MULTIFUNCTIONAL STIMULI-SENSITIVE PHARMACEUTICAL NANOCARRIERS

描述（由申请人提供）：目前水平的工程药物纳米载体允许药物传递系统（DOS）展示几种期望特性的组合。理想情况下，“智能”纳米粒子DOS应该能够：(a)在所需的器官或组织中积累，然后(b)穿透目标细胞内部，将其负载（药物或DNA）传递给目标细胞。器官或组织（肿瘤、梗死）的积累可以通过增强渗透性和保留（EPR）效应的被动靶向或抗体介导的主动靶向来实现，而细胞内递送可以通过内化配体或细胞穿透肽（CPPs）介导。为此，DOS应同时在其表面携带能够以某种精心安排的方式发挥作用的各种部分，并以这样一种方式构建，即在递送的第一阶段，细胞穿透功能被提供器官/组织积累的功能（立体保护聚合物或抗体）所屏蔽。在靶标内部，通过刺激敏感键附着在DOS表面的保护性聚合物或抗体应在局部病理条件（异常pH值或温度）的作用下迅速分离，并暴露先前隐藏的功能，允许载体及其货物进入细胞内。到目前为止，已多次尝试将各种药物载体（脂质体和胶束）绕过内吞途径直接递送到细胞质中，以保护药物和DNA不被溶酶体降解，但大多不成功。CPPs（如TAT肽、TATp）可直接进入细胞质，已成功用于小药物分子、大分子（酶、DNA）和纳米颗粒（量子点、氧化铁纳米颗粒、脂质体）的细胞内递送。目前，下一步需要的是开发基于cpp的系统，控制细胞内定位，并能够将药物和DNA输送到耐药癌细胞中。我们能够将大量的TATp分子偶联到一个脂质体/胶束上，并实现了它们在细胞内的有效递送；使用ttp -脂质体/DNA复合物进行高效、无毒的细胞转染；并构建了能在酸性条件下转换活性的多功能刺激敏感药物纳米载体。我们还拥有通过肿瘤细胞表面结合核小体（NS）识别多种肿瘤的单克隆2C5抗体（mAb 2C5）。我们还可以控制纳米载体的细胞内定位。我们的假设是，各种药物的治疗活性可以通过将它们装载到多功能刺激敏感的纳米颗粒DOS上，从而通过EPR效应和/或通过特异性抗体介导的识别延长循环和目标积累，然后在局部刺激作用下，在先前隐藏的CPP功能去保护之后，快速CPP（TATp）介导的内化进入靶细胞，从而增强。通过控制DOS的电荷和组成，atp介导的DOS（如脂质体和胶束）直接进入细胞质，可能进一步影响细胞内运输、命运和分布。脂质体和胶束装载着水溶性和不水溶性药物和DNA。我们建议：(a)制备和表征基于脂质体和胶束的多功能刺激敏感DOS；(b)利用可分离的PEG和/或癌症特异性单克隆抗体和TATp的组合，开发能够将药物/DNA递送到癌细胞和进入癌细胞的DOS；(c)证明通过控制atp脂质体/胶束的电荷和组成来调节细胞内命运和分布的可能性；(d)在体内和体外显示载药/ dna的tatp脂质体/胶束控制耐药癌细胞生长的效率。

DESCRIPTION (provided by applicant): The current level of engineering pharmaceutical nanocarriers allows for drug delivery systems (DOS) demonstrating a combination of several desired properties. Ideally, "smart" nanoparticular DOS should be able: (a) to accumulate in the required organ or tissue, and then (b) penetrate inside target cells delivering there its load (drug or DNA). Organ or tissue (tumor, infarct) accumulation could be achieved by the passive targeting via the enhanced permeability and retention (EPR) effect or by the antibody-mediated active targeting, while the intracellular delivery could be mediated by internalizable ligands or by cell-penetrating peptides (CPPs). For this, DOS should simultaneously carry on their surface various moieties capable of functioning in a certain orchestrated way, and be built in such a way that during the first phase of delivery, a cell-penetrating function is shielded by the function providing organ/tissue accumulation (sterically-protecting polymer or antibody). Inside the target, the protecting polymer or antibody attached to the surface of DOS via the stimuli- sensitive bond should rapidly detach under the action of local pathological conditions (abnormal pH or temperature) and expose the previously hidden function allowing for the delivery of the carrier and its cargo inside cells. So far, multiple and mainly unsuccessful attempts have been made to deliver various drug carriers (liposomes and micelles) directly into the cell cytoplasm bypassing the endocytic pathway, to protect drugs and DNA from the lysosomal degradation. CPPs (such as TAT peptide, TATp) can enter cell cytoplasm directly and have been successfully used for the intracellular delivery of small drug molecules, large molecules (enzymes, DNA) and nanoparticulates (quantum dots, iron oxide nanoparticles, liposomes). Currently, the next steps are required such as the development of CPP-based systems with controlled intracellular localization and the ability to deliver drug and DNA into drug resistant cancer cells. We were able to couple a large number of TATp molecules to a single liposome/micelle and achieved their efficient intracellular delivery; used TATp-liposomes/DNA complexes for an efficient and non-toxic cell transfection; and constructed multifunctional stimuli-sensitive pharmaceutical nanocarriers capable of switching their activity under acidic conditions. We have also in our possession monoclonal 2C5 antibody (mAb 2C5) recognizing a variety of tumors via tumor cell surface-bound nucleosomes (NS). We can also control the intracellular localization of nanocarriers. Our hypothesis is that the therapeutic activity of various Pharmaceuticals can be enhanced by loading them onto multifunctional stimuli-sensitive nanoparticular DOS allowing for prolonged circulation and target accumulation via the EPR effect and/or via specific antibody-mediated recognition, and subsequent fast CPP(TATp)-mediated internalization into target cells after the deprotection of previously hidden CPP function under the action of local stimuli. TATp-mediated delivery of DOS, such as liposomes and micelles, loaded with water-soluble and water-insoluble drugs and DNA, directly into the cell cytoplasm may be further influenced in terms of the intracellular trafficking, fate, and distribution by controlling DOS charge and composition. We propose: (a) to prepare and characterize liposome- and micelle-based multifunctional stimuli-sensitive DOS; (b) to develop DOS capable of drug/DNA delivery TO and INTO cancer cells using a combination of detachable PEG and/or cancer-specific monoclonal antibody and TATp; (c) to demonstrate the possibility of regulating the intracellular fate and distribution of TATp-liposomes/micelles by controlling their charge and composition; and (d) to show the efficiency of drug/DNA-loaded TATp-liposomes/micelles in controlling the growth of drug resistant cancer cells both in vitro and in vivo.