Preparation And Study Of The Proliposomes Of Pig Pulmonary Surfactant

Pulmonary surfactant (PS) is a lipoprotein complex mainly consisting of phospholipids and specific proteins. Downregulation of PS plays a important role in the development of respiratory distress syndrome (RDS), resulting in high morbidity and mortality, particularly in preterm infants. Administration of exogenous PS is a effective method for treating RDS and also used in other pulmonary diseases.At present, there is only Two kinds of PS preparation, the first one is Curosurf, manufactured by Kaisi Pharmacy Company in Italy, was approved to treat neonatal respiratory distress syndrome (NRDS) in our country. The price of it is very high, 4500 RMB per ampoule. The other PS reagents that developed by other domestic institutes are just get the certificate of new drug from SFDA in Nov, 2005. The price of it is 3750 RMB per ampoule. Proliposomes of pig pulmonary surfactant (PPPS) studied in this project is a new granules PS reagent. It is suitable for mass product and easy to transport and store. Moreover, because of the relatively low cost of manufacture, the price of PPPS will be only about 1500 RMB per ampoule, much lower than that of the above PS preparation. It can act as carrier of some drugs (ampule of Dexamethasone , ribavirin and penicillin, etc) administed by lung which maybe have some trearment value on the model of infective respiratory failure .The premature infants account for about 2% of the 20 millions newborns in our country every year, accordingly, there are tens of thousands of newborns suffering from NRDS should be received PS treatment. Therefore, the economic returns and social effects of PPPS are promising.Part 1 Purification of Pig Pulmonary Surfactant by Supereritical Carbon Dioxide ExtractionObjective To purify pig pulmonary surfactant(PPS) and calculate percentage of the purified PPS.Methods1 Preparation of the crude PPS: The crude PPS was obtained from bronchoaveloar lavage fluid(BALF), this method including a few steps: lavage, centrifugation of BALF(4℃, 1000g, 15min), centrifugation of the supernate(4℃, 13000g, 60min).2 Purification of the crude PPS: The 1g crude PPS was dissolved in mixed solution(dehydrated alcohol/ethylether is 1:3, v:v), then mixed with 1.5g diatomaceous earth(crude PPS/diatomaceous earth is 1:1.5, w:w). Putting the above mixture in the extractor, The crude PPS was extracted and separated by supercritical carbon dioxide extraction(SFE-CO₂). The best condition to extract and separate is pressure(25-30Mpa), temperature (35-40℃), static extraction 20 minute, dynamic extraction 4 hour. Finally the mixture is removed from the extractor and filtered with suction to separate diatomaceous earth, then be dried by nitrogen gas. The purified PPS is pallide-flavens and viscous, stored at temperature -20℃in nitrogen gas.ResultsWe purified 106.532g crude PPS in total. After extraction of SFE—CO₂, the purified PPS is pallide-flavens and viscous, weighing 60.316g totally, percentage of the purified PPS is 30.521±1.700(%). The result of this experiment has proved that the method that SFE is used to purify PPS, has a simple process of operation and a high efficiency of extraction. And the factors affecting the efficiency of extraction, such as pressure, temperature and so on, are controlled easily in the process. At the same time, the validity and reproducibility of choosing the extraction conditions by ZhaoQian for SFE is verified.Conclusions1 There are many advantages in the method using the technology of SFE-CO₂ to purify PS, such as an efficiency of extraction; a simple process of operation; a easy control of the factors affecting the efficiency of extraction, such as pressure, temperature; a little or no use of organic solvent; a pure extraction solvent of gas, and little possibility of absorbing impurity; a low temperature for extraction, benefiting the separation and extraction of thermal unstable compound. The method is significant in this field. In this paper, percentage of the purified PPS is 30.521±1.283(%).2 There are many factors affecting SFE-CO₂, such as pressure, temperature, dynamic time of extraction, the category and quantity of modifiers, dynamic volume of extraction, static time of extraction, the first three factors among which have the largest effect. The best condition of extraction for purifying PPS by SFE is as follows: pressure(25-30Mpa), temperature (35-40℃), static extraction 20 minute, dynamic extraction 4 hour. Part 2 Preparation of the Proliposomes of pig pulmonary surfactantObjective To prepare PPPS by revolving evaporating apparatus, and discuss and study the method and process of preparation of PPPS by Single Factor Test and Orthogonal Experiment.Methods1 The preparation method of PPPS: using revolving evaporating apparatus.2 Single Factor Test and Orthogonal Experiment: test individually the factors, such as the sieve aperture of steel meshes, the dosage of PPS, the seeding rate, the temperature of water bath, the location of capillary column, the velocity and inclination angle of revolving evaporating apparatus. Then according to the results of pre-experiment and Single Factor Test, the factors and range are fixed and the experiment can be carried out.3 Analysis the above data by SPSS13.0;Significance level is 0.05.ResultsIn Single Factor Test, the output rate of PPPS is all above 90% when the dosage of PPS(g) is between 1.6-1.8, while it decrease obviously when the dosage exceed 2.0g; It decrease along with increase of the seeding rate of PPS, especially when the seeding rate exceed 1.1ml/5min. The Orthogonal ANOVA indicate that there are statistical significance between output rate of PPPS in each the dosage of PPS (P= 0.004), and in each seeding rate of PPS (P=0.029), but not in each temperature (P =0.549). The degree in which the factors affect the output rate is : dosage＞seeding rate＞temperature. The best conditions of preparation of PPPS is as follows: dosage (1.8g), seeding rate (0.9ml/Smin), temperature of water bath (44℃). Conclusions1 In the process of preparation of PPPS, it is suitable for the grain amount of sodium chloride crystalloid (1.8g), the dosage of PPS (1.8g), and the seeding rate in the range of 0.8 to 0.9ml/5min. The best conditions of preparation are as follows: the dosage of PPS (1.8g), the feeding rate of PPS (0.9ml/5min), the temperature of water bath (44℃).2 In the process of preparation of PPPS, the degree in which the factors affect the output rate is: dosage＞seeding rate＞temperature.Part 3 chemical analysis of the proliposomes of pig pulmonary surfaetantObjective To analyze and compare the chemical component of PPPS and other PS preparation.Methods1 Mensuration of all components of PPPS: Method of malachite green is applied to measure the amount of total phosphatide; Method of Coomassie orchid is used to test the quantity of protein; Methods of assaying cholesterol and adtriglyeride are operated according to the introduction of test kit.2 Analysis of all component of phosphatide in PPPS: using high efficiency liquid chromatography.3 Analysis the above data by SPSS13.0; Significance level is 0.05.Results1 The amount of all chemical components of PPPS output is as follows: phosphatide (91.28±1.77)%, hypertriglyceridemia (0.76±0.14)%, cholesterin (0.98±0.14)%, protein (1.72±0.18)%. Compared to the relevant literature in the country and abroad, the content of hypertriglyceridemia, cholesterin and protein is low, but the proportion of phosphatide is more than 90%, which prove that the amount of phosphatide of PPPS output conforms the requirement of PS preparation.2 The proportion of all phosphatide component of PPPS is as follows: phosphatidyl choline (80.5±2.32%), phosphatidyl glycerol (7.48±0.80%), phosphatidyl ethanolamine (3.80±0.76%), phosphatidylinositol (2.98±0.85%), sphingomyelin (2.264±1.0%), phosphatidyl serine (1.69±0.58%). Compared to the relevant literature in the country and abroad, the proportion of phosphatidyl choline is about 80.5%, which is relatively high and abundant in kinds of phosphatide component. This shows that in the aspect of chemical components, the preparation of PPPS has met the command of PS preparation.ConclusionsThe amount of all chemical components and the proportion of all phosphatide ingredients in PPPS output conform the requirements of PS preparation.Part 4 Physical chemistry quality of Purifying pig pulmonary surfactantObjective To study PPPS' physical chemistry characters.Methods1 Mensurate and analyze PPPS' physical chemistry characters by the precise acidity indicator, the optical microscope, the transmission electron microscope, viscometer and so on.2 Analysis the above data by SPSS13.0; Significance level is 0.05. Results1 The appearance of PPPS is light yellow, loosen, and grain. The zero angle of PPPS is 34'C, inactive in fluid. If it is laid in different temperature, there is more changes in color and redispersion. When the temperature is above 20℃, the color of PPPS shows yellow, viscosity of PPPS is increasing in shot time. There are statistical significance between the time of color(x²=16.773, P= 0.001) and viscosity (x²=17.703, P=0.001) of PPPS changing in each group(4-8℃,20℃,40℃). There are statistical significance between the peroxide value of the each day within each temperature (4-8℃,20℃,40℃) (F =4584.027, P＜0.001). There are statistical significance between the peroxide value of the each day between each temperature (F=3091.756, P＜0.001). There are statistical significance between the peroxide value of the each day on the light and in 20℃.2 The liposomes were formed after PPPS has been watered by the the injection water. The diameter of liposomes particle is between 100-900nm. showing the shape of spheroid and vesicle. The pH of PPPS is 6.361±0.032. The viscosity value of PPPS is 1.36±0.12 mPas.ConclusionsPPPS possesses physical chemistry characters of liposomes and has met the command of PS preparation. It should be stored in low temperature and away from light. Part 5 Evaluation of PPPS' surface activity character in vitroObjective To survey and evaluate surface activity in vitro of PPPS.Methods1 To survey and evaluate PPPS' surfactant character by the kinetic membrane tension determinator of JM99A.2 Analysis the above data by SPSS13.0; Significance level is 0.05.ResultsThe indexes describing the characters of PPPS which depress the ability of surface tension and the stability of surface membrane are as following: the minimal surface tension r_min=5.40±1.36mN/m, the maximal surface tension r_max=34.56±3.05mN/m, stationary index SI=1.62±0.37, resilient index RI=41.87±8.12.PPPS can adsorb and expand automatically and speedily on the interface of gas to liquid then come into single-molecule velum by themselves. As the surface is compressed, the surface tension is decreased to less than 10mN/m, SI also meets the demand. It is proved that PPPS has a good surface activity in vitro.ConclusionsWhen PPPS has been watered by injection water, it has PS surface activity characters which accord with the requirements for substitutes preparation of exogenous PS. Part 6 Effects of PPPS on the model of the respiratory disdress syndrome in vivo lung-lavaged rabbitsObjective To observe effects of PPPS on the model of the respiratory disdress syndrome in vivo lung-lavaged rabbits.MethodsAfter preparing the the model of the respiratory disdress syndrome in vivo lung-lavaged rabbits, drop the PPPS (which has been watered by the injection water) into the lung of above model through the trachea. Then observe these indexes, such as the arteri blood gas, heart rate(HR), breathing rate(BR), systolic blood pressure (SBP), diastolic blood pressure (DBP), tidal volume (TV) and dynamic compliance (Cdyn) of the lung-lavaged rabbits at the experimental period, as well as the pneumonic pathology changes. Then Analysis the above data of Repeated Measures by ANOVA of SPSS13.0. Significance level is 0.05.ResultsIt exists the following phenomena: the Ph,PaO₂ of the model rabbits descending sharply, the PaCO₂ of the model rabbits increasing lightly, the HR of the model rabbits slowing down obviously but the BR of the model rabbits increasing, the SBP and DBP of the model rabbits descending markedly, and the PS content decreasing dramatically. The pneumonic pathology changes show pneumorrhagia, atelectasis, shrink of alveolus and pulmonary hyaline membrane. The above results indicate that it accords with the standard ofâ… -type of acute respiratory distress syndrome (ARDS). After applying the PPPS to the model rabbits, each index of it nearly reach the normal level, and pneumonic pathology changes are also alleviated distinctly, the analytic result of statistics indicate that there are statistical significance in pH (F=4.572, P =0.046),PaO₂ (F=557.586, P＜0.001),PaCO₂ (F=96.798, P＜0.001),TV (F=117.851, P＜0.001),Cydn (F值=276.996, P＜0.001),HR (F值=34.975, P＜0.001),BR (F=28.167, P＜0.001),SBP (F=124.751, P＜0.001) and DBP (F=108.672, P＜0.001) between control group and treated group. This shows PPPS has a great treatment effect on the model of the respiratory disdress syndrome in vivo lung-lavaged rabbits.ConclusionsPPPS has a great treatment effect on the model of the respiratory disdress syndrome in vivo lung-lavaged rabbits.Summary1 In this paper, the technology of SFE-CO₂ is applied to purify pig pulmonary surfactant, and the output of PPS with a high phosphatide is obtained. The best conditions of extraction to purify PPS by SFE are as follows: pressure (25-30Mpa), temperature (35-40℃), extraction time (static extraction by SFE-CO₂ for 20 minutes SFE-CO₂, and then dynamic extraction for 4 hours).2 We prepare PPPS by revolving evaporating apparatus, and study the method and process of preparation of PPPS by Single Factor Test and Orthogonal Experiment. The best conditions of preparation are as follows: the dosage of PPS (1.8g), the feeding rate of PPS (0.6ml/5min), the temperature of water bath (44℃).3 We study the chemical components of PPPS preparation and all ingredients of phosphatide. The amount of all chemical components of PPPS output is as follows: phosphatide (91.28±1.77) %, hypertriglyceridemia (0.76±0.14)%, cholesterin (0.98±0.14)%, protein (1.72±0.18)%. The proportion of all phosphatide component of PPPS is as follows: phosphatidyl choline (80.5±2.32%), phosphatidyl glycerol (7.48±0.80%), phosphatidyl ethanolamine (3.80±0.76%), phosphatidylinositol (2.98±0.85%), sphingomyelin (2.26±1.0%), phosphatidyl serine (1.69±0.58%). We survey and evaluate surface activity in vitro of PPPS. All of the above prove that the chemical components of PPPS preparation and all ingredients of phosphatide conform to the requirements of PS preparation.4 We analyse physical chemistry quality of PPPS. The appearance of PPPS is light yellow, loosen, and grain. If it is laid in different temperature, there is more changes in color and redispersion. When the temperature is above 20℃, the color of PPPS shows yellow, viscosity of PPPS is increasing in shot time. The light rays have a great effect on peroxide value of PPPS. In temperature(4-8℃,20℃,40℃), the peroxide value rises up obviously with the time increasing. Therefore, it should be stored in low temperature and away from light. The liposomes were formed after PPPS has been watered by the water for injection. The diameter of liposomes particle is between 100—900nm. The shape of liposomes show spheroid and ves. The pH of PPPS is 6.361±0.032. The viscosity value of PPPS is 1.36±0.12 mPas.5 The indexes describing the characters of PPPS which depress the ability of surface tension and the stability of surface membrane are as following: the minimal surface tension r_min=5.40±1.36mN/m, the maximal surface tension r_max =34.56±3.05mN/m, stationary index SI=1.62±0.37, resilient index RI=41.87±8.12. PPPS can adsorb and expand automatically and speedily on the interface of gas to liquid then come into single-molecule velum by themselves. As the surface is compressed, the surface tension is decreased to less than 10mN/m, SI also meets the demand. It is proved that PPPS has a good surface activity in vitro. 6 In this paper, the model of the respiratory disdress syndrome in vivo lung-lavaged rabbits is constructed. And then these indexes related to the model are observed, such as the arteri blood gas, HR, BR, SBP, DBP, TV and Cdyn of the lung-lavaged rabbits at the experimental period, as well as the pneumonic pathology changes. We find that PPPS has a great treatment effect on the model of the respiratory disdress syndrome in vivo lung-lavaged rabbits.