Data on synthesis and characterization of chitosan nanoparticles for in vivo delivery of siRNA-Npr3: Targeting NPR-C expression in the heart

This data article contains the data related to the research article ‘Transient silencing of Npr3 gene expression improved the circulatory levels of atrial natriuretic peptides and attenuated β-adrenoceptor activation-induced cardiac hypertrophic growth in experimental rats’ (Venkatesan et al., 2016 [1]). The siRNA-Npr3 loaded chitosan nanoparticles were synthesized using ionotropic gelation method, where the positive charge of the chitosan interacts with the negative charge of STPP and siRNA-Npr3. The physicochemical properties of the synthesized siRNA-Npr3 loaded chitosan nanoparticles were studied by dynamic light scattering, FE-SEM and HR-TEM analysis. In addition, the loading efficiency and stability of the nanoparticles were also studied. Further, the gene silencing efficacy, hemocompatibility and biocompatibility were studied using Wistar rats (in vivo), isolated red blood cells and H9c2 cardiomyoblast cells, respectively.


Subject area Biology
More specific subject area Nanotechnology, Molecular biology.
Type of data Figure This data validated the biocompatibility, hemocompatibility and gene silencing efficacy of siRNA-Npr3 loaded chitosan nanoparticles in in vitro and in vivo model system.
This method of siRNA-Npr3 loaded chitosan nanoparticles can be utilized as a drug delivery vehicle for in vitro and in vivo applications.

Data
The data provided here displays the synthesis, characterization, biocompatibility and hemocompatibility of siRNA-Npr3 loaded nanoparticles. Further, the gene silencing efficacy of the synthesized siRNA-Npr3 nanoparticles was demonstrated in the H9c2 cells in vitro and in rat hearts in vivo.

Materials
Chitosan (75-85% of deacetylation, low molecular weight), Npr3 specific siRNA - Target  [dT]-3 0 (CAT# SASI_Rn02_00260355) and sodium tripolyphosphate were procured from Sigma-Aldrich, USA. Dulbecco's Modified Eagle's Medium (DMEM), Trypsin-EDTA, fetal bovine serum (FBS) and antibiotic antimycotic solution were purchased from HiMedia, India. cDNA conversion kit was procured from Thermo scientific, USA. Red dye PCR master mix was purchased from Merck Millipore, German. Gene specific primers were purchased from Eurofins Scientific, Luxembourg. Primary antibody for NPR-C and HRP labeled secondary antibody were purchased from Santa Cruz biotechnology, USA.

Synthesis and characterization of siRNA-Npr3 loaded chitosan nanoparticles
The siRNA-Npr3 loaded chitosan nanoparticles were synthesized by mixing chitosan solution (1 mg/ml chitosan in 0.2 M sodium acetate buffer, pH 4.5) to a mixture containing STPP solution (2.5 mg/ml) and siRNA-Npr3 at 5:1 weight ratio of chitosan to STPP and 50:1 N:P ratio of chitosan and siRNA-Npr3. The contents were mixed and vortexed for 1 min on a vortex mixer and kept undisturbed for 30 min [2]. The synthesized nanoparticles were purified by centrifuging at 31,000g for 20 min. Further, the nanoparticles were washed with ultrapure DNase/RNase free water and centrifugation was repeated. Fig. 1A shows the particle size analysis (DLS -Malvern Nano ZS, UK) of synthesized nanoparticles, which revealed that the hydrodynamic size of the siRNA-Npr3 nanoparticles were in the range of 220 717.5 nm. From the zeta potential analysis, the nanoparticles were found to possess a surface charge of þ16.2 71.2 mV. It is evidenced from the FE-SEM - Fig. 1B (Hitachi SU6600, Germany) and HR-TEMinsert in Fig. 1B (FEI TECNAI G2) analysis of the nanoparticles that the synthesized particles were of fairly spherical in shape and evenly distributed.

Quantification of siRNA-Npr3 loading efficiency
The loading efficiency of the siRNA-Npr3 in the siRNA-Npr3 loaded chitosan nanoparticles were analyzed using UV-Vis spectrophotometer (Shimadzu UV 150-02, Japan) by comparing the A 260 of the supernatant solution obtained after the synthesis of siRNA-Npr3 loaded chitosan nanoparticles and the naked siRNA-Npr3 [3]. The loading efficiency was calculated by using the following formula: Loading efficiency ¼A 260 nm of siRNA present in the supernatant/A 260 nm of total amount of siRNA added for nanoparticles preparation*100 and the siRNA-Npr3 loading efficiency were observed to 100% (Fig. 1C).

Gel retardation assay
The stability and interaction strength between the chitosan and siRNA-Npr3 in the nanoparticles was carried out by gel retardation assay. Briefly, equal concentration of naked siRNA-Npr3, chitosan nanoparticles and siRNA-Npr3 loaded chitosan nanoparticles were loaded on different wells of 4% agarose gel for analyzing the stability of the nanoparticles [3]. The gel retardation assay showed that the nanoparticles loaded with siRNA-Npr3 gets retarded in the well as evidenced by the retarded movement of the nanoparticles, while the naked siRNA-Npr3 freely resolved in the agarose gel (Fig. 1D).

Biocompatibility of siRNA-Npr3 loaded chitosan nanoparticles
To assess the biocompatibility of the nanoparticles, MTT assay was carried out on H9c2 cell line. Briefly, 5000 cells/well were seeded in 96 well plate and maintained in 10% FBS containing DMEM for 24 h. After 24 h, the cells were treated with either chitosan nanoparticles or siRNA-Npr3 loaded chitosan nanoparticles or chitosan scrambled siRNA nanoparticles in serum free media for 48 h. Later, the media was removed and 10 ml of MTT solution (5 mg/ml) was added to each well and incubated for 4 h at dark. After the reaction, the MTT was removed and 100 ml of DMSO was added to all the wells. The A 570 was read by microplate reader [4]. Fig. 2A shows the results of the biocompatibility assay, where none of the nanoparticles (chitosan, siRNA-Npr3 loaded chitosan nanoparticles, and chitosan scrambled nanoparticles) tested exhibits cytotoxicity against H9c2 cells.

Hemolytic activity
The hemolytic activity of the nanoparticles was tested on isolated rat erythrocytes [5]. Briefly, the whole blood was processed and erythrocytes were collected in a vial. 5% v/v erythrocytes in PBS were distributed to each tube and the volume of the tube was made up to 1 ml with nanoparticles samples (chitosan nanoparticles or siRNA-Npr3 loaded chitosan nanoparticles or chitosan scrambled siRNA nanoparticles) and PBS. Erythrocytes treated with 1% triton X 100 served as the positive control and erythrocytes in PBS served as negative control. The reaction mixture was incubated for 1 h at 37°C in shaking condition. Then the tubes were centrifuged at 190g and the absorbance of the supernatant was measured at 540 nm. The percentage of hemolysis ¼[(A 540 nm in siRNA-Npr3 loaded chitosan nanoparticles supernatant solution -A 540 nm in PBS)/(A 540 in 1% Triton X-100 -A 540 in PBS)] Â 100. Fig. 2B, shows the results of the hemolytic activity assay. The nanoparticles (150 mg/ml) were tested individually on rat erythrocytes and found to be exhibit least toxicity as per ASTM standard which can be considered as compatible [6].

Validation the gene silencing efficacy of siRNA-Npr3 loaded chitosan nanoparticles
The in vivo gene silencing efficacy and effective dosage fixation of siRNA-Npr3loaded chitosan nanoparticles were performed by intramyocardial injection of the nanoparticles. Briefly, 2.5 or 5 mg of siRNA-Npr3 containing nanoparticles/kg body weight was administered to the hypertrophied Wistar rats. At the end of the experiment, the heart tissue was harvested and processed as described earlier [1]. Fig. 2C and D shows the Western blotting and densitometric analysis of NPR-C protein expression on the control and experimental group of rat hearts, where a 4 fold increase in the NPR-C protein expression was observed in the isoproterenol treated rat hearts, and treatment with 2.5 or 5 mg of siRNA-Npr3 decreased the expression of NPR-C by 45% and 70% respectively.