Safety, Tolerability, Pharmacokinetics, and Immunogenicity of a Novel Recombination Human Nerve Growth Factor in Healthy Chinese Subjects.

BACKGROUND: Nerve growth factor (NGF), the first-discovered member of the neurotrophin family, has long been regarded as a potential drug to combat acute and chronic neurodegenerative processes. However, the pharmacokinetic profile of NGF is poorly described. OBJECTIVES: The aim of this study was to investigate the safety, tolerability, pharmacokinetics, and immunogenicity of a novel recombinant human NGF (rhNGF) in healthy Chinese subjects. METHOD: The study randomized 48 and 36 subjects to receive (i) single-ascending dose (SAD group; 7.5, 15, 30, 45, 60, 75 µg or placebo) and (ii) multiple-ascending dose (MAD group; 15, 30, 45 µg, or placebo) rhNGF intramuscular injections, respectively. In the SAD group, all participants received rhNGF or placebo only once. In the MAD group, participants were randomly assigned to receive multiple doses of rhNGF or placebo once a day for 7 consecutive days. Adverse events (AEs) and anti-drug antibodies (ADAs) were monitored throughout the study. Recombinant human NGF serum concentrations were determined using a highly sensitive enzyme-linked immunosorbent assay. RESULTS: All AEs were mild, except for some injection-site pain and fibromyalgia, which were experienced as moderate AEs. Only one moderate AE was observed in the 15 µg cohort throughout the study and resolved within 24 hours of stopping dosing. Many participants (10% in 30 µg, 50% in 45 µg, and 50% in 60 µg in the SAD group; 10% in 15 µg, 30% in 30 µg, and 30% in 45 µg in the MAD group) experienced moderate fibromyalgia. However, all moderate fibromyalgia were resolved by the end of the subject's participation in the study. No severe AEs or clinically significant abnormalities were reported. All subjects in the 75 µg cohort experienced positive ADA in the SAD group, and one subject in the 30 µg dose and four subjects in the 45 µg dose also experienced positive ADA in the MAD group. Recombinant human nerve growth factor was absorbed (median Tmax, 4.0-5.3 h) and eliminated biexponentially (mean t1/2, 4.53-6.09 h) with a moderate speed. The Cmax and AUC increased in an approximately dose-proportional manner over the dose range of 7.5-45 µg, and at doses higher than 45 µg these parameters increased more than dose proportionally. There was no obvious accumulation after 7 days of daily dosing of rhNGF. CONCLUSION: The favorable safety and tolerability and predictable pharmacokinetic profile of rhNGF in healthy Chinese subjects support its continuing clinical development for the treatment of nerve injury and neurodegenerative diseases. The AEs and immunogenicity of rhNGF will continue to be monitored in future clinical trials. TRIAL REGISTRATION: This study was registered with Chinadrugtrials.org.cn (ChiCTR2100042094) on January 13th, 2021.

Microenvironment-responsive immunoregulatory electrospun fibers for promoting nerve function recovery.

The strategies concerning modification of the complex immune pathological inflammatory environment during acute spinal cord injury remain oversimplified and superficial. Inspired by the acidic microenvironment at acute injury sites, a functional pH-responsive immunoregulation-assisted neural regeneration strategy was constructed. With the capability of directly responding to the acidic microenvironment at focal areas followed by triggered release of the IL-4 plasmid-loaded liposomes within a few hours to suppress the release of inflammatory cytokines and promote neural differentiation of mesenchymal stem cells in vitro, the microenvironment-responsive immunoregulatory electrospun fibers were implanted into acute spinal cord injury rats. Together with sustained release of nerve growth factor (NGF) achieved by microsol core-shell structure, the immunological fiber scaffolds were revealed to bring significantly shifted immune cells subtype to down-regulate the acute inflammation response, reduce scar tissue formation, promote angiogenesis as well as neural differentiation at the injury site, and enhance functional recovery in vivo. Overall, this strategy provided a delivery system through microenvironment-responsive immunological regulation effect so as to break through the current dilemma from the contradiction between immune response and nerve regeneration, providing an alternative for the treatment of acute spinal cord injury.

Neuroprotective Effect of Nerve Growth Factor Loaded in Porous Silicon Nanostructures in an Alzheimer's Disease Model and Potential Delivery to the Brain.

Nerve growth factor (NGF) plays a vital role in reducing the loss of cholinergic neurons in Alzheimer's disease (AD). However, its delivery to the brain remains a challenge. Herein, NGF is loaded into degradable oxidized porous silicon (PSiO2 ) carriers, which are designed to carry and continuously release the protein over a 1 month period. The released NGF exhibits a substantial neuroprotective effect in differentiated rat pheochromocytoma PC12 cells against amyloid-beta (Aß)-induced cytotoxicity, which is associated with Alzheimer's disease. Next, two potential localized administration routes of the porous carriers into murine brain are investigated: implantation of PSiO2 chips above the dura mater, and biolistic bombardment of PSiO2 microparticles through an opening in the skull using a pneumatic gene gun. The PSiO2 -implanted mice are monitored for a period of 8 weeks and no inflammation or adverse effects are observed. Subsequently, a successful biolistic delivery of these highly porous microparticles into a live-mouse brain is demonstrated for the first time. The bombarded microparticles are observed to penetrate the brain and reach a depth of 150 µm. These results pave the way for using degradable PSiO2 carriers as potential localized delivery systems for NGF to the brain.

Optimized liposomes with transactivator of transcription peptide and anti-apoptotic drugs to target hippocampal neurons and prevent tau-hyperphosphorylated neurodegeneration.

Liposomes (lip) carrying pharmaceuticals have shown promise in their ability to advance the therapy for neurodegenerative diseases. However, the low nerve-targeting capacity and poor penetration rate of lip through the blood-brain barrier (BBB) are major hurdles to achieving successful treatment. Herein, we developed lip incorporating cardiolipin (CL) and phosphatidic acid (PA) to promote their capability against hyperphosphorylation of tau protein, and a transactivator of transcription (TAT) peptide to permeate the BBB for delivering nerve growth factor (NGF), rosmarinic acid (RA), curcumin (CURC) and quercetin (QU). We derived an optimization method to assess a better composition of phospholipids in the lip loaded with the four medicines. Experimental results revealed that this optimized lip increased the viability of SK-N-MC cells insulted with ß-amyloid peptide (Aß) fibrils and prevented Wistar rat brain from producing hyperphosphorylated tau. CL and PA and the grafted TAT peptide on the carrier surface improved the rescue efficiency by inhibiting Aß deposition and reducing the expressions of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), c-Jun N-terminal protein kinase, p38, tau at serine 202 and caspase-3. The lip also enhanced the expressions of p-ERK5 and p-cyclic adenosine monophosphate response element-binding protein. The amalgamated activity of NGF, RA, CURC and QU, and the effect of charged CL/PA on Aß deposits supported the therapeutic efficacy of lip. The optimized TAT-NGF-RA-CURC-QU-CL/PA-lip can be a capable drug delivery system to cross the BBB and protect Alzheimer's disease brains from tau hyperphosphorylation. STATEMENTS OF SIGNIFICANCE: The therapeutic efficiency of liposomes (lip) against neurodegenerative disorder depends on their nerve-targeting capacity and ability to permeate the blood-brain barrier (BBB). Lip was developed incorporating cardiolipin (CL) and phosphatidic acid (PA) to promote their target specificity against hyperphosphorylation of tau protein, and a transactivator of transcription (TAT) peptide to permeate the BBB. We have successfully derived an optimization method using a new mathematical expression for the first time to assess a better composition of phospholipids in lip loaded with nerve growth factor (NGF), rosmarinic acid (RA), curcumin (CURC) and quercetin (QU). The optimized TAT-NGF-RA-CURC-QU-CL/PA-lip efficaciously down-regulated the expressions of phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), c-Jun N-terminal protein kinase, p38, tau at serine 202 and caspase-3, and up-regulated the expressions of p-ERK5 and p-cyclic adenosine monophosphate response element-binding protein in Alzheimer's disease Wistar rat model.

Cholinergic striatal neurons are increased in HSAN V homozygous mice despite reduced NGF bioavailability.

The neurotrophin Nerve growth factor (NGF) plays a critical role in the mature and developing nervous system. A point mutation (R100W) in the NGFB gene was found in patients with Hereditary Sensory and Autonomic Neuropathy type V (HSAN V), which leads to pain insensitivity. In a previous work it has been shown that the mutation provokes a reduced secretion of mature NGF. In this study we generated and analyzed homozygous NGFR100W/R100W mice to understand whether the reduced NGF bioavailability can contribute to the clinical phenotype of the homozygous condition. We found that the majority of NGFR100W/R100W mice were born normal but failed to reach the first month of age. This early lethality was rescued by daily treatment with wild type NGF. In addition, we found that the density of cholinergic neurons of homozygous mice was unaffected in the medial septum and in the nucleus basalis of Meynert, whereas, suprisingly, it was increased specifically in the striatum. Due to the known action of the striatal cholinergic tone in modulating pain, our findings support the hypothesis that a central mechanism, linked to the NGFR100W-dependent increase of the striatal cholinergic tone, can contribute to the pain insensitivity observed in HSAN V patients.

Therapeutic Effect of Controlled Release of Dual Growth Factor Using Heparin-Pluronic Hydrogel/Gelatin-Poly (Ethylene Glycol)-Tyramine Hydrogel System in a Rat Model of Cavernous Nerve Injury.

The number of cases of erectile dysfunction (ED) caused after radical prostatectomy (RP) prostate cancer treatment is increasing steadily. Although various studies have been conducted for treatment of post-RP ED, there is still a need for more effective methods. A dual growth factor incorporated heparin-pluronic/gelatin-poly(ethylene glycol)-tyramine (HP/GPT) hydrogel, which consists of a basic fibroblast growth factor (bFGF)-loaded HP hydrogel and nerve growth factor (NGF)-loaded GPT hydrogel, can control dose and rate of growth factor release. In this study, we demonstrated that dual growth factor incorporated HP/GPT hydrogel could further improve erectile function in a rat model of bilateral cavernous nerve injury (BCNI). We showed that erectile function was decreased after BCNI, but it was further improved by treatment with a dual growth factor incorporated HP/GPT hydrogel compared with groups treated with single growth factor in a rat model of cavernous nerve injury. Also, we observed an increase in cyclic guanosine monophosphate (cGMP) levels in the dual growth factor group when compared with the groups treated with single growth factor. This effect was associated with greater upregulation of nitric oxide synthase and endothelial nitric oxide synthase expression in the penile tissue of the group treated with dual growth factor incorporated HP/GPT than in the other experimental groups. Apoptosis in the penile tissue treated with the dual growth factor incorporated HP/GPT hydrogel was lower than those treated singly with either bFGF or NGF incorporated GPT hydrogel. Both α-smooth muscle actin and CD31 expression increased in the group treated with dual growth factor incorporated HP/GPT hydrogel when compared to in the other experimental groups. Altogether, our results proved that the sequential and continuous release of growth factors from dual growth factor incorporated HP/GPT hydrogel prevented fibrosis and nerve damage induced by BCNI in the corpus cavernosum, and promoted the recovery of erectile function. Dual growth factor incorporated HP/GPT hydrogel may be a potent clinical application for the treatment of post-RP ED and could potentially be used various biomedical application in tissue regnerative medicine.

The application of control charts in regulated bioanalysis for monitoring long-term reproducibility.

In regulated bioanalysis, the acceptance of results is batch-wise. When during clinical development derived pharmacokinetic or pharmacodynamic results from different studies will be combined or compared, it is recommendable to monitor the long-term reproducibility of bioanalytical assays. Long-term reproducibility can be evaluated by control charts generated from control samples included in each batch. We present a methodology for the implementation, construction and evaluation of control charts next to the regular batch acceptance of bioanalytical results. Decision rules can be set up for a statistical evaluation of the results. Violation of a decision rule may lead to a root-cause investigation and corrective actions to improve assay robustness. Three examples of control charts, for pharmacokinetic and pharmacodynamic analytes are presented.

Establishment of nerve growth factor gradients on aligned chitosan-polylactide /alginate fibers for neural tissue engineering applications.

Nerve conduits containing aligned fibrous fillers with gradiently distributed signal molecules are essential for long-gap nerve repair. This study was to develop an approach for establishing nerve growth factor (NGF) gradients onto the aligned chitosan-polylactide (CH-PLA) fibers. CH-PLA containing 37wt% of PLA was spun into fibers using a wet-spinning technique. CH-PLA fibers showed much higher wet-state tensile strength, enhanced degradation tolerance and significantly lower swelling degree in comparison to chitosan fibers. The CH-PLA fibers with diameters from 40 to 60µm were selected and segmentally coated in bundles using NGF-contained alginate solutions to establish NGF gradients lengthwise along fibers. The diameter of resulting NGF-loaded CH-PLA/alginate fibers was well controlled within a range between 60 and 120µm. Calcium ion crosslinked alginate coating layers on fibers showed abilities to administer the sustainable NGF release in a gradient distribution manner for at least 5 weeks. NGF-induced neurite outgrowth of PC12 cells confirmed that bioactivity of NGF released from fibers was well retained.

Guangxi cobra venom-derived NGF promotes the osteogenic and therapeutic effects of porous BCP ceramic.

Neuro-osteological interactions have an important role in the regulation of bone metabolism and regeneration. Neuropeptides combined with porous biphasic calcium phosphates (BCP) using protein adsorption may contribute to the acceleration of bone formation. In the present study, we investigated the effect of BCP combined with nerve growth factor (NGF) on the growth of osteoblasts in vitro and the combinational therapeutic effect on the repair of calvarial defects in vivo. NGF was separated and purified from Chinese cobra venom using a simplified three-step chromatography method. BCP combined with NGF exerted a potent effect on osteoblast differentiation, as evidenced by enhanced cell proliferation, increased ALP activity and the up-regulated expression of osteogenesis-related genes and proteins. Further, combinational therapy with BCP and NGF improved calvarial regeneration, which was superior to treatment with therapy alone, as observed using imageological and morphological examination and histological and immunohistochemical staining. The results confirmed the effect of neuro-osteological interactions through combinatorial treatment with NGF and BCP to promote osteogenesis and bone formation, which may provide an effective and economical strategy for clinical application.

Heparin/collagen encapsulating nerve growth factor multilayers coated aligned PLLA nanofibrous scaffolds for nerve tissue engineering.

Biomimicing topological structure of natural nerve tissue to direct axon growth and controlling sustained release of moderate neurotrophic factors are extremely propitious to the functional recovery of damaged nervous systems. In this study, the heparin/collagen encapsulating nerve growth factor (NGF) multilayers were coated onto the aligned poly-L-lactide (PLLA) nanofibrous scaffolds via a layer-by-layer (LbL) self-assembly technique to combine biomolecular signals, and physical guidance cues for peripheral nerve regeneration. Scanning electronic microscopy (SEM) revealed that the surface of aligned PLLA nanofibrous scaffolds coated with heparin/collagen multilayers became rougher and appeared some net-like filaments and protuberances in comparison with PLLA nanofibrous scaffolds. The heparin/collagen multilayers did not destroy the alignment of nanofibers. X-ray photoelectron spectroscopy and water contact angles displayed that heparin and collagen were successfully coated onto the aligned PLLA nanofibrous scaffolds and improved its hydrophilicity. Three-dimensional (3 D) confocal microscopy images further demonstrated that collagen, heparin, and NGF were not only coated onto the surface of aligned PLLA nanofibrous scaffolds but also permeated into the inner of scaffolds. Moreover, NGF presented a sustained release for 2 weeks from aligned nanofibrous scaffolds coated with 5.5 bilayers or above and remained good bioactivity. The heparin/collagen encapsulating NGF multilayers coated aligned nanofibrous scaffolds, in particular 5.5 bilayers or above, was more beneficial to Schwann cells (SCs) proliferation and PC12 cells differentiation as well as the SC cytoskeleton and neurite growth along the direction of nanofibrous alignment compared to the aligned PLLA nanofibrous scaffolds. This novel scaffolds combining sustained release of bioactive NGF and aligned nanofibrous topography presented an excellent potential in peripheral nerve regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1900-1910, 2017.

Nanotechnological strategies for nerve growth factor delivery: Therapeutic implications in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder associated with amyloid-ß peptide misfolding and aggregation. Neurotrophic factors, such as nerve growth factor (NGF), can prevent neuronal damage and rescue the cholinergic neurons that undergo cell death in AD, reverse deposition of extracellular amyloid plaques and improve cognitive deficits. However, NGF administration is hampered by the poor pharmacokinetic profile of the therapeutic protein and its inability to cross the blood-brain barrier, which requires specialised drug delivery systems (DDS) for efficient NGF delivery to the brain. This review covers the main therapeutic approaches that have been developed for NGF delivery targeting the brain, from polymeric implants to gene and cell-based therapies, focusing on the role of nanoparticulate systems for the sustained release of NGF in the brain as a neuroprotective and disease-modifying approach toward AD. Lipid- and polymer-based delivery systems, magnetic nanoparticles and quantum dots are specifically addressed as promising nanotechnological strategies to overcome the current limitations of NGF-based therapies.

Protein-releasing conductive anodized alumina membranes for nerve-interface materials.

Nanoporous anodized alumina membranes (AAMs) have numerous biomedical applications spanning from biosensors to controlled drug delivery and implant coatings. Although the use of AAM as an alternative bone implant surface has been successful, its potential as a neural implant coating remains unclear. Here, we introduce conductive and nerve growth factor-releasing AAM substrates that not only provide the native nanoporous morphology for cell adhesion, but also induce neural differentiation. We recently reported the fabrication of such conductive membranes by coating AAMs with a thin C layer. In this study, we investigated the influence of electrical stimulus, surface topography, and chemistry on cell adhesion, neurite extension, and density by using PC 12 pheochromocytoma cells in a custom-made glass microwell setup. The conductive AAMs showed enhanced neurite extension and generation with the electrical stimulus, but cell adhesion on these substrates was poorer compared to the naked AAMs. The latter nanoporous material presents chemical and topographical features for superior neuronal cell adhesion, but, more importantly, when loaded with nerve growth factor, it can provide neurite extension similar to an electrically stimulated CAAM counterpart.

Using NGF heparin-poloxamer thermosensitive hydrogels to enhance the nerve regeneration for spinal cord injury.

OBJECTIVE: Nerve growth factor (NGF) has potential in spinal cord injury (SCI) therapy, but limited by the poor physicochemical stability and low ability to cross the blood spinal cord barrier. Novel heparin-poloxamer (HP) thermo-sensitive hydrogel was constructed to enhance the NGF regeneration on SCI. METHOD: NGF-HP thermo-sensitive hydrogel was prepared and related characteristics including gelation temperature, rheological behavior and micromorphology were measured. Local NGF delivery to the injured spinal cord was achieved by in situ injection in the injured space. The cellular uptake of NGF-HP hydrogel was evaluated with PC12 cells in vitro. Pathologic characteristics and neuron regeneration effects on the SCI rats were studied to evaluate the enhanced therapy of NGF-HP hydrogel. Endoplasmic reticulum (ER) stress-induced apoptosis was analyzed to explore the related mechanism in SCI regeneration. RESULTS: NGF-HP hydrogel showed good morphology and stable bioactivity of NGF in vitro. NGF-HP hydrogel combined treatment significantly enhanced the efficiency of NGF cellular uptake (P<0.05) without obvious cytotoxicity. Significant improvements in both neuron functions and tissue morphology on the SCI rats were observed in NGF-HP hydrogel group. Compared with free HP hydrogel and NGF treatment groups, NGF-HP hydrogel group showed significant inhibition on the formation of glial scars in the extreme crushed rat SCI model. The neuroprotective effects of NGF-HP were related to the inhibition of chronic ER stress-induced apoptosis. CONCLUSIONS: HP hydrogel combined with orthotopic injection technique might be an effective method to deliver NGF into the injured site, which will provide an effective strategy for SCI regeneration. STATEMENT OF SIGNIFICANCE: Spinal cord injury (SCI) is a devastating condition that can lead to sudden loss of sensory and autonomic function. Current treatment includes decompression surgery, injury stabilization, secondary complications prevention and rehabilitation. However, neurological recovery is limited. Nerve growth factor (NGF) has potential in SCI therapy, but limited by the poor physicochemical stability and low ability to cross the blood spinal cord barrier. Hydrogels have good affinity and compatibility to biological tissue. In this study, we developed a novel heparin-poloxamer (HP) thermo-sensitive hydrogel to enhance the spinal cord regeneration of NGF. From SCI rat experiment, HP hydrogel combined with orthotopic injection technique showed best neuroprotective effects among experimental groups. This novel combined technique will provide an effective strategy for SCI regeneration.

Enhanced bioavailability of nerve growth factor with phytantriol lipid-based crystalline nanoparticles in cochlea.

PURPOSE: Supplementation of exogenous nerve growth factor (NGF) into the cochlea of deafened animals rescues spiral ganglion cells from degeneration. However, a safe and potent delivery of therapeutic proteins, such as NGF, to spiral ganglion cells remains one of the greatest challenges. This study presents the development of self-assembled cubic lipid-based crystalline nanoparticles to enhance inner ear bioavailability of bioactive NGF via a round window membrane route. METHODS: A novel nanocarrier-entrapped NGF was developed based on phytantriol by a liquid precursor dilution, with Pluronic(®) F127 and propylene glycol as the surfactant and solubilizer, respectively. Upon dilution of the liquid lipid precursors, monodispersed submicron-sized particles with a slight negative charge formed spontaneously. RESULTS: Biological activity of entrapped NGF was assessed using pheochromocytoma cells with NGF-loaded reservoirs to induce significant neuronal outgrowth, similar to that seen in free NGF-treated controls. Finally, a 3.28-fold increase in inner ear bioavailability was observed after administration of phytantriol lipid-based crystalline nanoparticles as compared to free drug, contributing to an enhanced drug permeability of the round window membrane. CONCLUSION: Data presented here demonstrate the potential of lipid-based crystalline nanoparticles to improve the outcomes of patients bearing cochlear implants.

Magnetic NGF-releasing PLLA/iron oxide nanoparticles direct extending neurites and preferentially guide neurites along aligned electrospun microfibers.

Nerve growth factor releasing composite nanoparticles (NGF-cNPs) were developed to direct the extension of neurite outgrowth from dorsal root ganglia (DRG). Iron oxide magnetic nanoparticles were incorporated into poly-l-lactic acid (PLLA) nanoparticles in order to position the NGF-cNPs in a culture dish. Neurites growing from DRG extended toward the NGF released from the NGF-cNPs. DRG were then cultured on aligned PLLA microfibers in the presence of NGF-cNPs, and these biomaterials combined to align DRG neurite extension along one axis and preferentially toward the NGF-cNPs. This combinatorial biomaterial approach shows promise as a strategy to direct the extension of regenerating neurites.

Dual growth factor-immobilized microspheres for tissue reinnervation: in vitro and preliminary in vivo studies.

Growth factors (GFs) (basic fibroblast growth factor (bFGF) and/or nerve growth factor (NGF))-immobilized polycaprolactone (PCL)/Pluronic F127 microspheres were prepared using an isolated particulate-melting method and the sequential binding of heparin and GFs onto the microspheres. The GFs immobilized on the microspheres were released in a sustained manner over 28 days, regardless of GF type. From the in vitro culture of muscle-derived stem cells, it was observed that the NGF-immobilized microspheres induced more neurogenic differentiation than the bFGF-immobilized microspheres, as evidenced by a quantitative real-time polymerase chain reaction using specific neurogenic markers (Nestin, GFAP, ß-tubulin, and MAP2) and Western blot (Nestin and ß-tubulin) analyses. The dual bFGF/NGF-immobilized microspheres showed better neurogenic differentiation than the microspheres immobilized with single bFGF or NGF. From the preliminary animal study, the dual bFGF/NGF-immobilized microsphere group also showed effective nerve regeneration, as evaluated by immunocytochemistry using a marker - ß-tubulin. The dual bFGF/NGF-immobilized PCL/Pluronic F127 microspheres may be a promising candidate for nerve regeneration in certain target tissues (i.e. muscles) leading to sufficient reinnervation.

Polyethlyene glycol microgels to deliver bioactive nerve growth factor.

Delivery of bioactive molecules is a critical step in fabricating materials for regenerative medicine, yet, this step is particularly challenging in hydrated scaffolds such as hydrogels. Although bulk photocrosslinked poly(ethylene glycol) (PEG) hydrogels have been used for a variety of tissue engineering applications, their capability as drug delivery scaffolds has been limited due to undesirable release profiles and reduction in bioactivity of molecules. To solve these problems, this article presents the fabrication of degradable PEG microgels, which are micron-sized spherical hydrogels, to deliver bioactive nerve growth factor (NGF). NGF release and activity was measured after encapsulation in microgels formed from either 3 kDa or 6 kDa PEG to determine the role of hydrogel mesh size on release. Microgels formed from 6 kDa PEG were statistically larger and had a higher swelling ratio than 3 kDa PEG. The 6 kDa PEG microgels provided a Fickian release with a reduced burst effect and 3 kDa microgels provided anomalous release over ≥20 days. Regardless of molecular weight of PEG, NGF bioactivity was not significantly reduced compared to unprocessed NGF. These results demonstrate that microgels provide easy mechanisms to control the release while retaining the activity of growth factors. As this microgel-based delivery system can be injected at the site of nerve injury to promote nerve repair, the potential to deliver active growth factors in a controlled manner may reduce healing time for neural tissue engineering applications.

Extracellular pH and neuronal depolarization serve as dynamic switches to rapidly mobilize trkA to the membrane of adult sensory neurons.

Activation of the nerve growth factor (NGF) receptor trkA and tissue acidosis are critically linked to inflammation-associated nociceptor sensitization. This study explored how increased acidity is linked to sensory neuron sensitization to NGF. Adult Wistar rat primary sensory neurons grown at physiological pH 7.4, then either kept at pH 7.4 or challenged for 30 min in pH 6.5 medium, provided a model of acidosis. Nonpermeabilizing trkA immunofluorescence revealed a significant increase in trkA mobilization to the plasma membrane from intracellular stores in response to proton challenge. This was confirmed using a surface protein biotinylation assay and Brefeldin A disruption of the rough endoplasmic reticulum-Golgi-trans-Golgi network. Mobilization of trkA to the membrane at pH 6.5 was abolished in neurons treated with the acid-sensitive ion channel blocker, amiloride. While elevated levels of NGF-independent trkA phosphorylation occurred at pH 6.5 alone, the level of activation was significantly increased in response to NGF challenge. Exposure of sensory neurons to pH 6.5 medium also resulted in strong calcium (Ca(2+)) transients that were reversible upon reintroduction to physiological pH. The pH 6.5-induced mobilization of trkA to the membrane was Ca(2+) dependent, as BAPTA-AM Ca(2+) chelation abrogated the response. Interestingly, KCl-induced depolarization was sufficient to induce mobilization of trkA to the cell surface at pH 7.4, but did not augment the response to pH 6.5. In conclusion, increased mobilization of trkA to neuronal membranes in response to either acidosis or neuronal depolarization provides two novel mechanisms by which sensory neurons can rapidly sensitize to NGF and has important implications for inflammatory pain states.

Mechanism of polymeric nanoparticle-based drug transport across the blood-brain barrier (BBB).

In 1995 it was reported for the first time that nanoparticles could be used for the delivery of drugs across the blood-brain barrier (BBB) following intravenous injection. In vitro and in vivo experiments show that the underlying mechanism is receptor-mediated endocytosis followed by transcytosis. No opening of the tight junctions was observed. Due to the overcoating of the nanoparticles with polysorbate 80 or poloxamers 188, apolipoproteins A-I and/or E are adsorbed from the blood on to the particle surface after injection. These apolipoproteins mediate the interaction with LDL or scavenger receptors on the BBB followed by the above brain uptake processes. Likewise, covalent attachment of these apolipoproteins or of transferrin, insulin or antibodies against the respective receptors also enables a similar nanoparticle-mediated drug transport across the BBB. From these results it can be concluded that the nanoparticles act as "Trojan Horses" taking advantage of physiological receptor-mediated transport processes across the BBB.

Sustained local delivery of bioactive nerve growth factor in the central nervous system via tunable diblock copolypeptide hydrogel depots.

Biomaterial vehicles that can provide sustained, site-specific molecular delivery in the central nervous system (CNS) have potential for therapeutic and investigative applications. Here, we present in vitro and in vivo proof of principle tests of diblock copolypeptide hydrogels (DCH) to serve as depots for sustained local release of protein effector molecules. We tested two DCH, K(180)L(20) and E(180)L(20), previously shown to self-assemble into biocompatible, biodegradable deposits that persist four to eight weeks after injection into mouse forebrain. In vitro tests demonstrated sustained release from dialysis cassettes of the representative protein, lysozyme, dissolved in K(180)L(20) or E(180)L(20) hydrogels. Release time in vitro varied in relation to DCH charge and mechanical properties, and ionic strength of the media. To evaluate bioactive protein delivery in vivo, we used nerve growth factor (NGF) and measured the size of mouse forebrain cholinergic neurons, which respond to NGF with cellular hypertrophy. For in vivo tests, the storage modulus of DCH depots was tuned to just below that of CNS tissue. In comparison with NGF injected in buffer, depots of NGF dissolved in either K(180)L(20) or E(180)L(20) provided significantly longer delivery of NGF bioactivity, maintaining hypertrophy of local forebrain cholinergic neurons for at least 4 weeks and inducing hypertrophy a further distance away (up to 5 mm) from injection sites. These findings show that depots of DCH injected into CNS can provide sustained delivery within the blood-brain barrier of a bioactive protein growth factor that exerts a predicted, quantifiable effect on local cells over a prolonged subacute time.