Manipulating the function of tumor-associated macrophages by siRNA-loaded lipid nanoparticles for cancer immunotherapy

Purpose: Tumor-associated macrophages (TAMs) are largely M2-like and promote tumor growth, angiogenesis, metastasis, and immune suppression. There is a need for systemic delivery systems that can target TAMs and manipulate their pro-tumor functions with siRNA for cancer immunotherapy.

Hypothesis: If siRNA is delivered to TAMs using optimized pH-sensitive CL4H6 lipid nanoparticles, then TAM function can be manipulated by silencing M2-related genes such as STAT3 and HIF-1α, leading to reduced angiogenesis, altered macrophage phenotype, and an anti-tumor therapeutic response.

Aims: Prepare and optimize siRNA-loaded CL4H6-LNPs for TAM targeting. - Evaluate blood stability, biodistribution, tumor accumulation, and TAM-selective uptake. - Confirm gene silencing in TAMs using CD45, STAT3, and HIF-1α siRNAs. - Assess anti-tumor efficacy and changes in the tumor microenvironment after silencing STAT3 and HIF-1α in TAMs.

Delivery system: Platform: pH-sensitive cationic lipid nanoparticles (LNPs). - Key lipid: CL4H6, pKa 6.30. - Optimized formulation: CL4H6:cholesterol:DSG-PEG2000 = 60:40:1 mol%. - Payloads: siRNA against CD45, STAT3, HIF-1α, or Factor VII; DiD/DiI labels for tracking. - Physicochemical properties: >90% siRNA encapsulation efficiency, PDI 0.0–0.2, neutral surface charge (−5 to +5 mV), ~90–100 nm diameter for the optimal formulation. - Targeting ligand: None; TAM uptake was passive/selective in the OS-RC-2 tumor model. - Route: Intravenous administration.

Approach: In vitro: Murine macrophage RAW264.7 cells and bone marrow-derived macrophages for LNP screening. - In vivo: OS-RC-2 human renal cell carcinoma xenografts in BALB/c AJcl-nu/nu mice; TAMs are the dominant leukocyte population in this model. - Biodistribution/uptake: DiD-labeled LNPs; flow cytometry of tumor single-cell suspensions; multi-color gating for TAMs, neutrophils, other leukocytes, non-leukocytes. - Gene silencing: siCD45 at 2 mg/kg × 2 doses; siSTAT3/siHIF-1α at 2 mg/kg single dose. - Anti-tumor study: Groups included PBS, doxorubicin-loaded liposomes (positive control, 3 mg/kg), empty CL4H6-LNPs, and siSTAT3:siHIF-1α-loaded CL4H6-LNPs (1:1, 1 mg siRNA/kg), given IV every 3 days for up to 3 doses; n = 5. - Endpoint: Tumor volume, qRT-PCR of TME genes, body weight/toxicity.

Key methods: Dynamic light scattering and RiboGreen assay for size, PDI, zeta potential, and siRNA encapsulation. - RNase protection assay for siRNA stability. - FluorVivo/ImageJ for organ and tumor biodistribution. - Multi-color flow cytometry for LNP uptake and CD45 silencing in TAMs. - Cell sorting of TAMs (CD45⁺F4/80⁺) and qRT-PCR for STAT3, HIF-1α, CD31, VEGFR2, TGF-β, MMP-9, ARG1, IFN-γ, TNF-α, IL-6, IL-10, PD-1, etc. - Tumor volume measurement and body-weight monitoring.

Key results: Optimized CL4H6-LNPs had >90% siRNA encapsulation efficiency, neutral surface charge, and 90–100 nm size. - DSG-PEG2000 modification increased tumor accumulation 1.74-fold compared with DMG-PEG2000. - TAM uptake was significantly higher than non-leukocyte uptake: 6.89-fold higher for leukocytes and 8.67-fold higher for TAMs; TAM uptake also exceeded neutrophils and other leukocytes. - siCD45-loaded LNPs induced ~70% CD45 silencing in TAMs at 2 mg/kg × 2 doses. - siSTAT3/siHIF-1α LNPs significantly silenced HIF-1α (~80%) in TAMs; STAT3 silencing was not significant in that single-dose experiment. - In the anti-tumor study, siRNA-loaded CL4H6-LNPs reduced tumor volume by ~50% vs PBS. - In the TME, STAT3 and HIF-1α were downregulated by 37% and 48%; CD11b⁺ macrophage infiltration increased 59%; CD169⁺ M1-like macrophages increased 50% (not significant); CD163⁺ M2-like macrophages were unchanged; M1/M2 ratio increased 50%. - Pro-tumor markers decreased: CD31 37%, VEGFR2 12%, TGF-β 43%, ARG1 29%. IFN-γ increased 130%, TNF-α increased 91%. MMP-9 increased 16%; IL-10 unchanged; IL-6 and PD-1 increased. - No significant body-weight loss or toxicity was reported.

Interpretation: The authors conclude that siRNA-loaded CL4H6-LNPs can selectively deliver siRNA to TAMs and manipulate their function. Silencing STAT3 and HIF-1α in TAMs inhibited angiogenesis and tumor cell activation, increased macrophage infiltration and M1-like tendency, and produced a significant anti-tumor response, supporting TAM-targeted LNP immunotherapy as a promising cancer treatment strategy.

Limitations: Immune-deficient mouse model lacking T cells; cannot assess T cell–macrophage interactions or full immune contribution. - No direct evidence of definitive M2-to-M1 reprogramming; CD163⁺ M2 marker was unchanged and M1 increase was not statistically significant. - Only one tumor model (OS-RC-2 xenograft); no syngeneic/immune-competent efficacy data in the main study. - STAT3 silencing was not confirmed in the single-dose TAM silencing experiment. - Gene expression changes were modest for some markers; MMP-9 increased, and immune-suppressants IL-6 and PD-1 also increased. - No survival study, long-term safety, repeated-dose toxicity, or large-animal validation. - No active targeting ligand; TAM selectivity relied on passive tumor accumulation and the immune-deficient TME.