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Abstract
Melanoma is a severe metastatic skin cancer with poor prognosis and no effective treatment. Therefore, novel therapeutic approaches using nanotechnology have been proposed to improve therapeutic effectiveness. Lipid-core nanocapsules (LNCs), prepared with poly(ε-caprolactone), capric/caprylic triglyceride, and sorbitan monostearate and stabilized by polysorbate 80, are efficient as drug delivery systems. Here, we investigated the effects of acetyleugenol-loaded LNC (AcE-LNC) on human SK-Mel-28 melanoma cells and its therapeutic efficacies on melanoma induced by B16F10 in C57B6 mice. LNC and AcE-LNC had z-average diameters and zeta potential close to 210 nm and -10.0 mV, respectively. CytoViva® microscopy images showed that LNC and AcE-LNC penetrated into SK-Mel-28 cells, and remained in the cytoplasm. AcE-LNC in vitro treatment (18–90×109 particles/mL; 1 hour) induced late apoptosis and necrosis; LNC and AcE-LNC (3–18×109 particles/mL; 48 hours) treatments reduced cell proliferation and delayed the cell cycle. Elevated levels of nitric oxide were found in supernatant of LNC and AcE-LNC, which were not dependent on nitric oxide synthase expressions. Daily intraperitoneal or oral treatment (days 3–10 after tumor injection) with LNC or AcE-LNC (1×1012 particles/day), but not with AcE (50 mg/kg/day, same dose as AcE-LNC), reduced the volume of the tumor; nevertheless, intraperitoneal treatment caused toxicity. Oral LNC treatment was more efficient than AcE-LNC treatment. Moreover, oral treatment with nonencapsulated capric/caprylic triglyceride did not inhibit tumor development, implying that nanocapsule supramolecular structure is important to the therapeutic effects. Together, data herein presented highlight the relevance of the supramolecular structure of LNCs to toxicity on SK-Mel-28 cells and to the therapeutic efficacy on melanoma development in mice, conferring novel therapeutic mechanisms to LNC further than a drug delivery system.
Supplementary materials
Figure S1 Acetyleugenol infrared spectrum compared to clove oil infrared spectrum (eugenol).
Note: Major component of clove oil is eugenol (78%).
Abbreviation: AcE, acetyleugenol.
Figure S2 Swelling profiles of the polymer films as a function of time.
Notes: (A) Eugenol/CCT (1:1). (B) AcE/CCT (1:1). (C) eugenol/CCT (9:1). (D) AcE/CCT (9:1). Data are presented as mean ± SD (n=3).
Abbreviations: CCT, capric/caprylic triglyceride; AcE, acetyleugenol; SD, standard deviation.
Figure S3 LNC or AcE-LNC induces toxicity in animals after intraperitoneal treatment.
Notes: B16F10 cells (8×105/100 µL) were subcutaneously injected into dorsal region of C57Bl6 mice. After the tumors had reached ~90 mm3 (3 days), the animals were daily treated for 7 days. The animals received saline, LNC (1×1012 particles/day), AcE (50 mg/kg/day), AcE vehicle (DMSO), or AcE-LNC (50 mg/kg/day of AcE; 1×1012 particles/day) by intraperitoneal (A and B) or oral (C and D) route. Cumulative weight (A and C) and food intake (B and D) of animals were monitored daily during all treatment periods. Representative image of LNC and AcE-LNC agglomeration in abdominal cavity of animals is indicated by white arrows (B). The values are represented as mean ± SEM for ten different animals. Significant differences from saline (control) are *P<0.05, and ***P<0.001 assessed by one- or two-way analysis of variance followed by the Tukey’s post hoc test.
Abbreviations: LNC, lipid-core nanocapsule; AcE-LNC, acetyleugenol-loaded lipid-core nanocapsule; AcE, acetyleugenol; DMSO, dimethyl sulfoxide; SEM, standard error of the mean.
Figure S4 LNC or CTT by oral route did not alter cumulative weight or food intake of mice.
Notes: B16F10 cells (8×105/100 µL) were subcutaneously injected into dorsal region of C57Bl6 mice. After the tumors had reached ~90 mm3 (3 days), the animals were daily treated for 7 days. The animals received saline, free CCT, or LNC (1×1012 particles/day). Cumulative weight (A) and food intake (B) of animals were monitored daily during all treatment periods. The values are represented as mean ± SEM for seven different animals. Values were assessed by one- or two-way analysis of variance followed by the Tukey’s post hoc test, and no significant differences were found.
Abbreviations: LNC, lipid-core nanocapsule; CCT, capric/caprylic triglyceride; SEM, standard error of the mean.
Acknowledgments
This work was supported by grants from São Paulo Research Foundation (FAPESP, São Paulo), National Counsel of Technological and Scientific Development (CNPq, Brasília), and Coordination of Improvement of Higher Education Personnel (CAPES, Brasília). The authors thank Dr Viviane Ferraz de Paula and Dr Francisco Rafael Martins Laurindo for technical assistance with histological and NO analysis, respectively, and Paula Raddatz for technical assistance with LNC and AcE-LNC preparation.
Disclosure
Carine C Drewes, Celina G Bexiga, Mayara K Uchiyama were fellows from FAPESP (projects 2010/19802-1, 12/01257-2, 10/50072-0). Luana A Fiel is a fellow from National Counsel of Technological and Scientific Development (CNPq). Bruno Cogliati, Koiti Araki, Adriana R Pohlmann, Sílvia S Guterres, and Sandra HP Farsky are research fellows from CNPq. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was used in producing this manuscript.