Self-assembling glucagon-like peptide 1-mimetic peptide amphiphiles for enhanced activity and proliferation of insulin-secreting cells
Graphical abstract
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
References (60)
- et al.
Cytoprotection of pancreatic islets before and early after transplantation using gene therapy
Kidney Int
(2002) - et al.
Self-assembling nanostructures to deliver angiogenic factors to pancreatic islets
Biomaterials
(2010) - et al.
Anti-inflammatory peptide-functionalized hydrogels for insulin-secreting cell encapsulation
Biomaterials
(2010) - et al.
Mechanisms of action of glucagon-like peptide 1 in the pancreas
Pharmacol Ther
(2007) - et al.
NMR studies of the aggregation of glucagon-like peptide-1: formation of a symmetric helical dimer
FEBS Lett
(2002) - et al.
Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor
J Biol Chem
(2010) - et al.
The long-acting glucagon-like peptide-1 analogue, liraglutide, inhibits beta-cell apoptosis in vitro
Biochem Biophys Res Commun
(2005) - et al.
Synthesis, bioactivity and specificity of glucagon-like peptide-1 (7–37)/polymer conjugate to isolated rat islets
Biomaterials
(2005) - et al.
Self-assembling peptide amphiphile nanofiber matrices for cell entrapment
Acta biomater
(2005) - et al.
Peptide amphiphile nanostructure-heparin interactions and their relationship to bioactivity
Biomaterials
(2008)
Development of bioactive peptide amphiphiles for therapeutic cell delivery
Acta biomater
Supramolecular crafting of cell adhesion
Biomaterials
Dynamic in vivo biocompatibility of angiogenic peptide amphiphile nanofibers
Biomaterials
Incretin-based therapies in type 2 diabetes: a review of clinical results
Diabetes Res Clin Pract
A set of constructed type spectra for the practical estimation of peptide secondary structure from circular dichroism
Anal Biochem
Insulinotropic actions of exendin-4 and glucagon-like peptide-1 in vivo and in vitro
Metab Clin Exp
The reversal of hyperglycaemia in diabetic mice using PLGA scaffolds seeded with islet-like cells derived from human embryonic stem cells
Biomaterials
Type 1 diabetes: etiology, immunology, and therapeutic strategies
Physiol Rev
Effect of intensive therapy on the microvascular complications of type 1 diabetes mellitus
JAMA
Society IoD. Current advances and travails in islet transplantation
Diabetes
Five-year follow-up after clinical islet transplantation
Diabetes
Overcoming the challenges now limiting islet transplantation: a sequential, integrated approach
Ann N Y Acad Sci
Interventional strategies to prevent beta-cell apoptosis in islet transplantation
Diabetes
Beta cell apoptosis in diabetes
Apoptosis
Vascular endothelial growth factor as a survival factor for human islets: effect of immunosuppressive drugs
Diabetologia
Factors influencing the loss of beta-cell mass in islet transplantation
Cell Transplant
Implantation site-dependent dysfunction of transplanted pancreatic islets
Diabetes
Biological and biomaterial approaches for improved islet transplantation
Pharmacol Rev
Extracellular matrix protein-coated scaffolds promote the reversal of diabetes after extrahepatic islet transplantation
Transplantation
Cited by (33)
Biodegradable self-assembled nanocarriers as the drug delivery vehicles
2022, Nanoparticle Therapeutics: Production Technologies, Types of Nanoparticles, and Regulatory AspectsDesign of functional peptide nanofibers based on amyloid motifs
2020, Artificial Protein and Peptide Nanofibers: Design, Fabrication, Characterization, and ApplicationsProtein-mimetic peptide nanofibers: Motif design, self-assembly synthesis, and sequence-specific biomedical applications
2018, Progress in Polymer ScienceCitation Excerpt :Although great progress of PMP nanofibers in biomineralization has been made, their practical application faces challenges in many respects, such as weak mechanical properties and long-term instability. Specific cell-interacting motifs are necessary for cells to sense the surroundings and convert the information to modulate the cellular behavior [148,183,184]. In peptide-based self-assembly systems, different motifs can be potentially organized into different nanostructures (spherical micelles, nanorods and nanofibers), achieving promotion or inhibition of cell growth.
A novel GLP-1 analog, a dimer of GLP-1 via covalent linkage by a lysine, prolongs the action of GLP-1 in the treatment of type 2 diabetes
2017, PeptidesCitation Excerpt :These modifications mainly employed nonhomologous parts to prevent the recognition of DPP-IV [36]. Dimerization is an effective strategy to improve the stability or receptor binding affinity of proteins or polypeptide [37–39]. As previously reported, the potency of the PEG-dimerized Erythropoietin (a hormone involved in red blood cell production) peptide mimetics both in vitro and in vivo was improved up to 1000-fold compared to the corresponding peptide monomers [40].
The molecular basis for the prolonged blood circulation of lipidated incretin peptides: Peptide oligomerization or binding to serum albumin?
2016, Journal of Controlled ReleaseCitation Excerpt :One widely used approach to improve the pharmacokinetic properties of bioactive peptides is through lipidation of the peptides, i.e. covalent linking of the peptides to lipid conjugates [5–11]. The lipidated peptides often acquire superior pharmacological properties, e.g., higher metabolic stability, long circulating time, and the ability to interact with serum proteins and cell membranes. [7–14]. Although lipidation is widely used in the development of peptide drugs, the molecular basis for the long circulating lifetime of lipidated peptides is poorly understood.