| Lamivudyl Palmitate Solid Lipid Nanoparticles (LAP-SLN) and the Nanoparticles modified by galactosides (LAP-GSLN) were prepared and their liver targeting effect in mice was investigated.1. Synthesis and identification of Lamivudiyl palmitate(LAP) and galactosides (GDE)LAP was synthesized by the reaction of lamivudine (LA), Palmitic acid, and dicyclohexylcarbodiimide at the catalysis of 4-dimethylamino pyridine. HO-(CH:CH;O)n-C,:H:5(n=0,l,2,3) , isolated by silica-gel column chromatography from a surfactant MOA, were reacted with tetraacetyl-galactosyl bromide to give galactosides. The chemical structure of LAP and GDE was identified by 'H-NMR spectroscopy.2. Stability and lipophiliry of LAP2.1 LAP stability in phosphate buffer solutions (PBS), mouse serum and tissue homogenatesLAP solution was added to diluted serum, homogenates of liver, kidney, lung or spleen and PBS of different pH respectively, which had beenincubated at 37癈 in a water bath. At various time intervals, samples were withdrawn and analyzed by RP-HPLC. The degradation of LAP was shown to display pseudo-first-order kinetics in biological media and buffer solutions. LAP was more stable in w^eak acid PBS than in weak basic PBS, indicating that pH influenced the stability of LAP markedly. The results showed that enzymes in serum and tissue homogenates could accelerate hydrolysis of LAP, especially in liver. 2.2 Determination of partition coefficient(P) of LAP and LALAP was dissolved in n-octanol at a concentration of 32 u g ?mL~'. 5 mL of the solution was transferred to a test tube and 5 mL PBS was then added into the tube which was plugged and shaken on a votex mixter for 5 min subsequently. The concentration of LAP in n-octanol and PBS were determined by RP-HPLC. P of LA was measured by the same method. P of LAP and LA was 521.5 and 0.11 respectively, indicating that LA lipophility was improved dramatically after esterification with pamitic acid. 3. Preparation and characteristics of LAP-SLN and LAP-GSLN 3.1 Preparation of LAP-SLN and LAP-GSLNThe formulation was optimized by Uniform Design experiment. Stability of SLN in PBS, distillated water and 6%(m/v) mannitol solution was studied, which demonstrated that 6% mannitol solution was the best disperse medium. To prepare LAP-SLN, 11.3mg of LAP and lOOmg of soybean lecithin (11.3mg of LAP, 95mg of soybean lecithin and 5mg of GDE for LAP-GSLN) were dissolved in 15mL of chloroform in a lOOmL of round-bottomed flask. After removal of the solvent by rotary evaporation in vacuo, a lipid film was obtained on the flask wall and subsequently re-suspended in 5mL of 6% mannitol solution. The suspension was sonicated for 1 min twice and filtrated bv 0.45 u m filter.3.2 Particle sizeLAP-SLN was a sky-blue colloid, while LAP-GSLN was a milk-white colloid. Under TEM, spherical particles with an average diameter of 280.8 + 98.2nm for LAP-SLN, or 257.4?2.3nm for LAP-GSLN were observed. There was no significant difference in diameter between these two types of SLN.3.3 Determination of drug loading(DL) and entrapment efficiency(EE)Concentration of LAP before and after dialysis in the SLN was measured by RP-HPLC. DL for LAP-SLN and LAP-GSLN was 9.63% and 9.11% respectively. EE for LAP-SLN and LAP-GSLN was 97.69% and 95.82% respectively. There was no significant difference in DL and EE between these two types of SLN. 4. Body distribution of LAP-SLN and LAP-GSLN 4.1HPLC analysis of LA0.1 mL of serum or tissue homogenates and 0.5mL of methanol were vortexed in a plastic tube for SOsec and then centrifuged at ISOOOg for lOmin. 20 u L of the resulting supernatant was directly injected and LA was chromatographed on a reversed-phase Kromasil C18 column using a mixture of phosphate buffer (pH6.8) and methonal (91:9 v/v) and monitored at 270nm. The standard curves were linear over a range from 0.25 u g "mL"1 to 50 u g ?mL~'. The minimal detectable drug concentration was 60ng ?mL"' (S/N=3) . The mean methodological recovery of LAM was from 97% to 100%.
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