Andrew J. Ditto

The antimicrobial efficacy of sustained release silver–carbene complex-loaded l-tyrosine polyphosphate nanoparticles: Characterization, in vitro and in vivo studies

The pressing need to treat multi-drug resistant bacteria in the chronically infected lungs of cystic fibrosis (CF) patients has given rise to novel nebulized antimicrobials. We have synthesized a silver-carbene complex (SCC10) active against a variety of bacterial strains associated with CF and chronic lung infections. Our studies have demonstrated that SCC10-loaded into L-tyrosine polyphosphate nanoparticles (LTP NPs) exhibits excellent antimicrobial activity in vitro and in vivo against the CF relevant bacteria Pseudomonas aeruginosa. Encapsulation of SCC10 in LTP NPs provides sustained release of the antimicrobial over the course of several days translating into efficacious results in vivo with only two administered doses over a 72 h period.

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.

Anticancer Activity of Ag(I) N-Heterocyclic Carbene Complexes Derived from 4,5-Dichloro-1H-Imidazole

A class of Ag(I) N-heterocyclic carbene silver complexes, 1–3, derived from 4,5-dichloro-1H-imidazole has been evaluated for their anticancer activity against the human cancer cell lines OVCAR-3 (ovarian), MB157 (breast), and Hela (cervical). Silver complexes 1–3 are active against the ovarian and breast cancer cell lines. A preliminary in vivo study shows 1 to be active against ovarian cancer in mice. The results obtained in these studies warrant further investigation of these compounds in vivo.

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.

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.

The Interactions between L-Tyrosine Based Nanoparticles Decorated with Folic Acid and Cervical Cancer Cells Under Physiological Flow

Many anticancer drugs have been established clinically, but their efficacy can be compromised by nonspecific toxicity and an inability to reach the desired cancerous intracellular spaces. In order to address these issues, researchers have explored the use of folic acid as a targeted moiety to increase specificity of chemotherapeutic drugs. To expand upon such research, we have conjugated folic acid to functionalized poly(ethylene glycol) and subsequently decorated the surface of l-tyrosine polyphosphate (LTP) nanoparticles. These nanoparticles possess the appropriate size (100-500 nm) for internalization as shown by scanning electron microscopy and dynamic light scattering. Under simulated physiological flow, LTP nanoparticles decorated with folic acid (targeted nanoparticles) show a 10-fold greater attachment to HeLa, a cervical cancer cell line, compared to control nanoparticles and to human dermal fibroblasts. The attachment of these targeted nanoparticles progresses at a linear rate, and the strength of this nanoparticle attachment is shown to withstand shear stresses of 3.0 dyn/cm(2). These interactions of the targeted nanoparticles to HeLa are likely a result of a receptor-ligand binding, as a competition study with free folic acid inhibits the nanoparticle attachment. Finally, the targeted nanoparticles encapsulated with a silver based drug show increased efficacy in comparison to nondecorated (plain) nanoparticles and drug alone against HeLa cells. Thus, targeted nanoparticles are a promising delivery platform for developing anticancer therapies that overexpress the folate receptors (FRs).

Nanoparticle Deposition onto Biofilms

We develop a mathematical model of nanoparticles depositing onto and penetrating into a biofilm grown in a parallel-plate flow cell. We carry out deposition experiments in a flow cell to support the modeling. The modeling and the experiments are motivated by the potential use of polymer nanoparticles as part of a treatment strategy for killing biofilms infecting the deep passages in the lungs. In the experiments and model, a fluid carrying polymer nanoparticles is injected into a parallel-plate flow cell in which a biofilm has grown over the bottom plate. The model consists of a system of transport equations describing the deposition and diffusion of nanoparticles. Standard asymptotic techniques that exploit the aspect ratio of the flow cell are applied to reduce the model to two coupled partial differential equations. We perform numerical simulations using the reduced model. We compare the experimental observations with the simulation results to estimate the nanoparticle sticking coefficient and the diffusion coefficient of the nanoparticles in the biofilm. The distributions of nanoparticles through the thickness of the biofilm are consistent with diffusive transport, and uniform distributions through the thickness are achieved in about four hours. Nanoparticle deposition does not appear to be strongly influenced by the flow rate in the cell for the low flow rates considered.

Receptor-Mediated Attachment and Uptake of Hyaluronan Conjugates by Breast Cancer Cells

Chemotherapy, a mainstay modality for cancer, is often hindered by systemic toxicity and side effects. With the emergence of nanomedicine, the development of drug therapy has shifted toward targeted therapy. Hyaluronan (HA) is an ideal molecule as a targeted delivery system because many carcinomas overexpress HA receptors. We have conjugated resveratrol, a natural polyphenol, and 3-(5-methoxy, 2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), a chalcone, to HA with the goal of enhancing drug bioavailability and targeting triple negative breast cancers. We demonstrate the ability of HA conjugates to accumulate in the tumor interstitium within 6 h after tail vein injections. In vitro, these conjugates interact with their target receptors, which are overexpressed by triple negative breast cancer cells under static and physiological flow. These interactions result in enhanced uptake and efficacy of the therapeutic, as demonstrated by a reduced IC50 over that of nonconjugated drugs. Thus, HA offers a platform to solubilize, target, and enhance the efficacy of chemotherapeutics.

Targeting Ovarian Cancer Cells With Rapidly Biodegradable L-Tyrosine Polyphosphate Nanoparticles Decorated With Folate

Chemotherapy employs toxic chemicals to kill rapidly dividing cells. Examples of FDA approved antineoplastic drugs include cisplatin, doxorubicin, and paclitaxel. Since most of these drugs are nonspecific, they also damage normal tissues as well as the aberrant tumors. As a result, non-specific therapies have multiple side effects, which include myelosuppression, mucositis, alopecia, nephrotoxicity, and genotoxcity. In order to minimize these issues, researchers have begun to conjugate antineoplastic chemicals with targeting moieties or encapsulate drugs into nanoparticles decorated with compounds, peptides, or proteins that recognize specific cellular receptors, which are upregulated by the neoplastic cells. The targeting moieties aid in the accumulation of these drugs within the blood vessels of carcinomas, while keeping concentrations low in the systemic circulation. Thus, targeted delivery systems are able to minimize the unwanted side effects and increase the efficacy of chemotherapies.© 2011 ASME