Novel branched amphiphilic peptides for nucleic acids delivery

Branched peptides are a new type of peptide-based nucleic acid delivery vectors. It has been reported that branched and dendrimeric cationic peptides have better transfection efficiencies than linear peptides due to their superior capacity for DNA condensation (Ageitos et al., 2015, Luo et al., 2011). Ageitos et al. synthesized linear cationic peptides and branched cationic peptide conjugates with tris(2-aminoethyl)amine in a chemo-enzymatic method, and showed that the branched peptides have improved DNA complex formation (Ageitos et al., 2015). Leng et al. synthesized both linear and branched peptides, H2K (linear) and H2K4b-20 (branched), and found that the polyplexes formed by the branched H2K4b-20 had better delivery efficiency than the linear H2K peptide in vitro (Leng et al., 2014). However, the linear H2K peptide carrier was more effective than the branched H2K4b-20 peptide in transfecting the luciferase-expression plasmids to tumors in vivo. Further optimization of the branched peptide structures indicated that the H2K4b-14 was the most effective plasmid delivery vectors to tumors (Xu et al., 2022). These peptides with high charge density allow for effective condensation of negatively charged nucleic acids and can facilitate the polyplexes interact with cell membranes and escape from the endosomes. Avila et al. used two branched amphiphilic peptides (bis(FLIVIGSII)-K-KKKK and bis(FLIVI)-K-KKKK) with a hydrophilic oligo lysine segment and two identical hydrophobic tails, which mimic diacyl glycerol phospholipids in architecture, to deliver pDNA and showed a superior transfection efficiency than the commercial transfection agent Lipofectin (Avila et al., 2015). These studies showed great promise for the application of branched peptides as nucleic acid delivery vectors. However, there are still lack of documents about the branched peptides as nucleic acids delivery vectors, and the structure–activity relationship of the branched peptides needs further investigation.

Nucleic acid delivery technology has attracted increasing attention for its importance in gene therapy, gene editing and vaccination (El-Aneed, 2004, Olefsky, 2000). As naked nucleic acids could not directly enter cells because of the negative charges, appropriate vectors are essential to facilitate their delivery (Wolff and Budker, 2005). Viral vectors have been proven to be efficient for both gene delivery and expression, especially for in vitro applications like in stem cells (Brown et al., 2017, Schwarzer et al., 2021). Although many viral vectors have been successfully applied in clinical trials (Bartel et al., 2012), their broad application is restricted by concerns on safety risks like undesirable immunogenicity and inflammatory reactions, as well as the associated high cost (Lehrman, 1999, Sun et al., 2003). Thus, many recent researches have focused on the development of novel non-viral vectors with high nucleic acid delivery efficiencies and low toxicity (Remes and Williams, 1992).

Various types of materials have been evaluated as non-viral vectors for efficient and safe nucleic acid delivery, including liposomes (dos Santos Rodrigues et al., 2019, Rasoulianboroujeni et al., 2017, Zhao et al., 2018), polymers (Chen et al., 2020, Fan et al., 2017, Olden et al., 2018, Verbraeken and Hoogenboom, 2017) and their combinations, which form lipoplexes/polyplexes with nucleic acids through electrostatic interaction to facilitate nucleic acid delivery. Lipoplexes are self-assembling delivery nano-systems composed of cationic branched lipids and nucleic acids. Although lipoplexes are highly efficient in nucleic acid delivery, there are also problems like large particle sizes, low biocompatibility and stability, and it needs to be functionalized with other targeting moieties to reach target cells (Akabori and Nagle, 2015, Barratt, 2003, Daraee et al., 2016, Kyriakides et al., 2021, Li et al., 2015, Martin et al., 2005, Nakhaei et al., 2021, Sercombe et al., 2015). As for polyplexes, which are formed by polymers and nucleic acids, can generate diverse nano-structures. The most common polyplexes for nucleic acid delivery are polyethyleinimine (PEI), poly-L-lysine (PLL), and poly(lactide-co-glycolide) (PLGA) (Gomes dos Reis et al., 2020). These polymers are stable and easy to be functionalized to achieve multiple functions, but also raise safety concerns like accumulation in the body (Lee et al., 2015). Additionally, the endosomal entrapment of polyplexes after internalization often result in the insufficient delivery of nucleic acids (Liu et al., 2013). Some researchers combined the advantages of liposomes and polymer to construct lipopolyplexes, which are ternary nanocomplexes composed of cationic liposomes, polymers, and nucleic acids, and they can achieve promising transfection efficiencies and safety (Rezaee et al., 2016).

Peptides, as a kind of multi-functional molecule, can fulfill the requirements for each step of nucleic acid delivery, such as condensation of nucleic acids, cell targeting, cell internalization, endosomal escape, and so on. Therefore, peptides not only can be used as functional moieties of lipoplexes/polyplexes, but also can carry out the nucleic acid delivery tasks by themselves (Geng et al., 2022). Peptiplexes (peptide-nucleic acid complexes) as potential gene delivery systems have attracted some attention from researchers, because they would have several advantages over the other systems, such as better stability than lipids, ease to synthesize and modify, and ability to target specific receptors (Åmand et al., 2012, Avila et al., 2015). Such molecules made of amino acids have diverse structures like polypeptides, dendrimers, linear and branched peptides, and all can serve as nucleic acid delivery vectors (Avila et al., 2015, Wang et al., 2016, Xu et al., 2022). Among these types of peptide molecules, cell-penetrating peptides (CPPs) have been widely studied for gene delivery (Lehto et al., 2016, Tai and Gao, 2017). With concentrated positive charges, CPPs can cross the cell membranes and carry various “cargoes”, including nucleic acids, into cells. CPPs can directly interact with nucleic acids to form complexes, or conjugated with polymers, fatty acids, etc. to facilitate other types of vectors for nucleic acid delivery (Chen et al., 2017, Geng et al., 2022).