Team:REC-CHENNAI/Project-Background
PROJECT BACKGROUND
Transfection is the process of introducing foreign nucleic acids into eukaryotic cells. It is an analytical tool to study gene expression, regulation and protein function. The transfected nucleic acid can be transiently expressed for a short period of time, or become incorporated into the genomic DNA, where the change is passed on from cell to cell as it divides. In stable transfection, introduced genetic materials might have a marker gene for selection (transgenes) and the transfected DNA is either integrated into the host chromosomal DNA or maintained as an episome and sustain transgene expression even after host cells replicate. The transiently transfected genetic material does not integrate with the host genome and is expressed only for a limited period of time. Transfection methods can be broadly classified into 3 classes – Biological method, Chemical method and Physical method.
Biological method is virus-mediated transfection known as transduction.
Chemical methods use cationic polymers, calcium phosphate, cationic lipid and cationic amino acid to carry out transfection into mammalian cells. These transfecting reagents form a complex with the negatively charged nucleic acid and the complex is attracted to the negatively charged cell membrane. This complex is believed to pass through the cell membrane by endocytosis or phagocytosis.
The most recent physical methods of transfection include direct microinjection, biolistic particle delivery, electroporation and laser-based transfection. These methods are labor-intensive and there are high chances of cell death.
Transfection finds applications in gene silencing, stable cell line generation, virus production, large-scale protein production, stem cell reprogramming and differentiation.
Lipofectamine or Lipofectamine 2000, a cationic liposome formulation is commonly used for non-viral lipid-based transfection. It forms a complex with nucleic acid molecules, allowing them to overcome the electrostatic repulsion of the cell membrane and to be taken up by the cell.
The cationic lipid interacts with the negatively charged nucleic acid backbone to form a complex that enhances the oligonucleotide uptake. The cationic lipid molecule is often formulated with a neutral co-lipid (helper lipid). Together they form unilamellar liposomes (of 100nm in diameter). [1] The positive charge on the surface of the liposome generates an electrostatic interaction with nucleic acids and facilitates contact with the negatively charged cell membrane. The neutral co-lipid mediates fusion of the liposome with the cell membrane effecting entry of the nucleic acid. The mechanism of lipofectamine mediated transmembrane gene delivery is found to take place by the clathrin-mediated and caveolae-mediated pathway of endocytosis. [2]
Cell penetrating peptides (CPP) are short and usually basic amino acids-rich peptides originating from proteins able to cross biological barriers, such as the viral Tat protein, or are rationally designed. [3] CPPs represent a viable alternative for methods that have been elaborated earlier for the cellular delivery of bioactive macromolecules, like lipid- or polymer-based, viral methods and physical methods. [4] They are a new class of non-viral vectors that facilitate cellular uptake of various molecular equipment ranging from nanosize particles to small chemical molecules and large fragments of DNA. [5] They are often cationic with a net positive charge and have an inherent ability to enter cells. CPPs can either be tissue specific or non-tissue specific and do not cause significant membrane damage during their entry into cells. The "cargo" is associated with the peptide either through chemical linkage by covalent bonds or through non-covalent interactions. The function of CPPs is to deliver the cargo into cells, a process that can be in lieu of the usual endocytosis mediated transfection. [6]
The cell penetrating peptides are classified
CPP-assisted drug delivery can be realized through three means: [7]
(1) Via covalent conjugation of the drug molecule with a CPP;
(2) Encapsulation of the therapeutics into CPP-linked nano-carriers; and
(3) By physical adsorption of the therapeutic agents with CPPs via charge complexation.
The mechanisms of CPP-mediated cytoplasmic translocation have not been fully elucidated. But the identified pathways can be broadly classified into two types:
(1) energy-independent translocation of CPPs across the cell membranes by electrostatic interaction with the negatively charged lipid bilayers or through hydrogen bonding and
(2) energy-dependent macropinocytosis.
The inverted/converted micelle model: The interaction of the CPP with the cell membrane causes disturbance of the lipid bilayer and subsequently leads to the formation of inverted hexagonal structures termed as inverted micelles. The CPP is trapped in the hydrophilic environment of the micelle core and further interaction of the CPP with the membrane components leads to destabilization of the micelles releasing CPP into the cytosol.
Formation of membrane pores: In this mechanism, translocation of CPPs and cargo across the membrane takes place by the formation of transient pores generated by the insertion of CPP into the membrane in a ring-shaped structure.
Carpet model: CPPs with the associated conjugates transiently destabilize the biological membrane due to extensive association of the CPP to the membrane leading to the subsequent event of phospholipid reorganization.
Endocytosis: This mechanism is prevalent especially when CPP is conjugated with macromolecular cargoes like nano-carriers or large proteins large than 30 kDa. Cell translocation of CPPs occurs via several endocytic pathways, such as caveolae-mediated endocytosis, clathrin-mediated endocytosis, lipid raft-mediated endocytosis and macropinocytosis. The endocytic uptake pathways for CPPs mediated delivery is strongly depended on its attached cargoes. These pathways of internalization correlate with the chemical and physical variability of the peptide sequences, CPP concentrations, and characteristics of the drug cargoes.
CPPs have been extensively used for the delivery of chemotherapeutic agents to increase their efficiency. Methotrexate (MTX) covalently attached to the N-terminal of the CPP Penetratin or R8 via peptide bond more-efficiently enter MTX-resistant breast cancer cells. The CPP poly-L-arginine could increase the cellular uptake of doxorubicin (DOX), as well as its cytotoxicity towards human prostate cancer cells. TP10-cisplatin, a non-covalent carrier-cargo complex formed with a metal-affinity-based linkage, significantly improves the anti-cancer effect of cisplatin in human cervical tumor and osteosarcoma cells. [8]
Nucleic acid-based macromolecules such as a plasmid, antisense oligonucleotide, decoy DNA and siRNA have been realized as promising biological and pharmacological therapeutics in the regulation of gene expression. However, unlike other small-molecular drugs, their development and applications are limited by high molecular weight and negative charges, which results in poor uptake efficiency and low cellular traffic. CPPs have been successfully utilized for the delivery of nucleic acids since their discovery in 1994. CPPs can vectorize nucleic acid cargo via covalent conjugation or nanoparticle formation and cause efficient intracellular delivery of the same. [9]
CPPs are used for the development of vaccine delivery systems i.e. to deliver antigenic peptides. Penetratin linked to cytotoxic T lymphocyte epitopes derived from ovalbumin or mucin-1 tumor-associated antigens has been successful in inducing a stimulation of CD4+ and CD8+ T cells in vitro. In vivo, the secretion of cytokines due to T cell response inhibits B16.OVA melanoma cell growth. CPP-mucin 1-T cell epitope complex has been proved to have therapeutic potential as an anti-tumor agent. [8]
RNA-mediated interference (RNAi) is a simple and rapid method of silencing gene expression in a range of organisms. It silences a functional gene by the exogenous application of RNA and activates a sequence-specific RNA degradation.
Small (or short) interfering RNA (siRNA) is the most commonly used RNA interference (RNAi) tool for inducing short-term silencing of protein-coding genes. siRNA is a synthetic RNA duplex, usually about 21 nucleotides long, with 3' overhangs (two nucleotides) at each end that interferes with the translation of proteins by binding to and promoting the degradation of messenger RNA (mRNA) at specific sequences. [10]
On the molecular level, RNA interference is mediated by a family of ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs), which can be programmed to target virtually any nucleic acid sequence for silencing. [11]
It takes place in two stages.
The siRNA for gene silencing studies are produced [13]
