- 學位論文
探討辨認聚乙二醇抗體於聚乙二醇修飾奈米藥物療效與藥物動力學之影響及創新奈米藥物定量系統之研發
Using the anti-polyethylene glycol antibody to estimate the influence of the therapeutic efficacy and pharmacokinetics of PEGylated nanomedicine and to develop the novel drug quantification system
摘要
聚乙二醇 (polyethylene glycol, PEG) 是常用於修飾奈米藥物或奈米粒子的高分子，透過聚乙二醇修飾可增強其生物相容性、穩定性與生物半衰期。基於此特點，近年聚乙二醇修飾的相關製劑，更在市場上逐年增加。然而，近年研究指出，因過去幾十年大量使用PEG使人體產生可辨識PEG之抗體 (aPEG)，將可能對PEG奈米藥物之療效與藥物動力學造成影響。為了驗證此假設，在第一個研究主題中我們建立了兩種體內具有aPEG抗體的動物模式(主動生成與被動生成)，以評估aPEG抗體對臨床常用奈米藥物(LipoDox; liposomal doxorubicin) 在動物體中藥物動力學及療效之影響。結果發現aPEG抗體可加速藥物從血中排除，並縮短藥物的體內半衰期，進而降低皮下腫瘤的治療成效。故aPEG抗體的存在可能對於PEG修飾藥物的療效具有顯著影響，更突顯檢測PEG抗體之重要性。未來檢測體內aPEG抗體的存在與否，應可作為評估是否使用PEG修飾藥物進行個人化治療的重要指標。另外在第二個研究主題中，我們將發展一套通用型的PEG奈米藥物的定量系統，以改善傳統PEG奈米藥物定量，樣品需經複雜前處理及因藥物類別不同而需更換不同定量方法的缺點。透過基因工程將aPEG抗體Fab片段表現於細胞膜表面形成「aPEG bioparticle」，並結合另一株aPEG抗體，以發展出一套可用於定量各式PEG修飾奈米藥物與奈米分子的酵素連結免疫吸附分析系統。透過此系統，PEG修飾奈米藥物與奈米分子，可免去複雜的前處理(如金屬奈米需以強酸分解萃取後分析或微脂體藥物需先行分解後純化等) 快速得知樣品濃度。結果顯示此方法可成功定量常見的各式PEG修飾奈米製劑，如奈米量子點、SPIO造影劑、微質體藥物(LipoDox)與長效型干擾素(PEGASYS)，且敏感度分別可達0.01 nM、 0.1 nM、15.63 ng/mL、0.48 ng/mL。另外本系統可不受樣品血清干擾，在人類或老鼠10%的血清中，亦可穩定定量PEG修飾奈米藥物與分子。同時本系統也與藥物開發常用的放射線法進行藥物動力學的檢測比較，實驗結果顯示本定量系統與放射線法結果相似，證明本系統具備良好的敏感度。基於上述結果本研究已成功建立出一個創新、敏感，不受血清干擾的定量系統。可透過單一方法定量各式常見的奈米藥物或分子。相信此系統將可滿足PEG修飾奈米藥物或分子研究開發的市場需求。
並列摘要
Attachment of polyethylene glycol (PEG) molecules (PEGylation) to nanomedicine or nanoparticle (NPs) is a widely-used method to improve their biocompatibility, stability and half-life. Thus, the PEGylated agents may be continuing increase in the market. However, the recent study found increasing frequency of human long-term exposed to PEGylated agent cause the generation of anti-PEG antibody (aPEG Ab) against PEG in last decades. The PEG Ab may have affected the pharmacokinetic (PK) and the therapeutic efficacy of the PEGylated nanomedicine. In chapter one, we generate two mice model (endogenous or passive induced aPEG Ab titer) which have the aPEG Ab titer for evaluated the influence of aPEG Ab on the therapeutic efficacy and PK of PEGylated nanomedicine (LipoDox) in vivo. We found the PEG Ab alter the pharmacokinetic (PK) and reduce the therapeutic efficacy of LipoDox in vivo. It may have indicated the aPEG Ab could as the potential marker for developing personalized therapy of the LipoDox. In chapter two, we expressed aPEG Ab Fab on the cell surface to form aPEG bioparticles and combined them with aPEG Abs to generate a quantitative ELISA for universally measure the PEGylated nanomedicine or NPs without compound purification. Through the ELISA system, there is need to extract the ionic NPs by strong acid or to extract the active drug from liposome by detergent. The ELISA directly quantify PEG-quantum dots (PEG-QD), PEG-stabilizing super-paramagnetic iron oxide (PEG-SPIO), Lipo-Dox and PEGASYS and the detection limits were 0.01 nM, 0.1 nM, 15.63 ng/mL and 0.48 ng/mL, respectively. Furthermore, this aPEG bioparticle-based ELISA tolerated samples containing up to 10% human or mouse serum. There was no significant difference in pharmacokinetic studies of radiolabeled PEG-NPs (Nano-X-111In) through aPEG bioparticle-based ELISA and a traditional gamma counter. In chapter two, we developed a novel approach for evaluating the PK and concentration of the PEGylated nanomedicine or NPs by a universal quantification system. The aPEG bioparticle-based platform meets a need in the development of novel PEGylated nanomedicines or NPs in the market.