Development of antibacterial agents and materials

抗菌剂及材料的开发

• 批准号: 8763449
• 负责人: Joel Schneider
• 金额: $ 37.17万
• 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/8763449
• 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; 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; dextran; 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制。迄今为止，我们的数据表明抗菌作用机制涉及膜破坏，细胞与凝胶表面接触后会导致细胞死亡。采用激光扫描共聚焦显微镜进行的活死分析表明，凝胶的表面具有杀菌作用，细菌在接触表面时会很快被杀死。此外，我们在监测乳糖通透酶缺陷的大肠杆菌 ML-35 细胞质中释放 β-半乳糖苷酶的实验中发现，凝胶表面会导致内膜和外膜破裂。我们还表明，虽然可溶性、非凝胶化的 MAX1 在凝胶状态下在高浓度下显示出抗菌活性，但可用的可溶性肽很少，并且发挥活性的是凝胶的表面。目标 2：定义氨基酸组成和序列如何影响凝胶抗菌活性。当自组装时，MAX1 的赖氨酸侧链从其原纤维的溶剂暴露区域显示出来。我们的机制假设表明，这些侧链首先与细菌细胞表面结合，并可能解释了凝胶的大部分活性。回顾 AMP 文献表明，虽然许多 AMP 中都含有赖氨酸，但精氨酸 (Arg) 和色氨酸 (Trp) 也大量存在。在一些 AMP 中，当赖氨酸被精氨酸取代时，可能会产生更有效的肽，因为精氨酸的胍基侧链能够与细菌膜中包含的脂质头基团形成更强的相互作用。我们系统地用 Arg 替换了 MAX1 中的所有 Lys 残基，以研究 Arg 含量如何影响抗菌活性。我们发现含有精氨酸的肽形成凝胶，能够极其有效地杀死革兰氏阳性和革兰氏阴性耐药菌株。 SAR 研究表明，Arg 含量的增加不仅与抗菌活性的增加相关，而且与溶血潜力的增加相关。然而，通过平衡精氨酸相对于赖氨酸含量的比例，我们表明可以制备有效且具有选择性的凝胶。请参阅附录了解序列。重要的是，这些研究表明凝胶的抗菌活性可以通过肽设计来调节。目标 3：开发用于治疗递送的细胞穿透肽。我们正在开发一系列 CPP，能够根据癌细胞膜和细胞表面成分的改变优先向癌细胞输送药物。这一目标源自我们最近的工作，我们设计了一种能够优先杀死癌细胞的小裂解肽（SVS-1），如下所述。用于蛋白质局部递送的新型生物粘附凝胶的设计目标 1：探索多糖成分的内聚性、粘附性和蛋白质释放特性。由于其生物相容性和可用性，我们最初正在探索葡聚糖作为多糖成分。在我们的初步研究中，凝胶是通过改变三个参数来制备的，即葡聚糖分子量、醛含量和醛/胺 (CHO/NH2) 网络比率，以测量它们对所得凝胶的内聚和粘合性能的影响以及凝胶形成速率。通过评估时间、频率和应变扫描实验中的储能模量，以流变学方式测量内聚性和材料形成速率。使用猪皮的拉伸动态机械分析来测量凝胶粘合强度。葡聚糖分子量为 15-70 kD，其中 25-40 kD 葡聚糖提供具有最高储能模量 (G') 和粘合强度的凝胶。我们还优化了合成方法，使二醛含量在 25-50% 范围内变化，发现虽然 G' 和粘合强度都随着二醛含量的增加而增加，但影响最大的是粘合性能。最后，使用绿色荧光蛋白（GFP）作为模型蛋白质交联剂，我们发现储能模量、粘合强度和凝胶化速率均随着 CHO/NH2 比例的降低而增加。该数据表明，具有更多可用于交联的溶剂可及胺的制剂将产生更强、更快凝固的凝胶。这可以通过增加特定蛋白质的重量％或使用具有更多赖氨酸残基的蛋白质来实现。总之，我们可以改变这三个参数中的每一个来制备生物粘附凝胶，其储能模量为 102-105 Pa，粘附强度为 1-6 kPa，与临床粘合剂纤维蛋白胶相当。我们已经确定，模量约为 105 且粘合强度为 4 kPa 或更大的凝胶可以很好地粘附到组织上，当引入体内时可提供明确的形状。此外，从注射器输送后，材料形成的速率可以从几秒调整到几分钟。凝胶形成过快会产生问题，会堵塞注射器，但凝胶在 10-20 秒内凝固是最佳的。我们在评估新材料时使用这些机械特性作为基准。我们刚刚开始在批量释放研究和体内实验中使用 GFP 作为模型蛋白来研究这些凝胶的释放特性。在这些早期实验中，仅研究了一种凝胶成分（40 kD 葡聚糖，25% 氧化，CHO/NH2 = 8）。大量释放实验表明，蛋白质先爆发式释放，然后缓慢持续释放，蛋白质保持折叠状态并发出功能性荧光。当 50 微升 GFP-凝胶在裸鼠胁腹内原位形成时，生物荧光测量表明蛋白质在 14 天的时间内释放，具有与离体测量相似的释放曲线。我们的初步实验表明，蛋白质可以直接用于形成生物粘附凝胶以进行自身递送。据我们所知，这些凝胶将成为一流的生物粘附蛋白递送载体。目标 2：确定可输送的蛋白质范围。为此，我们研究了使用该技术可以输送的蛋白质的范围。我们还研究了制备由两种蛋白质成分组成的复合凝胶的可能性，其中用于交联的大部分蛋白质是惰性、廉价的填充蛋白，次要成分是生物活性蛋白质。这使得配方中可以使用较低浓度的高活性蛋白质。最后，我们提出了一种独特的材料类型，用聚胺聚合物完全取代蛋白质成分来构建抗菌、可注射伤口填充物。迄今为止，我们仅研究了三种模型蛋白来快速评估该技术的可行性：白细胞介素-2 (IL-2)、GFP 和肌红蛋白。这些蛋白质的分子量略有不同 (15-30 kD)，并且具有相似的 pIs (6-7)，但其折叠数（富含 α 和 β）和可接近的赖氨酸残基数量（分别为 11、16 和 20）不同。 。我们表明，可以使用这些蛋白质中的任何一种来形成生物粘附凝胶，并且溶剂可及的赖氨酸的数量影响凝胶的内聚性和粘附性。我们还通过 CD 和功能测定表明释放的蛋白质保持折叠和功能。尽管数据很有希望，但所有这些蛋白质都是单体且结构稳定。我们建议用更复杂的蛋白质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。