Conformational changes of GDNF-derived peptide induced by heparin, heparan sulfate, and sulfated hyaluronic acid - Analysis by circular dichroism spectroscopy and molecular dynamics simulation.

Glial-cell-line-derived neurotrophic factor (GDNF) is a protein that has therapeutic potential in the treatment of Parkinson's disease and other neurodegenerative diseases. The activity of GDNF is highly dependent on the interaction with sulfated glycans which bind at the N-terminus consisting of 19 residues. Herein, we studied the influence of different glycosaminoglycan (i.e., glycan; GAG) molecules on the conformation of a GDNF-derived peptide (GAG binding motif, sixteen amino acid residues at the N-terminus) using both experimental and theoretical studies. The GAG molecules employed in this study are heparin, heparan sulfate, hyaluronic acid, and sulfated hyaluronic acid. Circular dichroism spectroscopy was employed to detect conformational changes induced by the GAG molecules; molecular dynamics simulation studies were performed to support the experimental results. Our results revealed that the sulfated GAG molecules bind strongly with GDNF peptide and induce alpha-helical structure in the peptide to some extent.

Decellularized nerve matrix hydrogel and glial-derived neurotrophic factor modifications assisted nerve repair with decellularized nerve matrix scaffolds.

Nerve defects are challenging to address clinically without satisfactory treatments. As a reliable alternative to autografts, decellularized nerve matrix scaffolds (DNM-S) have been widely used in clinics for surgical nerve repair. However, DNM-S remain inferior to autografts in their ability to support nerve regeneration for long nerve defects. In this study, we systematically and clearly presented the nano-architecture of nerve-specific structures, including the endoneurium, basement membrane and perineurium/epineurium in DNM-S. Furthermore, we modified the DNM-S by supplementing decellularized nerve matrix hydrogel (DNMG) and glial-derived neurotrophic factor (GDNF) and then bridged a 50-mm sciatic nerve defect in a beagle model. Fifteen beagles were randomly divided into three groups (five per group): an autograft group, DNM-S group and GDNF-DNMG-modified DNM-S (DNM-S/GDNF@DNMG) group. DNM-S/GDNF@DNMG, as optimized nerve grafts, were used to bridge nerve defects in the same manner as in the DNM-S group. The repair outcome was evaluated by behavioural observations, electrophysiological assessments, regenerated nerve tissue histology and reinnervated target muscle examinations. Compared with the DNM-S group, limb function, electrophysiological responses and histological findings were improved in the DNM-S/GDNF@DNMG group 6 months after grafting, reflecting a narrower gap between the effects of DNM-S and autografts. In conclusion, modification of DNM-S with DNMG and GDNF enhanced nerve regeneration and functional recovery, indicating that noncellular modification of DNM-S is a promising method for treating long nerve defects.

Long-gap peripheral nerve repair through sustained release of a neurotrophic factor in nonhuman primates.

Severe injuries to peripheral nerves are challenging to repair. Standard-of-care treatment for nerve gaps >2 to 3 centimeters is autografting; however, autografting can result in neuroma formation, loss of sensory function at the donor site, and increased operative time. To address the need for a synthetic nerve conduit to treat large nerve gaps, we investigated a biodegradable poly(caprolactone) (PCL) conduit with embedded double-walled polymeric microspheres encapsulating glial cell line-derived neurotrophic factor (GDNF) capable of providing a sustained release of GDNF for >50 days in a 5-centimeter nerve defect in a rhesus macaque model. The GDNF-eluting conduit (PCL/GDNF) was compared to a median nerve autograft and a PCL conduit containing empty microspheres (PCL/Empty). Functional testing demonstrated similar functional recovery between the PCL/GDNF-treated group (75.64 ± 10.28%) and the autograft-treated group (77.49 ± 19.28%); both groups were statistically improved compared to PCL/Empty-treated group (44.95 ± 26.94%). Nerve conduction velocity 1 year after surgery was increased in the PCL/GDNF-treated macaques (31.41 ± 15.34 meters/second) compared to autograft (25.45 ± 3.96 meters/second) and PCL/Empty (12.60 ± 3.89 meters/second) treatment. Histological analyses included assessment of Schwann cell presence, myelination of axons, nerve fiber density, and g-ratio. PCL/GDNF group exhibited a statistically greater average area occupied by individual Schwann cells at the distal nerve (11.60 ± 33.01 µm2) compared to autograft (4.62 ± 3.99 µm2) and PCL/Empty (4.52 ± 5.16 µm2) treatment groups. This study demonstrates the efficacious bridging of a long peripheral nerve gap in a nonhuman primate model using an acellular, biodegradable nerve conduit.

Cryo-EM analyses reveal the common mechanism and diversification in the activation of RET by different ligands.

RET is a receptor tyrosine kinase (RTK) that plays essential roles in development and has been implicated in several human diseases. Different from most of RTKs, RET requires not only its cognate ligands but also co-receptors for activation, the mechanisms of which remain unclear due to lack of high-resolution structures of the ligand/co-receptor/receptor complexes. Here, we report cryo-EM structures of the extracellular region ternary complexes of GDF15/GFRAL/RET, GDNF/GFRα1/RET, NRTN/GFRα2/RET and ARTN/GFRα3/RET. These structures reveal that all the four ligand/co-receptor pairs, while using different atomic interactions, induce a specific dimerization mode of RET that is poised to bring the two kinase domains into close proximity for cross-phosphorylation. The NRTN/GFRα2/RET dimeric complex further pack into a tetrameric assembly, which is shown by our cell-based assays to regulate the endocytosis of RET. Our analyses therefore reveal both the common mechanism and diversification in the activation of RET by different ligands.

Alpha lipoic acid attenuates the long-term effects of lead exposure in retinal ischemic injury mouse model.

Lead (Pb) exposure is reported to be unsafe for humans. There have been several studies documenting acute and chronic Pb toxicity on the organ systems. New studies suggest that early-life exposure to such environmental toxins may increase the susceptibility to late-onset degenerative disorders. We aimed to examine the long-term effects of early-life postnatal exposure of Pb on retinal degeneration. Pb exposure (200 ppm) was provided either at postnatal day 1 through lactation (early-life exposure) or at 7th week of age (adulthood exposure) directly through drinking water for 20 days. The Pb-treated mice were followed till 20 weeks of age. At 20th week, ischemia/reperfusion (I/R) injury was induced in these mice by pterygopalatine artery ligation. Further, alpha lipoic acid (ALA) was administered to examine its neuroprotective effects against retinal damage. Histological and molecular analysis revealed that Pb-treated mice had greater retinal damage after I/R injury as compared to untreated or ALA treated mice, suggesting that ALA protects the early-life Pb exposure and its consequent impact on later life. The elevated levels of glial derived neurotrophic factor (GDNF) and ciliary neurotrophic factor (CNTF) and reduced levels of glial fibrillary acidic protein (GFAP) upon ALA pre-treatment suggest that it probably exerts anti-inflammatory effects via upregulation of neurotrophic factors.

Optimization of Serum-Free Culture Conditions for Propagation of Putative Buffalo (Bubalus bubalis) Spermatogonial Stem Cells.

Spermatogonial stem cells (SSCs) self-renew and produce a large number of differentiated germ cells to maintain normal spermatogenesis. However, the growth factors crucial for SSC self-renewal and the mechanism underlying this process remain unclear. In the present study, a serum-free culture media was used to evaluate the effect of several growth factors on the expression of some SSC markers and self-renewal related genes. The putative SSCs were cultured on buffalo Sertoli cell feeder layer in KO-DMEM +10% KOSR. The colony formation was observed between 7 and 10 days. The putative SSC colonies also expressed markers specific for undifferentiated type A spermatogonia and pluripotency markers. After 15 days, relative mRNA expression study revealed that 20 ng/mL concentration of Glial cell line-derived neurotrophic factor (GDNF) upregulated the expression of PLZF, TAF4B, and THY1. Furthermore, supplementation of a combination of 20 ng/mL GDNF, 10 ng/mL basic fibroblast growth factor (bFGF), 1000 IU/mL leukemia inhibitory factor (LIF), and 1 ng/mL colony stimulating factor 1 (CSF1) upregulated the expression of PLZF, TAF4B, BCL6B, and ID4 genes. These results demonstrated that our defined culture media in combination with GDNF, bFGF, LIF, and CSF1 well supported SSC self-renewal.

Supplementation of Glial Cell Line-Derived Neurotrophic Factor, Fibroblast Growth Factor 2, and Epidermal Growth Factor Promotes Self-Renewal of Putative Buffalo (Bubalus bubalis) Spermatogonial Stem Cells by Upregulating the Expression of miR-20b, miR-21, and miR-106a.

In this study, we investigated the effect of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF) 2, and epidermal growth factor (EGF) on the expression of some self-renewal-related microRNAs (miRs) in putative buffalo spermatogonial stem cells (SSCs). The SSCs were cultured on a buffalo Sertoli cell feeder layer, colony formation was observed between 7 and 10 days. The SSC colonies expressed markers specific for undifferentiated type A spermatogonia and pluripotency markers. After 15 days of initial culture, the colonies were subcultured as treatment (supplemented with 20 ng mL-1 GDNF +10 ng mL-1 FGF2 + 10 ng mL-1 EGF) and control groups. The number and area of SSC colonies were significantly (p < 0.05) higher in the treatment group than in the control group. The relative abundance of miR-20b, miR-21, and miR-106a in SSCs supplemented with growth factors was significantly higher (p < 0.001) than that in the control. The results indicate that supplementation of SSC culture medium with growth factors (GDNF, FGF2, and EGF) may promote the expression of miR-20b, miR-21, and miR-106a, which is essential for self-renewal and maintenance of SSCs.

Spatio-temporal release of NGF and GDNF from multi-layered nanofibrous bicomponent electrospun scaffolds.

Scaffolds capable of providing dual neurotrophic factor (NTF) delivery with different release kinetics, spatial delivery of NTFs at different loci and topographical guidance are promising for enhanced peripheral nerve regeneration. In this study, we have designed and fabricated multi-layered aligned-fiber scaffolds through combining emulsion electrospinning, sequential electrospinning and high-speed electrospinning (HS-ES) to modulate the release behavior of glial cell line-derived growth factor(GDNF) and nerve growth factor (NGF). GDNF and NGF were incorporated into poly(lactic-co-glycolic acid) (PLGA) fibers and poly(D,L-lactic acid) (PDLLA) fibers, respectively. Aligned fibers were obtained in each layer of multi-layered scaffolds and relatively thick tri-layered and tetra-layered scaffolds with controlled layer thickness were obtained. Their morphology, structure, properties, and the in vitro release of growth factors were examined. Dual and spatio-temporal release of GDNF and NGF with different release kinetics from multi-layered scaffolds was successfully demonstrated. High separation efficiency by PDLLA fibrous barrier layer for spatial neurotrophic factor delivery from both tri-layered scaffolds and tetra-layered scaffolds was achieved.

Incorporation and release of dual growth factors for nerve tissue engineering using nanofibrous bicomponent scaffolds.

Electrospun fibrous scaffolds have been extensively used as cell-supporting matrices or delivery vehicles for various biomolecules in tissue engineering. Biodegradable scaffolds with tunable degradation behaviors are favorable for various resorbable tissue replacements. In nerve tissue engineering, delivery of growth factors (GFs) such as nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) from scaffolds can be used to promote peripheral nerve repair. In this study, using the established dual-source dual-power electrospinning technique, bicomponent scaffolds incorporated with NGF and GDNF were designed and demonstrated as a strategy to develop scaffolds providing dual GF delivery. NGF and GDNF were encapsulated in poly(D, L-lactic acid) (PDLLA) and poly(lactic-co-glycolic acid) (PLGA) nanofibers, respectively, via emulsion electrospinning. Bicomponent scaffolds with various mass ratios of GDNF/PLGA fibers to NGF/PDLLA fibers were fabricated. Their morphology, structure, properties, and the in vitro degradation were examined. Both types of core-shell structured fibers were evenly distributed in bicomponent scaffolds. Robust scaffolds with varying component ratios were fabricated with average fiber diameter ranging from 307 ± 100 nm to 688 ± 129 nm. The ultimate tensile stress and elastic modulus could be tuned ranging from 0.23 ± 0.07 MPa to 1.41 ± 0.23 MPa, 11.1 ± 3.0 MPa to 75.9 ± 3.3 MPa, respectively. Adjustable degradation was achieved and the weight loss of scaffolds ranged from 9.2% to 44.0% after 42 day degradation test. GDNF and NGF were incorporated with satisfactory encapsulation efficiency and their bioactivity were well preserved. Sustained release of both types of GFs was also achieved.

Sustained delivery of glial cell-derived neurotrophic factors in collagen conduits for facial nerve regeneration.

Facial nerve injury caused by traffic accidents or operations may reduce the quality of life in patients, and recovery following the injury presents unique clinical challenges. Glial cell-derived neurotrophic factor (GDNF) is important in nerve regeneration; however, soluble GDNF rapidly diffuses into body fluids, making it difficult to achieve therapeutic efficacy. In this work, we developed a rat tail derived collagen conduit to connect nerve defects in a simple and safe manner. GDNF was immobilized in the collagen conduits via chemical conjugation to enable controlled release of GDNF. The GDNF delivery system prevented rapid diffusion from the site without impacting bioactivity of GDNF; degradation of the collagen conduit was inhibited owing to the chemical conjugation. The artificial nerve conduit was then used to examine facial nerve regeneration across a facial nerve defect. Following transplantation, the artificial nerve conduits degraded gradually without causing dislocations and serious inflammation, with good integration into the host tissue. Functional and histological tests indicated that the artificial nerve conduits were able to guide the axons to grow through the defect, reaching the distal stumps. The degree of nerve regeneration in the group that was treated with the artificial nerve conduit approached that of the autograft group, and exceeded that of the other conduit grafted groups. STATEMENT OF SIGNIFICANCE: In this study, we developed artificial nerve conduits consisting of GDNF immobilized on collagen, with the aim of providing an environment for nerve regeneration. Our results show that the artificial nerve conduits guided the regeneration of axons to the distal nerve segment. GDNF was immobilized stably in the artificial nerve conduits, and therefore retained a sufficient concentration at the target site to effectively promote the regeneration process. The artificial nerve conduits exhibited good biocompatibility and facilitated nerve regeneration and functional recovery with an efficacy that was close to that of an autograft, and better than that of the other conduit grafted groups. Our approach provides an effective delivery system that overcomes the rapid diffusion of GDNF in body fluids, promoting peripheral nerve regeneration. The artificial nerve conduit therefore qualifies as a putative candidate material for the fabrication of peripheral nerve reconstruction devices.

Research on the repairing effect of polylactic acid-trimethylene carbonate/GNDF slow-release catheter on the injured femoral nerve fiber.

Present research aims to investigate the repairing effect of polylactic acid-trimethylene carbonate/GNDF slow-release catheter on the injured femoral nerve fiber. Adult SD male rats as the subjects were divided into two groups, the GDNF group and the control group, and received the surgery to remove the nerve from the exposed left femoral nerves. Thereafter, rats in the GNDF group and the control group received the GNDF or normal saline, and we evaluated the changes in rats, including the morphological, functional and electrophysiological changes of regenerated nerves. Regenerated axons were found in each group, but enormous regeneration of axons was only identified in GDNF group. Further analysis showed that: At the 4th, 8th and 12th weeks, areas of the regenerated nerves in GDNF group were (0.95±0.06) mm2, (1.14±0.07) mm2 and (1.22±0.06) mm2, respectively; in the control group, these were (0.15±0.01) mm2, (0.25±0.07) mm2 and (0.52±0.05) mm2, respectively. These showed that the outcome of GDNF group was superior to that of control group. In GDNF group, quantities of the myelinated fiber were (0.8119×104±0.0637×104), (1.3371×104±0.0460×104) and (1.7669×104±0.0542×104); while in control group, these were (0.2179×104±0.0097×104), (0.3490×104±0.0329×104) and (0.7737×l04±0.0788×104). Again, these results also indicated that the outcome of GDNF group was superior to that of the control group (p<0.05). In GDNF group, the average diameters of myelinated fibers were (2.25±0.17) µm, (2.42±0.14) µm and (2.80±0.10) µm, which were significantly better than (1.24±0.08) µm, (1.43±0.14) µm and (1.82±0.14) µm in the control group. Degrees of fiber myelination in the GDNF group were (0.71±0.03), (0.64±0.03) and (0.6l±0.0l), respectively, which were also significantly higher than (0.02±0.01), (0.04±0.01) and (0.06±0.02) in the control group (p<0.01). At the 12th week after surgery, HE staining was performed to observe the histological changes in quadriceps femoris for evaluation of atrophy in each group. In the GDNF group, significant amelioration was found in the atrophy of quadriceps femoris with an average area of myofiber of (84.95±3.92) %, while the area of the control group was (57.95±5.78) %, suggesting that the outcome of the GDNF group was better than that of the control group (p<0.05). Electrophysiological examination of nerves was employed to detect the recovery of neurological functions after repair of nerve defect. At the 4th, 8th and 12th weeks after surgery, CMAP amplitudes in the GDNF group were (9.34±0.52) mV, (14.40±0.69) mV and (19.18±0.48) mV, significantly better than (0.39±0.07) mV, (1.44±0.41) mV and (9.27±0.40) in the control group (p<0.01). Polylactic acid-trimethylene carbonate/GNDF slow-release catheter can accelerate the functional recovery of injured nerves, thus promoting the regeneration efficiency of femoral nerves.

Ultrasound-triggered effects of the microbubbles coupled to GDNF- and Nurr1-loaded PEGylated liposomes in a rat model of Parkinson's disease.

The purpose of this study was to investigate ultrasound-triggered effects of the glial cell line-derived neurotrophic factor (GDNF) + nuclear receptor-related factor 1 (Nurr1)-polyethylene glycol (PEG)ylated liposomes-coupled microbubbles (PLs-GDNF + Nurr1-MBs) on behavioral impairment and neuron loss in a rat model of Parkinson's disease (PD). The unloaded PEGylated liposomes-coupled microbubbles (PLs-MBs) were characterized for zeta potential, particle size, and concentration. 6-hydroxydopamine (6-OHDA) was used to establish the PD rat model. Rotational, climbing pole, and suspension tests were used to detect behavioral impairment. The immunohistochemical staining of tyrosine hydroxylase (TH) and dopamine transporter (DAT) was used to assess the neuron loss. Western blot and quantitative real-time PCR (qRT-PCR) analysis were used to measure the expression levels of GDNF and Nurr1. The particle size of PLs-MBs was gradually increased, while the concentration and absolute zeta potential were gradually decreased as the time prolongs. 6-OHDA increased amphetamine-induced rotations and loss of dopaminergic neurons as compared to sham group. Interestingly, PLs-GDNF-MBs or PLs-Nurr1-MBs decreased rotations and increased the TH and DAT immunoreactivity. Combined of both genes resulted in a robust reduction in the rotations and a greater increase of the dopaminergic neurons. The delivery of PLs-GDNF + Nurr1-MBs into the brains using magnetic resonance imaging (MRI)-guided focused ultrasound may be more efficacious for the treatment of PD than the single treatment.

A coarse-grained model for DNA origami.

Modeling tools provide a valuable support for DNA origami design. However, current solutions have limited application for conformational analysis of the designs. In this work we present a tool for a thorough study of DNA origami structure and dynamics. The tool is based on a novel coarse-grained model dedicated to geometry optimization and conformational analysis of DNA origami. We explored the ability of the model to predict dynamic behavior, global shapes, and fine details of two single-layer systems designed in hexagonal and square lattices using atomic force microscopy, Förster resonance energy transfer spectroscopy, and all-atom molecular dynamic simulations for validation of the results. We also examined the performance of the model for multilayer systems by simulation of DNA origami with published cryo-electron microscopy and atomic force microscopy structures. A good agreement between the simulated and experimental data makes the model suitable for conformational analysis of DNA origami objects. The tool is available at http://vsb.fbb.msu.ru/cosm as a web-service and as a standalone version.

Encapsulation of primary dopaminergic neurons in a GDNF-loaded collagen hydrogel increases their survival, re-innervation and function after intra-striatal transplantation.

Poor graft survival limits the use of primary dopaminergic neurons for neural repair in Parkinson's disease. Injectable hydrogels have the potential to significantly improve the outcome of such reparative approaches by providing a physical matrix for cell encapsulation which can be further enriched with pro-survival factors. Therefore, this study sought to determine the survival and efficacy of primary dopaminergic grafts after intra-striatal delivery in a glial-derived neurotrophic factor (GDNF)-loaded collagen hydrogel in a rat model of Parkinson's disease. After intra-striatal transplantation into the lesioned striatum, the GDNF-enriched collagen hydrogel significantly improved the survival of dopaminergic neurons in the graft (5-fold), increased their capacity for striatal re-innervation (3-fold), and enhanced their functional efficacy. Additional studies suggested that this was due to the hydrogel's ability to retain GDNF in the microenvironment of the graft, and to protect the transplanted cells from the host immune response. In conclusion, the encapsulation of dopaminergic neurons in a GDNF-loaded hydrogel dramatically increased their survival and function, providing further evidence of the potential of biomaterials for neural transplantation and brain repair in neurodegenerative diseases such as Parkinson's disease.

Development and characterization of cores-shell poly(lactide-co-glycolide)-chitosan microparticles for sustained release of GDNF.

The microencapsulation of bioactive neurotrophic factors in biodegradable poly(lactide-co-glycolide) (PLGA) microspheres has been a promising tool in the treatment of various nervous system disorders. However, challenges still exist; the PLGA burst drug release and acidic degradation products often limit clinical application. In this study, cores-shell PLGA-chitosan microparticles (MPs) were fabricated with a single shell of chitosan and multi-cores of PLGA using a re-emulsification method. The glial cell line-derived neurotrophic factor (GDNF) was encapsulated at the PLGA cores of the cores-shell MPs. The cores-shell MPs prepared by different chitosan concentrations showed a rough surface, and the particle mean size varied between 32.3 and 45.2µm. The fluorescence images indicated that Nile red-stained PLGA microspheres were uniformly distributed in the cores-shell MPs. Compared with PLGA microspheres, the cores-shell MPs were able to reduce the initial burst release of GDNF and neutralize the acidity of PLGA degradation products, which could be modulated by changing the chitosan concentrations. Further differentiation of PC12 cells toward a neuronal phenotype in vitro indicated that the cores-shell MPs were capable of maintaining the bioactivity of GDNF during preparation. Taken together, these findings highlight the possibility of using cores-shell PLGA-chitosan MPs for the sustained release of GDNF, which offers potential applications in nerve injury repair.

A combination of GDNF and hUCMSC transplantation loaded on SF/AGs composite scaffolds for spinal cord injury repair.

Spinal cord injury (SCI) is a severe trauma for which no effective treatment is currently available. In this study, a composited treatment system was prepared using a silk fibroin/alginates/glial cell line-derived neurotrophic factor (SF/AGs/GDNF) scaffold seeded with human umbilical cord mesenchymal stem cells (hUCMSCs) and the combined therapeutic effects of the composite scaffold to repair SCI rats were evaluated. The use of SF as a scaffold material could act as a biomimetic platform allowing neurons to properly accommodate and rebuild the target tissue. The SF/AGs/GDNF scaffold had the best sustained-release function and the AGs were the key determining factor in the controlled release of GDNF. After 8weeks of treatment, the hUCMSCs on SF/AGs/GDNF composite scaffolds could significantly enhance the scar expansion of spinal cord tissue and increased the number of surviving neurons. The combination of GDNF and hUCMSCs transplantation loaded on SF/AGs composite scaffolds exhibited better therapeutic and repair effects to the SCI of rats, compared with the SF/AGs group or GDNF alone on SF/AGs scaffolds. The composite scaffold, GDNF and stem cells could build a bioactive material to form the micro-environment of growth and repair of the neurons. These results may provide a theoretical basis and beneficial exploration for clinical treatment of SCI.

Six month delivery of GDNF from PLGA/vitamin E biodegradable microspheres after intravitreal injection in rabbits.

Local long-term delivery of glial cell line derived neurotrophic factor (GDNF) from vitamin E/poly-lactic-co-glycolic acid microspheres (MSs) protects retinal ganglion cells in an animal model of glaucoma for up to 11weeks. However, the pharmacokinetics of GDNF after intravitreal injection of MSs is not known. We evaluated the GDNF levels after a single intravitreal injection of GDNF/VitE MSs. Biodegradable MSs were prepared by the solid-oil-in-water emulsion-solvent evaporation technique and characterized. Rabbits received a single intravitreal injection (50µL) of GDNF/VitE MSs (4%w/v; 24 right eyes; 74.85ng GDNF), blank MSs (4%w/v; 24 left eyes), and balanced salt solution (4 eyes). Two controls eyes received no injections. At 24h, 1, 4, 6, 8, 12, 18, and 24weeks after injection, the eyes were enucleated, and the intravitreal GDNF levels were quantified. Pharmacokinetic data were analysed according to non-compartmental model. Intraocular GDNF levels of 717.1±145.1pg/mL were observed at 24h for GDNF-loaded MSs, followed by a plateau (745.3±25.5pg/mL) until day 28. After that, a second plateau (17.4±3.7pg/mL) occurred from 8 to 24weeks post-injection, significantly higher than the basal levels. Eyes injected with GDNF/vitE and Blank-MSs did not show any abnormalities during the six-months follow up after administration. The single injection of GDNF/VitE MSs provided a sustained controlled release of the neurotrophic factor in a controlled fashion for up to six months.

Functional regeneration of the transected recurrent laryngeal nerve using a collagen scaffold loaded with laminin and laminin-binding BDNF and GDNF.

Recurrent laryngeal nerve (RLN) injury remains a challenge due to the lack of effective treatments. In this study, we established a new drug delivery system consisting of a tube of Heal-All Oral Cavity Repair Membrane loaded with laminin and neurotrophic factors and tested its ability to promote functional recovery following RLN injury. We created recombinant fusion proteins consisting of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) fused to laminin-binding domains (LBDs) in order to prevent neurotrophin diffusion. LBD-BDNF, LBD-GDNF, and laminin were injected into a collagen tube that was fitted to the ends of the transected RLN in rats. Functional recovery was assessed 4, 8, and 12 weeks after injury. Although vocal fold movement was not restored until 12 weeks after injury, animals treated with the collagen tube loaded with laminin, LBD-BDNF and LBD-GDNF showed improved recovery in vocalisation, arytenoid cartilage angles, compound muscle action potentials and regenerated fibre area compared to animals treated by autologous nerve grafting (p < 0.05). These results demonstrate the drug delivery system induced nerve regeneration following RLN transection that was superior to that induced by autologus nerve grafting. It may have potential applications in nerve regeneration of RLN transection injury.

Intranasal Administration of chitosan-Coated Nanostructured Lipid Carriers Loaded with GDNF Improves Behavioral and Histological Recovery in a Partial Lesion Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder, but current therapies are only symptomatic. A promising alternative to address the neurodegenerative process is the use of neurotrophic factors, such as the glial cell-derived neurotrophic factor (GDNF). However, its clinical use has been limited due to its short half-life and rapid degradation after in vivo administration, in addition to difficulties in crossing the blood-brain barrier (BBB). This barrier is a limiting factor in brain drug development, making the future progression of neurotherapeutics difficult. In the past few years, intranasal drug delivery has appeared as an alternative non-invasive administration route to bypass the BBB and target drugs directly to the CNS. Thus, the aim of this work was to study the in vivo neuroprotective effect of intranasally administered GDNF, encapsulated in chitosan-coated nanostructured lipid carrier (CS-NLC-GDNF), in a 6-OHDA partially lesioned rat model. The developed CS-NLC-GDNF showed a particle size of approximately 130 nm and high encapsulation efficiency. The in vitro study in PC-12 cells demonstrated the ability of the encapsulated GDNF to protect these cells against 6-OHDA toxin. After two weeks of daily intranasal administration of treatments, the administration of CS-NLC-GDNF achieved a behavioral improvement in rats, as well as a significant improvement in both the density of TH+ fibres in the striatum and the TH+ neuronal density in the SN. Thus, it can be concluded that the nose-to-brain delivery of CS-NLC-GDNF could be a promising therapy for the treatment of PD.

An engineered biocompatible drug delivery system enhances nerve regeneration after delayed repair.

Localized drug delivery strategies could greatly benefit patients with peripheral nerve injury and could be easy for surgeons to implement. We developed a local drug delivery system (DDS) using drug-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) embedded in a fibrin gel. In an in vitro study, we investigated the biocompatibility of this DDS by performing a toxicity assay in which we incubated PC-12 cells with the medium released from the DDS in vitro. In an in vivo study, this DDS was applied at the rat common peroneal (CP) nerve injury site to deliver exogenous glial cell line-derived neurotrophic factor (GDNF) to the regenerating axons after delayed nerve repair. In vitro, PC-12 cells incubated with released media samples from the DDS had similar viability to control cells cultured with normal media, demonstrating that the DDS was not toxic. In vivo, the numbers of motor and sensory neurons that regenerated their axons with empty MS treatment were the same as when there was no MS treatment. The DDS increased the numbers of regenerating motor- and sensory neurons to levels indistinguishable from those observed with immediate nerve repair. The DDS increased neuron regeneration to levels double those observed with negative control groups. This biocompatible, nontoxic, fibrin gel-based DDS enhances outcomes following severe peripheral nerve injuries.