Studies On Preparation Of Lappaconitine-Loaded Nanoparticles With Polylactic Acid And Their Pharmacokinetics And Pharmacodynamics

Lappaconitine?LA?, the national first class non-addictive analgesics of our country, was widely used in the treatment of moderate to severe pain, such as cancer and postoperative pain in china. The analgesic effect of LA is 7 times as much as that of aminopytine, and equivalents to pethidine. LA has neither serious side effects associated with the opioid drugs, such as teratogenic and mutagenic effects, nor accumulation of toxic and with a long duration efficacy. It was discovered that LA also has the anti-tumor and anti-arrhythmic effects. However, due to its high photochemical sensitivity, poor water solubility, low bioavailability and the tablets and injection of LA in the market need to be administered frequently, it is inconvenient and leads to poor compliance. To improve the water solubility, reduce the frequency of administration and improve its bioavailability. LA/PLA NPs were prepared by optimized emulsion-solvent evaporation method with biodegradable PLA as the carrier material and their pharmacodynamics and pharmacokinetics were studied, respectively. The antitumor activity of lappaconitine hydrochloride and lappaconitine sulfate in vitro were also studied.1. LA/PLA NPs were prepared by optimized emulsion-solvent evaporation method. The entrapment efficiency and drug loading of LA were used as the main evaluation indexes to optimize the preparation process by response surface methodology. The mean particle size and morphology were measured by laser particle size analyzer and atomic force microscope, respectively. The in vitro release behavior was studied by dynamic dialysis. The results showed that the optimal preparation technology conditions were as follows: the concentration of PLA and PVA were 40 mg · m L^-1and 10 mg · mL^-1 respectively, the rate of LA to PLA was 1:1, the rate of oil to water was 1:4. The LA/PLA NPs were spherical under this optimal condition. The mean particle size was?429 ± 9.19? nm, the entrapment efficiency and drug loading rate were?75.47 ± 4.47? % and?44.97 ± 2.92? %, respectively. The in vitro release study showed that the LA/PLA NPs could provide a continuous release of LA for 15 d. The drug release behavior could be well described by the First-order model and Weibull model.2. The concentration of LA in plasma sample was determined using RP-HPLC method. The data of drug concentration-time were processed with PKSolver program. Pharmacokinetics were studied in rats following intraperitoneal injection of LA / PLA NPs with LA injections as the reference drug. The results indicated that LA/PLA NPs could provide a continuous release of the entrapped LA for 8 days in vivo. The main pharmacokinetic parameters of LA/PLA NPs following intraperitoneal injection in rats were listed as follows: t_1/2=?103.16 ± 21.57? h, MRT=?165.14 ± 14.05? h, T_max=?3.6 ± 1.34? h, C_max=?3.50 ± 0.69? ?g·mL^-1, and AUC?0-t? =?455.14 ± 26.18? ?g·m L^-1·h.3. The pharmacodynamic evaluation in rats following intraperitoneal injection of LA, LH, and LA/PLA NPs were studied with thermal pain stimulus method. The significant analgesic effects were showed within 1 h to 6 h after the injection of LA and LH, then the analgesic effect began to weaken and disappeared after 8 h. The analgesic effects of LA/PLA NPs were gradually increased within 100 h and provide a stable analgesic effects, and the analgesic effect gradually weakened and substantially up to 200 h or so. The analgesic effects were more significant with a relatively high dose of nanoparticles(25 mg· kg^-1).4. The antitumor activities in vitro were evaluated in A549, HepG2 and Hela cancer cells by CCK-8 assay methed. The results indicated that the highest inhibition growth of human cancer cells such as A549, HepG2 and Hela cell were?35.50 ± 2.44?%,?24.01 ± 0.88? % and?22.07 ± 3.61? % of LA with the concentration of 0.48⁰.88 mg·mL^-1. In addition, the inhibition growth of A549, HepG2 and Hela cell were?32.88 ± 7.89? %,?29.99 ± 0.60? % and?27.34 ± 6.00? % of LH with the concentration of 0.88 mg·m L^-1. The results showed that the inhibition growth of A549, HepG2 and Hela were slight by LA and LH. The obvious antitumor activity were performed of lappaconitine hydrochloride. The inhibition growth of A549, HepG2 and Hela cell were?78.02 ± 1.11?%,?72.47 ± 0.05?% and?74.93 ± 5.82?% with the concentration 0.88 mg·mL^-1 and the IC50 were 0.711 mg·mL^-1?0.752 mg·mL^-1?0.669 mg·mL^-1, respectively. The stronger antitumor activity were showed of lappaconitine sulfate than lappaconitine hydrochloride. With the concentration of 0.56 mg·mL^-1, the inhibition growth of A549, HepG2 and Hela cell were?75.32 ± 6.00? %,?78.44 ± 4.98? % and?77.92 ± 1.96? %, and the IC50 were 0.422 mg·mL^-1, 0.414 mg·mL^-1 and 0.464 mg·m L^-1, respectively.The cell apoptosis and cell cycle change were demonstrated by flow-cytometric analysis. The results indicated that lappaconitine hydrochloride and lappaconitine sulfate exhibited apoptotic effect on A549, HepG2 and Hela cell, especially in late apoptotic effect. The cell cycle of A549, HepG2 and Hela cells were blocked in G₀/G₁ stage with the ettects of lappaconitine hydrochloride and lappaconitine sulfate.