Parth N. Shah

In vitro antimicrobial studies of silver carbene complexes: activity of free and nanoparticle carbene formulations against clinical isolates of pathogenic bacteria

OBJECTIVES Silver carbenes may represent novel, broad-spectrum antimicrobial agents that have low toxicity while providing varying chemistry for targeted applications. Here, the bactericidal activity of four silver carbene complexes (SCCs) with different formulations, including nanoparticles (NPs) and micelles, was tested against a panel of clinical strains of bacteria and fungi that are the causative agents of many skin and soft tissue, respiratory, wound, blood, and nosocomial infections. METHODS MIC, MBC and multidose experiments were conducted against a broad range of bacteria and fungi. Time-release and cytotoxicity studies of the compounds were also carried out. Free SCCs and SCC NPs were tested against a panel of medically important pathogens, including methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant Acinetobacter baumannii (MRAB), Pseudomonas aeruginosa, Burkholderia cepacia and Klebsiella pneumoniae. RESULTS All four SCCs demonstrated strong efficacy in concentration ranges of 0.5-90 mg/L. Clinical bacterial isolates with high inherent resistance to purified compounds were more effectively treated either with an NP formulation of these compounds or by repeated dosing. Overall, the compounds were active against highly resistant bacterial strains, such as MRSA and MRAB, and were active against the biodefence pathogens Bacillus anthracis and Yersinia pestis. All of the medically important bacterial strains tested play a role in many different infectious diseases. CONCLUSIONS The four SCCs described here, including their development as NP therapies, show great promise for treating a wide variety of bacterial and fungal pathogens that are not easily killed by routine antimicrobial agents.

Non-viral gene delivery using nanoparticles

Although the potential benefits of gene therapy for the treatment of acquired and inherited genetic diseases have been demonstrated through preclinical studies, the results of human gene therapy trials have been disappointing. Recombinant viruses are the primary vectors of choice because of their ability to protect genetic materials, cross cellular membranes, escape from endosomes and transport their genetic materials into the nucleus. Unfortunately, viral vectors have been unable to gain widespread clinical application because of their toxicity and immunogenicity. Consequently, the need for safer alternatives has led to the development of liposomes, cationic polyplexes, microparticles and nanoparticles. Although these alternative vectors have shown promise, degradable nanoparticles are the only non-viral vectors that can provide a targeted intracellular delivery with controlled release properties. Furthermore, the potential advantage of degradable nanoparticles over their non-degradable counterparts is the reduced toxicity and the avoidance of accumulation within the target tissue after repeated administration. In this article, current non-viral gene delivery devices are reviewed with a special emphasis on nanoparticle gene delivery systems. Also, the authors highlight their philosophy and efforts on the development of l-tyrosine-based polyphosphate nanoparticle-based non-viral gene delivery systems and assess the potential benefits and shortcomings of their approach.

Synthesis, characterization, and in vivo efficacy of shell cross-linked nanoparticle formulations carrying silver antimicrobials as aerosolized therapeutics.

The use of nebulizable, nanoparticle-based antimicrobial delivery systems can improve efficacy and reduce toxicity for treatment of multi-drug-resistant bacteria in the chronically infected lungs of cystic fibrosis patients. Nanoparticle vehicles are particularly useful for applying broad-spectrum silver-based antimicrobials, for instance, to improve the residence time of small-molecule silver carbene complexes (SCCs) within the lung. Therefore, we have synthesized multifunctional, shell cross-linked knedel-like polymeric nanoparticles (SCK NPs) and capitalized on the ability to independently load the shell and core with silver-based antimicrobial agents. We formulated three silver-loaded variants of SCK NPs: shell-loaded with silver cations, core-loaded with SCC10, and combined loading of shell silver cations and core SCC10. All three formulations provided a sustained delivery of silver over the course of at least 2–4 days. The two SCK NP formulations with SCC10 loaded in the core each exhibited excellent antimicrobial activity and efficacy in vivo in a mouse model of Pseudomonas aeruginosa pneumonia. SCK NPs with shell silver cation-load only, while efficacious in vitro, failed to demonstrate efficacy in vivo. However, a single dose of core SCC10-loaded SCK NPs (0.74 ± 0.16 mg Ag) provided a 28% survival advantage over sham treatment, and administration of two doses (0.88 mg Ag) improved survival to 60%. In contrast, a total of 14.5 mg of Ag+ delivered over 5 doses at 12 h intervals was necessary to achieve a 60% survival advantage with a free-drug (SCC1) formulation. Thus, SCK NPs show promise for clinical impact by greatly reducing antimicrobial dosage and dosing frequency, which could minimize toxicity and improve patient adherence.

Aerosolized Antimicrobial Agents Based on Degradable Dextran Nanoparticles Loaded with Silver Carbene Complexes

Degradable acetalated dextran (Ac-DEX) nanoparticles were prepared and loaded with a hydrophobic silver carbene complex (SCC) by a single-emulsion process. The resulting particles were characterized for morphology and size distribution using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The average particle size and particle size distribution were found to be a function of the ratio of the organic phase to the surfactant containing aqueous phase with a 1:5 volume ratio of Ac-DEX CH(2)Cl(2) (organic):PBS (aqueous) being optimal for the formulation of nanoparticles with an average size of 100 ± 40 nm and a low polydispersity. The SCC loading was found to increase with an increase in the SCC quantity in the initial feed used during particle formulation up to 30% (w/w); however, the encapsulation efficiency was observed to be the best at a feed ratio of 20% (w/w). In vitro efficacy testing of the SCC loaded Ac-DEX nanoparticles demonstrated their activity against both Gram-negative and Gram-positive bacteria; the nanoparticles inhibited the growth of every bacterial species tested. As expected, a higher concentration of drug was required to inhibit bacterial growth when the drug was encapsulated within the nanoparticle formulations compared with the free drug illustrating the desired depot release. Compared with free drug, the Ac-DEX nanoparticles were much more readily suspended in an aqueous phase and subsequently aerosolized, thus providing an effective method of pulmonary drug delivery.

Synthesis, characterization, and antimicrobial activity of silver carbene complexes derived from 4,5,6,7- tetrachlorobenzimidazole against antibiotic resistant bacteria †

Silver N-heterocyclic carbene complexes have been shown to have great potential as antimicrobial agents, affecting a wide spectrum of both Gram-positive and Gram-negative bacteria. A new series of three silver carbene complexes (SCCs) based on 4,5,6,7-tetrachlorobenzimidazole has been synthesized, characterized, and tested against a panel of clinical strains of bacteria. The imidazolium salts and their precursors were characterized by elemental analysis, mass spectrometry, (1)H and (13)C NMR spectroscopy, and single crystal X-ray diffraction. The silver carbene complexes, SCC32, SCC33, and SCC34 were characterized by elemental analysis, (1)H and (13)C NMR spectroscopy, and single crystal X-ray diffraction. These complexes proved highly efficacious with minimum inhibitory concentrations (MICs) ranging from 0.25 to 6 μg mL(-1). Overall, the complexes were effective against highly resistant bacteria strains, such as methicillin-resistant Staphylococcus aureus (MRSA), weaponizable bacteria, such as Yersinia pestis, and pathogens found within the lungs of cystic fibrosis patients, such as Pseudomonas aeruginosa, Alcaligenes xylosoxidans, and Burkholderia gladioli. SCC33 and SCC34 also showed clinically relevant activity against a silver-resistant strain of Escherichia coli based on MIC testing.

Nanospheres formulated from L-tyrosine polyphosphate as a potential intracellular delivery device.

Current delivery devices for drugs and genes such as films and microspheres are usually formulated from polymers that degrade over a period of months. In general, these delivery systems are designed to achieve an extracellular release of their encapsulated drugs. For drugs that require interaction with cellular machinery, the efficacies of both macroscopic and microscopic delivery systems are normally low. In contrast, nano-sized drug delivery vehicles could achieve high delivery efficiencies, but they must degrade quickly, and the delivery system itself should be nontoxic to cells. In this aspect, biodegradable nanospheres formulated from l-tyrosine polyphosphate (LTP) have been produced from an emulsion of oil and water for the potential use as an intracellular delivery device. Scanning electron microscopy (SEM) and dynamic laser light scattering (DLS) show that LTP nanospheres possess a diameter range between 100 and 600 nm. SEM reveals nanospheres formulated from LTP are spherical and smooth. Additionally, DLS studies demonstrate that nanospheres degrade hydrolytically in 7 days. Confocal microscopy reveals LTP nanospheres are internalized within human fibroblasts. Finally, the cell viability after exposure to LTP nanospheres and determined with a LIVE/DEAD Cell Viability Assay is comparable to a buffer control. In conclusion, our nanospheres have been shown to be nontoxic to human cells, possess the appropriate size for endocytosis by human cells, and degrade within 7 days. Therefore LTP nanospheres can be used for a sustained intracellular delivery device.

Nanospheres formulated from L-tyrosine polyphosphate exhibiting sustained release of polyplexes and in vitro controlled transfection properties.

Currently, viruses are utilized as vectors for gene therapy, since they transport across cellular membranes, escape endosomes, and effectively deliver genes to the nucleus. The disadvantage of using viruses for gene therapy is their immune response. Therefore, nanospheres have been formulated as a nonviral gene vector by blending l-tyrosine-polyphosphate (LTP) with polyethylene glycol grafted to chitosan (PEG-g-CHN) and linear polyethylenimine (LPEI) conjugated to plasmid DNA (pDNA). PEG-g-CHN stabilizes the emulsion and prevents nanosphere coalescence. LPEI protects pDNA degradation during nanosphere formation, provides endosomal escape, and enhances gene expression. Previous studies show that LTP degrades within seven days and is appropriate for intracellular gene delivery. These nanospheres prepared by water-oil emulsion by sonication and solvent evaporation show diameters between 100 and 600 nm. Also, dynamic laser light scattering shows that nanospheres completely degrade after seven days. The sustained release of pDNA and pDNA-LPEI polyplexes is confirmed through electrophoresis and PicoGreen assay. A LIVE/DEAD cell viability assay shows that nanosphere viability is comparable to that of buffers. X-Gal staining shows a sustained transfection for 11 days using human fibroblasts. This result is sustained longer than pDNA-LPEI and pDNA-FuGENE 6 complexes. Therefore, LTP-pDNA nanospheres exhibit controlled transfection and can be used as a nonviral gene delivery vector.

Cellular interactions with biodegradable polyurethanes formulated from L-tyrosine.

L-Tyrosine polyurethanes (LTUs) have been synthesized by structural modification of the poly (amino acid) backbone to circumvent the problems associated with the processing of poly (amino acids) arising from their high crystallinity, insolubility in common organic solvents, and high glass-transition and melting temperatures. Additionally, problems such as unpredictable swelling characteristics, change in conformation, and uncontrolled enzymatic degradation have severely restricted the use of poly (amino acids). In contrast, LTUs are designed to retain their superior physico-chemical properties, while incorporating biodegradability through enzymatic, hydrolytic, and oxidative pathways. The aim of this study is to evaluate initially the biocompatibility of LTUs and their degradation products. Studies involving primary dermal human fibroblasts cultured in contact with LTU films or degradation products suggest a lack of toxicity (cell viabilities >93% with p < 0.05 compared to the control for all studies). The diversity of LTU polymer chemistry and the ability of LTUs to phase separate seem to present a heterogeneous surface with variable wettability. This phenomenon influences the adhesion and proliferation of human fibroblasts on polymeric surfaces, wherein fibroblast adhesion on polycaprolactone diol (PCL) based LTUs is characterized by higher cell counts (81,250 ± 18,390 for PCL-C-DTH (desaminotyrosine–tyrosyl hexyl, DTH), 58,360 ± 7370 for PCL-L-DTH, 38,480 ± 12,680 for PEG-C-DTH (polyethylene glycol, PEG), and 46,430 ± 16,000 for PEG-L-DTH at 120 h with p < 0.001 for comparison between PCL-C-DTH and all other LTUs), more rapid cellular proliferation (doubling time of 37–49 h for PCL-based LTUs compared to 68–90 h for PEG-based LTUs), and a uniform cell distribution compared to PEG-based LTUs. However, immunofluorescence assay for F-actin suggests that the cells are well attached. Thus, the lack of cytotoxicity and the ability to control cellular adhesion through polymer chemistry make LTUs attractive candidates for tissue-engineering applications that require elastomeric, biodegradable, and biocompatible polymers.

Antimicrobial Activity of Ibuprofen Against Cystic Fibrosis Associated Gram–Negative Pathogens

ABSTRACT Clinical trials have demonstrated the benefits of ibuprofen therapy in cystic fibrosis (CF) patients, an effect that is currently attributed to ibuprofens anti-inflammatory properties. Yet, a few previous reports demonstrated an antimicrobial activity of ibuprofen as well, although none investigated its direct effects on the pathogens found in the CF lung, which is the focus of this work. Determination of ibuprofens in vitro antimicrobial activity against Pseudomonas aeruginosa and Burkholderia species strains through measurements of the endpoint number of CFU and growth kinetics showed that ibuprofen reduced the growth rate and bacterial burden of the tested strains in a dose-dependent fashion. In an in vitro Pseudomonas biofilm model, a reduction in the rate of biomass accumulation over 8 h of growth with ibuprofen treatment was observed. Next, an acute Pseudomonas pneumonia model was used to test this antimicrobial activity after the oral delivery of ibuprofen. Following intranasal inoculation, ibuprofen-treated mice exhibited lower CFU counts and improved survival compared with the control animals. Preliminary biodistribution studies performed after the delivery of ibuprofen to mice by aerosol demonstrated a rapid accumulation of ibuprofen in serum and minimum retention in lung tissue and bronchoalveolar lavage fluid. Therefore, ibuprofen-encapsulated polymeric nanoparticles (Ibu-NPs) were formulated to improve the pharmacokinetic profile. Ibu-NPs formulated for aerosol delivery inhibited the growth of P. aeruginosa in vitro and may provide a convenient dosing method. These results provide an additional explanation for the previously observed therapeutic effects of ibuprofen in CF patients and further strengthen the argument for its use by these patients.