Study On Brain-targeting Ginkgolide B Nanoparticles And Its Amphiphilic Carriers

The brain is a delicate organ, and evolution built very efficient ways to protect it. Unfortunately, the same mechanisms that protect it against intrusive chemicals can also frustrate therapeutic interventions. Many existing pharmaceuticals, such as hydrophilic solute like nerve growth factors et al, are rendered ineffective in the treatment of cerebral diseases due to our inability to effectively deliver and susutain them within the brain. It is now well established that the BBB is a unique membranous barrier that tightly segregates the brain from the circulating blood. The BBB is comprised of a contiguous layer of endothelial cells connected by tight junctions that circumferentially surround the entire cell margin at the brain capillaries. These tight endothelium junctions can be 100-fold times tighter than junctions of other capillary endothelium. These capillaries are lined with a layer of special endothelial cells that lack fenestrations and are sealed with tight junctions. Thus, the barrier provide efficient barriers to the diffusion of drugs from the blood stream into the central nervous system (CNS) especially of polar drugs such as peptides and proteins. The BBB also has an additional enzymatic aspect. Solutes crossing the cell membrane are subsequently exposed to degrading enzymes present in large numbers inside the endothelial cells. Furtheremore, the BBB is further reinforced by a high concentration of P-glycoprotein (Pgp), active-drug-efflux -transporter protein in the luminal membranes of the cerebral capillary endothelium. This effiux transporter actively removes a broad range of drug molecules from the endothelial cell cytoplasm before they cross into the brain parenchyma.While the characteristics of the BBB provide a formidable obstacle for drug therapy in the CNS, they are not insurmountable. Attempts to overcome the barrier in vivo have focused on altering barrier integrity or characteristics, or changing the characteristics of the drug. They are described as below:1) Tight junctions at the BBB have been opened by artificially created osmotic pressure and the administration of bradykinin analogs such as RMP-7. However, opening the barrier allows CNS entry of toxins and unwanted molecules, potentially resulting in significant damage.2) Another alternative for brain drug delivery has been made to modify drugs to more readily cross the barrier. Pro-drugs are an excellent example of such drug manipulation. While prodrugs work well, not all compounds (i. e., nerve growth factor) may be manipulated in this way and still maintain therapeutic efficacy. Furthermore, increased lipid solubility may significantly alter pharmacokinetic parameters such that clearance and half-life become undesirable.3) One promising approach to compensate permeability restrictions is the application of targeted release systems that are recognized by a BBB-located receptor that mediates transcytosis. While carriers are an attractive means of CNS delivery, the drugs must have carrier mediated specificity, thus limiting their molecular characteristics.4) While these transporters function in the direction of influx from blood to brain, efflux transporters are also present. These efflux transporters (P-glycoprotein, multi-drug resistance protein, and others) are likely located at the BBB for detoxification and/or prevention of nonessential compounds from entering the brain. It is yet another obstacle in delivering drugs to the CNS as many agents that readily cross the BBB are substrates for efflux transporters.5) Direct injection into the brain is another approach to circumvent the BBB. While these techniques can be successful to achieve certain therapeutic goals, disadvantages exist for direct injection for this method. The primary disadvantage is the requirement of extremely invasive neurosurgery. Furthermore, diffusion of the drug from the injection site may limit therapy. 6) there are other strategies as well. For example, intracarotid infusion delivers the drug directly to the BBB; after nasal administration drugs can be transported directly from the nasal cavity to the CNS via the olfactory epithelium and/or the trigeminal nerve system thereby bypassing the BBB.Currently, NPs are gaining interest as therapeutic drug carriers across the blood-brain barrier (BBB). Nanoparticles (NPs) are solid colloidal particles, ranging in size from 1 to 1000 nm, consisting of various macromolecules in which therapeutic drugs can be adsorbed, entrapped, or covalently attached. One utility of NPs is to serve as novel drug delivery carriers to tissues throughout the body. This is accomplished by masking the membrane barrier, limiting characteristics of the therapeutic drug molecule, as well as retaining drug stability, with that of the properties of the colloidal drug carrier. This disguising of the drug may allow access across the previously impermeable membrane. Once the NP reaches the desired tissue, release of the drug may occur by desorption, diffusion through the NP matrix or polymer wall or NP erosion, or some combination of any or all mechanisms.It is demonstrated that polybutylcyanoacrylate, a good biodegradable drug-loading carrier, can transport large quantities of drugs(i. e., dalargin, doxorubicin, loperamide) to brain after coated Tween-80, apolipoprotein B and E, et al. Its possible mechanism includes: passive diffusion, receptor mediated endocytosis, carrier mediated transport or inhibition of effiux transporters, and so on. Other studies on the brain trargeting NPs cariers are focused on the amphiphilic polymers with no or low toxicity. It is also showed that amphiphilic carrier, PEG-PHDCA et al, can sucesfully transport the BBB entry into the brain. In this study safe and biodegradable polymer poly(ethylene glycerol) and poly-e-caprolactone, as starting material, were selected to synthesize the amphiphilic multi-block copolymers, which acted as the brain-targeting NPs carrier.It is well known that the Ginkgo Biloba extract showed widely pharmacological activity against neurosensory disturbances,depressive symptoms, cerebral insufficien-cy, et al. The main constituents in its extract include flavonoid and ginkgolide, which involved in the free radical scavenging and antioxidant effects. Ginkgolide B, one kind of the ginkoglides, is endowed with significant pharmacological properties and exhibits potent antagonist activity against platelet -activating factor (PAF), which plays a key role in inflammatory processes. But in vitro pharmacological studies have provided evidence that the efficiency of ginkgolide B is linked to pH and that its anti-PAF activity is enhanced in acidic medium. This result is clearly related to the existence of three lactone groups which may undergo hydrolysis, leading to the rings opening. In vivo study revealed that GB existed two forms in the body: the original GB with its lactone rings closed and a second form with one of the rings opened. The original GB in plasma is taken up rapidly by various organs including the liver, the intestine and possibly the stomach, but in the brain it showed extremely low concentration.Material and methods1 Study on preparation of GB-PBCA-NPsThe GB-NPs carrier, PBCA, was synthesized by interfacial polymerization, which charaterized in anion polymerization. The affected factors in the preparing technology, including surfacant concentration, stirring velocity, reaction temperature, organic solvent volumn, PH value and the ratio between GB and monomer were investigated. The zeta potential, particle size and size distribution(polydispersion) of GB-PBCA-NPs was determined on a Zetasizer HSA 3000; the morphology was observed by transmittion electron microscopy; the encapsulation efficieny and drug-loading efficieny was measured using LC/MS/MS. To prepare the lyophilized NPs powder, different cryoprotectors were selected in the study. The animal acute toxicity of the nanopartiles formulation was conducted using kunming mice by tail intravenous administration.2 Preparation of GB-PEG-PCL-NPsThe muliblock polyethylene-polycaprolactone copolymer was synthesized, which PEG and PCL was used as starting material, succinic anhydride acted as cross-linking agent, DCC and DMAP as condensation agent and catalyst respectively. The cytotoxicity of the copolymers was evaluated with L929 cells through MTT and LDH methods.The GB copolymers NPs was prepared by using organic solvent. As afore-mentioned, the parameters such as particle size and size distribution, drug-loading efficiency et al, were measured.3 In vitro drug release studies of GB-NPsIn vitro drug release studies were carried out as follows. Drug-loaded nanoparticle suspensions were placed in a dialysis membrane bag with molecular weght cut-off 3000g/mol, tied, and dropped into 250ml of a phosphate buffer solution media(PH=7.4). The entire system was kept at 37℃with magnetic stirring at 100 rpm. At predetermined time intervals, 1 ml of aqueous solution was withdrawn from the release media. And the drug content of the sample was assayed by using LC/MS/MS. Futheremore, the drug release mechanism of GB -NPs was fitted with mathematics model including weibull distribution model, Ritger -Peppas model and so on.4 Body distribution studies of GB-NPsPreparation of GB-PBCA-NPs coated Tween-80 injection solution: the lyophilized GB-PBCA-NPs was resuspended in saline, which the GB concentration was adjusted to 0.5mg/ml. Under magnetic stirring, 1% Tween-80 was added and incubated 30min. Then the GB-PBCA-NPs coated with Tween-80 and other GB-NPs formulations with a dosage of 1mg/kg were administrated to mice via the tail vein. At predetermined time interval, the mice was sacrificed and dissected, the organs were ringsed, blotted and homogenized. The drug in plasma and the tissues sample were extracted with ethyl acetate, then measured on LC/MS/MS.5 Pharmacological study of GB-NPsMale Wistar rats were divided randomly into experimental and control groups. Then middle cerebral artery occlusion (MCAO) models of transient focal cerebral ischemia in rats were established. Repeffusion was carried out after 2h of ischemia, at the same time different GB formulations were administrated by tail vein with a dosage of 3mg/kg, wheras in the control group only saline was injected. After 24h reperfusion, the effects of GB on the focal volume of cerebral infraction, neurotic behavior, water content and the pathologic changes of brain were evaluated and analyzed.Result1 Study on GB-PBCA-NPsIt is showed that the surfacant concentration, stirring velocity, reaction temperature, organic solvent volumn, PH value can influence the particle size and size distribution. So the affected factors should be kept in the suitable range. The optimal parameter is 0.5% Dextran-70, stirring rate at 800 rpm, 20ml acetone, room teperature and PH=7. The mean particle sizes obtained by dynamic light scattering of the nanoparticles were 110.4 nm, zetal potential and the polydispersity is -29.3mv, 0.094, respectively. From the observation of transmission electron microscopy, the NPs exhibited a regular spherical shape. The encapsulation efficiency and drug-loading efficiency was 96.74% and 5.74%. Furtheremore, the lyophilized powder of GB- PBCA-NPs embodied good redissolving ability, in which 2.5% trehalose used as cryoprotector. In animal acute toxicity test, at a dosage of 90mg/kg, no poisonous effect and side-effects were found.2 Preparation of GB-PEG-PCL-NPsThe result of cytotoxicity showed that the multiblock PEG-PCL copolymers had good biocompatibility. The same as PBCA-NPs, the copolymers NPs also had a regular spherical shape. With differet molecular weight of hydrophilic fragment PEG and hydrophobic fragment PCL, the GB-NPs showed different zetal potential, the higher Mw of PEG, the higher zetal potential was; but the effect of PCL on the zeta potential is on the contrary. GB was sucessfully incorporated in nanoparticles with a mean diameter aroud 200 nm, its relative emcapsulation efficiency beyond 94%, and the drug-loading efficiency varied from 3.39% to 6.45%.3 In vitro drug release studies of GB-NPsIn vitro drug release studies, all NPs showed sustained release characteristics. The release mechanism of NPs was fitted to Weibull model, and it showed that the drug release process included passive diffusion and matrix-eroded procedure.4 Body distribution studies of GB-NPsIn body distribution studies, only PBCA-NPs coated with Tween-80, PEG₄₀₀₀ -PCLz00o-NPs and PEG400o-PCL₁₂₅₀-NPs groups showed brain-targeting characteristics. Compared with the GB aqeous solution, the content of GB in brain tissues of the aforementioned groups was significantly higher than that of GB control group at predetermined interval after administrated (p＜0.05), notwithstanding all NPs formulations showed liver targeting feature.5 Pharmacological study of GB-NPs Results showed that all GB-NPs could ameliorate the cerebral ischemia, decrease water content and brain blood capillary permeation, improve neurotic sympotoms, reduce the volume of focal infraction. Compared with control groups, a sinificance was found (P＜0.05).ConclusionAll GB-NPs exhibited a regular spherical shape and narrow distribution, which also had high encapsulation efficieny. Compared with the original GB, NPs can enhance GB solubility in aqeous solution. On animal acute toxicity test, no posionous result or other side-effects were found after kunming mice administrated at a dosage of 90mg/kg by vail tein; the same the cyotoxicity study showed that PEG-PCL copolymers had good biocompatibility. In vitro drug-release studies showed that all GB-NPs embodied sustained release characteristics, the drug-release behavior was conformed to Weibull equation; its release mechanism may be cooperated by passive diffusion and matrix-eroded procedure. In vivo body distribution studies testified that only GB-PBCA-NPs coated with Tween-80, GB -PEG₄₀₀₀-PCL₂₀₀₀-NPs and GB-PEG₄₀₀₀-PCL₁₂₅₀-NPs can transport the BBB effectively, which can enhace the content of GB in the brain compared with GB aqeous solution group (P＜0.05). Moreover, the data demonstrated that multiblock PEG₄₀₀₀-PCL₂₀₀₀ and PEG₄₀₀₀-PCL₁₂₅₀ copolymers can act as drug brain-targeting carriers also. In the pharmacological study, it was showed that all GB-NPs formulations had protective effects on cerebral ischemia by ameliorating cerebral ischemia, counteracting the brain damage induced by cerebral ischemia.