[Study on preparation of breviscapine liposomes and chemicophysical properties].

OBJECTIVE: To prepare breviscapine pro-liposomes and evaluate its properties and stability, as well as its interaction with the mimic-membrane. METHODS: Breviscapine liposomes were prepared by thin film-lyophilization method. Phase inversion temperature was measured by electrical conductance method and coalescence kinetics was studied. Water/n-octanol trans-membrane diffusion model was designed to study the dynamic distribution behavior between two phases, through the determination of diffusion rate of breviscapine and liposomes. RESULTS: The phase inversion temperature was 63 degrees C, the activity energy for coalescence was 14.66 kJ/mol. The results suggested that breviscapine from liposomes staying on the interface were found more than the breviscapine infusion. CONCLUSION: Breviscapine liposomes prepared with thin film-lyophilization method have good physicochemical properties and stability, which is beneficial to treatment.

[Formulation and process optimization of doxorubicin-loaded PLGA nanoparticles and its in vitro release].

Doxorubicin-loaded PLGA nanoparticles (DOX-PLGA NPs) was prepared by double emulsion (W/O/W) solvent evaporation method with the biodegradable materials-poly (lactic-co-glycolic acid) (PLGA) used as carrier materials. Single-factor test was used to investigate the influence of the type and ratio of the organic phase, the amount of surfactant, PLGA concentration, the ratio of external water phase and oil phase (W/O), the ratio of doxorubicin and PLGA, ultrasonic time and stirring time on the preparation of nanoparticles. The best formulation and preparation conditions were optimized by orthogonal test based on single-factor test, evaluation indicator as particle size and entrapment efficiency, and the results were analyzed by overall desirability. And the in vitro release behaviors of the nanoparticles were studied as well. The size distribution, zeta potential, morphology of DOX-PLGA NPs were characterized by laser light scattering and transmission electron microscopy; encapsulation efficiency and releasing behavior of DOX-PLGA NPs in vitro were investigated by ultraviolet spectrophotometry. The results show that the DOX-PLGA NPs are regularly spherical in shape with the mean size of (189.2 +/- 5.3) nm, and the zeta-potential of the NPs is about (-28.32 +/- 0.52) mV. Drug loading and encapsulation efficiency are estimated to be (73.16 +/- 0.43) % and (1.51 +/- 0.07) %, respectively. The cumulative percentage of the drug released is 90.34%, and the in vitro release behavior made up of initial burst release and sustained-release could be described by the bidirectional kinetic equation. The results indicate that hydrophilic small-molecule drugs could be successfully entrapped into PLGA-NPs. With optimization of the formulation and preparation conditions, we obtained uniform and stable DOX-PLGA NPs with sustained release character in vitro and pH-sensitive property, which could provide the experimental basis for the development of a new anti-tumor sustained-release formulation.

Synthesis of a novel polymer bile salts-(polyethylene glycol)2000-bile salts and its application to the liver-selective targeting of liposomal DDB.

PURPOSE: The objective of this study was to achieve a sustained and targeted delivery of liposome to the liver, by modifying the phospholipid [phosphatidylcholine (PC)/cholesterol (10 : 1) liposomes with a novel polymer bile salts-(polyethylene glycol)(2000)-bile salts (BP(2)B). METHODS: First, we generated a novel BP(2)B by N,N'-dicyclohexylcarbodiimide/4-dimethylaminopyridine esterification method and confirmed by Fourier transform infraredand (1) H-NMR spectra. Second, we prepared the BP(2)B-modified liposomes (BP(2)BL) that included BP(2)B, and the effect of the weight ratios of BP(2)B/PC on entrapment efficiency was investigated and BP(2)B/PC = 3% (w/w) was determined as the optimum ratio for the 4,4'-dimethoxy-5,6,5',6'-bi (methylenedioxy)-2,2'-bicarbomethoxybiphenyl liposomes. And then, the ability of the liver target of BP(2)BL was studied by calculating the targeted parameters. RESULTS AND DISCUSSION: All the results revealed that the introduction of polyoxyethylene chains could control interactions of bile salt moieties on liposome surfaces with the receptor compared with traditional liposomes (CL), marking BP(2)BL as a suitable carrier for hepatic parenchymal cell-specific and sustained targeting. It was suggested that liposomes containing such novel BP(2)B have great potential as drug delivery carriers for the liver-selective targeting that has targeted and sustained drug delivery.

[Evaluation with compression equations of compression behavior of pellets with different intragranular pore volumes].

Microcrystalline cellulose (MCC), calcium phosphate (DCP)/MCC (4:1, w/w) and lactose (Lac)/MCC (4:1) pellets with different intragranular porosity were prepared in an extrusion-spheronizator and three volume ratios of ethanol/water were used as binder agents to prepare pellets. The compression behaviors of these pellets with different intragranular pore volume were evaluated with the parameters of Kawakita model. The results showed that high pore volume of pellets made up of MCC had the best compressibility and low pore volume of pellets had a poor compactibility. However, the compressibility of different porosity of pellets made up of DCP/MCC (4:1) or Lac/MCC (4:1) was good, but they were not significantly different. The reason might be the main compression mechanism of high porosity of MCC pellets was plastic deformation and that of DCP/MCC pellets or Lac/MCC pellets was not plastic deformation but fragmentation. These results can be observed directly by the SEM photographs. According to these results, the conclusion could be drawn that high porosity MCC pellets and different porosity DCP/MCC pellets and Lac/MCC pellets can be used as cushion granules to maintain the original shape and release characteristics of drug pellets when pellets were tabletted.

[Electrophoresis and fluorospectrophotometry methods to determine the content and entrapment efficiency of siRNA in cationic liposomes].

To develop different methods for determining siRNA content and the entrapment efficiency of siRNA loaded liposomes, SYBR Gold electrophoresis method and Ribogreen fluorospectrophotometry method were used respectively. SYBR Gold electrophoresis method has a good linear relation in a range at 0.2-2.0 micromol x L(-1) (R = 0.9930), and the recovery at the high, middle and low concentrations were 96.35%, 96.92%, and 100.74%, respectively (n = 3). The intra-day and inter-day RSD were far below 5% (n = 5). Ribogreen fluorospectrophotometry method has a good linear relation in a range at 10-50 nmol x L(-1) (R = 0.9971), and the recovery at the high, middle and low concentrations were 98.22%, 99.88% and 99.64%, respectively (n = 3). The intra-day and inter-day RSD were far below 5% (n = 5). The content and the entrapment efficiency of three batches of siRNA cationic liposomes were 98.52%, 97.85% and 99.20%, 96.45%, respectively, with these two methods. And there is no significant difference by ANOVA. Both of the two methods are accurate, sensitive, convenient method for determination of the siRNA content and the entrapment efficiency of siRNA loaded cationic liposomes.

Synthesis and application of poly(ethylene glycol)-cholesterol (Chol-PEGm) conjugates in physicochemical characterization of nonionic surfactant vesicles.

Vesicles possessing poly(ethylene glycol) (PEG) chains on their surface have been described as a blood-persistent drug delivery system with potential applications for intravenous drug administration. In this research with different molecular weights (400-10,000g/mol) of PEG, a series of Chol-PEG(m) conjugates were generated by the DCC (N,N'-dicyclohexylcarbodiimide, DCC)/(4-dimethylaminopyridine, 4-DMAP) esterification method, and confirmed by FT-IR and (1)H NMR spectrum. Then their properties in aqueous solution were studied by electron microscopy images, associative behavioral and systematic tensiometric studies over a wide concentration range. In order to elucidate the application of this Chol-PEG(m) in vesicles, conventional nonionic surfactant vesicles (niosomes) composed of span 60 and cholesterol were prepared and the influence of various hydrophilic chains of the Chol-PEG(m) conjugates was investigated. Results indicated that all the niosomes prepared, with or without Chol-PEG(m) composition were similar in micrograph with diameter between 120 nm and 180 nm. The fixed aqueous layer thickness (FALT) around niosomes increased as Chol-PEG(m) chain length increase, particularly in the Chol-PEG(10,000) modified niosomes with 9.33+/-0.67 nm. In vitro release experiments indicated that release rate of nimodipine from Chol-PEG(m) modified niosomes was enhanced. Chol-PEG(m) modified niosomes showed greater accumulative release than that of plain niosomes over a period of 24 h. These studies have shed some light on the suitability of Chol-PEG(m) containing niosome preparation.

[Preparation of verapamil hydrochloride core-in-cup tablets with double-pulsatile and multi-phasic release].

To prepare verapamil hydrochloride (VH) core-in-cup tablets with tri-layered tablet and four-layered tablet as core tablets, separately, which can provide biphasic release with double-pulsatile and multi-phasic release, core tablets were prepared by direct compression method, and core-in-cup tablets by dry-compression coated technology. The parameter, time-lag (T(lag)), was used to evaluate the influence of factors, such as the weight of the top cover layer, the amount of hydroxypropylmethylcellulose (HPMC), and the compression load on VH release. With the increase of the weight and HPMC amount of the top cover layer, the first lag time T(lag1) was prolonged. The second lag time T(lag2) of core-in-cup tablet with four-layered tablet as core tablet increased with the increasing amount of HPMC K100M. With the increase of compression load among the range (6 - 10 kg x cm(-2)), the two lag times were prolonged. Core-in-cup tablets with double-pulsatile and multi-phasic release released VH after the first lag time (4 -5 h), then kept sustained release for 12 h or 13 h, finally released rapidly. The drug in the core-in-cup tablet only released from the top cover layer. T(lag) is determined by the erosion rate of the inhibitor layers (the top cover layer and the sustained-release layer of the multi-layer core tablet).

[Preparation of tablets containing enteric-coated diclofenac sodium pellets].

Fluidized-bed manufactured enteric-coated diclofenac sodium pellets were compressed into tablets. The blend of two aqueous acrylic resins dispersion in different ratios, Eudragit NE30D and Eudragit L30D-55, were used to prepare enteric-coated diclofenac sodium pellets of different particle sizes and coating level. The cushioning pellets with different properties and these enteric-coated pellets were compressed into tablets in different proportions. The drug release of the tablets containing these pellets would be lower than 10% in 2 h in simulated gastric fluid, but reach (83 +/- 2.42)% in 1 h in simulated enteric fluid. The mixture of Eudragit NE30D and Eudragit L30D-55 could be used to prepare enteric pellets which are suitable for compression. The cushioning pellets which were composed of stearic acid/microcrystalline cellulose (4:1, w/w) could avoid rupture of the coating of pellets during the compression.

[Preparation and in vitro corneal retention behavior of novel cationic microemulsion/in situ gel system].

The aim was to prepare a novel ocular cationic microemulsion-in situ gel (CM-ISG) system with vitamin A palmitate (VAP) as model drug, and investigate the corneal retention behavior and corneal irritation of the system. VAP/CM was prepared by a process based on supply of energy, and the before-and-after gelation rheology of VAP/CM-ISG was investigated. In vitro VAP release and gel dissolution of both VAP/CM-ISG and Oculotect Gel was determined. And in vitro corneal retention behavior of both formulations was evaluated by captive bubble technique. Ocular irritation test was carried out based on the Draize method. Images of TEM showed that homogenous VAP/CM was made, and no significant differences of particle size were found between the VAP/CM and VAP/CM in Poloxamer 407 gel. Rheology study illustrated that VAP/CM reduced the phase transition temperature of Poloxamer 407 gel by 1.5 degrees C, and the elastic modulus increased about 15.7 times. The in vitro release and gel dissolution profile of both formulations exhibited the characteristics of zero order kinetics. Comparing with Oculotect Gel, desorption kinetics study of VAP/CM-ISG exhibited longer corneal retention time and smaller contact angle. Irritation test showed a good ocular compatibility of VAP/CM-ISG. Therefore, VAP/CM-ISG combined both advantages of the cationic microemulsion and in situ gel system, provided better wettability and longer ocular retention time. It might be a promising ocular drug delivery system.

A simple HPLC method for the determination of bifendate: application to a pharmacokinetic study of bifendate liposome.

A rapid, sensitive and simple high-performance liquid chromatographic (HPLC) method with ultraviolet detector (UV) has been developed for the determination of bifendate in 100 microl plasma of rats. Sample preparation was carried out by deproteinization with 100 microl of acetonitrile. A 20 microl of supernatant was directly injected into the HPLC system with methanol-double distilled water (65/35, v/v) as the mobile phase at a flow rate of 1.0 ml/min. Separation was performed with a microBondapak C(18) column at 30 degrees C. The peak was detected at 278 nm. The calibration curve was linear (r(2)=0.9989) in the concentration range of 0.028-2.80 microg/ml in plasma. The intra- and inter-day variation coefficients were not more than 6.55% and 6.07%, respectively. The limit of detection was 5 ng/ml. The mean recoveries of bifendate were ranged from 94.53% to 99.36% in plasma. The present method has been successfully applied to the pharmacokinetic study of bifendate liposome in rats.

Stability and pharmacokinetic studies of O-palmitoyl amylopectin anchored dipyridamole liposomes.

Modified polysaccharides have been used widely to increase physico-chemical stability of liposomes. However, the stability and pharmacokinetic studies on the polysaccharides modified anchored liposomes containing hydrophobic drugs which exist in lipid bilayer membranes were insufficient as compared with the liposomes carrying hydrophilic or ionic drugs in inner aqueous phase. In the present study, a hydrophobic drug, dipyridamole (DIP), was entrapped into liposomes through film hydration. Amylopectin was palmitoylated and anchored on the surface of plain DIP liposomes. Subsequently, the stabilities of DIP ethanol solution, plain DIP liposomes (PDL) and anchored DIP liposomes (ODL) against irradiation, disperse medium, biofluid, long-term storage were determined and compared. The concentrations of DIP in plasma of rats and its pharmacokinetic behaviors after intravenous administration of DIP injection, PDL and ODL were studied by RP-HPLC. The pharmacokinetic parameters were computed by software 3p97 programme. The results showed that ODL could increase stabilities more of DIP in vitro as compared with PDL. The plasma concentration-time curves of DIP after intravenous administration of DIP injection, PDL and ODL were all in accordance with open two-compartment model. Pharmacokinetic parameters of DIP injection, PDL and ODL in rats were significantly different. The present findings suggest that anchored liposomes could increase stabilities of DIP in vitro as compared with plain liposomes. Furthermore, the difference of pharmacokinetic profiles was due to the targetability of anchored liposomes.

[Preparation of amylopectin modified dipyridamole liposome and its tissue distribution in mice].

AIM: To prepare amylopectin anchored dipyridamole (DIP) liposome and to study its tissue distribution in mice. METHODS: The regular DIP liposomes were prepared by film-scatter method. The amphiphilic O-palmitoyl amylopectin was synthesized and added to modify the surface of liposome. The entrapping efficiency, zeta potential, mean diameter, span of modified and regular liposomes were assayed. The RP-HPLC was used for the determination of DIP concentration in mice tissue. RESULTS: After modification, the entrapping efficiency depressed, zeta potential was raised, mean diameter and span had no obvious change. The level of DIP in lung, liver and spleen for regular liposomes were higher than that of injections. Compared with regular liposomes, the modified liposomes increased the DIP level in lung, and decreased the DIP level in liver, spleen, moreover, lengthened the retention time of DIP in lung. CONCLUSION: The distribution of modified liposome in mice was markedly changed as compared with regular liposomes and injections. The modified liposomes had obvious lung targeting property.

[Preparation of verapamil hydrochloride controlled-onset extended-release pellets and its pharmacokinetics in dogs].

AIM: To prepare verapamil hydrochloride controlled-onset extended-release pellets (VH-COERP) and study its release behavior in vitro. To compare the pharmacokinetic characteristics and bioavailability in six Beagle dogs after oral administration of VH-COERP and verapamil hydrochloride delayed-release pellets (VH-DRP) as reference. METHODS: The core of VH-COERP were prepared in the fluidized bed (mini-glatt) by spraying water solution containing drugs onto sucrose-starch pellets with hydroroxy propyl methyl cellulose (HPMC) as the inner coating swelling layer and ethylcellulouse aqueous dispersion as the outer coating controlled layer. Through modifying the coating level of inner and outer layer, the VH-COERP with the optimized cumulative release profile was obtained. The concentration of VH in plasma of six dogs and its pharmacokinetic behaviors after oral administration of VH-COERP and VH-DRP at different times were studied by RP-HPLC. The pharmacokinetic parameters were computed by software program 3P97. RESULTS: The lag time, the release behavior and the amount of VH from VH-COERP within 24 hours were not influenced by the pH of dissolution medium and post-process, but obviously influenced by the different kinds of added material in swelling layer and the coating level of the inner swelling layer and the outer controlled layer. In vitro the lag time of release profile of VH from VH-COERP was 5 h and then VH was extended release from VH-COERP in the following time. Compared with the VH-DRP, VH-COERP in vivo has an obviously lag time (4 h) , Tmax was also delayed (8 h) and the relative bioavailability was (94.56 +/- 7.64)%. CONCLUSION: The release profile of VH from VH-COERP was shown to be extended-release after an conspicuous lag time in vitro and in vivo. So the drug can be taken by the patient before bed time and begin to work at the morning.

[Optimization of formulation of Fufang Danshen immediate release tablet by colligation score].

OBJECTIVE: To optimize the formulation of immediate release tablet. METHOD: The immediate release tablet was prepared by using dry granules. The preparation was optimized by using orthogonal design which took the flow property of granules, the hardness, the disintegrating time and the dissolution rate of the tablet as indices. RESULT: The optimized formulation contained 40% microcrystalline cellulose, 10% sodium carboxymethyl starch and 15% dextrin. The hardness disintegrating time and T50 of the tablet were 4.5 kg, 3 min, 5 min respectively. CONCLUSION: It is successful to prepare on immediate release tablet using the optimized formula above.

[Preparation of lung targeting azithromycin liposomes and its tissue distribution in mice].

AIM: To prepare lung targeting azithromycin cationic liposomes and to observe its tissue distribution in mice. METHODS: The azithromycin cationic liposomes were prepared by thin film method with freeze-thawing steps. HPLC method was established and validated for the determination of azithromycin in tissues of mice. RESULTS: The particle size of the liposomes was 6.582 microm with zeta potential of +19.5 mV. The entrapment efficiency was more than 75%. The liposomes was stable in 6 months stored at 4 degrees C. The release in vitro was characterized by Higuchi equation. Azithromycin liposomes and free azithromycin solution were injected intravenously at a dose of 80 mg x kg(-1) to mice. Compared with solution, liposomes were characterized by slower clearance, increased half-life and the AUC increased by 7.4 fold in lung. CONCLUSION: Thin film method with freeze-thawing steps could increase the entrapment efficiency and increase the particle size of azithromycin liposomes. After modification of lipid membrane with stearylamine, the cationic liposomes were prepared. The azithromycin concentration and AUC increased in lung after iv administration to mice of the cationic liposomes. This offered a good information for preparing liposomes targeting on the lung.

[Preparation of acyclovir liposome and study on its stability].

AIM: To prepare acyclovir liposome for improvement the entrapment efficiency and stability. METHODS: Acyclovir liposome was prepared by the reverse evaporating method. Surfactants such as sodium deoxycholate and oleic acid were added to optimize the conditions and technology of preparing acyclovir liposome. The entrapment efficiency and particle size of the acyclovir liposome were determined. The liposome stability was proved by centrifugal acceleration experiment. RESULTS: The particle size of the acyclovir liposome was 219.8 nm with the polydispersity index of 0.158. The entrapment efficiency reached 65%. The liposome was stable. CONCLUSION: The results suggest that the conditions and technology are stable and practical to prepare the liposome with high entrapment efficient and stability.

[Preparation of diltiazem hydrochloride delayed-onset, sustained release tablet].

AIM: To prepare dilitazem hydrochloride delayed-onset, sustained release tablet, which can not only provide the delay in release start, but also the constant release rate after a lag time. To analyze release mechanism and investigate the effect of outershell compositions on release behavior. METHODS: The delayed-onset, sustained release tablets were prepared by dry-compression coated technology. The release profiles of uncoated cores and presscoated tablets were compared. Two parameters, time-lag (Tlag) and release rate (k), were used to evaluate the influence of factors, such as the amount of hydroxypropylmethylcellulose (HPMC) and poly-vinyl-pyrrolidinone (PVP) K30, the viscosity of ethylcellulose (EC) and HPMC, and the compression load on diltazam hydrochloride (DIL) release. Higuchi equation and Peppa's equation were used to analyze release mechanism. RESULTS: With the increase of HPMC amount or HPMC viscosity, Tlag was prolonged and k was decreased; With the increase of PVP K30 amount, Tlag was shortened and k was increased; EC viscosity and compression load above certain degree showed no effect on DIL release. CONCLUSION: The drug release from delayed-release tablet is controlled by erosion mechanism, Tlag is determined by the erosion rate of outer-shell.

[Determination of entrapment efficiency of breviscapine nanoliposomes].

AIM: To determine the entrapment efficiency of breviscapine nanoliposomes. METHODS: Retrodialysis method was used to determine the entrapment efficiency of breviscapine nanoliposomes. The drug recovery and the sample recovery were considered to verify the feasibility of the method. RESULTS: The drug recovery was (99.6 +/- 0.6)%. The sample recovery was (99.9 +/- 0.8)%. The entrapment efficiency of breviscapine nanoliposomes was (75.3 +/- 2.3)%. CONCLUSION: The method used in this study is simple, applicable and accurate for determination of the entrapment efficiency of breviscapine nanoliposomes.

[Studies on the preparation of camptothecin niosomes].

AIM: To study the non-ion surfactant vehicle (niosome) entrapped-camptothecin. METHODS: The niosome loaded with camptothecin was prepared from Span and cholesterol using aqueous dispersion of film. The vehicles were visualised by transmission electron microscopy and sized by laser particle analyzer on a Malvern Mastersizer. An HPLC analysis method of the camptothecin was established by fluorescence detection. The entrapment efficiency of the niosomes containing camptothecin was determinated after the ultracentrifugation of the niosome. The antitumor activities of the vehicles on S180 sarcoma in mouse were studied. RESULTS: The given niosomes were the suspension finely dispersed in aqueous solution. They were spherical vehicles with the single lamellar bilayers similar to phospholipid vehicles. The mean sizes of the vehicles were (565 +/- 6) nm. The recovery of the HPLC analysis method was 100.3% with 0.4% RSD. The entrapment efficiency of the camptothecin encapsulated by the niosome was 61%. The inhibition (%) of the niosome loaded with camptothecin on S180 sarcoma in mouse were 76.1% (P < 0.05). After the given dose the weight of the mouse of the niosome groups were 92.7% (P > 0.05) and 134.7% of blank control groups and compatothecin solution groups, respectively. CONCLUSION: The camptothecin niosomes were spherical in shape and similar to phospholipid vehicles with singlelamellar bilayers. Their size distributions were narrow. Their entrapment efficiency were higher. Its antitumor activity was better than camptothecin.

[Studies on combined release behaviors of versatile mini-tablets in capsule systems and fittings of their mathematical model].

AIM: To prepare nifedipine (NP) rapid release mini-tablet, sustained release mini-tablets, pulsed release mini-tablets and delayed-onset sustained release mini-tablets and develop multiplied pulsed drug delivery system (DDS), site-specific DDS, zero-order DDS and quick/slow DDS by various ways. METHODS: Velocity-time (v-t) equation of each mini-tablet was deduced by non-linear least square model fit. The difference of combinations in v-t profiles between theoretical value and test value was compared. RESULTS: According to the v-t equations, the combined release behaviors were observed directly from v-t profiles and the test values coincided with the theoretical profiles. CONCLUSION: The programmed DDS, which consist of a variety of mini-tablets with different dosages and combinations in capsules, could be predicted by summing up the v-t equation of each tablet.