Accelerated Polymer–Polymer Click Conjugation by Freeze–Thaw Treatment

Abstract

Herein, we report a unique technique to accelerate polymer-SNA conjugation based on copper-free click chemistry: gradual freeze-thawing of the reaction solution substantially increases the conjugation rate possibly because of the reactant concentration at the microenvironment scale. This technique was applied to the conjugation between a small interfering RNA (siRNA) and PEG in an aqueous buffer at/below room temperature.

Density-tunable conjugation of cyclic RGD ligands with polyion complex vesicles for the neovascular imaging of orthotopic glioblastomas

“…To facilitate the cRGD conjugation reactions, freezing and thawing was performed in the presence of an excess amount of cRGD (10 eq. per aldehyde group in the block aniomers of the PICsomes; see supporting information) [28]. Excess amount of Me–NH 2 was added to the PICsome solution to quench any residual aldehyde groups.…”

“…Finally, cRGD peptide (10 eq. versus the aldehyde group on the PICsomes) was added, and the reaction mixture was gradually frozen and thawed [28] to facilitate the formation of a thiazolidine ring between the N -terminal cysteine and the aldehyde group that converted from the acetal group [26] on the PICsomes’ surface. The unbound cRGD was removed by filtration (Vivaspin 6, MWCO: 300 000), and the cRGD introduction rates were analyzed with 1 H nuclear magnetic resonance (NMR) spectroscopy (solvent, D 2 O; 80 °C; JEOL LNM-ECS 400, JEOL, Tokyo, Japan) by comparing the relative peak integration values of the aromatic protons in the cRGD residues (7.2–7.5 ppm) with those of the internal standard (0 ppm, 1% wt sodium 3-(trimethylsilyl)-1-propanesulfonate: TMS), which are estimated to be 23.4% (20%-cRGD PICsomes).…”

Acidic pH‐Responsive siRNA Conjugate for Reversible Carrier Stability and Accelerated Endosomal Escape with Reduced IFNα‐Associated Immune Response

“…Figure 1 b shows the chemical structure of siRNA-releasable/endosome-disrupting conjugate (REC), in which several siRNA molecules are grafted into the endosome-disrupting polymer side chains by the MAA linkage. [10] This successful conjugation at the quite high rate allows the use of the obtained conjugate without further purification. [9] A precursor polyanion was synthesized from PAsp(DET) to have a dibenzyl cyclooctyne (DBCO) group by MAA linkage as a conjugation site for siRNA.…”

Reductive Decationizable Block Copolymers for Stimuli-Responsive mRNA Delivery

“…This molecule was finally conjugated to P(MEO 3 MA) 35 ‐ b ‐P(H 2 N‐Cys‐MA) 95 via SPAAC. Thereby, three freeze‐thaw cycles according to Takemoto et al were applied to enhance click reaction efficiencies (Figure A; Scheme S11, Supporting Information). End group modification with cRGD could successfully be verified by a complete peak shift to higher molecular weights in the SEC elugram (eluent: HFIP) (Figure B; Figure S70 in the Supporting Information – the cRGD derivative has a molecular weight of about 1200 g mol −1 ; also some peak broading occurred probably due to cRGD‐triggered repellent interaction with the SEC column material).…”