Towards next generation polymers for Gene-Delivery applications

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Coronavirus disease 2019 (COVID-19) is the historical landmark for the expanding development of mRNA therapeutics. More specifically, nucleic acid-based therapies have emerged as promising and efficient approaches to treat diseases, including genetic disorders, cancers, neurological disorders. Indeed, nucleic acids can be used to correct a disease-related inherited gene by adding, removing, or replacing genetic material, or to make cells and tissues behave in a certain way by modifying gene expression (1). Importantly, nucleic acid-based therapeutics must act intracellularly, although most of them are rapidly cleared and degraded after systemic administration and do not really cross the plasma membrane.
As a result, the use of gene-delivery vectors to protect and enable their transport inside cells with therapeutic efficiency and with minimal adverse effects to patients, are essential. To date, gene-delivery vectors are mainly classified into two major categories, including viral and nonviral vectors. The use of viral vectors has raised considerable safety concerns, such as immune responses, inflammatory responses, toxicity problems, and insertional mutagenesis. Consequently, non-viral vectors, which offer non-immunogenicity, low cost, improved loading capacity and high chemical versatility, have gained significant attention for gene delivery in recent years.
While lipid nanoparticles (LNPs) as non-viral vectors, have shown distinct advantages, they face stability and storage issues (2), strong immune stimulation, complex manufacturing and formulation heterogeneity (3). Polymeric vehicles have emerged as a promising solution due to their versatile structure, ease of functionalization, and stability. To be successful, polymeric vehicles must perform multiple functions. They must efficiently load and protect nucleic acid cargo, evade the immune system and premature clearance mechanisms, achieve cellular uptake and endosomal escape, and disappear without toxicity. Designing vehicles to fulfill all of these criteria has been challenging, but recent approaches aim to enhance vehicle performance through the modification of polymer physicochemical properties (eg. composition, molecular weight, and polydispersity), architecture, surface properties, and the combination of traditional polymers to create versatile carriers. One common feature in most polymers designed for nucleic acid delivery is the incorporation of cationic groups, with two purposes: first, to aid with the loading of negatively charged nucleic acid cargo, and second, to facilitate the interaction with negatively charged glycoproteins on the cell membrane (4).

 

Despite obvious safety and toxicity concerns, polymer biodegradability represents one of the most critical features affecting the clinical applicability of final biomedical products because it substantially influences their biocompatibility and biosafety. In addition, the need of repeated administration in gene-based therapies, reinforces the importance of biodegradability for the design of gene-delivery vectors. Nevertheless, there is a lack of standardized biodegradation testing methods (5), especially for polymers characterization, which could reasonably rely on libraries of existing biodegradation studies of polymers in a near future.

Both naturally-derived and synthetic polymers can be biodegradable, making them potentially safer options for gene delivery. However, the challenge for reaching also high performance, requires adjusting structural physicochemical functions. The best compromise is to consider the diversity of existing polymers and tune physicochemical properties, such as:

• Balancing the charge density and molar mass of polymers,
• Modulating the chemical structure,
• Controlling the topological structure of the polymer,
• Introducing stimuli-responsive groups into polymers.

 

Among naturally-derived polymers, structural proteins and polysaccharides (6), such as cationic collagen, or chitosan derivatives (7) have been studied. They generally offer biocompatibility and biodegradability but suffer from batch-to-batch variation and strong immunogenic responses. In contrast, synthetic polymers provide precise control in high quality synthesis, but for ensuring efficiency, such as interactions with cells, partial or non-biodegradable polymeric functionalities might need to be grafted.

 

Synthetic polymers with hydrolytically-labile bonds, like polyesters, have been extensively studied. Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) are biodegradable polymers known for their biocompatibility and encapsulation properties, allowing safe metabolism in the body. Several innovative formulations have been developed (8).

Indeed, biodegradable aliphatic polyesters have found widespread application in various biomedical products, including sutures, tissue-engineering scaffolds, and drug-delivery carriers. In recent years, diverse types of polyesters with varying monomer units have been extensively used for gene-delivery purposes (9). Various polymeric combinations based on amines and bisacrylates have been employed to create a library of poly(beta-amino ester) (PBAE) derivatives (10).

 

Poly(ethyleneimine) (PEI) is an extensively studied synthetic polymer for the encapsulation and delivery of genetic cargo. Different PEIs have been modified that are either based on low molecular weight PEI with biodegradable linkages or linear PEI that are less toxic than their branched counterparts. Modification with PEG chains enhance their in vivo performance and also imparts targeting as well as biodegradation capability to PEIs. Interestingly, polymer pegylation can drastically reduce hepatic clearance eg. cardiovascular treatments (11). Indeed, factors that influence clearance and biodistribution include, the composition, size, surface modifications of polymeric vehicles.
Polycarbonates (PCs) are gaining attention for gene-delivery applications due to their high biocompatibility, low cytotoxicity, and tunable mechanical properties. Polyurethanes (PUs), known for their elasticity, flexibility, and biodegradability, have shown promising results in tissue-engineering applications and gene delivery (12).
Finally, by incorporating a bioreducible disulfide function into the polymer backbone, which self-degrade via bonding cleavages, offer a unique approach for gene delivery, as cytotoxicity can be greatly enhanced while maintaining a good transfection efficiency. Representative bioreducible polymers include degradable PEI derivatives (13).

 

Despite rational and effective chemical structural designs, smarter vectors will need to be developed for enhancing in vivo stability and controlled gene release in gene-based therapies.
More investigations on clearance, biodegradability, and biocompatibility of polymeric components are still needed for accelerating clinical translation of polymeric mRNA delivery systems. Next generation of polymers need to be more biodegradable and biocompatible delivery vehicles (8).

 

Synthetic polymer families & general overview of factors influencing biodegradability

 

References and notes

1. Piotrowski-Daspit AS et al. Polymeric vehicles for nucleic acid delivery. Adv Drug Deliv Rev. 2020;156:119-132. doi: 10.1016/j.addr.2020.06.014
2. Dapeng Zhang et al. Journal of the American Chemical Society 2021 143 (31), 12315-12327, DOI: 10.1021/jacs.1c05813.
3. Friesen JJ, Blakney AK. Trends in the synthetic polymer delivery of RNA. J Gene Med. 2024; 26(2):e3672. doi:10.1002/jgm.3672
4. Ni R, Feng R, Chau Y, Synthetic Approaches for Nucleic Acid Delivery: Choosing the Right Carriers, Life (Basel), 9 (2019).
5. Joonrae Roger Kim et al. Exploring structure-activity relationships for polymer biodegradability by microorganisms, Science of The Total Environment, Volume 890, 2023, 164338.
6. Kuperkar K et al. Degradable Polymeric Bio(nano)materials and Their Biomedical Applications: A Comprehensive Overview and Recent Updates. Polymers (Basel). 2024 Jan 10;16(2):206.
7. Cosco D, Cilurzo F, Maiuolo J, Federico C, Di Martino MT, Cristiano MC, Tassone P, Fresta M, Paolino D, Delivery of miR-34a by chitosan/PLGA nanoplexes for the anticancer treatment of multiple myeloma, Sci Rep, 5 (2015) 17579.
8. a) Zabihi, F. et al. Synthesis of poly(lactide-co-glycerol) as a biodegradable and biocompatible polymer with high loading capacity for dermal drug delivery. Nanoscale 2018, 10, 16848–16856; b) Ren, B et al. Anti-inflammatory effect of IL-1ra-loaded dextran/PLGA microspheres on Porphyromonas gingivalis lipopolysaccharide-stimulated macrophages in vitro and in vivo in a rat model of periodontitis. Biomed. Pharmacother. 2021, 134, 111171; c) Lu Y, Cheng D, Niu B, Wang X, Wu X, Wang A. Properties of Poly (Lactic-co-Glycolic Acid) and Progress of Poly (Lactic-co-Glycolic Acid)-Based Biodegradable Materials in Biomedical Research. Pharmaceuticals (Basel). 2023 Mar 17;16(3):454.
9. LiPei Huang, Hongzhang Deng, Yongfeng Zhou, Xiaoyuan Chen, The roles of polymers in mRNA delivery, Matter, Volume 5, Issue 6, 2022, 1670-1699.
10. Ooi YJ, Huang C, Lau K, Chew SY, Park JG, Chan-Park MB. Nontoxic, Biodegradable Hyperbranched Poly(β-amino ester)s for Efficient siRNA Delivery and Gene Silencing. ACS Appl Mater Interfaces. 2024 Mar 6. doi: 10.1021/acsami.3c10620.
11. Xiyu Ke et al. Surface-Functionalized PEGylated Nanoparticles Deliver Messenger RNA to Pulmonary Immune Cells, ACS Applied Materials & Interfaces 2020, 12 (32), 35835-35844
12. Mahdieh A, Motasadizadeh H, Maghsoudian S, Sabzevari A, Khalili F, Yeganeh H, Nyström B. Novel polyurethane-based ionene nanoparticles electrostatically stabilized with hyaluronic acid for effective gene therapy. Colloids Surf B Biointerfaces. 2024 Feb 16;236:113802. doi: 10.1016/j.colsurfb.2024.113802.
13. Fattahi N, Gorgannezhad L, Masoule SF, Babanejad N, Ramazani A, Raoufi M, Sharifikolouei E, Foroumadi A, Khoobi M. PEI-based functional materials: Fabrication techniques, properties, and biomedical applications. Adv Colloid Interface Sci. 2024 Mar;325:103119. doi: 10.1016/j.cis.2024.103119.