Liposomes loaded with unsaponifiable matter from amaranthus hypochondriacus as a source of squalene and carrying soybean lunasin inhibited melanoma cells

Authors: CASTANEDA-REYES, ERICK - NATIONAL POLYTECHNIC INSTITUTE; GONZALEZ DE MEJIA, ELVIRA - UNIVERSITY OF ILLINOIS; Eller, Fred; BERHOW, MARK; PEREA-FLORES, MARIA - NATIONAL POLYTECHNIC INSTITUTE; DAVILA-ORTIZ, GLORIA - NATIONAL POLYTECHNIC INSTITUTE

Submitted to: Nanomaterials
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/23/2021
Publication Date: 7/30/2021
Citation: Castaneda-Reyes, E.D., Gonzalez De Mejia, E., Eller, F.J., Berhow, M.A., Perea-Flores, M., Davila-Ortiz, G. 2021. Liposomes loaded with unsaponifiable matter from amaranthus hypochondriacus as a source of squalene and carrying soybean lunasin inhibited melanoma cells. Nanomaterials. 11(8), Article 1960. https://doi.org/10.3390/nano11081960.
DOI: https://doi.org/10.3390/nano11081960

Interpretive Summary: Amaranth grain is a good source of bioactive molecules with proven bioactivities such as antioxidant activity and cancer prevention. This research is being conducted to develop new ways to provide food ingredients containing squalene, an important fat-soluble nutritional terpene, from processed amaranth seeds. The objective of this research was to optimize the conditions for the preparation of nanoparticle liposomes (protein vessels that encapsulate fats in water suspension) with amaranth fat-soluble squalene preparations along with the bioactive peptide lunasin, which is used to treat cancer cells, to minimize particle size and maximize the encapsulation efficiency of the squalene nutrient for use as a drug delivery agent for the prevention of cancer. A method was developed to prepare optimized liposomes containing squalene extracted from amaranth seeds. The extraction conditions affected the squalene extraction efficiency. The optimization aimed at the minimization of the particle size and maximization of encapsulation efficiency. The liposomes were tested with cultured animal cell viability assays. Cell treatment with squalene seems to play a protector role in melanoma cancer cells. The cell cytotoxicity of lunasin with the squalene in melanoma cells improved its incorporation into liposomes. For future studies, it is recommended to characterize the unsaponifiable matter with the different sterol and tocopherol profiles to obtain more accurate encapsulation efficiency to develop a more effective natural product cancer cell treatment.

Technical Abstract: Amaranthus hypochondriacus is a source of molecules with reported health benefits such as antioxidant activity and cancer prevention. The objective of this research was to optimize the conditions for preparing a liposome formulation using amaranth unsaponifiable matter as a source of squalene in order to minimize the particle size and to maximize the encapsulation efficiency of liposomes for carrying and delivering soybean lunasin into melanoma cell lines. Amaranth oil was extracted using supercritical dioxide carbon extraction (55.2 MPa pressure, 80 °C temperature, solvent (CO2)-to-feed (oil) ratio of 20). The extracted oil from amaranth was used to obtain the unsaponifiable enriched content of squalene, which was incorporated into liposomes. A Box–Behnken response surface methodology design was used to optimize the liposome formulation containing the unsaponifiable matter, once liposomes were optimized. Soybean lunasin was loaded into the liposomes and tested on A-375 and B16-F10 melanoma cells. The squalene concentration in the extracted oil was 36.64 ± 0.64 g/ 100 g of oil. The particle size in liposomes was between 115.8 and 163.1 nm; the squalene encapsulation efficiency ranged from 33.14% to 76.08%. The optimized liposome formulation contained 15.27 mg of phospholipids and 1.1 mg of unsaponifiable matter. Cell viability was affected by the liposome formulation with a half-maximum inhibitory concentration (IC50) equivalent to 225 µM in B16-F10 and 215 µM in A-375. The liposomes formulated with lunasin achieved 82.14 ± 3.34% lunasin encapsulation efficiency and improved efficacy by decreasing lunasin IC50 by 31.81% in B16-F10 and by 41.89% in A-375 compared with unencapsulated lunasin.