In-Vivo fluorescent nanosensor implants based on hydrogel-encapsulation: investigating the inflammation and the foreign-body response

Abstract

Nanotechnology-enabled sensors or nanosensors are emerging as promising new tools for various in-vivo life science applications such as biosensing, components of delivery systems, and probes for spatial bioimaging. However, as with a wide range of synthetic biomaterials, tissue responses have been observed depending on cell types and various nanocomponent properties. The tissue response is critical for determining the acute and long term health of the organism and the functional lifetime of the material in-vivo. While nanomaterial properties can contribute significantly to the tissue response, it may be possible to circumvent adverse reactions by formulation of the encapsulation vehicle. In this study, five formulations of poly (ethylene glycol) diacrylate (PEGDA) hydrogel-encapsulated fluorescent nanosensors were implanted into SKH-1E mice, and the inflammatory responses were tracked in order to determine the favorable design rules for hydrogel encapsulation and minimization of such responses. Hydrogels with higher crosslinking density were found to allow faster resolution of acute inflammation. Five different immunocompromised mice lines were utilized for comparison across different inflammatory cell populations and responses. Degradation products of the gels were also characterized. Finally, the importance of the tissue response in determining functional lifetime was demonstrated by measuring the time-dependent nanosensor deactivation following implantation into animal models.

Keywords: Carbon nanotube; Hydrogel; Implants; Inflammation; Nanosensor.

Fig. 1

In vitro sensor characterization. (a) Fluorescence emission spectrum at 785 nm excitation of SM8-3-wrapped HiPCO SWNT encapsulated in PEGDA-8000 hydrogel. (b) Fluorescence emission spectrum at 785 nm excitation of ss(AAAT)₇-wrapped (6,5) CoMoCAT SWNT encapsulated in PEGDA-8000 hydrogel. (c)SM8-3-wrapped HiPCO SWNT hydrogel fluorescence increases with an addition of 100 μm progesterone in 1X PBS solution (n = 3). (d) (AAAT)₇-wrapped (6,5) CoMoCAT SWNT fluorescence decreases with an addition of 100 μm riboflavin in 1X PBS solution. (e) Schematics of synthesis of the PEGDA hydrogels: monomer mixed with SWNT solution and initiator, degassed, pipped between two glass slides to control for thickness, and exposed to UV light to initiate free radical polymerization

Fig. 2

(a) Hydrogel pore sizes obtained via swelling experiments. (b) Compressive moduli (data presented as mean ± SEM, n = 2 for Formulation 3, and n = 3 for the rest, P-values are calculated using two-tailed Students’ t-test, *P < 0.05) of hydrogels obtained from the linear regions of dynamic mechanical analysis (Figure S1)

Fig. 3

Images of mice implanted with PEGDA 8000 and PEGDA 1000 hydrogels. Mice with PEGDA 8000 hydrogels showed observable swelling in the vicinity of the implants on day 7, whereas none were noticed with the PEGDA 1000 hydrogels

Fig. 4

H&E stained tissue samples of SKH1-E taken from implant sites of hydrogels. Hydrogels of formulations 1–5 were explanted at days 1, 7, 14, and 28. The hydrogel itself or locations of the hydrogels are marked by arrows. In all formulations, we see heavy neutrophilic infiltration on day 1, with less on day 14, and resolution by day 28. The severity of acute inflammation is higher in formulations 1 and 2 compared to 4 and formulation 3 relative to formulation 5. Formulation 3, however, has fewer neutrophils than formulations 1 and 2. All formulations show an increase in edema, neovascularization, and fibrosis with time. Images were taken at 20x magnification, and gels are indicated by red arrows

Fig. 5

Tissue response scores for the (a) implant site, (b) tissue surrounding the implant, (c) fibrosis, (d) edema, (e) neovascularization, and (f) total adverse tissue reaction. The inflammation at and surrounding the implant site, edema, and neovascularization were rated on a scale of 0 to 4: 0 is absent, 1 is minimal, 2 is mild, 3 is moderate, and 4 is severe. Fibrosis was rated on a scale of 1 to 3, with 1 being only a mild fibrous encirclement, 2 being moderate or poorly organized fibrous encirclement, and 3 being a well-organized and epithelioid histiocytic cap. The total adverse tissue reaction was obtained by summing all the components except fibrosis. (data presented as mean, n = 3 for formulation 3, and n = 2 for the other formulations)

Fig. 6

Masson’s Trichrome stained tissue samples imaged at 4x magnification. The progression of healing can be seen by observing the regions of blue, representing fibrous tissue, increasing from day 7 to day 28. Formulations 3–5 appear to have slightly more organized fibrous regions at day 7 compared to formulations 1 and 2, indicating faster healing with smaller pore sizes, as well as lower SWNT concentrations. The hydrogel itself or locations of the hydrogels are marked by arrows

Fig. 7

(a) Summary of the functional and dysfunctional inflammatory cells in five mice lines used in this study. Hydrogel sensors were implanted for a time period (1 min, 2 h, 24 h), explanted, and tested outside the mice against 100 µM progesterone. (b) The maximum sensitivity decreased with increasing implantation time in general. (c) For all mice lines, the kinetics of response slowed with longer implantation time