Engineering extracellular vesicles for targeted drug delivery

Ph.D.Extracellular vehicles (EVs) are double-layered membranous nanovesicles secreted by cells, also known as micro-vesicles (MVs) or exosomes. EVs mediate local and systemic intercellular communication and molecular transportation, which play important roles in both physiological and pathological processes. As an endogenous natural carrier for material transportation, the use of exosomes for drug delivery has unique advantages including biocompatibility, better permeability, low immunogenicity, and toxicity. Thus, exosomes have been explored as carriers for small molecules, proteins, and nucleic acid drugs.As a promising drug carrier, exosomes still face many challenges. For example, natural exosomes cannot differentiate healthy versus diseased tissues or cells. Rendering these natural carriers with specific cell-targeting properties, thereby, represents one of the urgent needs. This thesis provides solutions to this request through systematic exploration of the engineering of exosomal surface to endow exosomes with a targeting functionality. One approach is to display ligand peptides on the surface of exosomes through genetically engineering to realize chondrocyte-specific drug delivery. Also, to increase the capability and performance of the natural exosomes, I developed hybrid exosomes by fusing liposomes with chondrocyte-targeting exosomes and realized the delivery of CRISPR–Cas9 system in chondrocytes for genome editing. To enhance the chondrogenic of synovial fluid-mesenchymal stem cells (SF-MSCs), a mesenchymal stem cell affinity peptide E7 was fused with the exosomal membrane protein Lamp 2b, allowing the exosomes with E7 peptide displayed on the surface (E7- exosome) to deliver a small molecular KGN. All these delivery strategies were employed to repair cartilage degeneration and damage, with potential to develop into new treatments of osteoarthritis.In first part, I sought to determine whether estrogen regulates MMP-13 expression and interleukin-1 beta (IL-1β)-induced catabolic responses in human chondrocytes. Our results demonstrate that miR-140 acts as an intermediary transcriptional factor to suppress the catabolic activity of proteases within the chondrocyte extracellular matrix. Then I generated specifically targeted exosomes able to deliver miR-140 to chondrocytes. miR-140 can be effectively delivered into chondrocytes into deep articular tissue, and was strictly confined within the cartilage without noticeable diffusion to other tissues. Alleviation of the OA progress in a rat model demonstrates the efficacy of this therapy. As targeted ablation of MMP13 will reduce the expression of cartilage-degrading enzymes and attenuate IL-1beta induced cartilage deterioration, I also constructed hybrid engineered exosomes containing CAP-Lamp2B to specifically recognize chondrocytes. Loss-of-function of MMP-13 by the hybrid nanosystem mitigates adverse joint damages in the surgically induced OA rat. Furthermore, targeted delivery of a chondrogenic small molecule KGN to SF-MSCs is achieved by engineering surface protein of exosomes with an E7 peptide, and transplantation of SF-MSCs with in situ chondrogenesis is presented here as a new cell therapy for OA.Collectively, these results presented in this thesis indicate that harnessing cell-selective binding peptides, genetic engineering of the exosome surface protein grants these vesicles cell-specific targeting effect and enables potential therapies of osteoarthritis.細胞外囊泡(Extracellular vesicle EV)是一種由細胞分泌的雙層膜包被的囊泡，通常也被稱為微囊泡 (Microvesicle MV)或外泌體 (Exosome)。 EV通過介導細胞間的信號交流及物質轉運，參與到很多生理過程及病理進程。作為天然內源性的物質轉運載體，利用 EV負載藥物具有生物相容性、滲透性良好、低免疫原性、低毒性低等優勢。目前， EVs已成功負載並遞送化學藥物、蛋白質及基因等多種藥物 , 在藥物載體的領域具有巨大的應用潛力。作為一種新興的藥物載體 , EV在應用方面還面臨著諸多 挑戰，這其中包括靶向性給藥。提高外泌體的藥物靶向遞送能力非常必要，通過外泌體的靶向給藥可增強外泌體的治療作用及減少對正常細胞的毒副作用。 EV生物膜結構的特殊性質決定了外泌體的靶向策略 , 通過修 飾外泌體表面膜蛋白質可以在外膜表面展示配體與受體細胞受體相互作用，而賦予改造過的外泌體具備優良的趨向作用。本研究論文主要集中在外泌體膜的工程化改造，提高外泌體靶向功能。通過在外泌體表 b面展示基因工程可編碼的靶向性多肽，並應用於軟骨細胞靶向藥物遞送。同時為了控制和改變外泌體納米載體的生物功能，融合脂質體和靶向軟骨細胞的 外泌體從而獲得雜交型靶向外泌體。此雜交外泌體兼備軟骨靶向性及遞送CRISPR/Cas9a質粒至軟骨細胞進行基因編輯的功能。為了增強滑膜 間充質幹細胞（ SF-MSC）的成軟骨性，將間充質幹細胞親和 肽 E7與外泌體膜蛋白 Lamp 2b融合表達，使外泌體表面上展示 E7多肽，為幹細胞誘導成軟骨分化的小分子化合物 KGN提供新型靶向遞送方案。在第一部分，主要從機制方面探討雌激素調節人軟骨細胞中 MMP-13的表達和白介素 1β IL-1β）所誘導的軟骨基質分解代謝。結果表明， miR-140在其中充當仲介轉錄因數，參與抑制軟骨細 胞胞外基質蛋白酶的分解代謝活性。此後，本論文中構建了一種特異靶向軟骨細胞的外泌體能將 miR-140遞送到軟骨細胞的，使 miR-140可以有效地傳遞到軟骨細胞中並進入深層關節組織，並在大鼠 OA模 型能抑制關節的降解。由於抑制 MMP13表達可以阻止 IL-1β誘導的軟骨退化，因此本論文中構建了含有 CAP-Lamp2B的雜交型工程外泌體，可特異性靶向軟骨細胞。此雜交型工程外泌遞送 CRISPAR-Cas9能抑制 MMP-13，減輕了 OA模型中的關節損傷。此外，作為 OA的新細胞療法，本論文通過工程化外泌體的攜帶軟骨分化的小 分子化合物靶向遞送至 SF-MSC，在移植 SF-MSC後促進軟骨形成。綜上所述，利用細胞選擇性的結合肽與外泌體表面蛋白融合表達的基因工程改造方案 , 從而賦予的外泌體的特異性靶向作用，為骨關節炎 的潛在治療方法指供新方案。Liang, Yujie.Thesis Ph.D. Chinese University of Hong Kong 2020.Includes bibliographical references (leaves 137-160).Abstracts also in Chinese.Title from PDF title page (viewed on February 18, 2022)