Synthetic polymer nanoparticles with antibody-like affinity for a hydrophilic peptide

Abstract

Synthetic polymer nanoparticles with antibody-like affinity for a hydrophilic peptide have been prepared by inverse microemulsion polymerization. Peptide affinity was achieved in part by incorporating the target (imprint) peptide in the polymerization reaction mixture. Incorporation of the imprint peptide assists in the creation of complementary binding sites in the resulting polymer nanoparticle (NP). To orient the imprint peptide at the interface of the water and oil domains during polymerization, the peptide target was coupled with fatty acid chains of varying length. The peptide--NP binding affinities (ca. 90-900 nM) were quantitatively evaluated by a quartz crystal microbalance (QCM). The optimal chain length was established that created high affinity peptide binding sites on the surface of the nanoparticles. This method can be used for the preparation of nanosized synthetic polymers with antibody-like affinity for hydrophilic peptides and proteins ("plastic antibodies").

Figure 1

Preparation of hydrophilic imprinted NPs by inverse microemulsion polymerization. (a) Peptides utilized in this study: C5-P, C13-P and C15-P. The peptide was coupled with fatty acids (five, thirteen and fifteen carbons) at the N-terminus. (b) Schematic of the preparation of imprinted NPs. An aqueous monomer solution was dispersed into nano-sized water droplets in hexane. The droplets were stabilized by the surfactants AOT and Brij30 (Green). Modified peptides (yellow) were added into the microemulsion. The polymer nanoparticles (dark blue) were formed by addition of APS/TEMED initiator. Processing consisted of washing with EtOH and dialysis against water. (c) Size distribution of the three imprinted NPs and a control non-imprinted nanoparticle (NIP) as determined by DLS. Spectra of the NPs (1mg/mL) were taken in water after dialysis. Three measurements were taken and averaged for each NP.

Figure 1

Preparation of hydrophilic imprinted NPs by inverse microemulsion polymerization. (a) Peptides utilized in this study: C5-P, C13-P and C15-P. The peptide was coupled with fatty acids (five, thirteen and fifteen carbons) at the N-terminus. (b) Schematic of the preparation of imprinted NPs. An aqueous monomer solution was dispersed into nano-sized water droplets in hexane. The droplets were stabilized by the surfactants AOT and Brij30 (Green). Modified peptides (yellow) were added into the microemulsion. The polymer nanoparticles (dark blue) were formed by addition of APS/TEMED initiator. Processing consisted of washing with EtOH and dialysis against water. (c) Size distribution of the three imprinted NPs and a control non-imprinted nanoparticle (NIP) as determined by DLS. Spectra of the NPs (1mg/mL) were taken in water after dialysis. Three measurements were taken and averaged for each NP.

Figure 2

Interaction between nanoparticles and immobilized peptide by QCM. (a) Schematic of QCM experiments for monitoring interactions between NPs (left: NIP or MIP(C5-P) and right: MIP(C13-P) or MIP(C15-P))and GFP-9 peptide immobilized on QCM electrode. (b) Representative time courses of frequency change of the 27 MHz QCM. GFP-9 peptide was immobilized on the QCM electrode. Solutions of MIP (C5-P) (black line) and MIP (C13-P) (red line) were injected at the time points indicated by the arrows into two separate QCM cells. (c): Frequency Shift upon injections of NIP, MIP (C5-P), MIP (C13-P) and MIP (C15-P) to QCM sensor cells with GFP-9 immobilized on the surface. Data represent the mean frequency change±standard deviation (n=3) after injection of 64 μg/mL polymeric NP solutions in GFP-9-immobilized QCM cells.

Figure 3

(a) Binding isotherms of MIP (C15-P)-GFP-9 (pink line) and NIP-GFP-9 (cyan line), MIP (C15-P)-SAM (green line) and NIP-SAM (blue line) and MIP (C15-P)-peptide A (red line) and NIP-peptide A (black line). GFP-9, SAM and peptide A were immobilized on the surface of QCM electrode. Frequency shifts were recorded upon injections of solutions of NPs into the sensor cells. (b) Competitive study of MIP (C15-P)-GFP-9 interaction. MIP(C15-P) was preincubated with 0.1 mM GFP-9 overnight. The mixture solution was injected into the sensor cells with GFP-9 immobilized on the surface (black line). Binding was suppressed as compared to the MIP (C15-P) solution (pink line).

Figure 4

(a) 1 mL water with 1 mg Brij 30 (vial 1), 1mg C15-P (vial 2), 1mg C13-P (vial 3), 1mg C5-P (vial 4) and control (no peptide) (vial 5). (b) Mixed solvent (1mL water and 1mL hexane) with 1 mg Brij 30 (vial 6), 1mg C15-P (vial 7), 1mg C13-P (vial 8), 1mg C5-P (vial 9) and control (no peptide) (vial 10).