Development of antibacterial agents and materials

抗菌剂及材料的开发

• 批准号: 9153859
• 负责人: Joel Schneider
• 金额: $ 48.44万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9153859
• 关键词: Accounting; Addendum; Adhesives; Aldehydes; Amines; Amino Acids; Animal Model; Anti-Bacterial Agents; Arginine; Bacteria; Benchmarking; Biological Assay; Cancer cell line; Cell Death; Cell surface; Cells; Characteristics; Clinical; Collaborations; Crosslinker; Cytoplasm; Data; Development; Dextrans; Drug Formulations; Drug resistance; Equilibrium; Escherichia coli; Excision; Extracellular Matrix; Family; Family suidae; Fibrin Tissue Adhesive; Filler; Frequencies; Gel; Green Fluorescent Proteins; Head; Hydrogels; Hydrolysis; Immune; Implant; In Situ; Infection; Infection prevention; Injectable; Interleukin-2; Laser Scanning Confocal Microscopy; Life; Lipids; Literature; Lysine; Lytic; Malignant Neoplasms; Mammalian Cell; Maryland; Materials Testing; Measurement; Measures; Mechanics; Mediating; Membrane; Membrane Lipids; Mentors; Methods; Minor; Modeling; Molecular Weight; Monitor; Multi-Drug Resistance; Myoglobin; Nosocomial Infections; Nude Mice; Operative Surgical Procedures; Patients; Peptides; Pharmaceutical Preparations; Polyamines; Polymers; Polysaccharides; Property; Proteins; Relative (related person); Reporting; Research Personnel; Shapes; Side; Site; Skin; Solutions; Solvents; Structure-Activity Relationship; Sum; Surface; Syringes; System; Technology; Therapeutic; Time; Tissues; Tryptophan; Universities; Work; antimicrobial; bactericide; base; beta-Galactosidase; biomaterial compatibility; cancer cell; cancer therapy; crosslink; design; direct application; drug resistant bacteria; graduate student; implant coating; implantation; in vivo; killings; lactose permease; novel; open wound; oxidation; protein complex; protein crosslink; research study; resistant strain; therapeutic protein; tumor; wound

Aim 1: Investigate the mechanism of antibacterial action of the MAX1 hydrogel. To date, our data suggest a mechanism of antibacterial action that involves membrane disruption that leads to cell death upon cellular contact with the gel surface. Live-dead assays employing laser scanning confocal microscopy show that the gel's surface is bactericidal and that bacteria are quickly killed when they engage the surface. In addition, we showed that the gel surface causes inner and outer membrane disruption in experiments that monitor the release of beta-galactosidase from the cytoplasm of lactose permease-deficient E. coli ML-35. We have also shown that although soluble, non-gelled MAX1 shows antibacterial activity at high concentration in the gel state, there is little soluble peptide available and it is the gel's surface that exerts activity. Aim 2: Define how amino acid composition and sequence influences gel antibacterial activity. When self-assembled, the lysine side chains of MAX1 are displayed from the solvent exposed regions of its fibrils. Our mechanistic hypothesis suggests that these side chains are first to engage the bacterial cell surface and may account for much of the gel's activity. Review of the AMP literature indicates that although lysine is found in many AMPs, arginine (Arg) and tryptophan (Trp) are also found to a large extent. In some AMPs, when lysine is substituted with arginine, a more potent peptide is generated presumably due to the guanido side chain of Arg being able to form stronger interactions with lipid head groups contained in the bacteria's membrane. We have systematically replaced all of the Lys residues in MAX1 with Arg to study how Arg-content influences antibacterial activity. We found that peptides containing Arg form gels that are extremely effective at killing both gram-positive and gram-negative drug-resistant strains of bacteria. SAR studies show that an increase in Arg content correlates not only with an increase in antibacterial activity, but also an increase in hemolytic potential. However, by balancing the ratio of Arg relative to Lys content, we showed that potent, but selective, gels could be prepared. See addendum for sequences. Importantly, these studies show that the gel's antibacterial activity can be modulated by peptide design. Aim 3: Develop cell penetrating peptides for therapeutic delivery. We are developing a family of CPPs capable of preferentially delivering drugs to cancer cells based on their altered membrane and cell surface composition. This Aim evolved from our recent work where we designed a small lytic peptide (SVS-1) capable of preferentially killing cancer cells as described below. Design of Novel Bioadhesive Gels for the Local Delivery of Proteins Aim 1: Explore polysaccharide composition on cohesive, adhesive, and protein-release properties. We are initially exploring dextran as the polysaccharide component due to its biocompatibility and availability. In our initial studies, gels were prepared varying three parameters, namely, dextran molecular weight, aldehyde content, and the aldehyde/amine (CHO/NH2) network ratio to measure their effects on the cohesive and adhesive properties of resulting gels as well as the rate of gel formation. Cohesiveness and the rate of material formation were measured rheologically by assessing the storage moduli in time-, frequency-, and strain-sweep experiments. Gel adhesive strength was measured using tensile dynamic mechanical analysis employing porcine skin. Dextran molecular weight was varied from 15-70 kD, with 25-40 kD dextran providing gels with the highest storage moduli (G') and adhesive strength. We also optimized synthetic methods to vary the dialdehyde content from 25-50 % and found that although both G' and the adhesive strength increases with dialdehyde content, it is the adhesive properties that are most influenced. Lastly, using green fluorescent protein (GFP) as a model protein crosslinker, we showed that the storage modulus, adhesive strength, and rate of gelation all increase as the CHO/NH2 ratio decreases. This data indicates that formulations having more solvent accessible amines available for crosslinking will result in stronger, faster setting gels. This can be accomplished by increasing the wt% of a particular protein or using a protein having more Lys residues. In sum, we can vary each of these three parameters to prepare bioadhesive gels that range in storage moduli from 102-105 Pa and adhesive strengths from 1-6 kPa, which is on the order of fibrin glue, a clinical adhesive. We have determined that gels having moduli on the order of 105 and adhesive strengths of 4 kPa or greater adhere nicely to tissue affording well-defined shapes when introduced in vivo. In addition, the rate of material formation can be tuned from seconds to minutes after delivery from syringe. Gels that form too quickly are problematic, clogging the syringe, but gels that set in the regime of 10-20s are optimal. We use these mechanical characteristics as benchmarks when evaluating new materials. We have just begun to investigate the release properties of these gels using GFP as a model protein in bulk release studies and in vivo experiments. In these early experiments, only one gel composition was investigated (40 kD dextran, 25% oxidation, CHO/NH2 = 8). Bulk release experiments indicate that protein is released with a burst followed by a slow sustained release profile, with the protein remaining folded and functionally fluorescent. When 50 microliter of GFP-gel is formed in situ within the flanks of nude mice, biofluorescence measurements indicate that protein is released over a period 14d, with a similar release profile as that measured ex vivo. Our initial experiments suggest that proteins can be used directly to form bioadhesive gels for their own delivery. To our knowledge, these gels will be first-in-class as bioadhesive protein delivery vehicles. Aim 2: Determine the scope of proteins that can be delivered. In this aim, we investigate the scope of proteins that can be delivered using this technology. We also investigate the possibility of preparing composite gels comprised of two protein components where the majority of protein used for crosslinking is an inert, inexpensive filler protein and the minor component is a bioactive protein. This allows a lower concentration of highly active protein to be used in the formulation. Lastly, we propose a distinct material type in which we replace the protein component altogether, with polyamine polymers to construct antimicrobial, injectable wound fillers. To date, we have studied only three model proteins to quickly assess the feasibility of the technology, interleukin-2 (IL-2), GFP, and myoglobin. These proteins vary slightly in molecular weight (15-30 kD) and have similar pIs (6-7), but differ in their folds (alpha and beta-rich) and number of accessible Lys residues (11, 16, and 20 respectively). We showed that bioadhesive gels could be formed using any of these proteins and that that the number of solvent accessible lysines influences gel cohesive and adhesive properties. We also showed via CD and functional assays that released proteins remain folded and functional. Although the data is promising, all of these proteins are monomeric and structurally stable. We propose to challenge our delivery system with more complex proteins while exploring protein attributes that might influence the material's mechanical and release characteristics.

目标1：研究Max1水凝胶的抗菌作用机理。迄今为止，我们的数据提出了一种抗菌作用的机制，涉及膜破坏，导致细胞与凝胶表面接触时细胞死亡。使用激光扫描共聚焦显微镜进行的实时死亡测定表明，凝胶的表面是杀菌性的，并且细菌在参与表面时会很快杀死。此外，我们表明凝胶表面在监测β-半乳糖苷酶从乳糖渗透酶缺陷型大肠杆菌ML-35的细胞质中释放的实验中引起内膜和外膜破坏。我们还表明，尽管可溶的，非细胞的Max1在凝胶状态下高浓度下显示出抗菌活性，但几乎没有可溶肽可用，并且凝胶表面发挥了活性。目标2：定义氨基酸组成和序列如何影响凝胶抗菌活性。自组装时，从其原纤维的溶剂暴露区域显示了Max1的赖氨酸侧链。我们的机械假设表明，这些侧链是首先吸收细菌细胞表面的，并可能占凝胶的大部分活性。对AMP文献的评论表明，尽管在许多AMP，精氨酸（ARG）和色氨酸（TRP）中发现了赖氨酸，也很大程度上发现了赖氨酸。在某些放大器中，当赖氨酸被精氨酸取代时，可能会产生更有效的肽，这可能是由于guanido侧链的ARG能够与细菌膜中包含的脂质头基形成更强的相互作用。我们已经系统地替换了Max1中的所有LYS残基，以研究Arg-Content如何影响抗菌活性。我们发现，含有ARG形成凝胶的肽在杀死革兰氏阳性和革兰氏阴性抗药性细菌菌株方面非常有效。 SAR研究表明，ARG含量的增加不仅与抗菌活性的增加相关，而且还与溶血潜力的增加相关。但是，通过平衡ARG相对于LYS含量的比率，我们表明可以制备有效但有选择性的凝胶。有关序列，请参见附录。重要的是，这些研究表明，可以通过肽设计调节凝胶的抗菌活性。 AIM 3：开发细胞穿透性肽进行治疗。我们正在开发一个能够根据其改变的膜和细胞表面组成的癌细胞优先将药物输送到癌细胞的家族。这个目标是从最近的工作中演变而来的，我们设计了一个小型裂解肽（SVS-1），能够优先杀死癌细胞，如下所述。蛋白质局部递送的新型生物粘附凝胶的设计：探索在粘性，粘合剂和蛋白质释放特性上的多糖组成。最初，由于其生物相容性和可用性，我们最初将葡萄糖作为多糖成分。在我们的最初研究中，制备了三个参数的凝胶，即葡聚糖分子量，醛含量和醛/胺（CHO/NH2）网络比率，以测量其对产生凝胶的凝聚力和粘合性能的影响，以及凝胶形成的速率。通过在时间，频率和扫描实验中评估储存模量，通过评估凝聚力和材料形成速率。使用猪皮肤的拉伸动态机械分析测量凝胶粘合强度。右旋葡萄糖分子量从15-70 kD变化，其中25-40 kD葡萄糖提供具有最高储存模量（G'）和粘合强度的凝胶。我们还优化了合成方法将二醛含量从25-50％改变，但发现G'和粘合强度都随透气含量而增加，但受影响最大的粘合特性。最后，使用绿色荧光蛋白（GFP）作为模型蛋白交联链，我们表明储存模量，粘合强度和凝胶速率都随着CHO/NH2比的降低而增加。该数据表明，具有更溶剂可访问的胺可用于交联的配方将导致更强，更快的设置凝胶。这可以通过增加特定蛋白质的WT％或使用具有更多LYS残基的蛋白质来实现。总而言之，我们可以改变这三个参数中的每一个，以制备储存模量范围从102-105 PA的生物粘附凝胶，粘附强度从1-6 kPa（纤维蛋白胶（一种临床粘合剂））的1-6 kPa。我们已经确定，具有105次的模量的凝胶和4 kPa或更大的粘合强度粘附在体内引入时提供良好定义形状的粘附力。另外，从注射器输送后几秒钟到几分钟，材料形成速率。形成太快的凝胶是有问题的，堵塞了注射器，但是设定在10-20s的机制下的凝胶是最佳的。评估新材料时，我们将这些机械特性用作基准。我们刚刚开始使用GFP作为大量释放研究和体内实验中的模型蛋白来研究这些凝胶的释放特性。在这些早期实验中，仅研究了一个凝胶组成（40 kD葡萄糖，25％氧化，cho/nh2 = 8）。大量释放实验表明，蛋白质被释放，随后释放，随后释放缓慢的持续释放曲线，蛋白质保持折叠且功能性荧光。当裸鼠侧面的原位形成50微晶GFP凝胶时，生物荧光测量表明蛋白质在14d期间释放，其释放曲线与测量的离体相似。我们的最初实验表明，蛋白质可直接用于形成生物粘附凝胶以进行自身递送。据我们所知，这些凝胶将作为生物粘附蛋白递送车的第一类。目标2：确定可以传递的蛋白质范围。在此目标中，我们研究了可以使用该技术传递的蛋白质范围。我们还研究了制备由两个蛋白质成分组成的复合凝胶的可能性，其中大多数用于交联的蛋白质是惰性，廉价的填充蛋白，而次要成分是生物活性蛋白。这允许在制剂中使用较低的高度活性蛋白。最后，我们提出了一种不同的材料类型，在其中完全取代了蛋白质成分，并用多胺聚合物构建抗菌剂，可注射的伤口填充剂。迄今为止，我们仅研究了三种模型蛋白，以快速评估该技术，白介素-2（IL-2），GFP和肌红蛋白的可行性。这些蛋白质的分子量略有不同（15-30 kD），并且具有相似的PI（6-7），但折叠（富含α和β）和可访问LYS残基的数量（分别为11、16和20）。我们表明，可以使用任何这些蛋白质中的任何一种形成生物粘附凝胶，并且溶剂可及赖氨酸的数量会影响凝胶凝聚力和粘附性能。我们还通过CD和功能测定表明，释放蛋白质保持折叠和功能。尽管数据是有希望的，但所有这些蛋白质都是单体且在结构上稳定的。我们建议使用更复杂的蛋白质来挑战我们的递送系统，同时探索可能影响材料机械和释放特性的蛋白质属性。