Front-of-Mind:- Cell-Penetrating Peptides as Efficient Intracellular Drug Delivery Tools

According to the World Health Organisation, cancer is the second most common cause of death across the world (approximately 1 death in 6). Significant research has been directed towards discovering innovative therapies against cancer in recent times, but many challenges remain, such as drug-resistance, toxicity towards non-malignant cells and side effects, and inefficiency of drug delivery. Concerning the latter, one cause could be the inability of pharmaceutical compounds to cross the plasma membrane, which a semi-permeable hydrophobic barrier that ensures the integrity of cells. Therefore, several recent studies have focussed on the development of alternative drug delivery systems, such as viral based-vectors, nanoparticles, or cell-penetrating peptides that enhance cell internalisation.

Cell-penetrating peptides are classified as relatively short amphipathic and cationic peptides (7–30 amino acid residues) with the ability to cross biological membranes in an energy-dependent or -independent manner. Since the late 1980s, more than 1,700 cell-penetrating peptides have been characterised and listed in the CPPsite 2.0 database [1]. They have been experimentally validated for in vitro and in vivo delivery of small or large (≤120 kDA) bioactive cargo inside cells.

Cell-penetrating peptides have the unique ability to transport various cargos inside cells with limited toxicity. They are now considered as a powerful tool for both fundamental biology and medical applications. For example, they can deliver contrast agents (e.g. Quantum dots or metal chelates) for cell imaging purposes. Additionally, cell-penetrating peptides can transport nucleic acids (e.g. siRNA, antisense oligomers, plasmids, decoy DNA), for which intracellular delivery is often restricted by high molecular weight and negative charges, making the regulation of gene expression straightforward. Finally, they can provide effective drug delivery, ranging from nanoparticles to therapeutic proteins, and have been successfully used in many in vitro and in vivo studies. Critically, while cell-penetrating peptides can cross cellular membranes, many studies have shown that the majority of cell-penetrating peptides cannot cross the blood-brain barrier, which protects the central nervous system from toxicity. Currently, there are over 30 cell-penetrating peptides -conjugated drugs in development for clinical applications ranging from inflammation, pain, chronic wound management, bone regeneration, cancer and cardiovascular diseases.

The multiplicity of pathways and cell types investigated shows there is considerable opportunity for cell-penetrating peptide-based therapies. The potential clinical success of these therapies is not only because of their excellent intracellular delivery performance but also due to their adaptability; they are relatively simple to synthesise, to change and to improve. Nevertheless, currently, there are still no clinically approved cell-penetrating peptide-conjugated drugs commercially available. Potential reasons for this are:

  1. issues concerning in vivo stability, due to regular sensitivity to proteolysis;
  2. immunogenicity issues;
  3. poor efficiency due to the cargo’s failure to escape from endosomes following cell internalisation;
  4. poor efficiency due to the absence of specificity of the cell-penetrating peptide; and
  5. toxicity due to excipient degradation.

Finally, the cost and ability to manufacture at scale must also be considered, as well as hazards associated off-target effects.

In our respective labs (Prof Dunne and Prof McCarthy), our research is centred on the design and development of novel cell-penetrating peptide delivery systems for nucleic acids and anionic small molecules [2-18]. These are peptide (RALA and CHAT) delivery systems that are purposely designed to solve key criteria for controlled and effective intracellular delivery. We have used our patented cell-penetrating peptide technology for genetic therapies in cancer, chronic wounds and bone regeneration). Prof McCarthy is the Founder and Chairperson and Prof Dunne is the Scientific Advisor for pHion Therapeutics (www.phiontx.co.uk), a university spin-out company utilising the cell-penetrating peptide technology for nucleic acid vaccines and ex vivo therapies.

Please share your views and opinions on your experiences in developing cell-penetrating peptides and where you see future research opportunities that will accelerate the clinical translation of cell-penetrating peptides for efficient single/multi-cargo intracellular delivery.

References:
1. Agrawal et al. CPPsite 2.0: a repository of experimentally validated cell-penetrating peptides. Nucleic acids research. 2016 Jan 4;44(D1):D1098-103.
2. McErlean et al. Rational Design and Characterisation of an amphipathic cell-penetrating Peptide for Non-Viral Gene Delivery. International Journal of Pharmaceutics. 2021 Jan 27:120223.
3. O’Doherty et al. Improving the Intercellular Uptake and Osteogenic Potency of Calcium Phosphate via Nanocomplexation with the RALA Peptide. Nanomaterials. 2020 Dec;10(12):2442.
4. McErlean et al. Rational design and characterisation of a linear cell-penetrating peptide for non-viral gene delivery. Journal of Controlled Release. 2020 Nov 21.
5. Yan et al. Collagen/GAG scaffolds activated by RALA-siMMP-9 complexes with potential for improved diabetic foot ulcer healing. Materials Science and Engineering: C. 2020 Sep 1;114:111022.
6. Mulholland et al. Delivery of RALA/siFKBPL nanoparticles via electrospun bilayer nanofibres: An innovative angiogenic therapy for wound repair. Journal of Controlled Release. 2019 Dec 28;316:53-65.
7. Cole et al. DNA vaccination via RALA nanoparticles in a microneedle delivery system induces a potent immune response against the endogenous prostate cancer stem cell antigen. Acta Biomaterialia. 2019 Sep 15;96:480-90.
8. Gonzalez-Fernandez et al. Pore-forming bioinks to enable spatio-temporally defined gene delivery in bioprinted tissues. Journal of Controlled Release. 2019 May 10;301:13-27.
9. Sathy et al. Hypoxia mimicking hydrogels to regulate the fate of transplanted stem cells. Acta Biomaterialia. 2019 Apr 1;88:314-24.
10. McCrudden et al. Gene therapy with RALA/iNOS composite nanoparticles significantly enhances survival in a model of metastatic prostate cancer. Cancer Nanotechnology. 2018 Dec;9(1):1-5.
11. Cole et al. DNA vaccination for cervical cancer: Strategic optimisation of RALA mediated gene delivery from a biodegradable microneedle system. European Journal of Pharmaceutics and Biopharmaceutics. 2018 Jun 1;127:288-97.
12. Pauly et al. Hierarchically structured electrospun scaffolds with chemically conjugated growth factor for ligament tissue engineering. Tissue Engineering Part A. 2017 Aug 1;23(15-16):823-36.
13. Gonzalez-Fernandez et al. Mesenchymal stem cell fate following non-viral gene transfection strongly depends on the choice of delivery vector. Acta Biomaterialia. 2017 Jun 1;55:226-38.
14. Ali et al. DNA vaccination for cervical cancer; a novel technology platform of RALA mediated gene delivery via polymeric microneedles. Nanomedicine: Nanotechnology, Biology and Medicine. 2017 Apr 1;13(3):921-32.
15. McCrudden et al. Systemic RALA/iNOS nanoparticles: a potent gene therapy for metastatic breast cancer coupled as a biomarker of treatment. Molecular Therapy-Nucleic Acids. 2017 Mar 17;6:249-58.
16. Sathy et al. RALA complexed α-TCP nanoparticle delivery to mesenchymal stem cells induces bone formation in tissue-engineered constructs in vitro and in vivo. Journal of Materials Chemistry B. 2017;5(9):1753-64.
17. McBride et al. Development of TMTP-1 targeted designer biopolymers for gene delivery to prostate cancer. International journal of pharmaceutics. 2016 Mar 16;500(1-2):144-53.
18. McCarthy et al. Development and characterization of self-assembling nanoparticles using a bio-inspired amphipathic peptide for gene delivery. Journal of Controlled Release. 2014 Sep 10;189:141-9.