(M1230-12-69) Cytotoxicity, Safety and Mammary Tissue Retention of Nanostructured Lipid Carriers Co-encapsulating Paclitaxel and Tributyrin as a Novel Approach for Local Breast Cancer Treatment

Purpose: Breast cancer is one of the most common types of cancer in women worldwide, but there is a lack of well-accepted and effective alternatives for the local management of non-invasive forms of the disease, such as ductal carcinoma in situ (DCIS). We have developed and optimized nanostructured lipid carriers (NLC) co-encapsulating paclitaxel and the triglyceride tributyrin (for potentiation of cytotoxicity) for intraductal delivery and local treatment of non-invasive forms of breast cancer, which presented diameter of 350 nm and negative zeta potential (-14.3 mV). The present study aims at assessing the cytotoxic effects of the NLC in vitro in breast cancer cells and evaluate the mammary tissue retention after intraductal administration in rats.

Methods: The influence of nanoencapsulation on the cytotoxicity of drugs was evaluated through the MTT assay in breast cancer cell lines (MCF7 and T47D). A safety assay using Galleria mellonella larvae was employed to evaluate the toxicity of the formulation prior to its administration in rats. The nanocarrier (10 μL) was administered in the last left pro-leg and larvae survival was assessed for 5 days. To assess mammary tissue retention, a fluorescent marker (rhodamine B 0.5%, w/w) was incorporated into the nanoparticles and, using an in vivo imaging system (IVIS Spectrum System, Perkin-Elmer Life Sciences, Waltham, MA), the influence of the nanocarrier on the retention time in the mammary ducts was evaluated for 5 days. The NLC was also incorporated in a thermosensitive poloxamer 407 gel (11%, w/w) to further improve retention. Local irritation was also assessed by histological examination of the mammary tissue surrounding the administration site.

Results: The presence of tributyrin in the nanocarrier increased cytotoxicity in both tumor cell lines, when compared to a control carrier containing tricaprylin as a liquid lipid (~3-fold reduction in the IC₅₀ value). The addition of paclitaxel also increased the cytotoxicity and reduced IC₅₀ of the formulation by 8.1 and 7.9-fold in MCF7 cells compared to NLC with tributyrin alone; the reduction was 2.7 and 2.9-fold in T47D cells. The nanocarrier didn't increase drug cytotoxicity (IC50 27 - 30 μM) compared to drug solution (IC50 28 – 33 μM), which can possibly be attributed to the slow release of the drug from the lipid core and reduced free and soluble concentration of the drug to exert its effect. The unloaded nanocarrier was considered safe as indicated by high survival rates (~80%) of Galleria mellonella larvae exposed to the formulation. A longer retention of the fluorescent marker in breast tissue was observed after intraductal administration when the NLC was incorporated into the poloxamer gel (65.7 ± 18.9%) compared to its solution (28.2 ± 10.4%) and to the NLC dispersed in water (30.7 ± 12.1%). The architecture of the mammary tissue and its typical structures were maintained in all treatment groups. In addition, no inflammatory cell infiltrates were observed in the tissue, which suggests the absence of local irritation and the safety of the administered formulations.

Conclusion: In this study, we demonstrated that the co-encapsulation of paclitaxel and the triglyceride tributyrin in nanostructured lipid carrier was able to increase cytotoxic effect compared to control carriers. Moreover, the nanocarrier improved formulation retention in the mammary tissue after intraductal administration in rats. These results support the potential applicability of nanostructured lipid carries incorporated into thermosensitive gels for intraductal treatment of breast cancer.

References: Carvalho, V.F.M., et al., Co-encapsulation of paclitaxel and C6 ceramide in tributyrin-containing nanocarriers improve co-localization in the skin and potentiate cytotoxic effects in 2D and 3D models. Eur J Pharm Sci, 2017. 109: p. 131-143.
Giuliano, E., et al., Drug-Loaded Biocompatible Nanocarriers Embedded in Poloxamer 407 Hydrogels as Therapeutic Formulations. Medicines (Basel), 2018. 6 (1).
Heidor, R., et al., Anticarcinogenic actions of tributyrin, a butyric acid prodrug. Curr Drug Targets, 2012. 13(14): p. 1720-9.
Kuang, X.W., et al., Intraductal Therapy in Breast Cancer: Current Status and Future Prospective. J Mammary Gland Biol Neoplasia, 2020. 25(2): p. 133-143.

Acknowledgments: FAPESP grants #2020/01208-8, #2021/12664-7, CAPES (001).

Influence of the concentration of control (NLC-TC), unloaded (NLC-TB), paclitaxel-loaded NLC or paclitaxel solution on the viability of MCF7 and T47D cells. A) Comparisons of loaded and unloaded NLC in MCF7 cells; B) Comparisons of loaded and unloaded NLC in T47D cells; C) Comparisons of paclitaxel-loaded NLC and drug solution in MCF7 cells; D) Comparisons of paclitaxel-loaded NLC and drug solution in T47D cells; and E) IC50 values of paclitaxel (μM, in the NLC vs in the solution) and formulation (mg/mL) determined in MCF7 and T47D cells after 72 hours of treatment. Data represents average ± SD, n = 9-12, in 3 separate experiments. Concentrations of the drug and formulation are presented in μM and mg/mL, respectively.



Evaluation of formulation safety in Galleria mellonella larvae treated with unloaded-NLC. A) Health index of larvae treated with unloaded-NLC or PBS (control) and B) Survival rate of larvae treated with unloaded-NLC or PBS (control). Data represents average ± SD using 16 larvae for each group.



In vivo images of Wistar rats treated intraductally with different formulations containing Rhodamine B. A) Images at different times after intraductal administration of unloaded-NLC (negative controle), rhodamine-loaded NLC dispersed in water (NLC i.d.), rhodamine-loaded NLC incorporated in poloxamer gel (NLC + Gel i.d.) and rhodamine solution (solution i.d.); rhodamine-loaded NLC was administered intraperitoneally as a positive control. The colors indicate the fluorescent intensity according to the scale on the right; B) Fluorescent decay along 5 days after treatments administration; and C) Histology of breast tissue in the experimental groups. HE staining and scale bar = 100 μm. Data represents average ± SD, n = 2-3 animals.