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Spinal cord injury interferes with the impulse transmission between the brain and the peripheral nerves, leading to partial of complete loss of sensory, motor and autonomic functions below the injury site. Patients with SCI suffer lifelong disability and require continuous physical and medical care. SCI in the United States is one of the leading causes of disability, with approximately 11,000 new cases reported each year .
On the basis of pathology, there are four general types of spinal cord injury: 1) cord maceration; 2) cord lacerations; 3) contusion injury; and 4) solid cord injury. The first two injuries cause prominent and irreversible damage to the spinal cord, while the latter two cause less direct damage to connective tissue [3, 4]. In all SCI cases, the contusion injury has 25 to 40 % occupancy among the four injury types, and the ratio is still increasing. Commonly, primary damage of contusion injury model happens in gray matter. However, without any treatment in 60 days, the damage will expand to white matter and furthermore cause loss of the whole tissue and function . Our project mainly focuses on contusion injury model.
As mentioned above, the pathophysiology of contusion injury includes primary and secondary mechanisms . Primary injury refers to physical and mechanical trauma occurring to the spinal cord. Axons or blood vessels will suffer irreversible injury due to shear forces . Damage to the spinal cord does not stop after the initial trauma, but continues with a cascade of downstream event which exhibits a series of reactive changes causing the injury to expand the initial wound size many times, such as degeneration of white matter tracts and neuronal apoptosis. This event termed secondary injury .
Secondary injury takes place at the cellular level and neuronal level. At the cellular level, the irreversible physical damage caused by initial trauma leads to normal intercellular responses to the trauma, such as inflammation and oxidative damage. These responses then cause series of adverse reaction, including, but not limited to, ischemia, oxidative damage, cytoskeletal degradation, inflammation, glutamate excitotoxicity, and resulting more cell apoptosis and necrosis.
Immune and inflammatory responses are main syndromes of the secondary injury. These responses cause to release regulatory messenger proteins, called cytokines. Some cytokines, such as TNF-α, have a deleterious effect on neural repair of the secondary injury . They also have potential in inducing cell apoptosis and programmed cell death. Furthermore, some immune cell like neutrophils, have the adverse function to produce free radicals which lead to oxidative damage .
Oxidative damage is due to excess free radicals which have extra electron. Free radicals lead to lipid peroxidation of cell membranes, disrupt the electron transport chain, affect ATPase activity [10, 11], as well as cytoskeletal degradation [12, 13], and therefore cause the cell to simply fall apart [14, 15]. Large amount products of lipid peroxidation were found in the gray matter area following contusion injury . Furthermore, more lipid peroxidation decreases glutamate uptake, which may exacerbate cells excitotoxicity injury . Other sources also showed free radicals in mitochondria could form super oxide anions (O2-) , which could react with NO- to generate peroxynitrite to cause more cellular damage.
Glutamate excitotoxicity occurs due to the excessive release of glutamate, which is one of excitatory amino acids, after severe central nervous system (CNS) injury. Glutamate binds to the NMDA and AMPA receptors and activates calcium (Ca2+) and sodium (Na+) channel, respectively[20, 21]. Too much calpain is activated by large unwarranted amounts of Ca2+ ions entering into cell. This process through the NMDA receptor (located on neurons) results apoptosis . In addition, Ca2+ ions detrimentally affect mitochondria by introducing more free radicals, consequently causing oxidative damage. In the meantime, the entrance of Na+ through the AMPA receptor into the neuron disturbs the osmotic balance . Thus the cells begin to spill out of essential regulatory proteins and finally necrosis.
Apoptosis is a normal biological function. However, researches have recently shown that both necrotic and apoptotic cells lead more healthy cells around them death and dysfunction at the stage of secondary damage. Studies have suggested that by blocking the cell apoptosis, the extent of secondary damage can be moderately lessened.
Therefore therapies for secondary injury are essential to reduce further damage and improve functional recovery.
2.2 Choice of Drug
Inflammation plays an important role in the secondary injury cascade. Currently there is no effective therapy for SCI. A standard anti-inflammatory drug, methylprednisolone, is thought to reduce the size of the hemorrhage and to inhibit free-radical induced lipid peroxidation . However, the effectiveness of methylprednisolone treatment is highly controversial due to a lack of conclusive evidence from both animal studies and clinical trials . Also, lazaroids, thyrotropin-releasing hormone and some other treatments continue to improve for curing spinal cord injury in some other ways. However, all of them are not a standard of care but only a treatment option.
Minocycline Hydrochloride (MH) is a semi-synthetic tetracycline derivative, clinically available antibiotic and anti-inflammatory drug that also demonstrates neuroprotective properties in a variety of experimental models of neurological diseases [29, 30]. With its anti-inflammatory, anti-apoptotic, and anti-oxidant properties, MH is a very promising drug for SCI. MH exerts its anti-inflammatory action by inhibiting activation of microglia and subsequent release of inflammatory molecules contributing to progression of secondary injury. In recent studies, MH has been shown to inhibit inducible nitric oxide synthase (iNOS) that produces NO to reduce oxidative damage , and may also attenuate glutamate toxicity inducing neuronal cell death by inhibiting activation and proliferation microglia. Furthermore, MH was reported to reduce cell apoptosis and scavenge free radicals that cause cell damage after SCI [34-36]. Thus MH can serve as a multifaceted agent that targets both cell death and development of secondary injury in SCI. A number of studies have shown that systemic administration of MH for 1-6 days reduces secondary injury and improves functional recovery in various animal models of SCI[37-40]. In addition, a comparison study showed that MH treatment resulted in significant functional recovery, while methylprednisolone injections didn't lead to behavioral improvement .
2.3 Choice of Drug Delivery System
Application of MH is limited because the optimized functionality is in a high local concentration, which is much higher than the maximum IV dose. MH is unstable in aqueous solution, especially at body temperature. Therefore the drug delivery system must be capable of sustained release as well as preservation of the stability of MH. Treatment is also administered for a minimum of three days up to four weeks indicating a need for sustained delivery for maximum efficacy.
There are various ways of fabricating drug delivery system, such as double emulsion to make hydrophobic particles, complex coacervation by electrostatic interactions, etc. Both mechanisms have been used to MH release. MH is reported that some recent works was to investigate different methods of producing PLGA polyelectrocyte complex containing MH which suitable for periodontal infections. Minocycline-PEGylated PLGA polyelectrocyte complex prepared by the ion pairing method had the best drug loading and entrapment efficiency compared with other prepared polyelectrocyte complexes. They also showed higher in vitro antibacterial activity than the free drug. But the products after biodegradation in mouth lead an acid environment, which endanger teeth. For the PIC micelles, MH release only lasted for 24 hours, while a clinical trial showed that MH treatment for 7 days resulted in significant recovery. In addition, a synthetic polymer is used for micelle fabrication, and its safety and inflammatory property remain to be tested.
3. Specific Aim
We proposed to use minocycline for spinal cord repair. The goal of this study is to develop an injectable hydrogel-based MH delivery system to release high
We developed nanoparticles (NP) made from a biocompatible natural polymer (polysaccharide) capable of sustained release of bioactive MH. Further, we developed biocompatible, biodegradable, and injectable hydrogel systems for NP encapsulation. This hydrogel system can be injected in the subdural space between dura and spinal cord tissue to cover the injured cord during spine surgery. No additional tissue damage will be caused by this treatment because the hydrogel is located outside the spinal cord tissue. The efficacy of local topical drug delivery to spinal cord has been reported [42, 43]. In addition, our drug delivery system is capable of preservation of the stability of MH because MH is stable in solid form, and encapsulation of MH in solid nanoparticles ensures that MH remains as solid.
4. Materials and Methods
4. Materials and Methods
Dextran sulfate, minocycline hydrochloride, alginic acid sodium salt, calcium chloride and magnesium chloride were purchased from Sigma. Agarose was obtained from FMC (Philadelphia, PA). All chemicals were used without further purification. Scanning electron microscopy (SEM) was performed by Zeiss Supra 50VP SEM. UV-vis spectroscopy was assessed by Tecan M200 plate reader.
4.2 Incorporation of nanoparticles in alginate hydrogel
Dextran sulfate and calcium chloride were dissolved in deionized (DI) water at the concentration of 2.5mg/ml and 7.2mM respectively. Nanoparticles were formed after dissolving minocycline at the concentration of 1mg/ml in the solution of DS and calcium ions. Magnesium chloride was added in this suspension of nanoparticles before alginate was added into this solution at the concentration of 7.5 mg/ml, and was mixed by vortex. After overnight shaking in 37 °C, alginate hydrogel was formed by calcium ion crosslinking.
4.3 Incorporation of nanoparticles in agarose hydrogel
Dextran sulfate and magnesium chloride were dissolved in DI water. Agarose was dissolved in this solution at the concentration of 3% w/v at 70 °C. Nanoparticles were formed as soon as mixing this agarose solution with minocycline solution (2mg/ml). The mixture was cooled down in 4 °C for 30min before agarose gelled.
4.4 In vitro MH Release assay
Hank's Balanced Salt Solution (HBSS) was employed as release medium for invitro MH release. For alginate hydrogel, 500ul gel was injected by 27 gauge needle into 1.5 ml tube. For agarose hydrogel, 100ul gel was injuected by 27 gauge needle into 24-well plate. 500ul HBSS was incubated with each sample at 37 °C. Every 24 h, the HBSS was removed and replaced with fresh HBSS. The amount of MH released every 24 h was determined by UV absorbance at 245nm.
RAW264.7 murine macrophages (kindly provided by Dr. Narayan Avadhani, University of Pennsylvania) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were seeded in 96-well tissue culture plates at a density of 60,000 cells per well. 48 h after seeding, MH released during a 24 h period was diluted with cell culture medium and added to the macrophage culture together with LPS. Fresh MH at the same concentration was used as controls to compare the bioactivity with released MH. After 48 h, the accumulated levels of nitrite in the cell culture medium, as an indication of nitric oxide (NO), was measured with Griess reagent (Promega).
4.5 Statistical Analysis
Three replicates were used for all the other experimental groups. Pairwise comparisons were conducted using a general linear ANOVA model and Tukey test, and P < 0.05 was considered statistically significant. Data are presented as mean ± standard deviations.
5. Preliminary Data