Lee Woo-kyoung, Park Jong-yeun, Jung Sangho, Yang Chul Woo, Kim Wan-Uk, Kim Ho-Youn, Park Jae-Hun, Park Jong-sang
School of Nano Engineering, Inje University, Kimhae, Korea.
J Control Release. 2005 Jun 20;105(1-2):77-88. doi: 10.1016/j.jconrel.2005.03.009.
PEG-conjugated immunodominant peptides for collagen-induced arthritis (CIA) were prepared for oral tolerance induction instead of whole Type II collagen (CII), because a small peptide can be converted to a macromolecule soluble in methylene chloride by the coupling of poly-ethylene glycol (PEG). PEG-pep1 was synthesized from a peptide and mPEG-NH2 (Mw approximately 5000) using SPDP as a linker, whereas PEG-pep2 was prepared by the direct disulfide coupling between PEG-OD (Mw approximately 10,000) and the peptide. PEG-pep1 and PEG-pep2 were purified by gel permeation chromatography (GPC), and the peak fractions of GPC were identified by GPC and MALDI-TOF mass spectroscopy. The peptide coupling gave much earlier retention times for PEG-pep1 (11.26 min) and PEG-pep2 (10.61 min) than for mPEG-SPDP (15.63 min) and mPEG-OD (14.58 min). The Mw's of mPEG-NH2, mPEG-SPDP, PEG-pep1, mPEG-OD and PEG-pep2 were 5451, 5588, 7035, 10,360 and 11,826, respectively, suggesting that PEG-pep1 and PEG-pep2 of high purity could be obtained. The nanoparticles entrapping PEG-pep1 and PEG-pep2 (NP/PEG-pep1 and NP/PEG-pep2) were prepared by the o/w solvent evaporation method, whereas the peptide-loaded nanoparticles (NP/pep) were prepared by the w/o/w double emulsion method. Although all the nanoparticles had a similar spherical morphology under scanning electron microscopy, NP/pep showed up as having a larger mean size than the others, which was confirmed by dynamic light scattering analysis (NP/pep, 499.7+/-27.2 nm; NP/PEG-pep1, 333.0+/-16.8 nm; NP/PEG-pep2, 342.4+/-15.1 nm). The lower encapsulation efficiency of NP/pep (21.0+/-1.6%) than NP/PEG-pep1 (66.5+/-5.0%) and NP/PEG-pep2 (73.8+/-5.5%) can also be attributed to the preparation method. In in vitro release studies, NP/PEG-pep1 and NP/PEG-pep2 displayed a similar release profile, close to a linear release pattern, whereas NP/pep displayed a tri-phasic release profile. From these results, it was demonstrated that nanoparticles entrapping a PEG-conjugated peptide could be an alternative delivery method for the induction of oral tolerance rather than CII and peptide.
制备了用于胶原诱导性关节炎（CIA）的聚乙二醇（PEG）缀合的免疫显性肽，用于诱导口服耐受，而非使用完整的II型胶原（CII），因为通过聚乙二醇（PEG）偶联，小肽可转化为可溶于二氯甲烷的大分子。PEG - pep1是使用SPDP作为连接剂，由肽和甲氧基聚乙二醇（mPEG - NH2，分子量约5000）合成的，而PEG - pep2是通过PEG - OD（分子量约10,000）与肽之间的直接二硫键偶联制备的。PEG - pep1和PEG - pep2通过凝胶渗透色谱（GPC）纯化，GPC的峰级分通过GPC和基质辅助激光解吸电离飞行时间质谱（MALDI - TOF）鉴定。肽偶联使PEG - pep1（11.26分钟）和PEG - pep2（10.61分钟）的保留时间比mPEG - SPDP（15.63分钟）和mPEG - OD（14.58分钟）早得多。mPEG - NH2、mPEG - SPDP、PEG - pep1、mPEG - OD和PEG - pep2的分子量分别为5451、5588、7035、10360和11826，表明可获得高纯度的PEG - pep1和PEG - pep2。包封PEG - pep1和PEG - pep2的纳米颗粒（NP/PEG - pep1和NP/PEG - pep2）通过水包油溶剂蒸发法制备，而载肽纳米颗粒（NP/pep）通过油包水/水包油双乳液法制备。尽管在扫描电子显微镜下所有纳米颗粒都具有相似的球形形态，但动态光散射分析证实NP/pep的平均尺寸比其他纳米颗粒大（NP/pep，499.7±27.2纳米；NP/PEG - pep1，333.0±16.8纳米；NP/PEG - pep2，342.4±15.1纳米）。NP/pep（21.0±1.6%）的包封效率低于NP/PEG - pep1（66.5±5.0%）和NP/PEG - pep2（73.8±5.5%），这也可归因于制备方法。在体外释放研究中，NP/PEG - pep1和NP/PEG - pep2呈现相似的释放曲线，接近线性释放模式，而NP/pep呈现三相释放曲线。从这些结果表明，包封PEG缀合肽的纳米颗粒可能是诱导口服耐受的一种替代递送方法，而非CII和肽。
