In gene delivery systems, nonviral systems have gained wide attention because of the activation of oncogenes and excessive immunogenicity of viral vectors [1]. To absorb genes first and then penetrate the cell membrane, cationic polymers such as polyethyleneimine (PEI) [2,3,4,5] and poly-(l-lysine) (PLL) [6] have been proposed. However, significant toxicity can be observed in cells or animals after the administration of PEI. PEI was reported to activate Fasl-mediated antigen-induced cell death in the spleen [7]. Systemic administration of PEI caused liver necrosis and death, as well as the increase of small aggregates of both platelets and CD11-b-positive cells in the lung [8]. PLL also shows its very toxicity, which increases with the increment of its molecular weight [9]. Polyamidoamine (PAMAM) dendrimer, whose synthesis is complicated and costly [10], has potential cytotoxicity [11]. Thus, cationic nanocarriers with better biocompatibility such as peptide-based vehicles are wiser choices. Peptide-based self-assembly has been proposed to deliver cargos such as cyclic peptides [12,13], linear short peptides [14], and amphiphilic peptides [15]. Among them, di-phenylalanine (FF) is extracted from the Alzheimer’s β-amyloid polypeptides and can self-assemble in aqueous conditions. It is reported to have easy production, functional versatility, biodegradability, biocompatibility, and non-immunogenicity [16,17,18]. FF has been decorated with other subunits from amino acids such as Cys [19], to polymers such as PCL [20]. It can boost the self-assembly of those systems.

More than 1700 CPPs have been reported, and the most common are TAT and PLL [21,22]. However, through only electronic interactions, off-targeting effects are downsides in types of cationic CPPs [23]. Thus, some targeted CPPs are proposed, such as octarginine (R8) targeting neuropilin-1 receptors [24]. RTMIWY(d-P) GAWYKRI is a continuous sequence of 14 amino acids designed by our lab to blockade the PD-1/PD-L1 process [25], which can be more facile to synthesize through solid-phase peptide synthesis technology. Meanwhile, its net charge is above 3 from pH 7.4 to 6.0, which means it is likely to absorb anionic nucleic acids and penetrate the cell membrane.

Cationic peptides such as PLL and TAT were first used to absorb anionic nucleic acids, but their low transduction efficiency, low loading capacity, and high cell toxicity restrained their applications considerably. Recent research focused mainly on using them as one of the components in the delivery systems. For example, PLL for RNA absorption [26] and TAT for cell-penetrating [27] are the most common applications. Qiu et al.has reported cell-penetrating peptide (CPP3-CLP) for targeting A549 cells [26]. Zhang et al., conjugated TAT to chitosan [28]. However, these polysomes with unclear metabolism pathways still need to be explored. Therefore, tapping the potential of peptides themselves has appealed to great attention.

Peptides are pure amino acids without any additions such as chitosan, PEG, or DSPG, but can still target PD-L1. We hope to construct a system whose components are all from endogenous elements, which can be metabolized more easily. Though many pure peptide-based nano-systems have been proposed, very few of them can both absorb nuclei acids through electronic interactions and target a specific protein simultaneously. For instance, several cyclic peptide sequences can make the self-assembly by alternating α- and β-amino acids, β-amino acids, and δ-amino acids by molecular stacking and H-bonds between backbones [29]. However, they are quite rigid in structure, which makes it difficult to add ligands. Additionally, some artificial peptide-based viruses are cationic for both penetrating cell membranes and absorbing RNA [30]. . Thus, we proposed a flexible system by connecting FFAA to a cationic targeting sequence. In this way, a pure peptide-based functional vehicle can be gained readily.

We suggest the capacity of FFAA to be a hydrophobic force even in an 18 continuous sequence. It preserves targeting ability after assembly. This formula of the combination of hydrophobic force (FFAA) and targeting sequence (with cationic amino acids) can be further explored since there are many peptide sequences screened by affinity. Furthermore, it is very facile to synthesize or can even be pre-designed in peptide display technology such as T4 bacteriophage, which is hard to realize when using non-amino acids components or cyclic peptides. Using this approach, many types of nanoparticles can be gained simultaneously, and the objects screened will change from small molecular peptides to nanostructures.[31]

Reference

1. Ganju, A.; Khan, S.; Hafeez, B.B.; Behrman, S.W.; , M.M.; Chauhan, S.C.; Jaggi, M.RNA nanotherapeutics for cancer. Drug Discov. Today 2016, 22, 424–432, doi:10.1016/j.drudis.2016.10.014.
2. Jia, F.; Li, Y.; Deng, X.; Wang, X.; Cui, X.; Lu, J.; Pan, Z.; Wu, Y. Self-assembled fluorescent hybrid nanoparticles-mediated collaborative lncRNA CCAT1 silencing and curcumin delivery for synchronous colorectal cancer theranostics. Nanobiotechnology 2021, 19, 238, doi:10.1186/s12951-021-00981-7.
3. Li, Y.; Chen, G.; He, Y.; Yi, C.; Zhang, X.; Zeng, B.; Huang, Z.; Deng, F.; Yu, D. Selenomethionine-Modified Polyethylenimine-Based Nanoparticles Loaded with miR-132-3p Inhibitor-Biofunctionalized Titanium Implants for Improved Osteointegration. ACS Biomater. Sci. Eng. 2021, 7, 4933–4945, doi:10.1021/acsbiomaterials.1c00880.
4. Zhang, H.; Cao, Y.; Xu, D.; Goh, N.S.; Demirer, G.S.; Cestellos-Blanco, S.; Chen, Y.; Landry, M.P.; Yang, P. Gold-Nanocluster-Mediated Delivery of siRNA to Intact Plant Cells for Efficient Gene Knockdown. Nano Lett. 2021, 21, 5859–5866, doi:10.1021/acs.nanolett.1c01792.
5. Delyanee, M.; Akbari, S.; Solouk, A. Amine-terminated dendritic polymers as promising nanoplatform for diagnostic and therapeutic agents’ modification: A review. J. Med. Chem. 2021, 221, 113572, doi:10.1016/j.ejmech.2021.113572.
6. Shi, M.; Wang, Y.; Zhao, X.; Zhang, J.; Hu, H.; Qiao, M.; Zhao, X.; Chen, D. Stimuli-Responsive and Highly Penetrable Nanoparticles as a Multifunctional Nanoplatform for Boosting Nonsmall Cell Lung Cancer siRNA Therapy. ACS Biomater. Sci. Eng. 2021, 7, 3141–3155, doi:10.1021/acsbiomaterials.1c00582.
7. Regnstrom, K.; Ragnarsson, E.G.; Koping-Hoggard, M.; Torstensson, E.; Nyblom, H.; Artursson, P. PEI—A potent, but not harmless, mucosal immuno-stimulator of mixed T-helper cell response and FasL-mediated cell death in mice. Gene Ther. 2003, 10, 1575–1583, doi:10.1038/sj.gt.3302054.
8. Chollet, P.; Favrot, M.C.; Hurbin, A.; Coll, J.L. Side-effects of a systemic injection of linear polyethylenimine-DNA complexes. Gene Med. 2002, 4, 84–91, doi:10.1002/jgm.237.
9. Ferruti, P.; Knobloch, S.; Ranucci, E.; Duncan, R.; Gianasi, E. A novel modification of poly(L-lysine) leading to a soluble cationic polymer with reduced toxicity and with potential as a transfection agent. Chem. Phys. 1998, 199, 2565–2575.
10. Tomalia, D.A.; Fréchet, J.M.J. Discovery of dendrimers and dendritic polymers: A brief historical perspective. Polym. Sci. Part A Polym. Chem. 2002, 40, 2719–2728, doi:10.1002/pola.10301.
11. Sun, Y.; Jiao, Y.; Wang, Y.; Lu, D.; Yang, W. The strategy to improve gene transfection efficiency and biocompatibility of hyperbranched PAMAM with the cooperation of PEGylated hyperbranched PAMAM. J. Pharm. 2014, 465, 112–119, doi:10.1016/j.ijpharm.2014.02.018.
12. Yang, M.; Yuan, C.; Shen, G.; Chang, R.; Xing, R.; Yan, X. Cyclic dipeptide nanoribbons formed by dye-mediated hydrophobic self-assembly for cancer chemotherapy. Colloid Interface Sci. 2019, 557, 458–464, doi:10.1016/j.jcis.2019.09.049.
13. Yang, J.; Song, J.I.; Song, Q.; Rho, J.Y.; Mansfield, E.D.H.; Hall, S.C.L.; Sambrook, M.; Huang, F.; Perrier, S. Hierarchical Self-Assembled Photo-Responsive Tubisomes from a Cyclic Peptide-Bridged Amphiphilic Block Copolymer. Chem. Int. Ed. 2020, 59, 8860–8863, doi:10.1002/anie.201916111.
14. Schnaider, L.; Brahmachari, S.; Schmidt, N.W.; Mensa, B.; Shaham-Niv, S.; Bychenko, D.; Adler-Abramovich, L.; Shimon, L.J.W.; Kolusheva, S.; DeGrado, W.F.; et al. Self-assembling dipeptide antibacterial nanostructures with membrane disrupting activity. Commun. 2017, 8, 1365, doi:10.1038/s41467-017-01447-x.
15. Hamley, I.W. Self-assembly of amphiphilic peptides. Soft Matter. 2011, 7, –4138, doi:10.1039/c0sm01218a.
16. Reches, M.; Gazit, E. Casting Metal Nanowires within Discrete Self-Assembled Peptide Nanotubes. Science 2003, 300, 625–627, doi:10.1126/science.1082387.
17. Li, Q.; Jia, Y.; Dai, L.; Yang, Y.; Li, J. Controlled Rod Nanostructured Assembly of Diphenylalanine and Their Optical Waveguide Properties. ACS Nano 2015, 9, 2689–2695.
18. Ariga, K.; Li, J.; Fei, J.; Ji, Q.; Hill, J.P. Nanoarchitectonics for Dynamic Functional Materials from Atomic-/Molecular-Level Manipulation to Macroscopic Action. Mater. 2016, 28, 1251–1286, doi:10.1002/adma.201502545.
19. Reches, M.; Gazit, E. Formation of Closed-Cage Nanostructures by Self-Assembly of Aromatic Dipeptides. Nano Lett. 2004, 4, 581–585.
20. Liberato, M.S.; Kogikoski, S.; da Silva, E.R.; de Araujo, D.R.; Guha, S.; Alves, W.A. Polycaprolactone fibers with self-assembled peptide micro/nanotubes: A practical route towards enhanced mechanical strength and drug delivery applications. Mater. Chem. B 2016, 4, 1405–1413, doi:10.1039/c5tb02240a.
21. Agrawal, P.; Bhalla, S.; Usmani, S.S.; Singh, S.; Chaudhary, K.; Raghava, G.P.; Gautam, A. CPPsite 2.0: A repository of experimentally validated cell-penetrating peptides. Nucleic Acids Res. 2016, 44, D1098–D1103, doi:10.1093/nar/gkv1266.
22. Pujals, S.; Fernandez-Carneado, J.; Lopez-Iglesias, C.; Kogan, M.J.; Giralt, E. Mechanistic aspects of CPP-mediated intracellular drug delivery: Relevance of CPP self-assembly. Biophys. Acta Biomembr. 2006, 1758, 264–279, doi:10.1016/j.bbamem.2006.01.006.
23. Taylor, R.E.; Zahid, M. Cell Penetrating Peptides, Novel Vectors for Gene Therapy. Pharmaceutics 2020, 12, , doi:10.3390/pharmaceutics12030225.
24. Liu, Y.; Mei, L.; Xu, C.; Yu, Q.; Shi, K.; Zhang, L.; Wang, Y.; Zhang, Q.; Gao, H.; Zhang, Z.; et al. Dual Receptor Recognizing Cell Penetrating Peptide for Selective Targeting, Efficient Intratumoral Diffusion and Synthesized Anti-Glioma Therapy. Theranostics 2016, 6, 177–191, doi:10.7150/thno.13532.
25. Wang, K.; Song, Y.; Su, Y.; Liang, Y.; Wang, L. Effect of the hairpin structure of peptide inhibitors on the blockade of PD-1/PD-L1 axis. Biophys. Res. Commun. 2020, 527, 453–457, doi:10.1016/j.bbrc.2020.04.018.
26. Qiu, M.; Ouyang, J.; Wei, Y.; Zhang, J.; Lan, Q.; Deng, C.; Zhong, Z. Selective Cell Penetrating Peptide-Functionalized Envelope-Type Chimeric Lipopepsomes Boost Systemic RNAi Therapy for Lung Tumors. Healthc. Mater. 2019, 8, e1900500.
27. Pan, L.; He, Q.; Liu, J.; Chen, Y.; Ma, M.; Zhang, L.; Shi, J. Nuclear-targeted drug delivery of TAT peptide-conjugated monodisperse mesoporous silica nanoparticles. Am. Chem. Soc. 2012, 134, 5722–5725, doi:10.1021/ja211035w.
28. Zhang, S.; Liu, Y.; Gan, Y.; Qiu, N.; Gu, Y.; Zhu, H. Conjugates of TAT and folate with DOX-loaded chitosan micelles offer effective intracellular delivery ability. Dev. Technol. 2019, 24, 253–261, doi:10.1080/10837450.2018.1469147.
29. Chapman, R.; Danial, M.; Koh, M.L.; Jolliffe, K.A.; Perrier, S. Design and properties of functional nanotubes from the self-assembly of cyclic peptide templates. Soc. Rev. 2012, 41, 6023–6041, doi:10.1039/c2cs35172b.
30. Ni, R.; Chau, Y. Nanoassembly of Oligopeptides and DNA Mimics the Sequential Disassembly of a Spherical Virus. Chem. Int. Ed. 2020, 59, 3578–3584, doi:10.1002/anie.201913611.
31. Guo, F.; Ke, J.; Fu, Z.; Han, W.; Wang, L. Cell Penetrating Peptide-Based Self-Assembly for PD-L1 Targeted Tumor Regression. International Journal of Molecular Sciences 2021, 22, doi:10.3390/ijms222413314.