Nanocapsules embedded in microparticles for enhanced oral bioavailability and efficacy of Lopinavir as an anti-AIDS drug.

Lopinavir (LPV), an efficient drug for HIV infection treatment, was incorporated into biodegradable PLGA nanocapsules (NCs) embedded in microparticles (MCPs) using the spray-drying technique in an attempt to bypass the P-gp efflux and protect the drug from CYP3A pre-systemic metabolism without ritonavir (RTV). SEM observations confirmed the formation of NCs and their entrapment in the MCPs. LPV-loaded NCs and free LPV were released from the MCPs at pH of 7.4 as evidenced by in vitro release studies. Results obtained from rat studies showed a two-fold higher bioavailability of LPV following oral administration of the optimal formulation than Kaletra®, the marketed drug, showing that when properly entrapped, LPV can be effectively protected from CYP degradation in the gut as well as from the liver following systemic absorption. It was also shown that serum derived from rats following LPV oral administration in two formulations and Kaletra® significantly decreased the multiplication of HIV-1 in cultured SupT1 cells. Furthermore, the LPV formulations markedly restricted the titre of infectious HIV-1 production compared with Kaletra® confirming the improved antiviral activity of LPV delivered in the rat blood circulation by the nanocapsules embedded in microparticle formulations.

Reverting the molecular fingerprint of tumor dormancy as a therapeutic strategy for glioblastoma.

Glioblastoma is an aggressive and invasive brain malignancy with high mortality rates despite current treatment modalities. In this study, we show that a 7-gene signature, previously found to govern the switch of glioblastomas from dormancy to aggressive tumor growth, correlates with improved overall survival of patients with glioblastoma. Using glioblastoma dormancy models, we validated the role of 2 genes from the signature, thrombospondin-1 ( TSP-1) and epidermal growth factor receptor ( EGFR), as regulators of glioblastoma dormancy and explored their therapeutic potential. EGFR up-regulation was reversed using EGFR small interfering RNA polyplex, antibody, or small-molecule inhibitor. The diminished function of TSP-1 was augmented via a peptidomimetic. The combination of EGFR inhibition and TSP-1 restoration led to enhanced therapeutic efficacy in vitro, in 3-dimensional patient-derived spheroids, and in a subcutaneous human glioblastoma model in vivo. Systemic administration of the combination therapy to mice bearing intracranial murine glioblastoma resulted in marginal therapeutic outcomes, probably due to brain delivery challenges, p53 mutation status, and the aggressive nature of the selected cell line. Nevertheless, this study provides a proof of concept for exploiting regulators of tumor dormancy for glioblastoma therapy. This therapeutic strategy can be exploited for future investigations using a variety of therapeutic entities that manipulate the expression of dormancy-associated genes in glioblastoma as well as in other cancer types.-Tiram, G., Ferber, S., Ofek, P., Eldar-Boock, A., Ben-Shushan, D., Yeini, E., Krivitsky, A., Blatt, R., Almog, N., Henkin, J., Amsalem, O., Yavin, E., Cohen, G., Lazarovici, P., Lee, J. S., Ruppin, E., Milyavsky, M., Grossman, R., Ram, Z., Calderón, M., Haag, R., Satchi-Fainaro, R. Reverting the molecular fingerprint of tumor dormancy as a therapeutic strategy for glioblastoma.

Solid nano-in-nanoparticles for potential delivery of siRNA.

siRNA-based therapeutics possess great potential to treat a wide variety of genetic disorders. However, they suffer from low cellular uptake and short half-lives in blood circulation; issues that remain to be addressed. This work is, to the best of our knowledge, the first to report the production of solid nano-in-nanoparticles, termed double nano carriers (DNCs) by means of the innovative technology of nano spray drying. DNCs (with a median size of 580-770nm) were produced by spraying at low temperatures (50°C) to prevent damage to heat-sensitive biomacromolecules like siRNA. DNCs consisting of Poly (d,l-lactide-co-glycolide) used as a wall material, encapsulating 20% human serum albumin primary nanoparticles (PNPs) loaded with siRNA, were obtained as a dry nanoparticulate powder with smooth spherical surfaces and a unique inner morphology. Incubation of pegylated or non-pegylated DNCs under sink conditions at 37°C, elicited a controlled release profile of the siRNA for up to 12 or 24h, respectively, with a minimal burst effect. Prolonged incubation of pegylated DNCs loaded with active siRNA (anti EGFR) in an A549 epithelial cell culture monolayer did not induce any apparent cytotoxicity. A slow degradation of the internalized DNCs by the cells was also observed resulting in the progressive release of the siRNA for up to 6days, as corroborated by laser confocal microscopy. The structural integrity and silencing activity of the double encapsulated siRNA were fully preserved, as demonstrated by HPLC, gel electrophoresis, and potent RNAi activity of siRNA extracted from DNCs. These results demonstrate the potential use of DNCs as a nano drug delivery system for systemic administration and controlled release of siRNA and potentially other sensitive bioactive macromolecules.

Bio-imaging of colorectal cancer models using near infrared labeled epidermal growth factor.

Novel strategies that target the epidermal growth factor receptor (EGFR) have led to the clinical development of monoclonal antibodies, which treat metastatic colorectal cancer (mCRC) but only subgroups of patients with increased wild type KRAS and EGFR gene copy, respond to these agents. Furthermore, resistance to EGFR blockade inevitably occurred, making future therapy difficult. Novel bio-imaging (BOI) methods may assist in quantization of EGFR in mCRC tissue thus complementing the immunohistochemistry methodology, in guiding the future treatment of these patients. The aim of the present study was to explore the usefulness of near infrared-labeled EGF (EGF-NIR) for bio-imaging of CRC using in vitro and in vivo orthotopic tumor CRC models and ex vivo human CRC tissues. We describe the preparation and characterization of EGF-NIR and investigate binding, using BOI of a panel of CRC cell culture models resembling heterogeneity of human CRC tissues. EGF-NIR was specifically and selectively bound by EGFR expressing CRC cells, the intensity of EGF-NIR signal to background ratio (SBR) reflected EGFR levels, dose-response and time course imaging experiments provided optimal conditions for quantization of EGFR levels by BOI. EGF-NIR imaging of mice with HT-29 orthotopic CRC tumor indicated that EGF-NIR is more slowly cleared from the tumor and the highest SBR between tumor and normal adjacent tissue was achieved two days post-injection. Furthermore, images of dissected tissues demonstrated accumulation of EGF-NIR in the tumor and liver. EGF-NIR specifically and strongly labeled EGFR positive human CRC tissues while adjacent CRC tissue and EGFR negative tissues expressed weak NIR signals. This study emphasizes the use of EGF-NIR for preclinical studies. Combined with other methods, EGF-NIR could provide an additional bio-imaging specific tool in the standardization of measurements of EGFR expression in CRC tissues.

Phospholipids-embedded fully dilutable liquid nanostructures. Part 2: The role of sodium diclofenac.

Complex pseudo-ternary phase diagrams based on sucrose monolaurate (SE), propylene glycol (PG), and phosphatidylcholine (PC) as the "surfactant phase"; triacetin (TA) and decaglycerol ester (10G1CC) as the "oil phase"; and water as the aqueous phase were constructed, into which we solubilized the water-insoluble drug (sodium diclofenac, Na-DFC). In our previous study we demonstrated that the solubilization of Na-DFC in the oil+surfactant phases (prior to diluting it with water), was 90-fold greater than its dissolution in water, and that the system was pH-dependent. The greatest Na-DFC solubilization capacity was obtained at pH 7.2. In this study we examined the effect of the solubilization of Na-DFC in a phosphatidylcholine system using DLS, viscosity, electrical conductivity, SAXS, SD-NMR, and cryo-TEM measurements. It was found that: (1) the system remains micellar after aqueous dilution but with greater polydispersity and greater variety of shapes. We concluded that the structures in the absence of water (but in the presence of PG) were of direct spherical micelles (∼4 nm) mixed with elongated cylindrical micelles (12-140 nm); (2) the aqueous dilution causes fragmentation of the cylinders into smaller spherical micelles; (3) solubilization of Na-DFC behaving like a kosmotropic agent or "structure maker" yields mostly spherical swollen micelles and more ordered systems than in its absence; and (4) Na-DFC is solubilized at the interface of the micelles without swelling the droplets.

Phospholipids embedded fully dilutable liquid nanostructures. Part 1: compositions and solubilization capacity.

The use of phospholipids (PL) as surfactants in micellar systems and microemulsions offers many advantages as drug delivery vehicles. PL are commonly used in combination with other non-food surfactants with cosolvents and cosurfactants to form a cascade of delivery structures. In this work, we incorporated phosphatidylcholine (PC) in our unique U-type water-dilutable phase diagrams exhibiting large isotropic regions of nanostructures. The nanometric liquid structures were prepared from food-grade emulsifiers. We formed water-free concentrates with PC that are fully diluted with water to form a variety of unusually structured nanodroplets. Due to the uniqueness of their composition, the designed concentrates derived from the nature of the oil phase, type of surfactants, and cosurfactants were characterized and found to be direct micelles (rather than inverse micelles), with the surfactant headgroups convexed toward the hydrophilic phase away from the micelle's core, even in the absence of water. The concentrates tend to self-assemble upon adding water to form O/W microemulsions even with small amounts of water (water-poor compositions of 0-20 wt% water). Upon further dilution with water the swollen micelles retain the oil as the inner phase. Multi-component compositions with two types of hydrophilic surfactants were investigated. The most significant enhancement in the total isotropic region was obtained by decreasing the triacetin and PC content in the system. We explored, by varying the nature of the headgroups and the nature and length of the lipidic (lipophilic) tails of the PL, the dilution capabilities of each of the systems.