Basement Membrane-Based Glucose Sensor Coatings Enhance Continuous Glucose Monitoring in Vivo

Abstract

Background: Implantable glucose sensors demonstrate a rapid decline in function that is likely due to biofouling of the sensor. Previous efforts directed at overcoming this issue has generally focused on the use of synthetic polymer coatings, with little apparent effect in vivo, clearly a novel approach is required. We believe that the key to extending sensor life span in vivo is the development of biocompatible basement membrane (BM) based bio-hydrogels as coatings for glucose sensors.

Method: BM based bio-hydrogel sensor coatings were developed using purified BM preparations (ie, Cultrex from Trevigen Inc). Modified Abbott sensors were coated with Cultrex BM extracts. Sensor performance was evaluated for the impact of these coatings in vitro and in vivo in a continuous glucose monitoring (CGM) mouse model. In vivo sensor function was assessed over a 28-day time period expressed as mean absolute relative difference (MARD) values. Tissue reactivity of both Cultrex coated and uncoated glucose sensors was evaluated at 7, 14, 21 and 28 days post-sensor implantation with standard histological techniques.

Results: The data demonstrate that Cultrex-based sensor coatings had no effect on glucose sensor function in vitro. In vivo glucose sensor performance was enhanced following BM coating as determined by MARD analysis, particularly in weeks 2 and 3. In vivo studies also demonstrated that Cultrex coatings significantly decreased sensor-induced tissue reactions at the sensor implantation sites.

Conclusion: Basement-membrane-based sensor coatings enhance glucose sensor function in vivo, by minimizing or preventing sensor-induced tissues reactions.

Keywords: Trevigen; basement membrane; biocompatibility; continuous glucose monitoring; extracellular matrices; glucose sensor coatings; implantable glucose sensor.

Figure 1.

Crystal formation in predialyzed and postdialyzed Trevigen Cultrex BM preparations. Commercial Cultrex BM preparations were dialyzed against water, using a 3500 MW cutoff “slid-A-lizer” dialysis system from Life Technologies (Thermo scientific). Trevigen’s Cultrex BM preparations were evaluated directly or after dialysis against water dehydration and rehydration patterns in vitro on glass microscope slides. A total of 50 ul of Cultrex (a) or dialyzed Cultrex (e) was spotted on glass slides and allowed to dry at 37°C. Nondialyzed Cultrex dried with significant crystal formation (b), but no significant amount crystals formation was seen on the dialyzed Cultrex (f). Rehydration of both of the dehydrated Cultrex preparation resulted in formation of “gelatin” drops similar to the original Cultrex drops seen prior to dehydration (c and g). Dehydration of the rehydrated Cultrex preparations resulted in identical crystal formation as was seen after the first dehydration (b and f).

Figure 2.

Coating of glucose sensors in vitro with Cultex BM preparations. To allow simple and reproducible coating of glucose sensors with Cultrex for in vitro and in vivo studies a clamp based system which utilizes a standard magnetic office clamp (OC), a modified Abbott glucose sensor (GS), and a polytetrafluoroethylene sheet (PTFE) was utilized (A). The Abbott sensor is centered on top of the PTFE sheet (B), and 50 ul of dialyzed Cultrex is added on top of the sensor (C) and allowed to dry at 37°C, resulting in a thin protein layer on the sensor and associated PTFE sheet (D, red dotted line). The sensor is then removed from the PTFE sheet, flipped over and an additional 50 ul of dialyzed Cultrex is added (E) and allowed to dry (F). This process can be repeated as needed to form a Cultrex coating on the sensor. Finally the sensor with a dry Cultrex coating is removed from the PTFE sheet (g), dipped in sterile water (h) and then allow to dry at 37°C until dry (i). The resulting Cultrex sensor is then utilized for in vitro studies or implanted subcutaneously for in vivo studies.

Figure 3.

The Impact of Cultrex coating on continuous glucose monitoring (CGM) in vivo. To determine the impact of Cultrex coatings on sensor function we utilized our murine model of CGM. For these studies Cultrex coated Abbott sensors were compared with uncoated Abbott sensor implanted subcutaneously in mice over a 4-week time period. The resulting sensor output (nA) and actual blood glucose levels were evaluated using standard MARD analysis using Mann–Whitney U tests and Student t tests. Cultrex coated sensors preformed significantly better then uncoated sensors over the entire 4-week time period (total mean MARD P = .0114). Cultrex coated sensor performed statistically better then uncoated sensors at 2 weeks (P = .0058) and 3 weeks (P = .0303) post–sensor implantation. Although sensor performance was better in the Cultrex coated sensor at week 4, it was not statistically significant (P = .1774).

Figure 4.

Tissue reactions induced in murine models of CGM by Cultrex only, glucose sensors only, and Cultrex coated glucose sensors. Cultrex and Abbott sensors (with and without Cultrex coatings) were obtained as implantation sites at 7, 14, 21, and 28 days postimplantation, processed for standard histopathology using paraffin embedding and H&E staining. Tissue reactions to Cultrex only implantations demonstrated that Cultrex (smooth pink staining) was very biocompatible and it did not induces any significant tissue reactions over the 28-day test period (a-d). As expected sensor only (black bands are remnants of sensor) implantation sites displayed significant tissue reaction characterized by implantation and fibrosis (e-h). Tissue reactions to Cultrex coated sensors (i-l) demonstrated that the Cultrex coating minimized tissue reactions to the glucose sensors, particularly in the first 3 weeks (i-k), but by the 4 weeks post–sensor implantation there was significant degradation of the Cultrex, exposure of the glucose sensor and induction of tissue reactions (l). Glucose sensor performance generally followed the same pattern as was seen with the Cultrex coatings, that is, early time points (1-3 weeks) have intact Cultrex coatings and good sensor performance, but as the Cultrex degraded exposing the glucose sensor, resulting in increase tissue reactions and sensor performance decreased.