Preparation and Characterization of Nanoliposomal Beta- Cryptoxanthin and its Effect on Proliferation and Apoptosis in Human Leukemia Cell Line K562

Purpose: To prepare beta-cryptoxanthin-loaded nanoliposomes and evaluate their anti-proliferative activity in leukemia K562 cell line, compared to free beta-cryptoxanthin.Methods: Beta-cryptoxanthin-loaded nanoliposomes were prepared by extrusion method. Morphological characterization of the nanoliposomes was performed by cryo-transmission electron microscopy (cryo-TEM). The anti-proliferation effect of beta-cryptoxanthin (BC) in free and liposomal forms on K562 cell line was studied using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. Apoptotic activity, following treatment with beta-cryptoxanthin in the free and liposomal forms, was detected using flow cytometry.Results: Entrapment efficiency of beta-cryptoxanthin was 86.3 % ± 1.0. Cryo-TEM analysis revealed that the nanoliposomes have spherical shapes. In all conditions, beta-cryptoxanthin-loaded nanoliposomes exhibited greater anti-proliferative activity than than the free beta-cryptoxanthin (p < 0.001). Furthermore, in the presence of beta-cryptoxanthin-loaded nanoliposomes, the proportion of apoptotic cells was higher for free beta-cryptoxanthin (p < 0.001). Conclusion: The data obtained indicate that beta-cryptoxanthin, especially in the liposomal form, inhibits the growth of K562 cells and may therefore provide a basis for the development of leukemia therapies.Keywords: Beta-cryptoxanthin, Nanoliposome, Anti-proliferative, Apoptosis, Flow cytometry, Leukemi

Site-specific transgene integration in chimeric antigen receptor (CAR) T cell therapies

Chimeric antigen receptor (CAR) T cells and natural killer (NK) cells are genetically engineered immune cells that can detect target antigens on the surface of target cells and eliminate them following adoptive transfer. Recent progress in CAR-based therapies has led to outstanding clinical success in certain patients with leukemias and lymphomas and offered therapeutic benefits to those resistant to conventional therapies. The universal approach to stable CAR transgene delivery into the T/NK cells is the use of viral particles. Such approaches mediate semi-random transgene insertions spanning the entire genome with a high preference for integration into sites surrounding highly-expressed genes and active loci. Regardless of the variable CAR expression level based on the integration site of the CAR transgene, foreign integrated DNA fragments may affect the neighboring endogenous genes and chromatin structure and potentially change a transduced T/NK cell behavior and function or even favor cellular transformation. In contrast, site-specific integration of CAR constructs using recent genome-editing technologies could overcome the limitations and disadvantages of universal random gene integration. Herein, we explain random and site-specific integration of CAR transgenes in CAR-T/NK cell therapies. Also, we tend to summarize the methods for site-specific integration as well as the clinical outcomes of certain gene disruptions or enhancements due to CAR transgene integration. Also, the advantages and limitations of using site-specific integration methods are discussed in this review. Ultimately, we will introduce the genomic safe harbor (GSH) standards and suggest some appropriate safety prospects for CAR integration in CAR-T/NK cell therapies