Small interfering RNA (siRNA)具有專一性有效抑制特定基因表現的功能，然而它本身帶負電的緣故，無法有效接近帶負電的細胞膜、進去細胞質發揮正常功能。所以需要一個合適的載體把siRNA包覆，防止siRNA被降解和進入細胞。本研究利用Indolicidin(IL)以及其衍生物ILF89、PEI(M.W=25k da)-ILC、PEI(M.W=25k da)-CIL、PEI(M.W=750k da)-ILC以及PEI(M.W=750k da)-CIL作為基因載體。檢測出IL-siRNA和ILF89-siRNA粒徑大小分別為大約800-1000nm和300-600nm而在表面電位的部分IL-siRNA complex在小於N/P=20是呈現中性和負電，大於N/P=30以上則是18mV以上; ILF89其表面電位都呈現負電。以相同的配製方法對HEK293T這株細胞的基因-GAPDH(每個動物細胞都含有此基因)去做抑制實驗，IL、ILF89利用QPCR分析出來的實驗結果大約是30-40％和幾乎0％的mRNA抑制效果比預期的來的低。針對另一株細胞HT1080的基因-EGFP，以Flow cytometry分析螢光螢光蛋白質的表現，IL、ILF89當成載體攜帶siRNA其螢光幾乎沒有下降。原因是EGFP的半生期過長，長達26小時，蛋白質很穩定以致於抑制效果非常的不明顯。
以PEI(M.W=25k da)-ILC、PEI(M.W=25k da)-CIL、PEI(M.W=750k da)-ILC以及PEI(M.W=750k da)-CIL作為基因載體，分析的結果PEI接上IL的抑制效果都比PEI好; PEI(M.W=25k da)-IL的抑制效果則是比PEI(M.W=750 da)好。
IL、ILF89作為基因載體抑制效果沒有預期的高，然而PEI以共價鍵和IL相接，效果比PEI好，所以IL有發揮提高轉染的效果。

Small interfering RNA (siRNA) can specifically inhibit certain gene expression, but siRNA cannot approach to negatively-charged cell membrane and not even be functionalized into cytoplasm. To overcome the above problems, a suitable carrier to cover siRNA from degradation and to deliver it into cell is required.
In this study, we selected Indolicidin(IL), its derivative ILF89, PEI(M.W=25k da)-ILC、PEI(M.W=25k da)-CIL、PEI(M.W=750k da)-ILC以及PEI(M.W=750k da)-CIL as the gene carriers. Indolicidin may assemble into a complex with siRNA by electrostatic interaction at different amine over phosphate molar ratio (N/P ratio). First, we covered siRNA with IL and ILF89, and found that the sizes of siRNA-peptide complex are 800nm to 1000nm and 300nm to 600nm, respectively. On the other hand, the zeta potential of siRNA-IL complex at N/P ratio less than 20 is neutral and negatively charged while that of N/P ratio higher than 30 is above 18mV. In contrast to siRNA-IL complex, zeta potential of siRNA-ILF89 complex is completely negatively charged. We applied the above method to the inhibitory examination on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of HEK293T cell line. From the real-time polymerase chain reaction (QPCR) analysis, we found that the inhibitory efficiencies of siRNA-IL and siRNA-ILF89 complex are 30% to 40% of mRNA and 0％ of mRNA, respectively, which is relatively lower than the expected result.
To another gene, enhanced green fluorescent protein (EGFP), expressed from HT1080 cell line, there is little decrease in the fluorescence intensity from the green fluorescent protein expression of both siRNA-IL and siRNA-ILF89 complex, which was analyzed by QPCR. We attributed the above results to the long half-life of EGFP (26 hour) that makes the protein stable resulting in low inhibitory efficiency.
From results of PEI-IL, we found that the inhibitory efficiencies of PEI-IL are higher than PEI and the inhibitory efficiencies of PEI(M.W=25kda)-IL are higher than PEI(M.W=750kda)-IL. Thus, IL shows enhanced transfection ability.

Fire, A., et al., Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998. 391(6669): p. 806-811.
2. Rokitskaya, T.I., Indolicidin action on membrane permeability: Carrier mechanism versus pore formation. Biochimica et Biophysica Acta, 2011. 1808(1): p. 91-97.
3. Subbalakshmi, C., et al, Interaction of indolicidin, a 13-residue peptide rich in tryptophan and proline and its analogues with model membranes. Journal of biosciences, 1998. 23(1): p. 9-13.
4. C. E. Thomas, et al., Progress and problems with the use of viral vectors for gene therapy. Nature Reviews Genetics, 2003. 4(5): p. 346-358.
5. Walther, W.a.U.S., Viral Vectors for Gene Transfer-A Review of Their Use in the Treatment of Human Diseases. Drugs, 2000. 60(2): p. 249-271.
6. Yang, J.P.a.L.H., Direct gene transfer to mouse melanoma by intratumor injection of free DNA. Gene Therapy, 1996. 3(6): p. 542-548.
7. E. Neumann , et al., Gene transfer into mouse lyoma cells by electroporation in high electric fields. The EMBO Journal, 1982. 1(7): p. 841–845.
8. Fromm, M., et al., Expression of genes transferred into monocot and dicot plant cells by electroporation. Proceedings of the National Academy of Sciences of the United States of America, 1985. 82(17): p. 5824–5828.
9. Gao, X., et al., Nonviral gene delivery: What we know and what is next. Aaps Journal, 2007. 9(1): p. E92-E104.
10. Williams, R.S., et al., Introduction of Foreign Genes into Tissues of Living Mice by DNA-Coated Microprojectiles. Proceedings of the National Academy of Sciences of the United States of America, 1998. 88(7): p. 2726-2730.
11. Yang, N.S., et al., In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proceedings of the National Academy of Sciences of the United States of America, 1990. 87(24): p. 9568-9572.
12. Felgner, P.L., et al., Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proceedings of the National Academy of Sciences of the United States of America, 1987. 84(21): p. 7413-7417.
13. Farhood, H., et al., The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene transfer. Biochimica Et Biophysica Acta, 1995. 1235(2): p. 289-295.
14. Miller, A.D., The problem with cationic liposome/micelle-based non-viral vector systems for gene therapy. Current Medicinal Chemistry, 2003. 10(14): p. 1195-211.
15. Wagner, E.et al., Transferrin-polycation-DNA complexes: the effect of polycations on the structure of the complex and DNA delivery to cells. Proceedings of the National Academy of Sciences of the United States of America
1991. 88(10): p. 4255.
16. Kilk, K.et al., Evaluation of transportan 10 in PEI mediated plasmid delivery assay. Journal of Controlled Release, 2005. 103(2): p. 511-523.
17. Beyerle, A.et al., Comparative in vivo study of poly(ethylene imine)/siRNA complexes for pulmonary delivery in mice. Journal of Controlled Release, 2011. 151(1): p. 51-56.
18. Kim, S. H.et al., Comparative Evaluation of Target-Specific GFP Gene Silencing Efficiencies for Antisense ODN, Synthetic siRNA, and siRNA Plasmid Complexed with PEI-PEG-FOL Conjugate. Bioconjugate Chemistry, 2006. 17(1): p. 241-244.
19. Tseng, S. J. and Tang, S. C., Development of poly(amino ester glycol urethane)/siRNA polyplexes for gene silencing. Bioconjugate Chemistry, 2007. 18(5): p. 1383-1390.
20. Frankel, A. D. and Pabo, C. O.,, Cellular uptake of the tat protein from human immunodeficiency virus. Cell, 1988. 55(6): p. 1189-1193.
21. Joliot, A., et al., Antennapedia homeobox peptide regulates neural morphogenesis. Proceedings of the National Academy of Sciences of the United States of America, 1991. 88(5): p. 1864-1868.
22. Gupta, B., et al., Intracellular delivery of large molecules and small particles by cell-penetrating proteins and peptides. . Advanced Drug Delivery Reviews, 2005. 57(4): p. 637-651.
23. Endoh, et al., Cellular siRNA delivery using cell-penetrating peptides modified for endosomal escape. . Advanced Drug Delivery Reviews, 2009. 61(9): p. 704-709.
24. Sebbage, V., Cell-penetrating peptides and their therapeutic applications. Bioscience Horizons, 2009. 2(1).
25. Patel, L.N., et al., Cell penetrating peptides: intracellular pathways and pharmaceutical perspectives. Pharmaceutical Research, 2007. 24(11): p. 1977-1992.
26. Richard, J.P., et al., Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. . The Journal of Biological Chemistry, 2003. 278(1): p. 585-590.
27. Varkouhi, A.K., et al, Endosomal escape pathways for delivery of biologicals. Journal of Controlled Release, 2011. 151(3): p. 220-228.
28. Ladokhin, A., et al.,, Bilayer Interactions of
Indolicidin, a Small Antimicrobial Peptide Rich in Tryptophan, Proline, and
Basic Amino Acids. Biophysical Journal, 1997. 72(2, Part 1): p. 794-805.
29. Rozek, A., et al, Structure of the Bovine
Antimicrobial Peptide Indolicidin Bound to Dodecylphosphocholine and
Sodium Dodecyl Sulfate Micelles. Biochemistry, 2000. 39(51): p. 15765-15774.
30. Halevy, R., et al., Membrane binding and permeation by indolicidin analogs
studied by a biomimetic lipid/polydiacetylene vesicle assay. . Peptides, 2003. 24(11): p. 1753-1761.
31. Robinson, W., et al., Anti-HIV-1 activity of indolicidin, an antimicrobial
peptide from neutrophils. . Journal of Leukocyte Biology, 1998. 63(1): p. 94-100.
32. Schluesener, H.J., et al., Leukocytic antimicrobial peptides kill autoimmune
T cells. Journal of Neuroimmunology. Journal of Neuroimmunology, 1993. 47(2): p. 199-202.
33. Subbalakshmi, C., et al., Requirements for antibacterial and hemolytic
activities in the bovine neutrophil derived 13-residue peptide indolicidin. FEBS Letters, 1996. 395(1): p. 48-52.
34. Ahmad, I., et al., Liposomal entrapment of the neutrophil-derived peptide
indolicidin endows it with in vivo antifungal activity. Biophysica Acta (BBA) - Biomembranes, 1995. 1237(2): p. 109-114.
35. Ester , J.K., et al Application of an HIV gp41-Derived Peptide for Enhanced Intracellular Trafficking of Synthetic Gene and siRNA Delivery Vehicles. Bioconjugate Chemistry, 2008. 19: p. 920–927.
36. Brogden, K.A., Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nature Reviews Microbiology, 2005. 3(3): p. 238-250.
37. Yang, L., et al., Barrel-stave model or toroidal model? A case study on melittin pores. Biophysical Journal, 2001. 81(3): p. 1475-1485.
38. Chan, D.I., et al., Tryptophan- and arginine-rich antimicrobial peptides: structures and mechanisms of action. Biochimica et Biophysica Acta,, 2006. 1758(9): p. 1184-1202.
39. Biggin, P., et al.,, Interactions of alpha-helices with lipid bilayers: a review of simulation studies. . Biophysical Chemistry, 1999. 76(3): p. 161-183.
40. Miteva, M., et al., Molecular electroporation: a unifying concept for the description of membrane pore formation by antibacterial peptides, exemplified with NK-lysin. FEBS Letters, 1999. 462(1-2): p. 155-158.
41. Tieleman, D.P., The molecular basis of electroporation. Biochemistry, 2004. 5: p. 10.
42. Pokorny, A., et al., Permeabilization of raft-containing lipid vesicles by delta-lysin: a mechanism for cell sensitivity to cytotoxic peptides. Biochemistry, 2005. 44(27): p. 9538-9544.
43. Nakase, I., et al., Interaction of arginine-rich peptides with membrane-associated proteoglycans is crucial for induction of actin organization and macropinocytosis. . Biochemistry, 2007. 46(2): p. 492-501.
44. Adler, A., et al.,, Emerging links between surface nanotechnology and endocytosis: impact on nonviral gene delivery. . Nano Today, 2010. 5(6): p. 553-569.
45. Zamore, P.D., et al., RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. . Cell, 2000. 101(1): p. 25-33.
46. Elbashir, S.M., et al., RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes & Development, 2001. 15(2): p. 188-200.
47. Chatterjee-Kishore, M., et al., Exploring the sounds of silence: RNAi-mediated gene silencing for target identification and validation. . Drug Discovery Today, 2005. 10(22): p. 1559-1565.
48. Hannon, G.J., RNA interference. Nature 2002. 418(6894): p. 244-251.
49. Wang, J., et al., Delivery of siRNA therapeutics: barriers and carriers. AAPS Journals, 2010. 12(4): p. 492-503.
50. Timmons, L., et al., Specific interference by ingested dsRNA. Nature 1998. 395(6705): p. 854.
51. Williams, B.R., Role of the double-stranded RNA-activated protein kinase (PKR) in cell regulation. Biochemical Society Transactions. Biochemical Society Transactions, 1997. 25(2): p. 509-513.
52. Elbashir, S., et al., Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods, 2002. 26(2): p. 199-213.
53. Reynolds, A., et al., Rational siRNA design for RNA interference. . Nature Biotechnol, 2004. 22(3): p. 326-330.
54. Venkatesan, N., et al., Peptide conjugates of oligonucleotides: synthesis and applications. . Chemical Reviews, 2006. 106(9): p. 3712-3761.
55. Stetsenko, D., et al., Efficient conjugation of peptides to oligonucleotides by "native ligation". The Journal of Organic Chemistry, 2000. 65(16): p. 4900-4908.
56. Ede, N., et al., Routine preparation of thiol oligonucleotides: application to the synthesis of oligonucleotide-peptide hybrids. Bioconjugate Chemistry, 1994. 5(4): p. 373-378.
57. Zatsepin, T.S., et al., Synthesis of peptide-oligonucleotide conjugates with single and multiple peptides attached to 2'-aldehydes through thiazolidine, oxime, and hydrazine linkages. . Bioconjugate Chemistry, 2002. 13(4): p. 822-830.
58. Lee, J., et al., Self-assembled RNA interference microsponges
for efficient siRNA delivery. Nature materials, 2012. 11: p. 316–322.
59. Smith, T., et al., Attenuation of green fluorescent protein half-life in mammalian cells. Protein Engineering, 1999. 12(12): p. 1035-1040.
60. Swami, A., et al., Imidazolyl-PEI modified nanoparticles for enhanced gene delivery. International Journal of Pharmaceutics, 2007. 335: p. 180–192.