A series of linear poly(ethylenimine) (L-PEI) containing varying amounts of cationic charge density in its backbone was produced by controlled hydrolysis of poly(2-ethyl-2-oxazoline) (PEtOz) for using as a nonviral DNA transfection agent. The effects of cationic charge density and molecular weight of the L-PEI on the cytotoxicity and transfection efficiency were studied. Highly compacted L-PEI/DNA complex (~150 nm) was obtained with a surface charge value of around +28.4 mV. Cell cytotoxicity was affected to a great extent by the hydrolysis percent of L-PEI as well as by the molecular weight. Transfection efficiency of luciferase plasmid DNA against NIH 3T3 fibroblast was largely dependent upon the hydrolysis percent (charge density) in the polymer backbone and the molecular weight of the L-PEI, but independent of the total amount of cationic charges used for DNA condensation.
Poly(lactic-co-glycolic acid) [PLGA] grafted poly(L-lysine) [PLL] (PLL-g-PLGA) was synthesized to demonstrate its micelle-forming property in an aqueous solution. The micelles were used as a gene delivery carrier. The hydrodynamic diameter of PLL-g-PLGA micelles in an aqueous solution was ca. 149 nm with a narrow size distribution. Critical micelle concentration (cmc) was 9.6 mg/L. The PLL-g-PLGA micelles could be used to produce compact nanoparticulate complexes with plasmid DNA, which could efficiently protect the complexed DNA from enzymatic degradation by DNase I. The micelle/DNA complexes had highly compacted structure sized between 200 - 300 nm with a positive surface charge value. The PLL-g-PLGA micelles exhibited much higher transfection efficiency with lower cytotoxicity than PLL.
Biodegradable poly(D,L-lactic-co-glycolic acid) [PLGA] was chemically conjugated to oligonucleotide (ODN) to form an amphiphatic structure which is similar to an A-B type block copolymer. The ODN/PLGA conjugates self-assembled in aqueous solution to form a micellar structure by serving PLGA segments as a hydrophobic core and ODN segments as a surrounding hydrophilic corona. These micelles could release ODN in a sustained manner by controlled degradation of hydrophobic PLGA chains. Compared to unconjugated ODN, the ODN/PLGA micelles could be more efficiently transported within cells, presumably by endocytosis. This study proposes a potential delivery method of ODN into cells by forming hybrid ODN/PLGA micelles.
An anti-sense oligodeoxynucleotide (ODN), c-myb, was covalently conjugated to poly(ethylene glycol) (PEG) via an acid-cleavable phosphoramidate linkage to form a di-block copolymer-like structure. The phosphoramidate linkage between ODN and PEG was completely cleaved within 5 h in an endosomal acidic condition (pH 4.7). When complexed with a cationic fusogenic peptide, KALA, the ODN/PEG conjugate self-associate to form polyelectrolyte complex micelles in an aqueous solution. The anionic ODN segments were ionically interacted with cationic KALA peptide to form an inner polyelectrolyte complex core, while the PEG segments constituted a surrounding corona. Effective hydrodynamic volume of the micelles was ca. 70 nm with a very narrow size distribution. The polyelectrolyte complex micelles composed of c-myb ODN-PEG conjugate and KALA were transported into cells far more efficiently than c-myb ODN itself. They also exhibited superior anti-proliferative activity against smooth muscle cells. This study demonstrates the DNA/PEG hybrid micelles system can be applied for the delivery of antisense oligonucleotide.
The conjugate of anti-sense c-raf oligonucleotide (ODN) and poly(ethyleneglycol) (PEG) was synthesized for intracellular ODN delivery in the form of polyelectrolyte complex micelles. The ODN-PEG conjugate self-associated to form polyelectrolyte complex micelles in an aqueous solution when complexed with polyethylenimine (PEI). Effective hydrodynamic diameter of the micelles was ca. 70 nm with a narrow size distribution. Flow cytometry analysis indicated that the cellular uptake of the micelles within A2780 cell was much higher than that of ODN alone. They also showed a superior anti-proliferative activity against ovarian cancer cells in vitro as well as in vivo.
유전자 치료를 위하여 고분자 전달체를 사용하는 데에 있어 전달체 고분자의 세포독성은 사용 전에 반드시 고려해야 할 사항이다. 고분자 전달체의 양이온 밀도와 분자량이 transfection 효율과 세포생존에 미치는 영향을 알아보기 위해 poly(2-ethyl-2-oxazoline)를 제어적 산 가수분해를 통해 여러 양이온 밀도와 분자량이 다른 선형의 poly(ethylenimine)을 합성하였다. 합성 고분자는 DNA와 상호작용하여 150 nm 정도의 크기와 +28.4 mV의 표면전하를 지니는 나노입자를 형성함을 관찰하였다. 고분자의 양이온 밀도와 분자량이 커질수록 세포독성이 증가하였으며, transfection 효율 또한 DNA와의 상호작용에 필요한 양이온의 수에 상관없이 양이온 밀도와 분자량의 영향을 크게 받는 것을 알 수 있었다. 따라서 효율적인 transfection을 위해서는 세포독성과 transfection간의 적절한 균형을 맞추기 위해 고분자의 양이온 밀도와 분자량을 고려해야 함을 알 수 있었다. 수용액상에서 미셀형태를 지니는 유전자 전달용 양이온성 미셀을 poly(lactic-co-glycolic acid) [PLGA]을 poly(L-lysine) [PLL]에 그라프트형태로 접합하여 합성하였다. 이 합성고분자는 9.6 mg/L 정도의 낮은 미셀형성농도 (critical micelle concentration, cmc)를 지니며 일반적으로 많이 사용이 되어온 고분자인 PLL 보다 낮은 세포독성과 높은 transfection 효율을 나타냄을 관찰하였다.
안티센스 올리고뉴클레오티드의 세포 내로의 효율적인 전달을 위해 i) 소수성 고분자인 PLGA와의 접합체를 합성하여 미셀형태로 올리고뉴클레오티드를 전달할 수 있는 시스템과 ii) 올리고뉴클레오티드와 친수성 고분자인 polyethyleneglycol (PEG)의 접합체를 합성하여 이를 양이온성 펩타이드 또는 양이온서 고분자와 수용액상에서 상호 작용시킴으로써 고이온 복합체 미셀을 형성함으로써 올리고뉴클레오티드의 세포 내 전달을 향상시킬 수 있는 전달시스템을 제작하였다. PLGA-올리고뉴클레오티드 접합체의 경우 수용액상에서 70 nm 정도의 작은 크기의 미셀을 형성하였으며 일반 올리고뉴클레오티드에 비해 현격히 높은 세포 내 전달효율을 나타내었다. 안티센스 c-myb과 c-raf 올리고뉴클레오티드를 사용하여 PEG-올리고뉴클레오티드 접합체를 합성하고 fusogenic 펩타이드인 KALA 또는 PEI 와 상호작용시켜 고이온 복합체 미셀을 형성하여 세포에 적용한 경우, 세포 내 전달효율의 향상을 관찰할 수 있었다. 상기의 고이온 복합체 미셀형태의 formulation을 통해, 안티센스 c-myb을 포함한 formulation은 평활근 세포의 증식을 효율적으로 억제할 수 있었으며, 안티센스 c-raf를 포함한 formulation은 암세포의 증식을 효율적으로 억제할 수 있음을 관찰하였다. 또한, 안티센스 c-raf를 포함한 formulation을 암세포 동물모델에 적용하였을 때 효율적인 암 증식저해를 관찰할 수 있었다.
