2.1.

Preparation of OSMs

OSMs are made from an oxygen-sensitive dye embedded within a polystyrene matrix. Two types of OSMs were fabricated, each comprising a unique oxygen-sensing metalloporphyrin dye. The first is platinum(II) meso-tetraphenyl tetrabenzoporphine (PtTPTBP, Frontier Scientific, Logan) and the second is platinum(II) meso-tetra(pentafluorophenyl)porphine (PtTFPP, Frontier Scientific, Logan). OSMs are fabricated by first mixing 2 mg of dye with 30 mg of polystyrene ($M_{\mathrm{w}}$ 2500, Sigma, St. Louis) and then dissolving the mixture in $450\mu \mathrm{l}$ of chloroform (Sigma). The solution is pipetted and spread onto a glass slide to polymerize overnight in room air at 23°C. The resulting thin film is scraped off with a razor blade and crushed into small micron-sized particles using a glass mortar and pestle.

2.2.

Encapsulation of OSMs Within Alginate Beads

Clinical grade ultrapure low viscosity alginate (UPLVM, Novamatrix, Sandvika, Norway) is dissolved in deionized water at 3 wt. % and sterile filtered using a $0.22\mu \mathrm{m}$ syringe filter. OSMs are suspended in 1.5 mL of the UPLVM solution and loaded into an encapsulator (Nisco, Zurich, Switzerland) for the fabrication of alginate beads. Specifically, the solution is first loaded into a syringe and then driven through a 25 G needle under positive gas pressure (4 PSI, $N_2$). The needle is held at 9 V relative to a 120 mM ${\mathrm{CaCl}}_2$ solution within a Petri dish placed below the tip of the needle. Alginate beads form at the tip of the needle and fall into the ${\mathrm{CaCl}}_2$ solution for alginate polymerization. A typical OSM alginate bead is shown in Fig. 1. Bead size is dependent on gas pressure, voltage, and the distance between the needle and media in the dish. For these experiments, the average diameter of alginate beads was found to be $415\pm 47.68\mu \mathrm{m}$.

Fig. 1.

An alginate capsule containing oxygen-sensitive microparticle (OSM) sensors.

3.1.

Optical Probe for In Vitro Experiments

An optical probe was constructed to simultaneously measure both OSM types (Fig. 2). The two OSM types are spectrally separated. PtTFPP OSMs have multiple absorption peaks at 390, 504, and 538 nm and emission peaks at 647 and 710 nm.²² PtTPTBP OSMs have multiple absorption peaks at 430 and 614 nm and an emission peak at 770 nm.²² The optical probe can selectively excite either type of OSM with two pairs of light emitting diode (LEDs) (Rebel LED, Luxeon Star, Brantford, Canada) centered at 530 nm (green) and 617 nm (red) for PtTFPP and PtTPTBP, respectively. The LEDs are mounted onto a custom three-dimensional printed conical mount designed to pitch the LEDs such that emitted light converges onto samples placed just above the surface of the cone. The conical mount is glued onto the distal surface of a 2-in. long lens tube (Thorlabs, Newton) containing a 6.4-mm diameter image conduit (Edmund Optics, Barrington) and a focusing lens ($f=25\mathrm{mm}$). The distal end of the lens tube is mounted onto a filter cube holder (Thorlabs) containing a long-pass dichroic beam splitter (FF740-Di01, Semrock, Rochester) to reflect PtTFPP and transmit PtTPTBP emissions, respectively. Two amplified photodiode detectors (PDA100A, 9.8-mm diameter, $\text{Gain}=30$, Thorlabs) are mounted onto the cube to detect emissions from each OSM type. LED emission and digitalization of photodiode signals are conducted by a National Instruments data acquisition device (USB6361 DAQ, National Instruments, Austin) controlled by LabView (National Instruments).

Fig. 2

Illustration of optical probe used for in vitro measurement. Emitted light from the two OSM types is separated spectrally onto two separate detectors.

3.2.

Determining Oxygen Partial Pressure From OSM Emission

OSMs were calibrated from measurements acquired at different ${\mathrm{pO}}_2$ levels, as provided by four gas tanks containing different oxygen mixtures. OSM emission was analyzed using the method of luminescence lifetime. OSMs were optically excited by pulsing LEDs in a 100 Hz square-wave with a 50% duty cycle. Optical signals were sampled at 1 MHz. OSMs were probed every 20 s, where at each time point, 25 measurements were acquired at 100 Hz and averaged. The metalloporphyrin dye within OSMs continues to emit light after the cessation of each LED light pulse. This emission is modeled as an exponential decay as

3.3.

In Vitro Measurement of Oxygen Partial Pressure Within Alginate Beads

Alginate beads containing PtTPTBP OSMs and unencapsulated PtTFPP OSMs were suspended in 1 mL of media phosphate buffered saline (PBS) within a single well of a 24-well dish (Corning Inc., Corning) in which gas was bubbled (Fig. 3) with either compressed air (21% oxygen), or precalibrated mixtures of 10% oxygen/90% nitrogen, 5% oxygen/95% nitrogen, or argon gas (0% oxygen).

Fig. 3

(a) In vitro test system. Two spectrally distinct OSM types are suspended in a PBS bath within a well: one to monitor the PBS (green circles) and one to monitor the alginate capsules (red circles). (b) Close up view showing PBS and encapsulated OSMs within a well.

Figure 4 plots ${\mathrm{pO}}_2$ as reported by both the encapsulated and nonencapsulated OSMs versus time for a series of gas exchanges. The ${\mathrm{pO}}_2$ levels were calculated by first computing the mean steady state lifetime values for each gas concentration using Eq. (1). Regression of lifetime against gas concentration as computed by Eq. (2) ($R^2>0.99$) provides a calibration equation mapping lifetime to ${\mathrm{pO}}_2$. Data confirms oxygen sensitivity within the alginate bead and no significant dynamic delay between the PBS and the volume of the bead (Fig. 4).

Fig. 4

In vitro measurements. ${\mathrm{pO}}_2$ for a set of gases containing oxygen at 21%, 10%, 5%, and 0%. Black line: ${\mathrm{pO}}_2$ in PBS and red line: ${\mathrm{pO}}_2$ in alginate capsules.

4.1.

In Vivo Probe

A compact in vivo optical probe was constructed for PtTPTBP OSMs alone [Fig. 5(a)]. Since only one dye is being measured in vivo there is no need for the more complex optical setup used in the in vitro experiment. Two of the 617-nm centered LEDs were mounted on the conical mount, as described above. The conical mount was then placed directly on the photodiode module and aligned with the active area of the photodiode. A 715 nm high-pass optical filter (Semrock) is mounted above the surface of the photodiode. The conical mount is placed directly onto the skin during measurements.

Fig. 5

In vivo measurements. (a) Optical probe used for in vivo measurements. (b) Day 5 ${\mathrm{pO}}_2$ and ${\mathrm{SpO}}_2$. Animals were anesthetized during measurements with a mixture of isoflurane and 100% oxygen.

4.2.

In Vivo ${\mathrm{pO}}_2$

In vivo testing was performed to determine if PtTPTBP OSM signals can be related to ${\mathrm{pO}}_2$ within subcutaneously implanted alginate beads. A small incision was made on the anterior side of an anesthetized rat (Sprague–Dawley, Charles River Laboratories, Wilmington). Anesthesia was maintained with a gas mixture of 100% oxygen and vaporized isoflurane delivered to a nose cone placed over the snout. OSM-containing beads were then implanted subcutaneously via pipette injection in a small pocket formed in the subcutaneous space by blunt dissection. The incision was closed using a wound sealing adhesive (Gluture, Abbott, Abbott Park). ${\mathrm{pO}}_2$ within beads was then optically probed on demand through the skin. Rats were fixed with a rodent pulse oximeter (Kent Scientific, Torrington) placed on the front paw to simultaneously measure oxygen saturation of blood hemoglobin (${\mathrm{SpO}}_2$) and heart rate.

Figure 5(b) shows ${\mathrm{pO}}_2$ data acquired on day 5 post implantation during anesthesia. As expected, ${\mathrm{SpO}}_2$ was constant at normal physiological levels (95% to 100%) and ${\mathrm{pO}}_2$ within the beads was $\sim 100\mathrm{mm}\mathrm{Hg}$, indicating gas exchange both through the vasculature and skin.

The data collected in vivo was analyzed for its accuracy. Decay curves were collected as described above (measurements taken every 10 s with 25 curves for each measurement). After averaging the 25 curves, the variance at each point was found to be $<0.5\%$ of the mean value [Eq. (1)]. In the absence of OSMs, the detector returns an exponential decay of Tau $\sim 15\mu \mathrm{s}$. To determine the weight of this effect on OSM-mediated signals, the mean signal value between 0 and $50\mu \mathrm{s}$ of OSM-mediated signals was divided by that for the OSM-free signals. The ratio was $>7$, which we found was insignificant for fitting OSM lifetimes.

4.3.

Variable Inhaled Gas Experiment

We developed an assay to measure transport dynamics between the microbeads and local vasculature. Such an assay is important since it captures effects of both increase/decrease of local vasculature resulting from the implantation wound and subsequent healing as well as the deposition of new collagenous tissue, which forms an additional diffusional barrier. Inhaled gas is rapidly changed as ${\mathrm{pO}}_2$ and ${\mathrm{SpO}}_2$ are monitored. Oxygen dynamics after each gas change are governed by tissue architecture, with rise/fall times increasing or decreasing as the local tissue rejects or perfuses the implant, respectively.

For these tests, the 100% oxygen + isoflurane inhaled gas was transiently exchanged with room air (21% oxygen) + isoflurane and then returned to 100% oxygen + isoflurane. OSMs were probed every 10 s. Measurements were recorded on days 5 and 9 post implantation.

Figure 6(a) shows a typical variable inhaled gas experiment at day 5. ${\mathrm{pO}}_2$ and ${\mathrm{SpO}}_2$ exhibited dynamics in the early stages of anesthesia (first 30 min) and eventually reached a steady state when breathing the 100% gas mixture. This phenomenon was common to most of our experiments and is likely due to the physiological effects of isoflurane. The gas was changed to the room air mixture for $\sim 10\min$ and then returned to the 100% ${\mathrm{O}}_2$ mixture for $\sim 25\min$. ${\mathrm{pO}}_2$ within beads did not reach a steady state within the 10 min of room air, but did within the 25 min of 100% ${\mathrm{O}}_2$. Similar results were observed for the second room air challenge beginning at $\sim 72\min$.

Fig. 6

(a) Variable inhaled gas experiment. On day 5, ${\mathrm{SpO}}_2$ and ${\mathrm{pO}}_2$ within capsules consistently exhibit dynamics after the initiation of anesthesia, stabilizing after approximately 30 min. At 37 min, the percent oxygen mixed with isoflurane was changed from 100% to 21% for 11 min and then switched back. The same exchange in inhaled gas is repeated 26 min later. (b) On day 9, ${\mathrm{pO}}_2$ within capsules exhibits faster dynamics following a gas exchange as compared to day 5.

The variable inhaled gas experiment was repeated for the same rat on day 9 [Fig. 6(b)]. Here, ${\mathrm{pO}}_2$ reaches a steady state within the $\sim 10\min$ of room air gas indicating rapid ${\mathrm{O}}_2$ exchange between the beads and tissues as compared to day 5, an effect most likely mediated by tissue remodeling.