METHODS OF PREPARING STIMULI-RESPONSIVE MULTIFUNCTIONAL NANOPARTICLES

專利類型

發明

專利國別 (專利申請國家)

美國

專利申請案號

15/446,387

專利證號

US 10,117,837

專利獲證名稱

METHODS OF PREPARING STIMULI-RESPONSIVE MULTIFUNCTIONAL NANOPARTICLES

專利所屬機關 (申請機關)

國立清華大學

獲證日期

2018/11/06

技術說明

本技術目的為開發以透明質酸(hyaluronic acid)接枝 LHRH胜肽(LHRH peptide)作為主動標靶的奈米藥物載體(drug carrier)，此藥物載體為一雙親性(amphiphilic)物質，在水溶液中可自我聚合形成奈米顆粒(self-assembling nanoparticle)，在聚合過程可加入了顯影因子(imagine agent)，一同包覆在奈米載體內部，達到結合診斷與治療的雙重目的。抗癌藥物doxorubicin與透明質酸間利用順式烏頭酸鍵(cis-aconityl linkage)及雙硫鍵(disulfide bond)來做鍵結，使之分別具有酸鹼敏感性(pH sensitivity)與氧化還原敏感性(redox sensitivity)。透過上述方法能成功製備出以透明質酸為主成分的主動標靶自我聚合奈米藥物載體，該載體具有多功能性(multifunctional)：LHRH胜肽對於卵巢癌細胞有高度的專一性(specificity)，可有效增加癌細胞對奈米載體的胞吞作用(endocytosis)，並減少對於正常細胞的傷害，以減少副作用(side effect)；進入細胞後，癌細胞周圍及內部的酸性環境(pH<7.0)加上還原環境(reductive condition)，可促進釋放所包覆的抗癌藥物及顯影因子，達到緩效釋放(controlled drug release)、抗癌效果(anti-cancer effect)，及對於癌細胞的標定(labeling)。原位卵巢腫瘤動物模型(orthotopic ovarian tumor model)被建立用以評估活體動物治療與診斷效果，結果顯示搭載抗癌藥物且接枝有LHRH的奈米顆粒在活體動物中可有效抑制腫瘤增生及具有腫瘤顯影功能。 Ovarian cancers are the leading cause for mortality among gynecologic malignancies with five-year survival rate less than 30%. The purpose of this study is to develop a redox and pH-sensitive self-assembling hyaluronic acid nanoparticle with active targeting peptide for anticancer drug delivery. Anti-cancer drug is grafted onto hyaluronic acid (HA) via cis-aconityl linkage and disulfide bond to possess pH sensitivity and redox property, respectively. This conjugate is amphiphilic and can self-assemble into nanoparticle (NP) in aqueous solution. The results show that the nanoconjugate is successfully developed and the grafting ratio of cystamine (cys) is 17.8% with drug loading amount about 6.2% calculated by 1H NMR spectra. The particle size is approximately 229.0 nm using dynamic light scatting measurement, and the morphology of nanoparticles is observed as spherical shape by transmission electron microscope. The pH and redox sensitivities are evaluated by changing either pH value or concentration of dithiothreitol in the medium. It is proved that the drug carrier is capable of achieving sustained controlled release of anti-cancer drug to 95% within 150 hours. The intracellular uptake is observed by fluorescent microscope and the images show that conjugating luteinizing hormone-releasing hormone (LHRH) peptide can enhance specific uptake of nanoparticles by OVCAR-3 cancer cells; thus, resulting in inhibitory cell growth to less than 20% in 72 hours in vitro. Orthotopic ovarian tumor model is also established to evaluate the therapeutic and diagnostic efficacy using non-invasive in vivo imaging system. The representative results demonstrate that LHRH-conjugated NPs possess a preferable tumor imaging capability and an excellent antitumor ability to almost 30% of original size in 20 days.

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