Modulation of Dendritic Cell Function via Nanoparticle-Induced Cytosolic Calcium Changes

Calcium nanoparticles have been investigated for applications, such as drug and gene delivery. Additionally, Ca2+ serves as a crucial second messenger in the activation of immune cells. However, few studies have systematically studied the effects of calcium nanoparticles on the calcium levels and functions within immune cells. In this study, we explore the potential of calcium nanoparticles as a vehicle to deliver calcium into the cytosol of dendritic cells (DCs) and influence their functions. We synthesized calcium hydroxide nanoparticles, coated them with a layer of silica to prevent rapid degradation, and further conjugated them with anti-CD205 antibodies to achieve targeted delivery to DCs. Our results indicate that these nanoparticles can efficiently enter DCs and release calcium ions in a controlled manner. This elevation in cytosolic calcium activates both the NFAT and NF-κB pathways, in turn promoting the expression of costimulatory molecules, antigen-presenting molecules, and pro-inflammatory cytokines. In mouse tumor models, the calcium nanoparticles enhanced the antitumor immune response and augmented the efficacy of both radiotherapy and chemotherapy without introducing additional toxicity. Our study introduces a safe nanoparticle immunomodulator with potential widespread applications in cancer therapy.


Figure S3 .
Figure S3.Calcium release in solutions and inside cells.Quantification of calcium levels in solutions was achieved using an ion-selective electrode.In vitro quantification was based on a chromogenic calcium indicator, 0-cresolphthalein (OD: 575 nm).a) Standard calibration curve for potentiometry measurements, established with calcium salt (CaCl 2 , 150 ppm and 2000 ppm).b) Time-dependent Ca 2+ release from CHNPs, tested in ammonium acetate buffers at pH 7.4 and 5.5.c) Cytotoxicity of AnCHNPs, CaCl 2 , and aged AnCHNPs, tested with BMDCs using ATPlite-1step luminescence assay.d) Lysosomal pH changes after cells being treated with AnCHNPs (5 µg/mL), measured with BMDCs using LysoSensor™ Yellow/Blue DND-160 (PDMPO); the indicator shows predominantly yellow fluorescence (440 nm) in acidic organelles, and in less acidic organelles it shows a stronger blue fluorescence (540 nm).

Figure S4 .
Figure S4.Effect of degraded AnCHNPs on DC maturation.This was investigated by examining maturation markers including MHC-II, CD86, CD80, and CD40 when BMDCs were incubated with aged AnCHNPs.

Figure S6 .
Figure S6.Impact of AnCHNPs on antigen specific cellular immunity.Splenocytes taken from the three treatment groups, AnCHNPs, CaCl 2 , and PBS, were co-incubated with B16F10-OVA cells for 6 h ex vivo; the frequency of IFN-γ + CTLs was measured by flow cytometry.

Figure S10 .
Figure S10.Distribution of nanoparticles (DiR-labeled) in major organs after 24 hours (n=3).Distribution was measured by region of interest (ROI) analysis of photon intensity and the result was normalized to muscle signals.No significant increase in nanoparticle uptake by major organs was observed.

Figure S11 .
Figure S11.a,b) Serum concentrations of BUN and ALT.Samples were taken 7 days after i.t.injection of PBS or AnCHNPs (n=3).c) Serum concentration of calcium.Samples were taken 24 hours after i.t.injection of PBS or AnCHNPs (n=3)

Figure S13 .
Figure S13.Flow cytometry gating strategy for analyzing DC migration.

Figure S14 .
Figure S14.Flow cytometry gating strategy for examining populations of DCs in tumor and TDLNs.

Figure S15 .
Figure S15.Flow cytometry gating strategy for examining populations of T lymphocytes in tumor and spleen.