Journal of Shenyang Pharmaceutical University

29 12 2 0 1 2 1 2 Journal of Shenyang Pharmaceutical University Vol. 29 No. 12 Dec. 2012 p. 917 1006-2858 2012 12-0917-06 PEG HeLa 1 1* 2 1 1 1 1. 264005 2. 264005 - - monomethoxy polyethylene glycol-polyactic coglycolic acid-nanoparticles mpeg-plga-nps HeLa mpeg-plga-nps mpeg-plga-nps zeta mpeg-plga-nps -6 coumarin 6 HPLC HeLa mpeg-plga-nps 106. 2 ± 4. 3 nm Zeta - 12. 40 ± 0. 11 mv PLGA- NPs 35. 5% mpeg-plga-nps 92. 5% PLGA-NPs 3 HeLa mpeg-plga-nps PLGA-NPs 2 HeLa PLGA-NPs mpeg-plga-nps mpeg- PLGA-NPs HeLa mpeg-plga-nps PLGA-NPs P < 0. 01 - - HeLa R 94 A human papilloma virus HPV mucoadhesive polyethylene glycol PEG mucus penetrating particles MPP 3-6 - polyactic-coglycolic acid PLGA PEG PLGA 1 nanoparticles NPs PLGA-NPs mpeg-plga-nps 7-6 coumarin 6 PLGA-NPs mpeg-plga-nps HeLa 2 2012-06-25 30973949 ZR2009CM012 1988- E-mail qlf _hi@ 126. com * 1954- Tel. 0535-6706022 E-mail ldh@ ytu. edu. cn

918 29 1 Agilent 1100 Agilent Nicomp 380ZLS PSS A 5 g L - 1 NICOMP JY92-Ⅱ 10 g L - 1 B 20 g L - 1 S-4800 4 g L - 1 1 50 Olympus FV1000 1 C PM 50 100 200 300 71 Olympus B 51 400 500 mg L - 1 C Nikon Mircro 21R - 20 min Thermo Forma Series II 750 nm PM Thermo Synergy HT 1 d BioTek Nalge Nunc 2. 3 SEM PLGA-NPs mpeg-plga-nps 5% CO 2 37 50 40 S-4800 SEM 3 10 4 96 2. 4 2. 4. 1 - PM 3 3 d - PLGA Mr = 2. 4. 2 18 000 m LA m GA = 75 25 PLGA-NPs mpeg-plga-nps 1 ml - - mpeg-plga 1 g L - 1 PM 1 mg PBS 1 mpeg 2 000 ml 37 1 h 4 - pig gastric mucin PM -6 Sigma RPMI Medium 1640 GiBco MTT 40 000 r min - 1 30 min - PM 1 PM % = PM PM HeLa 100% 1 2. 5 2 1 ml 1 g L - 1 2 ml 2. 1 mpeg-plga 25 mg 2 ml ph 7. 4 37 60 r min - 1 0 1 2 4 6 8 12 24 h 30 ml 1 ml 14 000 r min - 1 4 1 h 0. 2% 60 HPLC mpeg-plga-nps PLGA-NPs -6 0. 1 mg -6-6 % = 2. 2-6 -6 100% 2 mpeg-plga-nps PLGA-NPs 2. 6 NICOMP 380 ZLS 2. 6. 1 HeLa Zeta MTT assay -6 HeLa HeLa 10% RPMI 1640 24 h PLGA-NPs mpeg-

12 PEG HeLa 919 PLGA-NPs -6 0. 4 mlpbs 2 5 10 20 25 40 50 100 μl HPLC 25 50 100 125 200 250 10 g L - 1 10 μl 4 h 96 BSA dimethyl sulfoxide DMSO 150 μl 10 min BSA 0. 625 1. 250 1. 875 2. 500 490 nm 3. 125 3. 750 5. 000 μg 10 μl CBB 200 μl % = 5 min 595 100% 3 nm BSA 2. 6. 2 HeLa HeLa 2 10 5 200 μl Lab - Tek 24 13 000 r min - 1 5 min 96 24 h 5 μl -6 5 μl CBB 200 μl PLGA-NPs mpeg-plga-nps 100 μl 5 min 595 nm 100 mg L - 1 C6 0. 3 mg L - 1 1 2 4 6 h HeLa PBS 3 4% 30 min % = - 6 488 /520 nm / HeLa 100% 4 2. 6. 3 HeLa 2. 6. 2 HeLa 24 h3. 1 zeta -6-6 PLGA-NPs mpeg-plga-nps 200 μl PLGA-NPs mpeg-pl- 200 mg L - 1 C6 GA-NPs zeta -6 0. 6 mg L - 1 0. 5 1. 0 2. 0 4. 0 6. 0 h -6 500 mg L - 1 3 Bradford 3 24 h MTT 1. 25 2. 50 3. 75 5. 00 6. 25 7. 50 10 μl 10 μl 3 1 PBS 3 0. 2 ml Table 1 Physico-chemical properties and PM anti-adhesive rate of nanoparticles n = 3 珔 x ± s 1 PM n = 3 珔 x ± s Formulation d /nm Polydispersity index V zeta /mv EE /% PM anti-adhesive rate /% PLGA-NPs 135. 0 ± 5. 25 0. 112 ± 0. 001-25. 79 ± 0. 28 74. 53 ± 1. 83 35. 5 ± 1. 9 mpeg-plga-nps 106. 2 ± 4. 3 0. 094 ± 0. 002-12. 40 ± 0. 11 80. 65 ± 2. 62 92. 5 ± 1. 4 mpeg-plga-nps mpeg-plga-nps zeta PLGA-NPs PEG 10 mv mpeg-plga PEG -6 > 70%

920 29 PLGA-NPs mpeg-plga-nps 3. 2 scanning electron microscope SEM SEM mpeg-plga-nps 1 mpeg-plga-nps 100 nm Nicomp 380ZLS 3. 5 HeLa 24 h -6 < 12% 6 h < 8% -6 MTT PLGA-NPs mpeg-plga-nps -6 HeLa 24 h 3 Fig. 1 1 SEM images of mpeg-plga nanoparticles mpeg-plga SEM 3. 3 PM 50 ~ 500 mg L - 1 Fig. 3 A = 0. 000 4ρ + 0. 004 5 r 2 = 0. 999 4 PLGA-NPS mpeg-plga-nps Fig. 2 In vitro release of coumarin-6 loaded PLGA and PEG-PLGA nanoparticles n = 3 珔 x ± s 2-6 PLGA-NPs mpeg-plga-nps n = 3 珔 x ± s In vitro inhibition of PLGA and mpeg-plga nanoparticles in HeLa cells n = 3 珔 x ± s RSD 0. 5% 3 PLGA-NPs mpeg-plga-nps HeLa RSD 1. 1% n = 3 珔 x ± s 1 PLGA- HeLa NPs PM 35. 5% mpeg-pl- 80% GA-NPs PM 92. 5% PLGA-NPs 3 mpeg-plga-nps 200 mg L - 1 HeLa 24 h > 90% 3. 4 HPLC -6 3. 6 HeLa 4 HeLa -6 2 PLGA-NPs 2 h HeLa mpeg-plga-nps HeLa mpeg-plga-nps PL- GA-NPs HeLa 4 h 6 h He- La 3. 7 HeLa BSA 0 ~ 5 μg A = 0. 069 7m + 0. 712 5 r 2 = 0. 998 8

12 PEG HeLa 921 HeLa 5 4 Fig. 4 PLGA-NPs mpeg-plga-nps Fig. 5 Uptake of PLGA and mpeg-plga nanoparticles in HeLa cell line n = 3 珔 x ± s A Coumarin-6 solution B PLGA-NPs C mpeg-plga-nps Confocal images of HeLa cells coincubated withnanoparticles HeLa PLGA-NPs mpeg-plga-nps 5 HeLa PLGA-NPs mpeg-plga-nps n = 3 珔 x ± s 5 HeLa mpeg-plga-nps PLGA-NPs 6 h HeLa PLGA-NPs 1 CU Y BOOTH C J SALTZMAN W M. In vivo distribution of surface-modified PLGA nanoparticles following 2. 49% mpeg-plga-nps 4. 71% PLGA-NPs 2 intravaginal delivery J. J Control Release 2011 156 2 258-264. 2. J. 4 b. mpeg-plga-nps HeLa mpeg- PLGA-NPs PLGA- NPs 11-13 PEG PEG 14 2010 37 6 435-438. a. mpeg-plga-nps 3 LAI S K O'HANLON D E HARROLD S et a1. Rapid PLGA-NPs transport of large polymeric nanoparticles in fresh undiluted human mucus J. Proceedings of National Acade- PEG my of Sciences 2007 104 5 1482-1487. 4 CU Y SALTZMAN W M. Controlled surface modification with poly ethylene glycol enhances diffusion of PL- 10 mpeg-plga-nps GA nanoparticles in human cervical mucus J. Molecu-

922 29 lar Pharmaceutics 2009 6 1 173-181. 5 YONCHEVA K LIZARRAGA E IRACHE J M. Pegylated nanoparticles based on poly methyl vinyl ether-comaleic anhydride preparation and evaluation of their bioadhesive properties J. Eur J Pharm sci 2005 24 411-419. 6 YANG M LAI S K HANES J et al. Biodegardable nanoparticles composed entirely of safe materials that rapidly penetrate human mucus J. Angew Chem Int Ed 2011 50 2597-2600. 7. - 2007 42 5 550-556. J. 2008 43 21 1665-1668. 9. -6 23 7 1553-1561. PLGA J. 2011 28 8 740-744. 10 CONE R A. Barrier properties of mucus J. Adv Drug Deliv Rev 2009 61 2 75-85. 11 PAMUJULA S HAZARI S BOLDEN G. Cellular delivery of PEGylated PLGA nanoparticles J. Journal of Pharmacy and Pharmacology 2012 64 1 61-67. 12. A549 J. 2004 39 11 876-880. 13 SADZUKA Y KISHI K HIROTA S et al. Effect of polyethyleneglycol PEG chain on cell uptake of J. PEG-Modified liposomes J. Journal of Liposome Research 2003 13 2 157-172. 8. 14 ZHANG Y KOHLER N ZHANG M Q. Surface modification of superparamagnetic magnetite nanoparticles and their intracellular uptake J. Biomaterials 2002 Anti-adhesive activity and uptake by HeLa cells of mpeg- PLGA nanoparticles QIN Li-fang 1 LIN Dong-hai 1* LI Gang 2 XIE Xin-xin 1 WANG Jun-teng 1 WEN Zhen 1 1. School of Pharmacy Yantai University Yantai 264005 China 2. School of Life Sciences Yantai University Yantai 264005 China Abstract Objective To prepare mpeg-plga nanoparticles discuss its properties and in vitro anti-adhesive activity and investigate the cellular uptake of them in human cervical cancer cells HeLa. Methods mpeg- PLGA-NPs w ere prepared by a solvent diffusion method. The particle size distribution and Zeta potential of nanoparticles w ere measured by light scattering. The anti-adhesive activity of nanoparticles w as evaluated by pig gastric mucin PM binding experiments. Coumarin 6 was incorporated into nanoparticles as fluorescent marker. The cellular uptake of PLGA-NPs and PEG-PLGA-NPs by HeLa cell lines w ere determined by laser scanning confocal microscope and HPLC. Results The mean size of mpeg-plga-nps was 106. 2 ± 4. 3 nm Zeta potential was - 12. 40 ± 0. 11 mv. The PM anti-adhesive rate of PLGA-NPs and PEG-PLGA- NPs was 35. 5% and 92. 5% respectively. The anti-adhesive activity of mpeg-plga-nps w as three times higher than PLGA-NPs. All nanoparticles showed low cytotoxicity. At the same incubation time the uptake of mpeg-plga-nps was two times higher than that of unmodified PLGA-NPs. The cellular uptake of PL- GA-NPs and mpeg-plga-nps w as dependent on the incubation time. Conclusions mpeg-plga-nps has much higher anti-adhesive activity. The uptake of mpeg-plga-nps by HeLa cells is much higher compared to the PLGA-NPs P < 0. 01. The mpeg-plga-nps shows better cell affinity than PLGA-NPs. Key words mpeg-plga nanoparticle anti-adhesive activity HeLa cell line cellular uptake