Create new genetic vectors from PLGA and PAMAM dendrimer nanoparticles. Physical studies.

Author: Tamaz Mdzinarashvili
Co-authors: Khvedelidze M., Shekiladze E., Lomadze E., Shengelia N.
Keywords: PLGA; PAMAM G4 dendrimers; complex with DNA.
Annotation:

There are complicated pathogenic diseases that are caused by cell genes that they are expressed or they are not sufficiently active enough for normal cell functioning. It is clear that fighting against such diseases is extremely difficult. Biologists are well known Gene vectors (e.g. plasmids, viruses) that are able to move healthy genes into the cell and their "sew" in the host cell DNA chain. Designed technology is hard and expensive, why we believe that nanoparticles can be used as gene vectors. The adventage lies in the fact that theyare easier to prepare and thechnology is cheap. In the presented work are offered two different types of nanoparticles that can be used as Gene vectors such as PLGA Dipalmitoylphosphatidylcholine) and PAMAM (poly-amidoamine) dendrimers. Were prepared PLGA and PAMAM nanoparticles to create a complex with DNA. We note that both types of nanoparticles are capable of penetrate a cell membrane and particles together DNA will be inside a cell. We conducted experiments with complex DNA-PLGA nanoparticles using various physical methods. The purpose of these studies was to determine the structure and stability of the complex of particles in time and in temperature. It was important to identified the number of DNA (ie the length of the gene) which is placed in the complex nanoparticle structure. We used the calf timuse DNA (SERVA) and two different type of PLGA nanoparticles (diameter both particles were d = 150 nm) with different values of surface potentials - one is negativ and the other is positive. Using Zetasizer (Malvern) method, we established that the PLGA particles have a negativ surface potential, while the PLGA nanoparticles with coating by citosan had a positive surface potencial. It is understood that the DNA surface charge in aqueous solution is due to the phosphoric acid is negative. We mixed the DNA and PLGA with various ratios of DNA / PLGA concentration, to obtain an effective correlation of complex. To determine this ratio , we used high-speed centrifuges and spectrophotometric methods. The experiments carried out showed that there was no possibility to obtain complex between the negative surface of the PLGA nanoparticles and DNA (both surface charge is negative), while we get complex between the PLGA with citosan (positive surface) and DNA. According to the study, we determined the effective DNA / PLGA ratio, which turned out 7:1(W/W). This result can be used to create a coplex of PLGA (with Citosan) and Gene (chain of DNA), that can be used in practical purposes. The recommendation is done on how to prepare dendrimer solutions for practical and safely use in gene delivery. DSC calorimeter have been also used to study the thermodynamic properties of DNA/PAMAM G4 dendrimer complexes. We showed that up to DNA/dendrimer ratio 43 ± 3 (w/w) the solution was homogeneous, but stable aggregates were formed at higher PAMAM G4 content. We note that the diametr of PAMAM G4 dendrimers compared to the PLGA (d=150nm) is very small and is 4.5nm. DSC experiments performed with homogeneous solution of dendriplexes revealed existence of the pH-dependent melting curves that contain several endothermic peaks associated with melting of GC-rich regions. In this study we have created a model of the complex of DNA and PAMAM G4 dendrimers, which will make it opossibile to determine the amount of concentration of the Gene and dendrimera, which can be used for practical purposes.