Various biocompatible polymeric delivery systems were designed by covalently conjugating specific functionalities to poly(amino acid) derivatives to address intracellular delivery barriers. The physicochemical properties of synthesized polymeric systems in aqueous solution were characterized, and the cellular uptake and therapeutic effect was mainly evaluated.
In the first part of this work, oligoarginine ($Arg_8$) as a cell-penetrating peptide was conjugated to poly(2-hydroxytethyl aspartamide) (PHEA) via a thioether linkage to enhance the intracellular delivery of macromolecular delivery systems. The cellular uptake was analyzed by confocal laser scanning microscopy and flow cytometry. $PHEA-Arg_8$ conjugate was effectively localized in both fixed and unfixed HeLa cells. The studies at endocytosis inhibition conditions suggested that the internalization of $PHEA-Arg_8$ into cells was a temperature- and energy-dependent process and the colocalization with an endocytosis marker revealed an endocytosis was a key mechanism in the internalization of $PHEA-Arg_8$. The fluorescence spectra of $PHEA-Arg_8$ in liposome solutions showed $PHEA-Arg_8$ was collectively adsorbed in negative liposome membrane due to the high cationic property of conjugated $Arg_8$, representing a surface adsorption was a first step in the internalization of $PHEA-Arg_8$. The membrane leakage activity of $PHEA-Arg_8$ was much lower than that of $Arg_8$ own, meaning $PHEA-Arg_8$ does not effectively disrupt the cell membrane integrity. The uptake of polymer conjugates was increased with incubation time and reached saturation after several hours. The increase of the number of peptide conjugated to one polymer chain could increase the collective adsorption of polymer conjugates and enhance the cellular uptake by an adsorptive-endocytosis. Oligoarginine $(Arg_8)$ was also successfully conjugated to alkyl chain grafted poly(2-hydroxyethyl aspartamide) $(PHEA-C_{18}-Arg_8)$ via a thioether linkage to prepare nanoparticle systems. $PHEA-C_{18}-Arg_8$ formed stable self-aggregates of the size of around 50 nm and had a positive surface charge due to $Arg_8$ located in aggregate surface. The $Arg_8$ increased the adsorption of aggregates on cell membranes and $PHEA-C_{18}-Arg_8$ was effectively internalized into HeLa cells by an adsorptive-endocytosis. The $Arg_8$ also increased the cellular uptake of doxorubicin (DOX) loaded aggregates $(PHEA-C_{18}-Arg_8(DOX))$. $PHEA-C_{18}-Arg_8(DOX)$ was truly localized in cytoplasm and nucleus after only 1 h incubation, on the other hand, $PHEA-C_{18}$ (DOX) was only distributed in cytosolic compartment even after 12 h incubation. The cell cytotoxicity of $PHEA-C_{18}-Arg_8$ (DOX) was higher than that of $PHEA-C_{18}$ (DOX) in same concentration of DOX and showed a quick cytotoxic action in early incubation time because of the fast cellular internalization and nuclear access by the conjugated Arg8. As a prodrug system, methotrexate (MTX) was conjugated to $PHEA-Arg_8$ and the cell proliferation inhibition study revealed $PHEA-MTX-Arg_8$ was about several orders of magnitude more active than PHEA-MTX.
In the second part, histidine was directly conjugated to poly(2-hydroxethyl aspartamide) (PHEA) and $C_{18}$ -grafted PHEA $(PHEA-g-C_{18})$ via an ester linkage using Nα-Boc-L-histidine, followed by the deprotection of Boc groups. PHEA-His series could be expected as an endosomolytic synthetic polymer because of the buffering capacity at physiological and endosomal pH by α-amine and imidazole groups in side chains. $PHEA-g-C_{18}-His$ series formed stable self-aggregates due to the hydrophobic interaction between grafted alkyl chains and had a positive surface charge originated from histidine moieties in the aggregate surface. The size, zeta potential, and micropolarity of $PHEA-g-C_{18}-His$ series greatly increased at pH 5.0, because aggregates swelled by the electrostatic repulsion of ionized histidine moieties in the aggregate surface. The confocal microscopy image revealed that $PHEA-g-C_{18}-His$ was more uniformly distributed than $PHEA-g-C_{18}$ in HeLa cells after 8 h incubation due to the endosomolytic ability of conjugated histidine moieties. In doxorubicin-loaded self-aggregate systems, the histidine conjugation improved the cell cytotoxicity by the fast release of loaded doxorubicin at low pH and endosomolytic ability of conjugated histidine, resulting in the easy nuclear access of doxorubicin.
In the last part, folate-poly(2-hydroxethyl aspartamide) (PHEA-folate) conjugates were synthesized through an ester linkage between folic acid and PHEA to enhance the folate receptor targeted delivery of macromolecular systems. Folic acid was also conjugated to hydrophobically modified PHEA $(PHEA-C_{18}-folate)$ for the study of nanoparticle systems and another conjugate system in which the folic acid was conjugated via a long PEG-spacer (PHEA-PEG-folate or $PHEA-C_{18}-PEG-folate$) was prepared to enhance the solubility and targeting to folate receptor. The chemical composition of each system was characterized by $^1H$ NMR and UV/vis spectroscopy. The cellular uptake of conjugates into KB cells was analyzed by the fluorescence microscopy and flow cytometry. The data revealed the PHEA-folate was more effectively internalized than PHEA and the cellular uptake was competitively decreased by free folic acid, indicating that the uptake is mediated specifically by the folate receptor. In hydrophobically modified system, however, we could achieve efficient internalization only by using PEG spacer because the folate may have been buried within the hydrophobic interior of the copolymer for direct conjugation of folic acid into polymer. To increase the intracellular delivery we conjugated oligohistidine that can act as proton sponges to trigger osmotic swelling and promote rupture of endosome/lysosome compartment. The fluorescence microscopy image indicated the oligohistidine modified PHEA-folate was more uniformly distributed in KB cell.
양친성 고분자를 이용한 수용액상에서의 자기회합 현상에 의한 나노전달체 제조는 소수성 도메인이 생리활성 물질을 포집하고 친수성 도메인이 입자를 효율적으로 감싸면서 인체 내에서의 여러 systemic barrier를 효율적으로 극복할 수 있고, 입자 크기, 구조와 여러 고분자의 장점을 활용할 수 있어 약물 전달 분야에서 각광을 받고 있다. 하지만 대부분의 생체 활성 물질이 세포내에서 약리작용을 나타낸다는 점을 고려할 때 상대적으로 세포 내로의 전달이 잘 이루어지지 않는 고분자 전달체는 다른 특정 기능기를 접목하여 세포 내로의 전달을 증가시켜야 한다. 본 연구에서는 폴리아미노산 (poly(amino acid)) 을 기본으로 하여 제조한 나노입자의 세포 내로의 전달을 증가시키기 위해 입자 표면을 세포막투과성 펩타이드 (oligoarginine), 아미노산 단량체(histidine), 리간드 (folic acid)로 각각 수식하고, 세포 내로의 전달과 메카니즘에 대한 연구를 수행하였으며, 항암제를 물리적 혹은 화학적으로 봉입하여 in vitro 약리효과를 관찰하였다.
thiol ether 결합을 통해 고분자 백본에 성공적으로 접목된 올리고알지닌 $(Arg_8)$ 은 수용성 고분자인 poly(2-hydroxyethyl aspartamide) (PHEA), 항암제가 접목된 PHEA (PHEA-MTX), 긴 알킬 체인으로 치환시킨 PHEA $(PHEA-C_{18})$ 로 이루어진 나노입자, 항암제가 봉입된 나노입자 $(PHEA-C_{18}(DOX))$ 의 세포 내로의 전달을 확연히 증가시켰으며, confocal laser scanning micrscopy와 flow cytometry를 이용하여 분석하였다. $PHEA-Arg_8$ 를 이용한 세포 내 전달 메카니즘 연구를 통해 endocytosis 에 의해 세포 내로 전달이 이루어지며, 강한 양이온성을 가지는 올리고알지닌이 세포막에서의 집합적 표면 흡착을 증가시켜 adsorptive-endocytosis를 촉진 시켜 세포 내로의 전달을 확연히 증가시킴을 liposome study를 통해 확인하였다. 나노입자 시스템의 경우 그 안에 봉입된 항암제의 세포질과 세포핵 내로의 전달을 증가시켜 in vitro cell cytotoxicity에서 짧은 배양 시간과 낮은 항암제 농도에도 불구하고 강한 cell cytotoxicity를 나타내었다.
Endosomolytic synthetic polymer를 제조하기 위해 히스티딘(His)을 에스터 결합을 통해 PHEA 혹은 $PHEA-C_{18}$ 에 성공적으로 접목하였으며, 이 물질은 히스티딘의 이미다졸 그룹과 α-아민 그룹에 의해 pH 4 이상의 넓은 영역에서 buffering ability를 보였다. $PHEA-C_{18}-His$ 의 나노입자는 pH 5.0에서 pH 7.4에 비해 입자의 사이즈, 제타포텐셜, micropolarity가 모두 증가하였으며, 이것은 pH 5.0 에서 입자 표면에 위치하는 히스티딘이 좀 더 이온화가 되면서 입자가 swelling이 되기 때문이다. 히스티딘이 접목된 나노입자는 그렇지 않은 입자에 비해 buffering ability에 의한 endosome붕괴 능으로 인해 세포질 내에 고루 분포되는 것이 확인되었다. 항암제인 doxorubicin을 물리적으로 나노입자에 봉입한 경우 히스티딘이 접목된 입자는pH 5.0에서 입자 swelling에 의해 약물방출이 증대 되었으며, 이것은 히스티딘의 endosome 붕괴 능과 합해져 세포와 배양 후, 세포핵 내로 약물의 전달을 증가 시켜 in vitro 상에서 강한 cell cytotoxicity를 나타내었다.
folate receptor를 통한 세포 내 전달을 증가시키기 위해 folic acid를 PHEA 혹은 $PHEA-C_{18}$ 에 에스터 결합을 통한 직접 접목, 혹은 PEG spacer를 통해 접목시킨 후 KB cell 내로의 전달을 fluorescence microscopy와 flow cytometry를 통해 관찰하였다. 직접 접목한 경우보다 PEG spacer를 사용한 경우 수용액상에서의 solubility와 세포로의 targeting 능력을 증가시켜 세포 내로의 전달이 잘 이루어졌다. 특히 PHEA-C$_{18}$에 직접 접목한 경우는 folate가 나노입자의 소수성 코어에 봉입되어 입자 표면에 노출 되지 못하여 targeting 능력을 가질 수 없었고, PEG spacer를 사용한 경우만 세포내로의 전달을 얻을 수 있었다. folate receptor를 통해 전달될 경우 endosome안에서 전달 물질이 붕괴 될 수 있으므로 endosomolytic agent로 올리고히스티딘을 접목하여 이용하였으며 이때의 경우 세포내에서의 고분자 분포가 확연히 증가하였다.
