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. 2025 Jan 27:16:1529007.
doi: 10.3389/fimmu.2025.1529007. eCollection 2025.

Liposomal encapsulation of cholecalciferol mitigates in vivo toxicity and delays tumor growth

Miriam Ezcurra-Hualde  1   2 , Sara Zalba  2   3 , Ángela Bella  1   2 , Leire Arrizabalaga  1   2 , Aline Risson  1   2 , Román García-Fuentes  1   2 , Celia Gomar  1   2 , Nuria Ardaiz  1   2 , Virginia Belsue  1   2 , David Ruiz-Guillamon  1   2 , Alejandro Serrano-Alcaide  2   3 , Ainara Salgado  2   3 , Fernando Aranda  1   2 , Maria J Garrido  2   3 , Pedro Berraondo  1   2   4
Affiliations

Liposomal encapsulation of cholecalciferol mitigates in vivo toxicity and delays tumor growth

Miriam Ezcurra-Hualde et al. Front Immunol. .

Abstract

Introduction: Vitamin D3 (cholecalciferol) has demonstrated potential anticancer properties, but its clinical application is limited by associated toxicity at effective doses. This study investigated the use of liposomal encapsulation to increase the therapeutic efficacy of vitamin D3 while mitigating its toxicity.

Methods: Liposomal vitamin D3 (VD-LP) was prepared via the film-hydration method and characterized for particle size, polydispersity index, encapsulation efficiency, and long-term stability. In vitro gene expression modulation was evaluated in monocytic THP-1 cells, and antiproliferative effects were assessed in HT29 (colorectal), BT474 (breast), and TRAMP-C1 (prostate) cancer cell lines. In vivo antitumor efficacy and toxicity were tested in a mouse model with subcutaneously implanted MC38 tumors. Tumor growth, survival rates, and serum calcium and phosphate levels were analyzed.

Results: VD-LP demonstrated high encapsulation efficiency and stability over 90 days, with a consistent particle size of approximately 83 nm. VD-LP modulated immune-related and metabolic gene expression in THP-1 cells, including upregulation of antimicrobial peptides and vitamin D receptor genes. VD-LP showed superior antiproliferative effects compared to free vitamin D3 in all tested cancer cell lines. In vivo, VD-LP delayed tumor growth and improved survival without causing hypercalcemia, highlighting its favorable toxicity profile.

Discussion: Liposomal encapsulation of vitamin D3 significantly improves its anticancer efficacy while mitigating toxicity, making it a promising strategy for future cancer therapies. VD-LP shows potential for enhanced therapeutic applications with reduced adverse effects, warranting further clinical exploration.

Keywords: anticancer efficacy; gene expression; liposomal encapsulation; tumor growth; vitamin D3.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. PB declared that he was an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
Characterization and stability of the VD-LP formulation. (A) Stability evaluation of VD-LP over 90 days at 4°C. The particle size (nm), polydispersity index (PDI), and encapsulation efficiency (EE%) were monitored and remained stable throughout the study period. The data represent the means ± SDs of three independent batches. (B) Transmission electron microscopy (TEM) images of the VD-LP showing spherical liposomes with sizes of less than 100 nm. The images confirmed the uniform morphology of the liposomes.
Figure 2
Figure 2
Modulation of gene expression by VD-LP in THP-1 cells. (A) A total of 300000 THP-1 cells were seeded in a 96-well plate and stimulated for 24 h with VD-LP, VD or Empty-LP. RNA was extracted for RNA-seq, and cDNA was generated for PCR. (B) Venn diagram representing the number of modulated genes in each experimental group. (C) Volcano plots representing down- and upregulated genes. (D) GSEA of differentially regulated pathways. (E) Real-time PCR analysis of cathelicidin antimicrobial peptide (hCAMP), vitamin D receptor (hVDR), ATP-binding cassette subfamily D member 2 (ABCD2), fructose-1,6-biphosphatase 1 (FBP1), and neuronal growth regulator 1 (NEGR1). One-way ANOVA with Sidak’s multiple comparisons test was used. *p ≤ 0.05, **p ≤ 0.01, ****p ≤0.0001.
Figure 3
Figure 3
Antiproliferative effects of VD-LP in cancer cell lines. (A–C) Evaluation of the antiproliferative effects of VD-LP on the HT29 (human colorectal cancer), BT474 (human breast cancer), and TRAMP-C1 (prostate cancer) cell lines. The cells were treated with VD-LP, free VD, or Empty-LP or left untreated, and cell proliferation was monitored via xCELLigence real-time cell analysis. One-way ANOVA with the Kruskal-Wallis test; ****p ≤ 0.0001.
Figure 4
Figure 4
In Vivo Antitumor Efficacy and Toxicity of VD-LP in a Mouse Model (A) Graphical representation of the in vivo assay. C57BL/6J mice were subcutaneously inoculated with 5 × 105 MC38 cells and treated with VD-LP, free VD, Empty-LP, or HEPES buffer (control). (B) Kaplan-Meier survival curve for mice treated with VD-LP, free VD, Empty-LP, or HEPES. VD-LP treatment resulted in improved survival rates compared with those of the other groups. Log-rank (Mantel-Cox) test. (C) Tumor volume over time in mice treated with VD-LP, free VD, Empty-LP, or HEPES. VD-LP significantly reduced tumor growth compared with that in the control groups. One-way ANOVA with Sidak’s multiple comparisons test was used. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. (D) Measurement of toxicity: serum calcium (Ca2+) and phosphate (PO4 3-) levels. (E) Volcano plot of differentially expressed genes in tumor tissues from VD-LP-treated versus control mice, highlighting upregulated and downregulated genes. (F) GSEA plot showing significant pathways that were activated or inhibited in tumor tissues following VD-LP treatment.
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References

    1. Reid IR, Bolland MJ, Grey A. Effects of vitamin D supplements on bone mineral density: A systematic review and meta-analysis. Lancet. (2014) 383:146–55. doi: 10.1016/S0140-6736(13)61647-5 - DOI - PubMed
    1. Feldman D, Krishnan AV, Swami S, Giovannucci E, Feldman BJ. The role of vitamin D in reducing cancer risk and progression. Nat Rev Cancer. (2014) 14:342–57. doi: 10.1038/nrc3691 - DOI - PubMed
    1. Carlberg C, Munoz A. An update on vitamin D signaling and cancer. Semin Cancer Biol. (2022) 79:217–30. doi: 10.1016/j.semcancer.2020.05.018 - DOI - PubMed
    1. Munoz A, Grant WB. Vitamin D and cancer: an historical overview of the epidemiology and mechanisms. Nutrients. (2022) 14:1448. doi: 10.3390/nu14071448 - DOI - PMC - PubMed
    1. Manson JE, Cook NR, Lee IM, Christen W, Bassuk SS, Mora S, et al. . Vitamin D supplements and prevention of cancer and cardiovascular disease. N Engl J Med. (2019) 380:33–44. doi: 10.1056/NEJMoa1809944 - DOI - PMC - PubMed

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