Studies Of Vesicular Phospholipid Gels Loaded With Cytaraine For The Brain-implant

Glioblastoma multiforme (GBM), a malignancy of the glial cells of the brain, is the most frequent primary brain tumor and is of high incidence rate. Removing all tumor cells is impossible through surgical operation. Additionally, conventional chemotherapeutic drugs are difficult to go across the blood brain barrier (BBB) and reach the effective concentration of treatment in the brain. So it is of high fatality rate and in situ recurrence rate. In this paper, this is first attempt to develop vesicular phospholipids gels loaded with cytarabine for GBM chemotherapeutic at the original tumor site after surgical operation. Some advantages are manifested: VPGs are semisolid phospholipids dispersions with high concentration of phospholipids intended for long time sustained release properties; VPGs were implanted in the site of removed tumor and bypass the BBB; phospholipids, as the main component, are of good biocompatibility and no toxicity.At preformulation study, determination methods of Ara-C, phospholipids and lysophosphatidylcholine (LPC) were established. The specificity, precision and reproducibility of the method were good. The solubility of Ara-C in different aqueous media are larger than452.7mg/ml; oil/water partition coefficient is low with poor lipophilicify and declines slightly with the increasing of pH, the value is0.1686for50mg/L Ara-C in pH7.4PBS; and liposome/water partition coefficient is2.51±0.53and is of better ablility of cell membrane affinity.Ara-C VPGs were prepared and characterized in term of the appearance, particle size, entrapment efficiency (EE), in vitro release, and rheologic propperties. The structure of small unilamellar vesicles was observed by a Transmission Electron Microscope (TEM). Micrograph of Freeze-fracture electron microscopy (FF-TEM) showed that each vesicle was tightly packed between neighbor-vesicles to be three-dimensional network structure, the structure is different from liposome and liposome gels. With the lipid concentration of300、400、500mg/g, EE of Ara-C VPGs were31.65%、53.99%、72.12%, respectively; in vitro cumulative release were 48.93%、37.37%、23.22%in432h, respectively. It showed that EE are increasing and in vitro release are slow with the increase of lipid concentration, it supposed that entrapment volume is increasing with the increase of lipid concentration and aqueous phase volume is constant, viscosity is increasing with increase of lipid concentration can lead to release slowly. The sterilization stability of cytarabine (Ara-C) loaded vesicular phospholipids gels (VPGs) were also examined. The particle size of VPGs after redispersion was119.6±66.2nm, and EE was32.6±2.1%. Comparatively, after autoclaved sterilization, increased particle size and EE were obtained as165.6±71.9nm and62.6±2.3%, respectively. In vitro cumulative release was from79.13%in80h for nonautoclaved VPGs to74.76%in432h for autoclaved VPGs. Viscoelasticity were strengthened. The change in viscosity and particle size distribution is closely related, it means that, being of larger polydispersity of particle size distribution, the viscosity is lower at the same lipid concentration. The reason might be that the smaller particles like ball bearings lubricate the larger ones. As a result, after sterilization polydispersity is smaller than that of nonautoclaved. Additionally, the increased viscosity can slow drug release. In total, after sterilization, the vesicle is integrity, EE is increased, in vitro release is slow, viscoelasticity was strengthened, the properties become more stable. And characteristics of in vitro drug release were slowed remarkablely. Also, the viscoelasticity was reinforced with clearly decreased fluidity. However, reduced Ara-C and increased LPC were observed. The content of Ara-C in VPGs was1.01±0.04mg, after sterilization the content declined to0.73±0.06mg; LPC in autoclaved VPGs was increased to2.32±0.01%from1.39±0.03%for non autoclaved VPGs. In order to enhance the stability of Ara-C and phospholipids, stabilizers were added and among them addition of sodium sulfite showed best effects with high stability of both Ara-C and phospholipids. It supposed that SO3-? free radicals producing from sodium sulfite during the process of sterilization can reduce the generation of HO?free radicals greatly, which inhibit the degradation of Ara-C and phospholipids.VPGs are with specific surface characteristics through the modification of the surface structure and change its in vitro and in vivo properties. For complexation of charged polyelectrolyte modified VPGs, TEM observed that the surface of vesicle was coated with a layer of film, particle size is about100nm. Zeta potential has no obvious different from Ara VPGs, which confirm that polyelectrolyte was adsorbed on the surface of charged VPGs with electrostatic interaction. In vitro cumulative release percent of CHss-PLys VPGs,OCA-PGlu VPGs and VPGs in432h is47.03%、45.65%、37.37%, respectively. It indicated that polyelectrolyte adsorbed-charged modified VPGs can speed up in vitro drug release. In the research of rheology, these parameters indicated that viscoelasticity of polyelectrolyte adsorbed-charged modified VPGs was strengthened. It supposed that the segment of polyelectrolyte interwove with mesh style around the vesicles, variables of shape are significantly larger under strain force, G’and G" has no change on the shape of the curve as a function angular frequency, the curve is approach to the creep phenomenon of the Voigt model. Despite the viscoelasticity are significantly strengthened, yet drug release were faster. So, it is inexplicable that from the point of the view of the increase in viscosity can slow release. However, We speculated that, for phospholipid molecules in the vesicular film with fluidity at37℃, polyelectrolyte can increase the permeability of the membrane, lead to drug release accelerating.The ultra performance liquid chromatography tandem mass spectrometry method (UPLC-MS/MS) has been developed for determination of Ara-C in biological sample. The brain tissue biodistribution study showed that drugs of Ara-C VPGs and solutions can penetrate into brain tissue5mm depth from implant site. The concentration of Ara-C with Ara-C VPGs intracerebral injection at28days is still higher than0.1ug/ml therapeutic concentrations, it showed that Ara-C VPGs can maintain therapeutic effects against glioma for28days. Ara-C VPGs were of significant sustained release properties and irregular biodistribution in brain tissue. Drug concentrations in brain tissue with Ara-C VPGs implanting were higher than that of Ara-C solutions injecting in normal SD rats. At the same time, flourescence sections were used to observe the relative distribution of drug in brain tissues, and the results of observation is in agreement with that of UPLC-MS/MS. In the safety study, the Ara-C VPGs were implanted into brain with infiltration of few inflammatory cells in 3to7days. No necrosis appears in brain tissue. Ara-C VPGs are of higher safety.The in vitro cytotoxicity of Ara-C VPGs was examined by microculture tetrazolium (MTT) colorimetric assay using two tumor cell lines, C6glioma cell line and human U87-MG glioma cell line. Cellular uptake of FITC formulations was assessed in C6and U87-MG glioma cells using Fluorescence Microscopy. As a result, Ara-C VPGs had a significant in vitro cytotoxicity against these two cell lines, which was concentration-dependent. OCA-PGlu VPGs exhibited more obvious cytotoxicity than any other formations. The result is consistent with that of cellular uptake observed with Fluorescence Microscopy.The in vivo antitumor effect of Ara-C VPGs was assessed using C6glioma-bearing wistar rats and human astrocytoma U87-MG tumor-bearing nude mice as models respectively. Experiment showed that tumor inhibition rate (95.87%) of Ara-C VPGs group on C6glioma-bearing wistar rats (p<0.01) was higher than that of Ara-C solutions group (45.52%)(p>0.05) with the same dose. However, for U87-MG glioma-bearing nude mice, all VPGs group showing tumor inhibition rate was higher(p<0.01)than that of Ara-C solutions group(p>0.01)with the same dose. Tumor inhibition rate of OCA-PGlu VPGs group (36.82%) is highest, Ara-C VPGs group (34.93%) is second, CHss-PLys VPGs group (28.58%) is minimum. This result was in agreement with the result of in vitro cytotoxicity and cellular uptake. The reason was supposed that, usually polyelectrolyte can promote drug permeation into cell, at the same time, OCA with positive charge on the surface of vesicle, have strong electrostatic interaction with a negatively charged cancer cellular membrane, this can contribute to cellular uptake. Yet, for CHss-PLys VPGs, CHss with Negative charge on the surface of vesicle, due to strong electrostatic repulsion with a negatively charged cancer cellular membrane, Vesicles can’t be uptaked. Meanwhile no obvious decrease were observed for the body weight of tumor-bearing wistar rats or nude mice. No obvious toxicity occurred in all group.Ara-C VPGs was of good physical and chemical stability and safe as implant in brain. Enhancing the concentration of drug and extending the action time of drug at the tumor region were successfully achieved. Moreover, the Ara-C VPGs can improve the brain distribution behavior and in vivo antitumor activity. This research provides scientific basis for development of Ara-C brain sustained released delivery system.