A functional tacrolimus-releasing nerve wrap for enhancing nerve regeneration following surgical nerve repair

Abstract

Axonal regeneration following surgical nerve repair is slow and often incomplete, resulting in poor functional recovery which sometimes contributes to lifelong disability. Currently, there are no FDA-approved therapies available to promote nerve regeneration. Tacrolimus accelerates axonal regeneration, but systemic side effects presently outweigh its potential benefits for peripheral nerve surgery. The authors describe herein a biodegradable polyurethane-based drug delivery system for the sustained local release of tacrolimus at the nerve repair site, with suitable properties for scalable production and clinical application, aiming to promote nerve regeneration and functional recovery with minimal systemic drug exposure. Tacrolimus is encapsulated into co-axially electrospun polycarbonate-urethane nanofibers to generate an implantable nerve wrap that releases therapeutic doses of bioactive tacrolimus over 31 days. Size and drug loading are adjustable for applications in small and large caliber nerves, and the wrap degrades within 120 days into biocompatible byproducts. Tacrolimus released from the nerve wrap promotes axon elongation in vitro and accelerates nerve regeneration and functional recovery in preclinical nerve repair models while off-target systemic drug exposure is reduced by 80% compared with systemic delivery. Given its surgical suitability and preclinical efficacy and safety, this system may provide a readily translatable approach to support axonal regeneration and recovery in patients undergoing nerve surgery.

Introduction

Approximately 43.8 per million people in the United States suffer traumatic nerve injuries annually (Karsy et al., 2019), most of which affect the upper extremity nerves that control crucial hand functions (Karsy et al., 2019; Tapp et al., 2019). Although the peripheral nervous system is capable of regeneration, functional recovery following surgical nerve repair is slow and often incomplete (Jaquet et al., 2001; Lan et al., 2019). Particularly in proximal nerve injuries, the inefficient rate of axonal regeneration of 1–2 mm/d (Seddon et al., 1943; Braam and Nicolai, 1993) represents a key issue. Society is in need of strategies that can accelerate axonal regeneration beyond what is currently achievable through surgery so that patients have the optimal chance of regaining their function after peripheral nerve injury.

To date, adjunct pharmaceutical therapies that can promote axonal regeneration following nerve surgery are clinically unavailable. The lack of clinical translation of promising experimental strategies may be attributable to concerns over the toxicity of bioactive agents necessitating comprehensive safety and efficacy studies. To overcome these concerns, we have focused on tacrolimus, an FDA-approved calcineurin inhibitor, which is widely used clinically as an immunosuppressant (Webster et al., 2005; Haddad et al., 2006; Baumgart et al., 2008; Lee et al., 2010; Cury Martins et al., 2015). Independent of its immunosuppressive actions, tacrolimus promotes axonal regeneration in vitro (Lyons et al., 1994; Quintá and Galigniana, 2012) and in vivo by 12% to 16% (Gold et al., 1995; Udina et al., 2002), which translates into faster functional recovery in rodents (Gold et al., 1994; Sulaiman et al., 2002; Udina et al., 2002). The neurotrophic effect of tacrolimus is mediated by FK506 binding protein (FKBP52; Steiner et al., 1997; Gold et al., 1999; Quintá and Galigniana, 2012; Daneri-Becerra et al., 2020) which forms heterocomplexes with the 90 kDa heat-shock protein and its co-chaperone p23 (Quintá and Galigniana, 2012) in the nucleus. In injured neurons, this complex is re-distributed to the growth cones of regenerating neurites upon cellular contact with tacrolimus, prompting their accelerated regeneration (Quintá and Galigniana, 2012). Further, FKBP52 mediates growth cone guidance in regenerating neurites in response to attractive and repulsive chemotactic signals (Shim et al., 2009).

However, side effects of systemically delivered tacrolimus, mainly nephrotoxicity (Bentata, 2020), often outweigh the expected benefits for patients undergoing nerve surgery (Zuo et al., 2020). Hence, localizing tacrolimus delivery to the nerve repair site through a biodegradable drug delivery system may allow tacrolimus to achieve its clinical potential while minimizing systemic toxicity.

Previously, our laboratory developed a composite microsphere-hydrogel system to localize the delivery of tacrolimus to injured axons (Tajdaran et al., 2015). We have shown that the controlled release of tacrolimus from this system over the course of 14 days is effective at promoting axonal regeneration whilst minimizing systemic exposure to other organs (Tajdaran et al., 2015, 2019a, b). However, this system faces challenges related to commercial translation and clinical usability as a result of laborious bedside preparation. Recognizing the need for convenience and effectiveness to achieve clinical translation, we have focussed on developing a novel off-the-shelf delivery system using a single-step electrospinning approach to encapsulate tacrolimus in core-shell electrospun fibrous nerve wraps made up of fully degradable polycarbonate urethanes. Here we present physical and bioactive drug release properties, demonstrate its microsurgical applicability, and determine its therapeutic efficacy and safety in a preclinical animal model of nerve injury and repair.

Methods

Electrospinning and physical characterization of the tacrolimus nerve wrap

Polycarbonate urethane (PCNU) was synthesized of hexane diisocyanate (52649, MilliporeSigma, Burlington, MA, USA), poly(hexamethylene carbonate) diol (461172, MilliporeSigma) and butanediol (309443, MilliporeSigma) in a 3:2:1 ratio as previously described (Wright et al., 2018). PCNU was mixed with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (105228, MilliporeSigma) to make the polymer solutions for electrospinning, in concentrations of 14% (w/v) for the inner core and 20% (w/v) for the outer shell. Tacrolimus (LC Laboratories, Woburn, MA, USA) was added as a dry powder to inner core PCNU solutions at a concentration of 5.2% (w/w). An electrospinning system (NanoSpinner, Inovenso Technology Inc, Boston, MA, USA) was used with a co-axial nozzle of two concentric 22- and 18-gauge blunt-tip needles for the inner core (drug load PCNU solution) and outer shell (PCNU solution alone), respectively. A flow rate of 0.5 mL/h and a constant voltage difference of 17 kV from nozzle tip to collector (1 kV/cm) was maintained. Electrospun matrices were collected and dried for 72 hours under vacuum at room temperature to remove residual solvent. The matrices were then wrapped in sterilization pouches and sterilized by gamma irradiation at a dose of 25 kGy (SOCAAR, University of Toronto, Ontario, CA, USA) and subsequently stored at 4°C until further use. Separate fibrous mats (n = 4) were electrospun for each experiment.

Scanning electron microscopy was used to characterize the morphological form of the nerve wrap (n = 6 tacrolimus encapsulated, n = 8 vehicle) from two electrospun matrices. Wraps were mounted, sputter coated with a 15 nm gold layer (Leica EM ACE200, Wetzlar, Germany), and imaged with a FEI XL30 ESEM (FEI, Hillsboro, OR, USA) using a beam energy of 20 kV. Average fiber diameter and porosity were determined using the DiameterJ plugin for ImageJ (Version 2.0.0., National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/; Rueden et al., 2017).

Using pull-to-break tensile testing (load cell = 23 N, 840L Servo-All-Electric Test System, TestResources Inc., Shakopee, MN, USA) the dry-state elastic modulus was determined for tacrolimus loaded and vehicle matrices (n = 3 each) as the slope of the linear region from each plotted stress-strain curve and normalized to the sample thickness.

Thermal gravimetric analyses of tacrolimus and vehicle control nerve wraps (n = 2 each) were conducted using a thermal gravimetric analyzer (TGA-Q500, TA Instruments, New Castle, DE, USA) under nitrogen gas flux. Samples were heated to 150°C with a ramp rate of 10°C/min and then held under isothermal conditions for 10 minutes before increasing to a final temperature of 700°C to determine the onset of thermal degradation (T_d). Differential scanning calorimetry (DSC) was conducted using a TA Instruments Q2000 differential calorimeter (TA Instruments) under nitrogen gas flux. Thermograms were recorded between –70°C and 200°C at a heating rate of 10°C/min with heat-cool-heat cycles.

Drug loading, encapsulation efficiency, and release profile

Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to determine tacrolimus loading in the PCNU wraps, and encapsulation efficiency for three different tacrolimus-loaded matrices. Samples were dissolved in 1 mL HFIP, the solvent was evaporated in a vacuum concentrator (SpeedVac, Thermo Fisher Scientific, Waltham, MA, USA) and the remaining drug was reconstituted in 1 mL acetonitrile (1401-7-40, Caledon Laboratories, Georgetown, ON, Canada) with cyclosporine A as an internal standard, and analyzed as previously described (Tajdaran et al., 2015; Tajdaran et al., 2019b). Briefly, a mass spectrometer (API4000, SCIEX, Framingham, MA, USA) with a spherisorb column (30 mm × 4.6 mm; 5 μm; Waters Corporation, Milford, MA, USA) was used with a mobile phase (65% aqueous acetonitrile, 2 mM ammonium acetate and 0.1% (v/v) formic acid). Quantification was based on ammonium‐adduct transition masses, and drug loading was determined as tacrolimus mass per nerve wrap mass. Encapsulation efficiency was defined as the ratio of detected tacrolimus loading over the theoretical maximum loading. Drug release was determined from six nerve wraps (10 × 15 mm) derived from three different matrices. Samples were incubated in sterile phosphate-buffered saline (PBS) in a water bath shaker (37°C, 50 r/min), and the solution was collected for tacrolimus content analysis via LC-MS/MS. Drug release was calculated as released tacrolimus mass per sample per day.

In vitro rat dorsal root ganglia neurite extension assay to determine bioactivity

Rat dorsal root ganglia (DRG) neurite extension assays were used. Tacrolimus nerve wraps (n = 8; 10 mm × 15 mm) from four different matrices were incubated in Neurobasal media (NBM, 21103049, Thermo Fisher Scientific), 2% (v/v) B-27 serum-free supplement, 1% (v/v) penicillin-streptomycin, and 1% (v/v) L-glutamine. A total of 16 DRGs were harvested using microforceps under a microscope from E15 rat embryos of our own stockbreeding, placed in a Matrigel-coated well (Corning, Steuben County, NY, USA), and subjected to four different experimental conditions: day 1 nerve wrap release media (n = 4), day 31 release media (n = 4), a negative control (NBM only, n = 4), and a positive control (50 ng /mL tacrolimus in NBM, n = 4). Following 24 hours of incubation, cultures were imaged using phase contrast microscopy (AxioVert, Carl Zeiss Microscopy GmbH, Jena, Germany). Mean neurite extension was measured using ImageJ (Tajdaran et al., 2015). After 48 hours, cultures were fixed in 4% paraformaldehyde (PFA), immunostained against a neuronal marker at room temperature for 1 hour each (rabbit anti-beta-3-tubulin; 1:500 dilution, 18207, Abcam, Cambridge, MA, USA; goat anti-rabbit Alexa Fluor 555 conjugated secondary antibody, 1:1000, A-21428, Invitrogen, Carlsbad, CA, USA) and imaged using confocal fluorescence microscopy (DMI8, Leica). The 48-hour time frame was based on preliminary experiments with this assay and defined by the neuronal growth rate and maximum regeneration distance this system allows for.

In vivo efficacy and safety testing in an experimental rat model

Treatment groups

The primary objectives of the animal experiments were to determine therapeutic efficacy and drug biodistribution of locally delivered (i.e., the drug loaded PCNU wraps) versus systemically delivered tacrolimus following nerve surgery. The number of rats used in each experimental group was determined by power analysis based on previous experience with rodent nerve injury models and local drug delivery (Tajdaran et al., 2019b). For labeled neuron quantifications we considered d = 1.5 for motoneurons (assuming a typical SD of 50) and d = 2.0 for sensory neurons (assuming an SD of 250) to be minimal relevant effect sizes. To achieve a power of 0.8 on an alpha level of 0.05 (normally distributed, two-tailed t-test) this required n = 6 and n = 9 animals per group respectively. For functional tests, we considered d = 2.5 to be worth being reported, corresponding to n = 4 animals per group. Animals were randomly allocated to experimental groups and the investigators were blinded for outcome assessments. No experimental animals or outliers were excluded from the analysis.

Experimental animals

A total of 76 adult (250–300 g, 8–10 weeks old), specific-pathogen-free (SPF) grade female rats with a genetic Sprague-Dawley background were included. All animals were housed in a central animal care facility with three to four animals per cage and fresh water and pellet food ad libitum. A constant room temperature (22°C) and a circadian rhythm of 12 hours per 24-hour illumination were automatically maintained. All procedures were performed in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (8^th ed, National Research Council, 2011), the Canadian Council on Animal Care (CCAC) guidelines and were approved by the Hospital for Sick Children’s Laboratory Animal Services Committee (AUP 49891, approval date November 2, 2019). All animal experiments are reported in accordance with the ARRIVE guidelines (Percie du Sert et al., 2020).

Hindlimb nerve injury and repair model

A total of 30 rats underwent unilateral common peroneal nerve cut and immediate epineural repair surgery. All surgical procedures were performed under aseptic conditions and inhalation anesthesia with a 2% isoflurane (Baxter, Deerfield, IL, USA) oxygen mixture for induction and maintenance and analgesia (4 mg/kg body weight extended-release Metacam, Boehringer Ingelheim, Ingelheim, Germany). The common peroneal nerve was exposed through a dorsolateral-gluteal muscle-splitting incision, cut 3 mm distal to the sciatic nerve bifurcation with micro scissors, readapted and repaired using three epineural single stitch sutures (10-0 Ethilon, Ethicon, Cincinnati, OH, USA). After nerve repair, the rats were allocated to the experimental groups (local tacrolimus n = 8, local vehicle n = 8, systemic tacrolimus n = 6, repair-only control n = 8) using permuted block randomization. In the local tacrolimus group, a 2.5 × 0.5 mm nerve wrap, loaded with 200 µg tacrolimus, was loosely wrapped around the nerve repair site and held in place by a single stitch (10-0 Ethilon, Ethicon). The vehicle group received an empty drug delivery system (PNCU electrospun polymer only) in an otherwise identical fashion. A repair-only control group received no additional treatment after nerve repair, and the systemic tacrolimus group (positive control) received daily subcutaneous injections of tacrolimus (2 mg/kg body weight). The wound was closed in three layers with 5-0 Vicryl (Ethicon) sutures, and all operated animals were allowed to recover in a warm environment prior to returning to the housing facility.

Preparation and systemic delivery of tacrolimus

For systemic delivery, a 10 mg/mL stock solution was prepared by dissolving tacrolimus (LC Laboratories) in 80% ethanol/20% Kolliphor (C5135, Sigma-Aldrich), stored at –20°C until use and replaced every 3 days (Yang et al., 2003). Immediately before injection, the stock solution was diluted with sterile double-distilled water to a final concentration of 2 mg/mL. The rats received 2 mg/kg tacrolimus daily via subcutaneous injection post-surgery (Yang et al., 2003; Jo et al., 2019).

Nerve fiber histomorphometry

Three weeks after nerve repair, a common peroneal segment 10 mm distal to the nerve repair site was harvested, immersed in 2.5% glutaraldehyde fixative (G6257, Sigma-Aldrich)/0.1 M sodium cacodylate trihydrate (C0250, Sigma-Aldrich) overnight at 4°C and postfixed in 2% osmium tetroxide (75632, Sigma-Aldrich) for 2 hours. Samples were dehydrated in ascending ethanol series, embedded in epoxy (45345, Sigma-Aldrich), sectioned into 1-μm cross-sections (ultramicrotome EM UC7, Leica Microsystems) and imaged (Axiovert 200M, Carl Zeiss Microscopy GmbH, Jena, Germany) using a 63×/1.4 oil objective. A custom-trained deep learning model based on the open-source software AxonDeepSeg (https://axondeepseg.readthedocs.io/en/latest/index.html; Zaimi et al., 2018; Daeschler et al., 2022a) determined the number of myelinated nerve fibers, axon diameter and myelin sheath thickness for entire cross sections.

Quantitation of regenerated motor and sensory neurons

Three weeks after nerve repair, the common peroneal nerve was cut 7 mm distal to the nerve repair site and the end of the proximal common peroneal nerve segment was placed in a well containing 5 µL 4% Fluoro-Gold (Fluorochrome LLC, Denver, CO, USA) in double distilled water. The wound bed was draped to prevent leakage of the fluorescent tracer. After 60 minutes of exposure, the wounds were thoroughly irrigated with saline, dried, and closed in three layers with 5-0 Vicryl (Ethicon) sutures. Seven days after the procedure, the spinal cord along with the ipsilateral DRG level L3 to L5 were harvested (Richner et al., 2017), fixed by immersion in 4% PFA at 4°C for 24 hours, and optically cleared using a modified FDISCO protocol (Qi et al., 2019; Daeschler et al., 2022b). Briefly, the tissue was immersed in an ascending series of precooled Tetrahydrofuran (186562, Sigma-Aldrich) in double distilled water solutions (v/v; 50%, 75%, 3× 100%) adjusted to pH 9.0 with triethylamine (471283, Sigma-Aldrich). After a subsequent step in 100% dichloromethane (270997, Sigma-Aldrich) the specimen was immersed in Ethanol/Dibenzyl ether (108014, Sigma-Aldrich) solutions for refractive index matching (Carro et al., 2015; Daeschler et al., 2022b). For image acquisition, we used a 3D light-sheet fluorescence microscope (Zeiss Lightsheet Z.1, Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with a 405 nm (20 mW) laser, a 20×/1.0 CLARITY objective, and a p.co edge 5.5 Camera (PCO AG, Kehlheim, Germany). For data processing, segmentation, and quantitative analyses, we used an Arivis vision 4D (version 3.0, arivis AG, Rostock, Germany) and Imaris (Version 9.5.1, Bitplane AG, Zurich, Switzerland). Selected images in this publication were created with BioRender.com.

Recovery of motor function

Following unilateral median nerve cut and immediate epineural repair, the time to onset of active finger flexion and grip strength at 6 weeks post-surgery were determined in 10 rats. Perioperative management was as described for the hindlimb model. The right median nerve was exposed by a medial incision along the medial bicipital groove, traced distally to its insertion into the forearm flexors, and transected 8 mm proximal to its muscle insertion point, followed by immediate epineural repair. Rats were block randomized into two groups, receiving either a tacrolimus-releasing nerve wrap or an empty nerve wrap (vehicle) as described above. Starting 7 days after surgery, the rats were tested daily for recovery of active long finger flexion using a horizontal ladder grasping test. The test was repeated 10 times per animal and session to reduce the risk of false negatives, and video-taped for post hoc analysis by a blinded investigator. Active flexion of the proximal interphalangeal joint was considered a positive response. Six weeks after nerve repair, the animals performed a grip strength test as previously described (Daeschler et al., 2018). Briefly, the rat was held at the base of its tail and a grip response was induced with the post-operative paw on a scale-coupled grip device. Then the rat was pulled away from the grid in a steady, upright motion and the maximum grip strength was determined as the average of the top 3 out of 10 repetitions to account for rounds with submaximal voluntary grip response.

Systemic drug exposure analysis

Systemic drug exposure was determined via LC-MS/MS. Twelve rats underwent unilateral common peroneal nerve repair and received either the tacrolimus-releasing nerve wrap (n = 6) or daily tacrolimus injections (n = 6) as described earlier in the methods section. Seven- and 28 days post-surgery the kidney, brain, liver, heart, the lumbar segment of the spinal cord, and both common peroneal nerves were harvested from 3 animals per group and snap-frozen in liquid nitrogen. Tissues were weighed, immersed in 300 µL lysis buffer containing 50 mM Tris (17926, Thermo Fisher Scientific), 150 mM NaCl, 2 mM EDTA (E9884, Sigma-Aldrich), 0.1% sodium dodecyl sulfate (L3771, Sigma-Aldrich), 1% NP40 (Sigma-Aldrich), 0.5% DOC and 1% Protease inhibitor and homogenized by sonication on ice for 30 seconds twice (Tajdaran et al., 2019b). Tacrolimus was extracted with acetonitrile (1401-7-40, Caledon Laboratories). The solution was homogenized for 30 seconds on ice twice, centrifuged at 4°C for 5 minutes at 16,000 × g and the supernatant was extracted for analysis with LC-MS/MS (Tajdaran et al., 2019b).

Biodegradation and biocompatibility in vivo

A total of 24 sterile nerve wraps (n = 12 tacrolimus loaded and n = 12 vehicle-only) were implanted around the uninjured sciatic nerve or repaired common peroneal nerve in n = 24 randomly allocated rats. The rats were further block-randomized into three sub-groups of four rats each for analysis 7, 60 or 120 days post-implantation. The wrapped nerve segment (8–10 mm) was harvested, immersion fixed in 4% precooled PFA overnight at 4°C, cryoprotected in 30% sucrose/4% PFA solution for 3–5 days, embedded in tissue freezing medium (23-730-571, Thermo Fisher Scientific), snap frozen with liquid nitrogen and stored in –80°C. Nerve cross sections (20 µm) were cut over the entire length (CM3050S cryostat, Leica Biosystems, Nuβloch, Germany), slide mounted and Hematoxylin & Eosin stained. Sections were imaged at 20× magnification (Panoramic 250 Flash II Slide Scanner, 3DHistech, Budapest, Hungary) and CaseViewer software (3DHistech, Budapest, Hungary) was used for measurements of nerve cross-sectional area and wrap thickness.

Statistical analysis

We used JMP (version 15.1.0, SAS Institute, Cary, USA) and GraphPad (Prism 9.0.0, GraphPad Software, San Diego, California USA) for statistical analysis. Descriptive statistics were calculated, and means are expressed with standard deviations ± SD or standard error of means ± SEM. To test for the normality of continuous variables, we used normal quantile plots and Anderson-Darling tests. For physical nerve wrap characterization, one-way analysis of variance with Tukey’s post hoc test was conducted. Kaplan-Meier curves were used to analyze time-to-event data for motor function recovery. For in vivo safety and efficacy testing we used one-way analysis of variance with Tukey’s post hoc test or two-sided Student’s t-test for pairwise comparison resepctively of normally distributed, continuous data. A significance level of 0.05 was used (P < 0.05).

Results

Design and characterization of the tacrolimus-releasing nerve wrap

For local tacrolimus delivery, we chose a nerve wrap design i.e. a biodegradable membrane wrapped around the peripheral nerve injury site. Similar designs are used clinically to prevent scarring around entrapped peripheral nerves by providing a biodegradable interface between the nerve and the surrounding tissue (Soltani et al., 2014; Papatheodorou et al., 2015). Biocompatible PCNU synthesized from hexane diisocyanate, poly(hexamethylene carbonate) diol and butanediol served as the encapsulation polymer. PCNU’s degradation products are considered non-toxic (Yeganegi et al., 2010) and PCNU fibers largely retain mechanical stability and form during biodegradation as they undergo surface degradation (Santerre et al., 2005; Wright et al., 2017, 2018), which minimizes the risk of nerve compression. The coaxial electrospun PCNU wraps allowed for open pores and cell migration through the construct as it would under degradation (Figure 1A). The nanofibers featured a core-shell architecture, with tacrolimus being encapsulated in the inner core to generate a drug reservoir for sustained release (Figure 1B). The electrospun matrix was then sectioned into nerve wraps, irradiation sterilized (25 kGy), and stored at 4°C until use (Figure 1A). Scanning electron microscopy revealed smooth and non-beaded fibers (Figures 1C and D and the inclusion of tacrolimus did not affect the matrix porosity (P = 0.64; Figure 1E), fiber diameter (P = 0.62; Figure 1F) or strength (P = 0.73; Figure 1G) compared with pure polymer fibers.

Figure 1.

Introduction of tacrolimus in electrospun polycarbonate urethane (PCNU) fibers does not negatively alter its physical characteristics. Composite electrospun tacrolimus-PCNU nerve wraps have suitable properties for clinical use.

(A) The manufacturing process of the nerve wrap includes co-axial electrospinning, sectioning, and gamma irradiation sterilization with subsequent storage at 4°C. (B) The nanofiber architecture follows a core-shell principle with tacrolimus being encapsulated in the core polymer to create extended drug release during biodegradation. The uniform fiber morphology of tacrolimus encapsulated (C) and pure polymer nanofibers (D); no beading was observed indicating successful electrospinning (scanning electron microscopy images, scale bars: 2 µm). The matrix porosity (E) and average fiber diameter (F) of pure polymer nerve wraps (vehicle, n = 8) did not differ from tacrolimus nerve wraps (n = 6). (G) The dry elastic modulus of the nerve wrap exceeds the tensile strength of human peripheral nerves (dashed line) indicating sufficient strength to withstand surgical manipulation and suturing. Graphs E–G show mean ± SEM (Student’s t-test). (H) PCNU has a soft polyol segment and a hard diisocyanate/chain extender segment (Wright et al., 2018). Thermal properties of vehicle and tacrolimus nerve wraps including glass transition temperature (T_g), segment melt temperatures (T_m1 and T_m2), and segment melt enthalpies (∆H₁ and ∆H₂), show similar thermal properties with the encapsulation of tacrolimus at the chosen concentration (5.2%, w/w), indicating stability at ambient and body temperature. Panels A and B were created with BioRender.com. ns: Not significant.

The clinical application requires the device to withstand surgical manipulation and suturing. We performed tensile testing, showing that the nerve wraps’ strength of 2.70 ± 1.05 MPa exceeded the tensile strength of human peripheral nerves averaging 0.54 MPa (Ma et al., 2013), indicating that the nerve wrap likely withstands manipulation during implantation. Differential scanning calorimetry revealed that tacrolimus encapsulation (5.2%, w/w) did not change the overall microstructure and thermal properties of the polymeric fiber scaffolds (Figure 1H). Thermogravimetric analysis indicated that the nerve wrap remains primarily in an amorphous state at room temperature (24°C > glass transition temperature [Tg]) and retains stability at physiological temperature (37°C > Tg and 37°C < segment melt temperatures [Tm1 and Tm2]). These properties may facilitate scale-up, long-term storage, and clinical application.

The nerve wrap efficiently encapsulates and releases bioactive tacrolimus

Based on our previously published work, we targeted a total drug loading of 200 µg tacrolimus per wrap with an extended release of greater than 14 days (Tajdaran et al., 2015, 2019a). We designed two different sizes of the nerve wrap, one representing dimensions for potential clinical application (10 mm × 15 mm, applicable for a nerve cross-sectional area up to 12.5 mm², e.g., ulnar nerve at the forearm level (Yalcin et al., 2013)), and one intended for preclinical testing in a rat model (2.5 mm × 0.5 mm). Mass spectrometry revealed an average drug loading of 223 ± 65 µg, with a mean encapsulation efficiency of 96 ± 8%, indicating minimal loss of tacrolimus during manufacturing. The 31-day drug release profile in vitro (Figure 2A) yielded a biphasic release profile with a maximum release rate of 11.7 ± 3.8 µg/d within the first 12 days and a subsequent maintenance dose averaging 60.7 ± 37.3 ng/d, sustained over 31 days.

Figure 2.

Loading of tacrolimus in electrospun polycarbonate urethane fibers results in a high drug loading efficiency and retention of drug bioactivity.

(A)The tacrolimus encapsulation and efficiency and in vitro release profile of tacrolimus-releasing nerve wraps over a 31-day period. (B) A neurite extension assay shows significantly longer neurites after a 24-hour culture when the dorsal root ganglia (DRG) were incubated in days 1 and 31 nerve wrap release media, compared with a pure media control. DRGs cultured in tacrolimus release media from the nerve wrap extended neurites to the same length as DRGs cultured in media with fresh tacrolimus, indicating that the bioactivity of tacrolimus is retained even after being incorporated into the electrospun polycarbonate urethane composite fibers. No exogenous nerve growth factor was added to the cultures. Graphs show mean ± SD. ***P < 0.001, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). (C–E) Representative DRG cultures after 48-hour immunostained against the neuronal marker beta 3 tubulin (red) show significantly greater neurite extension in tacrolimus-treated cultures (scale bars: 200 µm). A total of n = 16 DRGs were used, and the experiment was conducted once.

As encapsulation and release may affect drug bioactivity, we conducted a neurite extension assay with release media in cultured embryonic rat DRGs (Figure 2B–E). Compared with control DRGs (media only), DRGs that were incubated in nerve wrap release media from 1 to 31 days, displayed significantly greater 24-hour neurite outgrowth, comparable to a positive control (media containing 50 ng/mL fresh tacrolimus; Figure 2B). This indicates that the nerve wrap releases bioactive tacrolimus in therapeutic doses for at least 31 days.

The tacrolimus nerve wrap is biocompatible and biodegrades in vivo

Ideally, drug delivery implants degrade after they served their therapeutic purpose, to prevent potential long-term sequelae such as nerve compression. To test for biocompatibility and biodegradability, we implanted the nerve wrap around intact rat sciatic and repaired common peroneal nerves (Figure 3A and C). The postoperative nerve swelling was significantly reduced when a tacrolimus-releasing nerve wrap was implanted compared with an empty wrap (P = 0.03; Figure 3B). This may be due to the local immunosuppressive effects of tacrolimus potentially mitigating a local foreign body response. Fibrotic capsule formation was not observed. Following implantation, the thickness of the vehicle and tacrolimus nerve wraps decreased to (35.3 ± 10.6)% of the pre-implantation thickness by 7 days, and to (16.7 ± 6.1)% by 60 days (Figure 3D). The wraps were undetectable at 120 days post-implantation, indicating complete biodegradation (Figure 3E). There is a lack of T lymphocyte activity in nerves treated with tacrolimus nerve wraps over the course of 60 days compared with vehicle nerve wraps, supporting the idea that the immunosuppressive action of tacrolimus enhances device biocompatibility (Additional file 1 ^{(613.4KB, pdf)} ).

Figure 3.

Tacrolimus nerve wraps completely biodegrade and do not cause a major immune response and the localized immunosuppressive effects of tacrolimus may play a role in preventing swelling of the nerve in addition to its pro-regenerative effects.

(A) A nerve wrap following implantation around the nerve repair site in a rat the common peroneal nerve. CP: Common peroneal nerve. (B) Measurements of the nerve cross-sectional area (CSA) following nerve wrap implantation indicate significantly reduced post-operative nerve swelling after tacrolimus nerve wrap implantation compared with a vehicle nerve wrap. (C) At 60 days post-implantation, the nerve cross-sectional area was similar to healthy nerves. (D) A cross section through the nerve repair site was enclosed by a nerve wrap 7 days post-surgery. (E) The biodegradation profile of a tacrolimus nerve wrap (blue) and a vehicle nerve wrap (grey) indicates 65% thinning within the first week following implantation and complete degradation within 120 days. Graphs show mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test). ns: Not significant.

Tacrolimus nerve wrap implantation increases the number of regenerating nerve fibers following nerve repair

Based on the observation that the released tacrolimus from the nerve wrap promoted neurite outgrowth in vitro, we asked whether repaired nerves regenerate more axons when the nerve wrap is implanted around the coaptation site. We transected the common peroneal nerve in rats followed by immediate epineural repair, and either implanted a tacrolimus nerve wrap (200 µg per wrap) or subcutaneously injected tacrolimus daily after surgery (2 mg/kg body weight) as previously described (Yang et al., 2003; Jo et al., 2019). Rats that received a vehicle wrap or nerve repair only served as controls. In agreement with the in vitro results (Figure 2B–E), the tacrolimus treated rats regenerated significantly more myelinated nerve fibers 10 mm into the distal nerve segment (> 2.3 times more in locally treated nerves over control) within three weeks post-surgery compared with the control groups (Figure 4A–H). Similarly, regenerated axons in nerves that were exposed to the locally delivered tacrolimus were significantly larger compared with the control groups (P < 0.05; Figure 4I). As expected for this early time point, neither the presence of the delivery system nor the drug itself affected the myelination thickness (Figure 4J) or g-ratio of regenerating nerve fibers (Figure 4K). Notably, implanting a vehicle nerve wrap had no effect on the number and myelination of nerve fibers compared with nerve repair only (P = 0.78 and P = 0.98 respectively; Figure 4E and G), indicating that the physical presence of the carrier itself did not cause nerve compression, an important clinical endpoint of the intervention.

Figure 4.

Nerve fiber histomorphometry.

(A) A representative cross section of a locally tacrolimus-treated common peroneal nerve 10 mm distal to the nerve repair site 3 weeks post-surgery. (B–D) Representative cross sections in higher magnification of common peroneal nerve of a systemically tacrolimus treated rat (B), a vehicle nerve wrap treated rat (C), and a control rat receiving epineural nerve repair only (D). (E) A random region of interest of the locally tacrolimus-treated common peroneal nerve shown in A in the same magnification as images B–D. (F) A custom-trained deep learning model based on the open-source software AxonDeepSeg⁶³ was used to segment entire nerve cross sections in axon/myelin masks (red, G) and determine the number of myelinated axons (H), axon diameter (I), and myelin sheath thickness (J). Green dotted lines indicate physiological values. Graphs show mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance with Tukey’s post hoc test).

Tacrolimus nerve wrap implantation increases the number of regenerating neurons following nerve repair

Next, we asked whether the higher numbers of regenerating nerve fibers in rats that received a tacrolimus nerve wrap reflected a higher number of regenerating motor and sensory neurons. Three weeks following surgery, we used the fluorescent tracer Fluorogold to retrogradely label neurons that projected their axons at least 7 mm into the distal nerve segment (Figure 5A–J). Consistent with the increased number of regenerated nerve fibers (Figure 4), rats that had received tacrolimus either systemically or locally, had significantly more sensory (> 32% increase) and motoneurons (> 31% increase), that regenerated their axons, compared with the control groups (Figure 5K and L). Application of the vehicle nerve wrap did not affect the number of neurons that regenerated their axons compared with nerve repair only. When analyzing the L3 to L5 DRGs individually, we found that systemically treated rats predominantly regenerated sensory neurons from L3 and L4, whereas in locally treated rats a significantly larger proportion of L5 sensory neurons regenerated their axons (Figure 5M). The cell bodies of L5 neurons being the closest to the drug delivery site, this may suggest length-dependent mechanisms (e.g., retrograde axonal transport) to be involved in the growth-promoting effect of locally delivered tacrolimus.

Figure 5.

Tacrolimus released locally from nerve wraps results in better regeneration from sensory and motoneurons compared with the negative controls of injury alone and polycarbonate urethane (PCNU) nerve wraps without drug (vehicle). Local release of tacrolimus from nerve wraps has the same regenerative effect as the positive control of systemically-administered tacrolimus.

(A) Illustration of the localization of back-labeled motor and sensory neurons in relation to the rat common peroneal nerve repair site. (B) A representative intact, optically cleared lumbar spinal cord segment of a locally tacrolimus-treated rat, (C) a vehicle-treated rat and (D) a systemically treated rat showing fluorogold labeled motoneuron cell bodies (yellow) that project their axons at least 7 mm into the distal nerve segment 3 weeks post-surgery. (E) Spinal cord in higher magnification. (F and G) with a demonstration of the fluorescence intensity-based neuron segmentation process. (H) A representative intact, optically cleared L5 dorsal root ganglion (DRG) of a locally tacrolimus-treated rat, (I) a vehicle-treated rat and (J) a systemically treated rat showing fluorogold labeled sensory neuron cell bodies (yellow) that project their axons at least 7 mm into the distal nerve segment 3 weeks post-surgery. (K) Quantitation of regenerated motoneurons that project their axons at least 7 mm into the distal nerve segment 3 weeks post-surgery suggests accelerated motor axon regeneration in tacrolimus-treated rats. (L) Quantitation of regenerated sensory neurons (DRG L3–L5) that project their axons at least 7 mm into the distal nerve segment 3 weeks post-surgery. Tacrolimus-treated rats regenerated significantly more neurons compared with non-treated and vehicle-treated controls. Given the early time point post-surgery, this may indicate accelerated regeneration of sensory axons in vivo. (M) DRG level-specific presentation of regenerated sensory neurons showing that rats that received tacrolimus locally at the nerve repair site recruited significantly more neurons from L5 compared with systemically treated and non-treated rats. Green dotted lines indicate the physiological number of neurons that project their axons into intact control common peroneal nerves (mean, n = 6). Graphs show means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance with Tukey’s post hoc test). Panel A was created with BioRender.com.

Tacrolimus nerve wrap implantation accelerates functional recovery following nerve repair

We then asked whether these changes in neural regeneration translate into an earlier return of motor function. Clinically, most nerve injuries occur in the upper extremity and often affect the median nerve (Tapp et al., 2019) which is involved in hand motor control. The neuromuscular anatomy of the rat forelimb resembles the human anatomy, with the exception that the rat’s long finger flexors are exclusively median nerve innervated (Figure 6A; Bertelli et al., 1995). We transected and immediately repaired the median nerve 8 mm proximal to its muscle insertion (flexor digitorum profundus and superficialis) and implanted either a tacrolimus-releasing or a vehicle nerve wrap. Following nerve transection, the rats immediately lost active finger flexion and therefore the ability to grasp. Using daily forepaw grip function tests and video analysis by a blinded investigator (Figure 6B–E) we observed that rats which received the tacrolimus nerve wrap required less time to recover active finger flexion compared with animals that received vehicle wraps (P < 0.05; Figure 6F and G). Assuming a 10 mm average regeneration distance from the nerve repair site to the neuromuscular junction in this model, locally delivered tacrolimus accelerated axonal regeneration by 17.6% or 0.07 mm/day. Differences in grip strength 6 weeks post-surgery did not reach significance (P = 0.07; Figure 6H).

Figure 6.

Tacrolimus nerve wraps accelerate the recovery of muscle function.

(A) Illustration of the rat forelimb anatomy showing the long finger flexors are exclusively median nerve innervated; flexor digitorum profundus (FDP) and -superficialis (FDS). (B) Daily grip tests were used to monitor the return of active finger flexion following median nerve repair in local tacrolimus- and vehicle-treated rats. Rats that received a vehicle nerve wrap around the nerve repair site showed no signs of active finger flexion 23 days post-surgery. (C) In contrast, 4 of 5 rats that received a tacrolimus-releasing nerve wrap around the nerve repair site showed recurring active finger flexion 23 days post-surgery. (F) Kaplan-Meier curves for motor function recovery of locally tacrolimus- (n = 5) and vehicle-treated control rats (n = 5) showing the significantly accelerated return of motor function in rats that received the tacrolimus nerve wrap following nerve repair. (G) Bar graphs showing time to return of active finger flexion in rats that received the tacrolimus or the vehicle nerve wrap following median nerve cut and repair. (H) Grip strength of the operated paw 6 weeks post-surgery indicating no significant differences between tacrolimus-treated and vehicle-treated rats (P = 0.07). *P < 0.05 (Student’s t-test); ns: not significant. Graphs show mean ± SD. Panel A was created with BioRender.com.

Implantation of a tacrolimus nerve wrap reduces systemic drug exposure compared with systemic delivery

Systemic exposure to tacrolimus may result in nephro- and liver toxicity (Tao et al., 2017; Bentata, 2020), and occasionally, neurologic (Kemper et al., 2003) or psychiatric (Krishna et al., 2013) symptoms. In nerve patients, these side effects presently outweigh the potential benefits of tacrolimus (Zuo et al., 2020). We used liquid chromatography-tandem mass spectrometry to determine the biodistribution and drug exposure of vital organs following local and systemic delivery (Figure 7A). In rats that received the nerve wrap, the tacrolimus concentrations in the kidney, brain, liver, and heart at 7 and 28 days following implantation were significantly lower (–82.2% and –79.0% on average respectively, P < 0.05; Figure 7B and C) compared with rats that received tacrolimus systemically (2 mg/kg/d). Similarly, the 24-hour plasma concentration following tacrolimus injection averaged 15.2 ± 11.8 ng/mL with a peak concentration (C_max) of 30.2 ± 12.4 ng/mL 3 hours post-injection and a trough concentration (C_min) of 4.3 ± 0.5 ng/mL 24 hours post-injection. In contrast, 7 days following tacrolimus nerve wrap implantation, the 24-hour splasma concentration was significantly lower, averaging 0.3 ± 0.5 ng/mL (P = 0.013) with C_max of 0.9 ± 0.6 ng/mL and C_min of 0.09 ± 0.04 ng/mL (Figure 7F).

Figure 7.

Tacrolimus nerve wraps localize drug release to the nerve repair site and minimize accumulation of drug and potentially harmful effects on vital organs compared with systemically-administered tacrolimus.

(A) Organs known to be potentially susceptible to tacrolimus exposure, including heart, brain, kidney, and liver, together with neural tissue, including the lumbosacral spinal cord and both common peroneal nerves, and blood plasma were harvested from systemically and locally tacrolimus treated rats 7 and 28 days post-surgery. The snap-frozen tissue was homogenized, the tacrolimus was extracted and quantitatively analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). (B, C) Tacrolimus tissue concentration in vital organs 7 (B) and (C) 28 days post-surgery. Rats that received tacrolimus systemically via daily subcutaneous injections (2 mg/kg) showed significantly higher systemic drug exposure compared with rats that received the tacrolimus-releasing nerve wrap. (D, E) Tacrolimus tissue concentration in the lumbosacral spinal cord 7 (D) and 28 days (E) post-surgery. Local delivery of tacrolimus achieved comparable drug concentrations to systemic tacrolimus delivery (P > 0.05). (F) Tacrolimus 24-hour plasma concentration profile after subcutaneous injection (2 mg/kg) and 7 days post-implantation of the nerve wrap, indicating a significantly reduced circulating mass of tacrolimus when delivered locally. (G, H) Tacrolimus concentration in the regenerating common peroneal nerve and its contralateral counterpart show significantly higher drug concentration in the regenerating nerve 7 (G) and 28 days (H) post-surgery when tacrolimus is directly delivered to the nerve repair site. Graphs show mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t-test). Panel A was created with BioRender.com.

Notably, the tacrolimus concentration in the lumbosacral spinal cord of nerve wrap-treated rats was more than 4 times higher than the average drug concentration in their vital organs 7 and 28 days post-surgery (P < 0.05; Figure 7B–E) achieving similar drug levels to the systemically treated rats (Figure 7D and E). Further, the nerve wrap created a significantly higher tacrolimus concentration in the regenerating nerve compared with the systemic drug delivery at both 7 and 28 days post-surgery (P < 0.01; Figure 7G and H). The drug concentration in the contralateral nerve remained low in the locally treated rats whereas the systemically treated rats seemed to accumulate tacrolimus in healthy nerves over time (Figure 7E). These results indicate that the tacrolimus nerve wrap maintains a higher local tacrolimus concentration in the target tissue with reduced systemic exposure compared with systemically delivered tacrolimus.

Discussion

Here, we present a functional, easy-to-use, “off the shelf,” biodegradable drug delivery implant for tacrolimus that enables local and sustained neuroregenerative therapy following peripheral nerve surgery with minimal systemic drug exposure.

We used a PCNU polymer platform for its ability to biodegrade into non-cytotoxic byproducts within a clinically reasonable time frame (Santerre et al., 2005; Yeganegi et al., 2010). Coaxial electrospinning provided an efficient single-step method to encapsulate tacrolimus within a core-shell nanofiber matrix offering a large surface area-to-volume ratio, with uniform fiber morphology and structural flexibility. We validated the design and its drug compatibility by characterizing the morphology and physical properties with and without encapsulated tacrolimus. Scanning electron microscopy showed smooth PCNU-tacrolimus nanofibers and drug encapsulation did not affect fiber morphology. Because of the elastomeric nature of the PCNU (Santerre et al., 2005; Wright et al., 2017, 2018), the wrap provided sufficient pliability and tensile strength to minimize pressure along the nerve and yet withstand manipulation during the implantation process. Further, the tacrolimus nerve wrap demonstrated thermal stability at ambient and body temperature thereby facilitating manufacturing, distribution, storage, and clinical use.

Based on previously reported work of our laboratory (Tajdaran et al., 2015, 2019b) we encapsulated 200 µg tacrolimus per wrap, by incorporation into the nanofiber core (Perez and Kim, 2015). The nanofiber construct achieved higher encapsulation efficiencies of 96% compared with previously investigated systems (Li et al., 2010; Tajdaran et al., 2015), indicating minimal drug loss and thus enabling efficient large-scale manufacturing. Further, the construct released therapeutic doses of bioactive tacrolimus for at least 4 weeks, as demonstrated in neurite elongation assays, showing over 2.3-fold increased elongation of axons exposed to the nerve wrap release media. Within this time frame, regenerating nerves may be most receptive to local drug delivery at the repair site (Abram et al., 2006), as physical barriers including epi- and perineurium as well as the blood–nerve barrier (Bouldin et al., 1991; Hirakawa et al., 2003) and myelination (Bouldin et al., 1991) are gradually re-established and thus potentially hinder drug penetration (Liu et al., 2018) at later time points. We thus hypothesized a limited therapeutic window for local low-dose tacrolimus therapy early after nerve surgery and produced a biphasic release profile. A release of up to 12 µg/d within the first 12 days, immediately yielded a high local drug concentration gradient between the nerve and the surrounding implant, and therefore rapidly attained therapeutic drug levels within the target tissue, followed by a maintenance dose of 60 ng/d on average for up to 31 days. This is corroborated by the drug content analysis via liquid chromatography-tandem mass spectrometry showing high tacrolimus concentrations within the treated regenerating nerve 7 days after implantation that are maintained 28 days post-implantation.

For therapeutic efficacy testing of the tacrolimus-releasing nerve wrap, we used preclinical models of nerve injury and repair in the rat fore- and hindlimb. Given the relatively short nerve regeneration distances in small animal models, we used an early experimental endpoint of three weeks post-surgery to capture differences in axonal regeneration rates between treated and control nerves. Rats that received the tacrolimus-releasing nerve wrap implanted around the nerve repair site regenerated significantly more axons with a slightly larger diameter compared with control rats. However, higher axon counts may be a result of increased axonal sprouting at the nerve repair site and thus neither necessarily indicate the regeneration of more neurons nor automatically translate into functional benefits. We therefore exposed the regenerated axons to a fluorescent tracer that is retrogradely transported to the neuronal cell body. We found 40% more labeled moto- and 29% more labeled sensory neurons on average in rats that received the tacrolimus-releasing nerve wrap following nerve repair compared with nerve surgery alone. These effect sizes are comparable to rats that were systemically treated with tacrolimus (+26% motoneurons and +44% sensory neurons on average, P > 0.05), indicating a similar therapeutic efficacy. Notably, these results provide a snapshot of the early nerve regeneration progress, and thus a higher number of regenerating neurons that regenerate their axons distal to the repair site are believed to be the result of accelerated axon elongation as observed in tacrolimus treated neurons in vitro. From a clinical standpoint, a similarly massive expansion of the available neuron pool would greatly enhance the chances of meaningful sensory and motor recovery in human patients with nerve injuries.

To test whether these morphological effects translate into accelerated functional recovery following nerve surgery, we monitored the return of active finger flexion following median nerve transection and repair in rats. We found that rats which received the local drug delivery system regained their ability to grasp 17% earlier than rats that received an empty drug delivery system. This is in accordance with previously reported effect sizes for systemically delivered tacrolimus on axon elongation in vivo of 12% to 16% (Gold et al., 1995; Udina et al., 2002). Although, this study is limited to one species, and it remains to be determined as to whether this proportion holds true when axons traverse longer regeneration distances and progressively diverge from the drug delivery site, a 17% acceleration may represent weeks of earlier return-of-function for patients undergoing nerve surgery. Currently, such effect sizes can only be achieved through nerve transfer surgery, where a proximal nerve lesion is converted into a distal one thereby shortening the regeneration distance surgically (Novak and Mackinnon, 2002; Davidge et al., 2015; Flores, 2015; Chen et al., 2021). However, nerve transfers require sacrificing a healthy donor nerve which causes additional morbidity and necessitates relearning of functional motor patterns. Given the prolonged disability following nerve injuries, even with surgery, adjunct therapies that enable earlier return of function would provide major clinical and socioeconomic benefits to individuals and society.

For drug delivery implants in nerve surgery, potential safety concerns include local adverse reactions such as inflammation, fibrotic capsule formation, and nerve compression as well as systemic adverse effects including organ toxicity. Drug release systems that completely degrade after serving their therapeutic purpose overcome the need for secondary explantation surgery and reduce the risk of long-term sequalae. Within 7 days following implantation, the wrap thickness decreased by 65% due to hydrolytic degradation, while retaining its macroscopic shape and location for at least 60 days post-implantation before being entirely absorbed within 120 days. However, implant-induced local nerve compression may still be a reasonable concern for clinicians, given the susceptibility of axoplasmic transport (Dahlin et al., 1984) and intraneural blood flow (Rydevik et al., 1981) to external pressure. We therefore compared the neurohistomorphology of repaired nerves after vehicle nerve wrap implantation with repaired nerves, without additional treatment. Neither the number of regenerating axons nor their diameter or myelination state were affected by the empty implant compared with the current gold standard of epineural repair. The local release of tacrolimus further reduced the post-operative nerve swelling by 25% compared with vehicle implantation. This may be attributable to the immunosuppressive effects of tacrolimus, potentially supporting the biocompatibility of the implant, counteracting nerve edema, and thereby further reducing the risk of local nerve compression. Chronic tissue reactions, such as fibrotic capsule formation, were not observed.

Presently, systemic toxicity is a key limiting factor for the use of promising therapies such as tacrolimus within the context of peripheral nerve surgery (Zuo et al., 2020). By generating a local and sustained, low-dose release profile, the monthly cumulative tacrolimus dose was reduced by more than 98% (200 µg vs. 15.5 mg) compared with previously established systemic drug delivery regimen for promoting nerve regeneration (Yang et al., 2003; Jo et al., 2019). Accordingly, the tacrolimus plasma concentration following local delivery was significantly lower compared with systemic application, with maximum and average plasma levels remaining well below the C_min of systemically treated rats. Lower levels of circulating tacrolimus are reflected in the 80% reduced drug exposure of organs that are known to be susceptible to its systemic adverse effects. This indicates that adverse off-target effects may be reduced by local delivery of tacrolimus while its therapeutic efficacy is maintained. For clinical application, one must consider that the volume of distribution in preclinical rodent models is small compared with the human body and thus drug exposure levels are likely to be different. Nevertheless, these results indicate the potential of local tacrolimus delivery to enhance nerve regeneration in clinically relevant effect sizes with minimal systemic drug exposure.

This drug delivery system was designed for clinical translation, large-scale manufacturing, and regulatory approval. Future steps towards clinical translation include clinical feasibility studies aiming to capture preliminary information on safety and effectiveness in human subjects. Based on benefit/risk considerations such studies may be initially conducted in patients undergoing primary nerve repair. The evaluation may include longitudinal assessment of tactile thresholds and two-point discrimination, alongside thorough safety testing including pain assessments, regular drug plasma concentration measurements and high-frequency ultrasound imaging to monitor device degradation. Subsequent studies may enroll patients undergoing nerve transfer surgery with its high level of standardization, well-controlled regeneration distances, and rapid recovery of motor function allowing clinically meaningful outcome assessments with short trial duration. Two-stage procedures such as cross-face nerve grafting (stage one) and subsequent free-functioning muscle transfer (stage two) after 6–12 months may offer the added value of distal nerve graft biopsies at stage two (Braam and Nicolai, 1993), allowing for detailed histomorphometric analysis of regenerated nerve fibers. Based on these metrics, therapeutic effect sizes can be reliably determined for human subjects and compared with the herein-presented preclinical data.

Our study on a biodegradable tacrolimus-releasing implant for nerve regeneration, while promising, faces limitations such as its reliance on rat models and the early experimental endpoint, potentially limiting its direct applicability to humans. These constraints highlight the need for extended long-term studies and human clinical trials to validate efficacy and safety. Future research should explore different dosages, delivery timelines, and incorporate comprehensive assessments like tactile thresholds and drug plasma concentration measurements. Comparative studies with other nerve repair techniques are also crucial. Addressing these aspects will be vital for advancing neuroregenerative therapy in clinical settings.

In conclusion, given the extensive clinical safety data for local and systemically applied tacrolimus in various clinical conditions, this bioengineered nanofiber nerve wrap may be a suitable candidate for translation into clinical studies aiming to improve functional outcomes following peripheral nerve surgery.

Funding Statement

Funding: This work was supported by the German Research Foundation (DA 2255/1-1; to SCD); a SickKids Research Training Competition (RESTRACOMP) Graduate Scholarship (to KJWS), an Ontario Graduate Scholarship (to KJWS); a grant from Natural Sciences and Engineering Research Council of Canada (NSERC) (to KJWS) and a Kickstarter grant from the Institute of Biomedical Engineering (BME) at the University of Toronto (to KJWS); and the Abe Frank Fund from the Riley’s Children Foundation (GHB).