Editing Peptides for Transfection (section)

== pH responsive LAH4- L1 peptide&nbsp; ==

[https://pubs.acs.org/doi/abs/10.1021/acs.molpharmaceut.6b00338&nbsp https://pubs.acs.org/doi/abs/10.1021/acs.molpharmaceut.6b00338&nbsp];

https://www.nature.com/articles/s41598-017-10049-y

{| border="1" cellspacing="1" cellpadding="1" style="width:600px;"
|-
| Peptide<br/>
| NLS&nbsp;<br/>
| <br/>
| Diameter ± SD (nm)&nbsp;
| Zeta Potential ± SD (mV)&nbsp;<br/>
| Transfection Efficiency
|-
| LAH4-L1<br/>
| <br/>
| KKALLAHALHLLALLALHLAHALKKA&nbsp;<br/>
| 226.7 ± 31.7<br/>
| +2.2 ± 0.9&nbsp;<br/>
| low
|-
| PK1-LAH4-L1 (PK1)<br/>
| SV40&nbsp;<br/>
| PKKKRKVG CG KKALLAHALHLLALLALHLAHALKKA<br/>
| 164.9 ± 1.3<br/>
| +10.1 ± 2.1&nbsp;<br/>
| High
|-
| PK2-LAH4-L1 (PK2)&nbsp;<br/>
| Reversed SV40&nbsp;<br/>
| VKRKKKPG CG KKALLAHALHLLALLALHLAHALKKA<br/>
| 130.1 ± 1.1&nbsp;<br/>
| +13.3 ± 1.6&nbsp;&nbsp;<br/>
| High++
|-
| 
KR-LAH4-L1&nbsp; (KR)&nbsp;

| Nucleoplasmin&nbsp;<br/>
| 
KRPAATKKAGQAKKKKG CG KKALLAHALHLLALLAL HLAHALKKA

| 184.6 ± 6.8&nbsp;<br/>
| +24.2 ± 0.9&nbsp;&nbsp;<br/>
| high
|-
| NQ-LAH4-L1 (NQ)<br/>
| <br/>
| NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQG GYG CG KKALLAHALHLLALLALHLAHALKKA<br/>
| 354.5 ± 81.5<br/>
| N.A.&nbsp;&nbsp;<br/>
| <br/>
|-
| SV40<br/>
| <br/>
| PKKKRKV<br/>
| 563.3&nbsp;<br/>
| −3.67&nbsp;&nbsp;<br/>
| no
|-
| CSV40C
| <br/>
| CPKKKRKVC<br/>
| 235.8&nbsp;<br/>
| 4.1<br/>
| no
|-
| STR-CSV40C*<br/>
| <br/>
| CH3(CH2)16-CONH-CPKKKRKVC<br/>
| 85.0<br/>
| 33.0<br/>
| high
|-
| STR-CH2SV40H2C *<br/>
| <br/>
| CH3(CH2)16-CONH-CHHPKKKRKVHHC<br/>
| 89.2<br/>
| <span style="color: rgb(69, 69, 69); font-family: robotoregular, Meiryo; text-align: center;">29.1</span><br/>
| high+
|}



*STR was conjugated to the N-terminal of CSV40C and&nbsp;STR-CH2SV40H2C &nbsp;using the solid-phase peptide synthesis method.

DNA transfection. A549, Calu-3, RAW264.7 and JAWSII cells were transfected with peptide/DNA complexes at 1 µg luciferase plasmid per well in 24-well plates. Lipofectamine™ 2000 was used as control. The complexes were prepared at 5:1, 10:1 and 15:1 ratios (w/w) in Opti-MEM I reduced serum medium. The cells were transfected when they reached 70-80% confluency. After 5 h of incubation with the complexes at 37 °C, the transfection media were removed and replaced with serum supplemented cell culture media. At 24 h post-transfection, the cells were rinsed and lysed in 100 µl reporter lysis buffer. The luciferase expression was detected using the luciferase assay system according to the manufacturer's protocol. The results were expressed as relative light unit (RLU) per mg of total protein. For transfection with growth-arrested cells, the standard double thymidine block method was employed as previously reported.32 A549 cells were seeded at a density of 5 × 104 cells per well in 24-well plate for overnight. The cell culture medium was then replaced with one containing thymidine (2mM). After 19 h of incubation, the cells were washed and incubated with thymidine-free culture medium. After 9 h, thymidine was added to the culture medium and the cells were incubated for a further 17 h. The cells were washed again. Peptide/DNA complexes prepared in Opti-MEM I reduced serum medium containing thymidine were added to the cells. After 5 h of incubation, the transfection medium was removed and replaced with fresh culture medium. The luciferase expression was examined at 24 h post-transfection as described above.