Localized drug delivery graphene bioscaffolds for cotransplantation of islets and mesenchymal stem cells

Abstract

In the present work, we developed, characterized, and tested an implantable graphene bioscaffold which elutes dexamethasone (Dex) that can accommodate islets and adipose tissue–derived mesenchymal stem cells (AD-MSCs). In vitro studies demonstrated that islets in graphene–0.5 w/v% Dex bioscaffolds had a substantial higher viability and function compared to islets in graphene-alone bioscaffolds or islets cultured alone (P < 0.05). In vivo studies, in which bioscaffolds were transplanted into the epididymal fat pad of diabetic mice, demonstrated that, when islet:AD-MSC units were seeded into graphene–0.5 w/v% Dex bioscaffolds, this resulted in complete restoration of glycemic control immediately after transplantation with these islets also showing a faster response to glucose challenges (P < 0.05). Hence, this combination approach of using a graphene bioscaffold that can be functionalized for local delivery of Dex into the surrounding microenvironment, together with AD-MSC therapy, can significantly improve the survival and function of transplanted islets.

Fig. 1.. Bioscaffold fabrication using template-directed CVD method and characterizations.

(A) Photograph and (B and C) scanning electron microscopy (SEM) image of a nickel foam in low (B) and high (C) magnification. (D to F) Photos of our CVD chamber before (D), during (E), and after (F) the graphene growth on the nickel foam (the arrows indicate the nickel foam placed inside the chamber). The nickel foam was first annealed at 1000°C for 5 min under Ar and H₂ atmosphere with the flow rates of 500 and 200 standard cubic centimeters per minute (sccm), respectively, to clean its surfaces and eliminate the surface oxide layer. The CH₄ gas with the flow rate of 50 sccm was then introduced into the reaction chamber. After 5 min of reaction-gas mixture flow, the foam was rapidly cooled down to room temperature under Ar and H₂ atmosphere with the flow rates of 500 and 200 sccm, respectively. (G) Schematic representation showing graphene bioscaffolds before and after the transfer process of graphene bioscaffold from nickel-graphene foam. The nickel-graphene foam was first coated with a thin polymethyl methacrylate (PMMA) layer to support the graphene structure and prevent its structural failure when the nickel foams is etched away in the next step. Then, the nickel-graphene-PMMA foam was immersed into a mixture of FeCl₃ and HCl at 80°C for 72 hours to dissolve the nickel. Last, graphene bioscaffolds were obtained by dissolving the PMMA with acetone. (H to O) Photos [(H) and (L); showing a self-standing 3D porous structure] and SEM images [(I) to (K) and (M) to (O); showing a monolith of continuous and porous structure, which copied and inherited the interconnected 3D structure of the nickel foam template] of thin (I to K) and thick (M to O) graphene bioscaffolds. SEM images also indicate the Dex microparticles that has been immobilized on the surface of graphene bioscaffolds (K and O). (P and Q) Bioscaffold physicochemical characterization include Raman spectroscopy (P) and x-ray photoelectron spectroscopy (Q). Photo credit: Mehdi Razavi, Stanford University.

Fig. 2.. Bioscaffold coating with Dex.

(A) SEM images from graphene-Dex bioscaffolds with increasing Dex concentrations from 0 to 1 w/v% showing Dex particles that have been attached onto the surface of graphene bioscaffolds and by increasing the concentration of Dex from 0.25 to 1 w/v%, more Dex particles are seen. (B) Dex release profile showing the ability of graphene-Dex bioscaffolds to release Dex for at least 14 days with the release rate significantly increasing as the concentration of Dex increases from 0.25 to 1 w/v% (P < 0.05). Significant differences: (B) ^aP < 0.05: graphene–0.25 w/v% Dex bioscaffolds versus graphene–0.5 w/v% Dex bioscaffolds or graphene–1 w/v% Dex bioscaffolds, ^bP < 0.05: graphene–0.5 w/v% Dex bioscaffolds versus graphene–1 w/v% Dex bioscaffolds; *Day 1 versus days 3, 7, and 14.

Fig. 3.. Bioscaffold interactions with pancreatic islets in vitro.

(A) SEM images of the top surface, and center, of our graphene-Dex bioscaffold seeded with islets. (B) Bright-field images of islets cultured in conventional culture plates. (C) Confocal images of islets cultured in culture plates (islets only) or in graphene-alone bioscaffolds or graphene-Dex bioscaffolds with 0.25, 0.5, and 1 w/v% Dex at day 7. Green represents live cells and red represents dead cells. Results of (D) Live/Dead, (E) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and (F and G) GSIS assays for islets alone and islets in graphene bioscaffolds without, and with, Dex at day 7. Red, dead cells stained with propidium iodide. Green, live cells stained with fluorescein diacetate. Significant differences: (D to G) ^aP < 0.05: islets alone versus graphene and graphene–0.25, 0.5, and 1 w/v% Dex bioscaffolds; ^bP < 0.05: graphene bioscaffolds versus graphene–0.25, 0.5, and 1 w/v% Dex bioscaffolds; ^cP < 0.05: graphene–0.25 w/v% bioscaffolds versus graphene–0.5 and 1 w/v% Dex bioscaffolds; ^dP < 0.05: graphene–0.5 w/v% bioscaffolds versus graphene–1 w/v% Dex bioscaffolds; *Low glucose (LG) versus high glucose (HG).

Fig. 4.. Bioscaffold interactions with pancreatic islets in vivo.

(A) Experimental details of our in vivo experiment and schematic representation of our bioscaffold transplantation in the epididymal fat pad (EFP). Results of (B) nonfasting blood glucose measurements, (C) percentage of normoglycemia, (D) body weight, (E) IPGTT [i.e., changes of fasting blood glucose versus baseline (0-min time point)], and (F) results of calculation of area under the curve (AUC_0–120min) of IPGTT curves (i.e., glucose clearance rate). (G) Photographs of the transplantation procedure of islets alone and islet:AD-MSCs units in graphene–0.5 w/v% Dex bioscaffolds. Photo credit: Mehdi Razavi, Stanford University. Significant differences: (B to G) ^aP < 0.05: islets alone versus islets in graphene-alone bioscaffolds or islets in graphene–0.5 w/v% Dex bioscaffolds or islet:AD-MSCs units in graphene–0.5 w/v% Dex bioscaffolds; ^bP < 0.05: islets in graphene-alone bioscaffolds versus islets in graphene–0.5 w/v% Dex bioscaffolds or islet:AD-MSCs units in graphene–0.5 w/v% Dex bioscaffolds; ^cP < 0.05: islets in graphene–0.5 w/v% Dex bioscaffolds versus islet:AD-MSCs units in graphene–0.5 w/v% Dex bioscaffolds. (B) *P < 0.05: baseline (day −2) versus all other time points [two-way ANOVA (analysis of variance), Tukey post hoc test]. (C and D) *P < 0.05: post-transplant week 0 versus post-transplant weeks 1, 2, 3, and 4 (two-way ANOVA, Tukey post hoc test). (E) *P < 0.05: 0 min versus 30, 60, 90, and 120 min (two-way ANOVA, Tukey post hoc test). (F) One-way ANOVA, Tukey post hoc test.

Fig. 5.. Histological and molecular analyses.

(A) Representative histological [hematoxylin and eosin (H&E) staining] and immunohistochemical images [insulin, von Willebrand factor (vWF), and tumor necrosis factor–α (TNF-α) staining] of the EFP containing islets alone or islets into graphene-alone and graphene–0.5 w/v% Dex bioscaffolds or islet:AD-MSCs units into graphene–0.5 w/v% Dex bioscaffolds. Red stars, islets; black arrows, bioscaffolds; blue arrows, positive (brown) staining. (B) Cytokine expression profile within the EFP tissue. The level of insulin within the (C) EFP and (D) blood serum. Quantification of positive (E) insulin, (F) vWF, and (G) TNF-α staining. Results were analyzed with at least 15 to 20 islets from five different sections through the EFP of each animal. Significant differences: (B) ^aP < 0.05: islets in graphene-alone bioscaffolds versus islets in graphene–0.5 w/v% Dex bioscaffolds or islets:AD-MSCs units in graphene–0.5 w/v% Dex bioscaffolds; ^bP < 0.05: islets in graphene–0.5 w/v% Dex bioscaffolds versus islets:AD-MSCs units in graphene–0.5 w/v% Dex bioscaffolds; *P < 0.05: islets alone (control) versus islets in graphene-alone bioscaffolds or islets in graphene–0.5 w/v% Dex bioscaffolds or islet:AD-MSCs units in graphene–0.5 w/v% Dex bioscaffolds (one-way ANOVA, Tukey post hoc test). (C to G) ^aP < 0.05: islets alone versus islets in graphene-alone bioscaffolds or islets in graphene–0.5 w/v% Dex bioscaffolds or islet:AD-MSCs units in graphene–0.5 w/v% Dex bioscaffolds; ^bP < 0.05: islets in graphene-alone bioscaffolds versus islets in graphene–0.5 w/v% Dex bioscaffolds or islets:AD-MSCs units in graphene–0.5 w/v% Dex bioscaffolds; ^cP < 0.05: islets in graphene–0.5 w/v% Dex bioscaffolds versus islets:AD-MSCs units in graphene–0.5 w/v% Dex bioscaffolds.

Fig. 6.. Bioscaffold biodegradability and biocompatibility.

(A) Biodegradation profile and photographic of graphene–0.5 w/v% Dex bioscaffolds before and after incubation in PBS for 3 months. (B) Photographic and representative histological (H&E staining) of the (C) EFP and (D) subcutaneous tissue implanted with graphene–0.5 w/v% Dex bioscaffolds. Red arrow, bioscaffold; blue arrows, blood vessels (photographs and H&E staining images). Representative TNF-α staining of the (E) EFP and (F) subcutaneous tissue implanted with graphene–0.5 w/v% Dex bioscaffolds. Black arrow, bioscaffold; red arrows, positive (brown) staining. Photo credit: Mehdi Razavi, Stanford University.