Shape Specific, Enzyme-Responsive, Nano-Imprinted Particles for Drug Delivery

描述(申请人提供)：纳米技术是发展先进药物输送载体的最有前途的途径之一。传统上，该领域一直专注于基于自组装或基于乳液的概念来合成纳米载体。尽管聚合物或脂质体给药系统已经取得了重大进展，但仍然存在一些关键的根本性限制。这些问题包括无法(A)精确控制纳米载体的形状、长宽比、大小和多分散性，以及(B)将各种针对疾病的触发释放机制整合到纳米颗粒设计中。我们的目标是利用自顶向下、高通量的纳米制造技术，特别是一种改进的阶跃闪光压印光刻(S-FIL)方法，合成各种形状、尺寸和长径比的高度单分散的聚合物纳米载体。通过将疾病反应元件(如多肽)直接加入到颗粒基质中，我们建议将酶反应释放特性赋予这些纳米载体，这样药物或造影剂主要是响应于肿瘤相关信号而释放的。这两年的具体目标是：目标1：开发一种高通量、自上而下的纳米制造工艺，用于制造尺寸和几何形状(形状或长宽比)精确的肿瘤靶向、酶反应纳米载体。在这一目标中，将使用改进的阶跃和闪光压印光刻(S-FIL)技术来制备不同尺寸、横截面形状和长宽比的纳米颗粒。颗粒合成的基本材料将是聚乙二醇二丙烯酸酯(PEGDA)和对溶酶体半胱氨酸蛋白酶(如组织蛋白酶B)敏感的丙烯酸化五肽GFLGK(多肽-DA)。组织蛋白酶B在包括非小细胞肺癌(NSCLC)在内的多种癌症中高度过度表达。目的：研究纳米印迹载体的大小、形状、长宽比和大分子浓度对(A)细胞对印迹纳米载体的摄取和(B)模型药物和造影剂在细胞内的释放和转染酶反应纳米粒的影响。我们假设颗粒形状、长径比以及纳米尺寸应该显著影响颗粒内化的效率。此外，还将利用成纤维细胞和肺癌细胞研究包裹药物的细胞内递送以及模型siRNA药物的转染效率。目的：研究不同大小、形状和长径比的纳米印迹颗粒在鼻腔和荷瘤动物体内的生物分布。研究不同截面形状和长径比的纳米粒子的一个重要假设是，这些几何参数可能会对粒子在血管中的传输特性产生强烈的影响，因此应该对粒子的生物分布以及肿瘤的蓄积效果产生重大影响。总而言之，这些结果应该为未来在各种癌症动物模型中传递治疗和诊断药物的研究提供强有力的基础。这个为期两年的探索性项目的目标是开发一种用于制造智能药物递送纳米颗粒的纳米压印光刻方法。这是一种独特的自上而下的颗粒合成纳米制造方法。这些纳米颗粒被设计成具有特定的大小和形状，可以影响它们的生物分布。此外，这些颗粒将能够靶向肿瘤细胞，并将货物(药物或显像剂)主要响应于疾病特异性信号，例如酶上调。

DESCRIPTION (provided by applicant): Nanotechnology is one of the most promising avenues for the development of advanced drug delivery vehicles. Classically, the field has focused on synthesizing nanocarriers based on self assembly or emulsion based concepts. Although significant progress has been made in polymeric or liposomal drug delivery systems, there remain some key fundamental limitations. These include inability to (a) precisely control shape, aspect ratios, size and polydispersity of the nanocarriers and (b) integrate a variety of disease-specific triggered release mechanisms into the nanoparticle design. Our objective is to use top-down, high throughput nanofabrication technology, specifically a modified step and flash imprint lithography (S-FIL) method, to synthesize highly monodisperse polymer nanocarriers of various shapes, sizes and aspect ratios. By incorporating disease-responsive elements (e.g. peptides) directly into the particle matrix we propose to impart enzyme-responsive release properties into these nanocarriers such that drugs or contrast agents are released primarily in response to tumor-associated signals. The specific aims for this two year period are: Aim 1: To develop a high throughput, top-down nano manufacturing process for fabrication of tumor-targeted, enzyme responsive nanocarriers of precise size and geometry (shape or aspect ratio). In this aim, a modified Step and Flash Imprint Lithography (S-FIL) technique will be employed to produce nanometer size particles of various sizes, cross-sectional shapes, and aspect ratios. The basic material for particle synthesis will be polyethylene glycol diacrylates (PEGDA) and the acrylated penta peptide GFLGK (peptide-DA), which is sensitive to lysosomal cysteine proteases (e.g. Cathepsin B). Cathepsin B is highly over expressed in a variety of cancers including non-small cell lung cancer (NSCLC). Aim 2: To evaluate the effects of size, shape, aspect ratios and macromer concentration on (a) in-vitro cellular uptake of imprinted nanocarriers and (b) intracellular release and transfection of model drugs and contrast agents from enzyme-responsive nanoparticles. We hypothesize that particle shapes, aspect ratio as well as nanoscale dimensions should significantly influence the efficiency of particle internalization. In addition, intracellular delivery of the encapsulated drugs as well as transfection efficacy of a model SiRNA drug will be studied using both fibroblast and lung cancer cells. Aim 3: To study bio- distribution of nanoimprinted particles of various size, shapes and aspect ratios in naove as well as tumor bearing animals. A major hypothesis in studying nanoparticles of various cross sectional shapes and aspect ratios is that these geometric parameters might have strong influence on particle transport properties in blood vessels and therefore should have significant impact on particle bio-distribution as well as tumor accumulation efficacy. Collectively these results should provide a strong basis for future studies on the delivery of therapeutic and diagnostic agents in animal models of various cancers. The goal of this two year exploratory project is to develop a nanoimprint lithography method for fabrication of smart drug delivery nanoparticles. This is a unique top down nanofabrication method for particle synthesis. These nanoparticles are designed to have specific size and shape that can influence their bio distribution. In addition the particles would be able to target tumor cells and deliver the cargo (drugs or imaging agents) to the tumor primarily in response to a disease-specific signal, e.g. enzyme up regulation.