Oxygen sensitivity and biocompatibility of an implantable paramagnetic probe for repeated measurements of tissue oxygenation

Abstract

The use of oxygen-sensing water-insoluble paramagnetic probes, such as lithium octa-n-butoxynaphthalocyanine (LiNc-BuO), enables repeated measurements of pO(2) from the same location in tissue by electron paramagnetic resonance (EPR) spectroscopy. In order to facilitate direct in vivo application, and hence eventual clinical applicability, of LiNc-BuO, we encapsulated LiNc-BuO microcrystals in polydimethylsiloxane (PDMS), an oxygen-permeable and bioinert polymer, and developed an implantable chip. In vitro evaluation of the chip, performed under conditions of sterilization, high-energy irradiation, and exposure to cultured cells, revealed that it is biostable and biocompatible. Implantation of the chip in the gastrocnemius muscle tissue of mice showed that it is capable of repeated and real-time measurements of tissue oxygenation for an extended period. Functional evaluation using a murine tumor model established the suitability and applicability of the chip for monitoring tumor oxygenation. This study establishes PDMS-encapsulated LiNc-BuO as a promising choice of probe for clinical EPR oximetry.

Figures

Fig. 1

Effect of sterilization and high-energy irradiation on the LiNc-BuO:PDMS chip. The effect of sterilization (autoclaving and UV-treatment) and high-energy irradiation (X-rays and 60Co-γ rays) on the oxygen calibration and spin density of the LiNc-BuO:PDMS chip was evaluated using X-band EPR spectroscopy. (a) Effect of sterilization on the oxygen calibration of LiNc-BuO:PDMS chips (n=3). The oxygen response of the chip remained linear over the range of oxygen partial pressures (pO2: 0–160 mmHg) used, irrespective of the mode of sterilization. The oxygen sensitivity after autoclaving and UV-treatment was 7.14±0.04 mG/mmHg and 7.35±0.11 mG/mmHg respectively. These values were not significantly different from the sensitivity of unencapsulated (neat) LiNc-BuO (7.20±0.02 mG/mmHg). (b) Effect of sterilization on the spin density of the LiNc-BuO:PDMS chip. Spin density was estimated using a standard of known spin density, before and after the sterilization procedures. Results (mean ± SD, n=3), normalized to respective untreated control, indicate no significant difference in estimated spin density compared to control in both cases. (c) Effect of high-energy irradiation on the oxygen calibration of the LiNc-BuO:PDMS chip. The oxygen response of the LiNc-BuO:PDMS chip was unaffected by exposure to 30 Gy doses of ionizing radiation, viz. X-rays and 60Co γ-rays. Change in EPR linewidth was linear with increasing oxygen partial pressure, with no significant change in sensitivity (8.14±0.06 mG/mmHg for X-irradiation and 8.08±0.17 mG/mmHg for 60Co γ-irradiation, compared to 8.12±0.01 mG/mmHg for control) (d) Effect of high-energy irradiation on the spin density of the LiNc-BuO:PDMS chip. Spin density (mean ±SD, n=3), calculated using a known standard and normalized to control, revealed no significant difference between irradiated LiNc-BuO:PDMS chips and control (none irradiated) chips. Results show that the functionality of the LiNc-BuO:PDMS chip was unaffected by sterilization and exposure to high-energy radiation, thereby demonstrating its stability and durability

Fig. 2

Effect of oxidoreductant treatment on the LiNc-BuO:PDMS chip. (a) Effect of oxidoreductant treatment on the oxygen calibration of the LiNc-BuO:PDMS chip. LiNc-BuO:PDMS chips were exposed to superoxide (O2 •−) generated using 0.02 U/ml of xanthine oxidase and 0.2 mM of xanthine, nitric oxide (NO) generated using 5-mM S-nitroso-N-penicillamine (SNAP), 1-mM hydrogen peroxide (H2O2), 1-mM ascorbate (Asc) and 5-mM glutathione (GSH) for 30 min. After drying the samples in air, oxygen calibration of the samples was performed, using X-band EPR spectroscopy, for untreated control (Cont) and treated samples. Data are represented as mean ±SD (n=3). The calibration curves show that the response was linear with increase in oxygen partial pressure after treatment with all five agents. Inset shows oxygen sensitivities (slopes of calibration curves) of untreated control and chips treated with the oxidoreductants. Treatment with O2 •−, H2O2, Asc, and GSH did not significantly affect the oxygen sensitivity of the chip. A statistically significant (p<0.05) decrease in sensitivity, compared to control, was observed with NO treatment. However, this decrease was still within an acceptable range for potential applicability. (b) Effect of oxidoreductant treatment on the spin density the LiNc-BuO:PDMS chip. Active spin densities of LiNc-BuO:PDMS chips (untreated control and treated with oxidoreductants) were estimated using X-band EPR spectroscopy. Each treatment group had a separate control, and calculated spin density values were normalized to respective controls (expressed as mean ±SD, n=3). The oxidoreductant treatment did not have any significant effect on the spin density of the LiNc-BuO:PDMS chip. Overall, the results demonstrate that the LiNc-BuO:PDMS chip is biostable and unaffected by treatment with oxidoreductants

Fig. 3

In vitro biocompatibility of the LiNc-BuO:PDMS chip. In vitro biocompatibility was evaluated by assessing proliferation of cultured cells, using anti-BrdU colorimetric immunoassay. Chinese hamster ovary cells (CHO), cisplatin-sensitive human ovarian cancer cells (A2780), cisplatin-resistant human ovarian cancer cells (A2780 cDDP), and smooth muscle cells (SMC) were co-incubated with LiNc-BuO:PDMS chips, in their culture media for 48 h, after which cell proliferation was assessed. Data (mean ±SD, n=6) from cells exposed to chips, normalized to control values, showed no significant difference in cell proliferation (compared to control). Results indicate that the LiNc-BuO:PDMS chip exhibited excellent in vitro biocompatibility

Fig. 4

Long-term in vivo oxygen-sensing response of the LiNc-BuO:PDMS chip. (a) Measurements of pO2, from the gastrocnemius muscle tissue of mice reported by implanted LiNc-BuO:PDMS chips are shown. In vivo pO2 measurements were made using L-band EPR spectroscopy for up to 70 days. pO2 values obtained are expressed as mean ±SD (n=3). Data confirm the ability of the LiNc-BuO:PDMS chip to make repeated measurements of in vivo oxygenation. (b) Response of the LiNc-BuO:PDMS chip blood-flow constriction. Muscle pO2 , in the constricted state, was measured from each animal on three different days during the experiment (presented as mean ±SD). Data show that the implanted chip was capable of responding to changes in pO2. Constricted muscle pO2 values were significantly different (p<0.05) from normal muscle pO2 values (control). Collectively, the data demonstrate that the LiNc-BuO:PDMS chip was capable of reporting oxygenation of murine muscle tissue over a period of 70 days, and possibly longer

Fig. 5

In vivo biocompatibility of the LiNc-BuO:PDMS chip. (a) Images of the tissue sections, stained with hematoxylin and eosin, obtained using a light microscope at four different magnifications (40x, 100x, 200x, and 400x). The region where the implant was present is indicated as ‘C’ in the images, while the normal muscle tissue is depicted as ‘M’. The letters ‘P’ and ‘F’ denote the dark nuclei of polymorphonuclear leukocytes (PMNs), and the pink band of fibrous tissue encapsulating the implant area, respectively. Images demonstrate that the implant triggered an acute inflammatory response, with minimal fibrous encapsulation. Average capsule thickness, estimated using MetaMorph software, was 34.89±11.05µm. (b) Oxygen calibration of the LiNc-BuO:PDMS chip that was explanted from the gastrocnemius muscle tissue of mouse, after long-term implantation (70 days). Peak-to-peak linewidths at different levels of pO2, obtained using L-band EPR spectroscopy, were used to construct the calibration curves. It can be seen that the calibration was linear for both the unimplanted control and the explanted LiNc-BuO:PDMS chip. Data (mean ±SD, n=3) show that the slopes of the two curves (oxygen sensitivity) were not significantly different (8.6±0.64 mG/mmHg for control and 9.18±0.28 mG/mmHg for the explanted chip). Results show that the LiNc-BuO:PDMS chip exhibited good in vivo biocompatibility and biodurability

Fig. 6

In vivo functional testing of the LiNc-BuO:PDMS chip. The functional performance of the LiNc-BuO:PDMS chip was tested in a murine tumor model, which was developed by implanting RIF-1 tumor cells in the hind leg of the mice. (a) Representative MR image of a RIF-1 tumor implanted with a LiNc-BuO:PDMS chip, acquired on an 11.7 T MRI system. White arrow in the image indicates the location of the implanted LiNc-BuO:PDMS chip within the tumor mass. (b) Tumor pO2 measurement using the LiNc-BuO:PDMS chip. L-band EPR spectroscopy was used to monitor the pO2 reported by the LiNc-BuO:PDMS chip implanted in the tumor, as well as, the gastrocnemius muscle tissue on the contralateral side (control). Data are represented as mean ± SD (n=4). Measurements were made on the second day after surgical implantation of the chips in the tumor and muscle. It can be observed that the implanted chips were capable of sensing the hypoxia in the tumors, with a significant difference (p<0.05) compared to muscle (control). (c) Response of the LiNc-BuO:PDMS chip to changes in tumor pO2. The ability of the implanted chip to detect changes in tumor pO2 was tested by switching the breathing gas (delivering the isoflurane anesthesia to the mice) from room air to carbogen (95% O2, 5% CO2). The implanted chips reported a consistent increase in tumor pO2 upon carbogen-breathing. Overall, the results establish the suitability of the LiNc-BuO:PDMS chip for clinically relevant in vivo applications