The Preparation Of PAMAM Coated Compound Liposomes Of Chrysophanol And Berberine Hydrochloride And In Vivo Evaluation And Study Of New Nealand Rabbit

ObjectiveAged related macular degeneration(AMD)is one of the most serious coular disease which could caused irreversible blindness in people aged 50 years old.AMD is gradually to be the prevalence as the population ages in China.It takes the long-term and multi-faceted burdens on patients and their families as AMD can cause serious visual damage to the elderly.Therefore,looking for a drug that could effective in treating AMD and used to be preprared for suitable dosage form has become a research focus of ophthalmic.In this study,we choose chrysophanol and berberine hydrochloride as the model drugs to prepare ocular dug delivery and explored the preparation of liposomes to improve the bioavailability of berberine hydrochloride and chrysophanol;In addition,PAMAM was choosen as the tareting carrier as it has characteristic as enhanceing the cell permeality、histocompatibility and ect.Morever,the stability test、eye irritation、in vivo permeablity and tropical pharmacokinetics were study to evaluate the stability of the preparation、ocular toxicity、in vivo cornea permeability and drug absorption after administration,proving whether the PAMAM coated compound liposomes was conducive for delivering to the back of eyes.Methods:1.The establishment of HPLC method for determining content and entrapment efficiency of chrysophanol and berberine hydrochloride.Using the HPLC method to explore the chromatographic conditions which could isolated chrysophanol and berberine at the same time.Accoring to the Physical and chemical properties of chrysophanol and berberine hydrochloride,the method to evaluate encapsulation efficiency was investigated by the gel column、methodpetroleum ether method、protamine method and gel column centrifugation method in order to screen out the best method of entrapment efficiency which can be accurately measured the content of chrysophanol and berberine and operated conveniently.2.The study of compound liposome preparation and the technology research.Using the entrapment efficiency as the index to investige thin film dispersion and film ultrasonic method in order to determine the method of preparation of chrysophanol liposomes.Then the compound liposomes was prepared by thin film dispersion–p H-ultrasonic method and using the single factors method to investigate the ratio of drug to phospholipid and the prescription as the ratio of Lecithin to cholesterol based on entrapment efficiency and representative image.Moreover,with entrapment efficiency as evaluation index to optimize the preparation techniques as hydration time、p H and incubation time.3.The preparation and characterization of PAMAM coated compound liposomesPAMAM was labeled with fluorescein isothiocyanate(FITC)and then establish the fluorescence spectrophotometry to determine the coverage of FITC-PAMAM coated compound liposomes as FITC-PAMAM has good fluorescence characteristic.The chemical structure of FITC-PAMAM was confirmed by 1H-NMR.Then the molar ratio of PAMAM to Lecithin of 0.5%、0.1%、0.2%was investiged with the size、zeta potential and fluorescence intensity as evaluation index.Through using scanning electron microscope,oberve the morphology to confirm whether PAMAM cover the surface of compound liposomes successfully.Then,using the best ratio of PAMAM to Lecithin to prepare PAMAM coated compounbd liposomes and evaluate the its quality.With the size、the entrapment efficiency of chrysophanol and berberine hydrochloride、clarity as the index to evaluate the stability of PAMAM coated compound liposomes under the low temperature(4℃)and room temperature.Meanwhile,PAMAM coated compound liposomes was freezed and redissolved by normal saline.And then the liposomes suspension was investigated with size、the entrapment efficiency of chrysophanol and berberine hydrochloride as index.4.The evaluation of ocular irritation and in vivo permeabilityNew zealand rabbit was choosen as the experiment animal and then divided into three groups randomly.Each group was administrated into blank liposomes、compound liposomes and PAMAM coated compound liposomes and preformed three time once day.Then,the ocular irritation test for single dose was evaluated via the standard criteria of Draize ocular irritation test;Meanwhile,the long-term ocular irritation test of PAMAM coated compound liposomes was study by standard criteria and histopathological sections,which operated as irritation test of single dose;Through stained with Nile red to make formulations with the fluorescence characteristics and then rabbits were divided into each groups.After instilled the stained formulations in 15min、30min and 1h,the rabbit was sacrified;Then the cornea was obtained to make sections and observed the fluorescence distribution on the sections by the inverted fluorescence microscope to study penetration of different formulations.5.The establishment of recovery rate of chrysophanol and berberine hydrochlorideThe relatively recovery rate and relatively loss rate were study though using dialysis and retrodialysis with 30%ethanol saline as perfusion fluid,Meanwhile,the vivo recovery rate was investiged by retrodialysis method.6.The establishment of LC-MS-MS method for determining content of berberine hydrochloride in anterior chamber of eyeThough using the LC-MS-MS method to determine the concentration of Berberine hydrochloride and investiged the determining method as the HPLC-MS-MS could detect the concentration of Berberine hydrochloride in anterior chamber of eye and have high sensitivity.7.Tropical pharmacokinetics of New Nealand Rabbits that treated with chrysophanol and berberine hydrochloride aqueous solution、compound liposomes and PAMAM coated compound liposomes.twelve rabbits were randomly divided into three groups and the right eye of each group were as control and the left eyes were received a single dose instillation of 0.5%CMC-Na Chr-Bbr solutuion、CBLs and P-CBLs,respectively.Then 50μl of the formulations were adminstered into the lower conjunctival sac of right eyes in each rabbit with micropiopette.Samples(30μl)were collected at 20 min for 6h after instilled the formulations with microdialysis technology.All samples were stored at-20℃and analyzed by HPLC-MS-MS.Then the data were used to fit the concentration-time curve fited the concentration-time and analyzed by DAS software and to calculate pharmacokinetic parameters of each formulations.Results1.The chromatographic condition that could isolate chrysophanol and berberinehydrochloride was 0.1%phosphoric acid-mathanol as mobile phase with gradient elute;Morever,the entrapment efficiency of Chrysophanol was measured by protamine and sephadex chromatography used for Berberine Hydrochloride.2.The preparation technology of compound liposomes was prepared by two steps:First,the ASPC:chol:Chrysophanol(80:20:1,mass ratio)、DL-α-Tocopherol、Octadecylamine were dissolved in Anhydrous ethanol and then evaporated by rotary evaporator(Yamato,Japan)at 60℃under reduced pressure to produce the thin film.Then the thin film was dried at room temperature for 2 hours and then hydrated with 10 ml sodium citrate buffer(p H3.47)at 40℃to phase transition.The Chrysophanol liposome suspepensions were sonicated at 200 w for 3.3min and then filered by using 0.45μm、0.22μm microporus membrane filter to achive the desired size.Next the sorted Chrysophanol liposomes were added into 0.4mg/ml Berberine aquoue solution at the volume mass1:1.(ASPC:BBR=25:1,mass ratio),then the mixture were adjusted p H to 7.0.The p H adjusted mixture were incubation at water bath 60℃for 20min to produce the Chrysophanol-Berberine Hydrochloride compound liposomes.The compound liposomes were obtained and the entrapment efficiency of chrysophanol was 51.59±3.82 and 89.6±3.63of berberine hydrochloride,respetively.The liposomses was uniformed shape and the average size was 104.4±17.21 nm.Moreover,the preparation techniques was stable,which made the foundation of PAMAM coated compound liposomes.3.As shown on the ~1H-NMR spetrum,FITC was grafted onto PAMAM successfully.The morphology observed by scanning electron microscope indicated that PAMAM could coated on the surface of liposomes and the average size of PAMAM coated compound liposomes was a little bigger than the uncoated compound liposomes.Morever,the zeta potential of coated compound liposomes was negative and became stronger along with increasing of the molar ratio of Lecithin/PAMAM with coating the surface of liposomes.Considering the index evaluating the molar ratio of Lecithin/PAMAM,we choose the molar ratio of Lecithin/PAMAM:0.2%.The result showed that entrapment efficiency of chrysophanol was 80.69±0.96 and93.99±0.052 of berberine hydrochloride,represtively,indicating PAMAM coulad improve the entrapment efficiency of chrysophanol and berberine hydrochloride as coated on the surface of liposomesThe results showed that no obvious change of appearance and the change of size was found in different conditions at room temperature.Howere,the size was increased after 20 days in 4℃.The entrapment efficiency of chrysophanol and berberine hydrochloride were declined in both storage conditions,especially the entrapment efficiency of chrysophanol was the worse and chrysophanol could not detected in30days.In addition,The leakage of berberine hydrochloride was declined slowly when istoring in 4℃but worse in room temperature.However,Compared with the non-freeze liposomes,the size of liposomes that freezed and redissolved with normal saline was a little bigger and the entrapment efficiency of chrysophanol was essentially equal though the entrapment efficiency of berberine hydrochloride was little declined.4.It was no obvious ocular differences compared with the normal eyes after instilled at regular time for a day.Tissue were all normal,with no evidence of damage、injury in all formulation.no obvious injury and abnormal were observed in cornea、iris and conjunctiva after administrating P-CBLs for 14 days.All the formulations stained with Nile Red could permeate into cornea endothelium after administrating for 15 mins.However,the PAMAM coated liposomes and compound liposomes could well distribute into cornea endothelium after 30mins and1h and it can be seen that PAMAM coated liposomes have higher intensity in corneal layer and well-dispered than compound liposomes though0.5%CMC-Na solution had no significant proliferation.5.As flow rate increases,the probe rate was reduced;The probe rate and loss rate of Chrysophanol and berberine hydrochloride were essentially equal by investaged by dialysis method and retrodialysis method.Moever,the result of in vivo stability test,which was investigated by retrodialysis method showed that probe rate was stable in 7 h though fluctuation was occurred in period of time.6.Using LC-MS-MS to establish the method of determining content of berberine hydrochloride in aquaeous humour and the result showed that the method was good、simple and accurate,which meet the requirements of sample analysis.7.The area under the curve of compound liposomes and PAMAM-coated liposomes was 1.33 and 1.643 times that of 0.5%CMC-Na solution and the maximum concentration of compound liposomes and PAMAM-coated liposomes were1.719 and1.23 times that of 0.5%CMC-Na solution.Moreover,the peak time of compound liposomes and PAMAM-coated liposomes was later than 0.5%CMC-Na solution,which was proved that PAMAM-coated compound liposomes could promote corneal permeability and prolong the corneal retention time.Conclusion:the methods for determining content and entrapment efficiency of Chrysophanol and Berberine hydrochloride were established successfully;Using the single factor method to obtain the best prescription and technology of compound liposomes,which provide the foundation of preparation technology of PAMAM compound liposomes;PAMAM compound liposomes that produced by instillation method were stable when storing at low temperature;Morever,It could improve stability of PAMAM compound liposomes by lyophilization,No obvious ocular irritation was caused by PAMAM coated compoundliposomes,which proved that the preparation was safe.PAMAM coated compound liposomes had good permeability and PAMAM coated compound liposomes could improve drug availability and drug penetration,providng the basis that the preparation is conducive for delivering to the back of eyes.