Smart Titanium Dioxide Nanocomposites for Cellular Delivery of the Antisense Peptide Nucleic Acids

摘要

Peptide nucleic acids (PNAs) are one of the promising antisense drugs for the gene therapy. PNAs are DNA mimics with the peptide-like backbone [1]. PNAs form the most stable complementary duplexes with RNA among all known modified and non-modified oligonucleotides and have high exo- and endonuclease resistance [2, 3]. However, PNAs themselves cannot penetrate through cellular membranes [4]. Here we propose to use nanocomposites based on titanium dioxide (TiO_2) nanoparticles for the delivery of PNAs into living cells.After delivery of antisense PNA drugs into the cells there is still a problem with PNA molecules release from nanocomposite vehicles in cytoplasm inside of cells. Here we designed special nanocomposites on the basis of TiO_2 nanoparticles (4-6 nm in diameter) containing the hybrid PNADNA duplexes. The attachment of PNADNA duplexes to nanoparticles preliminary covered with polylysine (PL) occurs due to the electrostatic interaction between negatively charged internucleotide phosphate groups of DNA and positively charged amino groups of polylysine. It was demonstrated that the resulted TiO_2/PL/DNA/PNA nanocomposite can be delivered into the cells. Dissociation or release time of PNA from the DNA/PNA duplex attached to the nanocomposite was measured and easily could be regulated with the length of the overlap of complementary base pairs in the initially constructed DNA/PNA duplexes in nanocomposite TiO_2/PL/DNA/PNA. Thus, PNA/DNA complex with optimal overlap number or optimal half-release time of PNA from nanocomposite allows at least the half quantity of PNA still to remain in TiO_2/PL/DNA/PNA nanocomposite during the delivery and then able to be released from the nanocomposite after its delivery inside of the cells. These features of the constructed nanocomposites were demonstrated on the example of nanocomposites containing various overlap length of DNA/PNA duplexes (from 10 to 16 base pairs). It was found that the time of half-release of PNA from these TiO_2/PL/DNA/PNA nanocomposites changed from 10 to 70 min depended on overlap length of DNA/PNA duplexes in conditions close to physiological (35℃ in PBS buffer, pH 7.5). The greater the overlap of the DNA/PNA duplex in nanocomposite TiO_2/PL/DNA/PNA, the longer the time of PNA release from this nanocomposite. Thus, the adjustable reversible method of PNA immobilization on TiO_2 nanoparticles was developed. Created PNA containing nanocomposites were capable to penetrate cell membranes without transfectants or other adverse procedures.