Localized tacrolimus therapy: innovations in peripheral nerve regeneration through advanced drug delivery systems

1. Introduction

Peripheral nerve injury (PNI) can have a profound and devastating impact on a patient’s quality of life. Functional impairment, particularly in the upper extremities, frequently requires the patient to change his career or go on disability. Moreover, patients frequently encounter mental health challenges consequent to their PNI [1]. Despite decades of research dedicated to enhancing peripheral nerve repair, certain physiological processes following PNI significantly contribute to a poor outcome and present a barrier that available treatment options cannot overcome. One major challenge is when nerve regeneration is required over a long distance. The more proximal the nerve injury occurs, the larger is the distal segment that undergoes Wallerian degeneration and requires regeneration. Given that nerve fibers grow at a rate of approximately 1 mm/day, a significant amount of time passes before regenerating axons reach their intended muscle target at a distant location [2]. During this time period, changes occur in the denervated muscle. In the initial weeks following muscle denervation, an environment ideal for reinnervation is established. However, in later stages of denervation, the muscle becomes atrophic and undergoes increased fibrous tissue and fat deposition. In addition, neuromuscular junctions (NMJs), which represent the interface between the nerve and muscle, degenerate [3]. The more NMJs degenerate, the less than chance of successful reinnervation. At approximately 12–18 months, complete loss of NMJs is observed, making reinnervation impossible [4]. However, occasionally longer denervation periods have been reported with successful reinnervation in the past [5]. Thus, achieving successful muscle reinnervation in cases of long regeneration distances is extremely challenging, as the muscle tissue typically degenerates before the nerve fibers can reach it. If nerve regeneration rate could be accelerated, the possibility of muscle reinnervation in proximal nerve injuries is increased significantly.

Tacrolimus, also known as FK506, is a calcineurin inhibitory that is primarily administered as immunosuppressant to patients undergoing organ transplantation [6]. In 1994, tacrolimus was observed to promote sensory neurite outgrowth, initiating multiple studies to investigate its effect on nerve regeneration [7]. Although the full mechanism of action has not yet been elucidated, previous studies have shown that tacrolimus targets the injured neuron and increases neurotrophic growth factors via the FK506 binding protein [8].

Systemic administration of tacrolimus following a nerve crush injury demonstrated not only an earlier return of function but also a 2.75-fold increase in axon fiber quantity [9]. Further studies followed, verifying that tacrolimus enables an improvement in axonal regeneration by 12–15% as it increases the number, diameter and remyelination of regeneration axons [10]. More importantly, besides increasing axon quantity, tacrolimus was observed to also accelerate nerve regeneration [9]. This sparked further interest in FK506 treatment for nerve regeneration, as it presented as a possible solution to overcome the challenges of proximal nerve injuries in the extremities.

2. Current challenges in therapeutic delivery of tacrolimus

FK506 is a potent immunosuppressor. It affects the ABCB1 protein which expression is linked to inflammation and can lead to graft loss or acute rejection by triggering inflammatory markers like IL-2, IL-6 and TNF-α [11]. Tacrolimus can influence nerve regeneration by reducing the levels of these inflammatory cytokines. By lowering IL-2, tacrolimus can prevent excessive T-cell activation that might harm regenerating nerves. Reducing IL-6 and TNF-α levels can decrease inflammation, thus creating a more favorable environment for nerve repair.

However, tacrolimus is also known for having a narrow therapeutic window, which may lead to major side effects if not administered correctly or not well tolerated. Serious side effects include infections, diabetes, seizures, coma, fractures, malignancies and nephrotoxicity [12]. Even though recent studies demonstrated promising advances in nerve regeneration with the administration of also lower FK506 dosages, the systemic administration of FK506 for nerve regeneration remains ethically questionable, as the negative effects may outweigh the positive benefits, making it clinically not justifiable. Nevertheless, given the proven beneficial effect of tacrolimus, research has shifted toward novel local delivery systems in order to reduce the drug concentration but also limit its effects to a small local area only.

3. Novel strategies for therapeutic delivery of tacrolimus

3.1. Overview & putative advantages of novel drug delivery systems

Novel drug delivery systems (DDS) are platforms designed to transport a pharmaceutical compound to specific locations in the body. By enhancing bioavailability, DDS ensures that a higher percentage of the administered drug reaches the target site, allowing administration of lower concentrations while maintaining potency and reducing possible systemic side effects. Moreover, DDS can ensure sustained release, which allows for a continuous supply of the therapeutic agent over an extended period of time, mitigating the need for frequent dosing. This is particularly interesting in regard to improving patient compliance and maintaining consistent drug levels. The main aim of DDS is to reduce systemic toxicity, a vital consideration for drugs like FK506, which can have pronounced side effects [13]. A variety of different systems have found their way into applying FK506 for nerve repair. Among the first were osmotic pumps, which have shown promise in enhancing motor function post nerve repair; however, their non-biodegradability poses clinical challenges, leading to a shift toward biodegradable, biocompatible systems like nanoparticles (NPs) and biomaterials [14]. A common denominator and for FK506 especially interesting, is poly(lactic-co-glycolic acid) (PLGA). PLGA’s capacity for biphasic drug release can potentially align with the bimodal (double peak) dose-response of tacrolimus, which shows increased regenerative effects at both low and high concentrations, but not at intermediate doses [15]. This response is thought to arise from FK506 interacting with different immunophilin receptors, such as FKBP-52 which has two binding domains, suggesting two distinct mechanisms of action.

3.2. NP-based delivery systems

NPs are a diverse group of nanoscale objects with sizes typically ranging from 1 to 100 nm. These constructs are garnering significant interest in the realm of localized drug delivery due to their capability to enhance the biodistribution of therapeutic agents. This is achieved through improved drug solubility and stability, the ability to traverse biological barriers and targeted delivery to specific organs or cells [16]. Recent studies have explored various NP platforms, including lipid-based NPs, polymeric NPs and inorganic NPs, each providing distinct advantages in terms of biocompatibility, drug release kinetics and targeting capabilities [17]. Lipid NPs have been shown to improve the bioavailability and lymphatic delivery of FK506, potentially reducing systemic side effects while enhancing therapeutic outcomes at the site of nerve injury [18]. Similarly, polymeric NPs offer controlled release mechanisms that ensure sustained drug delivery, which is crucial for the prolonged treatment periods often necessary in nerve regeneration [19]. The potential efficacy of NP systems lies not only in their ability to deliver FK506 directly to the affected site but also in their potential to bypass physiological barriers that typically hinder drug delivery. This is particularly evident in strategies employing advanced designs that allow NPs to navigate the extracellular matrix (ECM) and cellular environments within injured tissues, thus ensuring that the drug reaches its intended target effectively and efficiently [20]. Further research and clinical trials will be critical to bridge the gap between promising preclinical results and successful therapeutic applications of NP-mediated FK506 delivery.

3.3. Hydrogel-based delivery systems

Hydrogels are cross-linked, water-rich polymer networks maintaining their gel-like consistency and tissue-like structure due to cross-linking of individual polymer chains [21]. Considered biomimetic scaffolds, they mimic the natural ECM, providing a supportive structure that closely resembles the natural environment of nerve tissues, thereby enhancing cell adhesion, proliferation and differentiation [22]. Hydrogel-based DDS have emerged as a versatile platform in the controlled release of therapeutic agents, such as FK506, for tissue repair and regeneration. Within regard to advanced drug delivery, various types of hydrogels stand out: injectable hydrogels for encapsulating drugs through minimally invasive means, thermosensitive hydrogels that form gels in response to body heat for sustained drug release and self-assembling peptide hydrogels that promote tissue regeneration by forming structures under physiological conditions [23]. Wang et al. [24] developed a chitosan-based thermosensitive hydrogel which allowed co-delivery of FK506 and ciliary neurotrophic factor to an injured rabbit optic nerve. They describe protective effects on retinal ganglion cells; however, thorough evaluation modalities are missing. A research group from Taiwan followed the same route and designed a mixed thermosensitive hydrogel (poloxamer [PLX]-poly[l-alanine-lysine with Pluronic F-127]) which significantly improved the functional recovery of injured sciatic nerves in mice [25]. Disadvantages of hydrogels include unprecise control over release kinetics as well as fluctuations in degradation rates. Furthermore, it is difficult to incorporate a hydrophobic drug such as FK506 within hydrogels which is why it needs to be encapsulated in its solid drug particulate form or/and in a combination of other biomaterials. The group under Gregory Borschel developed a local DDS in the form of FK506 encapsulated in PLGA microspheres that are incorporated into a fibrin hydrogel which provides sustained, local release of FK506 over 4 weeks, significantly improving the axon regeneration without evidence of systemic toxicity [26]. They applied this system through fresh nerve allografts to repair a 20 mm peroneal nerve gap in rat [27]. The local FK506 DDS matched systemic FK506 in enhancing axonal regeneration in nerve allografts and surpassed them by avoiding systemic drug levels and side effects, allowing for weight gain and reduced dosage. Additionally, both delivery methods significantly decreased serum IL-12, indicating that local FK506 effectively provides targeted immunosuppression necessary for improved nerve repair [27]. This suggests the dual advantage of local FK506 in facilitating nerve regeneration while also exerting an immunosuppressive effect.

3.4. Scaffold-mediated delivery systems

Scaffold-mediated DDS are structures that provide physical support for tissue regeneration while simultaneously enabling the localized release of therapeutic agents. These scaffolds are engineered from materials like polymers, ceramics or composites and are often porous to facilitate cell infiltration and tissue integration. Kim et al. [28] used commercially available collagen wraps soaked in FK506 in fibrin glue to treat a rat sciatic nerve transection, but found no significant differences between the sham group, the group treated with collagen wrap only and the experimental group. A study by Davis et al. [29] designed a micropatterned PLGA film which released sustained amounts of FK506 over 56 days. Davis et al. [30] further constructed nerve wraps by embedding poly(L-lactide-ε-caprolactone) films with low (0.1% w/w) and high doses (0.5% w/w) of FK506. They observed improved regeneration in the low dose wrap group but reduced end-target reinnervation in the high dose wrap group. This study highlights a dose-dependent effect of FK506 on nerve regeneration, with optimal outcomes at mid-range dosages while suggesting neurotoxicity at high concentrations. Xiao et al. [31] investigated the efficacy of a polyester urethane urea (PEUU) elastomer blended with FK506 (PEUU-FK506) on improving sensory recovery in an infraorbital nerve injury model in rats. By comparing two groups, one group undergoing daily 2.2 mg/kg intraperitoneal FK506 injections and one group receiving a 5 mm × 1.5 mm section of 10 mg PEUU-FK506 wrap around the nerve coaptation, they investigated the systemic as well as local drug concentrations. Their finding showed significant differences in the drug concentration with FK506 being significantly higher locally as well as lower systemically when using the nerve wrap. Sensory outcomes demonstrated an improved trend in nerve regeneration, however without any significant differences. This study is a great example for the beneficial effects of local delivery systems for FK506. Further studies are necessary to evaluate its efficacy in regard to motor functional outcomes.

3.5. Nerve conduit-based delivery systems

Nerve conduits are tubular structures used to guide the regeneration of damaged peripheral nerves [32]. Local delivery of FK506 using nerve conduits enables physical protection as well as a targeted method to bridge nerve gap defects. Compared with hydrogels and other scaffolds, nerve conduits offer precise customization in size and geometry as well as degradation rates and enhanced mechanical properties. This ensures structural support and facilitates a simpler surgical procedure by minimizing the need for subsequent removal surgery. Conduits can be constructed out of a variety of materials, both natural and synthetic. Chitosan conduits, for example, consist of the naturally occurring polysaccharide derived from chitin. Zhao et al. [33] used a FK506-loaded chitosan conduit to repair a rat nerve injury and reported enhanced nerve regeneration through upregulation of brain-derived neurotrophic factor and Tropomyosin receptor kinase B. Synthetic nerve conduits, on the other hand, offer the advantages of customizable properties, scalability and no risk of disease transmission, with a consistent quality that supports nerve regeneration without provoking an immune response. Labroo et al. [34] designed a PLGA conduit for controlled FK506 release which facilitated optimal dorsal root ganglia neuron outgrowth over 20 days. The group under Jayant Agarwal further constructed polytetrafluoroethylene conduits enabling controlled release of FK506 and glia-cell line-derived neurotrophic factor, respectively. They employed this conduit to treat a 10 mm mouse sciatic nerve gap and showed significant improvement in muscle mass, compound muscle action potential and axon myelination [35]. A study by Davis et al. [36] used a FK506-loaded poly(L-lactide-ε-caprolactone) nerve conduit to treat an 8.0 mm mouse sciatic nerve gap. They demonstrated that local FK506 delivery in nerve grafts is as effective as systemic delivery in promoting nerve regeneration for PNI, with both methods achieving outcomes similar to autografts despite an initial increase in CD4⁺ cell infiltration.

In summary, biodegradable, biocompatible systems such as PLGA have shown promise due to their biphasic drug release properties, aligning well with tacrolimus’s dose-response characteristics. Lipid and polymeric NPs improve drug solubility, stability and targeted delivery, essential for prolonged nerve regeneration treatments. Hydrogels, considered biomimetic scaffolds, mimic the ECM can be engineered for controlled FK506 release. Scaffold-mediated delivery systems and nerve conduits offer structural support and localized drug release, with materials like PLGA, chitosan and synthetic polymers providing customizable, effective solutions for nerve repair. These advanced biomaterials, by integrating tacrolimus, enhance nerve regeneration by reducing inflammation and supporting Schwann cell and neuronal functions, thus improving overall outcomes in nerve repair.

4. Challenges & future perspectives of novel delivery systems

Current research on the local delivery of FK506 for nerve regeneration is promising but harbors significant limitations that must be addressed. Some disadvantages around the drug’s toxicity are well known, such as pharmacokinetic challenges in achieving and maintaining therapeutic concentrations at the injury site without inducing systemic toxicity as well as the drug’s highly dose-dependent effects. Moreover, the long-term safety of FK506, particularly at higher concentrations necessary for nerve regeneration, remains poorly understood. However, a primary concern in the ongoing research which is rarely addressed is the ambiguity in differentiating the drug’s immunomodulatory effects from its direct regenerative capabilities. Often labeled as regenerative, the observed benefits of FK506 may largely be due to its potent immunosuppressive properties, potentially just decelerating nerve degeneration rather than actively promoting regeneration. This crucial distinction could significantly influence both the therapeutic approach and the evaluation of FK506’s effectiveness in clinical practice. Most studies do not include immune assays, focusing instead on macroscopic outcomes like nerve growth rates or functional recovery. This lack of immunological data is a significant omission, considering FK506’s primary mechanism of action involves modifying immune responses. Without these data, it is challenging to understand how FK506 might indirectly affect nerve healing through immunological pathways. This would not only clarify the direct and indirect benefits of FK506 but also enhance therapeutic strategies by tailoring DDS that optimize regenerative outcomes while minimizing immunosuppressive side effects.

5. Conclusion

Peripheral nerve injuries remain a significant clinical challenge, particularly when nerve regeneration distance is long and muscles degenerate before reinnervation is possible. FK506 has shown a promising potential in enhancing nerve regeneration by increasing axonal growth and promoting earlier functional recovery. Yet, systemic administration of FK506 is associated with severe side effects, highlighting the need for local delivery systems.

Recent advancements in NP, hydrogel, scaffold-mediated and nerve conduit-based delivery systems have shown promising approaches of localized delivery of FK506, thereby reducing systemic toxicity and enhancing therapeutic efficacy. Nevertheless, significant challenges remain, including achieving and maintaining therapeutic concentrations at the injury site as well as clarifying whether FK506 directly promotes nerve regeneration or rather slows nerve degeneration.

Optimizing FK506 DDS will possibly aid in developing more effective therapeutic strategies for PNI, ultimately improving patient outcomes and quality of life.

Financial disclosure

The authors have no financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Competing interests disclosure

The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, stock ownership or options and expert testimony.

Writing disclosure

No writing assistance was utilized in the production of this manuscript.