Preparation Of Nanostructured Lipid Curcumin Carriers And Its Targeting Evaluation

Alzheimer’s disease (AD) is the most common cause of dementia in the elderly population and the fourth leading cause of death in developed countries, after cancer, cardiovascular diseases, and stroke, which had brought Heavy burden to the society and family. The exact pathogenesis of the neuronal degeneration and cognitive impairment in AD still remains unclear. Although a number of drugs, which include several cholinesterase inhibitors and an NMDA receptor antagonist, have been approved for use, they have been shown to produce diverse side effects, short half-time and yield relatively modest benefits. To overcome these limitations of current therapeutics for AD, the development of new strategies is an active area of research.The natural phenolic compound curcumin, isolated from the roots of Curcumin longa (Zingiberaceae), which gives the yellow color to curry and mustard, is currently the only one containing phenol and uinine groups of natural medicines. Curcumin (commonly called diferuloylmethane) has been shown to exhibit antioxidant, anti-inflammatory, anticarcinogenic activities and protects against myocardial infarction properties. It also has nephroprotective activities (such as AD). The pharmacological safety and efficacy of curcumin makes it a potential compound for treatment and prevention of a wide variety of human diseases. In spite of its efficacy and safety, curcumin has not yet been approved as a therapeutic agent, and the poor absorption, rapid metabolism,, short elimination and the poor bioavailability of curcumin has been highlighted as a major problem for this.Nanostructured lipid carriers (NLCs) are the second generation of lipid nanoparticles, composed of a binary mixture of solid lipid and a spatially different liquid lipid as a carrier. Traditional lipid-based formulations include a broad range of lipid solution, emulsions, liposomes, lipid microparticles and polymer nanoparticles. Among the formulations above, NLCs are regarded as the second-generation of solid lipid nanoparticles (SLN), and are attracting major attention as alternative colloidal drug carriers. NLCs system possesses many advantages of SLN, such as good biocompatibility, controlled drug release, and the possibility of targeting transport to certain tissues. Drug wrapped in NLCs or adsorbed on the surface, targeted to the specific tissues, has pharmaceutical stability, slow speed of drug leakage, low toxicity, improving the oral bioavailability of a drug and reducing the side effects. Thus, NLCs is a promising drug delivery system because of its good physiological compatibility, controlled release, and the possibility of production on large industrial scale. Moreover, due to its lipid composition from biodegradable and biocompatible materials, protection of drugs from chemical degradation and enhanced drug stability, NLCs is expected to offer the possibility to improve new dosage form of drug by more and more scholars. The lipid formulations loaded with poorly water-soluble drugs have been investigated and reported to improve the oral bioavailability by many research Teams.Therefore, to overcome these difficulties, it is necessary to design a safe, controlled drug release, and targeting transport to certain tissues curcumin NLCs formulation, resolving the clinical application problem of phytochemicals, such as curcumin, to improve the bioavailability of curcuminn, leading to better therapeutic effect in the treatment of brain diseases such as AD. The main research and results are as following: Part One Preparation of nanostructured lipid carriers Section1Analytical method establishment of nanostructured lipid curcumin carriers content determinationObjective:To develop validated method of detection of curcumin in nanostructured lipid curcumin carriers using high performance liquid chromatography (HPLC).Methods:Our HPLC method with ultraviolet detection for the detection of curcumin in nanostructured lipid curcumin carriers. The samples were separated well on a Thermo Hypersil ODS C18Column (250mm*4.6mm,5μm). The mobile phase consisted of acetonitrile and0.5%phosphoric acid solution (58:42, v/v) at a flow rate of1mL per minute. The detection wavelength was set at423nm. The sample injection volume was20μL and the column temperature was maintained at30℃.Results:Curcumin could be successfully separated from nanostructured lipid carriers under the chromatographic conditions. The method showed good linearity (r=0.9998) over wide dynamic range (0.01-3.2μg/mL). Intra-and interday variabilities (RSD) never exceed3.2%and3.5%, respectively. Accuracies were more than99%.Conclusion:The HPLC method for the determination of curcumin in nanostructured lipid carriers is accurate and reliable, which could be applied to determine the curcumin concentration in the carriers. The method would provide a platform for preparation of curcumin nanoparticles.Section2Optimizing for preparation technique of nanostructured lipid curcumin carriers by orthogonal testObjective:To develop and optimize a process for obtaining nanostructured lipid curcumin carriers.Methods:Nanoparticles were produced by the method of solvent evaporation solidification at low temperature. Based on the single-factor experiment, CUR-NLCs was prepared using orthogonal design for the optimization and technology. The quality of the obtained CUR-NLCs was evaluated. Results:Based on the results obtained by single-factor experiment, factors including drug-to-lipid ratio, lecithin-to-poloxamer ratio, the ratio of nano-emulsion to ice water and the solidifying time were the four key factors. The optimal experimental conditions were obtained by orthogonal experiment as follows:mLecithin:mpoloxamer=1:1; mDrug:mLipid=1:20; VNano-emulsion: Vice water=1:8; The solidifying time was30min. The results showed that the optimal formulation of CUR-NLCs had entrapment efficiency (EE) of94.47±1.92%, drug loading (LC) of4.16±0.14%, particle size of132.3±16.13nm and Zeta potential of-29.6±3.7mV. Anaysis of variance showed that the effects of lecithin/poloxamer ratio and the drug to lipid ratio on the particle size of CUR-NLCs has significant statistical significance(p<0.05).Conclusion:The results of the present study demonstrated that CUR-NLCs with expected particle size, entrapment efficiency and stability can be obtained by the method of solvent evaporation—olidification at low temperature.Part Two Characterization of nanostructured lipid curcumin carriers and its oral pharmacokinetics in ratsSection1Preparation and property evaluation in vitro of CUR-NLCsObjective:To formulate and evaluate CUR-NLCs in vitro.Methods:The CUR-NLCs were characterized by the particle size analysis, Transmission Electron Microscopy (TEM) and Differential Scanning Calorimetery (DSC). The release of essential oil was determined using a dialysis membrane method.Results:The mean particle size, polydispersity index, zeta potential, EE, and LC of CUR-NLCs were131±17.5nm,-28.1±4.7mV,0.255±0.081,95.42%±1.63%, and4.14%±0.03%, respectively. The morphology of the CUR-NLCs was spherical as shown by transmission electron microscopy. The differential scanning calorimetry (DSC) analysis indicated that the curcumin incorporated into NLCs was in an amorphous form. The DSC curve of CUR-NLCs was different from curcumin, and it indicated that the curcumin can interact with the matrix of lipid during the preparation of the CUR-NLCs. The in vitro release study showed sustained release of curcumin from the CUR-NLC formulation, the CUR-NLC formulation released6.1%of its curcumin content after1hour and30.2%after24hours (p<0.01).Conclusion:In conclusion, results of the present study demonstrated that CUR-NLCs prepared by the method of solvent evaporation-solidification at low temperature exhibited expected particle size, entrapment efficiency, and sustained release in vitro.Section2Pharmacokinetics of CUR-NLCs in ratsObjective:To study the pharmacokinetics of CUR-NLCs in rats.Methods:HPLC analytical method was established in rat plasma in order to determine the content of CUR-NLCs in blood, and then pharmacokinetic parameters were calculated.Results:The results showed the HPLC analytical method for content determination of CUR-NLCs in rat plasma was stable, reliable, and repetitiveness, specificity is good. The good line range was emerged from0.01to3.2μg/mL, and RSD of mean recovery, mean retention time, intra-day precision, inter-day precision respectively was98.67%-99.56%,1.46%-2.74%, and2.09%-3.27%. Result of pharmacokinetic studies showed that CUR-NLCs and curcumin suspension were fit for the two room open model in rats. Elimination half life of CUR-NLCs was20.62h, and it was3.19times to curcumin suspension. AUC of CUR-NLCs was2.38times to curcumin suspension (p<0.01).Conclusion:Based on these data, the CUR-NLC formulation significantly prolonged the time of curcumin in the blood circulation and improved the bioavailability of curcumin in rats.Part Three Distribution of nanostructured lipid curcumin carriers in rats in vivo and evaluation on its targeting performanceObjective:To study the tissue distribution of CUR-NLCs in heart, liver, spleen, lung, kidney and brain of rat and to evaluate the tissue targeting property of CUR-NLCs.Methods:An HPLC method for separation and quantification of CUR-NLCs was developed and validated by studying rat tissues at different times following intragastric administration. The targeting in vivo of CUR-NLCs was estimated by targeting index (TI), targeting efficiency (TE) and relative total targeting efficiency (RTE).Results:The AUC values in the brain, liver, heart, spleen, kidney, and lung of the two formulations were11.93,1.75,1.08,3.12,1.26, and2.32, respectively. The MRT in each tissue of rats administrated with CUR-NLCs was longer than that administrated with curcumin suspension, except brain. The brain MRT for CUR-NLCs (2.77h) was approximately3.45fold greater than curcumin suspension (9.56h)(p<0.01). The RTE in the brain, heart, liver, spleen, lung and kidney for rats administered with CUR-NLCs were515.53%、-45.27、-10.64、58.41、17.61and-36.08, respectively. The drug targeting index (TI) are calculated:brain (6.1553), heart (0.5473), liver (0.8936), spleen (1.5841), lung (1.1761), kidney (0.6392). All the targeted values in the CUR-NLCs group were far higher than that in curcumin susupension group (p<0.0l).Conclusion:CUR-NLCs changed the drug distribution in the rats and showed good brain targeting property.