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Combination antiretroviral therapy (cART) has been proven effective in inhibiting human immunodeficiency virus type 1 (HIV-1) infection and has significantly improved the health outcomes in acquired immune deficiency syndrome (AIDS) patients. The therapeutic benefits of cART have been challenged because of the toxicity and emergence of drug-resistant HIV-1 strains along with lifelong patient compliance resulting in non-adherence. These issues also hinder the clinical benefits of non-nucleoside reverse transcriptase inhibitors (NNRTIs), which are one of the vital components of cART for the treatment of HIV-1 infection. In this study, using a computational and structural based drug design approach, we have discovered an effective HIV -1 NNRTI, compound I (Cmpd I) that is very potent in biochemical assays and which targets key residues in the allosteric binding pocket of wild-type (WT)-RT as revealed by structural studies. Furthermore, Cmpd I exhibited very potent antiviral activity in HIV-1 infected T cells, lacked cytotoxicity (therapeutic index >100,000), and no significant off-target effects were noted in pharmacological assays. To address the issue of non-adherence, we developed a long-acting nanoformulation of Cmpd I (Cmpd I-NP) using poly (lactide-coglycolide) (PLGA) particles. The pharmacokinetic studies of free and nanoformulated Cmpd I were carried out in BALB/c mice. Intraperitoneal administration of Cmpd I and Cmpd I-NP in BALB/c mice revealed prolonged serum residence time of 48 h and 30 days, respectively. The observed serum concentrations of Cmpd I in both cases were sufficient to provide >97% inhibition in HIV-1 infected T-cells. The significant antiviral activity along with favorable pharmacological and pharmacokinetic profile of Cmpd I, provide compelling and critical support for its further development as an anti-HIV therapeutic agent.

The cellular fate of nanoparticles in the liver is not fully understood. Because the effectiveness and safety of nanoparticles in liver therapy depends on targeting nanoparticles to the right cell populations, this study aimed to determine a relative distribution of PLGA-nanoparticles (sizes 271±1.4 nm) among liver cells in vivo. We found that Kupffer cells were the major cells that took up nanoparticles, followed by liver sinusoidal endothelial cells and hepatic stellate cells. Nanoparticles were found in only 7% of hepatocytes. Depletion of Kupffer cells by clodronate liposomes increased nanoparticle retention in liver sinusoidal endothelial cells and hepatic stellate cells, but not in hepatocytes. It is importantly suggested that studies of drug-loaded nanoparticle delivery to the liver have to demonstrate not only uptake of nanoparticles by the target cell type but also non-uptake by other cell types to assess their effect as well as ensure their safety.

Triplex-forming peptide nucleic acids (PNAs) facilitate gene editing by stimulating recombination of donor DNAs within genomic DNA via site-specific formation of altered helical structures that further stimulate DNA repair. However, PNAs designed for triplex formation are sequence restricted to homopurine sites. Herein we describe a novel strategy where next generation single-stranded gamma PNAs (γPNAs) containing miniPEG substitutions at the gamma position can target genomic DNA in mouse bone marrow at mixed-sequence sites to induce targeted gene editing. In addition to enhanced binding, γPNAs confer increased solubility and improved formulation into poly(lactic-co-glycolic acid) (PLGA) nanoparticles for efficient intracellular delivery. Single-stranded γPNAs induce targeted gene editing at frequencies of 0.8% in mouse bone marrow cells treated ex vivo and 0.1% in vivo via IV injection, without detectable toxicity. These results suggest that γPNAs may provide a new tool for induced gene editing based on Watson-Crick recognition without sequence restriction.

Nanomaterials have emerged as important and versatile platforms for the delivery of therapeutics to the brain. This article describes the complex anatomy and physiology of the brain, which create challenges in treating diseases of the central nervous system, and current strategies using nanomedicines to address these challenges. We discuss various approaches to overcome the blood-brain barrier, examples of successful incorporation of therapeutics with nanomaterials, and strategies to achieve efficient delivery of nanomedicines into specific cells.

Multilayer nanofilms, formed by the layer-by-layer (LbL) adsorption of positively and negatively charged polyelectrolytes, are promising substrates for tissue engineering. We investigate here the attachmemt and function of hepatic cells on multilayer films in terms of film composition, terminal layer, rigidity, charge, and presence of biofunctional species. Human hepatocellular carcinoma cells (HepG2), adult rat hepatocytes (ARH), and human fetal hepatoblasts (HFHb) are studied on films composed of the polysaccharides chitosan (CHI) and alginate (ALG), the polypeptides poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA), and the synthetic polymers poly(allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS). The influence of chemical cross-linking following LbL assembly is also investigated. We find HepG2 to reach confluence after seven days of culture on only 2 of 18 candidate multilayer systems: (PAH-PSS)n (i.e. n PAH-PSS bilayers) and cross-linked (PLL-ALG)n-PLL. These two systems, as well as cross-linked (PLL-PGA)n-PLL, support attachment and function (in terms of albumin production) of ARH, provided collagen is adsorbed to the top of the film. (PAH-PSS)n, cross-linked (PLL-ALG)n, and cross-linked (PLL-PGA)n-PLL films all support attachment, layer confluence, and function of HFHb, with the latter film promoting the greatest level of function at 8 days. Overall, film composition, terminal layer, and rigidity are key variables in promoting attachment and function of hepatic cells, while film charge and biofunctionality are somewhat less important. These studies reveal optimal candidate multilayer biomaterials for human liver tissue engineering applications.

Peptide nucleic acids (PNAs) can bind duplex DNA in a sequence-targeted manner, forming a triplex structure capable of inducing DNA repair and producing specific genome modifications. Since the first description of PNA-mediated gene editing in cell free extracts, PNAs have been used to successfully correct human disease-causing mutations in cell culture and in vivo in preclinical mouse models. Gene correction via PNAs has resulted in clinically-relevant functional protein restoration and disease improvement, with low off-target genome effects, indicating a strong therapeutic potential for PNAs in the treatment or cure of genetic disorders. This review discusses the progress that has been made in developing PNAs as an effective, targeted agent for gene editing, with an emphasis on recent in vivo, nanoparticle-based strategies.

Triplex-forming oligonucleotides (TFOs) are capable of coordinating genome modification in a targeted, site-specific manner, causing mutagenesis or even coordinating homologous recombination events. Here, we describe the use of TFOs such as peptide nucleic acids for targeted genome modification. We discuss this method and its applications and describe protocols for TFO design, delivery, and evaluation of activity in vitro and in vivo.

This pilot study examines noninvasive MR monitoring of tissue-engineered vascular grafts (TEVGs) in vivo using cells labeled with iron oxide nanoparticles. Human aortic smooth muscle cells (hASMCs) were labeled with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. The labeled hASMCs, along with human aortic endothelial cells, were incorporated into eight TEVGs and were then surgically implanted as aortic interposition grafts in a C.B-17 SCID/bg mouse host. USPIO-labeled hASMCs persisted in the grafts throughout a 3 wk observation period and allowed noninvasive MR imaging of the human TEVGs for real-time, serial monitoring of hASMC retention. This study demonstrates the feasibility of applying noninvasive imaging techniques for evaluation of in vivo TEVG performance.—Nelson, G. N., Roh, J. D., Mirensky, T. L., Wang, Y., Yi, T., Tellides, G., Pober, J. S., Shkarin, P., Shapiro, E. M., Saltzman, W. M., Papademetris, X., Fahmy, T. M., Breuer, C. K.. Initial evaluation of the use of USPIO cell labeling and noninvasive MR monitoring of human tissue-engineered vascular grafts in vivo.

Epithelial ovarian cancer is the most lethal gynecologic malignancy. It is treated through up front surgery followed by chemotherapy or with neoadjuvant chemotherapy before surgical debulking. Achieving complete or optimal cytoreduction improves both progression free and overall survival. In the operating room, no matter the care taken, there is the potential for areas of macroscopic, microscopic and occult metastases to remain unvisualized. The development of a sensitive and specific intraoperative system for the visualization and removal or destruction of metastatic disease may improve patient outcome. Fresh ovarian cancer samples were recently analyzed by our group with genetic fingerprinting. This analysis revealed high expression of claudin-3 and -4, the epithelial receptors for Clostridium Perfringens Enterotoxin (CPE). Although the administration of the full length CPE in mice is toxic, the injection of the only carboxi-terminal fragment (c-CPE) avoids toxicity while preserving the binding to the receptors. Our previous data showed specific binding of FITC conjugated c-CPE (FITC-c-CPE) to primary ovarian cancer cell lines in vitro as well as preferential accumulation of the labeled peptide into ovarian cancer xenografts in vivo. This study evaluates the in vivo distribution of FITC-c-CPE after intraperitoneal (IP) injection of the peptide as well as the kinetics and tumor binding capacity of c-CPE conjugated to the NearInfraRed Dye CW800 (CW800-c-CPE), focusing on the ability of CW800-c-CPE to identify metastases of chemotherapy-resistant ovarian cancer overexpressing claudin-3 and -4 in vivo. We found that fluorescence uptake by the tumor starts 30 minutes after FITC-c-CPE injection with negligible staining of healthy organs. When the abdominal cavity of FITC-c-CPE injected mice was visualized using a fluorescence microscope, strong signal was detected in near microscopic metastatic nodules and in malignant tumor spheroids isolated from the ascites. Similarly, CW800-c-CPE also accumulated in tumors in vivo following IP or systemic (IV) injection. Ex vivo distribution analysis demonstrated a significantly higher mean fluorescence intensity (MFI) in tumor compared to healthy organs (MFI: mean ± STDV: 156.55 ± 23.73, 95.72 ± 18.19, 30.68 ± 5.88, 23.33 ± 4.05, 34.71 ± 12.71, 28.16 ± 6.1413.46 ± 1.35, 19.78 ± 5.43 in the tumor, kidney, liver, spleen, bowel, lungs and brain, respectively; p Citation Format: Emiliano Cocco, Sara Gasparrini, Erik M. Shapiro, Carlton Schwab, Stefania Bellone, Ileana Bortolomai, Salvatore Lopez, Natalia J. Sumi, Elena Bonazzoli, Roberta Nicoletti, Yang Deng, William M. Saltzman, Caroline J. Zeiss, Dan-Arin Silasi, Thomas J. Rutherford, Peter E. Schwartz, Alessandro D. Santin. Fluorescence imaging using Clostridium Perfringens Enterotoxin carboxi-terminal fragment (c-CPE) to target metastatic chemotherapy-resistant human ovarian cancer in xenograft mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4937. doi:10.1158/1538-7445.AM2014-4937

A stagnant batch filtration system, in which saline is filtered through an unstirred bed of red cells at constant pressure, was employed to study the hydraulic permeability of red cell beds as well as the extent of cellular blockage of membrane pores. Since red cells are highly deformable, the compressibility of the cell bed was characterized by porosity measurements in centrifuged cell beds. Red cell deformation was also examined by microscopic observation of the cell bed fixed during filtration. Photomicrographs provided evidence that the red blood cells deform into a hexagonal close-packed structure. A simple two-dimensional model for cell bed compressibility developed on the basis of this observation was in good agreement with porosity data taken in the centrifugal system. Filtration data were analyzed using an integrated form of Darcy's law which permits the effects of cell bed hydraulic resistance of pore blockage to be evaluated separately. Functional relationships between hydraulic permeability, porosity, and compressive pressure were developed which can be incorporated into a detailed model for the filtrate flux in cross-flow microfiltration of red cell suspensions.