ABSTRACT
Sudden aggravations of chronic inflammatory airway diseases are difficult-to-foresee life-threatening episodes for which advanced prognosis-systems are highly desirable. Here we present an experimental chip-based fluidic system designed for the rapid and sensitive measurement of biomarkers prognostic for potentially imminent asthma or COPD exacerbations. As model biomarkers we chose three cytokines (interleukin-6, interleukin-8, tumor necrosis factor alpha), the bacterial infection marker C-reactive protein and the bacterial pathogen Streptococcus pneumoniae-all relevant factors in exacerbation episodes. Assay protocols established in laboratory environments were adapted to 3D-printed fluidic devices with emphasis on short processing times, low reagent consumption and a low limit of detection in order to enable the fluidic system to be used in point-of-care settings. The final device demonstrator was validated with patient sample material for its capability to detect endogenous as well as exogenous biomarkers in parallel.
Subject(s)
Biomarkers , Point-of-Care Systems , Pulmonary Disease, Chronic Obstructive , Streptococcus pneumoniae , Humans , Pulmonary Disease, Chronic Obstructive/diagnosis , Streptococcus pneumoniae/isolation & purification , C-Reactive Protein/analysis , C-Reactive Protein/metabolism , Cytokines/metabolism , Asthma/diagnosis , Lab-On-A-Chip Devices , Interleukin-6 , Prognosis , Tumor Necrosis Factor-alpha/analysisABSTRACT
Polylysine-b-p[HPMA] block copolymers containing a redox-responsive disulfide bond between both blocks are synthesized by RAFT polymerization of pentafluorphenyl-methacrylate with a macro-CTA from Nϵ-benzyloxycarbonyl (Cbz) protected polylysine (synthesized by NCA polymerization). This polylysine-b-p[PFMA] precursor block copolymer is converted to polylysine(Cbz)-b-p[HPMA] by postpolymerization modification with 2-hydroxypropylamine. After removal of the Cbz protecting group, cationic polylysine-b-p[HPMA] copolymers with a biosplittable disulfide moiety became available, which can be used as polymeric transfection vectors. These disulfide linked polylysine-S-S-b-p[HPMA] block copolymers show low cytotoxicity and increased transfection efficiencies (HEK-293T cells) compared to analogous blockcopolymers without disulfide group making them interesting for the transfection of sensitive immune cells.
Subject(s)
Polymers/chemistry , Transfection/methods , HEK293 Cells , Humans , Polylysine/analogs & derivatives , Polylysine/chemical synthesis , Polymerization , Polymers/chemical synthesis , Polymethacrylic Acids/chemical synthesisABSTRACT
Herein we describe the synthesis of poly-L-lysine-b-poly[N-(2-hydroxypropyl)-metha-crylamide)] (poly[HPMA]) block copolymers by combination of solid phase peptide synthesis or polymerization of α-amino acid-N-carboxy-anhydrides (NCA-polymerization) with the reversible addition-fragmentation chain transfer polymerization (RAFT). In the presence of p-DNA, these polymers form polyplex micelles with a size of 100-200 nm in diameter (monitored by SDS-PAGE and FCS). Primary in vitro studies with HEK-293T cells reveal their cellular uptake (FACS studies and CLSM) and proof successful transfection with efficiencies depending on the length of polylysine. Moreover, these polyplexes display minimal toxicity (MTT-assay and FACS-measurements) featuring a p[HPMA] corona for efficient extracellular shielding and the potential ligation with antibodies.
Subject(s)
DNA/drug effects , Methacrylates/chemistry , Polylysine/chemistry , Polymerization , DNA/chemistry , HEK293 Cells/drug effects , Humans , Methacrylates/adverse effects , Methacrylates/pharmacology , Polylysine/adverse effects , Polylysine/pharmacology , TransfectionABSTRACT
This paper describes the synthesis of semitelechelic maleimide-modified N-(2-hydroxypropyl)methacrylamid) (HPMA) based polymers of narrow dispersity that can be conjugated e.g. to anti-DEC-205 antibodies affording "star-like" topologies (one antibody decorated with several polymer chains). FCS revealed a hydrodynamic diameter of R(h) = 7.9 nm and SEC narrow dispersity (1.45). Primary in vitro studies with bone marrow derived dendritic cells (DC) show higher cellular binding and uptake rates compared to control samples. Moreover, incubating these conjugates to primary splenocytes demonstrates a much higher affinity to the primary DCs than to any other immune cell population within the spleen. This differentiation is, thereby, much more pronounced for the star-like conjugates than for conjugates made from polymers statistically modified with anti-DEC-205.
Subject(s)
Antibodies/chemistry , Dendritic Cells/metabolism , Drug Carriers/metabolism , Immunoconjugates/metabolism , Methacrylates/chemistry , Animals , Antibodies/metabolism , Antigens, CD/metabolism , Biological Transport , Cell Survival/drug effects , Dendritic Cells/cytology , Dendritic Cells/drug effects , Drug Carriers/chemistry , Drug Carriers/pharmacology , Humans , Immunoconjugates/chemistry , Immunoconjugates/pharmacology , Immunotherapy , Lectins, C-Type/antagonists & inhibitors , Lectins, C-Type/metabolism , Lymphocytes/cytology , Lymphocytes/drug effects , Mice , Minor Histocompatibility Antigens , Molecular Targeted Therapy , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cell Surface/metabolism , Spleen/cytology , Spleen/drug effectsABSTRACT
Herein the synthesis of antibody-polymer conjugates, with a quite narrow dispersity based on the polymer HPMA, are reported. These conjugates are synthesized by coupling antibodies to maleimide-functionalized poly(N-(2-hydroxypropyl)-methacrylamide) (poly-HPMA) copolymers derived through reversible addition-fragmentation chain transfer (RAFT) polymerization of pentafluorophenyl methacrylate via the intermediate step of an activated ester polymer. We develop a protocol that allows the attachment of two different model antibodies, monoclonal anti-RAGE (receptor for advanced glycation end-products) antibody, and polyclonal human immunoglobulin (huIgG). Modification of the antibody and conjugation is monitored by SDS-PAGE electrophoresis. Preserved affinity is demonstrated by Western Blott and cell-uptake analysis, for example, to cells of the immune system.
