There are many complicated pathogenic diseases caused by cell genes that are incorrectly expressed or not sufficiently active for normal cell functioning. It is clear that the fight against such diseases is extremely difficult. Gene vectors (e.g., plasmids, viruses) are able to move healthy genes into cells and “sew” them into the host DNA. Such designed technologies are hard and expensive, that is why we believe that nanoparticles can be used as gene vectors. The advantage lies in the fact that they are easier to prepare and cheap to produce. In the presented work, two different types of nanoparticles are proposed as gene vectors poly lactic co-glycolic acid (PLGA) and (poly-amidoamine) dendrimers (PAMAM). PLGA and PAMAM nanoparticles were prepared to create complexes with DNA. It should be noted that both types of nanoparticles are able to penetrate cell membranes.
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 terms of time and temperature. At the outset, tt was important to identify the length of DNA molecule or gene which is placed inside the nanoparticle complex. We used the calf timuse DNA (SERVA) and two different types of PLGA nanoparticles (diameter both particles were d = 150 nm) with different values of surface potentials (negative and positively charged).
Using ZetaSizer (Malvern) method, we established that the PLGA particles have a negative surface potential, while the PLGA nanoparticles coating with chitosan had a positive surface potential. It is clear that the negative charge of DNA in an aqueous solution is due to the phosphoric acid. We mixed the DNA and PLGA with various ratios of DNA:PLGA to obtain an effective correlation of complex. To determine the DNA:PLGA ratio, we used high-speed centrifugal and spectrophotometric methods. The study indicated that there was no possibility to obtain complexes between the negative surface of the PLGA nanoparticles and the negatively charged DNA. Complexes do form between the PLGA with chitosan and DNA. According to the study, we determined the effective DNA:PLGA ratio to be 7:1 (W/W). This result can be used to create a complex of PLGA (with chitosan) and DNA, that can be used in practical purposes.
We have a number of recommendations for the preparation of dendrimer solutions for their practical and safe usage as gene delivery systems. DSC calorimeter has also been employed to study the thermodynamic properties of DNA/PAMAM G4 dendrimer complexes. We showed that up to a DNA:dendrimer ratio of 43:3 (w/w) the solution was homogeneous, with stable aggregates formed at higher PAMAM G4 content. We note that the diameter of PAMAM G4 dendrimers compared to the PLGA (d = 150 nm) is very small and is 4.5 nm. DSC experiments performed with homogeneous solution of dendriplexes revealed the existence of the pH-dependent melting curves that contain several endothermic peaks associated with melting of GC-rich regions. In this study we created a model of the complex of DNA and PAMAM G4 dendrimers, which make it possible to determine the amount of DNA and dendrimer that can be used for practical purposes.
