PH-Responsive, Cell-Penetrating, Core/Shell Magnetite/Silver Nanoparticles for the Delivery of Plasmids: Preparation, Characterization, and Preliminary In Vitro Evaluation

Abstract

Over the past decade, gene therapies have attracted much attention for the development of treatments for various conditions, including cancer, neurodegenerative diseases, protein deficiencies, and autoimmune disorders. Despite the benefits of this approach, several challenges are yet to be solved to reach clinical implementation. Some of these challenges include low transfection rates, limited stability under physiological conditions, and low specificity towards the target cells. An avenue to overcome such issues is to deliver the therapies with the aid of potent cell-penetrating vectors. Non-viral vectors, such as nanostructured materials, have been successfully tested in drug and gene delivery. Here, we propose the development and in vitro evaluation of a nanostructured cell-penetrating vehicle based on core/shell, magnetite/silver nanoparticles. A subsequent conjugation of a pH-responsive polymer was used to assure that the vehicle can carry and release circular DNA. Additionally, the translocating peptide Buforin II was conjugated with the aid of a polyether amine polymer to facilitate translocation and endosome escape. The obtained nanobioconjugates (magnetite/silver-pDMAEMA-PEA-BUFII) were characterized by UV-Vis spectrophotometry, dynamic light scattering (DLS), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), scanning electron microscope equipped with energy dispersive spectroscopy (SEM+EDS), and transmission electron microscopy (TEM). They were also encapsulated in lecithin liposomes to form magnetoliposomes. The cell viability of Vero cells in the presence of the nanobioconjugates was above 95% and declined to 80% for the magnetoliposomes. The hemolytic tendency of nanobioconjugates and magnetoliposomes was below 10%, while the platelet aggregation approached that of the negative control (i.e., 35%). Cytoplasm coverage values of about 50% for both Vero and neuroblastoma cells confirmed significant cell penetration. Pearson’s correlation coefficients for both cell lines allowed us to estimate 20–40% colocalization of the nanobioconjugates with lysotracker green, which implied high levels of endosomal escape. The developed vehicles were also capable of loading around 16% of the added DNA and releasing such cargo with 8% efficiency. The developed nanoplatform holds a significant promise to enable highly efficient gene therapies as it overcomes some of the major issues associated with their eventual translation to the pre-clinical and clinical scale.

Tailoring Iron Oxide Nanoparticles for Efficient Cellular Internalization and Endosomal Escape

“…After one hour of exposure, approximately 75% of the nanobioconjugates were internalized and a colocalization with endosomal compartments of about 27% was observed, as calculated from the Pearson Correlation Coefficient. Similarly, BUF II conjugation to iron oxide/silver nanoparticles coated with (poly(2-dimethylamino)ethyl methacrylate) methyl chloride (pDMAEMA) and PEA, in our most recent work, demonstrated high internalization rates in neuroblastoma cells (SH-SY5Y) due to the combined action of BUF II and the polycationic coating pDMAEMA, as well as similar endosomal colocalization degree as previously synthesized nanobioconjugates [ 161 ]. PEG-coated IONs conjugated with BUF II and Frenatin 2.3S CPPs, independently, have also colocalized with endosomal compartments in lung carcinoma (A549), neuroblastoma (SH SY5Y), gastric adenocarcinoma (AGS) and breast adenocarcinoma (MDA) cells (unpublished results).…”

“…gH625-ION nanobioconjugates not only showed an effective pass of the blood-brain barrier but a homogeneous cytosolic accumulation in astrocytes and pericytes [ 266 ]. Moreover, Perillo and colleagues showed that gH625-PEG-IONs have high cytoplasmic distribution, making them good candidates for cancer theranostic treatments [ 161 ]. BUF II-conjugated IONs have also demonstrated great translocating abilities due to the high arginine content of BUF II, as we have shown in previous studies [ 131 , 132 ].…”

Patchy Core/Shell, Magnetite/Silver Nanoparticles via Green and Facile Synthesis: Routes to Assure Biocompatibility

“…Figure 3 A shows the characteristic spectra of silver nanoparticles; the absorbance between 300 and 400 cm −1 increased compared to bare magnetite [ 22 , 32 ]. Still, the magnetite spectrum is visible in the range below 300 cm −1 , indicating a discontinuous silver coverage [ 22 , 35 ]. Figure 3 B presents the results of the hydrodynamic radii.…”

“…Moreover, the stretching vibration at 1740 cm −1 corresponds to the carbonyl C=O group. At 1428 cm −1 , the deformational stretching vibration of the -N(CH 3 ) 2 also belongs to the pDMAEMA [ 35 , 43 ]. Silanization, in this case, is the process by which the APTES binds covalently to the magnetite core of the patchy nanoparticles [ 45 ].…”

“…The peaks at 1065 and 950 cm −1 are for the Si-O-Si (due to silanization), and Fe-O bonds can be attributed to the magnetite core, respectively [ 30 ]. The silane chemistries on the nanomaterials’ surface are widely used in the medical field for several applications ranging from delivering nucleic acids or detecting biological markers [ 35 , 39 ]. As previously discussed, one can highlight the importance of the purification process by evaluating DLS and UV-Vis results.…”