Cell penetrating peptide (CPP) has been highlighted for promising potential in cytosolic delivery, enabling applications as carriers to penetrate across biological barriers such as cell membrane, mucosa, stratum corneum, and blood-brain barrier [1], [2], [3], [4], [5], [6]. These peptides were extensively employed as delivery vehicles for intracellular protein transport [7], [8], [9], [10], [11], via genetic engineering [1], [12], or chemically conjugation [13], [14], [15] onto cargo proteins. The CPP-tagged proteins have shown increased cellular uptake, but their delivery efficacy is usually unsufficient due to endosomal entrapment. In addition, the conjugated peptides may affect the bioactivity of cargo proteins due to possible steric effect or strengthened intramolecular interactions [13], [16], [17].
Besides the covalent strategy, CPPs could directly complex with cargo proteins to form nanoparticles via ionic and hydrophobic interactions for cytosolic delivery [18]. However, the formed nanoparticles via non-covalent interactions are prone to disassembly due to abundant ions and biomacromolecules in physiological conditions [19], [20], [21], [22], [23], [24]. To strengthen the binding interactions and nanoparticle stability, polymerized CPPs with linear or branched topological structures were designed to achieve a multivalent effect [25], [26], [27], [28]. Alternatively, hydrophobic motifs such as alkyl chains or cholesterol were conjugated to CPPs to prepare amphipathic cationic peptides (ACPs), which could effectively improve nanoparticle stability via supramolecular assembly [10], [22], [29], [30]. To improve the endosomal escape capability, pH-responsive CPPs enabling conformational changes when entrapped in acidic vesicles, such as the influenza HA fusion domain, were adopted to design the delivery carrier [31], [32]. Furthermore, acidic amino acids such as glutamic acid or aspartic acid residues were incorporated into CPPs to construct the lipid-sensitive endosomolytic peptide carriers [33], [34].
Our previous studies have demonstrated that fluorination on carriers is an effective strategy to promote the cytosolic delivery of genes and proteins [35], [36], [37]. Conjugation of a fluorous tag to cargo peptides also achieve efficient and robust cytosolic peptide delivery via enhanced supramolecular assembly, cellular uptake and endosomal escape [38], [39], [40]. Here, we propose to design a type of fluorinated CPPs for cytosolic protein delivery (Fig. 1). A library of amphiphilic cationic peptides (ACPs) was constructed by conjugating a fluorous tag bearing thirteen fluorine atoms at the C-terminus to obtain fluorinated ACPs (FACPs). The ACPs in the library initiately designed with arginine- and tryptophan-rich peptides for efficient cytosolic protein delivery. The cationic amino acid arginine can interact with cargo protein via salt-bridge interactions [41], [42], [43], [44], and promote intracellular delivery via the known “arginine magic” [45], while the hydrophobic tryptophan can strongly interact with cell membranes via multiple interactions [19], [46]. The selection of the hexa-arginine motif is rationalized by the established principle that 6–8 arginine residues are optimal for cellular internalization, as demonstrated by Futaki and our group [41], [47]. Herein, we aim to develop a novel type of hexa-peptide carriers for efficient and robust intracellular protein delivery. The structure-function relationships of the FACPs in cytosolic protein delivery are intensively investigated. The lead material FACP2 efficiently deliver ovalbumin (OVA) into dendritic cells (DCs), stimulate DCs maturation and promote antigen cross-presentation both in vitro and in vivo (Fig. 1). The prepared FACP2/OVA nanovaccine significantly inhibited tumor growth, stimulated DC maturation, facilitated antigen cross-presentation, and activated antigen-specific T cells.