A paramagnetic implant containing lithium naphthalocyanine microcrystals for high-resolution biological oximetry

Abstract

Lithium naphthalocyanine (LiNc) is a microcrystalline EPR oximetry probe with high sensitivity to oxygen [R.P. Pandian, M. Dolgos, C. Marginean, P.M. Woodward, P.C. Hammel, P.T. Manoharan, P. Kuppusamy, Molecular packing and magnetic properties of lithium naphthalocyanine crystal: hollow channels enabling permeability and paramagnetic sensitivity to molecular oxygen J. Mater. Chem. 19 (2009) 4138-4147]. However, direct implantation of the crystals in the tissue for in vivo oxygen measurements may be hindered by concerns associated with their direct contact with the tissue/cells and loss of EPR signal due to particle migration in the tissue. In order to address these concerns, we have developed encapsulations (chips) of LiNc microcrystals in polydimethyl siloxane (PDMS), an oxygen-permeable, bioinert polymer. Oximetry evaluation of the fabricated chips revealed that the oxygen sensitivity of the crystals was unaffected by encapsulation in PDMS. Chips were stable against sterilization procedures or treatment with common biological oxidoreductants. In vivo oxygen measurements established the ability of the chips to provide reliable and repeated measurements of tissue oxygenation. This study establishes PDMS-encapsulated LiNc as a potential probe for long-term and repeated measurements of tissue oxygenation.

Copyright (c) 2009 Elsevier Inc. All rights reserved.

Figures

FIGURE 1. Encapsulation of LiNc in PDMS

LiNc:PDMS chips were made by cast-molding and polymerization of the uncured polymer base-catalyst mixture, in which LiNc microcrystals were thoroughly dispersed. Control (pure) PDMS films, lacking LiNc crystals, were made using the same procedure, but without adding any particulates. (a) Pure PDMS film without particulates (b) A LiNc:PDMS chip. Photographs show fairly even distribution of LiNc particles in the PDMS matrix.

FIGURE 2. Oxygen calibration of LiNc:PDMS chips

LiNc:PDMS chips (n=3) were exposed to different oxygen partial pressures (0–160 mmHg) and first-harmonic EPR spectra were recorded, using a X-band (9.8 GHz) EPR spectrometer, for the construction of calibration curves. Oxygen response of LiNc:PDMS chips was linear and reproducible with an oxygen sensitivity (slope of the calibration curve) of 32.6±0.4 mG/mmHg. Data are represented as mean±SD. The chip response was not significantly different from unencapsulated LiNc, which exhibited a sensitivity of 32.3±0.1 mG/mmHg. Data demonstrate that the encapsulation of LiNc in PDMS did not adversely affect the oxygen-sensing properties of LiNc:PDMS chips.

FIGURE 3. Effect of sterilization and oxidoreductant treatment on LiNc:PDMS chips

X-band EPR spectroscopy was used to evaluate the effect of sterilization and oxidoreductant treatment on LiNc:PDMS chips (a) Effect of sterilization on the oxygen calibration and spin density of LiNc:PDMS. Oxygen response of LiNc:PDMS chips (n=3, mean±SD), sterilized by autoclaving and UV-treatment, was not significantly different from the response of unsterilized control (Cont), with comparable oxygen sensitivities (autoclaving: 33.6±0.9 mG/mmHg; UV-treatment: 33.9±1.1 mG/mmHg; unsterilized control: 32.6±0.4 mG/mmHg). Inset shows that the spin densities of autoclaved or UV-treated LiNc:PDMS chips (n=6, mean±SD), calculated using a standard of known spin density, were not significantly different from the spin density of unsterilized controls. (b) Effect of oxidoreductant treatment on the oxygen calibration and spin density of LiNc:PDMS chips. Chips were treated with nitric oxide (NO), 1-mM hydrogen peroxide (H2O2), and 5-mM glutathione (GSH) for 30 min. After air-drying, the oxygen responses of treated chips and untreated controls (Cont) were evaluated using X-band EPR spectroscopy. Data (n=3, mean±SD) show that the response was linear with increase in oxygen partial pressure after treatment with all three agents. Oxygen sensitivity of the chips treated with NO (32.6±1.3 mG/mmHg), H2O2 (32.8±0.1 mG/mmHg), or GSH (32.8±0.4 mG/mmHg) was not significantly different from the sensitivity of untreated control (32.6±0.4 mG/mmHg). Inset shows that treatment with biological oxidoreductants did not significantly affect the spin density of LiNc:PDMS chips (n=6, mean±SD). Collectively, results indicate that LiNc:PDMS chips were durable and biostable.

FIGURE 4. In vivo oxygen measurements using LiNc:PDMS chips

LiNc:PDMS chips were implanted in the gastrocnemius muscle tissue of mice, and in vivo oxygen measurements were performed using an L-band EPR spectrometer for up to 30 days. Muscle pO2 from three mice, in the normal (filled symbols) and constricted (open symbols) condition of the muscle, demonstrate that implanted LiNc:PDMS chips were capable of reliable measurements of in vivo tissue pO2, and were also responsive to changes in pO2.