Hossein Hosseinkhani

DESIGN OF TISSUE-ENGINEERED NANOSCAFFOLD THROUGH SELF-ASSEMBLY OF PEPTIDE AMPHIPHILE

In order to mimic in vivo topography of the native tissue created by extracellular matrix (ECM) components, which make up all soft tissues, the surface features of each biomaterial should be considered as a nanodimensional structure. In this study, an artificial ECM was designed to mimic the nanostructured topography created by ECM components in native tissue. The proliferation and differentiation of mesenchymal stem cells (MSCs) was investigated in a three dimensional (3-D) network of nanofibers formed by the self-assembly of peptide amphiphile (PA) molecules. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. The sequence of arginine-glycine-aspartic acid (RGD) was included in peptide design as well. A 3-D network of nanofibers was formed by mixing MSC suspensions in a media with dilute aqueous solution of PA. The attachment, proliferation and osteogenic differentiation of MSCs were influenced by the self-assembled PA nanofibers as the cell scaffold and the values were significantly high compared with those in the static culture (2-D tissue culture plate).

Micro and nano-scale in vitro 3D culture system for cardiac stem cells

Despite the success to prevent or limit cardiovascular diseases, the restoration of the function of a damaged heart remains a formidable challenge. Cardiac stem cells (CSCs), with the capacity to differentiate into cardiomyocytes, hold great potential as a source of cells for regenerative medicine. A major challenge facing the clinical application of differentiated CSCs, however, is theability to generate sufficient numbers of cells with the desired phenotype. We previously established cell lines of CSCs using a c-kit antibody from adult rat hearts for use in regenerative medicine. C-kit -positive cardiac cells are well recognized as CSCs and have the potential to differentiate into cardiomyocytes. Here, before implant these cells in vivo, we first developed three-dimensional culture system (3D) using micro- and nano-scaled material. Sheets of poly(glycolic acid) (PGA) were fabricated by electrospinning. Composites of collagen-PGA were prepared that contained 0, 1.5, 3 or 6 mg of electrospun PGA nanofibers. The nanofibers were added as a sheet that formed a layer within the collagen sponge. The sponges were freeze-dried and then dehydrothermally crosslinked. A scanning electron microscopy (SEM)-based analysis of the surface of the sponges demonstrated a uniform collagenous structure regardless of the amount of PGA nanofibres included. The PGA nanofibers significantly enhanced the compressive strength of the collagen sponge. More CSCs attached to the collagen sponge incorporating 6 mg of PGA nanofibers than the sponge without PGA nanofibers. The attachment and proliferation of CSCs in the 3D culture was enhanced by incubation in a bioreactor perfusion system compared with 3D static and two-dimensional (2D; i.e. tissue culture plates) culture systems. The use of micro- and nano-scale materials in the fabrication of composites together with a 3D culture system is a very promising way to promote the culture of stem cells. (c) 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Liver targeting of plasmid DNA by pullulan conjugation based on metal coordination.

Liver targeting of plasmid DNA was achieved through conjugation of pullulan derivatives with chelate residues based on metal coordination. Triethylenetetramine (Ti), diethylenetriamine pentaacetic acid (DTPA), and spermine (Sm) were chemically introduced to pullulan, a polysaccharide with an inherent affinity for the liver, to obtain various pullulan-Ti, pullulan-DTPA, and pullulan-Sm derivatives. Irrespective of the type of pullulan derivatives, intravenous injection of the pullulan derivatives-plasmid DNA conjugates with Zn2+ coordination significantly enhanced the level of gene expression only in the liver to a significant greater extent than that of free plasmid DNA. The enhanced gene expression by the pullulan-DTPA-plasmid DNA conjugate was specific to the liver and the level was significantly higher than that of the pullulan-DTPA-plasmid DNA mixture. The level of gene expression depended on the percentage of chelate residue introduced, the mixing ratio of the plasmid DNA-DTPA residue in conjugate preparation, and the plasmid DNA dose. The gene expression induced by the conjugate lasted over 12 days after injection. A fluorescent-microscopic study revealed that the plasmid DNA was localized at the liver after injection of the pullulan-DTPA-plasmid DNA conjugate with Zn2+ coordination. Pre-injection of both arabinogalactan and galactosylated albumin suppressed significantly the liver level of gene expression, in contrast to that of mannosylated albumin, indicating that the plasmid DNA in the conjugate was transfected at hepatocytes. We conclude that the Zn2+-coordinated pullulan conjugation is a promising way to enable the plasmid DNA to target to the liver for gene expression as well as to prolong the time duration of gene expression

Silk fibroin nanoparticle as a novel drug delivery system.

Design and synthesis of efficient drug delivery systems are of vital importance for medicine and healthcare. Nanocarrier-based drug delivery systems, in particular nanoparticles, have generated great excitement in the field of drug delivery since they provide new opportunities to overcome the limitations of conventional delivery methods with regards to the drugs. Silk fibroin (SF) is a naturally occurring protein polymer with several unique properties that make it a suitable material for incorporation into a variety of drug delivery vehicles capable of delivering a range of therapeutic agents. SF matrices have been shown to successfully deliver anticancer drugs, small molecules, and biomolecules. This review will provide an in-depth discussion of the development of SF nanoparticle-based drug delivery systems.

Proliferation and differentiation of mesenchymal stem cells using self-assembled peptide amphiphile nanofibers

The objective of this study is to enhance the proliferation and differentiation of mesenchymal stem cells (MSC) in nanodimensional scaffolds. The proliferation and differentiation of MSC was investigated in a three-dimensional network of nanofibers formed by self-assembly of peptide amphiphile (PA) molecules. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH(2) terminus of the peptide. The sequence of arginine-glycine-aspartic acid was included in the peptide design as well. A three-dimensional network of nanofibers was formed by mixing MSC suspensions in media with dilute aqueous solution of PA. The attachment, proliferation and osteogenic differentiation of MSC were influenced by the self-assembled PA nanofibers as the cell scaffold and the values were significantly high compared with those in the static culture. The alkaline phosphatase activity and osteocalcin content of MSC cultured in the PA nanofibers significantly increased compared with the static culture method. It may be concluded that PA nanofibers enable MSC to positively improve the proliferation and differentiation extent.

Biodegradable Polymer-Metal Complexes for Gene and Drug Delivery

The delivery of genes and drugs into cells has increasingly attracted attention for the generation of genetically engineered cells. Successful drug delivery will have enormous academic, clinical, and practical impacts on gene therapy, cell and molecular biology, pharmaceutical and food industries, and bio-production. The major aim of gene therapy is to deliver genetic materials into cells effectively, genetically modifying and repairing cell functions with the possibility of inducing therapeutic healing of disease. The genetic material includes DNA, RNA, antisense, decoy DNA, and ribozymes. The aim is that the appropriate transfection would allow diseased cells to return to a healthy condition. The genetic manipulation is often manifested in the mechanisms of intracellular actions of genes and proteins, and may play an important role in making clear the key genes associated with various diseases. Based on fundamental and scientific knowledge, the delivery technology of genetic material should be applicable to producing various proteins of pharmaceutical value (e.g. cytokines, growth factors, and antibodies) and also to producing seeds resistant to harmful insects and cold weather damage. This implies that the cells might be enhanced to produce valuable pharmaceutical and food products. For each approach, it is important, for successful gene expression, to select an appropriate gene to be delivered as well as to develop the gene delivery technology to enhance transfection efficiency. This review will provide an overview of the enhanced gene expression of plasmid DNA complexed with new non-viral gene delivery vehicles by biodegradable biopolymer-metal complex, introducing our recent research data to emphasize the technical feasibility of biopolymer-metal complexes in gene therapy and biotechnology.

Engineering three-dimensional collagen-IKVAV matrix to mimic neural microenvironment.

Engineering the cellular microenvironment has great potential to create a platform technology toward engineering of tissue and organs. This study aims to engineer a neural microenvironment through fabrication of three-dimensional (3D) engineered collagen matrixes mimicking in-vivo-like conditions. Collagen was chemically modified with a pentapeptide epitope consisting of isoleucine-lysine-valine-alanine-valine (IKVAV) to mimic laminin structure supports of the neural extracellular matrix (ECM). Three-dimensional collagen matrixes with and without IKVAV peptide modification were fabricated by freeze-drying technology and chemical cross-linking with glutaraldehyde. Structural information of 3D collagen matrixes indicated interconnected pores structure with an average pore size of 180 μm. Our results indicated that culture of dorsal root ganglion (DRG) cells in 3D collagen matrix was greatly influenced by 3D culture method and significantly enhanced with engineered collagen matrix conjugated with IKVAV peptide. It may be concluded that an appropriate 3D culture of neurons enables DRG to positively improve the cellular fate toward further acceleration in tissue regeneration.

Polysaccharide gene transfection agents

Gene delivery is a promising technique that involves in vitro or in vivo introduction of exogenous genes into cells for experimental and therapeutic purposes. Successful gene delivery depends on the development of effective and safe delivery vectors. Two main delivery systems, viral and non-viral gene carriers, are currently deployed for gene therapy. While most current gene therapy clinical trials are based on viral approaches, non-viral gene medicines have also emerged as potentially safe and effective for the treatment of a wide variety of genetic and acquired diseases. Non-viral technologies consist of plasmid-based expression systems containing a gene associated with the synthetic gene delivery vector. Polysaccharides compile a large family of heterogenic sequences of monomers with various applications and several advantages as gene delivery agents. This chapter, compiles the recent progress in polysaccharide based gene delivery, it also provides an overview and recent developments of polysaccharide employed for in vitro and in vivo delivery of therapeutically important nucleotides, e.g. plasmid DNA and small interfering RNA.

DNA nanoparticles for gene delivery to cells and tissue

The development over the past decade of methods for delivering genes to mammalian cells has stimulated great interest in the possibility of treating human disease by gene-based therapies. The major aim of gene therapy is to effectively deliver the genetic materials into cells, genetically modifying and repairing cell functions, which may induce therapeutic healing of disease conditions. This review provides a critical view of gene therapy with a major focus on advanced DNA nanoparticles technologies to control the in vivo location and function of administered genes.

Ultrasound Enhancement of In Vitro Transfection of Plasmid DNA by a Cationized Gelatin

In vitro transfection efficiency of a plasmid DNA for rat gastric mucosal (RGM)-1 cells was enhanced by ultrasound (US) irradiation. Ethylenediamine was introduced to the carboxyl groups of gelatin to prepare a cationized gelatin as the vector of plasmid DNA encoding luciferase. An electrophoresis experiment revealed that the cationized gelatin was mixed with plasmid DNA at the weight ratio of 5.0 to form a cationized gelatin-plasmid DNA complex. The complex obtained was about 200 nm in diameter with a positive charge. When incubated with the cationized gelatin-plasmid DNA complex and subsequently exposed to US, RGM-1 cells exhibited a significantly enhanced luciferase activity although the extent increased with an increase in the DNA concentration, in contrast to the cationized gelatin alone with or without US irradiation and US irradiation alone. US irradiation was also effective in enhancing the activity by free plasmid DNA although the extent was less than that of the complex. The US-induced enhancement of luciferase activity was influenced by the exposure time period, frequency, and intensity of US. The activity enhancement became higher to be significant at the irradiation time period of 60 s and thereafter decreased. A series of cytotoxicity experiments revealed that an increase in the irradiation time period and intensity of US decreased the viability of cells themselves. It is possible that US irradiation under an appropriate condition enables cells to accelerate the permeation of the cationized gelatin-plasmid DNA complex through the cell membrane, resulted in enhanced transfection efficiency of plasmid DNA. These findings clearly indicate that US exposure is a simple and promising method to enhance the gene expression of plasmid DNA.