In Vitro ADME Properties of Two Novel Antimicrobial Peptoid-Based Compounds as Potential Agents against Canine Pyoderma.

Antimicrobial peptides (AMPs) hold promise as the next generation of antimicrobial agents, but often suffer from rapid degradation in vivo. Modifying AMPs with non-proteinogenic residues such as peptoids (oligomers of N-alkylglycines) provides the potential to improve stability. We have identified two novel peptoid-based compounds, B1 and D2, which are effective against the canine skin pathogen Staphylococcus pseudintermedius, the main cause of antibiotic use in companion animals. We report on their potential to treat infections topically by characterizing their release from formulation and in vitro ADME properties. In vitro ADME assays included skin penetration profiles, stability to proteases and liver microsomes, and plasma protein binding. Both B1 and D2 were resistant to proteases and >98% bound to plasma proteins. While half-lives in liver microsomes for both were >2 h, peptoid D2 showed higher stability to plasma proteases than the peptide-peptoid hybrid B1 (>2 versus 0.5 h). Both compounds were suitable for administration in an oil-in-water cream formulation (50% release in 8 h), and displayed no skin permeation, in the absence or presence of skin permeability modifiers. Our results indicate that these peptoid-based drugs may be suitable as antimicrobials for local treatment of canine superficial pyoderma and that they can overcome the inherent limitations of stability encountered in peptides.

3-D tumor model for in vitro evaluation of anticancer drugs.

The efficacy of potential anticancer drugs during preclinical development is generally tested in vitro using cancer cells grown in monolayer; however, a significant discrepancy in their efficacy is observed when these drugs are evaluated in vivo. This discrepancy, in part, could be due to the three-dimensional (3-D) nature of tumors as compared to the two-dimensional (2-D) nature of monolayer cultures. Therefore, there is a need for an in vitro model that would mimic the 3-D nature of tumors. With this objective, we have developed surface-engineered, large and porous biodegradable polymeric microparticles as a scaffold for 3-D growth of cancer cells. Using the MCF-7 cell line as model breast cancer cells, we evaluated the antiproliferative effect of three anticancer drugs: doxorubicin, paclitaxel and tamoxifen in 3-D model vs in 2-D monolayer. With optimized composition of microparticles and cell culture conditions, a density of 4.5 x 10 (6) MCF-7 cells/mg of microparticles, which is an 18-fold increase from the seeding density, was achieved in six days of culture. Cells were observed to have grown in clumps on the microparticle surface as well as in their interior matrix structure. The antiproliferative effect of the drugs in 3-D model was significantly lower than in 2-D monolayer, which was evident from the 12- to 23-fold differences in their IC 50 values. Using doxorubicin, the flow cytometry data demonstrated approximately 2.6-fold lower drug accumulation in the cells grown in 3-D model than in the cells grown as 2-D monolayer. Further, only 26% of the cells in 3-D model had the same concentration of drug as the cells in monolayer, thus explaining the reduced activity of the drugs in 3-D model. The collagen content of the cells grown in 3-D model was 2-fold greater than that of the cells grown in 2-D, suggesting greater synthesis of extracellular matrix in 3-D model, which acted as a barrier to drug diffusion. The microarray analysis showed changes in several genes in cells grown in 3-D, which could also influence the drug effect. In conclusion, the cells grown in 3-D are more resistant to chemotherapy than those grown in 2-D culture, suggesting the significant roles of cellular architecture, phenotypic variations, and extracellular matrix barrier to drug transport in drug efficacy. We propose that our model provides a better assessment of drug efficacy than the currently used 2-D monolayer as many of its characteristic features are similar to an actual tumor. A well-characterized 3-D model can particularly be useful for rapid screening of a large number of therapeutics for their efficacy during the drug discovery phase.

Quantification of the force of nanoparticle-cell membrane interactions and its influence on intracellular trafficking of nanoparticles.

Understanding the interaction of nanoparticles (NPs) with the cell membrane and their trafficking through cells is imperative to fully explore the use of NPs for efficient intracellular delivery of therapeutics. Here, we report a novel method of measuring the force of NP-cell membrane interactions using atomic force microscopy (AFM). Poly(D,L-lactide-co-glycolide) (PLGA) NPs functionalized with poly-L-lysine were used as a model system to demonstrate that this force determines the adhesive interaction of NPs with the cell membrane and in turn the extent of cellular uptake of NPs, and hence that of the encapsulated therapeutic. Cellular uptake of NPs was monitored using AFM imaging and the dynamics of their intracellular distribution was quantified using confocal microscopy. Results demonstrated that the functionalized NPs have a five-fold greater force of adhesion with the cell membrane and the time-lapse AFM images show their rapid internalization than unmodified NPs. The intracellular trafficking study showed that the functionalized NPs escape more rapidly and efficiently from late endosomes than unmodified NPs and result in 10-fold higher intracellular delivery of the encapsulated model protein. The findings described herein enhance our basic understanding of the NP-cell membrane interaction on the basis of physical phenomena that could have wider applications in developing efficient nanocarrier systems for intracellular delivery of therapeutics.

Inhibition of apoptosis through localized delivery of rapamycin-loaded nanoparticles prevented neointimal hyperplasia and reendothelialized injured artery.

BACKGROUND: A significant fraction of vascular smooth muscle cells (VSMCs) undergo rapid apoptosis after balloon angioplasty. In this study, we tested the hypothesis that protecting VSMCs from undergoing apoptosis prevents the cascade of events that lead to intimal hyperplasia. METHODS AND RESULTS: Rapamycin-loaded gel-like nanoparticles (mean diameter, 54+/-5 nm) were infused locally in a rat carotid artery model of vascular injury. The drug has both antiapoptotic and antiproliferative effects on VSMCs and hence was selected for the current study. Localized delivery of nanoparticles sustained the drug level in the target artery for >2 weeks; demonstrated significant inhibition of hyperplasia (intima/media ratio, 1.5+/-0.02 versus 2.7+/-0.6; P<0.01); and most importantly, re-endothelialized the injured artery (endothelium coverage: treated 82% versus control 28%). We also demonstrated inhibition of activation of caspase-3/7 enzymes in the treated artery, preventing VSMCs from undergoing apoptosis and subsequent infiltration of macrophages. CONCLUSIONS: It may be postulated that the localized delivery of rapamycin inhibited apoptosis of VSMCs, minimizing the inflammatory response to the injury and, thus, creating conditions conducive to vascular repair (re-endothelialization). Unlike stenting, which can lead to thrombosis and increased risk for in-stent restenosis, our approach could eliminate or minimize long-term complications because the injured artery undergoes a natural process of re-endothelialization.

Preparation of biodegradable nanoparticles and their use in transfection.

INTRODUCTIONBiodegradable nanoparticles (NPs) are colloidal particles with a gene of interest encapsulated inside a polymeric matrix. They are typically 100 nm in diameter, and are formulated using FDA-approved, biodegradable, biocompatible polymers such as poly(D,L-lactide-co-glycolide) (PLGA) or polylactide (PLA). The NPs are taken up by cells via an endocytic process, and the encapsulated plasmid DNA entrapped in NPs is protected from degradation by both extra- and intracellular nucleases. It is released slowly, sustaining gene delivery and gene expression. In contrast, higher but transient gene expression is observed with lipid- or polymer-based complexes where most of the delivered DNA is available quickly for transfection. Thus, unlike other nonviral gene delivery systems, NPs thus constitute a sustained gene expression vector. Sustained gene expression is advantageous, especially when the half-life of the expressed protein is very low or when chronic gene delivery is required for therapeutic efficacy. This protocol describes a method for nanoencapsulation of DNA and the subsequent use of NPs for transfection.

Erythropoietin induces excessive neointima formation: a study in a rat carotid artery model of vascular injury.

A therapeutic strategy that would mitigate the events leading to hyperplasia and facilitate re-endothelialization of an injured artery after balloon angioplasty could be effective for a long-term patency of the artery. It is hypothesized that erythropoietin (EPO), which has both anti-inflammatory and antiapoptotic properties, will prevent hyperplasia, and its ability to proliferate and mobilize endothelial progenitor cells will re-endothelialize the injured artery. To test this hypothesis, EPO (5000 IU/kg) in solution was injected intraperitoneally 6 hours before vascular injury and then on every alternate day for a week or as a single dose (5000 IU/kg) in a sustained release gel formulation 1 week before the vascular injury. Morphometric analysis revealed nearly continuous re-endothelialization of the injured artery in EPO solution-treated animals (90% vs less than 20% in saline control); however, the treatment also caused excessive neointima formation (intima/media ratio, 2.10 +/- 0.09 vs 1.60 +/- 0.02 saline control, n = 5, P < .001). The EPO gel also induced similar excessive neointima formation. Immunohistochemical analysis of the injured arteries from the animals treated with EPO solution demonstrated a significant angiogenic response in adventitia and media, thus explaining the formation of excessive neointima. Although the results are in contrast to expectation, they explain a greater degree of stenosis seen in hemodialysis access fistulas in patients who are on EPO therapy for anemic condition. The results also caution the use of EPO, particularly in patients who are at a risk of vascular injury or are suffering from an atherosclerotic condition.

Biodegradable nanoparticles for cytosolic delivery of therapeutics.

Many therapeutics require efficient cytosolic delivery either because the receptors for those drugs are located in the cytosol or their site of action is an intracellular organelle that requires transport through the cytosolic compartment. To achieve efficient cytosolic delivery of therapeutics, different nanomaterials have been developed that consider the diverse physicochemical nature of therapeutics (macromolecule to small molecule; water soluble to water insoluble) and various membrane associated and intracellular barriers that these systems need to overcome to efficiently deliver and retain therapeutics in the cytoplasmic compartment. Our interest is in investigating PLGA and PLA-based nanoparticles for intracellular delivery of drugs and genes. The present review discusses the various aspects of our studies and emphasizes the need for understanding of the molecular mechanisms of intracellular trafficking of nanoparticles in order to develop an efficient cytosolic delivery system.

Polymeric nanoparticles for gene delivery.

Since the evolution of the concept of gene therapy, delivering therapeutic genes to the diseased cells has been a major challenge. Although viral vectors have been shown to be efficient in delivering genes, the issue of their safety is still to be solved. Meanwhile, the field of developing nonviral expression vectors has seen considerable progress. As compared with viruses, these are relatively safe but are confronted with the problem of poor transfection efficiency. With the growing understanding of the biology of gene transfection, and the continued efforts at enhancing the efficiency of nonviral expression vectors, it could soon become a preferred option for human gene therapy. In this review, the potential of polymeric nanoparticles as a gene expression vector is discussed. Furthermore, the importance of understanding the pathophysiology of disease conditions in developing gene expression vectors is discussed in Section 6.

Targeted drug delivery in cancer therapy.

Chemotherapy has been the main modality of treatment for cancer patients; however, its success rate remains low, primarily due to limited accessibility of drugs to the tumor tissue, their intolerable toxicity, development of multi-drug resistance, and the dynamic heterogeneous biology of the growing tumors. Better understanding of tumor biology in recent years and new targeted drug delivery approaches that are being explored using different nanosystems and bioconjugates provide optimism in developing successful cancer therapy. This article reviews the possibilities and challenges for targeted drug delivery in cancer therapy.

Bioadhesive microspheres as a controlled drug delivery system.

The concept of controlled drug delivery has been traditionally used to obtain specific release rates or spatial targeting of active ingredients. The phenomenon of bioadhesion, introduced by Park and Robinson [Park, K., Robinson, J.R., 1984. Bioadhesive polymers as platforms for oral controlled drug delivery: method to study bioadhesion. Int. J. Pharm. 198, 107-127], has been studied extensively in the last decade and applied to improve the performance of these drug delivery systems. Recent advances in polymer science and drug carrier technologies have promulgated the development of novel drug carriers such as bioadhesive microspheres that have boosted the use of "bioadhesion" in drug delivery. This article presents the spectrum of potential applications of bioadhesive microspheres in controlled drug delivery ranging from the small molecules, to peptides, and to the macromolecular drugs such as proteins, oligonucleotides and even DNA. The development of mucus or cell-specific bioadhesive polymers and the concepts of cytoadhesion and bioinvasion provide unprecedented opportunities for targeting drugs to specific cells or intracellular compartments. Developments in the techniques for in vitro and in vivo evaluation of bioadhesive microspheres have also been discussed.