An In Situ Gelling Drug Delivery System For Improved Recovery After Spinal Cord Injury

Backgroud:A spinal cord injury(SCI)is defined as an injury to the spinal cord resulting in a temporary or permanent change in its loeomotor,sensory,or autonomie function.There is an urgent need to explore new therapeutie strategies with long-term controlled delivery of therapeutic compounds to treat SCI patients.Objective:Therapeutic strategies for the spinal cord injury(SCI)are limited by the current available drug delivery techniques.The aim of this study was to develop a thermoresponsive in situ gelling DDS to inhibit the secondary injury and to improve the recovery of the injured spinal cord.The in vitro(rheological behaviors and in vitro GMI release)and in vivo(pharmacological efficiency and immunohistochemistry)tests were used to evaluate the potential of the thermoresponsive in situ gelling GM1-hydrogel for SCI therapy.Methods:in vitro:Here,an in situ gelling DDS,composed of a Poloxamer-407,a 188 mixturebased thermoresponsive hydrogel matrix and,an incorporated therapeutic compound(monosialoganglioside,GM1),is developed for SCI therapy.The desired hydrogel was prepared by a cold method on a weight basis under an aseptic environment.The viscoelastic properties of Pol-1 alone,Pol-2 alone,blank hydrogel,and GM1-hydrogel were measured with a stress-controlled rheometer.The structure of the dried hydrogel and GM1-hydrogel were studied using SEM(Ultra-55,Zeiss EVO)at room temperature.Blank hydrogel and GM1-hydrogel were freeze-dried to maintain the porous structure without any collapse during water sublimation.The in vitro release of GM1 from GM1-hydrogel was evaluated in the aCSF to simulate the central nervous system environment.in vivo:A low-thoracic hemisection in rats is used as SCI model to evaluate therapeutic efficiency.All animal procedures were conducted according to the guidelines for the care and use of laboratory animals,which was approved by the Animal Care and Use Committee of Nanjing Medical University.Sprague-Dawley male rats weighing 170-220 g were provided by the Animal Experimental Center of Nanjing Medical University.Animals were randomly assigned into the following four groups:aCSF,"blank hydrogel,GM1 solution,and GM1-hydrogel(n=15).The laminectomy was performed to expose the thoracic spinal cord at T10.Then,a dorsal hemisection was made at the left side of T10 with fine microdissection scissors to remove a block?(2 mm)of the spinal cord tissue.After hemostasis,aCSF,blank polymer solution,GM1 solution(5 mg/mL),and GM1-incorporated polymer solution were topically administrated(20?L/rat)to the lesion sites,respectively.After spinal cord surgery,the recovery of locomotor capacity was measured by the BBB open field locomotor test procedure,as described elsewhere.The animal behaviors of the trunk,tail,and hindlimbs were evaluated at day 1 after spinal cord hemisection and weekly thereafter for 4 weeks.Animals were anesthetized with a lethal dose of chloral hydrate at certain time points(days 3,7,and 28)after surgery and treatment.TUNEL staining was performed using an in situ cell death detection kit(Roche Diagnostics Corp.Switzerland),according to the protocol provided by the manufacturer.Spinal cord sections harvested at days 3 and 7 after the surgery were analyzed.Spinal cord sections harvested at day 28 posttrauma were used for immunohistochemical analysis.Results are expressed as the mean±standard deviation(s.d.)for at least three independent experiments.Data were analyzed using one-way analysis of variance followed by a Bonferroni's posthoc test for multiple comparisons(SPSS 20;IBM Corn.,USA).Results:in vitro:1)Characterization of the hydrogel.a)The elastic and viscous moduli of Pol-1 and Pol-2 in terms of temperature.GT is around 15 ? for Pol-1 and no GT was observed for Pol-2 until 45?.b)Impact of the formulation compositions on the GT of blank hydrogel.The higher concentration of Pol-1,the lower GT of hydrogel.The GT increased to a maximum and then decreased as a function of the Pol-2 concentration.The color bar from light gray to black on the right side represents temperature magnitude(0-100?).c)The elastic and viscous modulus of blank hydrogel and GM1-hydrogel as a function of temperature,showing how the addition of relatively hydrophobic GM1 reduced the GT of hydrogel.d)The thermoresponsiveness of the formulation of hydrogel and GM1-hydrogel,for which the polymer solution at room temperature reversibly transformed into a hydrogel at 37?.e)SEM results of dried hydrogel and GM1-hydrogel illustrated the incorporation of GM1 has no significant impact on the hydrogel structure.2)The complete release of GM1 was sustained from roughly 3 d at room temperature to around 28 d at 37?,presenting a thermoresponsive drug release profile.According to the power law model fitting,the GM1 release mechanisms from GM1-hydrogel changed from the super Case-? transport to anomalous transport by increasing the temperature from 25 to 37?.in vivo:1)The pharmacological efficiency of the GM1-hydrogel was assessed in a T10 dorsal hemisection SCI model in rats.At day 3 and up to day 28 posttrauma,the animals were functionally graded using the BBB grading scale.GM1-hydrogel significantly improved hindlimb motor functions from day 14 after injury and onward.The improvement persisted until 28 d post trauma.2)Representative images of sagittal sections of TUNEL positive apoptotic cells at day 7 post trauma.Cellular alteration of typical apoptosis is evident.d)Comparison of the number of TUNEL-positive cells in animals.GM1-hydrogel reduced the number of apoptotic cells in the injured spinal cord.3)Immunohistochemistry study.a)Representative immunohistochemical staining images of GFAP and NF200 in the injured spinal cord tissues at day 28 post trauma.GFAP were characterized by brown staining.White arrows indicate the representative NF200 positive cells.Quantification of GFAP and NF200 without nuclear counterstaining demonstrated a decrease of astrocytic,reactivity and an improvement of neurite outgrowth in the injured spinal cord for the GM1-hydrogel group.Conclusion:The GM1-incorporating Poloxamer-407 and 188 polymer solution is converted to a hydrogel(GM1-hydrogel)upon instillation to the injured spinal cord,due to the increased temperature.At body temperature,the thermoresponsive hydrogel prolongs the release of GM1 for about 1 month,due to the superposition of dissolution and swelling(anomalous transport)of the hydrogel matrix.The sustained release of the GM1-hydrogel enables the prolonged residence time of GMI at the injured spinal cord,decreases the frequency of administration and,consequently,may improve patient compliance.After SCI,the administration of GM1-hydrogel to the lesion site inhibits the apoptotic cell death and glial scar formation,enhances the neuron regeneration,provides neuroprotection to the injured spinal cord,and improves the locomotor recovery.Overall,this study opens future perspectives for the treatment of SCI with a prolonged drug release DDS.