Polymerized cyclodextrin microparticles for sustained antibiotic delivery in lung infections.

Pulmonary infections complicate chronic lung diseases requiring attention to both the pathophysiology and complexity associated with infection management. Patients with cystic fibrosis (CF) struggle with continuous bouts of pulmonary infections, contributing to lung destruction and eventual mortality. Additionally, CF patients struggle with airways that are highly viscous, with accumulated mucus creating optimal environments for bacteria colonization. The unique physiology and altered airway environment provide an ideal niche for bacteria to change their phenotype often becoming resistant to current treatments. Colonization with multiple pathogens at the same time further complicate treatment algorithms, requiring drug combinations that can challenge CF patient tolerance to treatment. The goal of this research initiative was to explore the utilization of a microparticle antibiotic delivery system, which could provide localized and sustained antibiotic dosing. The outcome of this work demonstrates the feasibility of providing efficient localized delivery of antibiotics to manage infection using both preclinical in vitro and in vivo CF infection models. The studies outlined in this manuscript demonstrate the proof-of-concept and unique capacity of polymerized cyclodextrin microparticles to provide site-directed management of pulmonary infections.

Periadventitial Delivery of Simvastatin-Loaded Microparticles Attenuate Venous Neointimal Hyperplasia Associated With Arteriovenous Fistula.

Background Venous neointimal hyperplasia and venous stenosis (VS) formation can result in a decrease in arteriovenous fistula (AVF) patency in patients with end-stage renal disease. There are limited therapies that prevent VNH/VS. Systemic delivery of simvastatin has been shown to reduce VNH/VS but local delivery may help decrease the side effects associated with statin use. We determined if microparticles (MP) composed of cyclodextrins loaded with simvastatin (MP-SV) could reduce VS/VNH using a murine arteriovenous fistula model with chronic kidney disease. Methods and Results Male C57BL/6J mice underwent nephrectomy to induce chronic kidney disease. Four weeks later, an arteriovenous fistula was placed and animals were randomized to 3 groups: 20 µL of PBS or 20 µL of PBS with 16.6 mg/mL of either MP or MP-SV. Animals were euthanized 3 days later and the outflow veins were harvested for quantitative reverse transcriptase-polymerase chain reaction analysis and 28 days later for immunohistochemistical staining with morphometric analysis. Doppler ultrasound was performed weekly. Gene expression of vascular endothelial growth factor-A (Vegf-A), matrix metalloproteinase-9 (Mmp-9), transforming growth factor beta 1 (Tgf-ß1), and monocyte chemoattractant protein-1 (Mcp-1) were significantly decreased in MP-SV treated vessels compared with controls. There was a significant decrease in the neointimal area, cell proliferation, inflammation, and fibrosis, with an increase in apoptosis and peak velocity in MP-SV treated outflow veins. MP-SV treated fibroblasts when exposed to hypoxic injury had decreased gene expression of Vegf-A and Mmp-9. Conclusions In experimental arteriovenous fistulas, periadventitial delivery of MP-SV decreased gene expression of Vegf-A, Mmp-9, Tgf-ß1 and Mcp-1, VNH/VS, inflammation, and fibrosis.

Resveratrol Delivery from Implanted Cyclodextrin Polymers Provides Sustained Antioxidant Effect on Implanted Neural Probes.

Intracortical microelectrodes are valuable tools used to study and treat neurological diseases. Due in large part to the oxidative stress and inflammatory response occurring after electrode implantation, the signal quality of these electrodes decreases over time. To alleviate this response, resveratrol, a natural antioxidant which elicits neuroprotective effects through reduction of oxidative stress, was utilized. This work compares traditional systemic delivery of resveratrol to the novel cyclodextrin polymer (pCD) local delivery approach presented herein, both in vitro and in vivo. The pCD displayed an extended resveratrol release for 100 days, as well as 60 days of free radical scavenging activity in vitro. In vivo results indicated that our pCD delivery system successfully delivered resveratrol to the brain with a sustained release for the entire short-duration study (up to 7 days). Interestingly, significantly greater concentrations of resveratrol metabolites were found at the intracortical probe implantation site compared to the systemic administration of resveratrol. Together, our pilot results provide support for the possibility of improving the delivery of resveratrol in an attempt to stabilize long-term neural interfacing applications.

Elucidating the Structure-Function Relationship of Solvent and Cross-Linker on Affinity-Based Release from Cyclodextrin Hydrogels.

Minocycline (MNC) is a tetracycline antibiotic capable of associating with cyclodextrin (CD), and it is a frontline drug for many instances of implant infection. Due to its broad-spectrum activity and long half-life, MNC represents an ideal drug for localized delivery; however, classic polymer formulations, particularly hydrogels, result in biphasic release less suitable for sustained anti-microbial action. A polymer delivery system capable of sustained, steady drug delivery rates poses an attractive target to maximize the antimicrobial activity of MNC. Here, we formed insoluble hydrogels of polymerized CD (pCD) with a range of crosslinking densities, and then assessed loading, release, and antimicrobial activity of MNC. MNC loads between 5-12 wt % and releases from pCD hydrogels for >14 days. pCD loaded with MNC shows extended antimicrobial activity against S. aureus for >40 days and E. coli for >70 days. We evaluated a range of water/ethanol blends to test our hypothesis that solvent polarity will impact drug-CD association as a function of hydrogel swelling and crosslinking. Increased polymer crosslinking and decreased solvent polarity both reduced MNC loading, but solvent polarity showed a dramatic reduction independent of hydrogel swelling. Due to its high solubility and excellent delivery profile, MNC represents a unique drug to probe the structure-function relationship between drug, affinity group, and polymer crosslinking ratio.

Local delivery polymer provides sustained antifungal activity of amphotericin B with reduced cytotoxicity.

IMPACT STATEMENT: Amphotericin B (AmB) is an effective and commonly used antifungal agent. However, nephrotoxicity and poor solubility limits its usage. The proposed polymerized cyclodextrin (pCD) system therefore is an attractive method for AmB delivery, as it retains the antifungal activity of AmB while decreasing toxicity, and confining drug release to the local environment. This system could potentially be used for both prevention and treatment of established fungal infections, as AmB is toxic to fungus whether associated or released from pCD.

Evaluation of an in vivo model for ventricular shunt infection: a pilot study using a novel antimicrobial-loaded polymer.

OBJECTIVE: Ventricular shunt infection remains an issue leading to high patient morbidity and cost, warranting further investigation. The authors sought to create an animal model of shunt infection that could be used to evaluate possible catheter modifications and innovations. METHODS: Three dogs underwent bilateral ventricular catheter implantation and inoculation with methicillin-sensitive Staphylococcus aureus (S. aureus). In 2 experimental animals, the catheters were modified with a polymer containing chemical "pockets" loaded with vancomycin. In 1 control animal, the catheters were polymer coated but without antibiotics. Animals were monitored for 9 to 11 days, after which the shunts were explanted. MRI was performed after shunt implantation and prior to catheter harvest. The catheters were sonicated prior to microbiological culture and also evaluated by electron microscopy. The animals' brains were evaluated for histopathology. RESULTS: All animals underwent successful catheter implantation. The animals developed superficial wound infections, but no neurological deficits. Imaging demonstrated ventriculitis and cerebral edema. Harvested catheters from the control animal demonstrated > 104 colony-forming units (CFUs) of S. aureus. In the first experimental animal, one shunt demonstrated > 104 CFUs of S. aureus, but the other demonstrated no growth. In the second experimental animal, one catheter demonstrated no growth, and the other grew trace S. aureus. Brain histopathology revealed acute inflammation and ventriculitis in all animals, which was more severe in the control. CONCLUSIONS: The authors evaluated an animal model of ventricular shunting and reliably induced features of shunt infection that could be microbiologically quantified. With this model, investigation of pathophysiological and imaging correlates of infection and potentially beneficial shunt catheter modifications is possible.

Infection prevention using affinity polymer-coated, synthetic meshes in a pig hernia model.

BACKGROUND: Given concern for hernia mesh infection, surgeons often use biologic mesh which may provide reduced risk of infection but at the cost of decreased repair durability. We evaluated mesh coating to provide sustained release of antibiotics to prevent prosthetic mesh infection and also allow a durable repair. MATERIALS AND METHODS: Cyclodextrin-based polymer was crosslinked onto multifilament polyester mesh and loaded with vancomycin (1.75 mg/cm2). Pigs received modified meshes (n = 6) or normal, untreated meshes (n = 4), which were implanted into acute 10 × 5 cm ventral hernia, then directly inoculated with 106 colony-forming unit (CFU) of methicillin-resistant Staphylococcus aureus (MRSA). These were compared to animals receiving normal, uninfected mesh. All mesh was secured in an underlay bridge manner, and after 30 d, the abdominal wall was removed for quantitative bacterial culture and biomechanical analysis. RESULTS: All animals survived 30 d. All six animals with coated mesh cleared MRSA infection. The four control animals did not clear MRSA (P = 0.005). Quantitative bacterial load was higher in standard mesh versus drug-delivery mesh group (2.34 × 104versus 80.9 CFU/gm). These data were log10-transformed and analyzed by Welch's t-test (P = 0.001). Minimum number of CFUs detectable by assay (300) was used instead of zero. Biomechanical analysis of controls (1.82 N/mm infected; 1.71 N/mm uninfected) showed no difference to the modified meshes (1.31 N/mm) in tissue integration (P = 0.15). CONCLUSIONS: We successfully prevented synthetic mesh infection in a pig model using a cyclodextrin-based polymer to locally deliver vancomycin to the hernia repair site and clearing antibiotic-resistant bacteria. Polymer coating did not impact the strength of the hernia repair.

Affinity interactions drive post-implantation drug filling, even in the presence of bacterial biofilm.

Current post-operative standard of care for surgical procedures, including device implantations, dictates prophylactic antimicrobial therapy, but a percentage of patients still develop infections. Systemic antimicrobial therapy needed to treat such infections can lead to downstream tissue toxicities and generate drug-resistant bacteria. To overcome issues associated with systemic drug administration, a polymer incorporating specific drug affinity has been developed with the potential to be filled or refilled with antimicrobials, post-implantation, even in the presence of bacterial biofilm. This polymer can be used as an implant coating or stand-alone drug delivery device, and can be translated to a variety of applications, such as implanted or indwelling medical devices, and/or surgical site infections. The filling of empty affinity-based drug delivery polymer was analyzed in an in vitro filling/refilling model mimicking post-implantation tissue conditions. Filling in the absence of bacteria was compared to filling in the presence of bacterial biofilms of varying maturity to demonstrate proof-of-concept necessary prior to in vivo experiments. Antibiotic filling into biofilm-coated affinity polymers was comparable to drug filling seen in same affinity polymers without biofilm demonstrating that affinity polymers retain ability to fill with antibiotic even in the presence of biofilm. Additionally, post-implantation filled antibiotics showed sustained bactericidal activity in a zone of inhibition assay demonstrating post-implantation capacity to deliver filled antibiotics in a timeframe necessary to eradicate bacteria in biofilms. This work shows affinity polymers can fill high levels of antibiotics post-implantation independent of biofilm presence potentially enabling device rescue, rather than removal, in case of infection. STATEMENT OF SIGNIFICANCE: Post-operative prophylactic antimicrobial therapy greatly reduces risk of infection, such as on biomedical implants, but does not totally eliminate infections, and the healthcare cost of these remaining infections remains a major concern. Systemic antimicrobial therapy to treat these infections can lead to tissue toxicity and drug-resistant bacteria. In order to treat only those patients who have developed infections, a customizable antimicrobial delivery system made of cyclodextrin-based affinity polymer has been developed that is capable of filling post-implantation and delivering the filled antibiotic in a sustained manner even when the delivery device covered in bacterial biofilm. These observations have the potential to be translated to a wide variety of applications, such as implanted or indwelling medical devices, and/or surgical site infections.

Antibiotic-releasing microspheres prevent mesh infection in vivo.

BACKGROUND: Infection remains a dreaded complication after implantation of surgical prosthetics, particularly after hernia repair with synthetic mesh. We previously demonstrated the ability of a newly developed polymer to provide controlled release of an antibiotic in a linear fashion over 45 d. We subsequently showed that coating mesh with the drug-releasing polymer prevented a Staphylococcus aureus (SA) infection in vivo. To broaden the applicability of this technology, the polymer was synthesized as isolated "microspheres" and loaded with vancomycin (VM) before conducting a noninferiority analysis. MATERIALS AND METHODS: Seventy-three mice underwent creation of a dorsal subcutaneous pocket that was inoculated with 104 colony forming units (CFU) of green fluorescent protein (GFP)-labeled SA (105 CFU/mL). Multifilament polyester mesh (7 × 7 mm) was placed into the pocket, and the skin was closed. Mesh was either placed alone (n = 16), coated with VM-loaded polymer (n = 20), placed next to VM-loaded microspheres (n = 20) or unloaded microspheres (n = 10), or flushed with VM solution (n = 7). Quantitative tissue/mesh cultures were performed at 2 and 4 week. Mice with open wounds and explanted mesh were excluded. RESULTS: Twenty-two of 23 (96%) tissue-mesh samples from mesh alone or empty miscrospheres were positive for GFP-labeled SA at 2 and 4 wk. Six of seven (86%) samples from the VM flush group were positive for GFP SA at 4 wk. Thirty-eight of 38 (100%) VM-loaded crosslinked cyclodextrin polymers-coated mesh or VM-loaded microspheres were negative for GFP SA at 2 and 4 wk. CONCLUSIONS: Slow affinity-based drug-releasing polymers in the form of microspheres are able to adequately clear a bacterial burden of SA and prevent mesh infection.

Improved conduction and increased cell retention in healed MI using mesenchymal stem cells suspended in alginate hydrogel.

INTRODUCTION: Mesenchymal stem cells (MSCs) have been associated with reduced arrhythmias; however, the mechanism of this action is unknown. In addition, limited retention and survival of MSCs can significantly reduce efficacy. We hypothesized that MSCs can improve impulse conduction and that alginate hydrogel will enhance retention of MSCs in a model of healed myocardial infarction (MI). METHODS AND RESULTS: Four weeks after temporary occlusion of the left anterior descending artery (LAD), pigs (n = 13) underwent a sternotomy to access the infarct and then were divided into two studies. In study 1, designed to investigate impulse conduction, animals were administered, by border zone injection, 9-15 million MSCs (n = 7) or phosphate-buffered saline (PBS) (control MI, n = 5). Electrogram width measured in the border zone 2 weeks after injections was significantly decreased with MSCs (-30 ± 8 ms, p < 0.008) but not in shams (4 ± 10 ms, p = NS). Optical mapping from border zone tissue demonstrated that conduction velocity was higher in regions with MSCs (0.49 ± 0.03 m/s) compared to regions without MSCs (0.39 ± 0.03 m/s, p < 0.03). In study 2, designed to investigate MSC retention, animals were administered an equal number of MSCs suspended in either alginate (2 or 1 % w/v) or PBS (n = 6/group) by border zone injection. Greater MSC retention and survival were observed with 2% alginate compared to PBS or 1% alginate. Confocal immunofluorescence demonstrated that MSCs survive and are associated with expression of connexin-43 (Cx43) for either PBS (control), 1%, or 2% alginate. CONCLUSIONS: For the first time, we are able to directly associate MSCs with improved impulse conduction and increased retention and survival using an alginate scaffold in a clinically relevant model of healed MI.

Identification of regulatory Hck and PAI-2 proteins in the monocyte response to PEG-containing matrices.

Mass spectrometry is a powerful proteomic tool enabling researchers to survey the global proteome of a cell. This technique has only recently been employed to investigate cell-material interactions. We had previously identified material scarcity and limited adherent cells as challenges facing mass spectrometric analysis of cell-material interactions. U937 adherent to tissue culture poly(styrene) was used as a model system for identifying proteins expressed by adherent monocytes and analyzed by HPLC coupled offline to MALDI-ToF/ToF (LC-MALDI). We identified 645 proteins from two cation fractions of crude U937 monocyte cell lysate. Forty three proteins of interest from the 645 were chosen based on literature searches for relevance to monocyte-material inflammation and wound healing. Proteins such as 40S ribosomal protein S19 and tyrosyl tRNA synthetase highlight the ability of LC-MALDI to identify proteins relevant to monocyte-material interactions that are currently unexplored. We used PEG-based semi-interpenetrating polymer networks and PEG-only hydrogels to investigate surface dependent effects on the Src family kinase Hck and plasminogen activator inhibitor-2 (PAI-2) using the pyrazolo pyrimidine small molecule inhibitor PP2 and exogenous urokinase plasminogen activator addition, respectively. Hck is well researched in cell adhesion while PAI-2 is virtually unknown in cell-material interactions. U937 on TCPS and PEG-only hydrogels secreted similar levels of inflammatory cytokines and gelatinase MMP-9. MCP-1 secretion from monocytes on PEG-only hydrogels was Hck independent in contrast to Hck-dependent MCP-1 secretion in U937 on TCPS. Overall, U937 adherent to sIPNs secrete low levels of soluble gelatinase MMP-9, IL-1beta, TNF-alpha, IL-6, and MCP-1 independent of Hck and PAI-2. This work demonstrates significant changes in surface dependent expression of proteins from monocytes adherent to PEG-based materials compared to TCPS.

LC/MS identification of 12 intracellular cytoskeletal and inflammatory proteins from monocytes adherent on surface-adsorbed fibronectin-derived peptides.

The extent and duration of the host response determines device efficacy, yet the mechanism is poorly understood. U937 promonocytic cells were cultured on peptide-adsorbed tissue-culture polystyrene to better understand surface-modulated intracellular events. Phosphotyrosine proteins were enriched by immunoprecipitation and analyzed by nanospray HPLC-coupled tandem mass spectrometry (LC/MS). Tyrosine-phosphorylated proteins were chosen based on physiological significance and previous densitometry results, which identified a set of proteins ranging from approximately 200 to approximately 23 kDa showing altered phosphorylation levels in response to various surface-adsorbed ligands and phosphorylation inhibitor AG18. Although LC/MS has been used for nearly a decade, its application to the field of biomaterials is relatively novel. Twelve intracellular proteins identified by nanospray LC/MS are potentially related to the host response. Eight of the twelve proteins are related to the cytoskeleton including: moesin, heat shock protein 90beta, alpha-tubulin, elongation factor 1alpha, beta actin, vimentin, plasminogen activator inhibitor 2, and heterogeneous ribonuclear protein A2. The remaining four proteins: high mobility group box 1, caspase recruitment domain 5, glycoprotein 96, and heterogeneous nuclear ribonucleoprotein D0 modulate inflammation. The specific effect each peptide has upon modulating the phosphorylation state of these proteins cannot be determined from this work; however, 12 viable targets have been identified for further investigation into the role each plays in the surface-mediated monocyte response.

Effect of surface-adsorbed proteins and phosphorylation inhibitor AG18 on intracellular protein expression in adherent macrophages.

Macrophages are believed to play an important role in the host inflammatory response to implanted biomaterials. However, the mechanism of macrophage adhesion to protein-adsorbed substrates and the subsequent activation and inflammation is unresolved. Previously the effect of various surface-adsorbed proteins and increasing concentrations of phosphorylation inhibitor AG18 on intracellular protein expression levels in adherent human monocytic cell line U937 was identified using SDS-PAGE and densitometry. The protein ligands and AG18 concentrations up or down regulated the expression of a set of proteins ranging from approximately 200 to approximately 23 kDa. In the present work, HPLC coupled tandem mass spectroscopy (LC/MS) was used to identify proteins in these bands. We hypothesized that key proteins in macrophage adhesion and activation could be identified by observing protein expression resulting from various surface-adsorbed ligands and AG18 concentrations. Increasing concentrations of AG18 down or up regulate protein expression in adherent U937 on PBS-adsorbed TCPS at approximately 52, approximately 42 and approximately 23 kDa. AG18 concentrations had no effect on cells on albumin (Alb)-adsorbed surfaces but regulated different protein expression in adherent U937 on fibronectin (FN)-adsorbed TCPS at 40 and 80 microm AG18. Both Alb and FN regulate distinct sets of proteins in adherent cells as surface-adsorbed ligands. Based on the data from LC/MS, both surface associated ligand and increasing concentrations of AG18 modulate shifts in intracellular signaling.

Intracellular protein phosphorylation in adherent U937 monocytes mediated by various culture conditions and fibronectin-derived surface ligands.

Macrophages play a central role in the normal healing process after tissue injury and the host response to foreign objects such as biomaterials. The process leading to macrophage adhesion and activation on protein-adsorbed substrates is complex and unresolved. While the use of primary cells offers clinical relevancy, macrophage cell lines offer unique advantages such as availability and relatively homogeneous phenotype as models to probe the molecular mechanism of cell-surface interaction. Our goal was to better characterize the effect of the culture condition and surface-associated ligands on the extent of U937 adhesion. Tyrosine phosphorylation of intracellular proteins was surveyed as a basis to seek a greater understanding of the molecular mechanism involved in mediating U937 adhesion on various ligand-adsorbed surfaces. U937 viability and adhesion on tissue culture polystyrene (TCPS) increased with (i) increasing serum level, (ii) decreasing tyrosine phosphorylation inhibitor AG18 concentration, or (iii) increasing culture time. The adsorption of various adhesion proteins such as fibronectin and peptide ligands (i.e., RGD, PHSRN) on TCPS did not significantly increase the adherent density of U937 when compared with albumin and PBS ligand controls. However, ligand identity and the presence of phorbol myristate acetate dramatically affected the extent (i.e., increase or decrease) and the identity (i.e., molecular weight) of phosphotyrosine proteins in adherent U937 in a time-dependent manner. The extent and identity of phosphotyrosine proteins did not exhibit a clear AG18 dose dependency, rather the level of tyrosine phosphorylation for a distinct group of proteins was either increased or decreased for a given AG18 concentration.