Combinatorial photothermal and immuno cancer therapy using chitosan-coated hollow copper sulfide nanoparticles - PubMed (original) (raw)

. 2014 Jun 24;8(6):5670-81.

doi: 10.1021/nn5002112. Epub 2014 May 13.

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Combinatorial photothermal and immuno cancer therapy using chitosan-coated hollow copper sulfide nanoparticles

Liangran Guo et al. ACS Nano. 2014.

Abstract

Near-infrared light-responsive inorganic nanoparticles have been shown to enhance the efficacy of cancer photothermal ablation therapy. However, current nanoparticle-mediated photothermal ablation is more effective in treating local cancer at the primary site than metastatic cancer. Here, we report the design of a near-infrared light-induced transformative nanoparticle platform that combines photothermal ablation with immunotherapy. The design is based on chitosan-coated hollow CuS nanoparticles that assemble the immunoadjuvants oligodeoxynucleotides containing the cytosine-guanine (CpG) motifs. Interestingly, these structures break down after laser excitation, reassemble, and transform into polymer complexes that improve tumor retention of the immunotherapy. In this "photothermal immunotherapy" approach, photothermal ablation-induced tumor cell death reduces tumor growth and releases tumor antigens into the surrounding milieu, while the immunoadjuvants potentiate host antitumor immunity. Our results indicated that combined photothermal immunotherapy is more effective than either immunotherapy or photothermal therapy alone against primary treated and distant untreated tumors in a mouse breast cancer model. These hollow CuS nanoparticles are biodegradable and can be eliminated from the body after laser excitation.

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Figures

Figure 1

Figure 1

Transmission electron microscopic imaging of (A) HCuSNPs composed of small CuS nanocrystals (arrows) and (B) small CuS nanoparticles as a result of disintegration of HCuSNPs following near-infrared laser (2.0 W/cm2, 40 s, 900 nm) irradiation.

Figure 2

Figure 2

(A) Scheme of assembly of HCuSNPs-CpG conjugates, near-infrared light-triggered disintegration of HCuSNPs, and system reassembly. “HCuSNPs-Chi” represents chitosan-coated HCuSNPs. “Chi-CpG-NPs” represents chitosan-CpG nanocomplexes. “SCuSNPs” represents small CuS nanoparticles. (B) Transmission electron microscopic imaging of HCuSNPs-CpG before and after near-infrared laser (2.0 W/cm2, 40 s, 900 nm) treatment and centrifugation (3000 rpm, 5 min). (C) Agarose gel electrophoresis of free CpG, HCuSNPs-CpG before (“NPs”), and after near-infrared laser treatment (2.0 W/cm2) for different times. Following centrifugation, the supernatant and pellet of each laser-treated sample was individually loaded for electrophoresis.

Scheme 1

Scheme 1. Diagram of HCuSNPs-CpG-mediated photothermal immunotherapy of both primary treated and distant untreated tumors. Under near-infrared laser irradiation, the intratumorally injected HCuSNPs-CpG transform to chitosan-CpG nanocomplexes (Chi-CpG-NPs) resulting in uptake into Toll-like receptor 9-rich endosomes of plasmacytoid dendritic cells (pDCs). Upon stimulation with CpG, the pDCs secrete interferon-α (IFN-α) to promote innate immunity through NK cell activation. Simultaneously, HCuSNPs-mediated photothermal ablation disrupts the tumor cells, relieving tumor burden and releasing tumor-associated antigens to myloid dendritic cells (mDCs). In the presence of IFN-α secreted by the activated pDCs, these mDCs become professional antigen-presenting cells and subsequently migrate to tumor-draining lymph nodes (DLNs), where they cross-prime tumor antigen-specific T cells. The antigen-specific CD8+ T cells enter the systemic circulation and are recruited to both primary tumor and untreated tumor at a distant site to trigger the “effector phase” of the adaptive immune response.

Figure 3

Figure 3

(A) Representative live fluorescence imaging of BALB/c mice bearing EMT6 tumors at different time points following intratumoral injection with (1) free fCpG; (2) HCuSNPs-fCpG; or (3) HCuSNPs-fCpG plus laser (2.0 W/cm2, 40 s, 900 nm). fCpG represents IRDye680-labeled CpG. The injected dose of fCpG in all three groups was 10 μg/mouse. Fluorescence intensity is expressed as p/s/cm2/sr. (B) Fluorescence intensity in the dissected tumors and tumor-draining lymph nodes is shown 24 h after administration. (C) Fluorescence micrographs of fCpG distribution in tumors 24 h after intratumoral injection. Pseudored, fCpG; green, mAb (clone 120G8.04)-labeled plasmacytoid dendritic cells; blue, 4′,6-diamidino-2-phenylindole (DAPI)-labeled cell nuclei. Scale bar, 50 μm.

Figure 4

Figure 4

EMT6 tumor-bearing mice were given intratumoral injections of saline or different formulations containing 100 μg of CpG or GpC (sham CpG control) with or without laser treatment (2.0 W/cm2, 40 s, 900 nm) at day 0 and day 6. (A) Tumor sections were stained for activated NK cells (CD69+CD49b+) at day 8. Green, FITC-conjugated anti-mouse CD69; pseudored, allophycocyanin-conjugated anti-mouse CD49b; blue, DAPI. Scale bar, 50 μm. (B) Flow cytometric analysis of activated myeloid dendritic cells (CD11c+CD86+) in tumors or draining lymph nodes is shown at day 8. “HCuSNPs-GpC” represents HCuSNPs-chitosan-GpC conjugates.

Figure 5

Figure 5

EMT6 tumor-bearing mice were given intratumoral injections of saline or different formulations containing 100 μg of CpG or GpC (sham CpG control) with or without laser treatment (2.0 W/cm2, 40 s, 900 nm) at day 0 and day 6. (A) Flow cytometric analysis of intracellular IFN-γ production by CD8+ T cells in tumor, tumor-draining lymph node or spleen at day 12 followed by CpG (20 μg/mL) with or without mitomycin C-treated tumor cell stimulation in vitro. (B) ELISA analysis of IFN-γ or IL-2 level in tumor or spleen following CpG (20 μg/mL) stimulation in vitro at day 12. Significant difference (**p < 0.01) between “HCuSNPs-CpG + Laser” group and other groups. Data shown are expressed as mean ± SD (n = 3). “HCuSNPs-GpC” represents HCuSNPs-chitosan-GpC conjugates.

Figure 6

Figure 6

(A) EMT6 tumor-bearing mice were given intratumoral injection of saline or different formulations containing 100 μg of CpG or GpC (sham CpG control) with or without laser treatment (2.0 W/cm2, 40 s, 900 nm) at day 0 and day 6. The splenocytes were collected at day 12 followed by mitomycin C-treated tumor cell and mIL-2 (5 U/mL) stimulation in vitro. Cytolytic activity of the splenocytes was determined by lactate dehydrogenase assay. “E/T ratio” represents effector/target cell ratio. Significant difference (**p < 0.01) between “HCuSNPs-CpG + Laser” group and other treatment groups at E/T ratio of 1:1, 5:1, or 20:1. Data shown are expressed as mean ± SD (n = 3). (B) Growth of the primary treated tumor and the distant (contralateral) untreated tumor over time. Data shown are expressed as mean ± SD (n = 5). Significant difference between the compared groups (*p < 0.05). “HCuSNPs-GpC” represents HCuSNPs-chitosan-GpC conjugates. “HCuSNPs + Laser + Free CpG” represents photothermal therapy plus free CpG treatment.

Figure 7

Figure 7

BALB/c mice bearing EMT6-OVA tumor in right flank were given the following treatment: group 1, intratumoral injection with saline at day 0 and day 6; group 2, intratumoral injection with OVA (50 μg/mouse) with complete Freund’s adjuvant (CFA) at day 0 and intratumoral injection with OVA (50 μg/mouse) with incomplete Freund’s adjuvant (IFA) at day 6; group 3, intratumoral injection with HCuSNPs-CpG (100 μg/mouse) at day 0 and intratumoral injection with OVA (50 μg/mouse) with IFA at day 6; group 4, intratumoral injection with HCuSNPs-CpG (100 μg/mouse) plus laser (900 nm, 2.0 W/cm2 for 40 s) at day 0 and intratumoral injection with OVA (50 μg/mouse) with IFA at day 6. At day 6, the mice were subcutaneously inoculated with 3 × 105 EMT6-OVA cells in left (contralateral) flank. Growth of primary treated tumor or contralateral untreated tumor over time is presented following the scheduled treatment with saline or different formulations. Data shown are expressed as mean ± SD (n = 5). Significant difference between the compared groups (**p < 0.01).

Figure 8

Figure 8

EMT6 tumor-bearing mice were given intratumoral injection of HCuSNPs-CpG containing 1 mg of Cu per mouse with or without laser treatment (2.0 W/cm2, 40 s, 900 nm) at day 0 and day 6. Biodistribution of Cu in tumors (A) and major organs (B) at day 14 following injection. “DLN” represents tumor-draining lymph node. Data are expressed as percentage of injected dose per tissue (% ID/tissue). Cumulative excreted Cu in feces (C) and urine (D) collected from day 0 to day 14. All data presented are the values subtracted from endogenous Cu content. Data are presented as mean ± SD (n = 5). Significant difference in groups between HCuSNPs-CpG and HCuSNPs-CpG plus laser (*p < 0.05; **p < 0.01).

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