Studies On The Design,Synthesis And Transfection Capability Of Amphiphilic Self-Assembling Peptide Derivative Based On Aromatic Conjugation

As a novel treatment, gene therapy has good prospects for clinical application,especially dealing with difficult diseases that conventional treatment methods showlow efficiency (such as cancer, immune deficiency diseases and certain geneticdiseases). To reach the goal of gene therapy, therapeutic nucleic acid (DNA or RNA)should be transferred into the pathological cells and tissues accurately, stably andefficiently prior to its correct expression inside. Considering the complicatedphysiological and biochemical conditions in human body, therapeutic nucleic acidexposed to the inner environment is likely to encounter different degrees ofdegradation, resulting in therapeutic effects unreached. Thus, seeking effective andconvenient means of gene delivery has become one of the prerequisite tasks for genetherapy’s clinical use.Gene transfer media are divided into three categories: physical means, viralvectors and non-viral vectors. Among the three means, non-viral vectors draw muchattention due to their advantages of no capacity constraints, structural diversity andcontrollability, low toxicity and mutagenic effects. Non-viral vectors require thefollowing features: stability of the therapeutic gene coating, efficiency to enter thetarget cells, capability to release the nucleic acid in the cell rapidly, disintegrating,can cause significant physiological or genetic toxicity. Currently most commercialtransfection reagents are generated from a cationic liposome or cationic polymer.Since their way to enter the cells is via endocytosis or cationic escape effect, a certaindegree of cell membrane’s damage has been observed in current research work.Besides, taking account of general use in research, the design lack in specific targetcells directivity.This means that if used in the clinical application, such reagents will largely rely on treatment dose and frequency of administration, making the treatmentprocess probably associated with serious side effects. Therefore, this paper designed ashort peptide derivative that could self-assemble into nanostructures as envisagedgene carrier with high loading efficiency of nucleic acid, lower cytotoxicity thanconventional reagents, and potential capability of targeted gene delivery.In this paper, the peptide FGPep derivative’s sequence is based on Fmoc-Phe-Phe,one of the typical motifs in π-π stacking mediated self-assembly. We chose a sequence(YSSPTQR) obtained from phage display screening against HER2in previousresearch as the C terminal sequence. Between them we used several alanine residuesas a linker and exposed the hydrophilic end for HER2targeting capability. Finally thesequence was determined as Fmoc-FFAAYSSPTQR-NH2. We used classical Fmocsolid-phase peptide synthesis method in gaining the crude peptide. HPLC purificationgave a yield of desired compound above80%. Under sonication FGPep can bedispersed in aqueous phase and self-assemble into ordered nanowires. We assessed thenucleic acid loading capability of FGPep using agarose gel electrophoresis. Undercertain concentration and mixing ratio FGPep gel coated plasmid can be completelytrapped. SK-OV-3was selected as an object in in-vitro cytotoxicity and transfectionexperiments. When FGPep dispersed in the medium reached a concentration of500μM, there was still no significant cell apoptosis, thus we believed FGPep showedlow cytotoxicity which met the requirements as a transfection vector. However, theactual effect of transfection was not ideal. Using equal amount of pEGFP-N1in thesame conditions, GFP expression in the group of FGPep was much lower than thepositive control group using Lipofectamine2000. We speculated that the size ofFGPep-plasmid complex might be too large, not suitable for inducing endocytosis,which led to low level of cell nucleic acid intake.In summary, this paper designed a peptide derivative based on novel ideas. Itcould self-assembly and coat nucleic acids; besides it showed low cytotoxicity. Yetthere’s still some gap before its practical use as a transfection material, it needs furtherresearch and improvement.