Study On T7 Modified Tanshinone IIA-encapsulated PEG-PAMAM Brain Targeting Nano Drug Delivery System

Stroke is a leading cause of high mortality and long-term morbidity worldwide.Unfortunately,current therapeutic effect of ischemic stroke by drug treatment is very unsatisfied.This is largely due to the difficulty in drug delivery to the brain,where the existence of blood brain barrier(BBB)renders many neuroprotective compounds ineffective in stroke intervention.Therefore,delivering potent neuroprotective drugs across the BBB is an urgent unmet medical need for stroke treatment.Nanoparticles modified with specific targeting ligands have been proved possessing an excellent ability to cross the BBB.Polyamidoamine(PAMAM)dendrimers are increasingly accepted as novel multifunctional nanocarriers for biologically active substances delivering and controlled drug releasing attributable to their remarkable capability of molecule attachment on the surface by covalent bonding or physical absorbing and capacity of therapeutic agent encapsulation in the hydrophobic interior.In this paper,we developed novel T7 peptide modified TSIIA-loaded PEG-PAMAM NPs(T7-TSIIA-PEG-PAMAM NPs).Polyamide-amine(PAMAM)dendrimers were used as matrix carriers and modified with by bifunctional polyethylene glycol(NHS-PEG-MAL).In an effort to ensure efficient delivery of TSIIA to the brain,we further functionalized the PEG terminal end with T7 peptide.The active targeting ability and pharmacodynamics of T7-TSIIA-PEG-PAMAM NPs were evaluatd.The concrete research content and result are as follows:1.5.0G PAMAM dendrimers were synthesized through repetitive Michael addition and amidation condensation reaction with ethylenediamine(EDA)and methyl acrylate(MA)as reactants,and then PEGylated PAMAM copolymers were synthesized by conjugating bifunctional NHS-PEG-MAL to the amine groups of the synthetic5.0 G PAMAM dendrimers.2.T7-TSIIA-PEG-PAMAM NPs were prepared through conjugating T7 peptide to PAMAM dendrimer via the maleimide function of bifunctional PEG.T7-TSIIA-PEG-PAMAM NPs are spherical particles as evidenced by SEM images.The result of dynamic light scattering(DLS)revealed particles with diameter equal to 138±0.229 and a zeta potential 2.44±0.62 m V.3.T7-TSIIA-PEG-PAMAM NPs were prepared by physical embedding method.The encapsulation efficiency(EE%)and drug loading capacity(LC%)of T7-TSIIA-PEG-PAMAM NPs are 75.14% and 23.17%,as determined by HPLC.In vitro drug release result suggest a significant sustained TSIIA release from T7-PEG-PAMAM/TSIIA NPs beyond 72 h compared with free TSIIA.4.To evaluate active brain targeting of T7-PEG-PAMAM NPs,we injected Di R-loaded different NPs intravenously into ICR mice.In vivo fluorescence distribution of Di R-labeled PAMAM NPs,PEG-PAMAM NPs and T7-PEG-PAMAM NPs were evaluated.The results showed that the fluorescence intensity of Di R-labeled T7-PEG-PAMAM NPs was significantly higher than that of unmodified PEG-PAMAM and PAMAM.The fluorescence intensity of Di R-T7-PEG-PAMAM NPs in the brain of mice rapidly reached the peak at 1.5 h after injection,indicating that T7-PEG-PAMAM NPs had good brain targeting.5.Ischemic stroke in SD rats was modelled by middle cerebral artery occlusion(MCAO).The pharmacodynamics of T7-TSIIA-PEG-PAMAM NPs were conducted in cerebral ischemia-reperfusion rats with free TSⅡA suspension as control.The infract volume,neurological deficit scores and the levels of superoxide dismutase(SOD),Na~+-K~+-ATP,glutathione Peroxidase(GSH-Px),lactate dehydrogenase(LDH)and nitric oxide(NO)in response to cerebral ischemic injury was measured at 24 h after MCAO.The results showed that T7-PEG-PAMAM/TSIIA NPs can significantly alleviate infarct size and neurological deficit scores,increase the levels of SOD,Na~+-K~+-ATP,GSH-Px and LDH,and reduce NO level.T7-PEG-PAMAM/TSIIA NPs demonstrated an even most potent effect salvaging the ischemia-reperfusion brain tissue.This article develops a new strategy for delivering TSIIA to the brain after stroke,and provids significant anti-stroke activity.