Pd-porphyrin-cross-linked implantable hydrogels with oxygen-responsive phosphorescence

Abstract

Development of long-term implantable luminescent biosensors for subcutaneous oxygen has proved challenging due to difficulties in immobilizing a biocompatible matrix that prevents sensor aggregation yet maintains sufficient concentration for transdermal optical detection. Here, Pd-porphyrins can be used as PEG cross-linkers to generate a polyamide hydrogel with extreme porphyrin density (≈5 × 10(-3) m). Dye aggregation is avoided due to the spatially constraining 3D mesh formed by the porphyrins themselves. The hydrogel exhibits oxygen-responsive phosphorescence and can be stably implanted subcutaneously in mice for weeks without degradation, bleaching, or host rejection. To further facilitate oxygen detection using steady-state techniques, an oxygen-non-responsive companion hydrogel is developed by blending copper and free base porphyrins to yield intensity-matched luminescence for ratiometric detection.

Keywords: hydrogels; imaging; implants; oxygen sensing; phosphorescence; porphyrins.

Figure 1. Pd-porphyrins as effective PEG crosslinkers to generate a hydrogel with extreme (~5 mM), spatially segregated porphyrin density

a) Chemical structures of Pd-mTCPP and PEG-diamines and the schematic polymer network formed following condensation. b) Rapid and complete polymerization of the PEG and porphyrin following addition of DIPEA to initiate the reaction. Mean +/− std. dev. shown for n=3. c) Photograph of an 8 mm hydrogel.

Figure 2. Characterization of Pd-porphyrin hydrogel formation

In otherwise equivalent conditions, polymer yield was affected by a) PEG diamine size; b) Ratio of Pd-mTCPP to PEG-diamine (6K); and c) Pd-mTCPP concentration (with constant ratios of PEG:porphyrin:HBTU). d) Water swelling as a function of Pd-mTCPP concentration during polymerization. Mean +/− std. dev. shown for n=3. e) SEM of a freeze-dried portion of Pd-mTCPP hydrogel.

Figure 3. Oxygen-responsive phosphorescence of the Pd-porphyrin hydrogel

a) Reversible steady-state phosphorescence response to oxygen in indicated conditions at 37 °C. b) Characterization of hydrogel phosphorescence lifetime response. c) Stern-Volmer lifetime analysis of hydrogel phosphorescence as a function of oxygen concentration.

Figure 4. Suitability of the Pd-porphyrin hydrogel for in vivo implantation and transdermal imaging

a) Luminescence emission spectrum in the near infrared of the Pd-porphyrin hydrogel in 20% oxygen. b) Hydrogel was implanted subcutaneously in a BALB/c mouse and imaged every 10 days. Luminescence images are shown in the top row and corresponding white light images are shown in the bottom row. No obvious degradation, photobleaching or toxicity was observed. Images were aquired on a Maestro imager.

Figure 5. A copper-doped, non-oxygen-responsive companion hydrogel for ratiometric imaging

a) Freebase mTCPP could be titrated with non-luminescent Cu-mTCPP to tune overall hydrogel luminescence. b) Luminescence imaging of Pd-mTCPP and freebase-Cu-mTCPP hydrogels in varying oxygen conditions.

Figure 6. In vivo detection of subcutaneous oxygenation

a) Transdermal luminescence imaging of dual-implanted Pd-mTCPP and 15% freebase-Cu-mTCPP hydrogels. Left images were taken following anesthetization and right ones taken following high pressure oxygen administration. b) Ratiometric changes in Pd and 2H/Cu luminescence over time following mouse exposure to high pressure inhaled oxygen (error bars show mean +/− std. dev. for n=3 separately implanted and treated mice).