| Background and objectThe blood brain barrier serves to protect the central nervous system，to preventinfections and other toxic agents. These features, that is tight junctions, accomplish the barrierfunction and maintain the microenvironment of the brain parenchymal. The physiologicalcharacteristics of BBB, make the medicine to treatment of systemic into brain particularlydifficult.Many approaches to open the BBB have been undertaken for facilitating drugdelivery into brain,the aim is such as opening the cranium. This action of mannitol causes theopening of the tight junctions because the endothelial cells shrink for lasting a few hours. Theother hand, nither chemical methods nor the osmotic and require invasive intra-arterialcatheterization and produce diffuse, transient BBB opening by the arterial branch that isinjected. Like wise, lipid soluble solvents, immune adjuvants, alkylating agents (likeetoposide and melphalan), have been used to disrupt the BBB.A lot of animal studies have demonstrated that: under burst ultrasound exposures localBBB disrupt is possible on the time intravascular micro-bubbles.It has been proved by a large number of researches that UCA can not only be used inenhanced imaging but also be broken by ultrasound on purpose and cause a series of reactionswith high intensity, such as local violent microjet, which improves the dispersion force at thelocal tissue. This feature makes UCA be studied widely in the fields of delivery of drugs andgenes and thrombolysis therapy.ZHIFUXIAN is a new type of lipid microbubbles UCA self-designed by our departmentwith independent intellectual property rights. It plays well in diagnosis and therapy both，which has been confirmed by numerous previous animal researches. ZHIFUXIAN combined with the exposure of diagnostic or therapeutic ultrasound at certain intensity can reversiblyopen blood brain barrier of rats。The relative low drug loading is one of the obstacles inresearches on drug delivery for ZHIFUXIAN. By reviewing the reports about microbubbles’application in drugs and genes delivery, it’s not difficult to find that there are some inevitablelimitations for microbubbles to be the carrier. On the one hand, the shell of ZHIFUXIANmicrobubbles is the thin lipid or protein membrane, which has been proved by transmissionelectron microscope, so the amount of drugs and genes loaded in shell is limited. On the otherhand, the cavity inside microbubbles is occupied by inert gas, which further weakens theability of drugs carrying.Nanoparticle is a new type of drug carrier that has been studied much recent years, andits drug loading efficacy and ability of drug controlled release have been confirmed by lots ofresearches, especially the liposome drug-loading nanoparticles, which are composed ofdouble-layer lipid membrane and encapsulated aqueous nuclei, the former of which is capableof loading lipophilic or amphiphilic drugs and the latter of which is capable of loadinghydrophilic drugs, so their drug loading is more extensive than that of microbubbles.Furthermore, taking lipid as drug loader can lower drug dosage, decrease drug toxicity andimprove drug stability. It has been proved that drug-loading liposome could make the drugamount aggregating in tumor reach up to50~100times that of using drug only. Althoughpresent studies showed that modified liposome nanoparticles could get the targeted release atthe objective therapeutic area, their preparation is more complicated than normal liposome.Basing on the characteristics and shortcomings of ZHIFUXIAN and drug-loadingnanoparticles, this research tried to coupling drug-loading liposome nanoparticles on thesurface of ZHIFUXIAN microbubbles on the basis of ZHIFUXIAN preparation. The size ofnanoparticles are so small that the size of microbubbles will change little when coupling withthem, and the physical properties will not be changed in the acoustic field either consequently,by which not only will the shortcoming of lower drug loading be overcome, targeted releasingof drugs loading in liposome nanoparticles will but also be achieved by microbubbles’destruction under ultrasound exposure at the target area. Polyethylene glycol (PEG) is one ofthe commonly used modification materials in liposome preparation, which can decrease theaffinity of opsonin to liposome, reduce the macrophages’ phagocytosis to liposome in liver,improve the targeting of drugs, and obstruct the protein components’ connection with phospholipids and thereby lengthen the circulating duation in blood, when introduced inliposome. At the same time, the introduction of PEG will provide effective space frame forthe membrane constitution of both liposome nanoparticles and microbubbles. On that basis,only certain modifications to PEG will accomplish the coupling of both, and thus the designof new type of UCA in this study will be realized.The chemotherapy drugs are difficult to reach the blind spots like meninges, eyes, etc,due to the blood-brain barrier. The residual leukemic cells in these blind spots are the majorsource for recurrence of central nervous system leukemia (CNSL). At present, cranialirradiation, intrathecal injection of chemotherapy drugs and high-dose MTX chemotherapyare the main measures for prevention of CNSL recurrence. However, the radiotherapy hasside effects, with toxic effects on the endocrine and neuropsychiatric system; intrathecalinjection of chemotherapy drugs may be traumatic and are easily cause infection; high-dose ofmethotrexate (HDMTX) is one of the more effective methods for prevention of CNSLcurrently, but it also has obvious side effects and can cause long-term neuropsychiatrictoxicity. Therefore, the way to achieve effective concentration of MTX in the brain tissuelesions and reduce the toxic side effects is key to the treatment and prevention of CNSL.The purpose of our research is to prepare a new kind of ultrasound contrast agent whichcould be a effective vector baed on the successful preparation of the UCA“ZHI FUXIAN”,and this new UCA would be conjuncted with drug loaded liposome. the new UCA could notonly efficently load drug but also targetedly release the loaded drug assisted with theultrasound, and this would not only overcome the shortcoming of the traditional UCA whichhas low dosage of drug load but also overcome the shorcoming of the untargeted drug releaseof the drug-loaded liposome. Meanwhile the new UCA will be used in enhance the delivery ofMTX through the Blood-Brain Barrier, assisted with the therapeutic ultrasound which couldopen the blood-brain barrier. To explore a new method which could non-invasively therapythe central nerveous system disease.Materials and Methods1. Preparation of biotinylated ZHIFUXIAN on the basis of ZHIFUXIAN preparation. Acertain proportion of biotinylated phospholipids were added into the primary lipid formula ofZHIFUXIAN and the proportion of phospholipids was changed accordingly. The particle sizeand concentration of microbubbles were detected with Coulter granulometer when the biotinylated ZHIFUXIAN was made.2. Preparation of biotinylated liposome nanoparticles loading methotrexate (MTX) onthe basis of ZHIFUXIAN preparation. The biotinylated liposome nanoparticles loading MTXwere prepared by twice freeze drying and mechanical oscillation method. Separation betweendrug-loading nanoparticles and free drugs was performed by column chromatography. Theentrapment rate, particle size and morphology of the nanoparticles were measured by highperformance liquid chromatography, Zetasizer3000and transmission electron microscope,respectively.3. Coupling of ZHIFUXIAN microbubbles and liposome nanoparticles loadingmethotrexate. The biotinylated ZHIFUXIAN microbubbles and methotrexate-loadingliposome nanoparticles of different contents were prepared separately and coupled together byavidin-biotin connection system. The microbbules coupling drug-loading nanoparticles weregot after uncombined nanoparticles were removed by rinse-centrifugation method. Theparticle size was measured by Coulter Counter, the capability of enhanced imaging wasconfirmed by CEU in rabbit livers, and the amount of loaded methotrexate was detected byhigh efficiency liquid chromatography.4. Inhibition of ZHIFUXIAN microbubbles coupling MTX-loaded liposomenanoparticles to tumor in vitro and its mechanism research. Tumor cell suspension addingMTX-loaded liposome nanoparticles was exposed under ultrasound of certain frequency, afterwhich the inhibition ratio to tumor cells were detected by MTT and apoptosis test facility, andthe change of cell morphology was observed by scanning electron microscope.5. Discuss the feasibility, safety of therapy ultrasound united with microbubbles couplingMTX-loaded liposome nanoparticles opening the BBB by Evans blue tracer experiment, anddetermine the parameters of the following experiment.6. MTX in vivo permeability, Discuss the feasibility of MTX to cross BBB and transportinto the brain through quantitative evaluation of MTX detected by HPLC. We used TEMto assess the microstructure of the brain.7. Establish an in vitro model of the blood-brain barrier, which comprising Co-culture ofprimary brain capillary endothelial cells (BCEC) on semi-permeable filter inserts, withastrocytes on the bottom of the filter. Conclusion1. Biotinylated UCA can be prepared by freeze drying and liposome nanoparticlesloading MTX can be prepared by twice freeze drying. The method is relatively simple.2. Separation of drug-loaded nanoparticles and free drugs can be accomplished bycolumn chromatography.3. Coupling of microbubbles and drug-loaded liposome nanoparticles can be realized byavidin-biotin connection system.4. Microbubbles coupling MTX-loaded liposome nanoparticles under ultrasonicexposure can effectively inhibit tumor cell growth, the mechanism of which is related withthat it can produce pores on cell surface and directly cause cells broken.5.(BBB) can be opened reversible using Metron AP-170therapeutic ultrasound andMicrobubbles coupling MTX-loaded liposome nanoparticles under optimum parameters. Theoptimum parameters were: frequency,1MHz; power,2.0W/cm2; duty cycle,20%; exposuretime,5min; MB dosage,0.5ml/kg.6. The concentration of MTX is the highest in the group of ultrasound united withmicrobubbles coupling MTX-loaded liposome nanoparticles, In this group, The blue ofLanthanum distribution about the BMVEC basal membrane.7. The established invitro model of BBB has basic characteristics of BBB in vivo, and issuitable for central nervous system (CNS) drug research... |
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