Study On β-elemene Solid Lipid Nanoparticles

One major task of the antitumor research is to explore new dosage forms to increase curative effect and targeting ability, and at the same time to reduce toxicity and side effects of the existing drugs thoroughly. As an effective composition of traditional anticancer Chinese medicine,β-elemene has many advantages such as broad antitumor spectra, low side effects, well protection on the immunity of the patients, and etc. However, its limited dosage forms and high incidence rate of phlebitis inhibited the curative effect seriously. The main objective of this thesis was to prepareβ-elemene solid lipid nanoparticles (SLN) owning better tumor targeting ability, higher antitumor effect and lower vein irritation after i.v. administration. The contents in detail are as follows:First of all, the feasibility of using microemulsion technique and probe sonication -membrane extrusion method to prepare SLN was investigated, respectively. Simultaneously, optimal SLN was selected. The results showed that although the SLN prepared by microemulsion technique own small volume mean diameter particles(20.6nm), it could not avoid large microparticles(account for 19.1% of the volume diameter) and solid content was low(1%). Moreover, probe sonication and membrane extrusion methods were combined to prepareβ-elemene SLN for the first time. Short time of sonication was used to realize preliminary dispersion of the lipid matrix followed by membrane extrusion at high temperature to further homogenize the particles and exclude the microparticles. In addition, bacterium and impurity could be excluded with the 0.22μm membrane. A self-assembled device was used to control the temperature and facilitated the preparation. However, preparation with higher entrapment efficiency and stability could not be obtained by using usual composition design route and preparation procedure, attempts were made on the following aspects: firstly for the procedure, two surfactant addition methods on the properties of SLN were compared for the first time. Compared with the method of adding the surfactant in to the water phase(SW), adding the surfactant in to the lipid phase (SL) reduced the concentration of surfactant in the water phase on the early stage and therefore reduced the partition of freeβ-elemene in the water phase, leading to higher entrapment efficiency despite the reduced particle size. Secondly for the design of composition, mixture of monostearin and precirol ATO 5 was used as lipid phase for the first time. Based on the investigation of long-term stability, the optimal SLN (SLN-1) composition were as followed: 250 mg poloxamer 188, 150 mg monostearin, 350 mg precirol ATO 5, 10 mL water and 5.6 mg.mL^-1β-elemene. It could remain stable at room temperature for at least 8months.Chemical and physical properties of SLNs were investigated by using different methods from different aspects. Among them, lipid crystal modification and transition were emphasized to explore main reasons of higher stability for SLN-1. Contents including: 1) Mean diameter: mean diameter of SLN-lwas detected by COULTER DELSA 440SX,Nicomp-380 and Coulter LS 230, respectively. Most of SLNs were smaller than 100nm, which confirmed to the diameter requirement for tumor targeting. 2)ζpotential:ζpotential of SLN preparations was determined by COULTER DELSA 440SX and Nicomp-380, respectively. It was negative and changed with the dilution medium. The absoluteζpotentials increased with increasing water dilution rate; under the same dilution conditions, the absoluteζpotential of SLN decreased with increasing poloxamer 188 and increased with increasing monostearin concentration in the SLN. 3) In vitro release: 36% alcohol solution was chosen as the dialysis medium for in vitro release study ofβ-elemene from SLN. The cumulative release percentage ofβ-elemene from the SLN were 6.3% and 53.6% at 1h and 24h respectively, and the drug could be released completely within 84h.The release data were fitted into First order, Higuchi and Weibull equations, respectively. It showed thatβ-elemene release from SLN follows Higuchi equation better than First-order and Weibull equation. 4)Morphology: transmission electron microscope (TEM) was used to observe the morphology of SLNs. It showed that SLN-1, SLN-2 and SLN-3 were plate-like particles. Oil seemed to be absorbed on the surface of SLN-2. The mean diameter of SLN-1 was around 50nm. 5)Chemicai and physical stability: compared to freshly prepared SLNs, no significance difference in appearance, mean particle size and entrapment efficiency ofβ-elemene were found for the SLN-1 after storage of 8months at room temperature under the protection of light(18-25℃). IR spectrum and the pH of SLN stored for 18 months were similar to that of freshly prepared SLN and no apparent indication of hydrolyzation. Therefore, higher chemical and physical stability of the SLN was confirmed. 6) Lipid crystal modification and transition: samples were prepared mainly by drying SLN at room temperature naturally and were investigated by DSC. It was noticed that supercooling phenomenon occurred in SLN-1, SLN-2 and SLN-3 just after preparation and recrystallized in a metastable state. However, SLN-2 and SLN-3 changed into stable state after two months storage. SLN-1 could maintain metastable state for at least 8 months. The lipids in SLN-1 could maintain metastable state for longer period, which might pay a significant role in the stability of SLN-1.GC and HPLC Methods were established to determine the content ofβ-elemene in the SLN for the first time. However, high noise of the baseline and problems of column contamination and quickly decrease efficiency remained unsolved for the GC. In contrast, HPLC could avoid these problems and can be used to control the quality of the preparation more conveniently. In addition, another HPLC method was developed to determine the content ofβ-elemene in vivo. A new method named flocculation and filtration was set up to separate freeβ-elemene from that incorporated in the SLN to determine the entrapment efficiency ofβ-elemene in the SLN. The method could be easily operated with accurate results. The entrapment efficiency ofβ-elemene in the SLN-1 was 96.0±0.41%.In order to target PEG-FA-SLN to the folio receptor on the surface of tumor cell, a novel folio acid complex named N-stearyl-N'-pteroylglutamyl-polyethylene glycol(3350) bis-amine (FA-PEG-S) was synthesized and its chemical structure was confirmed by TLC, IR and ¹H-NMR.The pharmacokinetie profiles and tissue distribution ofβ-elemene were investigated afterâ…°.â…´. administration of different formulations(SLN-1, PEG-FA-SLN and control emulsion)by determineβ-elemene levels in plasma and tissues. There was no statically difference between the elimination t_1/2 of SLN-1 and that of control emulsion.β-elemene was eliminated rapidly with t_1/2 of 15.6 min. However, SLN-1 showed higher affinity to different tissues compared with control emulsion. 5 min after SLN-linjection,β-elemene levels in liver, spleen and kidney were 1.5, 2.9 and 1.4 times higher than those ofβ-elemene control emulsion respectively, whereasβ-elemene concentrations decreased 30% in heart and lung. The t_1/2 of PEG-FA-SLN containing 4% CHS-PEG and 0.1% FA-PEG-S (molecular ratio) was 44.0 min which was longer than SLN-1. Furthermore, tissue distribution PEG-FA-SLN was different from that of SLN-1 and control emulsion.:β-elemene levels of PEG-FA-SLN were higher in the liver and kidney and lower in the spleen compared with that of SLN-1 5 min after injection; after 30 and 60 min administration,β-elemene concentration in tissues except lung was higher for PEG-FA-SLN compared with that of SLN-land control emulsion.Concentration ofβ-elemene in the tumor were investigated afterâ…°.â…´. administration ofβ-elemene SLNs and control emulsion (84.2 mg.kg^-1) to mice with H22 replanted in the oxter and tumor targeting ability of the preparations were compared. Simultaneously, in order to investigate the main factors that influence the tumor targeting ability afterâ…°.â…´. administration, saturate retention concentration ofβ-elemene in the tumor after subcutaneous administration to the tumor and concentration ofβ-elemene in the blood afterâ…°.â…´. administration of SLNs and control emulsion were determined, respectively. Results showed that the highestβ-elemene concentration, approximately 21μg.g^-1 for SLN group and 13μg.g^-1 for the control emulsion, was reached in less than 20 min and remained stable for at least 8h. Therefore, it is reasonable to assume thatβ-elemene SLNs own better tumor targeting ability afterâ…°.â…´. administration compared with the control emulsion. There was no evident difference between those of SLNs. The average saturate retention concentration ofβ-elemene in tumor samples was 200.22±45.67μg.g^-1 for control emulsion group and 29.25±4.49μg.g^-1 for the SLN group.β-elemene concentration in the blood afterâ…°.â…´. administration was not lower than that of SLN-lin 20 min. In conclusion, tumor affinity was the main factor to determine tumor targeting ability. Compared with control emulsion, higher tumor targeting ability afterâ…°.â…´. administration of SLN-1 should due to the higher tumor affinity.Cancer inhibition effects were observed afterâ…°.â…´. administration ofβ-elemene SLNs and control emulsion to mice with H22 replanted in oxter. The cancer growth inhibition rates of PEG-FA-SLN, PEG-SLN and SLN-1 were 49.50%,48.87% and 47.78%, respectively, which were markedly higher than that of control emulsion with 18.68%. All of the preparations could stimulate the immune tissues in different degree and had little harm to the body.Degree of vein irritation was investigated afterâ…°.â…´. administration of two preparations to rabbit helix vein. Compared with the control emulsion,β-elemene SLN decreased the irritation significantly.