Inclusion of TAT and NLS sequences in lipopeptide molecules generates homogenous nanoparticles for gene delivery applications

• Published in: International journal of pharmaceutics 2024-09, Vol.662, p.124492, Article 124492
• Main Authors: Tarwadi, Pambudi, Sabar, Sriherwanto, Catur, Sasangka, Ayu N., Bowolaksono, Anom, Wijayadikusumah, Acep R., Zeng, Weiguang, Rachmawati, Heni, Kartasasmita, Rahmana E., Kazi, Mohsin
• Format: Article
• Language: English
• Subjects: Cytotoxicity; Homogenous nanoparticle; Lipopeptide; NLS; TAT; Transfection
• ISSN: 0378-5173; 1873-3476; 1873-3476
• DOI: 10.1016/j.ijpharm.2024.124492

[Display omitted] The objective of this study is to develop a versatile gene carrier based on lipopeptides capable of delivering genetic material into target cells with minimal cytotoxicity. Two lipopeptide molecules, palmitoyl-CKKHH and palmitoyl-CKKHH-YGRKKRRQRRR-PKKKRKV, were synthesized using solid phase peptide synthesis and evaluated as transfection agents. Physicochemical characterization of the lipopeptides included a DNA shift mobility assay, particle size measurement, and transmission electron microscopy (TEM) analysis. Cytotoxicity was assessed in CHO-K1 and HepG2 cells using the MTT assay, while transfection efficiency was determined by evaluating the expression of the green fluorescent protein-encoding gene. Our findings demonstrate that the lipopeptides can bind, condense, and shield DNA from DNase degradation. The inclusion of the YGRKKRRQRRR sequence, a transcription trans activator, and the PKKKRKV sequence, a nuclear localization signal, imparts desirable properties. Lipopeptide-based TAT-NLS/DNA nanoparticles exhibited stability for up to 20 days when stored at 6–8 °C, displaying uniformity with a compact size of approximately 120 nm. Furthermore, the lipopeptides exhibited lower cytotoxicity compared to the poly-L-lysine. Transfection experiments revealed that protein expression mediated by the lipopeptide occurred at a charge ratio ranging from 4.0 to 8.0. These results indicate that the lipopeptide, composed of a palmitoyl alkyl chain and TAT and NLS sequences, can efficiently condense and protect DNA, form stable and uniform nanoparticles, and exhibit promising characteristics as a potential gene carrier with minimal cytotoxicity.