Photophysical, thermal and imaging studies on vancomycin functional branched poly(N-isopropyl acrylamide) of differing degrees of branching containing nile red for detection of Gram-positive bacteria

Abstract

Highly branched poly(N-isopropyl acrylamide) additives chain end functionalised with vancomycin have been designed to agglutinate and report on targetted Gram-positive strains of bacteria (S. aureus). These branched systems selectively desolvate with temperature or binding interactions depending on their chain architecture. We have prepared samples with three different degrees of branching which have incorporated Nile red acrylate as a low concentration of co-monomer to report upon their solution properties. A linear analogue polymer functionalised with vancomycin along the chain instead of the termini is presented as a control which does not bind to targeted bacteria. These samples were analysed by diffusion NMR spectrometry (DOSY), calorimetry, fluorescence lifetime measurements, optical microscopy and scanning electron microscopy to gain a full understanding of their solution properties. The branched polymers are shown conclusively to have a core-shell structure, where the chain ends are expressed from the desolvated globule even above the lower critical solution temperature - as demonstrated by NMR measurements. The level of desolvation is critically dependent on the degree of branching, and as a result we have found intermediate structures provide optimal body temperature bacterial sensing as a consequence of the Nile red reporting dye.

Scheme 1. Chemical structure of HB-PNIPAM-VAN polymer backbone.

Scheme 2. Polymer structure of highly branched polymers produced via self-condensing reaction mechanism.

Fig. 1. (A) Thermogravimetric analysis (TGA) mass loss profiles for polymers 1–4 (B) TGA analysis for precursor polymers 1_a–4_a (unmodified pyrrole/acid chain ended products of SCVP RAFT synthesis process prior to chain end modification).

Fig. 2. Stejskal–Tanner plots of high resolution DOSY (48 gradient decay steps) of HB-PNIPAM-Vanc (polymer 2) showing varying diffusions of different components of branching architecture in D₂O at 288, 298 and 313 K. These represent peaks at (A) 4.69 ppm (H₂O residue in solvent), (B) 1.1 ppm (–CH₃ in PNIPAM isopropyl side chain), (C) 3.8 ppm (–CH–(CH₃)₂ in PNIPAM isopropyl side chain), (D) 1.5 ppm (CH₂ in polymer backbone), (E) 7.4 ppm (aromatic peaks from benzyl branching group), (F) 6.47 ppm (vancomycin aromatic-H peak from functional chain ends).

Fig. 3. Apparent hydrodynamic radii of polymer 2 from high resolution DOSY NMR analysis, as a 1 mg mL⁻¹ solution in D₂O at 288, 298 and 313 K.

Fig. 4. Micro-DSC thermogram for HB-PNIPAM-Vanc in absence (solid lines) and presence (dashed lines) of d-Ala–d-Ala peptide in PBS. Contains vanc. functionalised polymers with 1 (DB 0.107 (green)), 2 (DB 0.038 (blue)), 3 (0.021 (red)) and 4 (linear polymer (black)).

Fig. 5. HOMO and LUMO orbitals of Nile red, Nile red acrylate and Nile red monomer when calculated in an aqueous solvent conditions (via CPCM).

Fig. 6. Fluorescence emission spectra (15 °C) of HB-P(NIPAM-co-NR)-VAN polymers with emission spectra (left axis) and emission wavelength specific excited state lifetime (right axis). λ_ex = 570 nm. Polymer 1 (DB 0.107) is black (); 2 (DB 0.065) is red (); 3 (DB 0.044) is blue ().

Fig. 7. (A) Shift in peak NR emission intensity of dilute (1 mg mL⁻¹ H₂O) polymer solutions following λ_ex 580 nm. (B) Shift in peak fluorescence emission wavelength (average mean of distribution) following λ_ex 580 nm, with temperature of pyrrole chain end polymers compared to NR solvent shifts (solvents listed from max. to min. λ_em at 15 °C using data from Plenderleith et al. ethylene glycol, glycerol, methanol, ethanol, DMSO, butanol, propan-2-ol).

Fig. 8. Relative fluorescence output of following addition of Ala–Ala peptide to solutions of 1–4 at 35 °C (5 mg mL⁻¹), compared to polymers initial emission once equilibriated to temperature in PBS.

Fig. 9. Fluorescence emission intensity plotted against polymer–bacteria (S. aureus) concentration. Sample recorded after 2 hour incubation at 37 °C, then measurement read at 32 °C at emission 639 nm. Ties indicate significance (ANOVA, general linear model, post hoc Tukey) between samples 1–3 (*p < 0.05, **p < 0.01, ***p < 0.001). Error bars show variation over 5 repeat measurements for each sample.

Fig. 10. SEM images of combined 10⁸ cfu S. aureus + HB-PNIPAM-Vanc solutions dried on a glass plate.