Cytokines are polypeptides or glycoproteins that help immune cells communicate to each other to induce proliferation, activation, differentiation, and inflammatory or anti-inflammatory signals in various cell types.Gonçalves, A., Machado, R., Gomes, A. C., & Costa, A. da. (2020). Nanotechnology Solutions for Controlled Cytokine Delivery: An Applied Perspective. NATO Advanced Science Institutes Series E: Applied Sciences, 10(20), 7098.Razeghian, E., Margiana, R., Chupradit, S., Bokov, D. O., Abdelbasset, W. K., Marofi, F., Shariatzadeh, S., Tosan, F., & Jarahian, M. (2021). Mesenchymal Stem/Stromal Cells as a Vehicle for Cytokine Delivery: An Emerging Approach for Tumor Immunotherapy. Frontiers of Medicine, 8, 721174. Studies utilizing cytokines for antitumor therapies has increased significantly since 2000, and different cytokines provide unique antitumor activities.Nash, A. M., Jarvis, M. I., Aghlara-Fotovat, S., Mukherjee, S., Hernandez, A., Hecht, A. D., Rios, P. D., Ghani, S., Joshi, I., Isa, D., Cui, Y., Nouraein, S., Lee, J. Z., Xu, C., Zhang, D. Y., Sheth, R. A., Peng, W., Oberholzer, J., Igoshin, O. A., … Veiseh, O. (2022). Clinically translatable cytokine delivery platform for eradication of intraperitoneal tumors. Science Advances, 8(9), eabm1032. Cytokines hinder tumor cell development mostly through antiproliferative or proapoptotic pathways but can also interrupt development indirectly by eliciting immune cells to have cytotoxic effects against tumor cells. Even though there are FDA-approved cytokine therapies, there are two main challenges associated with cytokine delivery. The first is that cytokines have a short half-life, so frequent administration of high doses is required for therapeutic effect. The second is that systemic toxicity could occur if the cytokines delivered cause an intense immune response, known as a cytokine storm.Park, J., Gao, W., Whiston, R., Strom, T. B., Metcalfe, S., & Fahmy, T. M. (2011). Modulation of CD4+ T lymphocyte lineage outcomes with targeted, nanoparticle-mediated cytokine delivery. Molecular Pharmaceutics, 8(1), 143–152.Wang, B., Tan, L., Deng, D., Lu, T., Zhou, C., Li, Z., Tang, Z., Wu, Z., & Tang, H. (2015). Novel stable cytokine delivery system in physiological pH solution: chitosan oligosaccharide/heparin nanoparticles. International Journal of Nanomedicine, 10, 3417–3427.Dholakia, J., Cohen, A. C., Leath, C. A., 3rd, Evans, E. T., Alvarez, R. D., & Thaker, P. H. (2022). Development of Delivery Systems for Local Administration of Cytokines/Cytokine Gene-Directed Therapeutics: Modern Oncologic Implications. Current Oncology Reports, 24(4), 389–397.
Pegylated cytokines Pegylation is the process of covalently binding polyethylene glycol (PEG) to proteins. Pegylation prolongs the half-life of the bound protein, leading to sustained delivery.Autio, K., & Oft, M. (2019). Pegylated Interleukin-10: Clinical Development of an Immunoregulatory Cytokine for Use in Cancer Therapeutics. Current Oncology Reports, 21(2), 19.Battista, M. R., Grigoletto, A., Tedeschini, T., Cellucci, A., Colaceci, F., Laufer, R., Pasut, G., & Di Marco, A. (2022). Efficacy of PEGylated ciliary neurotrophic factor superagonist variant in diet-induced obesity mice. PLOS One, 17(3), e0265749. This is advantageous because lower, less frequent dosing will be needed to have the same therapeutic effect in the patient, which will limit the cytotoxicity of the delivery system. Pegfilgrastim is a successful example of this delivery system. Pegfilgrastim is the pegylated form of the granulocyte-colony stimulating factor (G-CSF) filgrastim. Pegfilgrastim stimulates production and release of neutrophils in patients who experience bone marrow toxicity after receiving myelosuppressive anticancer drugs or radiation. Filgrastim has a half-life of 3-4 h, while Pegfilgrastim has a half-life of 45 h. This is much more convenient for patients, as they will only need one dose of Pegfilgrastim instead of multiple doses of Filgrastim drawn out over a long period. Ciliary neurotrophic factor (CNTF) is a cytokine used to combat diabetes symptoms such as appetite reduction and weight loss. A pegylated version of CNTF retained biological activity in vitro and had enhanced pharmacokinetics. The pegylated CNTF also reduced glycemia in diet-induced obese animals, with a dose 10-fold higher than unmodified CNTF. These studies demonstrate that pegylated cytokines can be used for sustained delivery of cytokines, increasing the therapeutic window of these treatments.
A major problem caused by pegylation is that the cytokine may change its molecular conformation, activity, and bioavailability upon PEG binding. Cytokines are advantageous due to their small sizes, which allow them to reach intracellular targets. PEG binding to cytokines could cause them to become too bulky to reach their specific targets, which should be taken into consideration when designing pegylated cytokines.
GAG-based biomaterials Glycosaminoglycans (GAGs) are naturally derived polysaccharides with distinct sequences of disaccharides. GAGs bind to cytokines and regulate cell recruitment, inflammation, and tissue remodeling by delivering cytokines to the extracellular matrix.Fernandez-Botran, R., Gorantla, V., Sun, X., Ren, X., Perez-Abadia, G., Crespo, F. A., Oliver, R., Orhun, H. I., Quan, E. E., Maldonado, C., Ray, M., & Barker, J. H. (n.d.). TARGETING OF GLYCOSAMINOGLYCAN-CYTOKINE INTERACTIONS AS A NOVEL THERAPEUTIC APPROACH IN ALLOTRANSPLANTATION1. Hachim, D., Whittaker, T. E., Kim, H., & Stevens, M. M. (2019). Glycosaminoglycan-based biomaterials for growth factor and cytokine delivery: Making the right choices. Journal of Controlled Release: Official Journal of the Controlled Release Society, 313, 131–147. Binding studies suggest GAGs have a natural affinity for cytokines, and cytokine binding to GAGs is mediated by nonspecific electrostatic interactions between positively charged domains on cytokines and negatively charged sulfate and carboxylic acid residues on GAGs. The GAG heparin has been explored extensively for cytokine delivery. Heparin-based hydrogels have shown to provide sustained delivery of IL-4 for more than 2 weeks, leading to a greater anti-inflammatory response than IL-4 alone. Another study utilized a starPEG-heparin hydrogel system to deliver vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF-2). The large concentration of heparin allowed loading and release of the cytokines to be independent of each other.Zieris, A., Chwalek, K., Prokoph, S., Levental, K. R., Welzel, P. B., Freudenberg, U., & Werner, C. (2011). Dual independent delivery of pro-angiogenic growth factors from starPEG-heparin hydrogels. Journal of Controlled Release: Official Journal of the Controlled Release Society, 156(1), 28–36. The codelivery of the cytokines from the hydrogels led to pro-angiogenic effects both in vitro and in vivo, with the effect being much greater than administration of the single growth factors
Immunocytokines Antibody conjugation is another promising method for cytokine delivery. Antibody conjugation to cytokines can be used to improve site-specific delivery and prolong the cytokine half-life. Immunocytokines are delivered systemically but can specifically target the tumor through overexpressed or unique tumor antigens, cryptic extracellular matrix epitopes found only in tumors, or neovasculature markers indicating tumor angiogenesis.Nguyen, K. G., Vrabel, M. R., Mantooth, S. M., Hopkins, J. J., Wagner, E. S., Gabaldon, T. A., & Zaharoff, D. A. (2020). Localized Interleukin-12 for Cancer Immunotherapy. Frontiers in Immunology, 11, 575597. Cergutuzumab amunaleukin (CEA-IL2v) is an antibody-cytokine conjugate that links the cytokine IL-2 with an antibody targeting the carcinoembryonic antigen (CEA). This conjugate preferentially targets the tumor microenvironment to increase local delivery of IL-2 and minimize off-target toxicity. CEA-IL2v is ongoing phase I clinical trials in treating solid malignancies expressing CEA. Another immunocytokine is huBC1-IL12, which was developed to target the ED-B domain of fibronectin. This domain is overexpressed in tumor tissues but undetectable in almost all normal adult tissues. Systemic administration of huBC1-IL12 eliminated experimental PC3 metastases and suppressed the growth of multiple human tumor lines in immunocompromised mice more effectively than IL-12 alone. A Phase I trial studied the safety of weekly infusions of huBC1-IL12 in renal carcinoma and malignant melanoma patients. The maximum tolerated dose was found to be 15 ug/kg, which is 30 times higher than the maximum tolerated dose of IL-12 alone
Systemically administered immunocytokines are likely to significantly reduce cytokine-related cytotoxicity, but not eliminate it. Immunocytokines still interact with immune cells to induce signaling outside of the tumor, and there are problems with non-specific binding in non-target tissues that could disrupt regular immune functions in the body. Since immunocytokines are foreign to the body, they could also cause an immune reaction that produces anti-immunocytokine antibodies leading to pharmacological abrogation, therapeutic alteration, or hypersensitivity reactions.
Nonviral nanoparticles Nanoparticle delivery systems are popular due to their ability to encapsulate compounds without affecting bioactivity, and they exhibit controlled and sustained release of the encapsulated compounds to target tissues. Nanoparticles can be made of organic or inorganic agents and possess the ability to stabilize cytokines in vivo, enhance activity at the target site, improve aqueous solubility, and reduce systemic toxicity. Solvent, pH, temperature, charge, and size are many parameters that influence the encapsulation efficiency, nanoparticle toxicity, and cytokine stability. Cytokines can be encapsulated, adsorbed, or conjugated into nanoparticle systems for their delivery, and there are multiple nanoparticle systems that can be utilized for cytokine delivery
Polymeric nanoparticles are biocompatible, have low toxicity, and are biodegradable. They can be used to improve circulation times, stability, and encapsulation capacity compared to other nanoparticle systems. Polylactic-co-glycolic acid (PLGA) is the most popular polymer for nanoparticle delivery systems due to its simple synthesis using the oil-in-water method, high stability, easy encapsulation or adsorption of both hydrophobic and hydrophilic molecules, and easy surface modification. PLGA-PEG nanoparticles encapsulating IL-10 were created to prevent plaque formation in advanced atherosclerosis lesions. PLGA was charged at its terminal portions for better electrostatic interaction with IL-10 while PEG was used to functionalize the nanoparticle. These PLGA-PEG-IL-10 nanoparticles allowed for greater stability and prolonged systemic circulation of IL-10 and did not show any in vitro or in vivo cytotoxicity.