Christopher R. Loose

Controlling the release of peptide antimicrobial agents from surfaces

Medical conditions are often exacerbated by the onset of infection caused by hospital dwelling bacteria such as Staphylococcus aureus. Antibiotics taken orally or intravenously can require large and frequent doses, further contributing to the sharp rise in resistant bacteria observed over the past several decades. These existing antibiotics are also often ineffective in preventing biofilm formation, a common cause of medical device failure. Local delivery of new therapeutic agents that do not allow bacterial resistance to occur, such as antimicrobial peptides, could alleviate many of the problems associated with current antibacterial treatments. By taking advantage of the versatility of layer-by-layer assembly of polymer thin films, ponericin G1, an antimicrobial peptide known to be highly active against S. aureus, was incorporated into a hydrolytically degradable polyelectrolyte multilayer film. Several film architectures were examined to obtain various drug loadings that ranged from 20 to 150 microg/cm2. Release was observed over approximately ten days, with varying release profiles, including burst as well as linear release. Results indicated that film-released peptide did not suffer any loss in activity against S. aureus and was able to inhibit bacteria attachment, a necessary step in preventing biofilm formation. Additionally, all films were found to be biocompatible with the relevant wound healing cells, NIH 3T3 fibroblasts and human umbilical vein endothelial cells. These films provide the level of control over drug loading and release kinetics required in medically relevant applications including coatings for implant materials and bandages, while eliminating susceptibility to bacterial resistance.

A linguistic model for the rational design of antimicrobial peptides

Antimicrobial peptides (AmPs) are small proteins that are used by the innate immune system to combat bacterial infection in multicellular eukaryotes. There is mounting evidence that these peptides are less susceptible to bacterial resistance than traditional antibiotics and could form the basis for a new class of therapeutic agents. Here we report the rational design of new AmPs that show limited homology to naturally occurring proteins but have strong bacteriostatic activity against several species of bacteria, including Staphylococcus aureus and Bacillus anthracis. These peptides were designed using a linguistic model of natural AmPs: we treated the amino-acid sequences of natural AmPs as a formal language and built a set of regular grammars to describe this language. We used this set of grammars to create new, unnatural AmP sequences. Our peptides conform to the formal syntax of natural antimicrobial peptides but populate a previously unexplored region of protein sequence space.

Vascular Catheters with a Nonleaching Poly-Sulfobetaine Surface Modification Reduce Thrombus Formation and Microbial Attachment

Poly-sulfobetaine surface modification on a vascular catheter inhibits microbial adherence and thrombosis. Surface Modification Pulls Double Duty Marketing experts know that consumers can’t resist a good deal: Two-for-one; buy one, get one (or BOGO, if you’re up-to-date on your lingo); twice as nice, half the price. Similarly, engineers know that, if they can package twice the functionality in one medical device, they have a valuable product that clinicians cannot ignore. In this issue, Smith et al. designed one polymer coating for catheters that resolves two major challenges in biomaterials by preventing both blood clot accumulation (thrombosis) and bacterial adhesion (infection). Smith and colleagues created the zwitterionic polymer “polySB” (poly-sulfobetaine) surface modification, which, they hypothesized, can coordinate water and therefore resist protein adsorption and cell adhesion. PolySB was used to modify the inner and outer surfaces of common polyurethane peripherally inserted central catheters (PICCs). In vitro, the polySB-modified catheter reduced adherence and activation of human red and white blood cells compared with commercially available PICCs without the polySB surface. In addition, modified PICCs that had been soaked in serum for 60 days displayed no thrombus accumulation when exposed to bovine blood, thus demonstrating the long-term activity of the polySB. In vivo, in a canine model, polySB-modified PICCs had little thrombus accumulation: a reduction of ~99% compared to unmodified control devices. Last, polySB-modified PICCs showed up to 99.9% reduction in microbial attachment (both Gram-positive and Gram-negative bacteria) compared to unmodified PICCs; in rabbits, this translated to less inflammation. By preventing both infection and thrombosis, this multifunctional polymeric coating is just the BOGO the doctor ordered. Adherence of proteins, cells, and microorganisms to the surface of venous catheters contributes to catheter occlusion, venous thrombosis, thrombotic embolism, and infections. These complications lengthen hospital stays and increase patient morbidity and mortality. Current technologies for inhibiting these complications are limited in duration of efficacy and may induce adverse side effects. To prevent complications over the life span of a device without using active drugs, we modified a catheter with the nonleaching polymeric sulfobetaine (polySB), which coordinates water molecules to the catheter surface. The modified surface effectively reduced protein, mammalian cell, and microbial attachment in vitro and in vivo. Relative to commercial catheters, polySB-modified catheters exposed to human blood in vitro had a >98% reduction in the attachment and a significant reduction in activation of platelets, lymphocytes, monocytes, and neutrophils. Additionally, the accumulation of thrombotic material on the catheter surface was reduced by >99% even after catheters were exposed to serum in vitro for 60 days. In vivo, in a highly thrombogenic canine model, device- and vessel-associated thrombus was reduced by 99%. In vitro adherence of a broad spectrum of microorganisms was reduced on both the external and the internal surfaces of polySB-modified catheters compared to unmodified catheters. When unmodified and polySB-modified catheters were exposed to the same bacterial challenge and implanted into animals, 50% less inflammation and fewer bacteria were associated with polySB-modified catheters. This nonleaching, polySB-modified catheter could have a major impact on reducing thrombosis and infection, thus improving patient health.

Rapid, nondestructive estimation of surface polymer layer thickness using attenuated total reflection fourier transform infrared (ATR FT-IR) spectroscopy and synthetic spectra derived from optical principles.

We have developed a rapid, nondestructive analytical method that estimates the thickness of a surface polymer layer with high precision but unknown accuracy using a single attenuated total reflection Fourier transform infrared (ATR FT-IR) measurement. Because the method is rapid, nondestructive, and requires no sample preparation, it is ideal as a process analytical technique. Prior to implementation, the ATR FT-IR spectrum of the substrate layer pure component and the ATR FT-IR and real refractive index spectra of the surface layer pure component must be known. From these three input spectra a synthetic mid-infrared spectral matrix of surface layers 0 nm to 10 000 nm thick on substrate is created de novo. A minimum statistical distance match between a process samples ATR FT-IR spectrum and the synthetic spectral matrix provides the thickness of that sample. We show that this method can be used to successfully estimate the thickness of polysulfobetaine surface modification, a hydrated polymeric surface layer covalently bonded onto a polyetherurethane substrate. A database of 1850 sample spectra was examined. Spectrochemical matrix-effect unknowns, such as the nonuniform and molecularly novel polysulfobetaine-polyetherurethane interface, were found to be minimal. A partial least squares regression analysis of the database spectra versus their thicknesses as calculated by the method described yielded an estimate of precision of ±52 nm.

Optimization of protein fusion partner length for maximizing in vitro translation of peptides.

Using protein fusion partners for in vitro translation may increase solubility, assist in purification, or allow detection of small proteins and peptides. Here we show that the molar yield of peptide in a batch reaction may be maximized by optimizing the length of the translated product, which is composed of the fusion partner plus the peptide. Using truncated versions of GFP as a series of fusion partners, the molar yield increased approximately 3‐fold as the length of the translated product was reduced from 250 to 100 amino acids. When the translated product was shortened below roughly 100 amino acids, molar yield fell as a result of proteolysis. This trend was verified using two fusion partners with different amino acid sequences. Furthermore, protease inhibitors were used to confirm that proteases were responsible for limiting accumulation of peptides below the optimal length.

Anti-infection trauma devices with drug release and nonfouling surface modification.

Objectives: By coupling an antimicrobial release with a highly nonfouling betaine modification on titanium, this approach innovatively addresses the initial bacterial challenge and the longer term biofilm formation on trauma devices. Methods: Titanium substrates were modified to obtain a polymer reservoir for chlorhexidine (CHX) and a polybetaine surface layer. The surface was characterized by infrared spectroscopy, scanning electron microscopy, laser confocal scanning microscopy, and a radiolabeled fibrinogen assay. The in vitro drug release profiles were measured using an ultraviolet-visible spectroscopy and a high-performance liquid chromatography. The efficacy to inhibit surface biofilm formation was determined by a bacterial adherence assay. The surface modifications bonding strength to the titanium substrate was measured, and its resistance to abrasion was tested ex vivo. Additionally, the biocompatibility was tested after ISO 10993 procedures. Results: Titanium surfaces were successfully modified with a conformal and strongly bound polymer layer. No scratches were observed when inserting the modified titanium wires into porcine femur, and preservation of modification was confirmed by infrared spectroscopy. Controlled release of CHX was demonstrated for more than 8 weeks, and different formulations were tailored for different release rates. Greater than 3 log (99.9%) reductions in bacterial adherence were achieved after serum exposure. Additionally, the nonfouling properties were retained after several weeks of CHX release. Modified materials passed ISO 10993 testing for permanent implant devices. Conclusions: By innovatively addressing the initial bacterial challenge and longer term biofilm formation on trauma devices, this approach may be a superior solution to the current biofilm control technology.

From student to entrepreneur

Graduate student breaks into biotechnology.