Cell-penetrating Peptides (CPPs) for Drug Delivery

What is the drug delivery?

Drug delivery denotes the administration of pharmacological agents to elicit a therapeutic response within the organism. It includes the formulation, delivery systems, and techniques employed to administer medications into the body, ensuring they arrive at their designated location of action in the correct concentration and duration. The effective delivery of medications to target cells continues to pose a significant issue. Macromolecular medications can be introduced into target cells through two methods: direct introduction by membrane rupture or delivery utilizing vehicles, which are categorized as either viral or non-viral vehicles.

Delivery systems for macromolecular drugs. (Sun Z., et al., 2023)

Value of CPP in drug delivery

Cell penetrating peptides (CPPs) have undergone development for more than 30 years and continue to be rigorously researched owing to their superior delivery capabilities. In comparison to alternative delivery routes, CPP demonstrates significant potential for the administration of several medication kinds, which is the non-viral vehicle. Pharmaceutical administration frequently necessitates traversing biological barriers. CPPs are typically brief sequences of amino acids that have been extensively utilized as delivery vehicles to surmount numerous application challenges, possessing a limited capacity to permeate cell membranes. Certain CPPS can selectively engage with target cells with remarkable precision and efficiency, even at little doses. Moreover, a particularly appealing feature of CPPs is their capacity to covalently bind to macromolecular cargo (including DNA, RNA, and proteins) and facilitate their entry into cells. Consequently, owing to the selective impermeability of the cell membrane, bigger macromolecules often barred from cellular entry can be transported into the cell with the assistance of CPPs. Preliminary evidence indicates that certain CPPs have superior absorption and distribution efficiency while demonstrating less cytotoxicity compared to other therapies, such nanoparticles or viral vectors.

Enhanced drug delivery system by the combination of CPPs. (Li H., et al., 2015)

CPP for therapeutic peptide and proteins delivery

There is mounting evidence that cell penetrating peptides (CPPs) enhance selectivity against tumor cells by delivering therapeutic peptide ligands and proteins to the right places in cancer cells, where they bind with particular receptors. Enabling effective drug penetration can be achieved, for instance, by using peptide nanocarriers made of cell-penetrating peptide R9 and cholesterol molecules to transport adriamycin (DOX). These nanocarriers can then be surface modified with FAP-α antibodies, allowing them to attach to the surface antigens of CAFs and shed the antibodies. The role of vascular endothelial growth factor receptor-1 (VEGFR-1) in tumor cell proliferation has been well-documented. Combining TAT with a tumor-targeting peptide A1 (WFLLTM) ligand that was selected from a phage library and has a high affinity for VEGFR-1, Fang's team created TAT-A1 complex CPPs. The rate of internalization of CPP based TAT-A1 on cells was much greater than that of free TAT; in particular, it was raised by over 50% in the HepG2 human hepatocellular carcinoma cell line. The research shown that TAT-A1 improved HepG2 penetration and suppressed VEGFR-1 expression in hepatocellular cancer therapy. The IMT-P8-KLA binding peptide was synthesized by Gautam et al. using new CPPs (IMT-P8) and a pro-apoptotic peptide (KLA). Treatment with fluorescently-labeled IMT-P8-KLA resulted in a high fluorescent signal in the mitochondria of HeLa human cervical cancer cells, showing that KLA was delivered to the mitochondria and internalized more effectively by the cancer cells. In addition, the MTT assay revealed a significant drop in cell viability at a dosage of 20 μM IMT-P8-KLA, suggesting that this compound might trigger cell death in cancer cells by focusing on their mitochondria.

CPP-based drug delivery systems for cancer treatment. (Guo S., et al., 2024)

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CPP for small therapeutic drugs and multimers delivery

Chemotherapeutic chemo-drug delivery systems (DDS) made of CPPs enhance drug penetration into tumor cells. Selective CPP-functionalized multimers (SCPP-PS) bearing the sequence RLWMRWYSPRTRAYGC were described by Yang's team. Penetration of A549 cells was made possible by the modest size of CPPs, which ranged from 63 to 65 nm. By loading 19.4 weight percent of the anticancer medication methotrexate (MTX), SCPP-PS effectively blocked the ability of cancer cells to survive. The accumulation in the tumor of SCPP-PS-MTX grew dramatically in A549 lung cancer mice after 8 hours of injection, reaching 5.3% ID/g. An activatable cell-penetrating peptide (ACPP) known as Angiopep-2 (ANG) targets an overexpressed protein in brain tumors called low-density lipoprotein receptor-related protein 1 (LRP1), was used to create ANG-TAT-anticancer drug paclitaxel (PTX), which could traverse the blood-brain barrier (BBB) with the aid of TAT. To better penetrate solid tumors, ANG and medicines were shown to be uptaken by U87 glioblastoma cells at a rate that was 1.8 times more than ANG and 5.8 times greater than free PTX, according to trials using brain uptake. In addition, compared to PTX chemotherapy, ANG-TAT-PTX treatment significantly reduced U87 cell viability and further increased the survival rate of U87 homozygous mice for 15 days, resulting in a unique and very effective DDS. Furthermore, Kumthekar's team created a new DDS (ANG1005) with three PTX molecules covalently linked to ANG-2 (ANG-PTX). Adult patients with recurrent breast cancer brain metastases (BCBM) showed encouraging results in a clinical study employing ANG1005. ANG1005 is engineered to release PTX upon LRP1 endocytosis by cleaving the enzyme cleavage linker. The results showed that ANG1005 was a successful therapy for BCBM illness, with 77% of intracranial patients and 86% of extracranial patients achieving stable control. Results showed that it protected the central nervous system of meningeal carcinoma patients, with disease control and an average overall survival of 8 months observed in 79% of patients with intracranial mild meningeal carcinomas in this experiment. By enhancing the concentration of chemotherapeutic drugs at the target location, these research methodologies maximized the therapeutic effectiveness of DDSs based on CPPs. A pair of coiled-coiled lipopeptides, "E4" [(EIAALEK)4] and "K4" [(KIAALKE)4], were introduced into liposomes and phospholipid bilayers targeting cell membranes, respectively, to facilitate the efficient delivery of medicinal drugs to live cells. The in vivo zebrafish embryo and in vitro cellular studies proved that CPE4/CPK4 facilitated the fusing of targeting membranes with liposomes, while simultaneously releasing fluorescent colors and the cytotoxic chemical DOX from liposomes. The problem of inefficient intracellular delivery of drugs due to the escape or degradation of the nuclear endosomes of lysosomes was effectively solved by this strategy, which improved the drug's penetration ability into the cell and the efficiency of intracellular delivery. This allowed for rapid drug administration in the clinic and rapid release of the lipid drug in vivo.

The adaptability of CPP applications in oncology and cancer treatment. (Desale K., et al., 2021)

Applications of cyclic cell-penetrating peptides for drug delivery

A relatively new family of peptides called cyclic cell-penetrating peptides has tremendous potential for the intracellular delivery of therapeutic medicines to treat difficult conditions including HIV infection, cancer, and multidrug-resistant bacteria. An alternate to linear CPPs, cyclic ones can be useful as well.

Cyclic CPPs have significant promise as molecular transporters, effectively addressing the difficulty of transferring macromolecules and other hydrophilic medicinal agents. Numerous studies indicate their efficacy in enhancing the potency of current anticancer and antibacterial medicines. Parang and colleagues have meticulously assessed the utility of several cyclic CPPs as molecular transporters. For example, [WK]₅ was demonstrated to be an excellent delivery vehicle for model anti-HIV pharmaceuticals. [WH]₅ was utilized for the effective transport of a cell-impermeable payload. [CR]₄ exhibited improved cellular absorption of a negatively charged phosphopeptide, whereas [HR]₄ proved to be a proficient molecular transporter. Parang and colleagues previously demonstrated that cyclic [WR]₅ and analogous cyclic cell-penetrating peptides including tryptophan and arginine may self-assemble into nanostructures, possibly serving to stabilize protein biomolecules and silver nanoparticles. Shirazi' team investigated the enhancement of doxorubicin's efficacy in drug-resistant cancer cells. It was discovered that doxorubicin conjugated with a cyclic peptide including arginine and tryptophan residues, [W(RW)₄], led to a substantial enhancement of antiproliferative activity. They noted enhanced transport of the drug to the nucleus and reduced efflux of the doxorubicin-cyclic CPP conjugate relative to the control free doxorubicin. Subsequently, Shirazi's team examined the utilization of cyclic [WR]₅ to enhance the intracellular absorption of curcumin, an extensively studied medicinal compound for many ailments. They utilized both the peptide-curcumin conjugate and the peptide/curcumin physical combination for the cellular uptake investigations. Flow cytometry analyses demonstrated that cellular internalization increased by 5.7-fold with a peptide/curcumin physical mixture compared to curcumin alone after 3 hours in the human leukemia cell line (CCRF-CEM), while the peptide-curcumin conjugate yielded a 4-fold enhancement in cellular uptake. The antiproliferative activity of curcumin was enhanced by 20% with the [WR]₅/curcumin physical combination, in contrast to an approximate 13% enhancement with the [WR]₅-curcumin conjugation, following a 72-hour incubation in CCRF-CEM cells. Subsequently, Darwish et al. coupled curcumin and doxorubicin with the cyclic peptide [C(WR)₄K₂(βA)] to augment the solubility of curcumin and amplify the antiproliferative efficacy of doxorubicin. The study's findings indicate the specificity of peptide conjugates for several malignant cell lines (e.g., CCRF-CEM and SKOV-3) while exhibiting minimal toxicity to a normal human embryonic kidney cell line (HEK-293). These findings indicate the promise of cyclic CPPs as cargo delivery mechanisms for medicinal medicines. Shirazi's team developed and produced selenium nanoparticles based on cyclic peptides and evaluated their efficacy as a medication delivery mechanism. The authors observed a notable enhancement in the administration of several anticancer agents, including gemcitabine, clofarabine, doxorubicin, etoposide, irinotecan, paclitaxel, fludarabine, epirubicin, dasatinib, and camptothecin when conjugated with [W₅R₄C]-SeNPs against human leukemia (CCRF-CEM) cells and SK-OV-3 cells. The researchers determined that this cyclic CPP-based drug delivery system might effectively serve as a nanoscale delivery mechanism for anticancer and other negatively charged medicinal agents.

Cyclic CPPs as efficient intracellular drug delivery tools. (Park S E., et al., 2019)

Summary of biomedical applications of cyclic CPPs. (Park S E., et al., 2019)

Advantages of cyclic CPPs for drug delivery

Enhanced cellular uptake: A wide variety of payloads, including as tiny molecules, proteins, and nucleic acids, can be transported across cell membranes with the help of cyclic CPPs. For treatments that have trouble penetrating cells owing to size or charge, this quality is crucial.

Versatility in cargo delivery: From tiny medicinal compounds to big macromolecules (such as proteins and nucleic acids), cyclic CPPs are able to carry it all. Because of its adaptability, multipurpose delivery methods may be created. Synergistic effects in therapies, especially in cancer therapy, are made possible by engineering cyclic CPPs to co-deliver several substances, such as medications and genes.

Targeted delivery: Conjugating cyclic CPPs with targeting moieties, such as antibodies or ligands, enhances the selectivity of drug delivery to certain cell types, including tumor cells or infected cells. This focused strategy reduces off-target effects and enhances treatment effectiveness. The targeted delivery can mitigate detrimental effects on healthy cells, hence improving the safety profile of medicines.

Improved bioavailability: Cyclic CPPs can enhance the solubility and stability of weakly water-soluble pharmaceuticals, hence improving their bioavailability in biological systems. Cyclic CPPs can protect therapeutic compounds from enzymatic breakdown in the circulation, hence extending their half-life and efficacy.

Higher resistance to proteolysis: The cyclization of cell-penetrating peptides (CPPs) to enhance their stiffness is an emerging notion in their design, with several studies indicating that this cyclization confers resistance to proteolysis. A study developed a series of cFΦR4 analogues by altering the stereochemistry and/or peptide sequence, examining their cellular uptake through the incorporation of unnatural amino acids and varying sequence lengths. The findings indicated that cyclic peptides abundant in arginine residues, specifically cFΦR4 peptide analogues [FΦRRRRQ], with Φ representing l-2-naphthylalanine, exhibited remarkable proteolytic stability as confirmed by pharmacokinetic studies conducted on mice. Heitz's team examined the influence of a cyclic backbone on the stability of a peptide framework and discovered that the stability of cyclic peptides is primarily linked to the cysteine knot framework, proposing that cyclization significantly diminishes the peptides' vulnerability to thermal unfolding. Cyclization is proposed to diminish proteolysis by eliminating reactive C- and N-termini and by offering protective elements against proteolytic enzymes.

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References

1. Desale K., et al., Cell-penetrating peptides (CPPs): An overview of applications for improving the potential of nanotherapeutics, Biomaterials Science, 2021, 9(4): 1153-1188.
2. Kurrikoff K., et al., Recent in vivo advances in cell-penetrating peptide-assisted drug delivery, Expert opinion on drug delivery, 2016, 13(3): 373-387.
3. Guo S., et al., Advances in peptide-based drug delivery systems, Heliyon, 2024.
4. Park S E., et al., Cyclic cell-penetrating peptides as efficient intracellular drug delivery tools, Molecular pharmaceutics, 2019, 16(9): 3727-3743.