Abstract:
:Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells protect vertebrates by killing infected or transformed cells using granzyme B (GrB) to induce apoptosis. However, GrB-induced apoptosis of target cells causes inflammatory disease and chronic transplant rejection and so is an important disease target. The aim of this study was to prevent apoptosis of the target cells by delivering a plasmid encoding GrB inhibitor proteinase inhibitor-9 (PI-9) using cationic polymers as a non-viral vector. Polyethyleneimine (PEI, branched, Mn 10 kDa) gives a high degree of gene transfection efficiency in many types of cell lines, but it is highly cytotoxic. To reduce this cytotoxicity, we modified PEI by blocking primary amine groups through nucleophilic addition between primary amine and a protected mannose-functionalized cyclic carbonate (MTC-ipman), generating a carbamate linkage through the ring-opening of the cyclic carbonate. Deprotection of the mannose yielded a PEI polymer that is decorated with the carbohydrate. PEI with 7 or 20 of 67 primary amine groups substituted by the carbohydrate had similar gene binding ability compared to unmodified PEI, leading to almost 100% transfection efficiency of a GFP-reporter plasmid in HEK293T human embryonic kidney cells. Furthermore, modification of PEI resulted in a decrease in the cytotoxicity of PEI/DNA complexes. However, PEI with all primary amine groups blocked was unable to form a complex with DNA, and so reporter transfection was negligible. The PI-9 encoding plasmid was transfected into HEK293T cells effectively using the modified PEIs with the optimal degree of primary amine substitution, protecting up to 80% HEK293T cells from killing by human natural killer-like leukemic YT cells. Therefore, these carbamate-mannose modified PEI/PI-9 encoding plasmid complexes have potential clinical utility in the prevention of chronic transplant rejection and inflammatory disease caused by GrB.
journal_name
Biomaterialsjournal_title
Biomaterialsauthors
Cheng W,Yang C,Hedrick JL,Williams DF,Yang YY,Ashton-Rickardt PGdoi
10.1016/j.biomaterials.2013.01.090subject
Has Abstractpub_date
2013-05-01 00:00:00pages
3697-705issue
14eissn
0142-9612issn
1878-5905pii
S0142-9612(13)00145-2journal_volume
34pub_type
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