Controlling the Aggregation of Conjugates of Streptavidin with Smart Block Copolymers Prepared via the RAFT Copolymerization Technique

Abstract

Block copolymers containing stimuli-responsive segments provide important new opportunities for controlling the activity and aggregation properties of protein-polymer conjugates. We have prepared a RAFT block copolymer of a biotin-terminated poly(N-isopropylacrylamide) (PNIPAAm)-b-poly(acrylic acid) (PAA). The number-average molecular weight (M(n)) of the (PNIPAAm)-b-(PAA) copolymer was determined to be 17.4 kDa (M(w)/M(n) = 1.09). The PNIPAAm block had an M(n) of 9.5 kDa and the poly(acrylic acid) (PAA) block had an M(n) of 7.9 kDa. We conjugated this block copolymer to streptavidin (SA) via the terminal biotin on the PNIPAAm block. We found that the usual aggregation and phase separation of PNIPAAm-SA conjugates that follow the thermally induced collapse and dehydration of PNIPAAm (the lower critical solution temperature (LCST) of PNIPAAm is 32 degrees C in water) is prevented through the shielding action of the PAA block. In addition, we show that the cloud point and aggregation properties (as measured by loss in light transmission) of the [(PNIPAAm)-b-(PAA)]-SA conjugate also depended on pH. At pH 7.0 and at temperatures above the LCST, the block copolymer alone was found to form particles of ca. 60 nm in diameter, while the bioconjugate exhibited very little aggregation. At pH 5.5 and 20 degrees C, the copolymer alone was found to form large aggregates (ca. 218 nm), presumably driven by hydrogen bonding between the -COOH groups of PAA with other -COOH groups and also with the -CONH- groups of PNIPAAm. In comparison, the conjugate formed much smaller particles (ca. 27 nm) at these conditions. At pH 4.0, however, large particles were formed from the conjugate both above and below the LCST (ca. 700 and 540 nm, respectively). These results demonstrate that the aggregation properties of the block copolymer-SA conjugate are very different from those of the free block copolymer, and that the outer-oriented hydrophilic block of PAA shields the intermolecular aggregation of the block copolymer-SA bioconjugate at pH values where the -COOH groups of PAA are significantly ionized.

Living Radical Polymerization by the RAFT Process - A Second Update

“…249 [408] MA [405] St [407] St [408] 250* [405] S S N N N S S 251 [404] MA [405] St [407,409] MA [405] St [404] 252* N N N S S 253 [283] S S N N St [409] 374 [410] MA/374 [410] St/374 [410] (NVCL) [384] N S S N N 255 [411] O S S N CN 256* MA [411] (St) [411] AA-b-NIPAM [412] 257 [283] S S N 258 [413] S S N O O St [413] (MA) [413] A See footnote A of Table 3. B See footnote B of Table 3.…”

“…[117,130,479] This methodology has been applied to the synthesis of three-armed stars by an in-situ thiol-ene reaction with a triacrylate [130] and has been applied to directly make biopolymer conjugates. [479] A recent study shows that dithioester and trithiocarbonate RAFT agents [R-SC(S)Z] undergo a radical-catalyzed reaction with thiols that results in oxidation of the thiol to a disulfide and concomitant reduction of the RAFT agent to R-H. [481] The relative rates of reaction suggested that dithioester RAFT agents [204] PEGA Ph (58) Butylamine Used in thiol-ene [204] NIPAM SCH 2 CH(CH 3 ) 2 (188) Butylamine/TCEP/HEA butylamine/TCEP/BA One-pot thiol-ene [373] NIPAM SCH 2 CH(CH 3 ) 2 (188) Butylamine/TCEP Used in thiol-ene [371,372] NIPAM Ph (25) LiB(C 2 H 5 ) 3 H Used in thiol-ene [151] NIPAM S(CH 2 ) 2 CO 2 H (170) NaBH 4 Used in thiol-ene [355] NIPAM SC 4 H 9 (166) NaOH/MeOH/EDTA Used in thiol-ene [412] NIPAM Ph (24) Octylamine/DMPP/AMA One-pot thiol-ene [132] NIPAM Ph (24) Octylamine/DMPP/PMA One-pot thiol-ene [132] DEAM Ph (24) Hexylamine/DMPP/MA One-pot thiol-ene [130] St Ph (24) Propylamine/PBu 3 /MA NaBH 4 /PBu 3 /MA One-pot thiol-ene [117] St Ph (24) Propylamine NaBH 4 [205] MMA Ph (82) Hexylamine Conditions to favour disulfide formation [196] MMA Ph (22) Propylamine One-pot methanethiosulfonate [480] NVP OC 2 H 5 (228) NaBH 4 Used in peptide/nucleotide conjugation [396] A Monomer unit adjacent to thiocarbonylthio group. B TCEP -tris(2-carboxyethyl) phosphine hydrochloride, EDTA -ethylenediaminetetraacetic acid, TCEP -tris(2-carboxyethyl phosphine), DMPPdimethylphenylphosphine.…”

“…[368] The biotin moiety has also been attached post polymerization by azide-alkyne click chemistry [364] or by thiol-ene chemistry. [412] (f) conversion of thiocarbonylthio groups of RAFT-synthesized polymers into thiol functionalities which are then used for conjugation. The thiol-end-groups were protected by reaction with Ellman's reagent [5,5 -dithiobis-(2-nitrobenzoic acid)] which also activates the thiol for reaction with a thio functional biopolymer (ssDNA, peptide) by disulfide exchange (Scheme 20).…”

“…Pyridyl disulfide, 2. azide-alkyne click [364] 166 (AA-b-NIPAM) Streptavidin Biotin conjugation [412] 189 (NIPAM/321) BSA (telechelic) Thiol-ene [374] 185 (NIPAM) Streptavidin, BSA (heterotelechelic) 1. Thiol-ene, 2. biotin conjugation [368] 228 (NVP) ssDNA RAFT end reduction/thiol coupling [396] A See footnote A of Table 3.…”

Effect of tamoxifen in RAFT miniemulsion polymerization during the synthesis of polymeric nanoparticles

“…In RAFT polymerizations, a chain transfer agent with the general structure ZC(=S) SR, where Z is an activating group and R is a transferred chemical group, is used to control the chain growth. The thio-carbonyl-thio terminal group present at the final polymer chains can be easily converted to a thiol group for posterior chemical modifications of the polymer material [25,29] . For instance, thiol groups can be used for bioconjugation and development of biomedical applications [29][30][31] .…”