Electrospun fiber-mediated delivery of neurotrophin-3 mRNA for neural tissue engineering applications

Peripheral nervous system (PNS) injury resulting from disease or trauma impacts over 20 million individuals in the United States alone and can significantly reduce the patient's quality of life [1]. Recovery from PNS injury is often incomplete due to limited regeneration of damaged peripheral axons and deficient reinnervation of surrounding tissues [2,3]. A complete nerve transection injury gap larger than 1 cm typically requires surgical placement of a bridging graft to support axon regeneration and functional recovery [4]. A nerve autograft or allograft are the gold standard surgical options to bridge a large injury gap in a peripheral nerve [5]. However, aligned electrospun fiber-containing artificial nerve grafts serve as an alternative approach to bridge large injury gaps with the potential to overcome the limitations faced by autografts and allografts [4], [5], [6], [7]. The aligned fibers provide topographical features that mimic the native peripheral nerve extracellular matrix (ECM) and can be engineered to locally deliver therapeutics such as small molecule drugs, proteins, and nucleic acids over an extended duration to support robust axon regeneration [5,[8], [9], [10], [11], [12]].

Delivery of neurotrophic factor proteins from biomaterials following nervous system injury is of interest due to their known benefits on the regeneration process [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]. Neurotrophin-3 (NT-3) is a neurotrophic protein that binds to tyrosine kinase (Trk) receptors present on cells throughout the PNS, including Schwann cells and dorsal root ganglia (DRG) neurons [23], [24], [25], [26]. This enables NT-3 to elicit a broad range of responses in vitro and in vivo, such as increasing Schwann cell migration, mediating Schwann cell myelination, enhancing neurite outgrowth from DRG explants, and improving sensory axon regeneration [17,18,[27], [28], [29], [30], [31], [32], [33], [34], [35], [36], [37], [38], [39]]. However, the use of NT-3 as a clinical therapeutic is limited by its short half-life [36,40]. Alternatively, viral or non-viral DNA or RNA delivery can induce local expression of the desired protein [28,36,[41], [42], [43], [44], [45], [46]].

Synthetic messenger RNA (mRNA) delivery offers a non-viral approach to induce transient expression in the cytosol, removing the risk of insertional mutagenesis and improving the transfection efficiency of hard-to-transfect primary cells compared to DNA-based gene therapies [47], [48], [49], [50]. The instability and immunogenicity of mRNA have slowed the progression of mRNA therapeutics to the clinic [47]. However, the use of cationic lipid- and polymer-based gene delivery vehicles and incorporation of optimized capping structures like anti-reverse cap analog (ARCA) and modified nucleotides like pseudouridine-5’-triphosphate (Ψ) improve the delivery efficiency of bioactive mRNA and subsequent translation into the desired protein while reducing the risk of a severe immune response to the foreign genetic material [48,[51], [52], [53], [54], [55], [56]]. Synthetic mRNAs have been successfully delivered via systemic and local injection to increase the production and secretion of neurotrophic factors [43], [44], [45], [46]. Still, the limited success of mRNA therapeutics in tissue engineering and regenerative medicine is due, in part, to the challenge of delivering an efficacious dose of mRNA to the target location to induce sustained secretion of the desired protein for the optimal duration [50,57]. Electrospun fiber-based drug depots enable local, sustained, non-viral delivery of genetic material in the forms of plasmid DNA, small interfering RNA (siRNA), and microRNA (miRNA) while also providing structural support and guidance cues to enable robust nerve regeneration [11,12,[58], [59], [60], [61]]. However, mRNA delivery from electrospun fibers to improve neurite outgrowth or axon regeneration via production of neurotrophic factors has yet to be explored.

Poly(L-lactic acid) (PLLA) is an FDA-approved material commonly used to fabricate electrospun fibers for neural repair due to its biocompatibility and slow biodegradability [62], [63], [64]. Anionic surface coatings like poly(3,4-dihydroxy-L-phenylalanine) (pDOPA) and dextran sulfate sodium salt (DSS) have been employed to functionalize the electrospun fiber surface and improve immobilization of biologics for local, sustained delivery [12,61,[65], [66], [67], [68], [69], [70], [71]]. pDOPA possesses carboxyl groups and reactive o-quinones that support the immobilization of cationic delivery vehicles carrying genetic material through a variety of possible physical and chemical interactions, including electrostatic interactions, hydrophobic interactions, hydrogen bonding, or Shiff base or Michael addition reactions, depending on the surface chemistry of the gene delivery vehicle [12,61,[65], [66], [67], [68], [69], [70]]. DSS possesses sulfonate groups and has been deposited onto electrospun fibers to enable immobilization of a cationic enzyme via electrostatic interactions [71]. However, DSS has yet to be employed to immobilize genetic material to the fiber surface.

Here, we aimed to develop the first aligned electrospun fiber platform that delivers modified mRNA encoding NT-3 to 1) sustain local delivery of mRNA, 2) induce secretion of NT-3 protein from primary rat Schwann cells, and 3) enhance neurite outgrowth from rat DRG explants. First, we synthesized pseudouridine-5’-triphosphate (Ψ)-modified mRNA encoding NT-3 (ΨNT-3mRNA). Next, we fabricated aligned PLLA electrospun fibers and functionalized the fiber surface with a pDOPA or DSS coating. We then complexed the ΨNT-3mRNA to the cationic delivery vehicle JetMESSENGER® to form lipoplexes and immobilized the ΨNT-3mRNA/JetMESSENGER® lipoplexes to the functionalized electrospun fiber platforms. Finally, we investigated the ability of the ΨNT-3mRNA/JetMESSENGER®-loaded aligned electrospun fiber platforms to induce NT-3 protein secretion from rat Schwann cells and subsequently assessed whether increased NT-3 secretion from Schwann cells promoted neurite outgrowth from rat DRG explants. This study introduces an approach that combines sustained delivery of mRNA with topographical guidance cues to stimulate and guide neurite outgrowth and serves as a basis for future construction and in vivo testing of mRNA-loaded, electrospun fiber-containing artificial nerve grafts.

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