title: Targeted intracellular protein delivery based on hyaluronic acid-green tea catechin nanogels.
abstract: A novel ternary nanogel based on the self-assembly of hyaluronic acid-epigallocatechin gallate conjugates (HA-EGCG), linear polyethylenimine (PEI) and Granzyme B (GzmB) in an aqueous environment was developed for the targeted intracellular delivery of GzmB into cancer cells. Lysozyme-encapsulated HA-EGCG nanogels were first prepared and characterized. HA-EGCG nanogels exhibited smaller particle sizes and a more homogeneous size distribution than the HA counterpart. Fluorescence quenching and lysozyme activity studies revealed that EGCG moieties facilitated protein binding through physical interactions and led to the formation of stable nanogels. When CD44-overexpressing HCT-116 colon cancer cells were treated with GzmB-encapsulated HA-EGCG nanogels in vitro, a significant cytotoxic effect was observed. Caspase assays and intracellular trafficking studies confirmed that cell death was due to apoptosis triggered by the delivery of GzmB to the cytosol of those cells. In comparison, little cytotoxic effect was observed in CD44-deficient cells treated with GzmB-encapsulated HA-EGCG nanogels. This study highlights the potential utility of HA-EGCG as effective intracellular protein carriers for targeted cancer therapy.
  
  
  CONCLUSIONS
  Intracellularly activated cytotoxic proteins can be used to kill cancer cells but viable carriers for such proteins are lacking. In this work, we developed novel nanogels based on selfassembly of hyaluronic acid (HA)-(-)-epigallocatechin-3-gallate (EGCG) conjugates, linear polyethylenemine (PEI) and the cytotoxic protein Granzyme B (GzmB) for the intracellular delivery of GzmB for cancer therapy. HA was exploited for its ability to target CD44 which are overexpressed in many types of cancer cells, while EGCG, the main component of green tea catechins, was chosen for its ability to bind to proteins. Characterization studies showed that EGCG facilitated protein complexation through physical interactions and led to the formation of stable nanogels. HA-EGCG nanogels were able to achieve CD44 targeted killing of HCT-116 cancer cells by delivering GzmB into the cytosol of these cells. We believe that the applications of the HA-EGCG nanogels can be expanded to the intracellular delivery of other cytotoxic protein drugs for cancer therapy.
authors.affiliation: Institute of Bioengineering & Nanotechnology (IBN), 31 Biopolis Way, The Nanos, #04-01, Singapore 138669, Singapore.
authors.forename: Kun
authors.initials: K
authors.lastname: Liang
authors.affiliation: Institute of Bioengineering & Nanotechnology (IBN), 31 Biopolis Way, The Nanos, #04-01, Singapore 138669, Singapore.
authors.forename: Shengyong
authors.initials: S
authors.lastname: Ng
authors.affiliation: Institute of Bioengineering & Nanotechnology (IBN), 31 Biopolis Way, The Nanos, #04-01, Singapore 138669, Singapore.
authors.forename: Fan
authors.initials: F
authors.lastname: Lee
authors.affiliation: Institute of Bioengineering & Nanotechnology (IBN), 31 Biopolis Way, The Nanos, #04-01, Singapore 138669, Singapore.
authors.forename: Jaehong
authors.initials: J
authors.lastname: Lim
authors.affiliation: Institute of Bioengineering & Nanotechnology (IBN), 31 Biopolis Way, The Nanos, #04-01, Singapore 138669, Singapore.
authors.forename: Joo Eun
authors.initials: JE
authors.lastname: Chung
authors.affiliation: Institute of Bioengineering & Nanotechnology (IBN), 31 Biopolis Way, The Nanos, #04-01, Singapore 138669, Singapore.
authors.forename: Su Seong
authors.initials: SS
authors.lastname: Lee
authors.affiliation: Institute of Bioengineering & Nanotechnology (IBN), 31 Biopolis Way, The Nanos, #04-01, Singapore 138669, Singapore. Electronic address: mkurisawa@ibn.a-star.edu.sg.
authors.forename: Motoichi
authors.initials: M
authors.lastname: Kurisawa
mesh_terms: D000818:Animals; D002392:Catechin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry; D002470:Cell Survival / Q000187:drug effects; D002645:Chickens; D019281:Dimerization; D016503:Drug Delivery Systems / Q000379:methods*; D000067493:Dynamic Light Scattering; D005434:Flow Cytometry; D053804:Granzymes / Q000378:metabolism; D045325:HCT116 Cells; D056945:Hep G2 Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D006820:Hyaluronic Acid / Q000138:chemical synthesis / Q000737:chemistry*; D042541:Intracellular Space / Q000378:metabolism*; D009113:Muramidase / Q000378:metabolism*; D000080385:Nanogels; D011092:Polyethylene Glycols / Q000737:chemistry*; D011094:Polyethyleneimine / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013662:Tea / Q000737:chemistry*
chemical_list: D018960:Hyaluronan Receptors; D000080385:Nanogels; D013662:Tea; C413692:polyethylene glycol polyethyleneimine nanogel; D011092:Polyethylene Glycols; D002392:Catechin; D011094:Polyethyleneimine; D006820:Hyaluronic Acid; C045651:epigallocatechin gallate; D009113:Muramidase; D053804:Granzymes
keywords: Cancer therapy; Epigallocatechin gallate; Hyaluronic acid; Nanogels; Protein delivery