Elsevier

Acta Biomaterialia

Volume 8, Issue 5, May 2012, Pages 1685-1692
Acta Biomaterialia

Self-assembling glucagon-like peptide 1-mimetic peptide amphiphiles for enhanced activity and proliferation of insulin-secreting cells

https://doi.org/10.1016/j.actbio.2012.01.036Get rights and content

Abstract

Current treatment for type 1 diabetes mellitus requires daily insulin injections that fail to produce physiological glycemic control. Islet cell transplantation has been proposed as a permanent cure but is limited by loss of β-cell viability and function. These limitations could potentially be overcome by relying on the activity of glucagon-like peptide 1 (GLP-1), which acts on β-cells to promote insulin release, proliferation and survival. We have developed a peptide amphiphile (PA) molecule incorporating a peptide mimetic for GLP-1. This GLP-1-mimetic PA self-assembles into one-dimensional nanofibers that stabilize the active secondary structure of GLP-1 and can be cross-linked by calcium ions to form a macroscopic gel capable of cell encapsulation and three-dimensional culture. The GLP-1-mimetic PA nanofibers were found to stimulate insulin secretion from rat insulinoma (RINm5f) cells to a significantly greater extent than the mimetic peptide alone and to a level equivalent to that of the clinically used agonist exendin-4. The activity of the GLP-1-mimetic PA is glucose-dependent, lipid-raft dependent and partially PKA-dependent consistent with native GLP-1. The GLP-1-mimetic PA also completely abrogates inflammatory cytokine-induced cell death to the level of untreated controls. When used as a PA gel to encapsulate RINm5f cells, the GLP-1-mimetic PA stimulates insulin secretion and proliferation in a cytokine-resistant manner that is significantly greater than a non-bioactive PA gel containing exendin-4. Due to its self-assembling property and bioactivity, the GLP-1-mimetic PA can be incorporated into previously developed islet cell transplantation protocols with the potential for significant enhancement of β-cell viability and function.

Introduction

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by immune-mediated cell death of insulin-producing β-cells of the pancreatic islets of Langerhans [1], [2]. Current treatment with daily insulin injections fails to achieve the strict glycemic control observed in healthy individuals, leading to progressive secondary pathologies that decrease patient quality of life and lead to adverse clinical outcomes including kidney failure, blindness and limb amputation [3]. To alleviate these sequelae of inadequate glycemic control and to free patients from the burden of daily insulin injections, islet cell transplantation (ICT) has been proposed as a permanent treatment for T1DM [4]. The Edmonton protocol for intrahepatic ICT has achieved insulin independence in up to 80% of patients for a median of 3 years [5], [6] but is limited by the loss of transplanted β-cell mass and function due to immune-mediated and inflammation-induced apoptosis [7], [8], lack of vascularization [9], decreased proliferative potential [10] and impaired insulin secretion [11]. Current approaches to preventing the loss of β-cell mass and function resulting from these deleterious phenomena include the use of biomaterial scaffolds to control the islet microenvironment [12] and the addition of biological functionality to islets through genetic modification [13], substrate immobilization [14] or ligand presentation [15], [16], [17].

One source of biological functionality for the enhancement of ICT is the action of glucagon-like peptide 1 (GLP-1). GLP-1 is an incretin hormone produced by the gut epithelium in response to nutrient delivery to the duodenum that exerts insulinotropic effects on the endocrine pancreas through activation of the GLP-1 receptor [18], [19]. The N-terminal residues of GLP-1 bind to the receptor core to stimulate activation, while the C-terminal residues of GLP-1 stabilize the coiled coil homodimeric active structure and bind to the receptor arm to enhance the binding energy [20], [21], [22]. The GLP-1 receptor is a G-protein coupled receptor that requires clustering in caveolin-1 lipid rafts for activity [23]. Receptor activation results in short-term glucose-sensitive insulin secretion via two distinct signaling pathways activated by cyclic adenosine monophosphate (cAMP): the protein kinase A (PKA) pathway and the endogenous protein activated by cAMP 2 (Epac2) pathway. Prolonged GLP-1 receptor activation stimulates long-term insulin production, inhibits apoptosis, induces proliferation and inhibits inflammatory cytokine-mediated β-cell apoptosis [18], [24]. Multiple groups have previously incorporated the biological functionality of GLP-1 into biomaterials for ICT through chemical conjugation of native GLP-1 to polyethylene glycol to produce biomaterials that demonstrate enhanced insulin secretion and enhanced survival in the presence of inflammatory cytokines [25], [26], [27].

In this work, we have utilized peptide amphiphiles (PAs) to generate a bioactive, cytoprotective and fully biodegradable scaffold for ICT. This scaffold supports the survival, proliferation and function of transplanted β-cells during the post-transplant period, in which the cells are susceptible to inflammatory and immune-mediated damage leading to transplant failure, while allowing for the eventual replacement with secreted native extracellular matrix (ECM) to support long-term engraftment. PAs are composed of an oligopeptide conjugated to a lipid tail [28], and our group first introduced peptide sequences that lead to the self-assembly of high aspect ratio cylindrical nanofibers and at the same time effectively display bioactive epitopes on their surfaces [29], [30]. Self-assembly, mediated by hydrophobic collapse of lipid tails and hydrogen bond formation among oligopeptides, is promoted by charge screening by ions [31], [32], [33], [34], [35]. Multivalent ions cross-link PA nanofibers to form a three-dimensional network that turns aqueous solutions into macroscopic gels [36]. Cells suspended in PA solutions can be easily encapsulated by these gels, forming an artificial ECM [37]. The biological activity of the PA is conferred by bioactive sequences that can bind soluble ligands or cell surface receptors [38], [39], [40], [41], [42]. Different PA molecules can be co-assembled to present multiple bioactive epitopes on a single PA nanofiber [43], [44], [45]. PA nanofibers have the capacity to signal for differentiation [46], proliferation [47] and biological adhesion [48] and have demonstrated in vivo biocompatibility with biodegradation [49]. Previous application of PA nanofibers to islet transplantation focused on addition of pro-angiogenic bioactivity to promote vascularization of transplanted islets. The heparin-binding PA developed by our group [41] demonstrated enhanced islet vascularization and cure rate in a murine model of ICT [50] and was subsequently shown to enhance sprouting of new blood vessels from islets in vitro [15].

In this work, we incorporate the insulinotropic and proliferative bioactivity of GLP-1 into a PA molecule using a GLP-1-mimetic peptide sequence. Multiple GLP-1-mimetic peptides have been identified [51], including the clinically used peptide drug exendin-4 (Byetta™, Amylin Pharmaceuticals). We chose the 9mer GLP-1-mimetic peptide Ser[2]exendin(1–9) with sequence HSEDTFTSD [52], which has demonstrated bioactivity both in vitro and in vivo and is resistant to enzymatic inactivation due to the substitution at the second residue [53]. By incorporating this peptide sequence into a functional GLP-1-mimetic PA, we seek to create a single-component biomaterial that forms a cell-encapsulating network of nanofibers under physiological conditions, contains GLP-1 biological functionality, and does not require secondary chemical reactions or non-biodegradable materials.

Section snippets

Peptide synthesis and purification

All PAs and peptides were synthesized by fluorenylmethoxycarbonyl (Fmoc) protected solid-phase peptide synthesis as previously reported by our group [29] using materials purchased from EMD Chemicals Inc. (Merck KGaA, Darmstadt, Germany). Briefly, the PAs/peptides were synthesized at 0.5 mmol scale on Rink Amide MBHA resin. For each amino acid addition, the resin was deprotected using 30% piperidine in dimethylformamide (DMF), and the amino acid was coupled using 4 eq. of Fmoc-protected amino acid

GLP-1-mimetic PA self-assembles into one-dimensional nanofibers with α-helical conformation

The GLP-1-mimetic PA was designed to present the bioactive GLP-1-mimetic peptide Ser[2]exendin(1–9) [52] on the surfaces of self-assembling nanofibers. The GLP-1-mimetic PA (Fig. 1a) contains this bioactive peptide added to the amino terminus of the non-bioactive PA backbone, subsequently referred to as the control PA (Fig. 1d). Both the GLP-1-mimetic PA and the control PA formed gels in the presence of calcium ions or acid. Cryogenic transmission electron microscopy (TEM) revealed that the

Conclusions

We have successfully incorporated the biological activity of the insulinotropic peptide GLP-1 into self-assembling PA nanofibers to produce a novel biomaterial that demonstrates enhanced bioactivity and forms a macroscopic gel for three-dimensional encapsulation and culture of β-cells. This GLP-1-mimetic PA stimulates insulin release from rat β-cells at a level that is significantly greater than the peptide alone and comparable to the clinically used agonist exendin-4. However, the

Acknowledgments

This work was funded by research grant 2 R01 EB003806-06A2 (NIH/NIBIB), and the primary author’s graduate studies were supported by training grant 5 T90 DA022881 from NIH/NIDA. The authors would like to acknowledge the following core facilities at Northwestern University: the Biological Imaging Facility, which operates the JEOL 1230 microscope used for TEM, the Cell Imaging Facility, which operates the Zeiss LSM 510 microscope used for confocal imaging, the Keck Biophysics Facility, which

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