Poly(cyclodextrin)-Polydrug Nanocomplexes as Synthetic Oncolytic Virus for Locoregional Melanoma Chemoimmunotherapy

Abstract

Despite the approval of oncolytic virus therapy for advanced melanoma, its intrinsic limitations that include the risk of persistent viral infection and cost-intensive manufacturing motivate the development of analogous approaches that are free from the disadvantages of virus-based therapies. Herein, we report a nanoassembly comprised of multivalent host-guest interactions between polymerized paclitaxel (pPTX) and nitric oxide incorporated polymerized β-cyclodextrin (pCD-pSNO) that through its bioactive components and when used locoregionally recapitulates the therapeutic effects of oncolytic virus. The resultant pPTX/pCD-pSNO exhibits significantly enhanced cytotoxicity, immunogenic cell death, dendritic cell activation and T cell expansion in vitro compared to free agents alone or in combination. In vivo, intratumoral administration of pPTX/pCD-pSNO results in activation and expansion of dendritic cells systemically, but with a corresponding expansion of myeloid-derived suppressor cells and suppression of CD8⁺ T cell expansion. When combined with antibody targeting cytotoxic T lymphocyte antigen-4 that blunts this molecule's signaling effects on T cells, intratumoral pPTX/pCD-pSNO treatment elicits potent anticancer effects that significantly prolong animal survival. This formulation thus leverages the chemo- and immunotherapeutic synergies of paclitaxel and nitric oxide and suggests the potential for virus-free nanoformulations to mimic the therapeutic action and benefits of oncolytic viruses.

Keywords: Cancer; Chemotherapy; Immunotherapy; Nitric oxide; Paclitaxel.

Figure 1.

Synthesis and characterizations of pPTX/pCD-pSNO NPs. (A) Schematic of pPTX/pCD-pSNO NP preparation and NO and PTX release mechanisms. (B) Dynamic light scattering (DLS)-based hydrodynamic size distributions of pPTX/pCD, pPTX/pCD-pSH and pPTX/pCD-pSNO NPs. (C) Transmission electron microscopy (TEM) images of pPTX/pCD-pSH NPs. (D) TEM images of pPTX/pCD-pSNO NPs. (E) Zeta potential-based surface charges of pPTX/pCD, pPTX/pCD-pSH and pPTX/pCD-pSNO NPs. (F) FT-IR of pPTX/pCD-pSH and pPTX/pCD-pSNO NPs. (G) Quantification of thiol content of pPTX/pCD-pSH and pPTX/pCD-pSNO NPs by Ellmans’ assay (black box) and quantification of –SNO groups in pPTX/pCD-pSNO NPs by Saville and Griess assays (red box). (H) Cumulative NO release graph under intracellular and extracellular redox conditions. (I) Cumulative PTX release graph at different pH with or without esterase. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05.

Figure 2.. In vitro effects of pPTX/pCD-pSNO NPs on cancer cells.

Alamar blue-assisted cytotoxicity testing of free PTX, pPTX/pCD-pSH NPs and pPTX/pCD-pSNO NPs in (A) LS174T, (B) EL4, (C) B16F10, and (D) E0771 cell lines. (E) Quantification of apoptosis and necrosis in B16F10 by Annexin V/PI assay-assisted flow cytometry ([PTX] = 0.05 μM, [-SNO] = 7.2 nM, 1 d incubation). (F) CLSM images of MPR 1 expression (green) in B16F10 ([PTX] = 1 μM, [-SNO] = 144.5 nM, 1 d incubation). Scale bar is 10 μm. (G) Quantification of intracellular NO in B16F10 ([PTX] = 1 μM, [-SNO] = 144.5 nM, 1d incubation). (H) Quantification of extracellular ATP released from B16F10 ([PTX] = 1 μM, [-SNO] = 144.5 nM, 1 d incubation). (I) Quantification of intracellular autophagy in B16F10 ([PTX] = 0.1 μM, [-SNO] = 144.5 nM, 1 d incubation) (J) Quantification of CRT in B16F10 ([PTX] = 0.1 μM, [-SNO] = 144.5 nM, 1 d incubation). ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05.

Figure 3.. Effects of pPTX/pCD-pSNO NPs on DCs in vitro and in vivo.

Dose dependent in vitro activation of BMDCs, evaluated by (A) MHCII and (B) CD86. Dose dependent in vitro cytokine production by BMDCs, (C) IL-6 and (D) TNF-α. (A-D) The GSNO, PTX+GSNO and pPTX/pCD-pSNO contains 144.5 nM -SNO groups per 1 μM PTX. (E-I) Number of immune cells in draining lymph nodes (dLNs) 1 d after dorsal injection into tumor-free mouse (C57BL/6J); (E) CD45⁺ cells, (F) CD45⁺CD11b⁺CD11c⁺ DCs, (G) CD40⁺ activated CD45⁺CD11b⁺CD11c⁺ DCs, (H) CD86⁺ CD45⁺CD11b⁺CD11c⁺ DCs, and (I) MHCII^+low, MHCII^+mid, and MHCII^+high CD45⁺CD11b⁺CD11c⁺ DCs. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05.

Figure 4.. Tumor growth curves and immune cell profiles in various tissues.

Relative tumor size of (A) 1° tumor and (B) 2° tumor after one time i.t. injection. An arrow indicates injection date. Immune cells were profiled 18 d after inoculations of 1° tumors. Number of (C) CD45⁺CD11b⁺CD11c⁻Gr1⁺ MDSCs in 1° tumor. Number of immune cells in 1° dLN; (D) CD45⁺CD11b⁺CD11c⁺ DCs, (E) MHCII^+low, MHCII^+mid, and MHCII^+high CD45⁺CD11b⁺CD11c⁺ DCs, (F) CD45⁺CD11b⁺CD11c⁻Gr1⁺ MDSCs, (G) CD45⁺CD3⁺CD4⁺CD25⁺Foxp3⁺ Treg, (H) LAG-3⁺ CD45⁺CD3⁺CD4⁺ T cells, (I) LAG-3⁺ CD45⁺CD3⁺CD8⁺ T cells, (J) PD-1⁺ CD45⁺CD3⁺CD8⁺ T cells, (K) Tetramer+ CD45⁺CD3⁺CD8⁺ T cells, (L) CD45⁺CD11b⁺F4/80⁺ macrophages, and (M) CD45⁺CD3⁻NK1.1⁺ NK cells. Number of immune cells in spleen; (N) CD45⁺CD11b⁺CD11c⁺ DCs, (O) CD86⁺ CD45⁺CD11b⁺CD11c⁺ DCs, (P) MHCII^+low, MHCII^+mid, and MHCII^+high CD45⁺CD11b⁺CD11c⁺ DCs, (Q) CD45⁺CD11b⁺F4/80⁺CD86⁺ M1 macrophages, and (R) ratio of M1 to M2 (CD45⁺CD11b⁺F4/80⁺CD206⁺). Number of immune cells in 2° dLN; (S) CD86⁺ CD45⁺CD11b⁺CD11c⁺ DCs, (T) MHCII^+low, MHCII^+mid, and MHCII^+high CD45⁺CD11b⁺CD11c⁺ DCs, (U) CD45⁺CD11b⁺F4/80⁺ macrophages, (V) CD45⁺CD11b⁺F4/80⁺CD86⁺ M1 macrophages, and (W) CD45⁺CD11b⁺F4/80⁺CD206⁺ M2 macrophages. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05.

Figure 5.. In vivo therapeutic effects of aCTLA-4 with pPTX/pCD-pSNO.

(A) Outline of the disease model and therapy regimen. Relative size of (B) 1° tumor (C) 2° tumors. (D) Relative body weight during therapy. (E) Survival curves during treatment. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05.

Scheme 1.. Schematic illustration comparing OV and synthetic OV based on pPTX/pCD-pSNO NPs.

OV self-replicates and generate GM-CSF in the cancer cells, which facilitates lytic effect on cancer cells and recruitments of antigen-presenting cells. Synthetic OV based on pPTX/pCD-pSNO NPs exert synergistic chemotherapy and induce immunogenic cell death on cancer cells, which allow expansion and activation of DCs.