[24] THERMAL THERAPY WITH MAGNETIC NANO PARTICLES FOR CELL DESTRUCTION

 

Adi Vegerhof¹, Menachem Motiei², Arkady Rudintzky¹, Rachela  Popovtzer³, Zeev Zalevsky¹

 

	1 Bar Ilan University; Bar Ilan University
	2 Faculty of Engineering, Bar-Ilan University, Ramat-Gan, Israel; Institutes of Nanotechnology & Advanced Materials, Bar-Ilan University, Ramat-Gan, Israel
	3 Faculty of Engineering, Bar-Ilan University; The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University

 

 

Thermal therapy with magnetic Nano Particles for cell destruction

Adi Vegerhof 1, Menachem Motei1, Arkady Rudinzky1, Zeev Zalevsky1 and Rachela Popovtzer1

 

1Faculty of Engineering & the Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan, Israel

 

Abstract

The main goal of this study involves developing a new concept for cell destruction based upon manipulating magnetic nano-particles (MNPs) along with illuminating a focused laser beam on the sample for its heating. Photo-thermal therapy (PTT) is a minimally-invasive therapy in which photon energy is converted into heat to destruct cells of malignant tissue. The manipulated MNPs technique that we present in this paper is realized by applying external magnetic excitation fields that oscillate the particles while the laser beam causes their temperature rising.  Laser heating combined with the additional MNPs motion causes to the heating of the cell culture to be more efficient and quicker, thereby it is making the manipulated MNPs technique as a superior agent for PTT.

Our method can specifically target cells or other live tissue, while herein we used head and neck cancer cells and formed a concentrated assembly yielding the improved cell destruction capabilities that our concept can offer.

Keywords: Magnetic nanoparticles (MNPs), thermotherapy, Photo-thermal therapy, head and neck cancer, cell destruction.

 

 

 

[29] TARGETED DRUG DELIVERY OF NEAR IR FLUORESCENT AND DOXORUBICIN-CONJUGATED POLY(ETHYLENE GLYCOL) BISPHOSPHONATE NANOPARTICLES FOR DIAGNOSIS AND THERAPY OF BONE TUMOR IN A MOUSE MODEL

 

Safra Rudnick-Glick¹, Enav Corem Salkmon², Igor Grinberg³, shlomo Margel⁴

 

	1 Department of Chemistry ; The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University,
	2 Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Ramat-Gan, Israel; Institue of Nanotechnology and Advanced Matireals
	3 Department of Chemistry; The Institute of Nanotechnology and Advanced Materials
	4 Biu; Department of Chemistry & The Institute of Nanotechnology and Advanced Materials

 

 

Abstract

Osteosarcoma is a commonly diagnosed bone tumor. Current treatment based on a combination of surgery and chemotherapy is associated with severe side effects due to high dosages and nonspecific uptake. Bisphosphonates have a strong affinity to Ca+2 ions and are widely used in the treatment of bone disorders. We have engineered a unique biodegradable bisphosphonate nanoparticles baring two functional surface groups: 1) primary amine groups for covalent attachment of a dye/drug (e.g. NIR dye Cy 7 or doxorubicin); 2) bisphosphonates groups for targeting and chelation to bone hydroxyapatite. In vitro experiments on Saos-2 human osteosarcoma cell line, demonstrated that at a tenth of the concentration doxorubicin-conjugated bisphosphonate nanoparticles achieved a similar uptake to free doxorubicin. In vivo Soas-2 human osteosarcoma xenograft mouse models experiments confirmed specific targeting by the NIR fluorescence bisphosphonate nanoparticles, and 40% greater inhibition of tumor growth by doxorubicin-conjugated bisphosphonate nanoparticle over free doxorubicin.

 

 

 

[30] ENGINEERING OF BASIC PROTEINOIDS & PROTEINOIDS-PLLA POLYMERS AND HOLLOW NANOPARTICLES FOR BIOMEDICAL APPLICATIONS

 

Stella Kiel¹, Enav Corem Salkmon², Igor Grinberg³, Michal Kolitz Domb⁴, shlomo Margel⁵

 

	1 Bar Ilan University; Bar Ilan University
	2 Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Ramat-Gan, Israel; Institue of Nanotechnology and Advanced Matireals
	3 Chemestry Department; Institute for Nanotechnology and Advanced Materials (Bina)
	4 Chemistry Department; Institute for Nanotechnology and Advanced Materials (Bina)
	5 Biu; Department of Chemistry & The Institute of Nanotechnology and Advanced Materials

 

 

This research deals with the unique class of protein-like polymers, called “proteinoids”. Proteinoids are thermal polymers synthesized by the method of condensation polymerization of α-amino acids. Most of the reported proteinoids are composed of a various selected amino acids, so that the final product resembles natural proteins, thus providing non-immunogenic and non-toxic characteristics. It was also observed that proteinoids are far more resistant to cleavage by digestive enzymes than natural proteins. The proteinoids may be acidic, basic or neutral, depending on the composition of polymerized α-amino acids. The synthetic proteinoids may have an enormous number of applications and could be a source for preparing proteinoid hollow microspheres via a self-assembly process, for use in encapsulation and controlled release, drug delivery, etc.

Here we present a new set of basic proteinoids and proteinoid nano/micro-hollow particles containing a fluorescent dye and/or a cancer drug for biomedical applications. We prepared and characterized a new set of basic proteinoids by thermal random condensation polymerization of lysine with phenylalanine, histidine and arginine in the presence or absence of poly-L-lactic acid (PLLA 2 kDa). The PLLA was inserted into the main backbone polymeric chains and provided additional way for biodegradation of the proteinoid polymers. In our study we have demonstrated that Doxorubicin (DOX)-encapsulated proteinoid particles penetrated different types of cancer cells and were found to be toxic to cancer cells in 24 h post-treatment.

Our next goal is to produce nano-sized proteinoid reactors, which may combine drugs, imaging reagents with a covalently bonded therapeutic delivery system. Furthermore, since the particles possess functional groups, they may be conjugated to targeting moieties. The obtained bioactive nanosystems can serve in various biomedical applications.

 

 

 

 

[32] BIODEGRADABLE BISPHOSPHONATE NANOPARTICLES FOR IMAGING AND THERAPEUTIC APPLICATIONS IN OSTEOSARCOMA

 

Enav Corem Salkmon¹, Safra Rudnick-Glick², Igor Grinberg³, shlomo Margel⁴

 

	1 Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Ramat-Gan, Israel; Institue of Nanotechnology and Advanced Matireals
	2 Department of Chemistry ; The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University,
	3 Chemestry Department; Institute for Nanotechnology and Advanced Materials (Bina)
	4 Biu; Department of Chemistry & The Institute of Nanotechnology and Advanced Materials

 

 

Abstract

Osteosarcoma (OS) is amongst the most commonly diagnosed bone tumors occurring in adolescence, young adults and adults over the age of 65. Current treatment is based on a combination of surgery and chemotherapy. Chemotherapy has improved the survival rate, however it is associated with severe side effects due to the use of high dosages, nonspecific uptake and poor bone blood supply. At present bisphosphonates (BP) are widely used in the treatment of bone disorders including OS. We have engineered a unique biodegradable BP nanoparticle that possesses a dual functionality: 1) covalent attachment of a dye (e.g., NIR dye) or drug to the nanoparticles through the primary amine groups on the surface of the nanoparticle; 2) chelation to the bone mineral hydroxyapatite through the BP on the surface of the nanoparticle. Due to a high concentration of PEG in the BP nanoparticles they possess a relatively long plasma half-life time. Therefore, the nanoparticle has potential for use both in diagnosis and therapy of OS. Doxorubicin was conjugated to the free amine on the surface of the BP nanoparticles. In vitro experiments on osteosarcoma cells demonstrated that the doxorubicin-conjugated BP nanoparticles possess a higher efficacy than the free doxorubicin. Further investigation in vivo in a chicken embryo model confirmed that the doxorubicin-conjugated nanoparticle was significantly more effective in inhibiting tumor growth compared to free doxorubicin at a similar concentration. Additionally, we have shown that these BP nanoparticles preferentially target OS tumor tissue, thus increasing anti-cancer drug bioavailability at targeted site.

 

 

 

[33] NEAR IR FLUORESCENT CONJUGATED POLY(ETHYLENE GLYCOL)BISPHOSPHONATE NANOPARTICLES FOR IN VIVO BONE TARGETING IN A CHICKEN EMBRYO AND A YOUNG MOUSE MODELS

 

Igor Grinberg¹, Enav Corem Salkmon², Safra Rudnick-Glick³, Eran Gluz⁴, shlomo Margel⁵

 

	1 Chemestry Department; Institute for Nanotechnology and Advanced Materials (Bina)
	2 Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Ramat-Gan, Israel; Institue of Nanotechnology and Advanced Matireals
	3 Department of Chemistry ; The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University,
	4 Department of Chemistry, The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan 52900, Israel.; The Institute of Nanotechnology and Advanced Materials
	5 Biu; Department of Chemistry & The Institute of Nanotechnology and Advanced Materials

 

 

Abstract

Bisphosphonate (BP) compounds are widely used in the treatment of bone disorders. This group of drugs with a high affinity to Ca⁺² ions is rapidly attracted to bone mineral, especially in areas of high resorption. We have engineered unique biodegradable BP nanoparticles (NPs) by dispersion co-polymerization of the monomers methacrylate-PEG-BP) and (3-Aminopropyl)mathacrylamide) with the crosslinker monomer tetra ethylene glycol diacrylate.  These NPs possess a dual functionality: 1) covalent attachment of a dye (e.g., near IR dye) or a drug to the nanoparticles through the primary amine groups on the surface of the NPs; 2) chelation to the bone mineral hydroxyapatite through the BP on the surface of the NPs. This study describes the uptake of the unique near IR fluorescent Cy 7-conjugated BP NPs in bone of a chicken embryo model as well as in a young mouse model. Blood half-life studies revealed a relatively long half-life, due to a high concentration of PEG in the BP NPs as well as a relatively long whole body clearance. Body distribution studies showed a specific uptake of the BP NPs in bone. These unique engineered BP NPs are planned to be utilized in future work for diagnostic and drug delivery systems that are targeted to bone disorders.

 

 

 

[40] ENGINEERING OF NOVEL PROTEINOIDS AND PLLA-PROTEINOID POLYMERS OF NARROW SIZE DISTRIBUTION AND UNIFORM NANO/MICRO-HOLLOW PARTICLES FOR BIOMEDICAL APPLICATIONS

 

Michal Kolitz Domb¹, Enav Corem Salkmon², Igor Grinberg³, shlomo Margel⁴

 

	1 Chemistry Department; Institute for Nanotechnology and Advanced Materials (Bina)
	2 Department of Chemistry, Bar-Ilan Institute of Nanotechnology and Advanced Materials, Ramat-Gan, Israel; Institue of Nanotechnology and Advanced Matireals
	3 Chemestry Department; Institute for Nanotechnology and Advanced Materials (Bina)
	4 Biu; Department of Chemistry & The Institute of Nanotechnology and Advanced Materials

 

 

The use of near-infrared (NIR) fluorescence imaging has gained great interest in the field of cancer detection due to the low autofluorescence that water and intrinsic biomolecules display in this region. The study describes the synthesis of new protein-like thermal polymers, proteinoids. Several types of proteinoids made of one to three different amino acids, in absence or presence, of low molecular weight PLLA, were synthesized. The ability to obtain several high-MW durable proteinoids, by using different amino acids, along with incorporating PLLA in their structure, yields a new perspective of biodegradable polymers and polymer nano/microparticles. The proteinoids reported are of unusual high molecular weights, along with a low polydispersity index. Under suitable gentle conditions, the proteinoids can self-assemble to form nano- and micron-sized hollow particles of relatively narrow size distribution. The proteinoid particles are non-toxic and stable; hence, they may be excellent candidates for various biomedical applications, e.g., cell labeling and separation, encapsulation, controlled release, drug targeting, etc. In this study, the proteinoid poly(glutamic acid-phenylalanine)-PLLA (P(EF)-PLLA) was used to form NIR fluorescent nanoparticles by encapsulation of the dye indocyanine green (ICG). The encapsulation process increases significantly the photostability of the dye. These NIR fluorescent nanoparticles were found to be stable and non-toxic. Leakage of the NIR dye from these nanoparticles into PBS containing 4% HSA was not detected. The NIR fluorescent nanoparticles were injected into a mouse and the biodistribution was monitored and quantified. Interestingly, the particles injected through the tail vein were found in various parts of the body, including the bones and brain, and were evacuated within 24 h completely. Tumor-targeting ligands such as peanut agglutinin (PNA) and anti-carcinoembryonic antigen antibodies (anti-CEA) were covalently conjugated to the NIR fluorescent P(EF-PLLA) nanoparticles’ surface, increasing thereby the fluorescent signal of tumors with upregulated corresponding receptors. Specific colon tumor detection by the NIR fluorescent P(EF-PLLA) nanoparticles was demonstrated in a chicken embryo model and a mouse model.

 

 

 

[62] SIZE CONTROLLED LAYER-BY-LAYER TECHNIQUE FOR DRUG DELIVERY

 

Lior Minkowicz¹, Karen  Adler¹, Ofra Benny¹

 

1 The Hebrew University of Jerusalem; Institute for Drug Research

 

 

 

The ability of drugs to efficiently treat cancer is dependent on their stability and ability to penetrate tumor tissue. Drug delivery systems are designed to improve drugs’ properties such as stability and specificity, and provide a means for slow release. One of the main challenges in drug delivery in cancer is the ability to control the exact properties of drug vehicle. It was demonstrated that particle/carrier size, charge, and stiffness all have a substantial effect on the bio-distribution of drug carriers and on their tumor uptake.

Layer-by-layer (LbL) deposition on template beads represents an appealing method for controlling drug carrier properties. The majority of studies in this field use silica beads as template, requiring harsh conditions for melt-down that can damage drug loading. An alternative template material is calcium carbonate (CaCO₃), but it has some significant drawbacks such as large heterogeneity due to polymorphism and difficulty in controlling size.

We hypothesize that controlling CaCO₃ particle size and morphology can improve properties of LbL drug carriers and provide a versatile platform for preparing a drug loaded particle library with different charges, sizes, and flexibility. 

The crystallization of CaCO₃ particles was performed by reacting sodium carbonate (Na₂CO₃) and calcium nitrate (Ca(NO₃)₂) in the presence of additives and under sonication. The particles were used as template for LbL deposition of oppositely charged fluorescently labeled polysaccharides, alginate and chitosan. The particles were visualized using fluorescent and confocal microscope. We demonstrated that the size of CaCO₃ templates ranging from sub-micron to a few microns can be controlled by modifying preparation conditions. Moreover, by changing the number of layers, we can vary the multi-layering width affecting the overall diameter of the particles.

On-going research is being done to optimize properties of particles with anti-cancer drugs to investigate the potential increase in tumor selectivity by adjusting the carrier to optimize affinity and cellular uptake.

 

 

 

[63] NANOPARTICLE-BASED CT IMAGING FOR STEM CELLS TRACKING WITHIN THE BRAIN: APPLICATION IN NEUROPSYCHIATRIC DISORDERS

 

Oshra Betzer¹, Amit Shwartz², Menachem Motiei³, Gal Yadid⁴, Rachela  Popovtzer⁵

 

	1 The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University; Gonda Brain Research Center, Bar-Ilan University, Ramat-Gan, Israel
	2 Everard and Mina Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel; Gonda Brain Research Center, Bar-Ilan University, Ramat-Gan, Israel
	3 Faculty of Engineering, Bar-Ilan University, Ramat-Gan, Israel; Institutes of Nanotechnology & Advanced Materials, Bar-Ilan University, Ramat-Gan, Israel
	4 Gonda Brain Research Center, Bar-Ilan University, Ramat-Gan, Israel; Everard and Mina Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel
	5 Faculty of Engineering, Bar-Ilan University; The Institute of Nanotechnology and Advanced Materials, Bar-Ilan University

 

 

Nanoparticle-based CT imaging for stem cells tracking within the brain: application in neuropsychiatric disorders

 

A critical problem in the development and implementation of stem cell-based therapy is the lack of reliable, non-invasive means to image and trace the cells post-transplantation and evaluate their bio-distribution, final fate and functionality. In this study, we developed a gold nanoparticle-based CT imaging technique for longitudinal mesenchymal stem cell (MSC) tracking within the brain. We applied this technique for non-invasive monitoring of MSCs transplanted in a rat models for depression and drug addiction. Our research reveals that cell therapy is a potential approach for treating neuropsychiatric disorders. Our results, which demonstrate that cell migration could be detected as early as 24 hours and up to one month post-transplantation, revealed that MSCs specifically navigated and homed to distinct depression-or addiction related brain regions. We further developed a non-invasive quantitative CT ruler, which can be used to determine the number of cells residing in a specific brain region, without tissue destruction or animal scarification. This technique may have a transformative effect on cellular therapy, both for basic research and clinical applications.

 

 

 

[72] TUMOR NECROSIS FACTOR RELATED APOPTOSIS INDUCING LIGAND-CONJUGATED NEAR IR FLUORESCENT IRON OXIDE/HUMAN SERUM ALBUMIN CORE-SHELL NANOPARTICLES OF NARROW SIZE DISTRIBUTION FOR CANCER TARGETING AND THERAPY

 

Itay Levy¹

 

1 Bar Ilan University; Biu

 

 

Tumor Necrosis Factor Related Apoptosis Inducing Ligand-Conjugated Near IR Fluorescent Iron Oxide/Human Serum Albumin Core-Shell Nanoparticles of Narrow Size Distribution for Cancer Targeting and Therapy

Itay Levy, Igor Grinberg, Benny Perlstein, Enav Corem-Salkmon and Shlomo Margel*.

Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel

*Corresponding author. E-mail: shlomo.margel@mail.biu.ac.il

 

Abstract

Although much progress has been made in the field of cancer therapy, cancer remains one of the leading causes of death in the western world. Here we have designed and studied a unique type of composite multi-functional near IR (NIR) fluorescent iron oxide (IO) nanoparticles (NPs) of narrow size distribution for tumor targeting and therapy. These NPs were prepared by nucleation followed by controlled growth of thin films of IO onto Cy7-conjugated gelatin nuclei, coated then with human serum albumin (HSA) by a thermal precipitation process.  The hydrodynamic diameter of these core-shell NPs could be easily controlled by varying the precipitation reaction temperature.

For targeting and an anti-cancer effect, we conjugated the Tumor Necrosis Factor Related Apoptosis Inducing Ligand (TRAIL) cytokine to the surface of the NIR fluorescent IO/HSA NPs via a polyethylene glycol (3 kDa) linker.  The conjugated TRAIL exhibited enhanced and prolonged anti-cancer activity in both human glioblastoma multiforme and colon cancer cell lines. Further, the combination of these IO/HSA-TRAIL NPs with the commonly used chemotherapeutic drug doxorubicin resulted in a synergistic anti-cancer effect on these cancer cell lines. In addition, we also clearly demonstrated by topically and IV administrations the specific targeting effect and the synergistic therapy effect of these NIR fluorescent NPs in-ovo, by using a chicken embryo model of tumors derived from the various human cancer cell lines.

 

Keywords:   Iron oxide nanoparticles, Near IR fluorescent iron oxide nanoparticles, Cancer targeting, Cancer therapy.

 

 

 

[117] NIR FLUORESCENT CORE AND CORE-SHELL ALBUMIN NANOPARTICLES FOR IN VIVO DETECTION OF COLONIC NEOPLASMS

 

Sarit Cohen¹, shlomo Margel¹

 

1 Biu; Department of Chemistry & The Institute of Nanotechnology and Advanced Materials

 

 

 

NIR Fluorescent Core and Core-Shell Albumin Nanoparticles for In vivo Detection of Colonic Neoplasms

 

According to scientific literature, near IR (NIR) dyes such as Indocyanine Green (ICG) and other structurally related cyanine dyes have been shown to have high affinity to albumin (HSA, BSA). We have exploited this fact for the preparation of albumin nanoparticles containing cyanine NIR dyes, entrapped by strong non-covalent interactions. This study demonstrates that the encapsulation of NIR fluorescent dye within the albumin nanoparticles significantly reduces the photobleaching of the dye. Leakage of the NIR dye from these nanoparticles into PBS containing 4% albumin and into human bowel juice was not detected. The particles may also be of core-shell structure, e.g., a core such as iron oxide with the NIR-dyed albumin coating thus forming the shell. The work presented here is a feasibility study to test the suitability of NIR fluorescent HSA nanoparticles for optical detection of colonic cancer. The tumor-targeting ligands, peanut agglutinin

(PNA), anti-carcinoembryonic antigen antibodies (anti-CEA) and tumor associated glycoprotein-72 monoclonal antibodies (anti-TAG-72) were covalently conjugated to the NIR fluorescent HSA nanoparticles. Specific colon tumor detection was demonstrated in chicken-embryo and mouse models for both the non-conjugated and the biomolecule-conjugated NIR fluorescent albumin nanoparticles. The conjugation of the PNA, or anti- CEA or anti-TAG-72 to the nanoparticles significantly increased the fluorescence intensity of the tagged colon tumor tissues relative to the non-conjugated nanoparticles. The main advantage of these particles is their fluorescence in the NIR region of the electromagnetic spectrum, allowing in vivo imaging with low tissue absorbance, increased tissue penetration and low auto fluorescence of bodily tissues. This allows for early specific detection of neoplasms in the gastrointestinal tract. In future work we also plan to encapsulate cancer drugs, e.g., doxorubicin, within these NIR fluorescent nanoparticles for both colon cancer imaging and therapy

 

 

 

[121] NANOMEDICINE FOR CANCER IMMUNOTHERAPY:

 TRACKING CANCER-SPECIFIC T CELLS IN VIVO

 

Rinat Meir¹

 

1 Bar Ilan University; The Institute for Nanotechnology Biu

 

 

NANOMEDICINE FOR CANCER IMMUNOTHERAPY: TRACKING CANCER-SPECIFIC T CELLS IN VIVO

Rinat Meir1, Katerina Shamalov2, Oshra Betzer1, Menachem Motiei1, Cyrille Cohen2, Rachela Popovtzer1

1 Faculty of Engineering, Bar-Ilan University, Ramat-Gan, Israel 2 Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel

Application of immune cell-based therapy in routine clinical practice is challenging, due to the poorly-understood mechanisms underlying success or failure of treatment. Development of accurate and quantitative imaging techniques for non-invasive cell tracking can provide essential knowledge for elucidating these mechanisms. We designed a novel method for longitudinal and quantitative in vivo cell tracking, based on the superior visualization abilities of classical X-ray computed tomography (CT), combined with state-of-the-art nanotechnology. Herein, T-cells were transduced to express a melanoma-specific T-cell receptor and then labeled with gold nanoparticles (GNPs) as a CT contrast agent. The GNP-labeled T-cells were injected intravenously to mice bearing human melanoma xenografts, and whole-body CT imaging allowed examination of the distribution, migration and kinetics of T-cells. Using CT, we found that transduced T-cells accumulated at the tumor site, as opposed to non-transduced cells. Labeling with gold nanoparticles did not affect T-cell function, as demonstrated both in vitro, by cytokine release and proliferation assays, and in vivo, as tumor regression was observed. Moreover, to validate the accuracy and reliability of the proposed cell tracking technique, T-cells were labeled both with green fluorescent protein for fluorescence imaging, and with GNPs for CT imaging. A remarkable correlation in signal intensity at the tumor site was observed between the two imaging modalities, at all time points examined, providing evidence for the accuracy of our CT cell tracking abilities. This new method for cell tracking with CT offers a valuable tool for research, and more importantly for clinical applications, to study the fate of immune cells in cancer immunotherapy.

 

 

 

[122] RED BLOOD CELLS AS A TOOL FOR TESTING OF NANOPARTICLES BIOACTIVITY

 

Gregory Barshtein¹, Saul Yedgar¹, Leonid D Shvartsman²

 

	1 The Faculty of Medicine; Campus Een Cerem
	2 The Racah Institution of Physics; Campus Givat Ram

 

 

 

 

 

Engineered man-made NPs have various applications in biomedical fields for improving diagnostic tools, drug delivery and cancer treatment. Independently from their use, source and exposure, NPs eventually enter the bloodstream. The entrance of NPs to bloodstream leads to their interaction with red blood cells (RBC), a central object in the blood circulation. This interaction of NPs with RBC may cause impairment of RBCs functionality, specifically, their hemolysis.

Hemolysis may occur if the integrity of RBCs membrane is broken and hemoglobin is released, which may lead to an adverse health effects (e.g., anemia, hypertension, renal toxicity). Thus, hemolytic activity of NPs have been suggested as key tests in determining the safety of NPs. Although hemolysis tests have been conducted with various NPs, comparing results across studies are difficult due to variability in the protocols for performing particle characterization and hemolysis testing. It is necessary to emphasize that according to all previously used protocols, the interaction between NPs and RBC was examined in static conditions. 

In the presented research, we tested the role of shear stress application on hemolytic activity of NPs.

For examination of presented hypothesis we studied interaction between polystyrene NPs (PS-NPs) and RBCs under flow-induced shear stress. We examined the role of following two factors on the level NPs-induced hemolysis: protein-corona formation and application of high shear stress. We demonstrated that shear stress application accelerated NPs-induced hemolysis.

The research characterize, for the first time, the role of the shear stress application in NPs-induced hemolysis. This knowledge will aid our understanding of the NPs toxicity. Finally, the fruits of this research could lead to novel applications in the field of nanomedicine – development of a new methodology for the assessment of potential toxicity of nanoparticles.

 

 

 

[127] IMPROVED ORAL ABSORPTION OF EXENATIDE USING AN ORIGINAL NANOENCAPSULATION AND MICROENCAPSULATION APPROACH

 

Liat Soudry-kochavi¹, Natalya  Naraykin¹, Taher Nassar¹, Simon Benita¹

 

1 The Institute of Drug Research ; The Hebrew University of Jerusalem, Ein Karem

 

 

Oral delivery is the most convenient and favorable route for chronic administration of peptides and proteins to patients. However, many obstacles are faced when developing such a delivery route. Nanoparticles (NPs) are among the leading innovative solutions for delivery of these drugs. Exenatide is a peptidic drug administered subcutaneously twice a day chronically as an add-on therapy for the world wide pandemic disease, diabetes. Many attempts to develop oral nanocarriers for this drug have been unsuccessful due to the inability to retain this hydrophilic macromolecule under sink conditions or to find a suitable cross-linker which does not harm the chemical integrity of the peptide. In this study, we report about an original oral delivery solution based on a mixture of albumin and dextran NPs cross-linked using sodium trimetaphosphate. Moreover, we suggest a second defense line of gastro-resistant microparticles composed of an appropriate ratio of Eudragit® L100-55 and hydroxypropylmethylcellulose, for additional protection to these NPs presumably allowing them to be absorbed in the intestine intact. Our results demonstrate that such a system indeed improves the relative oral bioavailability of exenatide to a level of about 77% compared to subcutaneous injection due to the presence of dextran in the coating wall of the NPs which apparently promotes the lymphatic uptake in the enterocytes. This technology may be a milestone on the way to deliver other peptides and proteins orally.

 

 

 

[156] THERAPEUTIC EFFICACY OF COMBINING PEGYLATED LIPOSOMALDOXORUBICIN AND RADIOFREQUENCY (RF) ABLATION:COMPARISON BETWEEN SLOW-DRUG-RELEASING,NON-THERMOSENSITIVE AND FAST-DRUG-RELEASING,THERMOSENSITIVE NANO-LIPOSOMES

 

Alexander  V. Andriyanov¹

 

1 Laboratory of Liposome and Membrane Research; Imric

 

 

Aims: To determine how the accumulation of drug in mice bearing an extra-hepatic tumor and its therapeutic efficacy areaffected by the type of PEGylated liposomal doxorubicin used, treatment modality, and rate of drug release from the

liposomes, when combined with radiofrequency (RF) ablation.

Materials and Methods: Two nano-drugs, both long-circulating PEGylated doxorubicin liposomes, were formulated: (1) PEGylated doxorubicin in thermosensitive liposomes (PLDTS), having a burst-type fast drug release above the liposomes’ solid ordered to liquid disordered phase transition (at 42uC), and (2) non-thermosensitive PEGylated doxorubicin liposomes (PLDs), having a slow and continuous drug release. Both were administered intravenously at 8 mg/kg doxorubicin dose to tumor-bearing mice. Animals were divided into 6 groups: no treatment, PLD, RF, RF+PLD, PLDTS, and PLDTS+RF, for intratumor doxorubicin deposition at 1, 24, and 72 h post-injection (in total 41, mice), and 31 mice were used for randomized survival studies.

Results: Non-thermosensitive PLD combined with RF had the least tumor growth and the best end-point survival, better than PLDTS+RF (p,0.005) or all individual therapies (p,0.001). Although at 1 h post-treatment the greatest amount of intra-tumoral doxorubicin was seen following PLDTS+RF (p,0.05), by 24 and 72 h the greatest doxorubicin amount was seen for PLD+RF (p,0.05); in this group the tumor also has the longest exposure to doxorubicin.

Conclusion: Optimizing therapeutic efficacy of PLD requires a better understanding of the relationship between the effect of RF on tumor microenvironment and liposome drug release profile. If drug release is too fast, the benefit of changing the microenvironment by RF on tumor drug localization and therapeutic efficacy may be much smaller than for PLDs having slow and temperature-independent drug release. Thus the much longer circulation time of doxorubicin from PLD than from PLDTS may be beneficial in many therapeutic instances, especially in extra-hepatic tumors.

 

 

 

[184] MICROFLUIDIC BASED FABRICATING OF  NANOPARTICLES

 

Ben-Zion Amoyav¹, Ofra Benny²

 

	1 Hebrew University; Idr- Institute Drug Reserch
	2 The Hebrew University of Jerusalem; Institute for Drug Research

 

 

Microfluidic based Fabricating of  nanoparticles:

Microfluidics is the science of fluid flows at the microscopic scale. Small volumes of solvent, sample, and reagents flowing through micro-channels embedded in the chip, providing a whole new world of improved powerful platform for physical and biological assays.

Miniaturizing into a microfluidic device offer many advantages, among others small requirements for solvents and reagents, low cost and faster parallel analysis.

 

One of the projects we are focusing at the lab is trying to develop new techniques with these useful microdevices templates and overcome a central challenge in the development of new modern anti-cancer drugs nowadays – synthesis of monodisperse nanoparticles.

PLGA poly(lactic-co-glycolic acid) has attracted considerable attention due to its attractive biodegradability property and its one of the most successfully developed nano-particles.

To synthesize particles with this process, we used two different types of microfluidic chips (T Junction and Focused flow) that able to create PLGA emulsions with a highly controlled structure and uniform droplets, which are then solidified to produce particles. 

By varying flow rates, polymer concentration and polymer type we are able to optimize the size, decrease polydispersity index, and as a result to improve the PK and PD characteristics.

The Microfluidics may find applications for modern development and optimization of polymeric nanoparticles in the newly emerging field of nanomedicine

 

 

 

[185] ORAL ABSORPTION OF SOLIDIFIED POLYMER NANOMICELLES

 

Adi Karsch-Bluman¹, Eva Abramov², Ouri Schwob¹, Mara Shapiro¹, Ofra Benny³

 

	1 School of Pharmacy; Hebrew University
	2 The Hebrew University of Jerusalem; School of Pharmacy
	3 The Hebrew University of Jerusalem; Institute for Drug Research

 

 

Oral absorption of solidified polymer nanomicelles

Oral delivery of poorly soluble drugs represents a significant challenge in drug development. Oral administration is the preferable route of drug delivery, especially in chronic disease,that requires prolonged treatments. However, the gastrointestinal track presents a significant physiological barrier for drugs with its wide range of pH and enzymatic activity. Encapsulation of drugs may help this problem by increasing drug absorption, protecting drugs from the external milieu, and providing a controlled release.

Our previous studies demonstrated that conjugation of lipophilic drugs to short polymer monomethoxy poly lactic acid polyethylene glycol (mPEG-PLA) improved drug solubility, stability and oral availability. Here we present our recent results characterizing mPEG-PLA nanomicelles (~20nm) as a vehicle for oral delivery of encapsulated compounds (without chemical conjugation).

To study the intestinal absorption of solid nanomicelles, we used the Caco-2 permeability assay. Caco-2 are human epithelial colorectal adenocarcinoma cells which are used as a standard cellular model for studying oral availability of drugs. In order to elucidate the molecular mechanism of mPEG-PLA nanomicelle endocytosis in Caco-2 cells, specific inhibitors of clathrin, caveolae and lipid raft mediated endocytosis were used. mPEG-PLA nanomicelles were found to internalize rapidly, in a 30 min initial kinetics, with an Apical to Basolateral apparent permeability coefficient (Papp) of 3.8 x10-6 cm/s (2 hr) and 5×10-6 cm/s (4 hr). Moreover, endocytosis was found to be mediated by clathrin in an energy-dependent manner. Finally, we found that the low-density lipoprotein (LDL) receptor is directly involved in the endocytosis of mPEG-PLA, as indicated by interruption in internalization after blockage of the receptor by anti-LDLR antibody and by siRNA knockdown of LDLR gene. Our results introduce mPEG-PLA nanomicelles as a platform for oral delivery of poorly absorbed drugs.

 

 

 

 

[219] MODIFIED STARCH BASED NANOPARTICLES AS PIP3 DELIVERY SYSTEM FOR WOUND HEALING

 

ETILI HOLLANDER¹, ramesh Chintakunta², Riki Goldbart³, Tamar  Traitel³, Assaf Rudich⁴, Joseph Kost³

 

	1 Ben Gurion University OF THE Negev; Ben Gurion University OF THE Negev
	2 Ben Gurion University OF THE Negev; –
	3 Ben Gurion University of the Negev; Department of Chemical Engineering
	4 Faculty of Health Sciences; Department of Clinical Biochemistry

 

 

Modified Starch Based Nanoparticles as PIP₃ Delivery System for Wound Healing

Etili Hollander¹, Ramesh Chintakunta¹, Riki Goldbart¹, Tamar Traitel¹, Assaf Rudich², Joseph Kost¹

¹Department of Chemical Engineering; ²Department of Clinical Biochemistry, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel

Wound healing is a natural restorative response to tissue injury. The primary function of the skin is to serve as a protective barrier against the environment. Once the protective barrier is broken, as a result of an injury, the normal process of wound healing is immediately set in motion. ¹

Phosphatidylinositol-3,4,5-trisphosphate (PIP₃), is a phosphorylated phospha- tidylinositol with phosphate moiety on positions 3,4 and 5 of the inositol ring. PIP₃ is a negatively charged, required lipid messenger that plays an important role in the regulation of many cellular processes, including proliferation, survival, migration and wound healing. ²

To heal a wound, cells surrounding the wound must survive, proliferate, migrate and directionally grow to close the wound.

One of the main challenges is creating an effective path for delivering negatively charged PIP₃ into keratinocytes. Starch is a natural polysaccharide that is considered advantageous for drug delivery due to its biodegradability, biocompatibility, low immunogenicity and minimal cytotoxicity^{. 3 In this study, we use modified cationic starch} (Q-Starch) as a PIP₃ carrier.

The positively charged Q-starch and the negatively charged PIP₃ interact electrostatically, to form a complex. PIP₃/Q-Starch complexes’ surface charge, radius and morphology were evaluated by zeta potential, Dynamic Light Scattering (DLS) and cryo-TEM in the range of 1.5-3 molar ratio of Q-Starch nitrogen groups and PIP₃ phosphate groups (N/P ratio). Results show that average surface charge increases with the increase of N/P ratio. The complexes were successfully obtained above 1.5 N/P ratio.

 

¹ Martin, et al. 1997, Science, 276, 75-81.

² Leslie, et al. 2007, Curr Biol. 17, 115–125.

³ Amar-Lewis, Eliz, et al. 2014, Journal of Controlled Release 185, 109-120.

 

 

 

[238] EXPLORING THE TRANSPORT MECHANISM OF RNAI AND QUATERNIZED-STARCH BASED NANOPARTICLES FOR REDUCING MULTIDRUG RESISTANCE IN OVARIAN CANCER CELLS

 

Eliz Lewis¹, Limor Cohen², Riki Goldbart³, Tamar  Traitel³, Levi A. Gheber², Joseph Kost³

 

	1 Ilse Katz Institute for Nanoscale Science and Technology; Department of Chemical Engineering
	2 Ilse Katz Institute for Nanoscale Science and Technology; Department of Biotechnology Engineering
	3 Ben Gurion University of the Negev; Department of Chemical Engineering

 

 

Drug resistance in ovarian cancer cells is inherited due to over expression of the multi drug efflux pump, P-glycoprotein (P-gp), which effectively reduces the concentration of anti-cancer drugs in the cells. We propose RNA interference (RNAi) approach to silence P-gp expression and induce sensitivity to chemotherapeutic drugs; however a major challenge is a successful siRNA delivery into the cancer cells and its release in the cytoplasm. Therefore, our goal is to develop a delivery platform that will allow overcoming siRNA delivery barriers. We have presented before a safe (biodegradable and biocompatible) and efficient carrier for siRNA based on modified potato starch with quaternary amine groups (Q-starch). Q-starch in the presence of siRNA undergoes self-assembly formation of Q-starch/siRNA nanoparticles and ovarian cancer cells transfected with Q-starch/siP-gp (downregulate P-gp) showed reduced expression of P-gp (50% reduction).

This study examines the nanoparticle’s cellular transport mechanism and its main barriers, cellular uptake and endosomal escape, using light-microscopy and biophysical approaches.

We demonstrated that the nanoparticles rapidly adhere to the cell surface and already following 15min of application accumulate in the cells. Using live-cell microscopy, by tracking nanoparticles’ mean square displacement, we showed significant changes in the dynamics of the nanoparticles in time. The uptake stage is presented by a kinetic study qualitatively and quantitatively showing that Q-starch is necessary for the internalization of siRNA. We elucidated the mechanism by which the nanoparticles enter the cells and revealed a dynamine-dependent endocytosis and partial dependency on clathrin-mediated mechanism. In correspondence, we have shown the following intracellular transport of the complexes through early endosomes (rab5a) up to 4hr, late endosomes (rab7a) and lysosomes following 24hr. Finally, we suggest that a fraction of the complexes are rendered by the endosomal escape barrier in delivering RNAi to the cell cytoplasm. Thus, we suggest in our future study to increase endosomal escape by additional chemical modifications of Q-starch that will promote efficiency of gene silencing.

 

 

 

[251] QUATERNIZED STARCH NANOPARTICLES FOR EXOGENOUS PI3P DELIVERY

 

Nitzan Marelly¹, Tali Vodonos², ramesh chintakunta¹, Riki Goldbart¹, Tamar  Traitel¹, Assaf Rudich², Joseph Kost³

 

	1 Ben Gurion University of the Negev; Department of Chemical Engineering
	2 Faculty of Health Sciences; Department of Clinical Biochemistry
	3 Ben-Gurion University; Department of Chemical Engineering

 

 

Quaternized starch nanoparticles for exogenous PI3P delivery

Nitzan Marelly¹, Tali Vodonos², Ramesh Chintakunta¹, Riki Goldbart¹, Tamar Traitel¹, Assaf Rudich², Joseph Kost¹

¹Department of Chemical Engineering; ²Department of Clinical Biochemistry, Ben-Gurion University of the Negev, Beer-Sheva, 84105, Israel

 

Autophagy, an intra-cellular process in eukaryotic cells, allows for the digestion and recycling of cytoplasmic contents. This is achieved through the formation of double-membrane vesicles (autophagosomes) that undergo degradation through fusion with lysosomes. Basal autophagy plays an important role in cellular homeostasis. Autophagy can also be induced as a cellular reaction to various situations, such as nutrient starvation or pathogen infection. Thus, dysfunction in autophagy has been implicated in the pathogenesis of various diseases, like cancer, infectious diseases and neurodegenerative disorders. For example, a decrease in autophagy flux is correlated with insulin resistance of hepatocytes in obesity. This may be solved by up-regulating autophagy flux.

Since PI3P mediates autophagosome biogenesis through membrane deformation and elongation, and therefore acts as an activator of autophagy, we decided to evaluate its exogenous delivery to the cells using modified starch as a carrier. A major obstacle in delivering exogenous PI3P into cells is overcoming its negative charge (derived from the phosphate groups on the inositol ring).

Starch, a natural polysaccharide, is considered advantageous for drug/gene delivery due to its biodegradability and biocompatibility. In this research potato starch was modified into cationic starch and was used as PI3P carrier. The positively charged quaternized ammonium groups on the modified starch and the negatively charged PI3P interact electrostatically, allowing for self-assembly complexation.

        Q-starch/PI3P complexes were characterized (size, surface charge and geometry) at different N/P ratios (molar ratio between positive nitrogen groups on Q-starch (N) and negative phosphate groups on PI3P (P)). Complexes uptake and effect on autophagy was assessed on HEK-293 cells. Up-regulation of autophagy was shown, suggesting a potential approach for overcoming insulin resistance.

 

 

 

 

[260] TOPICAL TREATMENT OF PSORIASIS BY MIRNA DELIVERY BASED ON ULTRASOUND AND MODIFIED STARCH

 

Rinat Lifshiz¹, Gali Lerman², Einat Elharrar², Tal Segman², Riki Goldbart¹, Tamar  Traitel³, Dror Avni², Yehezkel Sidi², Joseph Kost¹

 

	1 Ben-Gurion University; Department of Chemical Engineering
	2 Laboratory of Molecular Cell Biology, Center for Cancer Research and Department of Internal Medicine; Tel Hashomer
	3 Ben Gurion University of the Negev; Department of Chemical Engineering

 

 

 

     Psoriasis is a chronic inflammatory skin disease that affects 3% of the population, yet it is still without a cure. The most advanced drugs developed in recent years for psoriasis are systemic interventions aimed to inhibit one of the major cytokines which play important roles in the pathogenesis of the psoriatic inflammation. All of these agents are immunosuppressive and indicated only in patients with severe psoriasis or psoriatic arthritis, while safe and effective topical therapy, needed in most of the psoriatic patients, is still lacking. Lerman et al discovered specific micro-RNAs (miRNAs), which are silenced in the psoriatic epidermis and can down-regulate the expression of IL-22RA1 and IL-17RA two subunits of the receptors  to IL-22 and IL-17A respectively. Although miRNA offers new therapeutic potential for treating psoriasis, its topical delivery to the depth of the epidermis is challenging. There are several barriers to topical delivery of miRNAs: 1. The barrier properties of the top layer of the epidermis (stratum corneum): 2. Naked miRNA is unstable in vivo due to enzymatic degradation and immunological responses. 3. The efficacy of RNAi that penetrates the epidermis, is further limited by poor cellular uptake. To overcome these obstacles and allow topical delivery of miRNA to skin cells, we suggested the use of ultrasound (US) as a mean to enhance biological membrane and skin permeability, and quaternized starch (Q-starch) as an miRNA delivery carrier. In vivo experiments on humanized psoriatic mouse model verified the ability of US and modified starch carrier to enhance miRNA transdermal delivery, as well as subsequent decrease in the expression of the IL-22RA1 and IL-17RA, the miRNA target proteins. Moreover, a significant decrease in the psoriatic inflammatory markers has been visualized.

Our results suggest that ultrasound and the Q-starch carrier assisted topical delivery of miRNA could pave the way for future miRNA-based therapy.

 

 

 

[304] 3-D MULTI-CELLULAR MODEL FOR DRUG DELIVERY

 

Hila Shoval¹, Ouri Schwob², Ofra Benny³

 

	1 The Hebrew University; Institue for Drug Research
	2 School of Pharmacy; Hebrew University
	3 The Hebrew University of Jerusalem; Institute for Drug Research

 

 

3-D multi-cellular model for Drug Delivery

Hila Shoval,Ouri Schwob,Ofra Benny

Studying nanoparticle uptake in cells is being predominantly performed in monolayer cultured cells. However, the monolayer lack the presentation of complex cell-cell interactions and extra-cellular matrix. As a result, while the two-dimensional cellular models may be good for determine mechanism of nanoparticle uptake and kinetics of cellular interactions at the single cell level, they cannot provide information about transport of nanoparticles in tissues.

Our goalis to develop a tissue-like 3-D cellular models, which contain more components of the tumor microenvironment, to provide a prediction for the potential of drug nano-carriers to access into tissues.

            Cancer multicellular spheroids were grown from different tumor cell types and characterized to be used as a model for nanoparticle penetration in 3- dimension. Fluorescent-labeled Polyethylene glycol-Poly lactic acid (mPEG-PLA) polymer nanoparticles (~20 nm) were fabricated and characterized. The micelles were incubated with tumor spheroids and the kinetics of their “tissue” diffusion was determined by detecting the fluorescence inside spheroids. Our results indicated a time-dependent penetration of PEG-PLA nanoparticles into the spheroids cell layers. Moreover, we found that this model can differentiate between different tumor types according to the kinetic of nanoparticle transport. We concluded that multicellular spheroid can be used as an efficient model for nanoparticle interaction in tissues and our follow-up studies will compare our results to in-vivo.

 

Kay words: spheroids, tumor, micelles, PEG-PLA

 

 

 

 

 

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[308] ANIONIC NANOPARTICLES OF ALGS–CA2+-SIRNA ARE EFFICIENT CARRIERS OF SIRNA TO HUMAN HEPATOCYTE CELL LINE

 

Efrat Korin¹, Olga  Kryukov¹, Smadar  Cohen²

 

	1 Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev; Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev
	2 Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev; Regenerative Medicine and Stem Cell (Rmsc) Research Center and 2the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel

 

 

Anionic nanoparticles of AlgS–Ca²⁺-siRNA are efficient carriers of siRNA to human hepatocyte cell line.

 

Efrat Korin¹, Olga Kryukov¹, Smadar Cohen^{1,2, 3}

 

¹Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, ²Regenerative Medicine and Stem Cell (RMSC) Research Center and ³The Ilse Katz Institute for Nanoscale Science and Technology,  Ben-Gurion University of the Negev, Beer-Sheva, Israel.

 

Over the past decade, small interfering RNA (siRNA) has intensively been explored owing to its potential in disease therapy. Yet, siRNA delivery in vivo is still a major challenge due to its rapid degradation by nucleases, poor cellular uptake and rapid renal clearance following systemic administration. Thus, it is critical to develop an appropriate delivery system that will overcome these limitations and to improve the safety of potential RNAi-based therapeutics. We developed a novel siRNA delivery platform, based on the complexation of Alginate sulfate (AlgS) with siRNA, mediated by calcium ions bridges. Realizing the potential toxicity of cationic carriers, the AlgS–Ca²⁺-siRNA nanoparticles (AlgS-NPs) have a net negative surface charge of ~-8 mV. The chemical interaction between the three components constituting the AlgS-NPs was confirmed by XPS and TEM. Assessing the cellular uptake of AlgS-NPs in human hepatocyte cell line (HEPG2) by imaging flow cytometry analysis revealed a highly efficient uptake, resulting in siRNA internalization in 89% of cells. Furthermore, results obtained by quantitative Real-Time PCR (qPCR) showed the silencing of the STAT3 gene to an extent of 90% without being cytotoxic to cells. In summary, the results obtained so far, indicate that AlgS-NPs may offer a promising platform for efficient delivery of therapeutic siRNA.

 

 

 

 

 

[320] MODIFIED PECTIC GALACTAN CARRIER FOR GENE THERAPY DEMONSTRATED SIGNIFICANT CELLULAR UPTAKE IN VITRO

 

Nitsa Buaron¹, ramesh chintakunta¹, Tamar  Traitel¹, Riki Goldbart¹, Henry Brem², Joseph Kost¹

 

	1 Ben Gurion University of the Negev; Department of Chemical Engineering
	2 Johns Hopkins Medical Institutions ; Neurosurgery

 

 

 

                Brain tumors represent one of the most malignant forms of human cancers, where the most common and aggressive is the glioma. We explored a novel gene therapy approach based on natural polysaccharides for targeted delivery to cancerous cells. Galectin-3 is a cell protein that carries an active carbohydrate recognition domain (CRD) for β-galactoside sugars that is highly expressed in a variety of cancer cells, such as glioma. Since the natural polysaccharide pectin has galactose-rich side chains (galactans), it can be utilized as a carrier for delivering genes to glioma cells in a targeted manner, based on the highly specific carbohydrate interaction between galactan and galectin-3 receptors on the cell membrane. Moreover, pectin has been proven to be effective in inhibiting or blocking cancer cell aggregation, adhesion, and metastasis. Since pectin is a natural polysaccharide, it carries further advantages as a gene delivery carrier over the currently available synthetic ones, such as biodegradability, biocompatibility, low immunogenicity, and minimal cytotoxicity.     

                Modified pectin-based carrier was synthesized and explored. Q-galactan was prepared by modifying quaternary ammonium groups (Q=N+(CH₃)₃) on pectic galactan. Q-galactan was successfully synthesized and characterized. A globular condensed complexation with plasmid DNA was clearly observed. Q-galactan was found to form complexes with size ranging from 80 nm to 120 nm, which is suitable for internalization to the cell through endocytosis. The complexes were successfully proven to be non-toxic to C6 rat glioma cells line. Investigation of cellular uptake and cellular path indicated the complexes were able to penetrate the cell membrane and approach the nucleus within 24 hours. Cellular uptake of ~75% was observed at the best conditions. This investigation demonstrates that Q-galactan is a potential carrier for gene therapy. Further studies are required in order to investigate the intracellular barriers for establishing an efficient gene delivery system.  

 

 

 

[330] CALCIUM-SIRNA NANOCOMPLEXES: MECHANISM OF CELLULAR UPTAKE AND ENDOSOMAL RELEASE

 

Matan Goldshtein¹

 

1 Ben Gurion University of the Negev; N/A

 

 

Small interfering RNA (siRNA) represents a promising type of therapeutics exploiting the mechanism of RNA interference for silencing target genes. Yet, the clinical translation of siRNA has been limited due to delivery challenges. We recently described a novel Ca²⁺– siRNA nanocomplex capable of strong but reversible complexation, siRNA protection, cellular uptake, and cytoplasmatic unloading of its cargo. Here, we investigated the importance of Ca²⁺compared to other bi- or tri-valent cations in creating these nanocomplexes, and the cytocompatibility of the various nanocomplexes. Further, we elucidated cellular entry and endosome release mechanisms of Ca²⁺-siRNA nanocomplexes. The nanocomplexes were prepared by incubating siRNA (50 nM final) with either Ca²⁺, Mg²⁺, Zn²⁺, Ba²⁺, Mn²⁺,  Fe²⁺ or Fe³⁺ ions (5 mM for divalent and 3.33mM for Fe³⁺) Of these nanocomplexes, only those prepared with  Ca²⁺, Mg²⁺, Ba²⁺ andFe³⁺ were cytocompatible as judged by PrestoBlue® for cell viability. Effective eGFP silencing (~80%) in GFP expressing mouse colon carcinoma CT26 cells was achieved only with Ca²⁺– siRNA nanocomplexes. Cell uptake studies (using confocal microscopy) and silencing experiments (using flow-cytometry), were performed using different inhibitors of several possible entry mechanisms: Dynasore, Pitstop2®, EIPA, Nifedipine, Cadmium and Genistein. We revealed that the major endocytic pathways involved in the entry of Ca²⁺-siRNA nanocomplexes are clathrin and dynamin-dependent. Treatment with Bafilomycin, which inhibits endosome acidification after Ca²⁺ entry to endosomes, completely abolished siRNA-mediated silencing indicating that Ca²⁺ is critical for the endosomal unloading through a “proton sponge” effect. In conclusion, Ca²⁺ is a critical component for particle assembly, particle uptake and endosomal escape.

 

 

 

[338] SPECIFICITY IN CANCER THERAPY BASED ON CELL MECHANICS

 

Yifat Brill-Karniely¹, Nethanel Friedmann², Ben-Zion Amoyav³, Ofra Benny⁴

 

	1 Institute of Drug Research; The Hebrew University
	2 Institute of Drug Research; Institute of Drug Research
	3 Hebrew University; Idr- Institute Drug Reserch
	4 The Hebrew University of Jerusalem; Institute for Drug Research

 

 

Specificity in Cancer Therapy Based on Cell Mechanics

Yifat Brill-Karniely, Nethanel Friedman, Benzion Amoyav and Ofra Benny

School of Pharmacy, Institute for Drug Research, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel

The presented work is composed of a tight cross-talk between computational modelling and advanced experimental methods, aiming to optimize the design of drug carriers for cancer treatment. Our prime aim is to investigate the mechanical mechanism of micro-particle engulfment and uptake by cells. We hypothesize that physical properties of cells control the engulfment process in a manner that can enhance the specificity of cancer therapy. Our work includes a cooperative combination of in vitro experiments, advanced particle fabrication methods and physical modelling. In agreement with our model predictions, we found a binary behavior in particle uptake: cells internalize either many particles or none. Furthermore, according to our theoretical modelling, the rigidity of cells determines their engulfment abilities. Therefore, our findings can potentially lead to significant enhancement of treatment specificity that is based on differences in the physical rigidities between normal and malignant cells, rather than on the expression of specific molecules.

 

 

 

[340] SELF-ASSEMBLED COMPLEXES OF PLASMID DNA AND MODIFIED STARCH FOR ACTIVE DELIVERY INTO THE NUCLEUS

 

Shachar Gat¹, Ramesh Chintakunta¹, Dina Aronovich¹, Tamar  Traitel², Riki Goldbart², Anne Bernheim-Groswasser³, Joseph Kost²

 

	1 Department of Chemical Engineering ; Ben Gurion University
	2 Ben Gurion University of the Negev; Department of Chemical Engineering
	3 Department of Chemical Engineering ; Ilse Kats Institute for Nanoscale Science and Technology

 

 

Gene therapy is a novel clinical strategy whereby exogenous genetic material is introduced into human cells for the treatment of hereditary or acquired disorders. The main objective of gene therapy is the development of efficient, non-toxic gene carriers that can condense and deliver foreign genetic materials into specific cell types, such as cancerous cells. Non-viral carriers have advantages over viral carriers, since they have low toxicity and induce low immune response; however, their major disadvantage is their relatively low gene expression. This low productivity relates largely to the poor efficiency of transport the nanoparticle from the plasma membrane to the nucleus and their subsequent import into the nucleus. All trafficking across the nuclear membrane occurs through the nuclear membrane embedded nuclear pore complex (NPC). The import of molecules larger than 45 kDa into the nucleus is mediated by nuclear localization signals (NLS) which recruit import proteins (importins) to their surface as well as motor proteins, thereby mediating their active movement from the plasma membrane toward the nucleus and subsequent docking and import through the NPC.

The objective of this work is to design a carrier that can overcome this limitations; starch, a natural polysaccharide was modified into cationic starch (Q-starch) and was used as a pDNA carrier. Covalent coupling of polyethylene glycol (PEG)-thiol to the Q-starch was carried out in order to attach NLS peptides. Complexation of Q-starch-PEG-thiol with pDNA in different N/P ratios (molar ratio of Q-starch nitrogen groups to nucleic acid phosphate groups) was evaluated by gel electrophoresis. Results showed spherical nanoparticles that are more condensed and their average surface charge increases with the increase of N/P. NLS peptides attachment to the complexes was done by coupling the free end of the polymer (PEG-thiol) to a NLS peptide. Specific binding of the complexes to motor proteins was confirmed by western blot using anti-dynein antibodies and by cryogenic transmission electron microscopy.

 

 

 

[343] FEASIBILITY OF LEADLESS CARDIAC PACING USING INJECTABLE MAGNETIC MICROPARTICLES

 

Hemi Rotenberg¹, Hovav Gabay², Smadar  Cohen³, Yoram Etzion⁴

 

	1 Bgu ; Beer Sheva
	2 Regenerative Medicine & Stem Cell Research Center; Ben-Gurion University of the Negev
	3 Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev; Regenerative Medicine and Stem Cell (Rmsc) Research Center and 2the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel
	4 Regenerative Medicine & Stem Cell Research Center, ; Ben-Gurion University of the Negev, Beer-Sheva, Israel.

 

 

An approach for immediate, painless and non-invasive heart pacing may have invaluable implications in several clinical scenarios. We hypothesized that mechano-­electric feedback (MEF) can be utilized to evoke cardiac pacing using injectable magnetic micro or nano-particles. Such particles can be trapped in the right ventricle (RV) by an external electromagnetic force. Thereafter, magnetic pulses banging the particles over the RV wall can initiate leadless electrical pacing. We used ex­-vivo isolated rat heart model to demonstrate the ability to provoke MEF-induced pacing by iron microparticles (IMPs) inserted directly to the RV cavity. In vivo rat model was used to demonstrate the ability to localize IMPs and magnetite nanoparticles (MNPs) in the RV and provoking MEF induced pacing using external electromagnet. Thereafter, this modality was tested in a pig model, by inserting IMPs or MNPs directly to the RV cavity of a blood perfused isolated pig heart.

Isolated rat heart studies showed that MEF-induced overdrive pacing was consistently applied in 5 consecutive hearts by IMPs, subjected to magnetic pulses. The in-­vivo studies demonstrated that the electromagnet effectively captured IMPs and MNPs in the RV. Cryosections of the frozen heart show large aggregates of IMPs and MNPs when the electromagnet was used, while sham hearts were vacant (n=2 for each group). After we showed successful localization, we demonstrated that MEF induced overdrive pacing could temporarily revert bradycardia by applying magnetic pulses on localized IMPs. After appropriate optimizations, overdrive pacing of at least 5s was obtained in 13 out of 15 tested rats. Finally, we demonstrated the efficacy of this modality in a relevant large mammalian pig model; we induced overdrive pacing of an isolated pig heart by applying magnetic pulses on both IMPs and MNPs.

Our results demonstrate an effective modality for selective cardiac pacing in a completely non­invasive fashion. Additional optimization is needed to prolong pacing using this novel approach and to further evaluate its applicability in the intact large mammalian model.

 

 

 

 

 

[493] BI-FUNCTIONAL MAGNETO-FLUORESCENT NANOPARTICLES FOR MULTIMODAL IMAGING

 

Sandip Pahari¹, Shunit  Olszakier², Itamar Kahn³, Lilac Amirav⁴

 

	1 Israel Institute of Technology, Technion; Schulich Faculty of Chemistry
	2 The Ruth and Bruce Rappaport Faculty of Medicine ; Technion – Israel Institute of Technology
	3 The Ruth and Bruce Rappaport Faculty of Medicine; Technion – Israel Institute of Technology
	4 Schulich Faculty of Chemistry; Technion – Israel Institute of Technology

 

 

Bi-functional Magneto-Fluorescent Nanoparticles for Multimodal Imaging

Sandip Kumar Pahari¹, Shunit Olszakier², Itamar Kahn², and Lilac Amirav¹

¹Schulich Faculty of Chemistry,

²Department of Physiology and Biophysics, The Ruth and Bruce Rappaport Faculty of Medicine,

Russell Berrie Nanotechnology Institute, Technion – Israel Institute of Technology, Haifa, Israel.

Tel: +972-4-829-3715. E-mail: lilac@technion.ac.il

Abstract:

Designing effective nanoparticles that can passively or actively label fine biological structures in various functional states is a central goal of medicine-oriented nanotechnology development. Since each established imaging modality has its own drawbacks, integration of different techniques into multimodal imaging can provide complimentary information. Hybrid nanostructures can exhibit several features synergistically and deliver more than one function simultaneously.

In particularly, multifunctional magnetic nanoparticles can have unique advantages in biomedical applications. Here we present our strategy to fabricate magnetic nanoparticle-based multifunctional nanostructures, which are integrated with quantum dots. These hybrid nanostructures exhibit paramagnetism alongside fluorescence. Direct contact between the semiconductor and magnetic domains, typical of traditional core-shell or heterodimer structures, can lead to strong electronic coupling, diminishing the desired optical fluorescence. Hence, our structure comprises an optically active nanoparticle quantum dot core encapsulated in a hollow shell providing the MRI contrast agent. Such encapsulation of the quantum dot might also prove to be essential for biocompatibility and toxicity aspects. We expect that the combination of unique structural characteristics and integrated functions of multicomponent magnetic nanoparticles will lead to new opportunities in biological and medical imaging.

 

 

 

 

 

 

[494] SIRNA DELIVERY PLATFORM BASED ON CO-ASSEMBLING ANIONIC NANOPARTICLES OF SIRNA AND HYALURONAN SULFATE VIA CALCIUM BRIDGES

 

Efrat Forti¹

 

1 Ben-Gurion University of the Negev; Ben-Gurion University of the Negev

 

 

siRNA delivery platform based on co-assembling anionic nanoparticles of siRNA and hyaluronan sulfate via calcium bridges

 

Efrat Forti¹, Olga Kryukov¹, Edan Elovic¹, Matan Goldstein¹, Efrat Korin¹, Gal Margolis¹, Felder Shani ¹, Emil Ruvinov¹, Smadar Cohen^1-3

¹Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, ²Regenerative Medicine and Stem Cell (RMSC) Research Center, ³The Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Therapeutic implementation of gene silencing using small interfering RNA (siRNA) relies on the critical need for a safe and effective carrier for siRNA protection, capable of strong but reversible complexation, cellular uptake, and cytoplasmatic unloading of its cargo. We developed anionic siRNA nanoparticles (NPs) co-assembled by the electrostatic interactions of the semi-synthetic polysaccharide hyaluronan-sulfate (HAS), with siRNA, mediated by calcium ion bridges. Physical characterization of the HAS-Ca²⁺-siRNA NPs, using high resolution microscopy and dynamic light scattering (DLS), showed the formation of stable nanosized complexes ~130 nm in diameter, bearing mild (~−10 mV) negative surface charge. X-ray photoelectron spectroscopy (XPS) demonstrated the spatial organization of siRNA molecules in the particle core, surrounded by a layer of HAS. The anionic NPs efficiently encapsulated siRNA, were extremely stable in physiological-relevant environments and were cytocompatible, not affecting cell viability or homeostasis. The anionic siRNA NPs, successfully induced potent gene silencing (>80%) across multiple cell types, including murine primary peritoneal macrophages, human hepatocellular carcinoma cells, and human breast cancer cells. The potential toxic effects of anionic NP formulation were tested in mice, following single intravenous injection (IV) of HAS-Ca²⁺-siRNA. Results showed that acute administration of the HAS-Ca²⁺-siRNA NPs at a dose of 3.3 mg/kg siRNA via the intravenous (IV) route was not associated with toxic risk. Collectively, the developed anionic NPs were shown to be an efficient and cytocompatible platform for enhancing the therapeutic efficiency of siRNA.

 

 

 

 

 

 

[497] BIO-INSPIRED ANIONIC NANOPARTICLES FOR THE DELIVERY OF PLASMID DNA

 

Stav Shamir¹, Efrat Forti², Matan Goldstein¹, Smadar  Cohen³

 

	1 Ben Gurion University of the Negev; Ben Gurion University of the Negev
	2 Ben-Gurion University of the Negev; Ben-Gurion University of the Negev
	3 Avram and Stella Goldstein-Goren Department of Biotechnology Engineering, Ben-Gurion University of the Negev; Regenerative Medicine and Stem Cell (Rmsc) Research Center and 2the Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel

 

 

Bio-inspired anionic nanoparticles for the delivery of plasmid DNA

Stav Shamir¹, Efrat Forti¹, Matan Goldstein¹, Smadar Cohen^1-3

¹Avram and Stella Goldstein-Goren Department of Biotechnology Engineering,

²Regenerative Medicine and Stem Cell (RMSC) Research Center,

³The Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Gene therapy is a promising strategy for the treatment of many diseases including cancer and various genetic disorders by target-specific delivery of therapeutic genes. We are developing a novel anionic nanoparticle (NP) for targeted delivery of plasmid DNA (pDNA). The NPs are assembled by the electrostatic interactions of a modified natural polysaccharide, alginate sulfate (AlgS), with pDNA, mediated by interactions with calcium ion. Characterization of the NPs by dynamic light scattering (DLS) and zeta potential measurements showed an average size of 103±2.8 nm (SEM, n=5) and a mild negative surface charge of -5.8±0.3 (SEM, n=9). Encapsulation efficiently was estimated by ethidium bromide exclusion assay and found to be 62.2% ± 0.8% (SEM, n=9). The pDNA NPs did not affect cell viability, and efficient cellular uptake of fluorescently-labeled anionic pDNA NPs was observed (95%) in the human breast cancer cell line MDA-MB 231 by imaging flow cytometry. Collectively, anionic pDNA NPs show a promise to be an efficient and cytocompatible delivery system for pDNA.

 

 

 

 

 

[506] PHARMACOLOGIC STUDIES OF A PRO-DRUG OF MITONYCIN C IN PEGYLATED LIPOSOMES (PROMITIL)

 

Patricia Ohana¹, Hilary Shmeeda², Yogita Patil³, Yasmine Amitay⁴, Alberto Gabizon³

 

	1 Lipomedix Pharmaceuticals Ltd. ; Shaare Zedek Medical Center
	2 Shaare Zedek Medical Center; Shaare Zede Medical Center
	3 Shaare Zedek Medical Center; Shaare Zedek Medical Center
	4 Lipomedix Pharmaceuticals Ltd.; Shaare Zedek Medical Center

 

 

Pharmacologic studies of a prodrug of mitomycin C in pegylated liposomes (Promitil®)

Purpose: Pegylated liposomal (PL) mitomycin C lipid-based prodrug (MLP) has recently entered clinical testing. We studied the preclinical and clinical pharmacology of PL-MLP.

Methods: The stability, pharmacokinetics, biodistribution, and other pharmacologic parameters of PL-MLP were examined. Thiolytic cleavage of MLP and release of active mitomycin C (MMC) were studied using dithiothreitol (DTT), and by incubation with tissue homogenates. Pharmacokinetic data were collected from a dose escalation phase 1 study in cancer patients.

Results: MLP was incorporated in the bilayer at 10% molar ratio with nearly 100% entrapment efficiency, resulting in a formulation with high plasma stability. In vitro, DTT induced cleavage of MLP with predictable kinetics, generating MMC and enhancing pharmacological activity. A long half-life of MLP (10-15 hours) was observed in rodents and minipigs. PL-MLP was less toxic in vivo than equivalent doses of MMC. Studies in mice with H3-cholesterol radiolabeled PL-MLP demonstrated relatively greater tissue levels of H3 than MLP. MLP levels were highest in tumor and spleen, and very low or undetectable in liver and lung. Rapid cleavage of MLP in various tissues, particularly in liver, was shown in ex-vivo experiments of PL-MLP with tissue homogenates. Urine from PL-MLP injected rats revealed delayed but significant excretion of MMC indicating in vivo activation of MLP. Therapeutic studies in C26 mouse tumor models demonstrated improved dose-dependent efficacy of PL-MLP over MMC.

The pharmacokinetics of PL-MLP in a first-in-man study showed a median t_1/2 of 23 hours, with no trend by dose or cycle, while C_max and AUC_0-∞ increased linearly over the dose range 0.5-2.0 mg/kg, and greater than linearly from 2.5-3.5 mg/kg. No free MMC was detected. The results were consistent with preclinical observations.

Conclusions: Thiolytic activation of PL-MLP occurs in tissues but not in plasma. Liposomal delivery of MLP confers a favorable pharmacological profile and greater therapeutic index than MMC. Further clinical testing is ongoing.

 

 

 

[508] NANOPARTICULATE DELIVERY OF SIRNA/MIRNA TO MODULATE CARDIAC MACROPHAGES TOWARDS HEART REPAIR

 

tzlil bejerano¹, Sharon etzion², Sigal Elyagon ², Yoram etzion², Smadar Cohen³

 

	1 Avram and Stella Goldstein-Goren Department of Biotechnology Engineering; Ben Gurion University
	2 Regenerative Medicine and Stem Cell (Rmsc) Research Center; Ben-Gurion University of the Negev
	3 Avram and Stella Goldstein-Goren Department of Biotechnology Engineering; Regenerative Medicine and Stem Cell (Rmsc) Research Center

 

 

The wound healing process after myocardial infarction (MI) is tightly controlled by different macrophage sub-populations: inflammatory macrophages (M1 type) and reparatory macrophages (M2 type). An excessive activity of M1 macrophages resulting from a delay in the transition from M1 to M2 macrophage often leads to heart failure. We hypothesized that modulation of cardiac macrophages from M1 to M2 phenotype, at the proper time-point after MI, using miRNA delivery, may attenuate infarct expansion and adverse left-ventricular remodeling. For this purpose, we developed siRNA/miRNA carrying nanoparticles (NPs) formed by the co-assembly of siRNA/miRNA with the semi-synthetic glycosaminoglycan, hyaluronan-sulfate, mediated by calcium ion bridges. The NPs were characterized, and exhibited efficient siRNA entrapment, protection from enzymatic degradation, enhanced cellular uptake, and effective gene silencing across multiple cell types. The NPs have an average diameter of 125 nm, and a mild negative surface charge (-11.3mV). In vivo uptake studies of NPs containing fluorescently labeled Cy3-siRNA were conducted initially in mice stimulated with peritoneal sterile infection. One hour after intraperitoneal (IP) injection, FACS analysis showed NP uptake in 40% of the peritoneal macrophages. In a model of acute MI in mice, intravenous  (IV) injection of NPs 3 days after MI, resulted in accumulation of Cy3-siRNA in CD11b (monocyte/macrophage marker)-positive cells at the infarct area. An in vivo imaging system (IVIS) analysis showed ~30% higher signal of cy5-siRNA NPs in infarcted hearts, relative to healthy hearts. These results indicate the feasibility of our NPs to target macrophages at the infarct and efficiently deliver siRNA/miRNA to enable their immunomodulation.  The multidisciplinary approach presented in this research combines engineering of miRNA delivery system and cardiac macrophage biology. Ultimately we foresee this approach to lead to a novel bio-inspired therapeutic modality for heart repair after acute MI.

 

 

 

[537] LIPOSOME-BASED NANO-DRUGS FOR THE TREATMENT OF CANCER AND INFLAMMATION

 

Keren Turjeman¹, Ahuva Cern², Xiaohui Wei³, Yaelle Bavli⁴, Tal Berman⁵, Alexander  V. Andriyanov⁶, Moria Barlev-Gross⁷, Lisa Silverman⁸, Alexander Lyskin⁸, Yechezkel (Chezy)  Barenholz⁹

 

	1 Laboratory of Liposome and Membrane Research, Imric, The Hebrew University-Hadassah Medical School, Jerusalem, Israel; Hebrew University
	2 Biochemistry Department; Hebrew University
	3 Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada (Imric); The Hebrew University-Hadassah Medical School,
	4 Laboratory for Membrane and Liposome Research, Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada (Imric), The Hebrew University of Jerusalem, Hadassah Medical School; Hebrew University
	5 Department of Biochemistry and Molecular Biology, Institute of Medical Research Israel-Canada (Imric) The Hebrew University of Jerusalem, Hadassah Medical School ; The Hebrew University – Hadassah Medical School
	6 Laboratory of Liposome and Membrane Research; Imric
	7 Laboratory of Liposome and Membrane Research; The Hebrew University of Jerusalem
	8 Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada (Imric); The Hebrew University – Hadassah Medical School
	9 Institute of Medical Research Israel-Canada (Imric), ; The Hebrew University of Jerusalem

 

 

Liposome-based nano-drugs for the treatment of cancer and inflammation

 

Keren Turjeman, Ahuva Cern, Xiaohui Wei, Yaelle Bavli, Tal Berman, Alexander Andriyanov, Moria Barlev-Gross, Liza Silverman, Alexander Lyskin, Prof. Yechezkel Barenholz

 

Laboratory of Membrane and Liposome Research, Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada (IMRIC), The Hebrew University-Hadassah Medical School

 

Abstract

The Barenholz lab focuses on the development and application of liposome-based nano-drugs, ranging from basic aspects of drug-carrier design, through preclinical and clinical trials, all the way to FDA-approved drugs.

We have developed injectable nano-drug delivery systems based on pegylated, long-circulating, nano sterically stabilized liposomes (nSSL) remotely loaded with either amphipathic weak acid, anti-inflammatory steroid pro-drugs, or with doxorubicin; an amphipathic weak base, anti-cancer agent. We applied the bio-energetic strategy of transmembrane ion-gradient-driven remote loading, by which, liposomes encapsulate a salt containing either a weak base (e.g. ammonium) or a weak acid (e.g. acetate). This remote drug loading method has three main advantages over passive drug loading: high drug loading efficiency, a high drug-to-lipid mole ratio, and controlled drug release both in vitro and in vivo.  These nano-drugs are specifically designed to use the unique micro-anatomical vascular abnormality of inflamed and cancerous tissues: this phenomenon is called the “enhanced permeability and retention” (EPR) effect. The pegylation of the nano-liposomes by the lipopolymer PEG-DSPE provides steric stabilization, which results in prolonged circulation time, size-dependent passive targeting, and drug accumulation in the diseased tissue.

All lead to reduced toxicity and improved efficacy.

 

 

 

[539] ELECTRICALLY-CONTROLLED REDOX STATE MOIETY POPULATION ON NANOWIRE SURFACE AS A TOOL FOR MULTIPLEX, REAL-TIME AND CONTINUOUS MONITORING OF METABOLITES

 

Marina Zverzhinetsky¹, Vadim Krivitsky², Vladimir Naddaka³, Fernando Patolsky³

 

	1 Tel-Aviv University; School of Chemistry, the Raymond and Beverly Sackler Faculty of Exact Sciences
	2 Tel Aviv University; School of Chemistry, the Raymond and Beverly Sackler Faculty of Exact Sciences
	3 School of Chemistry; School of Chemistry

 

 

Electrically-Controlled Redox State Moiety Population on Nanowire Surface as a Tool for Multiplex, Real-Time and Continuous Monitoring of Metabolites

Marina Zverzhinetsky†, Vadim Krivitsky†, Vladimir Naddaka† and Fernando Patolsky†

†School of Chemistry, the Raymond and Beverly Sackler Faculty of Exact Sciences,  Tel-Aviv University, Israel

 

Abstract

The development of efficient sensors for the continuous monitoring of metabolic activity is of a critical importance in modern medicine and bio-sample analysis, primarily due to the fact that most bio-samples are mixtures of high diversity and complexity. We designed a redox-reactive nanowire biosensor for multiplex, real-time and continuous monitoring of metabolic activity in physiological environment. The surface of the nanowire sensor was covalently modified with redox-reversible moiety, while the reversible transformation can happen either by applying chemicals or voltage. In order to make continuous measurements of metabolic activity, the reversible redox properties of the modified moiety were used. Importantly, this represents the direct analysis of bio-samples on a single nanowire device, which is able to selectively detect specific metabolites, without the requirement of time-consuming steps, such as labeling and purification. Remarkably, first nanowire continuous sensing of metabolites in physiological solutions without preprocessing was realized. Typically, concentration-dependent sensing of metabolites covers physiological concentration ranges.

 

Extension of the Generic Amyloid Hypothesis to Non-Proteinaceous Metabolite Nano-Assemblies

 

Shira Shaham-Niv¹, Lihi Adler-Abramovich^1,2, Lee Schnaider¹ and Ehud Gazit^1,3

¹Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel. ²Department of Oral Biology, The Goldschleger School of Dental Medicine, Tel Aviv Univeristy, Tel Aviv 69978, Israel.

³Department of Materials Science and Engineering, Iby and Aladar Fleischman Faculty of Engineering, Tel Aviv University, Tel Aviv 69978, Israel.

 

Keywords: Amyloid fibril formation, metabolic disorders, supramolecular nano-assemblies, drug development, self-assembly.

 

The formation of ordered amyloid fibrils is associated with several notable human disorders. These nano-scale assemblies are predominantly rich in β-sheet secondary structure and specifically bind dyes, such as ThT and Congo-red. The formation of the amyloid fibrils or earlier pre-fibrillar forms correlates to an apoptotic effect in various tissues. While the formation of these cytotoxic supramolecular entities has previously been linked to proteins and peptides, it was later demonstrated that phenylalanine, as a single amino-acid, can also self-assemble to form amyloid-like fibrils possessing typical ultrastructural, biophysical and biochemical properties. Moreover, it was demonstrated that these phenylalanine assemblies are cytotoxic and antibodies raised against these species deplete fibril toxicity. The generation of antibodies in a phenylketonuria (PKU) mice model and identification of aggregate deposits post mortem in patients’ brains suggested a pathological role for these assemblies. To study whether these observations represent a general amyloid-like mechanism prevalent in other metabolic disorders, we have screened metabolites that accumulate in inborn error of metabolism disorders. Here, we reveal that several other metabolites can self-assemble to form ordered amyloid-like ultrastructure in solution with the molecular dimensions and dye-binding specificity similar to canonical amyloid fibrils. In addition, we show that these fibrillar self-assemblies are cytotoxic by the induction of apoptotic programmed cell death, as observed for many amyloid disorders. These results suggest that the formation of nano-scale amyloid-like self-assemblies by metabolites indicate to a general phenomenon of amyloid formation beyond proteins and peptides and offer a new paradigm for metabolic diseases. This may lead to new therapeutic directions of treatments for these disorders beyond a highly-restrictive diet.