For the efficient and specific delivery of nucleic acid drugs, reducible nanoparticle systems and surface engineered nanoparticle systems were investigated. Reducible PEG nanogels via thiol-crosslinking encapsulating plasmid DNA were prepared for the specific delivery of plasmid DNA only in environmental reductive condition, such as cytoplasm. DNA was dissolved in selected organic solvents in the presence of poly(ethylene glycol) (PEG). Nano-scale PEG/DNA complex (~100 nm) was produced in dimethylsulfoxide (DMSO) phase. Using a thiol-functionalized six-arm branched PEG for DNA solubilization, the PEG/DNA nanocomplex was crosslinked through the formation of disulfide linkages between the thiol groups, resulting in the production of stable PEG/DNA nanogels in aqueous solution. DNA release from the nanogels could be modulated by changing the concentration of an external reducing agent. While the released plasmid DNA from the nanogels maintained intact structural integrity, the transfection efficiency by PEG nanogels was lower than that by conventional cationic carriers such as PEI and $lipofectamine^{TM}$.
To enhance transfection efficiency, reducible polyelectrolyte nanocomplexes cleavable in reductive condition were prepared. Antisense oligodeoxynucleotide (ODN) was covalently conjugated to hyaluronic acid (HA) via a reducible di-sulfide linkage, and the HA-ODN conjugate was complexed with protamine to increase the extent of cellular uptake and enhance the gene inhibition efficiency of GFP expression. The HA-ODN conjugate formed more stable polyelectrolyte complexes with protamine as compared to naked ODN, probably due to its increased charge density. As alternative approach, self-crosslinked and reducible peptide was synthesized for stable formation of nanoscale complexes with an siRNA-PEG conjugate to enhance transfection efficiency in serum containing condition without compromising cytotoxicity. A fusogenic peptide, KALA, with two cysteine residues at both terminal ends was crosslinked via disulfide linkages under mild DMSO oxidation condition. The reducible crosslinked KALA (cl-KALA) was used to form nano-complexes with green fluorescent protein (GFP) siRNA. cl-KALA exhibited more reduced cell cytotoxicity and formed more stable and compact polyelectrolyte complexes with siRNA, compared to naked KALA and polyethylenimine (PEI), probably due to its increased charge density. Protamine/HA-ODN complexes and cl-KALA/siRNA complexes showed comparable gene silencing efficiency to those of cytotoxic PEI. Since HA, KALA peptide, and protamine are biocompatible materials, the current formulation based on a cleavable conjugation strategy could be potentially applied as safe and effective nonviral carriers for ODN and siRNA nucleic acid therapeutics.
Surface modified nanoparticles were investigated for the targeted intracellular delivery of them in an acidic pH-specific manner for tumor cells. Quantum dot (QD) and adenovirus (ADV) nanoparticles were surfaced modified with graft copolymers that exhibited a charge reversal behavior under acidic condition. The surface of streptavidin conjugated Qdot was modified in a quantitative manner with biotin-PEG-grafted poly-L-lysine (biotin-PEG-PLL). The primary amine groups in PLL were post-modified using citraconic anhydride, a pH-sensitive primary amine blocker, to generate carboxylate groups, producing net negatively charged QD nanoparticles. Under acid conditions, citraconylated amide linkages cleaved, resulted in the recovery of positively charged amine groups with subsequently altering surface charge values. Intracellular delivery of QD nanoparticles was greatly enhanced in an acid pH condition due to the surface charge reversal. The expression extent of GFP by surface engineered virus was significantly increased at more acidic pH than pH 7.4.
The surface of streptavidin conjugated quantum-dot (QD) nanoparticles was immobilized with a PEGylated and biotinylated peptide containing a specific matrix metalloproteinase-2 (MMP-2) cleavable sequence (biotin-mmp2-PEG conjugate). The conjugate was immobilized onto the surface of QD in a quantitative manner via biotin-streptavidin interaction. To facilitate cellular uptake of QD (~ 12 nm), biotinylated cell penetrating peptide (biotin-CPP) was bound onto the QD prior to biotin-mmp2-PEG coating. MMP-2 enzyme specific dePEGylation on the QD was confirmed by fluorescence resonance energy transfer (FRET)-based QD quenching effect under various enzyme concentrations. With increasing the number of immobilized PEG (MW: 3400) chains per QD up to 18, the intracellular uptake percent of QD was gradually reduced to ~ 20 %, compared to that of the control biotin-CPP modified QD. However, upon an exposure of increasing amount of MMP-2 enzyme, QD particles were taken up by cells to a greater extent, resulting from the dePEGylation effect with concomitant re-surfacing of CPP on the QD. The effect of dePEGylation on the cellular uptake of CPP-QD was quantitatively analyzed. These studies demonstrate that various nano-sized particles encapsulating nucleic acid drugs could be similarly surface engineered to enhance their cellular uptake specifically at a low pH microenviromnent and a MMP-2 enzyme sufficient environment like solid tumor tissue.
최근 유전자 약물을 이용한 난치성 질환 치료에 대한 관심이 증가됨에 따라 효율적으로 유전자 약물을 전달하는 약물전달체계의 개발이 요구되고 있다. 이러한 약물전달체계는 유전자 약물을 세포내로 효율적으로 전달할 수 있어야 하고, 특이적으로 원하는 세포에만 전달되도록 하여 약물의 부작용을 낮추도록 해야 한다. 나노 입자를 이용하여 유전자 약물을 전달할 경우, 외부 자극으로부터 유전자 약물을 보호하여 유전자 약물의 안정성을 높이고, 세포내로의 도입이 용이하며, EPR효과로 인해 암 조직으로 선택적으로 전달된다는 장점을 가지고 있다.
본 연구에서는 이러한 장점을 가진 나노 입자에 다양한 외부 환경 민감성 인자를 도입함으로 봉입된 유전자 약물의 전달 효율을 높이고자 하였다. 첫번째로 황황다리결합을 이용한 나노 입자의 제조를 통해 외부 환원 조건에 선택적으로 유전자를 방출하는 나노 입자를 제조하였다. 이는 높은 환원 조건이 유지되는 세포질내로 유전자를 선택적으로 전달할 수 있도록 할 것으로 생각된다. 또한, 황황다리결합을 이용하여 유전자에 음전하를 띤 히아루론산을 도입하거나, 양이온성 펩타이드를 연결하여 고분자량의 양이온성 전달체를 제조함으로 전하밀도를 높여서 유전자 전달 효율을 높이고자 하였다.
나노 입자가 선택적으로 타겟 세포에만 전달되도록 하기 위해, 나노 입자의 표면을 pH 민감성 고분자나 외부 효소 민감성 고분자로 코팅하여 나노 입자가 외부 자극에 선택적으로 세포내로 도입되도록 하였다. 약산성 pH 조건과 MMP-2 효소가 충분한 환경은 암조직에서 발견되는 특징이므로, 이러한 자극 조건에서만 세포내로 도입되도록 한 나노 입자는 다양한 유전자 약물들을 암세포에 선택적이고 효율적으로 전달할 수 있는 수송체로 사용될 수 있을 것으로 생각된다.
