% TFA 1. 0 ml rahp-pl- min nm GA AUFS 20 μl LC 20A Eclipse XDB-C mm 250 mm Ag- % = / + ilent ALPHA 2-4 PL

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1 - * 孙海燕, 方科伟, 刘冬制备重组降血压肽 ( rahp, 氨基酸序列为 VLPVPR) 缓释微球以不同单体比和相对分子质量的乳酸 - 羟基乙酸共聚物 ( PLGA) 为缓释材料, 利用正交设计优化微球制备的最佳工艺条件, 并考察微球的体外释药特性乳酸 - 羟基乙酸共聚物相对分子质量越小, 微球粒径越小 ( P < 0. 05), 孔隙率越高 ; 乳酸 - 羟基乙酸共聚物中乳酸比例越大, 微球粒径越大 ( P < 0. 05) ; 微球制备的最优工艺为 : 油相中乳酸 - 羟基乙酸共聚物质量浓度 ρ PLGA 为 12 g 100 ml - 1 初乳搅拌速度 V stir 为 r min - 1 内水相与油相体积比 R w/o 为 , 外水相聚乙烯醇 124( PVA) 质量浓度 ρ PVA 为 4 g 100 ml - 1, 按此工艺制得的重组降血压肽乳酸 - 羟基乙酸共聚物微球包封率在 90% 以上, 载药量 11% 以上, 微球粒径 70 ~ 90 μm; 载药微球在 PBS 缓冲液中 2 h 内的累积释药量在 40% 以内, 乳酸 - 羟基乙酸共聚物相对分子质量越小, 释药速度越快 ( P < 0. 05), 羟基乙酸含量越高释药速度也越快 ( P < 0. 05), 释药曲线符合 Higuchi 方程, 表明微球通过扩散机制进行缓释该微球制备工艺成熟, 包封率高, 具有缓释能力重组降血压肽 ; 聚乳酸 - 聚羟基乙酸 ; 缓释微球 ; 体外释放 R944 A Preparation and in Vitro Release of Recombinant Peptide-PLGA Sustained Release Microspheres SUN Hai-yan FANG Ke-wei LIU Dong * Shenzhen Polytechnic Shenzhen China ABSTRACT OBJECTIVE To prepare recombinant peptide rahp amino acid sequence is VLPVPR PLGA sustained release microspheres by double emulsion solvent evaporation method. METHODS The rahp-plga microspheres preparation process was optimized by orthogonal experiments using PLGA copolymers with different LA / GA ratios or different molecular weights as sustained material and in vitro drug release profiles of the microspheres were also investigated. RESULTS The microspheres were smaller P < and more porous with lower molecular weight of PLGA. The optimal preparation process of rahp-plga microspheres was as follows PLGA concentration was 12 g 100 ml - 1 the stirring rate of the first emulsion was r min - 1 the volume ratio of inner water phase to oil phase was and the PVA concentration was 4 g 100 ml - 1. The encapsulation efficiency of the rahp-plga microspheres prepared by the optimum process was more than 90% the drug loading was above 11% and the average particle diameter was in the range of μm. The cumulative in vitro release percentage in PBS buffer was less than 40% within 2 h the release speed increased P < with lower molecular weight or lower ratio of LA /GA. The drug release curve was fit to Higuchi equation suggesting that rahp released from the microspheres based on diffusion mechanism. CONCLUSION can be prepared easily with good morphology high entrapment efficiency and certain sustained release capacity. KEY WORDS recombinant peptide poly d l-lactide-co-glycolide sustained microsphere in vitro release The rahp-plga microspheres antihypertensive peptide AHP 3 1 re- combinant antihypertensive peptide rahp - PLGA % h 2009B K017BA * Tel liudongsz@ 163. com

2 % TFA 1. 0 ml rahp-pl- min nm GA AUFS 20 μl LC 20A Eclipse XDB-C mm 250 mm Ag- % = / + ilent ALPHA 2-4 PLGA 100% PLGA CHRIST Barnstead Nikon IX71 - A21PH O- lympus S3500 Microtrac S Hitachi IKA SK - 747R 3 50 ml RP- Amerex H550S HPLC rahp VLPVPR 50 mg PLGA 6 1 ml 99% ACN rahp TFA Sigma mg ml ml 3 RP- PVA -HPLC rahp PLGA LA /GA 50 /50 75 /25 X Y Y = X r 2 = n = mg ml - 1 RP-HPLC rahp ρ A rahp ~ 1 mg ml - 1 r 2 = n = 3 A = ρ RP-HPLC rahp VY- DAC 238EV54 C mm 200 mm 5 μm rahp % = - / 100% mg ml 3 ml 3 rahp 1% RP-HPLC LA /GA PLGA rahp rahp 5 min W 1 /O 800 r min ml % = / 100% PVA 3 min W 1 /O /W h rahp 1. 4 rahp Span rahp Span = D 90 - D 10 D 50 D 10 D 50 D 90 10% 50% 90% Span PLGA ρ PLGA V stir

3 R w /o PVA ρ PVA M w LA /GA = 75 /25 M w L ρ PLGA 12 g 100 ml - 1 V stir S 1 S 2 r min - 1 R w /o 1 /7. 5 PVA ρ PVA 4 g 100 ml - 1 S 3 S S 1 S 2 S 3 3 PVA > S > PLGA > S 1 S 2 S = 3 槡 S mg 10 ml 5 ml ph % min h r min min100 μl RP-HPLC rahp PLGA 100 μl Tab. 2 spheres LA /GA PL- PLGA rahp 3 ~ 6 LA / GA = 50 /50 A 3 B 2 C 2 D 2 PLGA PVA PVA PLGA 2. 2 rahp-plga GA % 11% 85% 1 rahp L ~ 90 μm Tab. 1 Factors and levels of orthogonal experiments LA/GA P <0. 05 Factors Levels ρ PLGA / g 100 ml - 1 V stir /r min - 1 R w /o ρ PVA / g 100 ml - 1 P >0. 05 LA/GA /5 2 LA P < / /10 6 P > The orthogonal design table for preparation of micro- rahp Factors ρ PLGA A / g 100 ml - 1 V stir B /r min - 1 R w /o C ρ PVA D / g 100 ml - 1 Experiment /5 2 Experiment / Experiment /10 6 Experiment / Experiment /10 2 Experiment /5 4 Experiment /10 4 Experiment /5 6 Experiment / PLGA LA / GA = 50 /50 M w rahp. n = 3 x 珋 ± s Tab. 3 The results of orthogonal test for microspheres preparation LA /GA = 50 /50 M w n = 3 x 珋 ± s No. S 1 /% S 2 /% S 3 S ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

4 4 rahp LA /GA = 1 50 /50 M w Tab. 4 The intuitive analysis table of the results of orthogonal PLGA test LA / GA = 50 /50 M w Line A ρ PLGA B V stir C R w /o D ρ PVA Experiment Experiment Experiment Experiment Experiment Experiment Experiment Experiment Experiment K K K R A 3 B 2 C 2 D 2 5 S PLGA LA /GA = 75 /25 M w rahp 79. 7% 72 h 144 h. n = 3 x 珋 ± s Tab. 5 The results of orthogonal test for microspheres preparation LA /GA = 75 /25 M w n = 3 x 珋 ± s No. S 1 /% S 2 /% S 3 S ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± R A 3 B 2 C 2 D 2 LA /GA = 50 / 50 1 M w M w M w LA /GA = 75 /25 PLGA 2. 3 rahp-plga rahp-pl- GA 2 2 h rahp 2 h 20. 1% ~ 35. 6% h 50. 9% ~ 71. 2% 55. 7% ~ 76. 3% 59. 1% ~ 66. 2% ~ 82. 4% LA /GA P < LA /GA GA P < PLGA GA Higuchi 3 r MSE 6 rahp LA /GA Higuchi 75 /25 M w Tab. 6 The intuitive analysis table of the results of orthogonal test LA /GA = 75 /25 M w A B C D Line S PLGA ρ PLGA V stir R w /o ρ PVA Experiment LA /GA Experiment PLGA Experiment Experiment Experiment % 11% 85% Experiment Experiment ~ 90 μm Experiment LA /GA Experiment K PLGA K K GA PLGA

5 7 Tab. 7 LA /GA rahp-plga. n = 3 x 珋 ± s Characteristics of microspheres prepared by different LA /GA ratios and molecular weights. n = 3 珋 x ± s Groups Encapsulation efficiency /% Yield rate /% Drug loading rate /% Average particle diamete /μm Span LA /GA = 50 /50 M w ± ± ± ± ± LA /GA = 50 /50 M w ± ± ± ± ± LA /GA = 50 /50 M w ± ± ± ± ± LA /GA = 75 /25 M w ± ± ± ± ± LA /GA = 75 /25 M w ± ± ± ± ± LA /GA = 75 /25 M w ± ± ± ± ± LA /GA = 50 / PLGA 2 h 35. 6% 24 h 71. 2% LA /GA = 75 / h 20. 1% 144 h 66. 2% 8 REFERENCES 1 LUSCHER T F YANG Z. Calcium antagonists and ACE inhibitors effect on endothelium and vascular smooth muscle J. Drugs suppl LIU D SUN H Y ZHANG L J et al. High-level expression of milkderived antihypertensive peptide in Escherichia coli and its bioactivity J. J Agric Food Chem SUN H Y FANG K W LIU D et al. The acute antihypersentive effects of recombinant angiotension converting enzyme inhibitory peptides on spontaneously hypertensive rats J. Chin J Hyper LEI L SUN H Y LIU D et al. Transport of Val-Leu-Pro-Val- Pro in human intestinal epithelial Caco-2 cell monolayers J. J Agric Food Chem HAIYAN S DONG L SHIMIN L et al. Transepithelial transport characteristics of the antihypertensive peptide Lys-Val-Leu-Pro- Val-Pro in human intestinal Caco-2 cell monolayers J. Biosci Biotechnol Biochem FANG K W SUN H Y LIU D et al. Stabilities and antihypertension efect of highly expressed anti-hypertensive peptide by genetic engineering J. J Clin Rehabil Tiss Eng Res WEI G LU L F CHEN W et al. Biodegradable microspheres loaded with recombinant human growth hormone. Ⅰ. Study on preparation and in vitro release J. J Chin Med Ind Ch. P 2012 Vol Ⅱ S

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g 16 6 2018 11 Chinese Journal of Bioprocess Engineering Vol. 16 No. 6 Nov. 2018 doi 10. 3969 /j. issn. 1672-3678. 2018. 06. 004 1 2 1 1 1. 330200 2. 330006 4 Box-Behnken 1 25 g /ml 121 92 min 2 6. 284%