Improved Gene Transfer with Functionalized Hollow Mesoporous Silica Nanoparticles of Reduced Cytotoxicity

Abstract

Gene therapy is a promising strategy for treatment of genetically caused diseases. Successful gene delivery requires an efficient carrier to transfer the desired gene into host cells. Recently, mesoporous silica nanoparticles (MSNs) functionalized with 25 kD polyethyleneimine (PEI) were extensively used as gene delivery carriers. However, 25 kD PEI could significantly reduce the safety of the modified MSNs although it is efficient for intracellular delivery of nucleic acids. In addition, limited drug loading remains a challenge for conventional MSNs drug carriers. Hollow mesoporous silica nanoparticles (HMSNs) with high pore volume, tunable pore size, and excellent biocompatibility are attractive alternatives. To make them more efficient, a less toxic 1.8 kD PEI polymer was used to functionalize the HMSNs which have large pore size (~10 nm) and form PEI-HMSNs. Scanning and transmission electron microscopic images showed that HMSNs were spherical in shape and approximately 270 nm in diameter with uniform hollow nanostructures. The maximum loading capacity of green fluorescent protein labeled DNA (GFP-DNA) in PEI-HMSNs was found to be 37.98 mg/g. The loading capacity of PEI-HMSNs was nearly three-fold higher than those of PEI modified solid nanoparticles, indicating that both hollow and large pores contributed to the increase in DNA adsorption. The transfection of GFP-DNA plasmid loaded in PEI-HMSNs was increased two-fold in comparison to that of 25 kD PEI. MTT assays in Lovo cells showed that the cell viability was more than 85% when the concentration of PEI-HMSNs was 120 µg/mL, whereas the cell viability was less than 20% when the 25 kD PEI was used at the same concentration. These results indicated that PEI-HMSNs could be used as a delivery system for nucleic acids due to good biocompatibility, high gene loading capacity, and enhanced gene transfer efficiency.

Keywords: PEI; cytotoxicity; gene transfer; hollow mesoporous silica nanoparticles.

Figure 1

The morphology of nanoparticles. (A,B): SEM images of HMSNs; (C,D): TEM images of HMSNs; (E,F): TEM images of as-synthesized HMSNs without removing the template.

Figure 2

Characterization of porous structure and surface charge. (A): Nitrogen adsorption/desorption isotherms of HMSNs; (B): Pore size distribution in HMSNs; (C): Zeta potentials of HMSNs and PEI-HMSNs.

Figure 3

The DNA adsorption by PEI-HMSNs. (A): DNA adsorption isotherms by PEI-HMSNs with various PEI-HMSNs/DNA weight ratios and PNHMSNs/DNA at WR60; (B): Total DNA (µg/mL) in the sediment after incubating DNA with PEI-HMSNs and PHMSNs/DNA at WR60 was calculated by subtracting DNA in the supernatant determined at different time intervals from initial total DNA added.

Figure 4

Cell viabilities of Lovo cells upon incubation with (A): 1.8 kD PEI; (B): HMSNs; (C): 25 kD PEI; and (D): PEI-HMSNs for 24 h, respectively. The number of independent determinations was at least three.

Figure 5

In vitro transfection efficiency of Lovo cells. The WR of PEI-HMSNs to DNA was fixed at 60 and the number of independent determinations was at least three (**, *** p < 0.05).

Figure 6

Confocal microscopy images of Lovo cells incubated with RHO-DNA and FITC-PEI-HMSNs for 4 h at 37 °C. Cell nuclei were stained blue with DAPI, RHO-DNA were stained red with rhodamine phalloidin, and FITC was shown as green fluorescence.