George R. Dakwar

Delivery of proteins to the brain by bolaamphiphilic nano-sized vesicles.

Bolaamphiphilic cationic vesicles with acetylcholine (ACh) surface groups were investigated for their ability to deliver a model protein-bovine serum albumin conjugated to fluorescein isothiocyanate (BSA-FITC) across biological barriers in vitro and in vivo. BSA-FITC-loaded vesicles were internalized into cells in culture, including brain endothelial b.End3 cells, at 37 °C, but not at 4 °C, indicating an active uptake process. To examine if BSA-FITC-loaded vesicles were stable enough for in vivo delivery, we tested vesicle stability in whole serum. The half-life of cationic BSA-FITC-loaded vesicles with ACh surface groups that are hydrolyzed by choline esterase (ChE) was about 2 h, whereas the half-life of vesicles with similar surface groups, but which are not hydrolyzed by choline esterase (ChE), was over 5 h. Pyridostigmine, a choline esterase inhibitor that does not penetrate the blood-brain barrier (BBB), increased the stability of the ChE-sensitive vesicles to 6 h but did not affect the stability of vesicles with ACh surface groups that are not hydrolyzed by ChE. Following intravenous administration to pyridostigmine-pretreated mice, BSA-FITC encapsulated in ChE-sensitive vesicles was distributed into various tissues with marked accumulation in the brain, whereas non-encapsulated (free) BSA-FITC was detected only in peripheral tissues, but not in the brain. These results show that cationic bolaamphiphilic vesicles with ACh head groups are capable of delivering proteins across biological barriers, such as the cell membrane and the blood-brain barrier (BBB). Brain ChE activity destabilizes the vesicles and releases the encapsulated protein, enabling its accumulation in the brain.

Colloidal stability of nano-sized particles in the peritoneal fluid: Towards optimizing drug delivery systems for intraperitoneal therapy

Intraperitoneal (IP) administration of nano-sized delivery vehicles containing small interfering RNA (siRNA) has recently gained attention as an alternative route for the efficient treatment of peritoneal carcinomatosis. The colloidal stability of nanomatter following IP administration has, however, not been thoroughly investigated yet. Here, enabled by advanced microscopy methods such as single particle tracking and fluorescence correlation spectroscopy, we follow the aggregation and cargo release of nano-scaled systems directly in peritoneal fluids from healthy mice and ascites fluid from a patient diagnosed with peritoneal carcinomatosis. The colloidal stability in the peritoneal fluids was systematically studied as a function of the charge (positive or negative) and poly(ethylene glycol) (PEG) degree of liposomes and polystyrene nanoparticles, and compared to human serum. Our data demonstrate strong aggregation of cationic and anionic nanoparticles in the peritoneal fluids, while only slight aggregation was observed for the PEGylated ones. PEGylated liposomes, however, lead to a fast and premature release of siRNA cargo in the peritoneal fluids. Based on our observations, we reflect on how to tailor improved delivery systems for IP therapy.

Decationized polyplexes as stable and safe carrier systems for improved biodistribution in systemic gene therapy

Many polycation-based gene delivery vectors show high transfection in vitro, but their cationic nature generally leads to significant toxicity and poor in vivo performance which significantly hampers their clinical applicability. Unlike conventional polycation-based systems, decationized polyplexes are based on hydrophilic and neutral polymers. They are obtained by a 3-step process: charge-driven condensation followed by disulfide crosslinking stabilization and finally polyplex decationization. They consist of a disulfide-crosslinked poly(hydroxypropyl methacrylamide) (pHPMA) core stably entrapping plasmid DNA (pDNA), surrounded by a shell of poly(ethylene glycol) (PEG). In the present paper the applicability of decationized polyplexes for systemic administration was evaluated. Cy5-labeled decationized polyplexes were evaluated for stability in plasma by fluorescence single particle tracking (fSPT), which technique showed stable size distribution for 48 h unlike its cationic counterpart. Upon the incubation of the polymers used for the formation of polyplexes with HUVEC cells, MTT assay showed excellent cytocompatibility of the neutral polymers. The safety was further demonstrated by a remarkable low teratogenicity and mortality activity of the polymers in a zebrafish assay, in great contrast with their cationic counterpart. Near infrared (NIR) dye-labeled polyplexes were evaluated for biodistribution and tumor accumulation by noninvasive optical imaging when administered systemically in tumor bearing mice. Decationized polyplexes exhibited an increased circulation time and higher tumor accumulation, when compared to their cationic precursors. Histology of tumors sections showed that decationized polyplexes induced reporter transgene expression in vivo. In conclusion, decationized polyplexes are a platform for safer polymeric vectors with improved biodistribution properties when systemically administered.

Nanomedicine-based intraperitoneal therapy for the treatment of peritoneal carcinomatosis — Mission possible?

ABSTRACT Intraperitoneal (IP) drug delivery represents an attractive strategy for the local treatment of peritoneal carcinomatosis (PC). Over the past decade, a lot of effort has been put both in the academia and clinic in developing IP therapeutic approaches that maximize local efficacy while limiting systemic side effects. Also nanomedicines are under investigation for the treatment of tumors confined to the peritoneal cavity, due to their potential to increase the peritoneal retention and to target drugs to the tumor sites as compared to free drugs. Despite the progress reported by multiple clinical studies, there are no FDA approved drugs or formulations for specific use in the IP cavity yet. This review discusses the current clinical management of PC, as well as recent advances in nanomedicine‐based IP delivery. We address important challenges to be overcome towards designing optimal nanocarriers for IP therapy in vivo. Graphical abstract Figure. No Caption available.

Disregarded Effect of Biological Fluids in siRNA Delivery: Human Ascites Fluid Severely Restricts Cellular Uptake of Nanoparticles

Small interfering RNA (siRNA) offers a great potential for the treatment of various diseases and disorders. Nevertheless, inefficient in vivo siRNA delivery hampers its translation into the clinic. While numerous successful in vitro siRNA delivery stories exist in reduced-protein conditions, most studies so far overlook the influence of the biological fluids present in the in vivo environment. In this study, we compared the transfection efficiency of liposomal formulations in Opti-MEM (low protein content, routinely used for in vitro screening) and human undiluted ascites fluid obtained from a peritoneal carcinomatosis patient (high protein content, representing the in vivo situation). In Opti-MEM, all formulations are biologically active. In ascites fluid, however, the biological activity of all lipoplexes is lost except for lipofectamine RNAiMAX. The drop in transfection efficiency was not correlated to the physicochemical properties of the nanoparticles, such as premature siRNA release and aggregation of the nanoparticles in the human ascites fluid. Remarkably, however, all of the formulations except for lipofectamine RNAiMAX lost their ability to be taken up by cells following incubation in ascites fluid. To take into account the possible effects of a protein corona formed around the nanoparticles, we recommend always using undiluted biological fluids for the in vitro optimization of nanosized siRNA formulations next to conventional screening in low-protein content media. This should tighten the gap between in vitro and in vivo performance of nanoparticles and ensure the optimal selection of nanoparticles for further in vivo studies.

Mapping of the Binding Landscape for a Picomolar Protein-Protein Complex through Computation and Experiment

Our understanding of protein evolution would greatly benefit from mapping of binding landscapes, i.e., changes in protein-protein binding affinity due to all single mutations. However, experimental generation of such landscapes is a tedious task due to a large number of possible mutations. Here, we use a simple computational protocol to map the binding landscape for two homologous high-affinity complexes, involving a snake toxin fasciculin and acetylcholinesterase from two different species. To verify our computational predictions, we experimentally measure binding between 25 Fas mutants and the 2 enzymes. Both computational and experimental results demonstrate that the Fas sequence is close to the optimum when interacting with its targets, yet a few mutations could further improve Kd, kon, and koff. Our computational predictions agree well with experimental results and generate distributions similar to those observed in other high-affinity PPIs, demonstrating the potential of simple computational protocols in capturing realistic binding landscapes.

Freeze-dried mucoadhesive polymeric system containing pegylated lipoplexes: towards a vaginal sustained released system for siRNA

Topical vaginal sustained delivery of siRNA presents a significant challenge due to the short residence time of formulations. Therefore, a drug delivery system capable to adhere to the vaginal mucosa is desirable, as it could allow a prolonged delivery and increase the effectiveness of the therapy. The aim of this project is to develop a polymeric solid mucoadhesive system, loaded with lipoplexes, able to be progressively rehydrated by the vaginal fluids to form a hydrogel and to deliver siRNA to vaginal tissues. To minimize adhesive interactions with vaginal mucus components, lipoplexes were coated with different derivatives of polyethylene glycol: DPSE-PEG2000, DPSE-PEG750 and ceramide-PEG2000. Based on stability and diffusion properties in simulated vaginal fluids, lipoplexes containing DSPE-PEG2000 were selected and incorporated in hydroxyethyl cellulose (HEC) hydrogels. Solid systems, called sponges, were then obtained by freeze-drying. Sponges meet acceptable mechanical characteristics and their hardness, deformability and mucoadhesive properties are not influenced by the presence of lipoplexes. Finally, mobility and stability of lipoplexes inside sponges rehydrated with vaginal mucus, mimicking in situ conditions, were evaluated by advanced fluorescence microscopy. The release rate was found to be influenced by the HEC concentration and consequently by the viscosity after rehydration. This study demonstrates the feasibility of entrapping pegylated lipoplexes into a solid matrix system for a prolonged delivery of siRNA into the vagina.

Design of smart GE11-PLGA/PEG-PLGA blend nanoparticulate platforms for parenteral administration of hydrophilic macromolecular drugs: synthesis, preparation and in vitro/ex vivo characterization.

Active drug targeting and controlled release of hydrophilic macromolecular drugs represent crucial points in designing efficient polymeric drug delivery nanoplatforms. In the present work EGFR-targeted polylactide-co-glycolide (PLGA) nanoparticles were made by a blend of two different PLGA-based polymers. The first, GE11-PLGA, in which PLGA was functionalized with GE11, a small peptide and EGFR allosteric ligand, able to give nanoparticles selective targeting features. The second polymer was a PEGylated PLGA (PEG-PLGA) aimed at improving nanoparticles hydrophilicity and stealth features. GE11 and GE11-PLGA were custom synthetized through a simple and inexpensive method. The nanoprecipitation technique was exploited for the preparation of polymeric nanoparticles composed by a 1:1weight ratio between GE11-PLGA and PEG-PLGA, obtaining smart nanoplatforms with proper size for parenteral administration (143.9±5.0nm). In vitro cellular uptake in EGFR-overexpressing cell line (A549) demonstrated an active internalization of GE11-functionalized nanoparticles. GE11-PLGA/PEG-PLGA blend nanoparticles were loaded with Myoglobin, a model hydrophilic macromolecule, reaching a good loading (2.42% respect to the theoretical 4.00% w/w) and a prolonged release over 60days. GE11-PLGA/PEG-PLGA blend nanoparticles showed good in vitro stability for 30days in physiological saline solution at 4°C and for 24h in pH 7.4 or pH 5.0 buffer at 37°C respectively, giving indications about potential storage and administration conditions. Furthermore ex vivo stability study in human plasma using fluorescence Single Particle Tracking (fSPT) assessed good GE11-PLGA/PEG-PLGA nanoparticles dimensional stability after 1 and 4h. Thanks to the versatility in polymeric composition and relative tunable nanoparticles features in terms of drug incorporation and release, GE11-PLGA/PEG-PLGA blend NPs can be considered highly promising as smart nanoparticulate platforms for the treatment of diseases characterized by EGFR overexpression by parenteral administration .

High-pressure nebulization as application route for the peritoneal administration of siRNA complexes

Peritoneal carcinomatosis is a severe form of cancer in the abdomen, currently treated with cytoreductive surgery and intravenous chemotherapy. Recently, nebulization has been proposed as a less invasive strategy for the local delivery of chemotherapeutic drugs. Also, RNA interference has been considered as a potential therapeutic approach for treatment of cancer. In this study, Lipofectamine RNAiMAX/siRNA complexes and cyclodextrin/siRNA complexes are evaluated before and after nebulization. Nebulization of the siRNA complexes does not significantly lower transfection efficiency when compared to non-nebulized complexes. After incubation in ascites fluid, however, the cyclodextrin/siRNA complexes show a drastic decrease in transfection efficiency. For the Lipofectamine RNAiMAX/siRNA complexes, this decrease is less pronounced. It is concluded that nebulization is an interesting technique to distribute siRNA complexes into the peritoneal cavity, providing the complexes are stable in ascites fluid which might be present in the peritoneal cavity.

PEGylated and Functionalized Aliphatic Polycarbonate Polyplex Nanoparticles for Intravenous Administration of HDAC5 siRNA in Cancer Therapy.

Guanidine and morpholine functionalized aliphatic polycarbonate polymers are able to deliver efficiently histone deacetylase 5 (HDAC5) siRNA into the cytoplasm of cancer cells in vitro leading to a decrease of cell proliferation were previously developed. To allow these biodegradable and biocompatible polyplex nanoparticles to overcome the extracellular barriers and be effective in vivo after an intravenous injection, polyethylene glycol chains (PEG750 or PEG2000) were grafted on the polymer structure. These nanoparticles showed an average size of about 150 nm and a slightly positive ζ-potential with complete siRNA complexation. Behavior of PEGylated and non-PEGylated polyplexes were investigated in the presence of serum, in terms of siRNA complexation (fluorescence correlation spectroscopy), size (dynamic light scattering and single-particle tracking), interaction with proteins (isothermal titration calorimetry) and cellular uptake. Surprisingly, both PEGylated and non-PEGylated formulations presented relatively good behavior in the presence of fetal bovine serum (FBS). Hemocompatibility tests showed no effect of these polyplexes on hemolysis and coagulation. In vivo biodistribution in mice was performed and showed a better siRNA accumulation at the tumor site for PEGylated polyplexes. However, cellular uptake in protein-rich conditions showed that PEGylated polyplex lost their ability to interact with biological membranes and enter into cells, showing the importance to perform in vitro investigations in physiological conditions closed to in vivo situation. In vitro, the efficiency of PEGylated nanoparticles decreases compared to non-PEGylated particles, leading to the loss of the antiproliferative effect on cancer cells.