Alireza Nomani

Release profile and stability evaluation of optimized chitosan/alginate nanoparticles as EGFR antisense vector.

Chitosan/alginate nanoparticles which had been optimized in our previous study using two different N/P ratios were chosen and their ability to release epidermal growth factor receptor (EGFR) antisense was investigated. In addition, the stability of these nanoparticles in aqueous medium and after freeze-drying was investigated. In the case of both N/P ratios (5, 25), nanoparticles started releasing EGFR antisense as soon as they were exposed to the medium and the release lasted for approximately 50 hours. Nanoparticle size, shape, zeta potential, and release profile did not show any significant change after the freeze-drying process (followed by reswelling). The nanoparticles were reswellable again after freeze-drying in phosphate buffer with a pH of 7.4 over a period of six hours. Agarose gel electrophoresis of the nanoparticles with the two different N/P ratios showed that these nanoparticles could protect EGFR antisense molecules for six hours.

Preparation and Characterization of Copolymeric Polymersomes for Protein Delivery

Biodegradable copolymeric polymersomes have been used for controlled drug delivery of proteins. These polymersomes important areas to overcome formulation associated problems of the proteins. The aim of this study was to develop polymersomes using biodegradable copolymers for delivery of bovine serum albumin (BSA) as a model protein. Encapsulated BSA by mPEG-PCL polymersomes led to formation of BSA-loaded mPEG-PCL polymersomes. The polymersomes synthesized with the protein-polymer ratio of 1:4 at 15 000 rpm gave maximum loading, minimum polydispersion with maximally sustained protein release pattern, among the prepared polymersomes. Investigation on FTIR and DSC results revealed that such a high encapsulation efficiency is due to strong interaction between BSA and the copolymer.The particles size and their morphology of polymersomes were determined by DLS and AFM.The encapsulation efficiency of BSA was 91.02%. The results of AFM showed that the polymersomes had spherical shapes with size of 49 nm.The sizes of BSA-loaded polymersomes ranged from 66.06 nm to 84.97 nm. The results showed that polymersomes exhibited a triphasic release, for BSA. Overall, the results indicated that mPEG-PCL polymersomes can be considered as a promising carrier for proteins.

Physicochemical and biological properties of self-assembled antisense/poly(amidoamine) dendrimer nanoparticles: The effect of dendrimer generation and charge ratio

To gain a deeper understanding of the physicochemical phenomenon of self-assembled nanoparticles of different generations and ratios of poly (amidoamine) dendrimer (PAMAM) dendrimer and a short-stranded DNA (antisense oligonucleotide), multiple methods were used to characterize these nanoparticles including photon correlation spectroscopy (PCS); zeta potential measurement; and atomic force microscopy (AFM). PCS and AFM results revealed that, in contrast to larger molecules of DNA, smaller molecules produce more heterodisperse and large nanoparticles when they are condensed with a cationic dendrimer. AFM images also showed that such nanoparticles were spherical. The stability of the antisense content of the nanoparticles was investigated over different charge ratios using polyacrylamide gel electrophoresis. It was clear from such analyses that much more than charge neutrality point was required to obtain stable nanoparticles. For cell uptake, self-assembled nanoparticles were prepared with PAMAM G5 and 5’-FITC labeled antisense and the uptake experiment was carried out in T47D cell culture. This investigation also shows that the cytotoxicity of the nanoparticles was dependent upon the generation and charge ratio of the PAMAM dendrimer, and the antisense concentration had no significant effect on the cytotoxicity.

Characterization of Chitosan/Alginate Self-Assembled Nanoparticles as a Protein Carrier

The aim of this study was to evaluate the effect of the polymeric ratios on the characteristics of chitosan/alginate (ch/alg) self-assembled nanoparticles and their potential as protein delivery vehicle. The nanoparticles were prepared using proper mixing of polymers in presence or absence of bovine serum albumin (BSA) as a protein model. Three formulations of nanoparticles comprising ch/alg ratios of 2:1, 1:1, and 1:2 were prepared. Size, shape and zeta potential of the formulations were studied by scanning electron microscopy (SEM) and nanosizer instruments. FTIR, and differential scanning calorimetery (DSC) studies were performed to investigate polymer-polymer or polymer-protein interactions. Release profiles and entrapment efficiencies of the nanoparticles were determined by calorimetric technique using appropriate techniques. Entrapment efficiency was 70% for ch/alg ratio of 1:1, 65% for 1:2, and 60% for 2:1. The z-average size of the nanoparticles were 403, 205, and 318 nm for ch/alg ratios of 2:1, 1:1, and 1:2, respectively. Average zeta potentials were −47, +15, −25 mV for 2:1, 1:1, and 1:2 as well. Considering the favorable features required for protein delivery systems, ch/alg (1:1) due to its smallest size, highest loading, and most homogenous shape was regarded as the best ratio.

Novel biotinylated chitosan-graft-polyethyleneimine copolymer as a targeted non-viral vector for anti-EGF receptor siRNA delivery in cancer cells

The major impediments to develop an efficient non-viral siRNA-mediated gene silencing method, as a therapeutic approach, are the low cellular uptake and intracellular delivery and release of non-viral vectors. To overcome these problems, designing a proper vector with high transfection efficiency is obviously under scrutiny of various studies. The present study, evaluate a novel biotinylated chitosan-graft-polyethyleneimine (Bio-Chi-g-PEI) copolymer as an appropriate non-viral vector for targeted delivery of siRNA to cancer cells. The composition of the synthesized Bio-Chi-g-PEI copolymer was thoroughly characterized using (1)H NMR and FTIR spectroscopy, besides the hydroxyazobenzene-2-carboxylic acid (HABA) assay. In vitro cytotoxicity assay of the Bio-Chi-g-PEI copolymers was performed by MTT assay. Cytotoxicity evaluations indicated that the new copolymer was markedly less toxic than PEI 25KD. Physicochemical properties of the Bio-Chi-g-PEI/siRNA complexes such as complex stability, size, zeta potential, and their morphology at various weight ratios, investigated by appropriate methods, revealed the suitability of the complexes for the transfection. The efficient cellular internalization of the complexes for HeLa and OVCAR-3 cells in culture media was confirmed by intracellular tracking of the prepared complexes using confocal laser scanning microscopy and Cy3-labeled anti-epidermal growth factor receptor siRNA. Finally, evaluation of the transfection efficiency and gene silencing by flow cytometry and real-time polymerase chain reaction highlighted the significantly higher efficiency of transfection and silencing for biotinylated copolymer compared with the PEI 25KD and non-biotinylated copolymer.

Evaluation of cationic dendrimer and lipid as transfection reagents of short RNAs for stem cell modification

Nowadays a large number of clinical trials suffer from lacking an efficient method for drug delivery into target cells with minimal side effects. Due to the great significance of this issue in novel and effective therapies, more attempts are required in order to distinguish better conditions for biomedical drug delivery. Since embryonic stem cells (ESCs) are under scrutiny of many new studies, development of novel methods for their genetical and functional modifications is of great value. On the other hand, the application of short nucleic acids in new therapeutic approaches is increasing. In this study the efficiency of small interfering RNA (siRNA) uptake with two transfection reagents generation five of polyamidoamine dendrimer (PAMAM G5) as a cationic dendrimer and N-[1-(2,3-dioleoyloxy)]-N,N,N-trimethylammonium propane methyl-sulfate (DOTAP) as a cationic lipid and one commercially available reagent were evaluated in mouse ESCs using flow cytometry. Prior to the cellular investigations; atomic force microscopy; gel electrophoresis; siRNA binding and release assays; and size and zeta potential measurements were utilized to characterize the physicochemical properties of reagent-siRNA nano-complexes. The safety of the nano-complexes was subsequently assessed by MTT assay. Functional effects of siRNA (complementary strand for OCT4 transcript) transfection in ESCs with the mentioned reagents were analyzed using a quantitative real-time polymerase chain reaction (qPCR). Surprisingly DOTAP at higher molar ratios and PAMAM at lower molar ratios could successfully knock down the OCT4 transcription relatively better than commercial reagent. Our findings supported the appropriate efficiency of the mentioned transfection reagents for short nucleic acid transfection. From a clinical point of view, this research helps allocation of short nucleic acids into stem cells therapies.

Cell-surface glycosaminoglycans inhibit intranuclear uptake but promote post-nuclear processes of polyamidoamine dendrimer-pDNA transfection.

BACKGROUND Interaction of cell-surface glycosaminoglycans (GAGs) with non-viral vectors seems to be an important factor which modifies the intracellular destination of the gene complexes. Intracellular kinetics of polyamidoamine (PAMAM) dendrimer as a non-viral vector in cellular uptake, intranuclear delivery and transgene expression of plasmid DNA with regard to the cell-surface GAGs has not been investigated until now. METHODS The physicochemical properties of the PAMAM-pDNA complexes were characterized by photon correlation spectroscopy, atomic force microscopy, zeta measurement and agarose gel electrophoresis. The transfection efficiency and toxicity of the complexes at different nitrogen to phosphate (N:P) ratios were determined using various in vitro cell models such as human embryonic kidney cells, chinese hamster ovary cells and its mutants lacking cell-surface GAGs or heparan sulphate proteoglycans (HSPGs). Cellular uptake, nuclear uptake and transfection efficiency of the complexes were determined using flow cytometry and optimized cell-nuclei isolation with quantitative real-time PCR and luciferase assay. RESULTS Physicochemical studies showed that PAMAM dendrimer binds pDNA efficiently, forms small complexes with high positive zeta potential and transfects cells properly at N:P ratios around 5 and higher. The cytotoxicity could be a problem at N:Ps higher than 10. GAGs elimination caused nearly one order of magnitude higher pDNA nuclear uptake and more than 2.6-fold higher transfection efficiency than CHO parent cells. However, neither AUC of nuclear uptake, nor AUC of transfection affected significantly by only cell-surface HSPGs elimination and interesting data related to the effect of GAGs on intranuclear pDNA using PAMAM as delivery vector have been reported in this study. CONCLUSION Presented data shows that the rate-limiting step of PAMAM-pDNA complexes transfection is located after delivery to the cell nucleus and GAGs are regarded as an inhibitor of the intranuclear delivery step, while slightly promotes transgene expression.

A novel diblock of copolymer of (monomethoxy poly [ethylene glycol]-oleate) with a small hydrophobic fraction to make stable micelles/polymersomes for curcumin delivery to cancer cells.

Curcumin is a potent natural anticancer agent, but its effectiveness is limited by properties such as very low solubility, high rate of degradation, and low rate of absorption of its hydrophobic molecules in vivo. To date, various nanocarriers have been used to improve the bioavailability of this hydrophobic biomaterial. This study investigates the encapsulation of curcumin in a novel nanostructure of monomethoxy poly(ethylene glycol)-oleate (mPEG-OA) and its anticancer effect. Tests were done to determine the critical micelle concentration (CMC), encapsulation efficiency, drug-loading efficiency, and cytotoxicity (against U87MG brain carcinoma cells and HFSF-PI3 cells as normal human fibroblasts) of some nanodevice preparations. The results of fluorescence microscopy and cell-cycle analyses indicated that the in vitro bioavailability of the encapsulated curcumin was significantly greater than that of free curcumin. Cytotoxicity evaluations showed that half maximal inhibitory concentrations of free curcumin and curcumin-loaded mPEG-OA for the U87MG cancer cell line were 48 μM and 24 μM, respectively. The Annexin-V-FLUOS assay was used to quantify the apoptotic effect of the prepared nanostructures. Apoptosis induction was observed in a dose-dependent manner after curcumin-loaded mPEG-OA treatments. Two common self-assembling structures, micelles and polymersomes, were observed by atomic force microscopy and dynamic light scattering, and the abundance of each structure was dependent on the concentration of the diblock copolymer. The mPEG-OA micelles had a very low CMC (13.24 μM or 0.03 g/L). Moreover, atomic force microscopy and dynamic light scattering showed that the curcumin-loaded mPEG-OA polymersomes had very stable structures, and at concentrations 1,000 times less than the CMC, at which the micelles disappear, polymersomes were the dominant structures in the dispersion with a reduced size distribution below 150 nm. Overall, the results from these tests revealed that this nanocarrier can be considered as an appropriate drug delivery system for delivering curcumin to cancer cells.

Specific gene delivery mediated by poly(ethylene glycol)-grafted polyamidoamine dendrimer modified with a novel HER2-targeting nanobody

As compared to other nanobiopolymers used in gene delivery applications, polyamidoamine dendrimers possess significantly improved physical and chemical properties such as monodispersity, well-defined size and shape, and biocompatibility, which make them ideal vectors for biomedical purposes. The aim of this study was fine-tuning of an anti–human epidermal growth factor receptor 2 (HER2) nanobody–polyethylene glycol (PEG)–G5 polyamidoamine gene delivery vector for potential in vivo applications. Polyethylene glycol was conjugated to polyamidoamine dendrimers at three different molar ratios of 10, 20, and 30 (polyethylene glycol:polyamidoamine). Anti-HER2 nanobody was chosen as our targeting agents and attached to polyethylene glycol–polyamidoamine conjugates. Cytotoxicity assays and transfection studies were carried out to find the most efficient conjugate in terms of both low cytotoxicity and high transfection rate. Our data indicated that PEGylation decreased the cytotoxicity of dendrimers alone and when complexed with DNA (dendriplexes). Among all of the polyethylene glycol–polyamidoamine dendrimers, polyethylene glycol(10)–polyamidoamine resulted in the most efficient gene transfection vector in both BT-474 and MCF-10A cell lines. Incorporation of anti-HER2 nanobodies could further increase their cellular uptake up to 1.7-fold compared to native dendrimers in HER2-overexpressing BT-474 cells but not in HER2-negative MCF-10A cells. It can be concluded from our results that nanobody–polyethylene glycol–polyamidoamine conjugates might be a promising new bioconjugate for the targeted gene delivery to HER2-positive tumor cells in vivo.

Targeted DNA delivery to cancer cells using a biotinylated chitosan carrier

A novel biotinylated chitosan‐graft‐polyethyleneimine (Bio‐Chi‐g‐PEI) copolymer was synthesized and evaluated as a nonviral gene delivery carrier for improvement of the transfection efficiency, endosomal escape, and targeted gene delivery of a plasmid encoding green fluorescent protein N1 (pEGFP‐N1) into two different biotin‐overexpressing cell lines including HeLa and OVCAR‐3 cells. The structure of the obtained copolymers was confirmed by 1H nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy. Physicochemical properties of the Bio‐Chi‐g‐PEI/plasmid DNA (pDNA) complexes such as complex stability, size, zeta potential, and their morphology were investigated at various weight ratios of copolymer to pDNA. Bio‐Chi‐g‐PEI copolymers could effectively condense pDNA into small particles with average diameters less than 164 nm and the zeta potential of +34.8 mV at the N/P ratio of 40/1. As revealed by flow cytometry, Bio‐Chi‐g‐PEI/pDNA complexes had lower cytotoxicity than that of PEI 25 kDa/pDNA complexes in both cell lines. In vitro experiments revealed that the Bio‐Chi‐gPEI/pDNA complexes not only had much lower cytotoxicity, but also displayed higher transfection efficiency than that of PEI 25kDa/pDNA complexes. High percentage of cancer cells was successfully transfected by Bio‐Chi‐g‐PEI/pDNA and properly expressed GFP protein. This study indicates that this copolymer complex can be a promising gene delivery carrier.