Novel targeted siRNA-loaded hybrid nanoparticles: preparation, characterization and in vitro evaluation

Abstract

Background: siRNAs have a high potential for silencing critical molecular pathways that are pathogenic. Nevertheless, their clinical application has been limited by a lack of effective and safe nanotechnology-based delivery system that allows a controlled and safe transfection to cytosol of targeted cells without the associated adverse effects. Our group recently reported a very effective and safe hybrid nanoparticle delivery system composing human IgG and poloxamer-188 for siRNA delivery to cancer cells. However, these nanoparticles need to be optimized in terms of particle size, loading capacity and encapsulation efficiency. In the present study, we explored the effects of certain production parameters on particle size, loading capacity and encapsulation efficiency. Further, to make these nanoparticles more specific in their delivery of siRNA, we conjugated anti-NTSR1-mAb to the surface of these nanoparticles to target NTSR1-overexpressing cancer cells. The mechanism of siRNA release from these antiNTSR1-mAb functionalized nanoparticles was also elucidated.

Results: It was demonstrated that the concentration of human IgG in the starting nanoprecipitation medium and the rotation speed of the magnetic stirrer influenced the encapsulation efficiency, loading capacity and the size of the nanoparticles produced. We also successfully transformed these nanoparticles into actively targeted nanoparticles by functionalizing with anti-NTSR1-mAb to specifically target NTSR1-overexpressing cancer cells, hence able to avoid undesired accumulation in normal cells. The mechanism of siRNA release from these nanoparticles was elucidated to be by Fickian diffusion. Using flow cytometry and fluorescence microscopy, we were able to confirm the active involvement of NTSR1 in the uptake of these anti-NTSR1-mAb functionalized hybrid nanoparticles by lung adenocarcinoma cells.

Conclusions: This hybrid nanoparticle delivery system can be used as a platform technology for intracellular delivery of siRNAs to NTSR1-overexpressing tumor cells.

Fig. 1

Diagrammatic representation and TEM micrograph of the produced nanoparticle. a Nanoparticles at lower magnification. b Nanoparticles at higher magnification to show the internal arrangement of the components

Fig. 2

-FI-IR spectra showing the conjugation of anti-NTSR1-mAb to hybrid nanoparticles (a) incomparison to non-functionalized nanoparticles (b)

Fig. 3

Comparison of in vitro siRNA release profile from siRNA encapsulated anti-NTSR1-mAb functionalized and non-functionalized nanoparticles at pH 5 and 7. siRNA was more efficiently released at pH 5 due to the superior solubility of human IgG at that pH. Functionalization of the nanoparticles did not seem to affect the release rate at each of the pH values. n = 3 for each sample point

Fig. 4

Reverse transcriptase PCR showing the expression of neurotensin receptor 1 (NTSR1) in A549 and H23 cell lines

Fig. 5

Fluorescence micrograph showing the delivery of siGLO into the cytosol of A549 cells using the NTSR1-mAb-functionalized hybrid nanoparticles. The upper panel shows the inhibition of siGLO delivery following an initial treatment of the cells with neurotensin

Fig. 6

Fluorescence micrograph showing the delivery of siGLO into the cytosol of H23 cells using the NTSR1-mAb-functionalized hybrid nanoparticles. The upper panel shows the inhibition of siGLO delivery following an initial treatment of the cells with neurotensin

Fig. 7

Probing the effect of inhibition of neurotensin receptor 1 (NTSR1) with neurotensin on the internalization of siRNA-loaded targeted hybrid nanoparticles in A549 and H23 cells using flow cytometry (n = 3)

Fig. 8

Reaction schemes for the attachment of anti-NTSR1-mAb to hybrid nanoparticles