**2.3.5 Transfection efficiency**

As observed in previous sections, compared to polyplexes formed with polycations chitosan and pDADMAC, the DNA-MEP lipoplex yielded transfection rates markedly higher. We speculate that the higher transfection efficiency of the MEP lipoplex must be related to a successful endosomal escape that is promoted simultaneously by a repulsive membrane acidolysis process and different conformational transitions adopted by DOPE upon pH changes (Bonetta 2005; Khalil et al., 2006; Tros de Ilarduya et al., 2010). Concerning the cell entrance mechanism our lipoplexes should display, we hypothesize mitosis as the most probable option given both the negative ζ-potential of the system and that, to our best knowledge, MEP does not contain any kind of cell-receptors. In this context, HeLa cells (the cells we worked with) are recognized as highly proliferating ones (Ota, et al.,2010). Importantly, as part of our protocols we seeded the cells at an 80-90% optical confluence so that transfection was practiced with the maximum possible number of healthy cells, assuring mitosis.4

### **2.3.6 Particular conclusions**

Emphasizing the importance of studying the lipoplex formation under the same conditions at which transfection is practiced, our results point to a ''beads on a string'' complex conformation as depicted by i) the TEM and AFM micrographs revealing coils of DNA coming out from vesicle surface, ii) the ζ-potential results showing that the transfection mass ratios are well below isoneutrality, and iii) the practically constant vesicle sizes after complexation depicted by DLS. On the other hand, a sharp increase in the intensity of light

<sup>3</sup> Mitosis is also accepted as an important factor in the nuclear translocation of transgenes given that the

integrity of the nuclear membrane is transiently lost, allowing their entrance (Khalil et al, 2006). 4 It has to be noted that this very reasoning should be applied to the chitosan- and pDADMACmediated polyplexes.

Polycation-Mediated Gene Delivery: The Physicochemical Aspects Governing the Process 409

in order to improve the transfection process. In the context of the DNA release, special attention needs be paid to the development of strategies assuring a high degree of DNA-vector de-compaction. Interesting pioneering results stem from publications by González-Pérez and coworkers (Carlstedt et al., 2010; Gonzalez-Perez & Dias, 2009; Gonzalez-Perez et al., 2008).

M. A.-M. thanks the Ramón Areces foundation for his Post-Doctoral fellowship (Research

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**4. Acknowledgment**

project: 2010/CL509).

0927-7765.

**5. References** 

Reproduced from (Alatorre-Meda et al., 2010a) by permission of the PCCP Owner Societies.

Fig. 11. Typical TEM (A and B) and AFM (C and D) images obtained for DNA-MEP lipoplexes at L/D = 1000.

scattered by samples marks (L/D)c ~ 600 as the zone from which lipoplexes exist, validating the mass ratio recommended for transfection of (L/D) ~ 700. Finally, DLS results reveal lipoplexes with an average size of 135 nm that were stable within at least 7 days.
