Targeted Therapy Of Propolis - Induced Spinal Cord Injury

Spinal cord injury (SCI), a pathological damage to the spinal cord resulting from trauma, inflammation, and many other causes, is a devastating condition that affects more than 130,000 people each year worldwide and often results in permanent functional and sensory deficits. The enormous impact, both in an individual and familial context and on the broader socioeconomical scale, is partly due to the early mean age (33 years) of the patient population, who are predominantly male (4:1 male/female ratio). Moreover, the lifelong supportive care needed to prevent complications (e.g., decubitus, respiratory and urinary tract infections.and others) means a substantial financial burden on both the patients and the society. Despite major progress in pharmacological and surgical treatment approaches, SCI remains a complex medical and psychological challenge, both for the patients and their relatives and for the involved physicians, with no curative therapy currently available. To date, methylprednisolone (MP) is the major recognized treatment for SCI, with neuroprotective effect that appear to be mediated through the inhibition of inflammatory reactions and lipid peroxidation. However, the therapeutic efficacy of MP is relatively minor, while its side effects, including gastrointestinal bleeding, gastritis, and Cushing’s syndrome, are significant. Claims of improvements in functional outcomes after high doses of MP have recently become controversial, given the growing concern over the elevated risk of infection and higher mortality among spinal cord patients that received MP, particularly in view of its modest beneficial effects.By combining three ingredients currently in clinical use or undergoing testing, we have designed a central nervous system targeted delivery system based on apaminmodified polymeric micelles (APM). Apamin, one of the major components of honey bee venom, serves as the targeting moiety, poly (ethylene glycol) (PEG) distearoylphosphatidylethanolamine (DSPE) serves as the drug-loaded material, and curcumin is used as the therapeutic agent.Apamin (CNCKAPETALCARRCQQH-NH2) was site-specifically conjugated with NHS-PEG3400-DSPE and purified by dialyzed against deionized water to remove the unreacted ligand. The conjugation of apamin with PEG3400-DSPE was confirmed by H nuclear magnetic resonance (NMR) spectroscopy and a matrix assisted laser desorption/ ionization time-of-flight (MALDI-TOF) MS.The experimental molecular weights of apamin-PEG-DSPE was determined by MALDI-TOF MS to be 5542 Da. The 1H NMR spectra of apamin-PEG-DSPE in CDC13 shown the disappearance of 82.60 in NHS-PEG-DSPE indicates that the conjugation was successful. The liposolubility CUR was used as a model drug. Apamin-PM-CUR and PM-CUR micelles were prepared by film hydration method.four conditions,the organic solvent type mixing ratio, hydration volume and temperature were test by single factor method for the best preparation conditions.The optimized formulation micelles were prepared and the micelle diameter was determined to achieve the desired 50 nm, uniform size, good dispersion. The encapsulation efficiency was 80.4%±1.2% and the drug loading content was determined to be 13.2%±1.2%. X-ray diffraction profiles of drug-loaded micelles (apamin-PM-CUR) and the physical mixture of CUR and blank micelle (apamin-PM) at the same drug-to-material ratio suggesting that the encapsulation of curcumin in micelles was successful. X- PS was used for the detection of nitrogen in apamin-PEGDSPE. X- PS was used for the detection of nitrogen in apamin-PEGDSPE. As evidenced by the absence of nitrogen in the native copolymers and blank samples, the nitrogen detected in the apamin-PEG-DSPE samples likely reflects the presence of covalently bound peptides.Placed the apamin-PM-CUR at 4℃ for four months later, there wasn’t found leaked CUR, particle size and dispersion of the micelle is also no significant change. In vitro release experiments showed that apamin-PM-CUR and PM-CUR have no significant difference. This result suggested that peptide apamin behavior modification does not affect the release CUR micelles.The SCI mouse model was implemented using Allen’s weight dropping (WD), with the success confirmed by BMS score, MEP and histological observation at multiple levels. SCI mice were divided into 4 groups and administered apamin-PM-CUR, PM-CUR, MP and physiological saline, respectively. The recovery of SCI mice of 4 groups were assessed in 3 levers:praxeology (BMS score), Electrophysiology (MEP) and histology (H&E and HRP) Experiments show that the recovery of PM-CUR group is similar with the physiological saline group. They were all not good. The survival rate of mice is low and a few number of mice remaining at 24 weeks are still can’t move freely. The incubation period the MEP-N1 of PM-CUR group is also more than 10ms indicating that mice neural pathways blocked. The damage of spinal cord tissue sites is still severely. The spinal cord of those mice are structural integrity and functional completeness. In contrast, mice of apamin-PM-CUR group were recovered well, even better than the positive control group. In those two groups the survival rate of SCI mice is about 80%. Mice can move freely, neural pathways is good recovery.in the tissues obtained from the animals administered MP and apamin-PM-CUR, the spinal cord tissue structure was clearer than in other groups. Neurons were normal in appearance, with maintained membrane integrity, visible cytoplasmic Nissl bodies, and glial scar tissue with regular appearance. Taken together, these results suggest that apamin-PM-CUR helped relieve secondary spinal cord injury and promoted neuronal regeneration.In terms of safety, A single dose of apamin-conjugated polymers was about 700-fold lower than the LD50 amount, suggesting that APM and apamin have potential for clinical applications as spinal cord targeting ligand for delivery of agents in treatment of diseases of the central nervous system.All of those suggesting that APM and apamin have potential for clinical applications as spinal cord targeting ligand for delivery of agents in treatment of diseases of the central nervous system.