| Objective Neurotoxin(NT)as an effective analgesic drug for advanced cancer patients could occur respiratory inhibition when it is injected at higher dosage levels.We developed Angiopep-2 modifying phospholipid-functionalized mesoporous silica nanoparticles(ANG-LP-MSN).To detect and evaluate NT preparation,high selectivity and high sensitivity analysis methods of NT were developed in vitro and in vivo.Methods Single-label derivatization of NT was prepared.In vitro analysis method was developed using HPLC.In vivo analysis method was developed using CE-LIF.We use film hydration method to prepare ANG-LP-MSN,and modified-Stober method to synthesize mesoporous silica nanoparticles(MSN).MSN morphology was observed through transmission electron microscope(TEM).Small angle X-ray diffraction(SAXRD)was used to determine the mesoporous structure;nitrogen adsorption method was used to calculate the surface area,pore diameter and pore volume.The drug release behavior of ANG-LP-MSN-FITC-NT was studied by dialysis method.Pharmacodynamics of ANG-LP-MSN-FITC-NT was studied by the hot-plate test and the writhing test.Pharmacokinetics behavior after intramuscular injection in rats was studied.Results Neurotoxin labelled with fluorescein isothiocyanate(FITC-NT)was prepared by high-performance liquid chromatography(HPLC)with a purity value higher than 99.29%and identified by MALDI-TOF/TOF-MS.The peak area in rats plasma varied linearly over the concentration ranges tested(0.5～100 μg·mL-1)in vitro.The dynamic range was over two orders of magnitude(0.01 to 1.00 μg mL-1)in vivo.Numerous demonstrations were carefully performed for the methodological validations of FITC-NT,including high specificity of method,good linearity,fair wide linear concentration range,low limit of quantity,less than 10%intra-and inter-day variabilities,and between 85%and 115%recovery of FITC-NT.Morphology of ANG-LP-MSN-FITC-NT was spherical and the mean particle size of ANG-LP-MSN-FITC-NT were 15.2±3.32 nm with PDI 0.045±0.02.FITC-NT was highly encapsulated into MSN(MSN-FITC-NT)and the drug loading efficiency was up to 10.75%.In vitro release,the results showed that about 75.7%of NT released from ANG-LP-MSN-FITC-NT after 48 h and burst release was effectively reduced compared with other group.In Pharmacodynamics study,the concentration-time profile of NT exhibited the characteristics of a two-compartment model.Compared with FITC-NT group,have T1/2β was significantly prolonged,AUC0-∞ was significantly increased(P<0.01)in ANG-LP-MSN-FITC-NT treatment group.In Pharmacodynamics study,ANG-LP-MSN-FITC-NT treatment group have significant analgesic action compared to other group.Conclusion A simple,robust and reliable in vitro and in vivo analysis methods to determine FITC-NT after single-label fluorescent derivatization was developed.Furthermore ANG-LP-MSN-FITC-NT was prepared with small size,higher EE and DL successfully.Release of FITC-NT from ANG-LP-MSN-FITC-NT showed sustained-release in vitro.In pharmacokinetics studies,ANG-LP-MSN-FITC-NT have long circulatory effect,which profit for FITC-NT enrichment in brain and entering brain as well as increasing bioavailability and exerting more effective analgesic effect.In pharmacodynamics studies,ANG-LP-MSN-FITC-NT is more beneficial to NT in the concentration of the brain and performs better analgesic effect.ANG-LP-MSN is a prospective drug delivery system for neurotoxin. |
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