Cell-penetrating peptides as facilitators of cargo-specific nanocarrier-based drug delivery.

Combination therapies of a small molecule drug combined with immunomodulatory therapy, nucleic acid cargo, and/or a secondary small-molecule drug are becoming increasingly favorable as treatment regimens for highly recalcitrant diseases, such as cancer. Encapsulating combination therapies into NCs can improve treatment efficacy by increasing accumulation and uptake. 74 One such method is the dual loading of both a chemotherapy drug and small-molecule protein inhibitors into CPP–NCs to block proteins associated with cancer cell survival and chemoresistance. Zhao et al. co-delivered doxorubicin with quercetin, a monocarboxylate inhibitor that limits lactate production to prevent crosstalk between tumor cells and their microenvironment. Conjugation of legumain-responsive KC26 (ke5Ne4GPTN2R9C, k: d -lysine; e: d -glutamate) peptides to the co-loaded liposome allowed selective internalization into 4T1 breast cancer cells overexpressing legumain and subsequent inhibition of lactate metabolism and angiogenesis. 75 As lactate promotes breast cancer progression, regulation of lactate metabolism caused significant cell death compared to free drug. 75 Liu et al. co-delivered doxorubicin with tariquidar, a P-glycoprotein inhibitor, using liposomes decorated with a novel peptide comprising octa-arginine incorporated into the C-terminus of an EGFR-specific targeting peptide. Their system significantly inhibited tumor growth of drug-resistant triple-negative breast cancer cells in vitro and in vivo compared to free drug, liposomes with DOX only, and liposomes without the CPP. This was a result of improved EGFR targeting and cell penetration and inhibited efflux. 76 In both cases, incorporation of both drugs into a CPP-conjugated NC improved the response to the chemotherapeutic by increasing tumor sensitivity to doxorubicin. Another promising approach is co-loading a chemotherapeutic and siRNA or microRNA (miRNA) to knockdown the expression of proteins critical to tumor growth and metastasis. While RNA interference (RNAi) is a potent anticancer therapy, the efficacy of free siRNA and miRNA is limited by their sensitivity to nuclease degradation in serum, poor cell uptake, and inefficient endosomal escape. 41 Thus, encapsulation strategies that protect the nucleic acids from nucleases and improve cellular delivery—such as CPP-conjugated NCs with a cell targeting and intracellular release mechanism—are crucial. Zhao T. et al. synthesized a dual-pH responsive cell-penetrating peptide with R8, a polyanionic shielding domain that exerts charge shielding on the polyarginine at physiological pH, with a pH-sensitive imine linkage between them. The CPP domain also facilitates the endosomal escape of siRNA complexed with stearylated octahistidine, which dissociates in the cytosol. This multistage mechanism effectively delivers siRNA against polo-like kinase-1 (siPLK-1), a mitotic regulator overexpressed in cancer cells, and docetaxel to tumors to downregulate expression of PLK-1 and inhibit tumor growth. This multistage delivery was observed to have the greatest tumor growth reduction and lowest toxicity when compared to siPLK-1-loaded liposomes, DTX-loaded liposomes, and the two cargos delivered in separate liposomes, highlighting the significance of combined therapies enhanced with CPP–NC platforms. 77 Huang et al. similarly combined small-molecule with nucleic acid cargo, developing PLGA-PEG NCs encapsulating etoposide (inactivates DNA topoisomerase II) and siRNA against PIK3CA (siPIK3CA, which inhibits PIK3CA to prevent small-cell lung carcinoma proliferation and tumor growth) and conjugated the CPP TAT externally. Co-loading in this carrier system demonstrated synergy between etoposide and siPIK3CA, as the IC 50 (half-maximal inhibitory concentration) of carriers loaded with both components was significantly lower than that of single-loaded carriers, indicating enhanced potency. The co-loaded system had significant anti-tumor effects. 78 In summary, multi-drug delivery has significant promise for improved outcomes of recalcitrant diseases by attacking the disease on multiple fronts. CPP–NC delivery of multi-drug therapies can enhance this efficacy further, taking therapeutics directly to their active site, thereby enhancing potency.

Small molecule drugs, natural or synthetic organic compounds that are <500 Da, 14 in particular face an array of challenges for systemic drug delivery. Free drugs tend to be quickly cleared and accumulate in off-target organs, yielding significant side effects, and many are only taken up by either simple diffusion or require transporters, which reduces intracellular bioavailability. 15,16 NC encapsulation of small molecules has been found to address some of these issues, including increasing circulation half-life, drug solubility (for hydrophobic molecules), and bioavailability, all while reducing off-target accumulation by providing a platform that can be modified for localized targeting. 15 However, NC drug delivery comes with its own set of unique challenges, including the blood endothelial barrier, which limits NC extravasation into the interstitial tissue, the charged extracellular matrix (ECM) that can trap oppositely charged NCs, and plasma membranes that can only be crossed via efficient interactions with membrane proteins or the membrane itself. 15 Conjugating CPPs to NCs can address these issues, improving cell penetration and uptake. Though conventional linear CPPs lack specificity and can be degraded by proteases or trapped within lysosomes upon endosomal uptake into cells, 17 modification of their sequences and structures to circumvent these issues and aid in cargo loading is ongoing. 18 The term CPP is broad and encompasses a variety of sequences with distinct structures that have been shown to enhance cell penetration. Each sequence can be categorized in terms of its amphiphilicity and derivation (natural vs. synthetic). CPPs are commonly cationic peptides that contain mostly positively charged amino acids, such as arginine and lysine. Amphiphilic CPPs contain both hydrophilic and hydrophobic residues and often adopt secondary α-helical structures. The most common CPPs described in literature that are used to enhance the cellular uptake of conjugated NCs are TAT (YGRKKRRQRRR), a cationic peptide derived from the HIV-1 virus; penetratin (RQIKIWFQNRRMKWKKGG), a secondary amphipathic peptide derived from the Antennapedia homeodomain of Drosophila; octa-arginine, a synthetic, highly cationic oligopeptide; and transportan (GWTLNS/AGYLLGKINLKALAALAKKIL), a chimeric amphipathic peptide derived from the neuropeptide galanin and the toxin mastoparan. 10,19,20 CPP structure and amino acid sequence dictate their ability to deliver conjugated drugs/NCs. A key example of this was recently observed by Gessner et al. , who saw that swapping positively charged arginines and lysines with alanine in an arginine-rich amphiphilic sC18 peptide reduced internalization, except for when swapped at position 15, which increased both charge and uptake. 7 Similarly, Habault et al. observed that replacement of aspartic acid with arginine in the amphiphilic hAP10 CPP significantly increased penetration efficiency, demonstrating 50% higher penetration than TAT. 6 These studies indicate that secondary peptide structure is essential for interacting with biological membranes. On the other hand, certain peptide modifications have negligible impacts on cell penetration: deletion of the 47 th amino acid in TAT had no significant effect on its translocation, and switching C–N or C–C linking (effectively flipping the peptide sequence) of antigens to CPP had no significant effect on uptake of peptide vaccines. 5,9 While these works represent key steps in understanding the structure–function relationships of CPPs, there is still a need to elucidate this relationship further, particularly in the context of NC–CPP conjugation. Two categories of internalization mechanisms utilized by CPP–NCs have been observed: direct translocation, an energy-independent process that relies on destabilizing the cell membrane temporarily to allow translocation, and endocytosis, an energy-dependent process that relies on interactions with membrane lipids or proteins. These mechanisms are further subdivided into several pathways. One of these pathways is known as micropinocytosis, of which clathrin- and caveolae-mediated endocytosis are prime examples. Clathrin-mediated endocytosis is mediated by multiple low-affinity protein–protein and protein–lipid interactions that lead to the formation of clathrin-coated pits that invaginate and pinch off into intracellular endocytic vesicles. Caveolae-mediated endocytosis is similar in that caveolin and accessory proteins are recruited during an invagination of the cell membrane that pinches off and traffics to an early endosome in the cell. 21 Macropinocytosis, different from micropinocytosis like clathrin- or caveolin-mediated endocytosis, occurs when an actin-driven expansion of the cell membrane encloses extracellular fluid and folds back onto the cell membrane into a macropinosome, which matures into an early endosome. 21 Although CPPs have been observed to enhance NC cargo uptake via these uptake mechanisms, there is a lack of understanding as to what is causing these interactions/mechanisms at the cellular level. There is a need for further elucidation on the relationship between cell type, CPP sequence, sequence modification (cyclization), molecule conjugation receptor expression, peptide concentration, and the binding strategy of the cargo, as all of these factors affect CPP–NC internalization. 19,20,22–24 Some notable trends in cellular uptake mechanisms have been observed; however, most CPPs have been found to leverage multiple mechanisms. Cationic peptides are translocated via energy-independent interactions with anionic membrane components, causing membrane instability. Three primary models have been proposed: the carpet model ( Fig. 1A ), pore formation model ( Fig. 1B ), and the membrane-thinning model ( Fig. 1C ). In the carpet model, hydrophobic residues embed into the membrane and hydrophilic sites orient to the polar face, causing a reorganization of the membrane that disrupts the bilayer and allows the CPP to enter. 25 In the pore formation model, amphiphilic peptides intercalate with the cell membrane and form direct pores that allow cargo to penetrate. The membrane-thinning effect is observed when negatively charged lipids in the outer membrane interact with cationic residues, allowing the CPP to intercalate within the laterally rearranged membrane. 19 Arginine has been found to be a crucial residue for achieving membrane thinning, as it electrostatically interacts with the plasma membrane to penetrate it without active transport. Heparin sulfate proteoglycan (HSPG), an anionic membrane component, appears to be a key component involved in the uptake of arginine-rich peptides and arginine-based CPP–NCs. This was recently observed in human glioblastoma U87 spheroids incubated with micellar NCs conjugated to octa-arginine, in enteroid models incubated with mesoporous silica NCs bound with PLA-PEG-protamine (VSRRRRRRGGRRRRC), and binding studies investigating membrane protein binding with arginine in CPPs. 6,24,26 Amphiphilic CPPs can directly translocate across or enter the cell through endocytosis. The former mechanism requires the formation of an inverted micelle in the plasma membrane (due to peptide–lipid interactions). Then, pore formation occurs in a “barrel-stave” (hydrophilic residues form the core of a large pore) or “toroidal” (lipids bend to interact with CPPs at their head groups) model upon reaching a certain peptide concentration ( Fig. 1B ). 19 This method depends on hydrophilic regions in the sequence that stabilize the peptide when interacting with membrane proteins, and there is an optimum proportion of hydrophilicity and hydrophobicity as the strength of the protein–peptide interactions can either prevent translocation or sufficiently destabilize the membrane to allow the peptide to pass through. 27 Endocytic mechanisms that translocate amphipathic peptides include clathrin- and caveolae-mediated endocytosis and macropinocytosis. For endocytic uptake in general, induction of membrane curvature by amphiphilic sequences promotes endocytic uptake via formation of an endocytic cup and then vesicle. This appears to be dependent at least in part on the secondary structure of the peptide; for an artificial nine-residue peptide (R6W3, RRWWRRWRR-amide), when α-helical structures and facial amphiphilicity were lost, the improved uptake was lost. 28 Beyond this, specific residues in amphiphilic peptides appear to promote certain endocytic mechanisms. Proline-rich amphipathic peptides are thought to interact with ECM glycosaminoglycans, and studies co-delivering cholera toxin found that uptake with these CPPs depended on caveolae-mediated endocytosis. 29 Combination of CPPs with NCs has also been found to influence uptake. Octa-arginine conjugation to polyethylene glycol-conjugated (PEGylated) liposomes, with a 1% peptide density and containing short serine-glycine repeat spacers, had a primary dependence on clathrin-mediated endocytosis in glioma cells. in another study, macropinocytosis was the predominant uptake mechanism used by U87 spheroids when incubated with elastin-like polypeptide NCs conjugated with octa-arginine. 24 These studies demonstrate that the introduction of NCs further complicates and affects the type of uptake, as uptake depends on the physiochemical properties of both the peptide and the cargo, and also that uptake mechanisms may be dependent on the type of cellular barrier that the CPP–NC is facing. 29 Once endocytosed peptides enter the cell, the next barrier to delivery is endosomal escape. Upon internalization, endocytosed NCs are contained within endosomes, which may mature into late endosomes and then lysosomes that degrade the CPP and its cargo ( Fig. 2 ). If the delivery system cannot escape the endosome, it will be rendered nonfunctional (unless it is not biodegradable). Cationic residues have been observed facilitating endosomal escape by inducing endocytic vesicle membrane leakage or disruption. Certain CPPs, then, can serve dual-purposes: cellular uptake and endosomal escape. For example, TAT induces endosomal leakage by interacting with anionic phospholipids, and oligo-arginines form ion pairs with lipids to partition the membrane. 19 The presence of bis(monoacylglycero)phosphate (BMP), which is uniquely abundant in late endosomes, and the distance between lipid bilayers in the endosomal wall are the most important factors in CPP-mediated endosomal escape. 30 To translocate out of the endosome, cationic CPPs must be encapsulated at sites where the lipid bilayers are in close contact so that BMP can interact with peptide chains, promoting encapsulation. This suggests that interactions between cargo and peptide do not affect endosomal escape. 30 However, recent work is in direct contradiction to this: Patel et al. incubated 10T1/2, HepG2, HeLa, and HEK cells with eight GFP–peptide conjugates containing linear, cyclic, or hemagglutinin-conjugated TAT, linear or cyclic octa-arginine, transportan, penetratin, or Xentry, and observed no significant endosomal escape. 23 Conjugation in general may reduce escape as other researchers have shown that nonlinear, low molecular weight PEG spacers can attach to the wall of the endosome and prevent associations between peptides and lipids. It is possible that the efficacy and method of endosomal escape may vary with CPP type and sequence, cargo, and other factors, which would explain the differences in these findings. However, the interactions between cargo, NC, and externally conjugated components are not fully understood for most designed, complex CPP–NCs. 22 This knowledge gap is due to a lack of investigation as most studies focus on improving therapeutic outcomes and do not include experiments measuring colocalization of CPP–NCs with endosomes, let alone the mechanisms by which the NCs exit these vesicles. With any drug, toxicological impacts are an obvious concern. Reports following administration of CPP conjugates in preclinical studies have demonstrated off-target and systemic toxicity in some cases. Cationic CPPs may be more prone to causing toxicity due to their strong penetrative capacity and positive charge, while electroneutral CPPs performed better in vivo . 31 For example, a recent study found that nuclear entry of arginine-rich CPPs can displace RNA- and DNA-binding proteins from chromatin and mRNA, impairing RNA processing and DNA repair. 32 However, this is not a hard rule as TAT, one of the most commonly used CPPs, is relatively nontoxic compared to other cationic CPPs. 31 Most studies have reported that, overall, CPP-mediated toxicity is low but varies with the physicochemical properties of the CPP and its cargo. 33 CPP–NCs engineered with these potential challenges in mind have been promising. In one study, the combination of a novel lactoferrin-derived CPP with a biodegradable PLGA nanoparticle caused no impacts on mitochondrial activity or membrane permeability when administered to two lung epithelial cell lines. 34 Compared to viral vectors, which are often used to intracellularly deliver macromolecules, CPPs demonstrate high delivery efficiency without notable cytotoxicity. 35 Thus far, no CPP-conjugated drugs have been approved by the FDA and the most significant limitations of these drugs is their short duration of action, low in vivo stability, and lack of specificity if not designed with a specific target in mind. 33,36 However, as described in the rest of this review, recent data is promising, and researchers have developed ways to overcome a significant number of these issues.

The investigation thus far of CPPs in conjugation with cargo-loaded NC systems has yielded promising results in terms of enhanced cellular uptake and therapeutic outcomes. Taking this a step further, recent works have developed novel CPPs designed for conjugation to NCs with a goal of enhanced conjugation, barrier penetration, and/or target cell population uptake. These additional functionalities have been shown to further enhance therapeutic outcomes. Despite these advances, most studies have emphasized only the impacts of CPP conjugation/novel CPP design on therapeutic outcomes. The large-scale impact is clear and an important indicator of success; however, the field would benefit from a more in-depth understanding of what is driving the success of these systems. Specifically, many of the studies cited herein failed to investigate what cellular mechanisms drove the targeting and uptake observed in CPP–NCs. An enhanced understanding of the mechanistic level of CPP–NC functionality, and how synergistic effects between system properties such as CPP structure, sequence and concentration, cargo type, and NC material properties, will allow for more informed future rational design of CPP–NCs and better understanding of why some of these formulations may fail. Further, few of the cited studies quantified or reported the peptide conjugation efficiency, degree of surface coverage, quantity of peptide present, or storage stability, all parameters required for rational design of future NC systems, as well as for optimization for clinical translation of these drug delivery vehicles. Future investigation of both of these areas, underlying mechanisms and NC characterization, will open the door for rationally designed NCs for personalized medicine, a long-standing goal of the nanotechnology field. 1 These rationally designed CPP–NC systems could drive solutions to improve therapeutic delivery for diseases beyond cancer, including autoimmune disorders and rare and chronic diseases.

Nanocarriers (NCs) have emerged as a top drug delivery vehicle due to their highly tunable properties allowing for targeting down to the organelle level, crossing of complex biological barriers, high surface area to volume ratio, increased therapeutic efficacy, and decreased off-target effects. 1 While originally developed with a focus on chemotherapeutic delivery, applications of NCs have stretched beyond cancer to include a vast array of diseases, including rare and chronic diseases, opening doors for new and improved treatment options to improve their clinical outcomes. 2 Despite significant promise in this work and early successes, there is still a need for more research and development: as of 2021, just over 25 drug-loaded s nanomedicine treatment platforms have been approved by the FDA and/or EMA, but all of them have limitations. 2,3 A primary reason for this is that despite significant efforts, targeting itself is still an issue. A comprehensive study found that in nanomedicines for cancer treatment, the average percent of administered NCs that make it to the target site is 0.7%, due to interactions with a wide variety of biological barriers, including immune clearance, nonspecific uptake, endosomal degradation, and cellular efflux, to name a few. 4 This indicates a need for systems with higher targeting yield, to in turn enhance clinical efficacy and reduce required dosages. Cell-penetrating peptides (CPPs) are now emerging as a popular peptide class which address this need. CPPs improve NC transport across biological barriers by improving nonspecific penetration at the cellular level, and in certain cases, passive and/or active targeting at the systemic level, enhancing the percentage of administered particles which make it to the site of action. CPPs are a diverse class of short (5–40 amino acid s(AA), long) and often linear peptides that vary greatly in size, charge, sequence, balance of hydrophobicity and hydrophilicity, and origin (natural or hybrid/synthetic). 5–7 The first CPP discovered, the transcription-transactivating protein of HIV-1, or TAT, was identified in 1988 after the domain was observed penetrating the cell membrane and nucleus. TAT was then conjugated to small molecule drugs and biologics to enhance uptake into cells and across biological barriers, and more recently to improve uptake of larger cargos, like NCs. A significant portion of newer CPPs is derived from TAT, using the key uptake sequence 47-YGRKKRRQRRR-57. 8 A wide array of natural and synthetic CPPs has also been created and used to enhance intracellular drug delivery. This diverse family of peptides can directly translocate through cell membranes or associate with membrane lipids/receptors to improve cargo uptake. Notably, CPP-conjugated delivery systems exhibit first-order kinetics with short half-lives and can have low cytotoxicity. 9,10 Conjugating CPPs to drug-loaded NCs leverages both the improved barrier penetration capabilities of the peptide and the stability of the NC, which can reduce drug degradation outside the cell. Novel sequences, rationally designed and synthesized specifically for NC formulation, include sequences with domains that intercalate within the lipid bilayer of liposomes or peptides that themselves act as NCs. While other notable reviews have delved into the efficacy of CPPs for NC delivery before, the focus of these are largely solely chemotherapeutics, and/or do not emphasize the key synergistic features of these novel cargo–CPP–NC systems which allow for barrier penetration and accumulation in target sites. 11–13 This review aims to cover the scope of these novel CPPs, as well as the uses of well-established CPPs for NC-based drug delivery. We highlight CPP–NC research, which investigates the relationship between CPPs, NCs, and cargo, and its synergistic effects on enhancing delivery of small molecules, nucleic acids, and proteins to their target sites in order to provide insights into design on a system level. Key examples are highlighted below, in Table 1 . We also discuss areas where more research is needed.

There are no conflicts to declare.