<p>Nano-Graphene Oxide-supported APTES-Spermine, as Gene Delivery System, for Transfection of pEGFP-p53 into Breast Cancer Cell Lines</p>

Abstract

Purpose Genetic diseases can be the result of genetic dysfunctions that happen due to some inhibitory and/or environmental risk factors, which are mostly called mutations. One of the most promising treatments for these diseases is correcting the faulty gene. Gene delivery systems are an important issue in improving the gene therapy efficiency. Therefore, the main purpose of this study was modifying graphene oxide nanoparticles by spermine in order to optimize the gene delivery system. Methods Graphene oxide/APTES was modified by spermine (GOAS) and characterized by FT-IR, DLS, SEM and AFM techniques. Then pEGFP-p53 was loaded on GOAS, transfected into cells and evaluated by fluorescent microscopy and gene expression techniques. Results FT-IR data approved the GOAS sheet formation. Ninety percent of the particles were less than 56 nm based on DLS analysis. SEM analysis indicated that the sheets were dispersed with no aggregation. AFM results confirmed the dispersed structures with thickness of 1.25±0.87 nm. STA analysis showed that GOAS started to decompose from 400°C and was very unstable during the heating process. The first weight loss up to 200°C was due to the evaporation of absorbed water, the second one observed in the range of 200–550°C was assigned to the decomposition of labile oxygen- and nitrogen-containing functional groups, and the third one above 550°C was attributed to the removal of oxygen functionalities. In vitro release of DNA demonstrated the efficient activity of the new synthesized system. Ninety percent of the cells were transfected and showed the GFP under fluorescence microscopy, and TP53 gene was expressed 51-fold in BT-20 cells compared to β-actin as the reference gene. Flow cytometry analysis confirmed the apoptosis of the cells rather than necrosis. Conclusion It could be concluded that the new synthesized structure could transfer a high amount of the therapeutic agent into cells with best activity.

Characteristics of Graphene Oxide for Gene Transfection and Controlled Release in Breast Cancer Cells

“…On the other hand, the wide range of lateral sizes is advantageous to meet different requirements for biological applications [ 49 , 50 ]. GO nanoparticles ranging from 50 to 200 nm have consistently been demonstrated in multiple investigations to bypass immune cells, diffuse through vessel walls and the ECM, and infiltrate breast cancer cells effectively [ 51 , 52 , 53 , 54 , 55 ].…”

“…These results indicated that depending on the application, fabricating a carrier that can diffuse the mentioned barriers to reach the targeted site of the body raises the demand for achieving a uniform size of GO particles using the mentioned approaches. In addition, different sizes of functionalized GO have been utilized for breast cancer therapy [ 17 , 51 , 53 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 ].…”

“…The intensity was increased, indicating that fragmentation of the GO had occurred. Moreover, even if it has been reported that the cell internalization of GO is size-dependent in different cell lines [ 52 , 53 ], different studies have proven that macrophages (i.e., PMƟ and J774A.1) are not always affected by the size and dosage during GO uptake [ 117 ]. All of these findings may demonstrate the potential for naked graphene-based materials to be degraded within phagocytic cells after the payload delivery [ 69 , 123 ].…”

“…In addition, GO nanocarriers have a high probability of being removed by macrophages before reaching their destination and may initiate an inflammatory reaction [ 24 , 164 ]. In particular, when in the bloodstream, larger nanoparticles (>200 nm) have a higher risk of being recognized and sequestered [ 44 , 53 , 123 , 137 ] by the phagocytosing cells of the immune system (i.e., macrophages, dendritic cells, neutrophils, and B lymphocytes), which are responsible for recognizing the foreigner and destroying it via enzymatic digestion, as explained previously [ 31 ]. When macrophages are activated by invader molecules, they start to release cytokines and chemokines, resulting in the accumulation of neutrophils and monocytes.…”

“…This would allow an increase in treatment efficiency, as a high percentage of nanocarriers would transport the therapeutic agent directly to the oncological sites [ 123 ]. This mechanism would lead to a decrease in the dose of nanocarriers necessary to administer, with consequent advantages of low toxicity and limited side effects [ 53 , 168 ].…”