Pore Formation

The molecular dynamic studies and electrophysiology experiments have already indicated the formation of transient pores in the membrane allowing passive diffusion of ariginine-rich and TAT-peptides across the plasma membrane (Figure 6.1B). ^{[203, 204]} The electrostatic interaction between the positively charged amino acid residues and negatively charged phosphate headgroups in lipids resulted in the accummulation of the peptides in the outer leaflet of the membrane, between the phosphate and carbon-chains of the lipids. The accummulation caused a perturbation and overall thinning of the plasma membrane. The electrostatic force of attraction between the positively charged side chains of arginine or lysines and the negatively charged phosphate groups in lipid led to the formation of transient toroidal pores in the membrane through which the peptides got actively translocated accross the plasma membrane. Further investigations on this mechanism with other type of CPPs, also suggsted that formation of transient pores were a direct translocation mechanism.[205, 206, 207, 208] Palm-Apergi et al.

proposed that perturbation of the plasma membrane by the formation of transient pores triggered the membrane-repair-response (MRR) process induced by calcium ion efflux into the cells. In due course the internal vesicles got fused together to serve as a patch to reseal the membrane pores. ^[209]

Figure 6.1: Schematic illustration of the possible mechanistic routes for direct membrane translocation of the cell penetrating peptides by forming inverted micelles (A), forming transient pores (B) and by adaptive translocation (C)^[210]

6.3 Design

It has repeatedly been indicated by the scientific reports that β-peptides were one of the most promising candidates to mimic natural α-peptides in different biological processes with enhanced proteolytic stability, there have been very few experimental investigations carried out to test the cellular uptake efficiencies of short-chain β-peptides. Sufficient efforts have not been made to shed light on the capabilities of different types of β-peptides in cell membrane penetration. One of the main reasons behind the lack of reports in this field was the synthetic challenges to access different types of β-peptides and their propensity to get folded into different types of helical secondary structures. So, a preliminary trial was carried out to test cytotoxicity at different peptide concetrations, cellular uptake efficiency of two different types of short-chain β-peptides and also to investigate on any differential behaviours by the two types of β-β-peptides in connection to cellular uptake efficiency.

Although it was previously discussed in section 6.2, that presence of overall positive charged charater in the candidate peptide had crucial roles in cellular uptakes by direct translocation accross the plasma membrane, but it was also been indicated in section 6-1, that one of the major physiological side effects caused by the accummulation of the candidate peptides in different organs, like, liver, evolved due to the positively charged nature of the peptides. On the other hand, most of the cell penetrating peptides requied very high dosage as only a smaller fraction of these could ultimately deliver the cargo to the intra-cellular environment and the rest were decomposed by the proteolytic enzymes. To overcome these two major disadvantages, the idea of testing the following two types of β-peptide sequences (P6, P7 and P8, P9) as initial candidates for cellular uptake came into play. A standard and historically known cell penetrating peptide, HIV-1 TAT and its fluorescently labeled analogue, 5-FAM-HIV-1 TAT, were also selected as standards for all the measurement done with the β-peptide sequences for comparison.

P6

P7

P8

P9

HIV-1 TAT-Peptide

5-FAM-HIV-1 TAT-Peptide

The candidate β-peptides were designed in such a way that both of the types were devoid of any positive charges and were composed of completely hydrophobic sequences. The peptide P6 consisted an alternate β²/ β³-(Val/Ala/Leu) triad sequences whereas the P8 was composed of only β³-(Val/Ala/Leu) triad sequence. P7 and P9 were the 5(6)-carboxyfluorescein labeled analogues of the peptides, P6 and P8, respectively. The peptide P6 folded into a rare alternate 10/12-helical secondary structure while the P8 into a 14-helical pattern. Although both the β-peptides were devoid of any positive charges, but there were some differences between them.

The peptide P8 folded into a 14-helix and had overall inverted helical macro-dipole moment in comparison to the α-peptides. This macro-dipole moment induced a charged character into the peptides P8 and its fluorescently labeld analogue, P9. On the other hand, the peptide P6 folded into an alternating 10/12-helix where the individual dipole moments of each amides were nullified due to the alternate parallel ans perpendicular orientations of the amide dipole moments with respect to the helical axis in 12- and 10-membered hydrogen bonded rings, respectively. Therefore the overall partially charged character was completely absent in case of the helical peptide, P6 and its fluorescently labeled analogue, P7. This made the 10/12-helical peptides even more hydrophobic in nature than that of the 14-10/12-helical peptides. Although there were many other structural differences between the 10/12- and 14-helical β-peptides, but the presence and absence of overall helical macro-dipole moment was definitely one of the major ones. It was already experimentally verified that the helical macro-dipole moment could have major function in transmembrane insertion and spanning into lipid bilayers. It was observed that the more the helical macro-dipole moment, the less was the hydrophobic character of the peptide due to induction of partial charged character in it and the less was its propensity to get inserted and spanned into lipid bilayers due to unfavourable interaction between the

hydrophobic lipid core and the less hydrophobic transmembrane segment. Now membrane insertion and spanning was a completely different procedure than that of membrane penetration and cellular uptake. So, it would be interesting to investigate whether there were any differences in cytotoxicity or cellular uptake efficiency between the two types of β-peptides and that would indicate a possible role of helical macro-dipole moment in cellular uptake. The results could further be compared to that of the membrane insertion and spanning to check whether the same trend was maintained.

6.4 Results: