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.