Nano-biomimetic carriers are implicated in mechanistic evaluation of intracellular gene delivery

Several tissue specific non-viral carriers have been developed for gene delivery purposes. However, the inability to escape endosomes, undermines the efficacy of these carriers. Researchers inspired by HIV and influenza virus, have randomly used Gp41 and H5WYG fusogenic peptides in several gene delivery systems without any rational preference. Here for the first time, we have genetically engineered two Nano-biomimetic carriers composed of either HWYG (HNH) or Gp41 (GNH) that precisely provide identical conditions for the study and evaluation of these fusogenic peptides. The luciferase assay demonstrated a two-fold higher transfection efficiency of HNH compared to GNH. These nanocarriers also displayed equivalent properties in terms of DNA binding ability and DNA protection against serum nucleases and formed similar nanoparticles in terms of surface charge and size. Interestingly, hemolysis and cellular analysis demonstrated both of nanoparticles internalized into cells in similar rate and escaped from endosome with different efficiency. Furthermore, the structural analysis revealed the mechanisms responsible for the superior endosomal escaping capability of H5WYG. In conclusion, this study describes the rationale for using H5WYG peptide to deliver nucleic acids and suggests that using nano-biomimetic carriers to screen different endosomal release peptides, improves gene delivery significantly.


Desalting, dialysis and determining the concentration
The purified carriers were desalted prior to cellular and structural assays. Briefly, the carrier solutions were dialyzed against phosphate buffered saline (PBS; pH 7.4) or acetate buffer (pH 5.4) at 4 °C for 48 h using a dialysis tubing (Sigma-Aldrich, MWCO 2kDa). The final concentrations of carriers were determined by measuring the UV absorption at 280 nm using a UV-vis spectrophotometer (Biochrom, Cambridge-UK). Carrier stocks were also prepared by addition of glycerol to 10% final concentrations and stored at -20 °C.

Serum stability
Serum stability analysis was performed to evaluate if nanocarriers are protected against serum nucleases. First, HNH and GNH nanocarriers were complexed with 0.5 µg plasmid at N/P ratio of 8, as described above. Then, fetal bovine serum (Invitrogen, CA, USA) at a final concentration of 10 % (v/v) was added to the two batches of nanoparticles and incubated for 1h at 37 °C.
Subsequently, SDS was added to the final concentration of 10% to release DNA from nanocarriers.
The nanoparticles were then electrophoresed on a agarose gel (1%) and plasmid mobility was visualized by ethidium bromide staining and UV illumination.

Transmission electron microscopy:
The HNH and GNH nanocarriers in complex with pGL3 plasmid at N/P ratio of 10 were drop-cast carefully onto a carbon-supported copper grids. The grids were dried in air for 20 min and imaged using Transmission electron microscope (TEM, Zeiss -EM10C -80 kV).
Atomic Force Microscopy :For observing topography of nanocarriers, 20 μl of HNH and GNH were deposited on mica and spray-dried. The images were acquired on a scanning probe microscope SPM (Veeco Instruments, Sunnyvale, CA, USA) using a Si cantilever in the tapping mode. Minimum image processing was employed and image analysis was done using Picoview software.

FITC labeling of nanocarriers
The HNH and GNH nanocarriers were labeled with Fluorescein isothiocyanate (FITC) according to manufacturer's protocol (Sigma-Aldrich, Wisconsin, USA). Briefly, FITC was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 1 mg/ml. The 50 µl of this solution was added into 950 µl of each nanocarrier with concentration of 0.5 mg/ml. The mixtures were then vortexed at 37 °C for 2 h and centrifuged at 12000 rpm for additional 2 min. The unbound FITC was separated by dialysis against PBS overnight at 4 °C. Finally, FITC labeled nanocarriers were store at -20 °C.

Modelling of nanocarriers structures
The three-dimensional structure of nanocarriers were predicted by I-Tasser server 53 . Briefly, the primary sequence of peptide based nanocarriers were deposited on I-Tasser web page. Using this server, the matched regions of sequences were modeled first, based on its similar PDB structure, and then the unmatched regions were produced with an ab initio modeling calculation. The server produced five models, and the best model was selected based its c-score. Finally, the PyMol software was used to illustrate the 3D structure of models (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrodinger, LLC).

Fluorescence characterization
The intrinsic fluorescence of HNH and GNH carriers were measured using a Spectrofluorometer (Perkin-Elmer, U.K). The emission spectra of carriers prepared at different pH in PBS and acetate buffers, were recorded over the wavelength ranges of 300-450 nm upon excitation of the tryptophan residues at 295 nm. Furthermore, hydrophobic patches of carriers were investigated by addition of a fluorescence probe, 8-Anilino-1-naphthalenesulfonic acid (ANS; Sigma-Aldrich).
The HNH and GNH nanocarriers at 30 µM concentration was incubated with the ANS at 1:30 molar ratio. After 5 min incubation, the emission spectra were recorded between 400 and 700 nm upon excitation at 350 nm.