Bioorthogonal layer-by-layer encapsulation of pancreatic islets via hyperbranched polymers

Abstract

Encapsulation of viable tissues via layer-by-layer polymer assembly provides a versatile platform for cell surface engineering, with nanoscale control over the capsule properties. Herein, we report the development of a hyperbranched polymer-based, ultrathin capsule architecture expressing bioorthogonal functionality and tailored physiochemical properties. Random carbodiimide-based condensation of 3,5-dicarboxyphenyl glycineamide on alginate yielded a highly branched polysaccharide with multiple, spatially restricted, and readily functionalizable terminal carboxylate moieties. Poly(ethylene glycol) (PEG) was utilized to link azido end groups to the structured alginate. Together with a phosphine-functionalized poly(amidoamine) dendrimer, nanoscale layer-by-layer coatings, covalently stabilized via Staudinger ligation, were assembled onto solid surfaces and pancreatic islets. The effects of electrostatic and/or bioorthogonal covalent interlayer interactions on the resulting coating efficiency and stability, as well as pancreatic islet viability and function, were studied. These hyperbranched polymers provide a flexible platform for the formation of covalently stabilized, ultrathin coatings on viable cells and tissues. In addition, the hyperbranched nature of the polymers presents a highly functionalized surface capable of bioorthogonal conjugation of additional bioactive or labeling motifs.

Figure 1. Characterization of hyperbranched alginate

(A) ATR-FT-IR spectra of (I) alginate, (II) 3,5-dicarboxyphenylglycineamide, (III) Hyperbranched alginate, (IV) hyperbranched alginate azide, and (V) fluorescent hyperbranched alginate azide. (B) Proton NMR of hyperbranched alginate azide.

Figure 2. Characterization of PAMAM dendrimers

(A) ATR-FT-IR spectra of (I) PAMAM 15/0, (II) PAMAM 30/0, (III) PAMAM 15/20, and (IV) PAMAM 15/40. (B) Proton NMR of PAMAM 30/0.

Figure 3

Assessment of particle size via dynamic light scattering (DLS) for PAMAM 15/0 (3 mg/mL), Hyp-Alg-Azide (3 mg/mL), and a mix of PAMAM 15/0 (3 mg/mL) with Hyp-Alg-Azide (0.15 mg/mL or 5%). Error=SD; N=3.

Figure 4. Layer-by-layer assembly of functionalized hyperbranched alginate and PAMAM polymers on planar substrates

(A) Selectivity and stability of azide-functionalized Si wafer illustrated via incubation of silicone azide surface with mPEG-N₃ (control), followed by incubation with mPEG-MDT (bioorthogonal conjugation). Wafer was subsequently washed with PBS (Wash 1–3), followed by 4 M NaCl (Wash 4–6). (B) Ellipsometry film thickness measurements on planar azide-functionalized Si surfaces after deposition of 12 alternating polymeric layers of either: (1) PAMAM 30/0 with hyperbranched Alg-N₃; (2) PAMAM 15/0 with hyperbranched Alg-N₃; and (3) PAMAM 15/40 with hyperbranched Alg-N₃. Controls consisted of (4) PAMAM 0/0 with hyperbranched alginate, (5) PAMAM 15/40 with hyperbranched alginate, and (6) PAMAM 15/40 with Alg-N₃ (not hyperbranched). Error = standard deviation. (C) ATR-FT-IR spectra of the deposited films (2) (solid line) and (3) (dashed line). (D) Elemental composition of surfaces of Si wafers by X-ray photoelectron spectroscopy.

Figure 5. Coating uniformity and cell viability of primary rat pancreatic islets following 3-layer encapsulation via PAMAM and alginate

Single plane, confocal fluorescent images of rat islets cross-section 24 h post-encapsulation with 3-layer coatings (Images). Islet viability, via MTT metabolic assay, normalized to control (Graph). Groups: uncoated (Control); 3-layer coating using either PAMAM 15/0 (1), 15/20 (2), 15/30 (3), or 15/40 (4), interlayered with fluorescently-labeled hyperbranched Alg-N₃; and NHS-PEG-N₃, followed by 3-layer coating using PAMAM 15/40 interlayered with fluorescently-labeled hyperbranched Alg-N₃. Error = standard deviation. Scale bar = 200 μm. * P < 0.05

Figure 6. Six-layer encapsulation of primary rat pancreatic islets via layer-by-layer assembly of alginate and PAMAM

Evaluation of capsule formation via fluorescein labeled alginate and confocal z-stack projection (A–C) or single plane (a–c) imaging of rat pancreatic islets 24 hr after coating. Groups: (1) electrostatic assembly via three bilayers of PAMAM 15/0 and fluorescently-labeled hyperbranched Alg-N₃; (2) primary layer of NHS-PEG-N₃, followed by three bilayers of PAMAM 15/20 and fluorescentlylabeled hyperbranched Alg-N₃; and (3)primary layer of NHS-PEG-N₃, followed by three bilayers of PAMAM 15/40 and fluorescently-labeled hyperbranched Alg-N₃. Scale bar = 50 μm

Figure 7. TEM images of primary rat pancreatic islets coated with 6 layers via layer-by-layer assembly of alginate and PAMAM

Groups: (1) electrostatic assembly via three bilayers of PAMAM 15/0 and fluorescently-labeled hyperbranched Alg-N₃; (2) primary layer of NHS-PEG-N₃, followed by three bilayers of PAMAM 15/20 and fluorescently-labeled hyperbranched Alg-N₃; and (3) primary layer of NHS-PEG-N₃, followed by three bilayers of PAMAM 15/40 and fluorescently-labeled hyperbranched Alg-N₃. Scale bar = 1 μm

Figure 8

Assessment of islet viability and function after 6-layer coating via MTT metabolic assay (A), glucose-stimulated insulin release (B), and live/dead confocal microscopy imaging (bottom panel; blue, nuclei; live, green; dead, red). Groups: (1) electrostatic assembly via three bilayers of PAMAM 15/0 and fluorescently-labeled hyperbranched Alg-N₃; (2) primary layer of NHS-PEG-N₃, followed by three bilayers of PAMAM 15/20 and fluorescently-labeled hyperbranched Alg-N₃; and (3)primary layer of NHS-PEG-N₃, followed by three bilayers of PAMAM 15/40 and fluorescently-labeled hyperbranched Alg-N₃. Note: nonspecific binding of the nuclei staining (ethidium homodimer-1, red fluorescence) was observed for (1). *P < 0.05 **P < 0.01 Scale bar = 50 μm

Figure 9. Bioorthogonal tethering of FITC-PEG-MDT to islets following layer-by-layer encapsulation

Confocal planar slice image of uncoated control islets incubated in FITC-PEG₅₀₀₀-MDT (A); islet coated with 6 layers of PAMAM 15/20 and hyperbranched alginate-N₃, followed by incubation with FITC-PEG₅₀₀₀-CH₃ (B); and islet coated with 6 layers of PAMAM 15/20 and hyperbranched alginate-N₃, followed by incubation with FITC-PEG₅₀₀₀-MDT (C). Scale bar = 50 μm.

Scheme 1

Synthetic steps for generation of hyperbranched Alg-N3 and MDT/GA PAMAM, specifically: (1) 3,5-dicarboxyphenyl glycineamide; (2) PAMAM functionalized with MDT and GA; and (3) hyperbranched alginate azide. Key: (a) Chloroacetyl chloride, NaOH; (b) NH₃; (c) EDC, NHS; (d) H₂N-PEG-N₃, EDC, NHS; and (e) 2-(diphenylphosphino)terephthalic acid 1-methyl-4-pentafluorophenyl diester, triethylamine, and glutaric anhydride.

Scheme 2. Schematic of ultrathin coating assembly

For electrostatic assembly, MDT functionalized PAMAM (2) is deposited directly onto the islet surface via electrostatic interactions. For covalent assembly, N₃-PEG-NHS is first covalently bound to free amines on the islet surface (1), followed by covalent linkage of MDT functionalized PAMAM (2). Subsequently, hyperbranched alginate azide is covalently linked to the exposed MDT functionalized PAMAM coating (3). Interlayer covalent bonds are formed between complementary azide and MDT groups via Staudinger ligation (inset reaction scheme). Additional layers can be built via step-wise incubation of MDT functionalized PAMAM (2) and hyperbranched alginate azide (3), until desired number of layers is achieved.