Experimental Study On Preparation Of MPS-PLGA Microspheres And Therapy Of Rat SCI Model

In traumatic spinal cord injury (SCI), the impact on the spinal cord induces the primary injury and the secondary damage. The secondary pathological changes following mechanical injury to the spinal cord contribute to the worsening of neurological functions. Methylprednisolone can cut down the disservice of the secondary damage and enhance the recovery of the neurofunction after SCI. The mechanisms probably included inhibiting lipid peroxidation, inflammatory reaction, lipid hydrolization and arachidonic acid releasing, improving microcirculation of the damaged spinal cord, preventing the disequilibrium of the Na, K and Ca between the internal and external, preventing apoptosis, suppressing excitatory amino acids, enhancing nervous excitation and synaptic transmission. MP is the one of those which has been certificated having effect with the SCI, and it is the only drug which having been approved to therapy the SCI by the FDA. At present, MP has been used to therapy the SCI with the high dose chemotherapy through intravenous injection. This drug delivery system usually has many adverse reactions. In this study, we try to inject the drug in the subarachnoid space in order to decrease the drug consumption and lessen the adverse reactions. First, we prepare the MPS-PLGA microspheres so as to therapy the SCI by one time injection without infection.Materials and Methods:Preparation of microspheres experiment: PLGA was choice as the capsule wall material and W/O/W double emulsion solvent evaporation-extraction method is used to prepare MPS-PLGA microspheres. The different variables of influencing factors on the preparation technology of PLGA microspheres were optimized by single factor analysis experiments and orthogonal design experiments. Prepare MPS-PLGA microspheres under the optimized conditions, and determine the shape, particle size, drug loading amount, encapsulation efficiency and release profile in vitro. Animal experiment: 240 healthy adult SD rats (weight: 230-250g) of either sex half and half were randomly divided into four groups. Group A, B and C were spinal cord compression model (50g/5min) according to the letter reported by Nystrom. Group A was injected MPS-PLGA microspheres (110mg/kg) though subarachnoid route, Group B was injected MPS (30mg/kg) by vena caudalis, and nothing has to do with Group C. Group was exposed the spinal cord without compression. The evaluating indicators in two weeks include: BBB score, motor evoked potentials, amount of nitric oxide in the damaged spinal cord, activity of nitric oxide synthetase in the damaged spinal cord, expression of Bcl-2 and NF200 in the spinal cord.Results:1. The optimized conditions of Preparation of MPS-PLGA microspheres: the phase volume ratio of the inner aqueous phase and oil phase is 1: 10, ultrasonic power of emulsicication is 150w, the density of MPS is 60mg/ml, frequency of stir is 2000rpm, stir time is 3min.2. The mirospheres prepared under the optimized conditions have nearly round integrated shape and slick appearance. Its average particle size is 12±9.41um, drug loading is 26.98%, drug trapping efficiency is 67.46%. The in vitro release profile was figured by Higuchi equation: y=3.203t1/2+40.526.3. BBB score of Group A, Group B and Group C is lower than Group D at every time point after surgery(p<0.01), and that of Group A and Group B is higher than Group C(p<0.01) at every time point after SCI except 8h. There are obviously differences between Group A and Group B 7d and 14d after SCI (P<0.05).4. The latent time of MEP N1 lengthen after SCI and then become short and short with the time increasing. The latent time of MEP N1 of Group A and Group B recovery nearly to the normal level at 7d and 14d after SCI.5. The amount of NO and the activity of NOS in the damaged spinal cord of Group A and Group B are higher than Group C at every time point after SCI(p<0.01). So do the Group A and Group B.6. The Area and AOD of the NF200 of the Group A and Group B are higher than the Group C 7d and 14d after SCI(p<0.01). There is no significant difference between Group A and Group B. 7. The expression of Bcl-2 increase and reach to the peak 1d after SCI, then it return to the normal level at the 7d time point. At the 8h,1d and 3d time point, the expression of Bcl-2 of the Group A and Group B is lower than Group C(p<0.01). There is also an obviously difference between Group A and Group B.Conclusions:1. W/O/W double emulsion solvent evaporation-extraction method is a excellent method to prepare MPS-PLGA microspheres.2. The MPS-PLGA microspheres prepared under the optimized conditions according to the orthogonal design experiments consistent with the requisition of the pharmaceutical sciences.3. Methylprednisolone can decrease the disservice of the secondary damage and enhance the recovery of the neurofunction after SCI.4. The therapeutic effect of MPS-PLGA microspheres injected through subarachnoid route to the SCI is as good as MPS injected though intravenous.