culture. On day 6, the non-


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adherent cells suspended in the medium were collected, centrifuged, and resuspended in a new medium and GM-CSF. The BMDCs were ready for use at day 7. Cell Viability. An in vitro cytotoxicity assay for Au@XL-MSN-CpG and Au@XL-MSNCpG/PEG was conducted using an AlamarBlue cell viability assay. Mouse mammary tumor cells (4T1, ATCC CRL-2539) were cultured at 37 oC in an RPMI medium with 10 % fetal bovine serum (FBS) and penicillin/streptomycin. Then, 4T1 cells were retrieved and seeded at a concentration of 3.0 × 103 cells/well in a 96 well plate until complete adherence of the cells onto the bottom of the plate. Au@XL-MSN-CpG and Au@XLMSN-CpG/PEG dispersed in an RPMI medium were added into a 96 well plate seeded with 4T1 cells. The final concentration of Au@XL-MSN-CpG and Au@XL-MSNCpG/PEG were 0, 100, 200, and 400 µg/mL, and the final volume in each well was 100 µL. After incubation of the cells for 24 h, an AlamarBlue solution (10 µL) was added into each well, and the cells were incubated for another 2 h. The absorbance at 560 nm was then measured using a UV-VIS spectrophotometer. The cytotoxicity was expressed as the percentage of living cells compared with that of the untreated control cells. In Vitro BMDC Activation. 1 × 106 BMDCs were seeded in a 6 well plate and stimulated with



PBS, CpG (3 µg/mL), Au@XL-MSN/PEG, and Au@XL-MSN-CpG/PEG (3 µg/mL equivalent SH-CpG-ODNs) for 24 h. The expression of the surface markers CD11c, MHC II, and CD86 on the BMDCs were then measured and calculated using a flow cytometer, and the secretion of TNF-α and IL-12 cytokines in the culture supernatant were measured using ELISA according to the manufacturer’s protocol. Measurement of Photothermal Effect. XL-MSNs and Au@XL-MSNs (2 µg/µL, 200 µL) were placed in a 96 well plate and irradiated using an NIR laser (808 nm, 0.15 W/cm2) for 10 min. The temperature of each sample was recorded every 2 min. Tumor Suppression Study. To prepare the tumor-bearing mouse model, B16-F10 cells dispersed in PBS (0.5 × 106 cells/50 µL) were subcutaneously injected into a 6-week old C57/BL6 mouse (Orient Bio, Seongnam, Republic of Korea). When the size of the tumor nodule reached 5 mm (8 days after injection), different materials (50 µL) were intratumorally injected into the mice according to the following grouping: PBS, CpG (5 µg), Au@XL-MSN/PEG, Au@XL-MSN/PEG + NIR, Au@XL-MSN-CpG/PEG (5 µg equivalent SH-CpG-ODNs), and Au@XL-MSN-CpG/PEG + NIR (5 µg equivalent SHCpG-ODNs). The second and third injections were conducted on day 12 and day 18.


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For the Au@XL-MSN/PEG + NIR and Au@XL-MSN-CpG/PEG + NIR groups, the nearinfrared laser (808 nm, 0.15 W/cm2) was irradiated for 5 min after each injection. The tumor size was measured using calipers, and the mice survival rate was observed during the experiment. All surviving mice were euthanized at day 40. The tumor volume was calculated according to the following equation: Volume = (Tumor Width)2 × (Tumor Length)/2. All animal experiments were conducted with approval by the Sungkyunkwan University Institutional Animal Care and Use Committee. Statistical Analysis. All values in this study were presented as the mean ± standard deviation as error bar. The statistical analysis was performed with the F-test and Student’s t-test (two-tailed). A p value of < 0.05 was considered as a significant difference.

Results and Discussion To increase the loading of the CpG-ODNs, we first designed mesoporous silica nanoparticles as a support to attach a high amount of AuNPs where thiolated CpGODNs can be subsequently conjugated later. As the conventional MSNs synthesized via



cationic micelle templating have small mesopores (average size of 3 nm), the loading of large biomolecules or nanoparticles is typically limited.46-47 Therefore, we synthesized MSNs with extra-large pores (XL-MSNs) according to the previous report.46-47 These MSNs with extra-large pores are known as good carriers for large biomolecules with an enhanced loading capacity compared to conventional MSNs with small mesopores.46-47 Transmission electron microscopy (TEM, Figure 2a) and scanning electron microscopy (SEM, Figure 2b) clearly show the spherical morphology of XL-MSNs with large mesopores distributed over all of the particles. The sizes of the XL-MSNs observed in TEM images were 130 nm on average. The pore size, pore volume, and surface area of the XL-MSNs were characterized using nitrogen sorption (Figure 2c), indicating that there are extra-large pores of approximately 20-30 nm. The pore volume and BrunauerEmmett-Teller (BET) surface area of the XL-MSNs were 1.58 cm3/g and 621.6 m2/g, respectively. The resulting XL-MSNs were then functionalized with amine groups via silane chemistry to endow positive charges on the surfaces of XL-MSNs. The 1-2 nm sized AuNPs with negative charges were synthesized and attached to the surface of amine-


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modified XL-MSNs. The AuNPs (black dots in the TEM image) were well-attached to the surface and large-mesopores of the XL-MSNs (designated as Au@XL-MSNs) (Figure 2d). Owing to the high loading of the AuNPs in the XL-MSNs, the porosity of the AuNPattached XL-MSNs was decreased (Figure 2e). Energy-dispersive X-ray spectroscopy (EDS) also confirmed the attachment of the AuNPs throughout the XL-MSNs (Figure 2f). Zeta-potential data show that the positive surface charge of the amine-modified XLMSNs was changed to a negative charge after the attachment of the Au NPs (Figure 2g). The loading of the AuNPs in the XL-MSNs was characterized by measuring the absorbance

of

the

XL-MSNs,

AuNPs

and

Au@XL-MSNs

using

a

UV-VIS

spectrophotometer (Figure S1). XL-MSNs showed no absorbance from a wavelength of 200 to 1,000 nm, whereas the AuNPs showed some absorbance at a wavelength of 520 nm. After the attachment of the AuNPs to the XL-MSNs, Au@XL-MSNs showed an absorbance at approximately 520 nm, representing the successful attachment of AuNPs on the XL-MSNs. Interestingly, the absorbance was observed even at a longer wavelength, such as approximately 800 nm, indicating that the surface plasmon resonance at a longer wavelength was derived from the close contact between the



AuNPs closely attached with neighbors, as shown in the TEM images.51 Based on this absorption within the near infrared (NIR) range of the Au@XL-MSNs, a photothermal effect can result from NIR laser irradiation to the particles. The temperature was increased up to 37 oC when the NIR laser was irradiated onto the Au@XL-MSNs, whereas no significant temperature changes observed in the XL-MSNs (Figure 2h). By taking advantage of the high loading of the AuNPs, we next investigated the loading of CpG-ODNs on the Au@XL-MSNs. We used thiolated CpG-ODNs (SH-CpGODNs) to induce stable gold-thiol bonding to load the CpG-ODNs in the Au@XL-MSNs. The loading amount of SH-CpG-ODNs was approximately 160 µg/mg XL-MSNs. The resulting CpG loading is significantly higher than that of the other nanocarriers reported previously.52-55 We further investigated the release kinetics of SH-CpG-ODNs from the Au@XL-MSNs (Figure 3a). The SH-CpG-ODNs underwent a burst release followed by a sustained release with a cumulative release of 17, 24, and 45 % at 12, 24, and 72 h, respectively (Figure 3a). This very slow release of CpG-ODNs is presumably due to more robust gold-thiol bonding in Au@XL-MSNs-CpG. The slower release of CpGODNs from Au@XL-MSNs will be beneficial under physiological conditions, without a


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loss of CpG before being internalized into the DCs. The X-ray fluorescence (XRF) analysis of the supernatants from release test obtained at 12 h, 24 h, and 72 h (data not shown), the comparison of absorbance spectrum of supernatant from release test (Figure S2a), and the TEM image of Au@XL-MSN-CpG after 72 h release test (Figure S2b) shows that there was almost no release of gold nanoparticles over the CpG release study. To further enhance the colloidal stability and decrease the potential cytotoxicity, thiolated poly(ethylene glycol) (SH-PEG) was subsequently conjugated. A TEM image shows that even after CpG and PEG modification, the AuNPs on the surfaces of the XLMSNs were well maintained (Figure 3b). The hydrodynamic sizes of Au@XL-MSN-CpG, and Au@XL-MSN-CpG/PEG dispersed in phosphate-buffered saline (PBS), measured in dynamic light scattering (DLS), were 132 and 212 nm, respectively (Figure 3c). The PEGylated sample maintained colloidal stability without aggregation in the PBS, indicating that PEGylation allowed the stable colloidal dispersion of the resulting CpGloaded particles in the physiological condition. The zeta potential of the particles showed that PEGylation can decrease the enhanced negative charge resulting from



CpG-loading, which will be beneficial for the intracellular delivery of CpG moieties (Figure 3d). We further investigated the influence of PEGylation on the cytotoxicity of CpGloaded particles by comparing Au@XL-MSN-CpG and Au@XL-MSN-CpG/PEG (Figure 3e). The cell viability was decreased with an increase in the concentration of both particles, but there was a significant influence of PEGylation on cytotoxicity at higher particle concentration. After a 24 h incubation with 100, 200, and 400 µg/mL of Au@XLMSN-CpG, the cell viability was 94.6, 60.0 and 59.0 %, respectively. When incubated with 100, 200, and 400 µg/mL of Au@XL-MSN-CpG/PEG, the cell viability was 105.4, 87.2, and 77.2 %, respectively. This observation indicates that the PEGylation reduces the cytotoxicity of Au@XL-MSN-CpG while maintaining the colloidal stability under the physiological conditions. After confirming the cytotoxicity of Au@XL-MSN-CpG/PEG, we conducted an in

vitro immunostimulatory activity experiment by treating BMDCs with PBS (control group), CpG (3 µg/mL), Au@XL-MSN/PEG, and Au@XL-MSN-CpG/PEG (equivalent to 3 µg/mL SH-CpG-ODNs). CD11c is a surface marker of DCs, and CD86 is a co-


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stimulatory molecule used to indicate the activation of DCs. A higher CD86 expression indicates that more activated DCs are ready for an antigen presentation to the T cells. Au@XL-MSN-CpG/PEG showed the highest activation on the BMDCs even when compared to soluble CpG (Figure 4a, b). The CD11c+CD86% BMDCs were 5, 15, 6, and 20 % for the control (PBS), CpG, Au@XL-MSN/PEG, and Au@XL-MSN-CpG/PEG groups, respectively. We also checked the CD11c+MHC II+ DCs, where MHC II is another indicator of DC activation (Figure 4c). The activations of BMDCs were 14, 27, 18, and 35 % for the PBS, CpG, Au@XL-MSN/PEG, and Au@XL-MSN-CpG/PEG groups respectively. These results indicate that Au@XL-MSN-CpG/PEG is extreamly effective in inducing the activation of DCs compared to soluble SH-CpG-ODNs. DCs are an important bridge connector between innate and adaptive immunity. As the activation of DCs is enhanced, both the subsequent innate and adaptive immunities will also be augmented, eventually strengthening the antitumor effect. Proinflammatory cytokines such as TNF-α and IL-12 released by the activated DCs are very important in term of the anti-tumor effect because these cytokines can help increase the number of recruiting T cells and NK cells.21-25 We thus measured the pro-



inflammatory cytokines (TNF-α and IL-12) in the supernatant collected from the BMDC culture using an enzyme-linked immunosorbent assay (ELISA) (Figure 4d, e). PBS and Au@XL-MSN/PEG only induced 101 and 22 pg/mL of TNF-α, respectively whereas the soluble CpG significantly induced a higher secretion of TNF-α (324 pg/mL). Au@XLMSN-CpG/PEG can induce an even higher secretion of TNF-α (541 pg/mL) compared to the soluble CpG. The secretion of IL-12 showed a similar result (Figure 4e); 261, 286, 1230, and, 1647 pg/mL IL-12 for PBS, Au@XL-MSN/PEG, soluble CpG, and Au@XLMSN-CpG/PEG, respectively. These data indicate that Au@XL-MSN is a good carrier for SH-CpG-ODN delivery and can successfully trigger a high activation of DCs. On the basis of immunostimulatory property of Au@XL-MSN-CpG/PEG in vitro, we further investigated the therapeutic effect of our delivery system with an in vivo antitumor activity. Since we used AuNPs as a part of the SH-CpG-ODN carrier, we expected that irradiation using an NIR laser can trigger heat and provide a synergetic effect in tumor suppression. The tumor cells were first inoculated, and eight days later a small tumor volume was observed (Figure 5a). Mice bearing a tumor were divided into six groups as follows: the control group treated by PBS, CpG, Au@XL-MSN/PEG,


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Au@XL-MSN/PEG with NIR laser irradiation, Au@XL-MSN-CpG/PEG and Au@XLMSN-CpG/PEG with NIR laser irradiation. Administration method, the number of injections, and the amount of CpG-ODNs were determined based on other previous reports on cancer immunotherapy combined with phototherapy.56-58 The amount of Au@XL-MSN/PEG injected was 100 µg and the amount of SH-CpG-ODNs attached was 5 µg. When the tumor size reached 5 mm (day 8 after the subcutaneous inoculation of B16F10 cells), different vaccine formulations were intratumorally injected into the mice. The second and third treatments were conducted at day 12 and day 18. For the Au@XL-MSN/PEG + NIR, and Au@XL-MSN-CpG/PEG + NIR groups, 5 min of NIR laser irradiation (808 nm, 0.15 W/cm2) was applied after each injection. Although the maximum photothermal temperature around 60 oC could be achieved (Figure S3), we maintained the photothermal temperature in a range between 45 oC and 50 oC in animal study to prevent undesired damage on normal tissue such as skin around the tumor. There were no severe skin problems on the irradiation site of all animals (Figure S4). The size of the tumor was monitored over time (Figure 5b), showing that Au@XL-MSNCpG/PEG is extremely effective in the inhibition of tumor growth when compared to the



PBS (control group), soluble CpG, or photothermal therapy groups. This indicates that the particles can deliver CpG into tumor-residing DCs and more effectively activate them. Furthermore, additional photothermal therapy combined with immunotherapy (Au@XL-MSN-CpG/PEG + NIR) showed a synergetic effect in tumor growth inhibition even compared to the vaccine group without NIR irradiation. NIR irradiation can generate a high heat that destroys the tumor cells, and the dying tumor cells can release tumor antigens at the tumor site. The tumor-residing DCs can then uptake the released tumor antigen along with the CpG-carrying nanoparticles, which may induce a higher activation of DCs and an enhanced antigen-presentation. At day 22, the tumor volume of mice treated with Au@XL-MSN-CpG/PEG + NIR was approximately 1.5times smaller than that of the Au@XL-MSN-CpG/PEG group without NIR irradiation. Consistent with the tumor growth inhibition results, mice treated with Au@XL-MSNCpG/PEG showed a high survival rate compared to the soluble CpG group and NIRtreated group (Figure 5c). Furthermore, mice treated with Au@XL-MSN-CpG/PEG + NIR showed the highest survival rate even after 37 days of tumor inoculation whereas all mice in the other groups died. These results clearly demonstrate the potent cancer


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therapeutic efficacy of Au@XL-MSN-CpG/PEG for the combination of immunotherapy and photothermal therapy.

Conclusions For effective cancer immunotherapy, extra-large pore mesoporous silica nanoparticles decorated with small gold nanoparticles were synthesized for the delivery of high amounts of SH-CpG-ODNs. Au@XL-MSNs loaded with SH-CpG-ODNs were successfully internalized into DCs, activating toll-like receptor 9 (TLR9) and therefore initiated the expression of IL-12 and TNF-α. These pro-inflammatory cytokines enhanced the proliferation, differentiation, and activation of T cells. After NIR irradiation, dead tumor cells can be the best source of tumor antigen. Professional APCs such as dendritic cells could uptake these antigens and antigen-specific adaptive immune response could be induced. Since Au@XL-MSN-CpG/PEG has shown good therapeutic results in both tumor growth inhibition and a long survival rate, it may become an alternative platform for cancer therapy in the future.



ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Absorbance spectra of AuNPs, XL-MSNs, and Au@XL-MSNs; TEM image of Au@XL-MSN-CpG after release test; heat map of photothermal therapy; photographs of mouse skin before and after NIR irradiation (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +82-31-290-7252. Fax: +82-31-290-7272. ORCID Jaeyun Kim: 0000-0002-4687-6732 Notes The authors declare no competing financial interest.


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ACKNOWLEDGEMENTS This work was supported by grants funded by the National Research Foundation (NRF) under the Ministry of Science and ICT, Republic of Korea (grants 2019R1A2C2004765, 2010-0027955), and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant HI17C0076).

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Figures

Figure 1. Schematic illustration of (a) Mechanism of Au@XL-MSN-CpG/PEG in cancer immunotherapy and photothermal therapy and (b) Synthesis of PEGylated mesoporous silica nanoparticles decorated with gold nanoparticles for delivery of thiol-modified CpGODNs (Au@XL-MSN-CpG/PEG).


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Figure 2. Mesoporous silica nanoparticles (XL-MSNs) and gold nanoparticles decorated mesoporous silica nanoparticles (Au@XL-MSNs). TEM images of (a) XL-MSNs, and (d) Au@XL-MSNs. SEM images of (b) XL-MSNs, and (e) Au@XL-MSNs. (c) N2 sorption analysis of XL-MSNs. (f) Energy-dispersive X-ray spectroscopy (EDS) of Au@XL-MSNs. (g) Zeta potential of XL-MSNs, and Au@XL-MSNs dispersed in PBS. (h) Photothermal conversion of XL-MSNs, and Au@XL-MSNs.


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Figure 3. (a) Cumulative release profiles of CpG-ODN from CpG-ODN-loaded Au@XLMSNs in DI water. (b) TEM image of Au@XL-MSN-CpG/PEG (c) Hydrodynamic sizes of Au@XL-MSN-CpG, and Au@XL-MSN-CpG/PEG dispersed in PBS measured in dynamic light scattering (DLS). (d) Zeta potential of Au@XL-MSN-CpG, and Au@XLMSN-CpG/PEG dispersed in PBS. (e) Cell viability of 4T1 cells accessed with the AlamarBlue cell viability assay after 24 h incubation with Au@XL-MSN-CpG, and Au@XL-MSN-CpG/PEG.



Figure 4. CpG-ODN loaded Au@XL-MSN/PEG triggers BMDCs activation in vitro. BMDCs were incubated with PBS, soluble CpG-ODN (3 µg/mL), Au@XL-MSN/PEG, and Au@XL-MSN-CpG/PEG (equivalent to 3 µg/mL CpG-ODN) for 24 h and different surface markers were checked. (a) Expression of CD11c and CD86 were analyzed using flow cytometry. (b) Percentages of CD11c and CD86-expressing BMDCs. (c) Percentages of CD11c and MHC II-expressing BMDCs. (d) TNF-α secretion by BMDCs analyzed using ELISA. (e) IL-12 secretion by BMDCs analyzed using ELISA. Data are representative of four independent experiments (n=4, mean ± SD) *p < 0.05, **p < 0.005.


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Figure 5. CpG-loaded Au@XL-MSN/PEG efficiently inhibiting growth of B16F10 in vivo with and without NIR irradiation. (a) Timeline for in vivo anticancer therapy. (b) Tumor volume changes of mice received with PBS (control), soluble CpG-ODN (three injections, 5 µg CpG per injection), Au@XL-MSN/PEG, Au@XL-MSN/PEG with NIR irradiation, Au@XL-MSN-CpG/PEG (three injections, equivalent to 5 µg CpG per injection) and Au@XL-MSN-CpG/PEG (three injections, equivalent to 5 µg CpG-ODN per injection) with NIR irradiation. For the NIR irradiation groups, 5 min of (1.5 W/cm2) power laser was applied after every injection. (c) Kaplan-Meier survival curve of mice treated with the above regimes. Data are representative of five independent experiments (n = 5, mean ± SD) *p < 0.05.



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Mesoporous Silica Nanoparticles Doped with Gold Nanoparticles for

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