Doxorubicin was chemically conjugated to a terminal end group of poly(D,L-lacticco-glycolic acid) [PLGA] by a carbamate linkage and an ester linkage. The doxorubicin-PLGA conjugate was formulated into nanoparticles. A hydroxyl terminal group of PLGA was activated by p-nitrophenyl chloroformate and reacted with a primary amine group of doxorubicin for the conjugation. A carboxylic acid end group of PLGA was conjugated to a primary hydroxyl group of doxorubicin. The primary amine group of doxorubicin was protected during the conjugation process and then deprotected. The nanoparticles containing the conjugate exhibited sustained doxorubicin release profiles over a one-month period, whereas those containing unconjugated free doxorubicin showed a rapid doxorubicin release within 5 days. Doxorubicin release patterns could be controlled by conjugating doxorubicin to PLGA having different molecular weights. The conjugated doxorubicin nanoparticles showed increased uptake within a HepG2 cell line, which was quantitated by a flow cytometry and visualized by a confocal microscopy. The nanoparticles exhibited slightly lower IC50 value against the HepG2 cell line compared to that of free doxorubicin. In vivo anti-tumor activity assay also showed that a single injection of the nanoparticles had comparable activity to that of free doxorubicin administered by daily injection. The conjugation approach of doxorubicin to PLGA was potentially useful for the formulation of nanoparticles that requires targeting for cancer cells as well as sustained release at the site. Doxorubicin was chemically conjugated to the terminal end of a di-block copolymer composed of poly(L-lactic acid) (PLLA) and methoxy-poly(ethylene glycol) (mPEG) via two acid-cleavable linkages. A hydrazone bond and a cis-acotinyl bond were formed between doxorubicin and the terminal group of PLLA segment in the block copolymer. Doxorubicin conjugated PLLA-mPEG di-block copolymers self-assembled to form micelles in aqueous solution. The doxorubicin-conjugated micelles were about 89.1 nm in diameter and their critical micelle concentration was 1.3 ？/ml. These values were comparable with those of unconjugated micelles. In an acidic condition, the conjugated doxorubicin in the hydrazone linkage was readily cleaved, releasing doxorubicin in an intact structure. Doxorubicin conjugated PLLA-mPEG micelles were more potent in cell cytotoxicity than free doxorubicin, suggesting that they were more easily taken up within cells with concomitant rapid. release of cleaved doxorubicin into the cytoplasm from acidic endosomes.
Lysozyme was hydrophobically modified with a fatty acid, sodium oleate, via an ion pairing mechanism. Ionic binding between an anionic carboxylic group of sodium oleate and basic amino groups in lysozyme was primarily utilized to form lysozymeloleate complex. The complex formation was pH-dependent. The lysozymeloleate complex dissolved in an organic solvent exhibited much higher conformational stability at elevated temperature compared to free lysozyme in the same solvent. The complex was formulated into biodegradable nanoparticles by a spontaneous emulsion and solvent diffusion method. The resultant formulation showed near 100 % encapsulation efficiency of lysozyme within nanoparticles having less than 100 nm in diameter with a narrow distribution. Lysozyme could be loaded into the nanoparticles up to 18.6 % (w/w) with concomitantly increased particle sizes. This study demonstrates a new formulation method of biodegradable nanoparticles with highly efficient encapsulation of proteins potentially useful for oral protein delivery including mucosal vaccination. Biodegradable salmon calcitonin (sCT) nanoparticles were formulated using protein-fatty acid complexes, and their in vitro transport against a Caco-2 cell monolayer and the extent of in vivo oral uptake were assessed. Positively charged sCT was hydrophobically ion-paired to form physical complexes with fatty acid, phospholipid, and surfactant. Among them, sodium oleate was used to form sCT oleate complexes, and they were characterized and formulated into biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles. Endocytosis of sCT nanoparticles by Caco-2 cells was studied by flow cytometry. Transcytosis of sCT across the Caco-2 monolayer was also quantitated by an ELISA method. The sCT nanoparticles were orally administered to Sprague-Dawley rats, and serum sCT was monitored. Biodegradable polymeric nanoparticles containing a loading amount of sCT as high as 2.7 % (w/w) were prepared based on the complexation of sCT with sodium oleate. A greater amount of sCT nanoparticles could be delivered into Caco-2 cells compared to free sCT, and sCT could also be transported from the apical side to the basolateral side of the Caco-2 monolayer. In vivo experiments showed the possibility of oral uptake of sCT. Physical complexation of sCT with amphiphilic molecules enabled the formation of sCT loaded PLGA nanoparticles at a high loading efficiency. sCT PLGA nanoparticles exhibited transcytosis against the Caco-2 cell monolayer and were readily taken up in an oral route in vivo.
항암 약물 치료의 목적으로 광범위하게 사용되는 항암제인 독소루비신은, 비특이적인 공격성으로 인해, 치료를 위한 투여량에 한계가 있다. 항암제의 비특이성을 극복하기 위해, 독소루비신을 나노 입자 및 미셀의 형태로 제제화하였다. 나노 입자 및 미셀의 형태는, 수백 나노미터에 이르는 특징적인 크기로 인해, 수동적인 암세포 지향성을 갖는다고 알려져 있다. 독소루비신을 폴리(D,L-락트-글리콜산)(PLGA)에 카바메이트 및 에스터 연결고리를 이용해 화학적으로 접합시켰다. 이 경우, 나노 입자 및 미셀내의 독소루비신 봉입률이 현격히 향상되었으며, 독소루비신의 방출은 생분해성 고분자인 PLGA의 분해속도에 따라 방출되어, 1개월동안 지속적으로 방출되었다. 나노 입자 및 미셀 입자 형태로 만든 독소루비신은 기존의 독소루비신에 비해 항암 효과가 높았으며, 실제 동물실험에서도 상당한 항암 효과가 있는 것이 관찰되었다. 또, 낮은 pH에서 단시간내에 절단가능한 연결을 사용해서, 독소루비신과 생분해성 고분자를 접합시켜 미셀의 구성했을경우에는, 고분자의 분해가 아닌, 연결 고리의 절단으로 6시간이내에 다량의 독소루비신(약 40%)이 방출되었다. 따라서 이 방법은 세포내 함입과정으로 암세포로 들어가는 미셀에 대해 적당하다.
라이소자임과 올레산의 소수성 이온결합을 이용한 제조한 이온복합체는, 유기용매에 대한 용해도가 증가했으며, 소화 효소인 트립신에 대한 저항성과 열에 대한 안정성이 보통의 라이소자임에 비해, 증가했다. 또, 이 이온복합체를 이용해 생분해성 고분자 나노 입자를 제조했다. 골다공증의 치료목적으로 사용되는 칼시토닌은, 단백질 자체의 불안정성으로 인해 경구투여의 어려움이 많았다. 이 연구에서, 칼시토닌을 올레산과 소수 이온 결합을 시켜 이온 복합체를 제조하였으며, 이 복합체로 경구 투여의 목적으로 사용할 생분해성 나노 입자를 제조하였다. 이 나노 입자는 장막 모델 투과 실험에서, 기존의 칼시토닌은 거의 투과하지 않은 반면, 칼시토닌 나노 입자는, 장막을 투과하는 것으로 나타났다. 실제로, 동물에 대한 경구 투여 실험에서도, 혈중 칼시토닌의 양이 증가하는 것을 확인하였으며, 혈중 칼슘의 감소도 확인하여, 칼시토닌 경구투여제 개발의 가능성을 확인하였다.
