2.1. Cationic CPPs

Cationic CPPs, such as TAT, penetratin, polyarginine, P22N, DPV3, and DPV6, are composed of peptide sequences that are rich in arginine, lysine, and histidine. The guanidinium group present in arginine plays a crucial role in forming hydrogen bonds with the negatively charged phosphate groups on cell membranes. This interaction facilitates CPP entry into cells under physiological pH conditions. These peptides often interact electrostatically with glycoproteins on the cell surface and are internalized independently of receptor-mediated mechanisms. The efficiency of cationic CPPs is influenced by the number and spatial arrangement of positively charged arginine residues within their structure [6]. Most cationic CPPs contain more than five positively charged amino acids, which are critical for their functionality [7]. Among these, polyarginine sequences show the highest efficiency for cell entry, making them particularly promising for therapeutic applications [8]. Studies by Chu et al. (2015) showed that the internalisation potential of oligoarginine increases with peptide length, with R8 to R10 being the optimal range for delivery applications [9]. Experimental analyses of oligoarginines ranging from three to twelve residues revealed that efficient membrane perforation requires at least eight arginine residues, with activity progressively increasing as the number of residues increases [2]. Although lysine shares a positive charge with arginine, it lacks the guanidinium group, resulting in comparatively lower membrane permeability when used in isolation [10]. Research by Futaki et al. (2001) further underlined that cationic CPPs with at least eight positively charged residues achieve superior membrane-penetrating efficacy [11].

Other amino acids also play critical roles in CPP functionality. For example, substituting tryptophan at position 14 with phenylalanine in penetratin results in a loss of membrane permeability [12]. Nuclear localization signal (NLS) sequences represent a subset of cationic CPPs. These are short peptides enriched in arginine, lysine, and proline that make possible the transport to nuclear pore complexes. NLS peptides are further classified as mono- or bi-partite based on whether they contain one or two clusters of basic amino acids. An example of a monotype NLS is PKKKRKV from Simian virus 40 (SV40), while bitype sequences are exemplified by nucleoplasmin. The smallest known NLS sequence with membrane-permeating ability is KRPAATKKAGQAKKKL [2]. However, due to their lower charge (fewer than eight positive residues), NLS peptides are not inherently effective CPPs [13]. When conjugated with hydrophobic sequences, NLS peptides can form amphiphilic CPPs with enhanced properties. Cationic CPPs are being actively explored for their versatility in biomedical applications, including drug delivery, gene therapy, and molecular imaging.

2.2. Amphiphilic CPPs

Amphiphilic CPPs are a group characterized by their ability to possess both hydrophilic and hydrophobic properties. They are further subdivided into four categories: primary amphipathic, secondary α-helical amphipathic, β-sheet amphipathic, and proline-rich amphipathic CPPs [14]. Primary amphipathic CPPs are either derived from natural protein sequences or designed by combining hydrophobic peptide domains with nuclear localization signals (NLSs). For instance, MPG (GLAFLGFLGAAGSTMGAWSQPKKKRKV) and Pep-1 (KETWWETWWTEWSQPKKKRKV) are synthetic examples of this. Both are based on the NLS sequence PKKKRKV from Simian Virus 40 (SV40) but incorporate hydrophobic domains to enhance their amphipathic nature. In MPG, the hydrophobic domain is derived from a fusion sequence, while in Pep-1, it is represented by the peptide segment KETWWETWWTEW. These hydrophobic sequences are joined to the NLS through a short linker peptide, WSQP [15]. Alternatively, some primary amphipathic CPPs originate from naturally occurring proteins. Examples include pVEC, ARF (1-22), and BPrPr (1-28), which demonstrate similar structural and functional features but are directly sourced from protein sequences rather than synthetic design. Secondary α-helical amphipathic CPPs function by interacting with the cell membrane through their α-helical conformation. These peptides possess distinct hydrophilic and hydrophobic regions distributed across different faces of the helix, allowing effective membrane association and penetration. A representative example is MAP (KLALKLALKALKAALKLA), which demonstrates significant transmembrane activity through this mechanism [2]. The functionality of β-sheet amphipathic CPPs is closely tied to their ability to form β-sheet structures. The alignment of amino acid residues in pleated β-sheets is essential for their interaction with and entry into cell membranes. Peptides unable to establish this configuration lose their membrane-permeating capabilities, highlighting the critical role of β-sheet formation in their mechanism of action [16].

Proline-rich amphipathic CPPs are characterized by their high proline content, which facilitates the adoption of a polyproline II (PPII) helical structure in aqueous environments. Unlike the typical right-handed α-helix with 3.6 residues per turn, PPII helices are left-handed and consist of approximately three residues per turn. Examples of proline-rich amphipathic CPPs include the bovine antimicrobial peptide Bac7 and synthetic peptides such as (PPR)n, where “n” can vary between 3 and 6 [2]. These unique structural features contribute to their efficient cellular uptake and potential therapeutic applications.

2.3. Hydrophobic CPPs

Hydrophobic CPPs are composed predominantly of nonpolar amino acid residues and possess a net positive charge of less than 20%. Their functionality relies on the presence of hydrophobic motifs or specific chemical groups essential for traversing the lipid bilayer. Despite being less extensively studied than other CPP classes, hydrophobic CPPs have notable examples, such as fibroblast growth factor-derived peptides like K-FGF and F-GF12, which are associated with Kaposi’s Sarcoma [17]. These peptides represent an alternative strategy for intracellular delivery, particularly in hydrophobic environments where traditional CPPs might face limitations.

3.1. Direct Membrane Penetration

Direct membrane penetration is an energy-free pathway, with proposed mechanisms including inverted micelle mode [23], carpet mode [24], perforation mode, i.e., “Barrel-Stave” model [25], and plasma membrane sparse mode [21]. Each one of these modes necessitate cationic CPPs to first engage with anionic components on the plasma membrane, such as phospholipid bilayers, causing modifications in plasma membrane integrity, and the successive internalisation process is dependent on the type and concentration of CPPs, the quantity of cargo, etc. attributes, cell lines treated, and storage conditions, etc. [2]. While these mechanisms can describe some facets of CPP transmembrane transport, none can establish a comprehensive internalisation pathway relevant to all types of CPPs. When nuclear magnetic resonance (NMR) was used to investigate the interaction between penetratin and phospholipid membranes, the inverted micelle model was proposed [26]. The cationic CPPs first interact with the anions on the phospholipid membrane, and then the phospholipid bilayer rearranges and shuttles due to the interaction between the CPPs’ hydrophobic amino acid residues and the phospholipid membrane, and finally, the CPPs are wrapped [27]. The micelle, entombed within the inverted micelle, moves to the other side of the lipid bilayer membrane and explicitly discharges CPP into the cytoplasm [2]. The mechanism cannot work for CPPs without hydrophobic amino acid residues such as TAT and oligoarginine, since this model assumes hydrophobic amino acid residues are required to form flipped micelles. Carpet structures have been employed to characterize not only the translocation mechanism of some antimicrobial peptides but also the cytotoxicity of CPPs at high concentrations. Throughout this mode, cationic CPPs carpet the surface of the negatively charged plasma membrane like on flooring, and then the hydrophobic amino acid residues engage with the plasma membrane’s hydrophobic core because the basic amino acid residues in the CPPs face the plasma membrane surface. When CPP concentrations reach a certain level, the phospholipid membrane becomes regionally compromised, permitting CPPs to penetrate the cell [28,29]. The sequence, also known as the barrel stave sequence, is a mechanism for antimicrobial peptide internalisation in bacteria. The α –helical structure of the amphiphilic CPPs is required for the formation of the pore-type channel. CPPs are grouped to the cell surface and implanted in parallel structures into the cell membrane to establish a barrel-like conduit [18]. The hydrophobic area of the α-helix adheres to the membrane lipid’s outer surface to construct the conduit, while the hydrophilic region binds to the phospholipid’s hydrophilic head, forming the conduit’s central core and facilitating CPP transmembrane translocation and internalisation. The CPPs are delivered into the cytoplasm [19]. The “plasma membrane thinning” impact was first used to explain magainin’s mechanism of action [30]. After CPPs form a carpet structure, the anomaly of the interaction between the outer leaflet charges induces the rearrangement of negatively charged membrane lipids and the thinning of the plasma membrane in this model. CPP interaction or aggregation on the plasma membrane surface lowers the surface tension of the plasma membrane’s local surface, allowing CPPs to insert into the plasma membrane and penetrate the cell [2]. However, since no plasma membrane permeability was observed, this mechanism is highly unlikely to be plausible. It is possible to explain why the cell membrane does not appear permeable by considering the transient properties of plasma membrane pores combined with the repair response of the cell membrane [31].

3.2. Endocytic Pathway

The involvement of endocytic pathways in CPP internalisation has been a central focus in understanding the mechanisms of CPP-mediated membrane penetration. Initial studies suggested that CPP uptake occurred through a non-receptor-mediated, non-transporter-dependent, and energy-independent direct internalisation process. Over time, however, four distinct endocytic pathways have been identified: clathrin-mediated endocytosis [32], caveolin-mediated endocytosis [33], macropinocytosis [34], and clathrin- and caveolin-independent endocytosis [35]. Early research by Vives et al. (1997) demonstrated that TAT could enter cells at 4°C or under energy-depleted conditions, supporting the idea of energy-independent internalisation and excluding the involvement of endocytosis [36]. However, subsequent studies revealed limitations in these early methods, such as the use of methanol or formaldehyde for cell fixation, which could redistribute CPPs on the cell surface, thereby obscuring the true internalisation process [37]. Cationic CPPs also exhibit strong membrane affinity, complicating the distinction between membrane-bound and internalized peptides when analyzed by flow cytometry. This limitation often led to overestimation of internalisation rates [2]. To address these concerns, Richard et al. (2003) utilized more precise techniques, confirming through flow cytometry and cell fixation that TAT internalisation occurs via endocytosis [38]. Similarly, Wadia et al. (2004) observed co-localization of TAT-Cre with the endocytic marker FM4-64, providing direct evidence for an endocytic mechanism underlying TAT-mediated transport of exogenous molecules [34].

While some CPPs can bypass membranes through direct translocation, the majority rely on endocytic processes for internalisation. This mode of entry, however, poses challenges for cargo release, as CPP-bound materials often remain sequestered in endosomes and are subsequently degraded in lysosomes unless they successfully escape the endosomal compartment [39,40]. In receptor-mediated endocytosis, CPPs or their cargo bind to membrane receptors, inducing curvature through interaction with epsin proteins. Clathrin and hetero-tetrameric adaptor proteins (AP-2) assemble to form clathrin-coated pits, which mature into vesicles encapsulating the CPP-cargo complex [35,41]. The Caveolin-mediated endocytosis pathway involves CPP binding to receptors located in lipid rafts—hydrophobic membrane regions enriched in sphingomyelin and cholesterol. The cavin-1 and caveolin proteins assist vesicle formation and endosome generation, mediating cargo internalisation [42,43]. The preference for caveolin-mediated pathways may vary based on the size and properties of the associated cargo [44].

When CPPs are bound to larger macromolecular cargos (e.g., >30 kDa), they often employ macropinocytosis, an actin-dependent endocytic pathway characterized by membrane ruffling and vesicle formation. This process is initiated by CPP interactions with membrane proteoglycans, which activate rac proteins. These proteins, in turn, trigger F-actin rearrangements that also assist vesicle formation and cargo uptake [35,45]. Unlike other pathways, macropinocytosis is receptor-independent and often lipid raft-dependent, making it particularly suited for CPPs carrying large payloads [2,46]. In specialized cells like macrophages, CPP uptake can occur through an alternative mechanism that does not involve clathrin or caveolin. Instead, CPPs are opsonized and recognized by cell surface receptors such as Fc receptors. Actin activation drives the internalisation of CPP-cargo complexes into membrane-coated vesicles [47]. Regardless of the endocytic pathway utilized, CPPs and their cargos must escape endosomes to avoid lysosomal degradation and exert their biological effects. This escape is facilitated by processes such as pH gradient alterations, vesicle accumulation, and CPP interaction with charged endosomal membranes. These interactions can lead to increased membrane rigidity and eventual rupture, enabling the release of CPPs and their associated cargos into the cytoplasm [6,7]. The choice of endocytic pathway for CPP internalisation is influenced by the peptide’s physicochemical properties and the characteristics of the bound cargo, including size, charge, and hydrophobicity. For example, TAT has been shown to use different pathways—lipid raft-mediated endocytosis for protein cargos and clathrin-mediated endocytosis for smaller fluorescent molecules.

4.1. Role of Cargo Molecules in CCP Uptake

The cargo coupled to the CPP is frequently a key internalisation component. The absorption of TAT conjugated to peptides and globular proteins in live cells was compared by Tünnemann et al., 2006 [51]. They discovered that the uptake process was significantly influenced by the size of the payload fused to TAT. While the TAT-peptide conjugates were dispersed widely throughout the cell, the bigger complexes comprising proteins were visible inside vesicular structures [51]. This suggests that a separate absorption method results from the size of the cargo, which naturally determines the size of the total complex. The likelihood of the complex being taken up by direct translocation increases with decreasing size. Endocytosis, however, predominates at larger dimensions. This is comparable to the situation where the presence of a cargo molecule chooses between two alternative endocytosis methods when comparing the uptake mechanism of unconjugated TAT versus TAT fused to a cargo [41]. Whereas its conjugated counterpart is more likely to adopt CvME, unconjugated TAT favours CME for cell entry [48]. Cargo impact can also affect how other arginine-rich peptides are taken up. The effects of cargo molecules on the absorption of R7 and R7W were examined by Maiolo et al., 2005 [52]. In the cytoplasm of the tested cells, the peptides alone displayed diffuse signals. The diffuse signal from endocytic vesicles was significantly reduced following fusing with cargo peptides, but the punctate signal barely changed.

4.2. Role of Concentration in CCP Uptake

Since multiple uptake routes can be stimulated by CPP concentration, it is a crucial component. It is thought that endocytosis often takes place at low peptide concentrations and that direct penetration takes over at greater concentrations. Fretz et al., 2007 [50] evaluated the dependency on concentration while examining the impact of temperature on the absorption of R8. Vesicular staining in the cytoplasm was seen at lower peptide concentrations, indicating endocytic absorption [48]. Vesicular and diffuse labelling was seen at greater concentrations, suggesting that direct penetration and endocytosis may take place concurrently [53]. The concentration-dependent absorption of R9 and TAT has also been investigated [53]. In addition, these peptides exhibit substantial cytosolic labelling at higher concentrations and mostly vesicular signals at lower concentrations. Yet things become more difficult since endocytic inhibitors had only a minimal impact on the absorption of R9 and TAT at low concentrations (<5 uM), but clathrin inhibitors appear to have a significant impact at higher concentrations [48]. In addition to the quick, nonendocytic absorption via nucleation zones, it was shown that the peptides are, to some extent, taken up by vesicular structures at greater concentrations [54].

4.3. Role of the Type of Cells Involved in CCP Uptake

The CPP uptake process is greatly influenced by the cell lines that were employed in the research. The nature of the extracellular matrix and the plasma membrane, in particular, can have a significant impact on CPP uptake. It is well known that the extracellular matrix’s negatively charged GAGs and the positively charged CPPs have their first interaction during internalisation. Hällbrink et al., (2004) looked at the impact of the peptide-to-cell ratio on the absorption of cells into Chinese hamster ovary (CHO) cells [55]. To be more precise, they were interested in how the absorption would alter if the number of peptides, rather than the concentration, were raised while maintaining the same number of cells. They also looked at how cell number and confluence affected uptake. They demonstrated that increasing the internal peptide concentration more effectively than increasing the exterior peptide concentration required double the incubation volume at a certain number of cells. Moreover, applying a constant peptide concentration to various cell densities showed that the uptake decreased with increased confluence. The variable membrane composition or the various endocytosis patterns of the developing cells may be the reason for this. Another hypothesis is that when confluence rises, membrane access diminishes [48]. These findings offer an illustration of how experimental variables in cell culture might affect uptake effectiveness. A thorough analysis of the absorption of 22 distinct CPPs in four different cell lines was conducted by Mueller et al., 2008 [56]. They investigated the internalisation of several of the most well-known CPPs, including penetratin, TAT, transportan, Pep-1, MPG, MAP, R7, and R9. They employed Cos-7, HEK293, HeLa, and MDCK as representative cell lines. According to their behaviour, the data allowed for the classification of CPPs into three groups: high (penetratin, transportan, MAP), medium (TAT, Pep-1, MPG), and low cellular uptake [48]. The results demonstrate that some of the peptides are taken up by cells in a cell-dependent manner, which is interesting. For instance, Cos-7 cells, which resemble fibroblasts and were isolated from monkey kidney tissue, took up MPG preferentially [48]. This could be because the virus that was utilised to create the Cos-7 cell line, SV-40 big T antigen, includes an NLS that was generated from MPG [57].

Research by Gronewold et al. (2017) showed how cell lines affected the absorption of a CPP with potential anticancer action [58]. The antimicrobial peptide CAP18’s C-terminal domain was used to create sC18, the CPP. In addition to HEK293, a noncancer cell line, its absorption was examined in the cancer cell lines HeLa, PC-3, HCT-15, and MCF-7 [58]. Interestingly, nuclear accumulation and a diffuse fluorescent signal with punctate distribution were seen in the cytoplasm of all cancer cell types. However only a punctate distribution and little to no peptide in the nucleus were found in the non-cancer cell line.

7.1. CPPs Facilitate Transmembrane Transport of Small Molecule Drugs

Because of their small size and lipophilicity, small molecule anticancer drugs can dissipate into tumor cells efficiently. Multidrug resistance (MDR) develops when tumor cells are subjected to the same drug continuously. To address this issue, some researchers have attempted to combine these drugs with CPPs to facilitate the entry of these small-molecule drugs into cells [71]. Dubikovskaya et al. formed an R8-taxol covalent compound by disulfide bonding an octamer arginine R8 to the anticancer drug paclitaxel [71]. The results revealed that the R8-taxol covalent compound exhibited a similar effect to paclitaxel alone in taxol-sensitive tumor models. However, in taxol-resistant tumor models, R8-taxol covalent drugs were more likely to induce tumor cell apoptosis compared to paclitaxel alone [2]. There are numerous advantages to combining small-molecule drugs and CPPs. It can increase drug water solubility and improve drug utilisation rate, in addition to overcoming multidrug resistance. Lee et al., (2011) used chemical methods to combine doxorubicin, TAT, and polymerized chitosan backbones to generate chitosan/doxorubicin/TAT chimaeras that were compatible with TAT-free chimaeras to enhance the cytotoxicity and targeted transport of anticancer drugs [72]. This chimaera has more efficient cell internalisation than doxorubicin or chitosan/doxorubicin, and it can change the distribution of doxorubicin in the organism, enhance tumor localization, and thus considerably constrain tumor growth [72].

7.2. CPPs Facilitate Transmembrane Transport of Peptides and Proteins

Numerous cancers can be caused by mutations in tumor suppressor genes [73,74]. To reinstate the activity of these proteins and accomplish the goal of cancer treatments, some researchers attempt to insert full-length proteins or polypeptides about tumor suppressor genes into cancer cells. To treat mice with advanced peritoneal cancer metastasis, Snyder et al. created a TAT-p53 chimeric peptide and administered it intraperitoneally. The average survival time of the mice in the experimental group was more than 6 times that of the control group, which had a mean survival time of 10 days [75]. Hosotani et al., 2002 covalently joined a short peptide made up of 20 amino acid residues of the p16 protein to penetratin by a disulfide bond to reinstate the functioning of the p16 protein, and they then tested its effectiveness in an animal model of pancreatic cancers [76]. According to the findings (tumor in treatment group: 79 ± 17 mg; tumor in control group: 149 ± 12 mg), the complex could greatly slow the growth of cancers [76]. Apoptosis induction of tumor cells was attempted by certain researchers, utilizing various therapeutic techniques. As a protein of mitochondrial origin, Second Mitochondrial-Derived Activator of Caspase (SMAC) is crucial for the regulation of apoptosis [77]. SMAC can deactivate apoptosis-inhibiting proteins when it is released from mitochondria, hence increasing apoptosis. The 7 amino acid residues at the amino terminals of SMAC and TAT were combined to create a chimeric peptide, which was identified by Fulda et al. Research conducted in vitro revealed that while this chimeric peptide was ineffective at stimulating tumor cell apoptosis, it did make tumor cells more susceptible to apoptosis effectors such tumor necrosis factor-related ligand that induces apoptosis (TNF-related apoptosis-inducing ligand, TRAIL) [78]. The conjunction of TRAIL and SMAC-TAT peptide can greatly slow the formation of tumors in a mouse model of human glioma xenografts, and when 0.6 or 2ug of TRAIL is employed, tumor cells can be eliminated [78]. The ribosome-inactivating protein (RIP) generated from plants known as gelonin can efficiently limit protein translation when its half maximum inhibitory concentration (IC50) is at the picomolar level [2]. Poor membrane permeability makes it difficult to treat tumors and frequently fails [79]. A chemical combination of gelonin and TAT or LMWP was used by Park et al. to overcome this obstacle [80]. In mouse colon cancer cells, the gelonin chimeric peptide was shown to have strong antitumor activity when it was injected. At a dose of 100 μg, the chimeric peptide can completely inhibit tumor growth [80].

7.3. CPPs Facilitate Transmembrane Transport of Genes

For the treatment of cancer, genes, antisense oligonucleotide strands, and small interfering RNA (siRNAs) may be useful tools [81]. A particularly alluring therapeutic approach, RNA interference technology is extremely selective in targeting genes and has a special tumor suppressor impact. Drugs containing siRNA have a minimal ability to penetrate cells due to their enormous size, strong charge, and size. CPPs have drawn a lot of attention due to their low toxicity in endeavors to facilitate siRNA entry into cells using cationic liposomes, cationic polymers, and CPPs. In a covalent or non-covalent manner, siRNA can attach to CPPs. Even though covalent bonding is the best type of binding, tight complexes and polymers are simple to produce because of the strong charge interaction between the two [2]. It is more difficult to cross the cell membrane than CPP when combined with another molecule in a 1:1 covalent manner [2]. Cationic CPPs’ charge can be easily neutralised by negatively charged siRNA, rendering both siRNA and CPPs ineffective for their intended biological purposes. Researchers have decided to combine siRNA with CPPs in a non-covalent form due to the several disadvantages of covalent binding. They are mostly focused on the viability of siRNA-CPP chimeric peptide research in vitro, however, animal research is also ongoing. In the angiogenesis requisite for tumor growth, vascular endothelial growth factor (VEGF) plays a significant role.

The VEGF gene can be silenced or VEGF receptors blocked to treat malignancies. A cholesterol/R9/siVEGF combination was created by Kim et al., 2006 using R9 chimaera as a carrier and anti-VEGF siRNA (siVEGF) to drastically reduce tumor development and angiogenesis [82]. Cyclin B is a mitotic cycle protein that is necessary for cell division, but tumor cells manifest uncontrolled expression of this protein [2]. To reach therapeutic goals, several researchers try to use RNA interference technologies. Cromez et al. formed an anti-cyclinB1 siRNA complex by combining anti-cyclinB1 siRNA with the shortened version of MPG (MPG-8), which can significantly inhibit cell proliferation by inhibiting the expression levels of cyclinB1 and causing cell cycle arrest [83].

7.4. CPPs-Facilitated Transmembrane Transport of Nanoparticles

Cell-penetrating peptides have been extensively used in the transmembrane transport of numerous nanoparticles, including magnetic nanoparticles, lipid nanoparticles, gold nanoparticles, micelles, and quantum dots, even though the transmembrane mechanism of these molecules is not fully understood. TAT and cross-linked iron oxide particles (CLIOs) were coupled by Josephson et al., (1999) to create a TAT/CLIO complex with an average particle size of 41 nm [84]. The efficiency of the transmembrane was higher than CLIO without TAT. It is advantageous for MRI or magnetic sorting since it is approximately 100 times higher and can mark cells effectively [84]. The transmembrane efficiency of TAT/CLIO increased nonlinearly as the ratio of TAT/CLIO increased, and when the ratio reached 15, the highest transmembrane efficiency was found [2].

The complex created when liposomes and CPPs combine may efficiently enter a variety of cells, including mouse heart muscle cells, mouse lung cancer cells, and human breast cancer cells, and the effectiveness of liposomes’ membrane penetration is related to the density of CPPs [85]. The penetratin/liposome complex has a higher rate of membrane penetration than the TAT/liposome complex and reaches its maximum absorption in under one hour. Researchers are very interested in employing CPP/nanoparticle complexes to transport diverse anticancer medicines since CPPs can successfully facilitate the transmembrane transport of different nanoparticles. Paclitaxel, a drug that can effectively enter malignant glioma cells and suppress tumor cell proliferation, was loaded into lipid nanocapsules with CPP modifications by Balzeau et al., 2013 [86].

7.5. CPPs’ role in Cancer Therapy

One challenge in treating cancer was that tumor microenvironment or other obstacles prevented medication transport to tumor cells, particularly in cases of duodenal and brain gliomas. CPPs provided a fresh viewpoint on how to get through a semi-permeable hydrophobic barrier and achieve excellent drug delivery in tissue and subcellular architecture. The majority of CPPs reacted with the high-density anionic charge on cell membranes and possessed positive side chains. Polyarginines of various lengths were often employed for medication delivery [35]. The CPPs’ cationic charge density was a significant factor in determining the feasibility of the cargos’ translocation. CPPs can be used to improve the specificity and selectivity of chemotherapy drugs by delivering them selectively to cancer cells while sparing normal cells. This approach not only reduces the side effects associated with traditional chemotherapy but also enhances the efficacy of the drugs by increasing their bioavailability and reducing the development of drug resistance. Extracellular vesicles altered with the hexadecanoyl-arginine (R16) peptide revealed moderately good anti-cancer efficacy [87]. Proteins were typically transferred via CPPs by covalent bonding. Table 1 shows some of the CPPs used in cancer treatment.

Unfortunately, because of changed biological activity and/or steric hindrance, covalent CPPs technology was not the most efficient method to transfer macromolecules [89]. Delivering oligonucleotides seems to be mostly accomplished by electrostatic adsorption. The linear structure of CPPs typically does not achieve highly satisfying oligonucleotide transfection efficiency due to low charge number, which leads to poor complexation and organizational volatility of nano-carriers [35]. To create a unique bio-reducible cationic network employing R9 as a vector, Yoo et al., (2017) synthesised a branched R9 using disulfide bonds [90]. Branching structures enable effective electrostatic adsorption of pDNA or siRNA. In vitro, B-mR9 demonstrated strong intracellular trafficking and biocompatibility. The EPR impact of B-mR9 also demonstrated a targeted effect on the tumor that lasted for 48 hours. In the NCI-H460 harbouring BALB/c nude mice model, B-mR9/siVEGF significantly suppressed tumor development by 56.5% when compared to control, and the therapeutic effectiveness was greater than PEI25k and R9 vector [35].

A fresh method to create a platform for gene delivery was offered by the cationic network formed from CPPs. ULK1 siRNA and the AMPK activator narciclasine have been delivered together in a pH-sensitive and biocompatible micelle system by Tai W., Gao X., (2017) to successfully prevent hepatocellular cancer in preclinical trials by controlling programmed cell death [89]. Subsequent studies confirmed that CPPs were to be the revolutionary siRNA oligonucleotide paradigm. Clinical uses for CPPs were restricted to positive charges that caused systemic and off-target toxicity. In MCF-7 cells, CsA (Cyclosporin A), with electronic neutral, a new highly hydrophobic cyclic CPP, outperformed both pentapeptide VPT (VPTLQ) and PFV by a factor of several times and conventional neutral CPPs by a large margin [35]. By administering a membrane-impenetrable pro-apoptotic peptide (PAD), the effectiveness and toxicity of cyclosporin A were compared to TAT. The uptake of PDA was increased 2.2–4.7-fold in the tumor cell lines examined by CsA when CsA was conjugated to PAD, and cellular uptake of CsA-PAD was often higher than TAT-PAD. Depending on the cell type, the cytotoxicity of CsA-PAD was comparable to or greater than TAT-PAD in four distinct tumor cell lines, but it was much more potent than PAD. CsA-PAD showed an equivalent anti-tumor effect to TAT-PAD in xenografted MCF-7 nude mouse models, but with less systemic toxicity [35]. Although the correct tissue distribution of electroneutral CPPs has to be further evaluated, cationic CPPs likely have more potential application value in vivo than electroneutral CPPs [91]. Hyaluronic acid (HA), a high-affinity ligand for tumor surface-specific overexpressed marker CD44, was one such polyanionic substance employed to coat nanoparticles to decrease toxicity and non-target of positive CPPs [35]. To administer 10-HCPT against hepatocellular carcinoma, Zhao et al., (2018) created a multifunctional liposome modified with TAT and HA (HA/CPPs-10-HCPT-NPs) [92]. Low-intensity focused ultrasound was employed to precisely regulate drug release to tumor tissue. In the multicellular tumor spheroid model, the liposome penetration depth was enhanced 2.76-fold following TAT modification. HA-coated nano-carrier was a useful and promising method for CPPs administration in vivo [35]. Liposome-coupled application of HA and CPPs with the assistance of ultrasound had a considerably greater tumor inhibition against hepatic carcinoma than other groups [92]. Figure 3 below shows the types of cancer that were tested with CPPs.

Poor prognosis, particularly for duodenal cancer, gliomas, and lymph metastases, was a significant obstacle in cancer treatment. Refractory cancers have a limited capacity to be treated since delivery methods couldn’t get medications to the treatment site due to the complicated tumor microenvironment. CPPs may serve as the molecular motor for tumor-deepening cargo. To create mixed micelles for siRNA systemic administration, two tandem peptides (pTP-PEG-iRGD and pTP-iRGD) were synthesised by Lo et al., 2018 to solve the target and tumor stroma penetration problems in PDAC [94]. It could successfully get beyond PDAC’s delivery obstacles to penetrate tumors in three-dimensional organoids and models of native tumors. Moreover, the mixed micelle complexed siRNA greatly slowed the development of the tumor [94]. To treat gliomas, CPPs may cause cargos to pass the blood–brain barrier (BBB). To transport siRNA against glioma, Liu et al., 2018, created an anionic random-coiled polypeptide (PLG) coated CPPs (PVBLG-8) micelle [95]. To create a stable structure in serum and turn the micelle’s surface potential negative, PLG became entangled with the PVBLG/8/siRNA complex [95]. Also, micelle could carry out PVBLG-8’s cell penetrating activity in response to low pH in the tumor extracellular microenvironment. CPPs were paired with glioma-homing peptides to precisely translocate siRNA to increase the target limit of CPPs in glioma applications [35,95]. To maximise tumor-specific targeting and gene knockdown impact, the bonding form of two CPPs (PF14, PF28) with targeting moieties via either covalent conjugation or non-covalent complex was adjusted [35]. Lymph nodes nearby were the site of the first tumor metastasis and eventually extended across the entire body, making lymph metastasis an important channel of tumor spread [35]. Because of the blood-lymph barrier, current medicines that target lymphatic metastasis by intravenous injection have limited non-target and low penetration capability. After being administered intravenously for a possible anti-metastasis treatment, a R9 modified cabazitaxel nanoparticle (R9-CN) with a 13 nm size and a little positive charge was shown to have a conspicuous lymph target and deep penetration impact [35]. R9-CN’s fluorescence signal persisted in primary tumor locations for at least 24 hours at a high intensity [96]. In a breast cancer lymphatic metastasis model, R9-CN significantly reduced the tumor growth rate by 1.4-fold and demonstrated a 63.3% inhibition rate of lung metastasis compared to CN. Deep lymphatic penetration made CPP-modified nanoparticles an excellent anti-metastasis platform [96].

7.6. Use of CPPs in the Treatment of Inflammatory Conditions

The stratum corneum and mucosa are the primary delivery-related challenges with transdermal administration, which has high compliance and is an efficient method of local delivery of anti-inflammatory medicines. Polyarginine peptides are frequently used in transdermal medication administration because of their ability to penetrate skin. To treat rat paw edema, Gao et al., (2019) created lornoxicam-loaded lipid gels that were R11 modified (LN-NLC-R11) [97]. Rat paw edema was eliminated, and LN-NLC-R11 dramatically reduced the generation of inflammatory cytokines as compared to NLC in vivo [97]. The distance between CPPs and nanoparticles will affect the effectiveness of cellular internalisation because of steric hindrance.

Due to steric hindrance, the separation between CPPs and nanoparticles will affect the effectiveness of cellular internalisation. To reduce pulmonary inflammation, CPPs modified gene carriers R9Gn-chitosan/siMIF (n = 0, 4, 10) were developed [35]. R9Gn-chitosan/in siMIF’s vivo cell uptake, gene silencing effectiveness, and anti-inflammatory efficacy were all enhanced by lengthening the Gn controlled spacer arm [35]. In a mouse model of particulate matter-induced airway inflammation, R9G10-chitosan/siMIF dramatically decreased inflammation and goblet cell hyperplasia of lung tissue compared to R9-chitosan/siMIF [98]. Similar to this, intranasal injection of phospholipase D1 (PLD1) conjugated with TAT enhanced the anti-asthmatic action [99]. Epidermal hyperplasia is a prevalent condition with a significant immune cell infiltration known as psoriasis. A significant contributor to the pathophysiology of psoriasis is signal transducer and activator of transcription 3 (STAT3). The high-affinity peptide designed to inhibit STAT3 is called APTstat3. R9 (APTstat3-9R) altered APTstat3 to increase stratum corneum penetration [35]. After topical intradermal therapy, APTstat3-9R showed positive efficacy in reducing localised psoriasis-like skin irritation [35]. APTstat3-9R, however, had minimal skin penetration after transcutaneous injection because the stratum corneum barrier inhibited it. APTstat3-9R complexed with DMPC/DHPC to create discoidal lipid nanoparticles (DLNPs) with great colloidal stability (20 nm size) and minimal side effects, improving transdermal administration [35]. In a mouse model of psoriasis, DLNPs were able to pass through the stratum corneum because of their lipophilicity and then pass through the openings between epidermal layers to reach the dermal layers [100]. The imiquimod-induced psoriatic mouse model’s skin edema and epidermal hyperplasia were successfully decreased by DLNPs [100].