Cytotoxicity and Transfection Efficiency

To overcome this problem, PAMAM dendrimers have been modified and/ or used at lower generations. Skoza and colleagues covalently attached GALA, a 30 amino acid synthetic peptide, with a glutamic acid-alanine-leucine-alanine repeat, to PAMAM dendrimers and observed that the transfection efficiency was enhanced. In another approach, PAMAM dendrimers were covalently linked with cyclodextrin macrocycles to create a synergic effect between dendrimer and cyclodextrin. To enhance PAMAM transport into cells, a series of artificial proteins were designed by 

Interestingly, one of the most well-known transfection reagents of genetic material into eukaryotic cells is SuperFect , which is based on amine-terminated PAMAM dendrimers. Moreover, it has been shown that more flexible fractured PAMAM dendrimers exhibited better DNA or RNA transfection properties compared with non-fractured dendrimers. To achieve higher transfection efficiencies, recent design studies have focused on dendrimer structures incorporating higher flexibility (e.g. softer dendrimers).  

PPI dendrimers were also tested for transfection due to their structural similarities with PAMAM dendrimers and their commercial availability. The use of high-generation PPI dendrimers as gene vectors was disqualified because their disappointing transfection and toxicity levels, although lower generations, specifically G2, were found to be suitable.  

Another class of potential transfecting agents is the phosphorus-containing dendrimers, which can be synthesized up to the 12th gener- 

Until now, carbosilane dendrimers have the best cytotoxicity profile, and also present an added value, since they can function in media containing serum and antibiotics, while the use of Lipofectintrade (a common reagent used for in vitro transfection in laboratories) is required to remove these additives. This is a fundamental advantage that will allow carbosilane dendrimers to make the transition to in vivo scenarios. 

---

Kean T, Roth S, Thanou M (2005) Trimethylated chitosans as non-viral gene delivery vectors cytotoxicity and transfection efficiency. J Control Release 103 643-653... 

T. Kean, S. Roth, and M. Thanou, Trimethylated chitosans as non-viral gene dehvery vectors Cytotoxicity and transfection efficiency, J. Control. Release, 103 (3), 643-653,2005. M. Gonzalez-Rodriguez, M. Holgado, C. Sanchez-Lafuente, A. Rabasco, and A. Fini, Alginate/chitosan particulate systems for sodium diclofenac release, Int. J. Pharm., 232(1), 225-234, 2002. 

Kean, T., Roth, S., and Thanou, M. (2005). Tri-methylated chitosans as non-viral gene delivery vectors Cytotoxicity and transfection efficiency. Journal of Controlled Release 103, 643-653. 

Multi-block copolymers were synthesized based on LMW pLL (1.5-6 kDa) copolymerized with difunctional 1.5 kDa PEG succinate ester [113]. Synthesized copolymers showed almost neghgible cytotoxicity and transfection efficiency was comparable to the PLL homopolymer with a MW of 26 kDa. Biodegradation of the succinate ester Hnkages under physiological conditions revealed that the molecular weight of copolymers decreased to 20% of the initial MW within 72 h. Transfection efficiencies of copolymers were not affected by the presence of serum, while that of PLL homopolymer decreased to the level of naked DNA in the presence of serum. 

Kean T, Roth S, Thanou M (2(X)5) Trimethyiated chitosans as non-vital gene delivery vectors cytotoxicity and transfection efficiency. J Control Release 103 643-653 Kim TH, Jiang HL, Jere D et al (2007) Chemical modification of chitosan as a gene carrier in vitro and in vivo. Prog Poiym Sci 32 726-753... 

The urocanic-acid-modified chitosan showed good DNA binding abihty, high protection of DNA from nuclease attack, and low cytotoxicity. The transfection efficiency of chitosan into 293T cells was much enhanced after coup-Hng with urocanic acid [96]. 

Composite NPs such as calcium phosphate NPs have also gained interest for nmi-viral gene delivery. Cao et al. [93] used calcium phosphate (CP) nanocomposite particles to encapsulate DNA for their delivery into MSCs. The CP-DNA NPs ( 100 nm) were reported to be less cytotoxic and more efficient in gene delivery into MSCs than those of Lipofectamine or a standard calcium phosphate transfection kit. 

This chapter will discuss the production and characterization of CLS obtained by different techniques, the formation of the complex between CLS and DNA, and, finally, the in vitro cytotoxicity on different cell lines and the transfection efficiency... 

Obika S, Yu W, Shimoyama A, et al. Properties of cationic liposomes composed of cationic lipid YKS-220 having an ester linkage adequate stability, high transfection efficiency, and low cytotoxicity. Biol Pharm Bull 1999 22(2) 187-190. 

Fischer, D., Bieber, T., Li, Y., Elsasser, H-P, Kissel, T. (1999). A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine effect of molecular weight on transfection efficiency and cytotoxicity. Pharm. Res., 16, 1273-12791. 

Polyfection was carried out with pCMVLuc (a 5 kb pDNA) on HepG2 cells for four hours at 37 °C. After 48 hours, the transfection efficiency was determined from Luciferase activity in cells measured by luminescence (RLU) and the cytotoxicity was evaluated by using the colorimetric MTT assay, dp is the poly lysine degree of polymerization. The transfection efficiency is scored on a 0 to 100 scale where 0 and 1 00 correspond to an absence of transfection and the most effective transfection, respectively compared to the pDNA alone. His is histidyl residue. 

Three families of polymers have been used to study transfection mechanisms polyamines, polyamides, and polyvinyl type polymers. The transfection efficiencies achievable with these systems vary widely, so an in-depth analysis of each polymer family and subsequent comparison of what affects gene delivery will be discussed in this chapter. In addition to high transfection efficiency, it is important for the polymeric systems to be relatively nontoxic to cells in vitro and not to elicit an immune response in vivo. Thus, the effect of transfection parameters on cytotoxicity and immunogenicity will also be examined. 

The design of a new polymer system should take into consideration all of the parameters identified to increase transfection efficiency and those that decrease cytotoxicity and immune response. 

The linker group that bridges the cationic lipid headgroup with the hydrocarbon moiety controls the biodegradability of a cationic amphiphile. Most of the linker bonds are ether, ester, or amide bonds (Fig. 1). Compounds with ether links generally render better transfection efficiency. However, they are more stable and may cause higher toxicity, while cationic lipids with ester links such as DOTAP are more biodegradable and less cytotoxic in cultured cells [28, 39]. Noteworthy,... 

This review deals with the syntheses of various hyperbranched polyamines that are prepared through a one-step polymerization process. Furthermore, we present the current status of polyamines as gene carriers and describe their versatility, and their properties such as structure-property dependency, gene transfection efficiency, and cytotoxicity profiles of hyperbranched polyamines. 

---