Application of mucus modulating multipurpose trypsin nanoparticles to overcome the mucus barrier and deliver mitochondria-targeted anticancer drugs in mucinous carcinoma peritonei

项目摘要 粘液性结直肠癌和阑尾癌（MCAC）是一种独特的组织学亚型， 转移到腹膜腔（称为腹膜粘液癌[MCP]）。MCP经常 不可切除，对标准静脉化疗反应差，且常在“治愈性”手术后复发 腹腔内（IP）化疗，导致肿瘤学结局较差。腹腔化疗 MCP面临两大挑战。首先，MCP的特征在于丰富的细胞外粘液， 癌细胞周围的保护屏障，阻碍IP化疗药物递送。我们先前已经 在患者来源的MCP体外和体内模型中，使用粘液溶解药物， (e.g.菠萝蛋白酶[BRO]、N-乙酰半胱氨酸[NAC]和胰蛋白酶[TRYP]）。我们还发现，基线净负 粘液溶解后粘液的电荷显著增加（在我们的研究中，粘液的电荷-电位：未消化的粘液-1.93 mV;消化粘液-17.2 mV）。第二，MCP的常用IP药物（例如阿霉素[DOX]和 丝裂霉素C [MITO]）通过腹膜快速吸收，导致IP保留时间短， 低肿瘤内（IT）渗透和全身毒性。治疗性纳米颗粒制剂具有更长的IP 保留和IT渗透比游离药物，因为增强的渗透性和保留效果，并提供 防止早期降解和预吸收。这项提案的目的是利用纳米技术 以及粘液溶解后粘液的显著负电荷，以增强IP保留、IT渗透和 在MCP中递送带正电荷的抗癌药物。为此，我们合成了粘液调节剂， 多用途TRYP纳米颗粒（MTN）由三种组分组成：（a）带负电荷的TRYP核 簇，由聚乙二醇（PEG）和TRYP的4个臂组成，用于酶促粘液溶解和药物递送; (b)纳米颗粒缀合的NAC，用于粘液破坏和粘膜粘附;和 带正电荷的靶向抗癌药物（mitocans），特别是三苯基磷（TPP）- 阿霉素（TPP-DOX）和TPP-丝裂霉素C（TPP-MITO），用于抗癌作用。我们假设我们的 MTN将破坏粘液的结构完整性，增强IP/IT保留和负载药物的渗透， 通过逐渐升高的负电荷梯度传递带正电的TPP-DOX或TPP-MITO， 纳米颗粒表面至消化粘液至线粒体（电位：消化粘液-17.2mV;细胞 膜-30至-60 mV;线粒体膜-160 mV）。我们的研究计划提供了一个新的 治疗策略，以克服细胞保护性粘液屏障和改善MCP中的药物递送。是 预期拟定MTN将提供优于非MTN的药代动力学和药效学优势。 药物的纳米载体制剂。值得注意的是，所提出的MTN是由生物相容性和生物相容性合成的。 生物可降解材料，增加其可翻译性; TRYP是一种天然合成的哺乳动物蛋白酶 能够水解肽，酰胺和酯键，不消化活组织，因为血清 和TPP-MITO/TPP-DOX，靶向带负电荷的粘液， 线粒体，是我们实验室新开发的，在我们的初步实验中对MCP有效。 问题研究这种方法可能适用于其他粘液性肿瘤（例如粘液性卵巢癌）， 分泌丰富的细胞外粘液。我们处于独特的地位，可以进行临床前研究， 鉴于我们已经开发了MCAC/MCP的体外3D培养和体内异种移植模型， 我们的机构是管理MCP患者的主要国际转诊中心之一。

PROJECT ABSTRACT Mucinous colorectal and appendiceal cancers (MCAC) are unique histologic subtypes that frequently metastasize to the peritoneal cavity (known as mucinous carcinoma peritonei [MCP]). MCP is frequently unresectable, responds poorly to standard intravenous chemotherapy, and often recurs after “curative” surgery with intraperitoneal (IP) chemotherapy, resulting in poor oncologic outcomes. Intraperitoneal chemotherapy for MCP faces two major challenges. First, MCP is characterized by abundant extracellular mucus that forms a protective barrier around cancer cells, hindering IP chemotherapeutic drug delivery. We have previously demonstrated robust mucolysis, in patient-derived in vitro and in vivo models of MCP, using mucolytic drugs (e.g. bromelain [BRO], N-acetylcysteine [NAC] and trypsin [TRYP]). We also found that the baseline net negative charge of mucus was significantly increased after mucolysis (ζ-potential in our studies: undigested mucus -1.93 mV; digested mucus -17.2 mV). Second, commonly administered IP drugs for MCP (e.g. doxorubicin [DOX] and mitomycin C [MITO]) are rapidly absorbed across the peritoneal membrane, resulting in short IP retention time, low intra-tumoral (IT) penetration, and systemic toxicity. Therapeutic nanoparticle formulations have longer IP retention and IT penetration than free drugs because of enhanced permeability and retention effect and provide protection from early degradation and pre-absorption. The aim of this proposal is to leverage nanotechnology and the significant negative charge of mucus following mucolysis to enhance IP retention, IT penetration, and delivery of positively charged anticancer drugs in MCP. To this end, we have synthesized mucus modulating multipurpose TRYP nanoparticles (MTN) comprised of three components; (a) a core of negatively charged TRYP clusters, consisting of 4 arms of polyethylene glycol (PEG) and TRYP, for enzymatic mucolysis and drug delivery; (b) nanoparticle-conjugated NAC, for mucus disruption and mucoadhesion; and (c) nanoparticle-loaded and positively-charged mitochondria-targeted anticancer drugs (mitocans), specifically triphenyl phosphonium (TPP)- doxorubicin (TPP-DOX) and TPP-mitomycin C (TPP-MITO), for anti-cancer effect. We hypothesize that our MTN will disrupt the structural integrity of mucus, enhance IP/IT retention and penetration of loaded drugs, and deliver positively charged TPP-DOX or TPP-MITO across a progressively higher negative charge-gradient from the nanoparticle surface to digested mucus to mitochondria (ζ-potential: digested mucus -17.2 mV; cell membranes -30 to -60 mV; mitochondrial membranes -160 mV). Our research proposal provides a novel therapeutic strategy to overcome the cytoprotective mucus barrier and improve drug delivery in MCP. It is expected that the proposed MTN will provide a pharmacokinetic and pharmacodynamic advantage over non- nanocarrier formulations of the drugs. Notably, the proposed MTN are synthesized from biocompatible and biodegradable materials, increasing their translatability; TRYP is a naturally synthesized mammalian protease capable of hydrolyzing peptide-, amide-, and ester-bonds, and does not digest living tissue since both serum and viable cells contain TRYP inhibitors; and TPP-MITO/TPP-DOX, targeted to negatively charged mucus and mitochondria, are newly developed in our laboratory and significantly effective against MCP in our preliminary studies. This approach is likely to be applicable for other mucinous tumors (e.g. mucinous ovarian cancer) that secrete abundant extracellular mucus. We are uniquely positioned to conduct the preclinical studies in this proposal given that we have already developed in vitro 3D cultures and in vivo xenograft models of MCAC/MCP and our institution is one of the major international referral centers for the management of patients with MCP.