Enhanced antitumor immunity by targeting dendritic cells with tumor cell lysate-loaded chitosan nanoparticles vaccine

Purpose: Whole tumor cell lysates (TCL) are a broad source of tumor antigens for cancer vaccines, but soluble TCL is unstable, poorly taken up by dendritic cells (DCs), and inefficient at inducing cytotoxic T lymphocyte (CTL) responses. A delivery system was needed to protect TCL antigens, target DCs, and enhance antigen presentation and antitumor immunity.

Hypothesis: If TCL from B16 melanoma cells is encapsulated in mannose-decorated chitosan nanoparticles, then the nanoparticles will target DCs via mannose receptors, enhance DC antigen uptake, maturation, and presentation, and generate stronger prophylactic and therapeutic antitumor immune responses than TCL alone or non-targeted chitosan nanoparticles.

Aims: Prepare and characterize mannose-decorated chitosan nanoparticles loaded with tumor cell lysates (Man-CTS-TCL NPs). - Assess antigen uptake, intracellular localization, and DC maturation in bone marrow-derived dendritic cells (BMDCs) in vitro. - Evaluate in vivo trafficking of the nanoparticles to draining lymph nodes and their effect on endogenous DC maturation and antigen presentation. - Test prophylactic and therapeutic antitumor efficacy, T cell responses, and CTL activity in B16 melanoma mouse models.

Delivery system: Platform: Chitosan nanoparticles (CTS NPs) surface-decorated with mannose for DC targeting. - Polymer: Chitosan (Mw = 50,000; deacetylation >95%). - Targeting ligand: Mannose, introduced via mannose-modified alginate (Man-ALG); mannose substitution degree 8.4%. - Payload: B16 melanoma tumor cell lysates (TCL); fluorescent labels used for tracking (FITC-TCL, Cy7-TCL). - Preparation: TCL mixed with chitosan to form CTS-TCL complexes; Man-ALG added by electrostatic interaction to form Man-CTS-TCL NPs. - Particle properties: Average diameter ~120 nm, spherical by TEM, overall surface charge ~ −12 mV. Protein release was higher and more sustained at acidic pH 5.0 than at pH 7.4.

Approach: In vitro: BMDCs from mice; B16 melanoma cells. - In vivo: C57BL/6 and Balb/c mice; B16 melanoma subcutaneous tumor model. - Prophylactic design: Mice immunized subcutaneously on days −14, −13, and −7 with PBS, TCL, CTS NPs, Man-CTS NPs, CTS-TCL NPs, or Man-CTS-TCL NPs, then challenged with 1 × 10⁵ B16 cells on day 0; n = 6 per group. - Therapeutic design: Mice inoculated with B16 cells on day 0, then immunized on days 7, 14, and 21; n = 6 per group. - TCL dose: 100 µg/mouse per immunization. - Trafficking: Balb/c mice injected subcutaneously at tail base with TCL-Cy7, CTS-TCL-Cy7 NPs, or Man-CTS-TCL-Cy7 NPs (50 µg TCL-Cy7).

Key methods: DLS and zeta potential for size/surface charge; TEM for morphology; FT-IR and ¹H NMR for Man-ALG characterization. - BCA protein assay for TCL release at pH 5.0 and 7.4. - Flow cytometry for DC surface markers (CD11c, CD80, CD86, CD40, MHC I, MHC II, CCR7) and T cell subsets (CD3, CD4, CD8). - Confocal microscopy for antigen uptake and lysosomal localization in BMDCs. - In vivo imaging (Maestro) for Cy7-labeled vaccine trafficking to draining lymph nodes. - ELISA for serum IFN-γ, IL-4, IL-12p70, and TCL-specific IgG. - CFSE dilution for T cell proliferation. - CTL assay by LDH release using B16 target cells. - Tumor volume and tumor weight measurements.

Key results: Nanoparticle characterization: Man-CTS-TCL NPs were ~120 nm, spherical, with a zeta potential of ~ −12 mV. Protein release was elevated at pH 5.0 compared with pH 7.4. - DC uptake/maturation: Man-CTS-TCL NPs increased BMDC uptake of TCL-FITC about 3-fold versus controls. They significantly upregulated CD80, CD86, CD40, MHC I, MHC II, and CCR7 on BMDCs and on CD11c⁺ DCs in draining lymph nodes. - In vivo trafficking: Robust Cy7 signal was detectable in the inguinal lymph node at 24 h only in the Man-CTS-TCL NP group; TCL and CTS-TCL NP groups showed no detectable signal at 24 h. - Immune response: Man-CTS-TCL NPs increased serum IFN-γ and IL-4, enhanced CD8⁺ and CD4⁺ T cell proliferation, and increased CD8⁺ T cells in lymph nodes and spleen. - Prophylactic efficacy: Man-CTS-TCL NPs significantly delayed tumor growth and reduced tumor weight versus controls. CTL lysis was ~35% at 50:1 E:T ratio versus ~12% in controls. - Therapeutic efficacy: In established B16 tumors, Man-CTS-TCL NPs significantly inhibited tumor growth and reduced tumor weight, increased serum IFN-γ, and increased splenic CD8⁺ T cells. No significant body-weight differences were observed among groups.

Interpretation: The authors conclude that mannose-decorated chitosan nanoparticles loaded with tumor cell lysates are an effective antitumor vaccine platform. Mannose targeting enhances DC maturation, antigen uptake, and presentation, leading to improved cellular and humoral antitumor immunity. Man-CTS-TCL NPs may be a feasible therapeutic approach for cancer vaccine development.

Limitations: Efficacy was tested only in the B16 melanoma mouse model; no human or large-animal validation. - Follow-up was short (~21–22 days); no long-term survival, memory response, or rechallenge data. - The specific contribution of mannose receptor-mediated uptake was not definitively isolated by receptor-blocking studies. - Long-term toxicity, immunogenicity, and safety of the chitosan/mannose formulation were not extensively evaluated. - The study reports prophylactic and therapeutic effects but not complete tumor eradication or survival benefit.