title: pH-sensitive degradable nanoparticles for highly efficient intracellular delivery of exogenous protein.
abstract: BACKGROUND
  Encapsulating exogenous proteins into a nanosized particulate system for delivery into cells is a great challenge. To address this issue, we developed a novel nanoparticle delivery method that differs from the nanoparticles reported to date because its core was composed of cross-linked dextran glassy nanoparticles which had pH in endosome-responsive environment and the protein was loaded in the core of cross-linked dextran glassy nanoparticles.
  
  
  METHODS
  In this study, dextran in a poly(ethylene glycol) aqueous two-phase system created a different chemical environment in which proteins were encapsulated very efficiently (84.3% and 89.6% for enhanced green fluorescent protein and bovine serum albumin, respectively) by thermodynamically favored partition. The structures of the nanoparticles were confirmed by confocal laser scanning microscopy and scanning electron microscopy.
  
  
  RESULTS
  The nanoparticles had a normal size distribution and a mean diameter of 186 nm. MTT assays showed that the nanoparticles were nontoxic up to a concentration of 2000 μg/mL in human hepatocarcinoma cell line SMMC-7721, HeLa, and BRL-3A cells. Of note, confocal laser scanning microscopy studies showed that nanoparticles loaded with fluorescein isothiocyanate-bovine serum albumin were efficiently delivered and released proteins into the cytoplasm of HeLa cells. Flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assays showed that nanoparticles with a functional protein (apoptin) efficiently induced significant tumor cell apoptosis, which was confirmed by DAPI staining.
  
  
  CONCLUSIONS
  Our findings indicate that these nanoparticles meet the high demands for delivering protein medicines and have great potential in protein therapy.
authors.affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Xu
authors.forename: Fei
authors.initials: F
authors.lastname: Wu
authors.forename: Yinghui
authors.initials: Y
authors.lastname: Chen
authors.forename: Liangming
authors.initials: L
authors.lastname: Wei
authors.forename: Weien
authors.initials: W
authors.lastname: Yuan
mesh_terms: D003692:Delayed-Action Preparations / Q000008:administration & dosage / Q000737:chemistry*; D011992:Endosomes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D053769:Nanocapsules / Q000008:administration & dosage / Q000737:chemistry* / Q000648:ultrastructure*; D011506:Proteins / Q000008:administration & dosage / Q000737:chemistry* / Q000493:pharmacokinetics*
chemical_list: D003692:Delayed-Action Preparations; D053769:Nanocapsules; D011506:Proteins
keywords: apoptosis; cellular uptake; nanoparticles; protein delivery
references.citation: Int J Nanomedicine. 2012;7:257-70
references.pmid: 22287838
references.citation: Langmuir. 2012 Jan 31;28(4):2056-65
references.pmid: 22188099
references.citation: Int J Pharm. 2009 Jan 21;366(1-2):154-9
references.pmid: 18835346
references.citation: Cancer Res. 2010 Sep 15;70(18):7242-52
references.pmid: 20719884
references.citation: Proc Natl Acad Sci U S A. 1997 May 27;94(11):5843-7
references.pmid: 9159162
references.citation: Int J Nanomedicine. 2012;7:4841-8
references.pmid: 22973103
references.citation: Biomaterials. 2010 Oct;31(29):7575-85
references.pmid: 20599266
references.citation: Nat Rev Drug Discov. 2006 Feb;5(2):147-59
references.pmid: 16424916
references.citation: Nanomedicine. 2012 May;8(4):424-7
references.pmid: 22366599
references.citation: Eur J Pharmacol. 2011 Mar 1;654(1):17-25
references.pmid: 21184754
references.citation: J Virol. 1991 Jun;65(6):3131-9
references.pmid: 1851873
references.citation: Adv Drug Deliv Rev. 2003 Feb 24;55(3):329-47
references.pmid: 12628320
references.citation: Int J Cancer. 2009 Jun 15;124(12):2973-81
references.pmid: 19326452
references.citation: Eur J Pharm Sci. 2011 Jul 17;43(4):334-42
references.pmid: 21621611
references.citation: J Am Chem Soc. 2005 Apr 27;127(16):6021-6
references.pmid: 15839702
references.citation: Trends Immunol. 2001 Apr;22(4):183-4
references.pmid: 11274916
references.citation: Drug Discov Today. 2003 Mar 15;8(6):259-66
references.pmid: 12623240
references.citation: Angew Chem Int Ed Engl. 2009;48(29):5309-12
references.pmid: 19294716
references.citation: Biomacromolecules. 2005 Jul-Aug;6(4):2172-81
references.pmid: 16004460
references.citation: Oncogene. 2004 Feb 5;23(5):1153-65
references.pmid: 14691460
references.citation: Nat Rev Cancer. 2002 Oct;2(10):727-39
references.pmid: 12360276
references.citation: J Control Release. 2008 May 22;128(1):50-9
references.pmid: 18417240
references.citation: J Biol Chem. 2002 Dec 27;277(52):50820-7
references.pmid: 12393903
references.citation: Biomaterials. 2012 May;33(15):3959-66
references.pmid: 22365810
references.citation: Colloids Surf B Biointerfaces. 2010 Sep 1;79(2):326-33
references.pmid: 20483570
references.citation: Adv Drug Deliv Rev. 2011 Nov;63(14-15):1247-56
references.pmid: 21605607
references.citation: Int J Nanomedicine. 2012;7:4559-69
references.pmid: 22923993
references.citation: Endocr Relat Cancer. 2002 Jun;9(2):75-85
references.pmid: 12121832
references.citation: Eur J Pharm Sci. 2009 Feb 15;36(2-3):212-8
references.pmid: 18832030
references.citation: Biomaterials. 2007 Aug;28(23):3427-36
references.pmid: 17482261
references.citation: Anal Chem. 1992 Aug 1;64(15):765A-773A
references.pmid: 1443621