A common human embryonic kidney cell line with uses in protein production, transformed with SV40 large T antigen.
1 Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. 2 Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran.
3 Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran. 4 Endocrinology and Metabolism Research Center, Tehran University of Medical Sciences, Tehran, Iran. Purpose: Non-viral vectors have been widely proposed as safer alternatives to viral vectors, and cationic polymers have gained increasing attention because they can form self-assembly with DNA. In the past decade, Cationic polymers have been proposed as an alternative approach to the viral vectors.
Among non-viral vectors, chitosan has been considered to be a good gene carrier candidate, since it is known as a biocompatible, biodegradable, and low toxic material with high cationic potential [8], and it has functional groups that allow simple coupling of extracellular and intracellular targeting ligands [9].
Alkylated chitosans synthesized for gene deliveries are, N-dodecylated chitosan (NDC) [15], alkyl bromide [16] and trimethlyated chitosan oligomers [17]. Human Embryonic Kidney Epithelial Cells (HEK 293 Line), are a specific cell line originally derived from human embryonic kidney cells grown in tissue culture.
The aim of the present work is to show the effect of another chemical modification of chitosan in gene delivery. The EGFP and Jred plasmids were used to monitor gene transfer and transgene expression after transfection. In order to optimize the system for gene delivery, we used Flourescein isothiocyanate-dextran (FD) (Figure 2).
Two kinds of plasmids (1 ug) were used for HEK cell transfection with TEC and DEMC: 1) pLEX-JRED (from stem cell technology institute (Tehran, Iran)) which produced red fluorescent protein and 2) pEGFP which produced green fluorescent protein after transfection. The H-NMR spectrum, data and polymer peaks for TEC and DEMC can be seen in the below figures.
For TEC, the triple signal at 1.2 ppm was attributed to the CH3 groups of the ethyl substituents, while the CH2 groups at the quaternized site are superimposed by the 2-H and 6-H protons of the polysaccharide backbone. According to DEMC H-NMR, the signal at 1.3 ppm was attributed to CH3 groups of the ethyl substituent and the signal at 3 ppm is related to the CH3 group of methyl. FITC-dextran (FD) is primarily used for studying permeability and transport in cells and tissues. Since the miRNA was fluorescein labeled so, like fluorescence dextran (FD), it was detected after 5 hours in the cell cytoplasm. The positively charged head group of polymers makes electrostatic complexes with the negatively charged phosphate ions on the base backbone on miRNA. As stated in the introduction many studies have been done on chitosan derivatives for gene delivery. So up to now, according to the transfection results, the polymer concentration required for cell transfection is 2%. Based on the assumption that quaternization may increase the DNA condensing ability of chitosan, we have prepared quaternized derivatives of chitosan polymer, Triethyl chitosan (TEC) and Diethylmethyl chitosan (DEMC) .These derivatives proved to transfect HEK 293T cells to a high extent. The authors would like to acknowledge the stem cell technology institute (Tehran, Iran) for generous use of equipment. Chitosan is also considered to be a good candidate for gene delivery systems, since it is already known as a biocompatible, biodegradable, and low toxic material with high cationic potential.
However, the low specificity and low transfection efficiency of chitosan must be overcome for its use in clinical trials. An important variant of this cell line is the 293T cell line that contains, in addition, the SV40 Large T-antigen, that allows for episomal replication of transfected plasmids containing the SV40 origin of replication.

Ethyliodide, methyl iodide, and sodium borohydride were obtained from Sigma (Vienna, Austria).
At the beginning, we distributed chitosan (DD=97%) in N-methyl pyrrolidone and it was mixed at room temperature. In the first step chitosan was dissolved into 1% acetic acid solution and formaldehyde solution was added.
HEK 293T cell line, which are the standard cells for transfection, were used for system optimization. Prior to transfection, culture medium was removed and culture medium without FBS was added. Prior to transfection, culture medium was removed and the cells were rinsed with phosphate-buffered saline (PBS, pH 7.4).
15-19, approximately 80-90% of the HEK cells are transfected with TEC and DEMC complexed with pEGFP and pJred.
Considering that HEK cells are the standard cell line for transfection evaluation the transfection results in this cell line could be used in order to transfect other epithelial cell lines including pancreatic cancer cells (the results will be published). However, low solubility and transfection efficiency need to be overcome prior to clinical trial.
These favorable characteristics of the hydrophobic units lead to higher transfection efficiency of chitosan than polymer systems using only ionic interactions. This allows for amplification of transfected plasmids and extended temporal expression of the desired gene products.
Sodium hydroxide, N-methyl pyrrolidone (NMP) and sodium iodide were purchased from Merck (Darmstadt, Germany).
Then sodium hydroxide, sodium iodide and ethyl iodide was added to the mixture and it was heated at 60 °C for 6 hours under stirring.
The integral of CH3 of ethyl groups versus the other protons was used to calculate the degree of quaternization. It has negative charge and because of its fluorescence part, it can easily be detected inside the cells.
From the gained results, it is concluded that TEC and DEMC could be used for miRNA delivery to epithelial cell lines. Compared with lipofectamine (positive control) and plasmid transfected (negative control), the results with pEGFP and pJred are seen as green flouresent protein and red flourecent protein. Triethyl chitosan (TEC) and Diethylmethyl chitosan (DEMC) are quaternized and more hydrophobic then TMC so we could use the advantages of both hydrophobic and hydrophilic modification of chitosan in gene delivery. Up to now these results indicate that these partially quaternized chitosan derivatives are promising agents to be used in gene and miRNA, which are used widely in cancer therapy [27, 28], delivery. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine.
A novel chitosan oligosaccharide-stearic acid micelles for gene delivery: properties and in vitro transfection studies. Poly(2-alkylacrylic acid) polymers deliver molecules to the cytosol by pH-sensitive disruption of endosomal vesicles.
Temperature-responsive hydroxybutyl chitosan for the culture of mesenchymal stem cells and intervertebral disk cells. Transfection efficiency increases by incorporating hydrophobic monomer units into polymeric gene carriers. Trimethylated chitosans as non-viral gene delivery vectors: cytotoxicity and transfection efficiency. Preparation and evaluation of thiol-modified gelatin nanoparticles for intracellular DNA delivery in response to glutathione. Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency.

In this work, we focus on alkyl modified chitosan which might be useful in DNA condensing and efficient gene delivery. Examples include diethylaminoethyl dextran [2], poly(L-lysine) (PLL) [3], polyethylenimine (PEI) [4], gelatin [5], polyamidoamine dendrimers [6], and chitosan [7]. These chemical modifications include hydrophilic (10), hydrophobic (11), pH-sensitive (12), thermosensitive (13) and cell-specific ligand (14) groups for enhancement of cell specificity and transfection efficiency of chitosan in vitro. Then the solution pH was adjusted to 10 by adding 1 M NaOH solution and a white precipitant was yield.
After 5 h the formulations were removed, the cells were rinsed with PBS and grown in culture medium for 24 h to allow for GFP expression and detected with fluorescent microscope.
Quaternizing the polymer increases gene –polymer intraction and increases its transfection efficiency and hydrophobic modifications of chitosan increases transfection efficiency by modulating complex interactions with cells, such as adsorption on cell surfaces and cell uptake.
Sequential chemical modifications at the C-6 positions of N-phthaloylchitosan and evaluation as a gene carrier.
Methods: N, N- Diethyl N- Methyl (DEMC) and N- Triethyl Chitosan (TEC) were synthesized from chitosan polymer.
Both PEI and the dendrimers are effective gene carriers, but both are synthetic and not biodegradable, which means that their potential toxicity is a concern. Fluorescent microscope was used, in order to evaluate the polymers’ efficiency for gene delivery to human embryonic kidney epithelial cells (HEK 293T). In order to separate the N-methyl pyrrolidone between the TEC chains and get a better NMR, the precipitate was left for one week in acetone under gentle stirring. In the second step, methyl chitosan was dispersed in N-methyl pyrrolidone and after 4h of sttiring, sodium hydroxide; ethyl iodide and sodium iodide were added. Although biodegradable, PLL forms polyplexes with lower transfection efficiency than that of PEI and the dendrimers. 800 ul of LB (liquid broth) was added to the cells with shaking for 1h, and then centrifuged. The polymer was precipitated with acetone, centrifuged and dried to obtain a white water-soluble powder.
According to results, because of their positive charge, both polymers are suitable for complex formation with negative charged Flourescein isothiocyanate-dextran and plasmids. The polymer structure, the degree of quaternization and zeta potential were characterized by H-NMR and Malvern zeta sizer.
After 16 h the transformed bacteria were centrifuged and the plasmid was isolated via Plasmid Miniprep Kit.
Results gained from fluorescent microscope showed that TEC and DEMC were able to transfer FD, DNA and miRNA (micro RNA) to HEK cell line.
The Plasmid concentration and purity were determined using BioPhotometer Eppendorf (Hamburg, Germany) and electrophoresis on 1.5% agarose gel.
Conclusion: We conclude that these chitosan derivatives present suitable characteristics to be used as non-viral gene delivery vectors to epithelial cells. The gels were stained with ethidium bromide and photographed on a UV transilluminator (Uvidoc, Bridgeville, UK). Compared to 1kb DNA Ladder (GeneRuler™, Fermentase) the plasmid is pure and its size is approximately 7 kbp.

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