Synthesis and characterization of GLP-1-based self-assembling peptide hydrogel.
To manufacture purposeful GLP-1 co-assembled hydrogels, we initially designed GLP-1 peptides with self-assembly properties. Particularly, an FFG sequence with self-assembly property we verified in our earlier research [26, 27], was launched on the C-terminus of the native GLP-1 peptide (GLP-1-FFG) (Fig. 2A). The self-assembly GLP-1-FFG serves two functions: firstly, to boost the soundness of GLP-1, and secondly, to allow its co-assembly binding with the supramolecular peptide hydrogels (SupraGel) we now have beforehand described [23]. The native GLP-1 and GLP-1-FFG peptides have been ready by commonplace stable part peptide synthesis (SPPS) utilizing Fmoc-amino acids. They have been characterised by high-resolution mass spectrums (HR-MS) and high-performance liquid chromatography (HPLC) (Determine S1).
Characterization of GLP-1-FFG peptide and SupraGel + GLP-1-FFG hydrogel. A Schematic illustration of the amino acid sequences of GLP-1 and GLP-1-FFG. B Chemical construction of the Biotin-DFYIGSRGD gelator (SupraGel). SupraGel kinds a hydrogel in aqueous buffer. C Affinity curves of GLP-1 and GLP-1-FFG to SupraGel. D TEM pictures of GLP-1, GLP-1-FFG, SupraGel, SupraGel + GLP-1 (Gel 1) and SupraGel + GLP-1-FFG (Gel 2) at a focus of 10 mM in 1 × PBS buffer (pH 7.4). GLP-1 and GLP-1-FFG are in resolution type, whereas SupraGel, SupraGel + GLP-1 (Gel 1), and SupraGel + GLP-1-FFG (Gel 2) are in hydrogel type. Scale bar, 100 nm. High proper nook, the optical {photograph} of Tyndall scattering. E Degradation habits of GLP-1 and GLP-1-FFG after incubation with DPP-4 for 0–24 h. F Dynamic frequency sweep of SupraGel, Gel 1 and Gel 2 at a pressure of 1% at 37 °C. G Dynamic time sweep of SupraGel, Gel 1 and Gel 2 at 37 °C. H Dynamic pressure sweep of SupraGel, Gel 1 and Gel 2 at a frequency of 10 Hz at 37 °C. I Gel-sol transition and self-healing of Gel 2
We beforehand reported a supramolecular hydrogel, SupraGel (Biotin-DFYIGSRGD), shaped by self-assembly of a local brief peptide, which can have an extracellular matrix-like operate [23,24,25]. (Fig. 2B). Due to this fact, we additional investigated whether or not the introduction of the FFG sequence may improve the interplay between GLP-1-FFG and SupraGel, thus co-assembling to type GLP-1-functionalized hydrogels. Microscale thermophoresis (MST) was used to find out the binding affinity of GLP-1 and GLP-1-FFG to SupraGel. SupraGel was fluorescently labeled to allow detection of binding interactions. GLP-1-FFG demonstrated a transparent dose-dependent binding to SupraGel, with a calculated Kd worth of 267.82 μM. In distinction, for native GLP-1, no dose-dependent change in fluorescence depth of the labeled SupraGel was detected with growing GLP-1 concentrations, indicating negligible affinity. In consequence, no Kd worth may very well be decided. These outcomes point out that the FFG self-assembly sequence may improve the binding affinity of GLP-1 to SupraGel (Fig. 2C). To visually verify the self-assembly habits of GLP-1-FFG and its interplay with SupraGel, we carried out TEM imaging on numerous compounds (Fig. 2D). There isn’t a apparent nano-structure of GLP-1 peptide resolution below TEM. In distinction, the Tyndall impact of the GLP-1-FFG peptide resolution below laser irradiation proves its self-assembly habits, with TEM displaying localized nanofiber buildings regardless of the absence of gel formation. The microstructure of the SupraGel hydrogel appeared as comparatively dense nanofibers. When GLP-1 was integrated into SupraGel (SupraGel + GLP-1, Gel 1), there was no apparent change within the microscopic construction, significantly when it comes to fiber density, in comparison with the SupraGel hydrogel. In distinction, when GLP-1-FFG was combined with SupraGel (SupraGel + GLP-1-FFG, Gel 2), the hydrogel confirmed a a lot denser nanofiber community, which means that enhanced interactions and attainable cross-linking between GLP-1-FFG and SupraGel, resulting in a extra strong and sophisticated gel construction. (Fig. 2D). These outcomes point out that GLP-1-FFG reveals self-assembly habits, and this self-assembly technique can improve the interplay of GLP-1 with SupraGel, thereby facilitating co-assembly. Furthermore, GLP-1-FFG itself has the flexibility to withstand degradation by dipeptidyl peptidase-4 (DPP-4), a extensively distributed endogenous protease within the human physique that performs a key function in degrading GLP-1 [28]. The DPP-4 was individually incubated with GLP-1 and GLP-1-FFG over a 24-h interval. At 1 h, GLP-1 retained roughly 79% of its authentic content material, in comparison with 98% for GLP-1-FFG, indicating a considerable enchancment in stability. At 4 h, GLP-1 retained 76% of its content material, whereas GLP-1-FFG retained 93%. By 12 h, GLP-1 retained about 73%, whereas GLP-1-FFG maintained about 86% of its authentic content material. Lastly, at 24 h, GLP-1 retained about 65% of its content material, whereas GLP-1-FFG retained about 85% (Fig. 2E). These outcomes point out that GLP-1-FFG reveals superior resistance to enzymatic degradation in any respect noticed time factors. This enhanced stability is probably going as a result of self-assembly habits of GLP-1-FFG, which can defend the peptide by burying the energetic websites of DPP-4 inside the nanofibrous construction, thereby lowering alternatives for enzymatic cleavage.
Additional, we carried out mechanical characterization of various gels. Underneath the dynamic frequency sweep mode, all three hydrogels have been in a position to preserve stability inside 100 Hz. Nevertheless, on the frequency of 100 Hz, SupraGel + GLP-1 (Gel 1) had reached the crucial level of gel destruction, whereas SupraGel + GLP-1-FFG (Gel 2) remained a steady gel state. Furthermore, the storage modulus (G’) of Gel 1 was the bottom, beneath that of the unique SupraGel, whereas Gel 2 exhibited the very best storage modulus (Fig. 2F). This means that the incorporation of native GLP-1 with lengthy amino acid sequence might disrupt the nanofiber community of SupraGel to a sure extent, leading to lowered elasticity and elevated susceptibility to deformation. The self-assembling sequence current in GLP-1-FFG can crosslink with the fibrous community of SupraGel to type a co-assembly, which reinforces the hydrogel’s resistance to deformation. Dynamic time scanning exhibits that the three sorts of hydrogels may type gel at a really quick charge (the gelation level of the hydrogel was not captured resulting from limitation of the testing gear), indicating that the addition of peptides doesn’t destroy the gelation efficiency of SupraGel. On the identical time, the distinction in G’ values among the many three gels is per the outcomes of dynamic frequency scanning (Fig. 2G). SupraGel reveals a typical shear-induced gel-sol transition habits and fast self-healing, which is crucial for cell tradition and in vivo cell supply. We used dynamic pressure scanning mode for rheological measurements to characterize whether or not the incorporation of GLP-1/GLP-1-FFG nonetheless possesses this property (Fig. 2H). We discovered that Gel 1 and Gel 2 retained the attribute gel-sol transition of SupraGel itself. Figure 2I additionally exhibits that Gel 2 may remodel into an answer by merely vortexing or vigorously guide shaking, and the ensuing resolution may type a hydrogel once more after roughly 5 min, which is handy for cell tradition and supply. As well as, we additionally discovered that the pressure values of gel-sol part transition of SupraGel, Gel 1 and Gel 2 elevated in flip (1.01%, 2.01% and 5.04%, respectively) (Fig. 2H), indicating a gradual enhance within the mechanical power they will face up to. These outcomes present that though the mixture of native GLP-1 and SupraGel can improve the mechanical bearing capability of SupraGel to a sure extent, ends in decreased elastic properties and elevated danger of deformation. In different phrases, Gel 1 is extra like a comparably inflexible and brittle substance, much like a “biscuit.” The dense fiber community shaped by Gel 2 cannot solely enhances the mechanical properties of hydrogel, but additionally enhance its elasticity, which isn’t simple to deform, extra steady and more durable. This property is relevant for delivering islet grafts to make sure the integrity of the hydrogel throughout transplantation and to withstand a few of strain from the physique on the transplantation website.
Protecting results of the SupraGel + GLP-1-FFG hydrogel on islets in vitro
We additional explored the results of the SupraGel + GLP-1-FFG (Gel 2) on islets by way of in vitro experiments. First, we confirmed that the SupraGel (2 wt%) hydrogel is biocompatible for islets (Determine S2). And the viability of mouse islets cultured in vitro for twenty-four h was nicely maintained utilizing both SupraGel or Gel 2 at 100 μM (Fig. 3A–C).
Protecting results of SupraGel + GLP-1-FFG (Gel 2) on mouse islets. A A 3D reconstructed picture of the CM-Dil fluorescently labeled mouse islets cultured in vitro. Scale bar, 50 μm. B Fluorescein diacetate (FDA)/propidium iodide (PI) fluorescence staining to guage cell viability of the mouse islets cultured in vitro for twenty-four h. Inexperienced: dwell cells. Pink: lifeless cells. Scale bar, 150 μm. C Quantitative evaluation of the islet viability (n = 3). D Insulin secretion ranges after low glucose and excessive glucose remedies throughout glucose-stimulated insulin launch assay (n = 3). E Glucose stimulation index (GSI) calculated based mostly on the insulin secretion ranges throughout low glucose and excessive glucose remedies (n = 3). F Consultant pictures of fluorescein diacetate (FDA)/propidium iodide (PI) fluorescence staining to guage the viability of mouse pislets cultured below hypoxic circumstances. Inexperienced: dwell cells. Pink: lifeless cells. Scale bar, 150 μm. G Quantitative evaluation of the islet viability (n = 3). H Detection of ROS ranges in mouse islets cultured below hypoxic circumstances (n = 3). All knowledge have been expressed as imply ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
We additional assessed the results of the Gel 2 on islet operate by glucose-stimulated insulin secretion experiments. The outcomes confirmed that below low glucose circumstances, mouse islets cultured in SupraGel or Gel 2 had basal insulin secretion ranges near these of the free-cultured management islets. In distinction, below excessive glucose stimulation, mouse islets cultured in Gel 2 had considerably greater insulin secretion ranges in comparison with the opposite two teams (Fig. 3D). The glucose-stimulated index (GSI) verified the outcomes (Fig. 3E), suggesting that Gel 2 has a trophic impact on the operate of islets.
To additional examine the protecting results of Gel 2 on islets, we induced hypoxic damage in vitro on mouse islets. Hypoxic damage is likely one of the main causes of early islet graft failure after transplantation [8, 29]. The viability of mouse islets was considerably decreased after 12 h of incubation below hypoxic circumstances, suggesting that hypoxia induces impaired islet survival. In distinction, the viability was improved when mouse islets have been handled with hypoxia whereas incubated with SupraGel. Notably, the viability of mouse islets cultured with Gel 2 below hypoxic circumstances was additional elevated in comparison with the SupraGel group (Fig. 3F, G). We additionally demonstrated that GLP-1-FFG alone exhibited a big enchancment within the viability of hypoxic islets (Determine S3), so the protecting impact of Gel 2 on hypoxic islets will be thought of as a superimposition of the results of SupraGel and GLP-1-FFG. The useful results of GLP-1 on islet viability and performance have been confirmed by our analysis and others [13, 30, 31]. SupraGel accommodates YIGSR and RGD purposeful motifs [23], the place the pentapeptide YIGSR is derived from the purposeful area of laminin [32], and RGD can bind to cell floor receptor integrin [33]. Our current research additionally demonstrates that SupraGel can improve the therapeutic results of mesenchymal stem cells (MSCs) by activating the α2β1 integrin signaling [24, 25]. Quite a few research have already proven the optimistic results of laminin and integrin signaling on islet survival [34,35,36,37]. Due to this fact, the protecting results of SupraGel in islet survival might contain the activation of extracellular matrix indicators.
Hypoxia normally causes reactive oxygen species (ROS) accumulation and induces oxidative stress damage. Our earlier research demonstrated that GLP-1 may inhibit oxidative stress in islets throughout chilly preservation [13]. Right here, we discovered hypoxic damage elevated ROS ranges in mouse islets, whereas each SupraGel and Gel 2 inhibited ROS accumulation (Fig. 3H). These outcomes recommend that the Gel 2 has the impact of selling islet survival and bettering islet operate.
SupraGel + GLP-1-FFG hydrogel protects islets from hypoxic damage by activating Akt sign pathway based mostly on RNA-Seq evaluation
To additional discover the mechanism of the protecting results of Gel 2 on hypoxic islets, we carried out RNA sequencing (RNA-seq) evaluation. Heatmap exhibits differentially expressed genes between teams (Determine S4A, Fig. 4A). The volcano plot illustrated the identification of 1,051 upregulated and 181 downregulated differentially expressed genes within the Hypoxia group in comparison with the Management group (Determine S4B). The applying of Gel 2 on hypoxic islets downregulated 184 genes and upregulated 260 genes (Fig. 4B). Genes of curiosity have been screened from the differentially expressed genes, and the normalized heatmap confirmed that classical hypoxic genes, represented by Ldha, have been considerably upregulated following induced islet hypoxia damage. On this context, Gel 2 downregulates the expression of hypoxia-related genes, indicating its inhibitory impact on the hypoxic response in islets (Fig. 4C). The inflammatory response induced by hypoxia additionally contributes to islet hypoxia damage [38, 39]. Our outcomes confirmed that the expression of pro-inflammatory cytokine genes, reminiscent of Tnf-α, was upregulated in hypoxic islets, whereas therapy with Gel 2 inhibited this upregulation (Fig. 4C). Gel 2 additionally inhibited the hypoxia-induced downregulation of β-cell function-related genes, reminiscent of Mafa (Fig. 4C). The RNA-seq outcomes have been subsequently confirmed through qRT-PCR experiments (Fig. 4D). The gene ontology (GO) enrichment evaluation was performed, and the outcomes point out that the optimistic regulation of PI3K-Akt sign transduction could be the potential pharmacological mechanism of Gel 2 on hypoxic islets (Fig. 4E). Extra importantly, this discovering was confirmed by western blot evaluation. The expression degree of p-Akt protein was considerably upregulated following therapy with Gel 2 (Fig. 4F). This result’s per our earlier research, which demonstrated that GLP-1 attenuates islet chilly ischemic damage by activating the Akt signaling pathway [13].
RNA-seq evaluation of mouse islets handled with SupraGel + GLP-1-FFG (Gel 2) below hypoxic circumstances. A Heatmap of differentially expressed genes within the Hypoxia + Gel 2 and Hypoxia teams. B Volcano diagram showcasing differentially expressed genes within the Hypoxia + Gel 2 teams examine to Hypoxia teams. C The normalized heatmap shows the response to hypoxia, irritation, and β-cell function-related genes within the Management, Hypoxia, and Hypoxia + Gel 2 teams. D qRT-PCR evaluation of mouse islets from hypoxic mannequin in vitro (n = 7). E Gene ontology (GO) evaluation was undertaken to delineate organic processes or pathways which may be implicated within the mediation of the therapy results. F Western blot evaluation of p-Akt and Akt in mouse islets from hypoxic mannequin in vitro (n = 5). All knowledge have been expressed as imply ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
The in vivo impact of the SupraGel + GLP-1-FFG hydrogel (Gel 2) on islet transplantation
We additional investigated the in vivo impact of the Gel 2 on islet transplant outcomes in a syngeneic islet transplant mannequin (Fig. 5A). Within the syngeneic islet transplant mannequin, diabetic mice acquired a marginal dose of 150 syngeneic islets, which have been transplanted below the renal capsule. When diabetic mice acquired a marginal dose of donor islets alone, blood glucose decreased slowly, exhibiting restricted efficacy, with a 50% diabetes reversal charge noticed after roughly 100 days post-transplantation (Fig. 5B–D). In distinction, when donor islets have been transplanted with SupraGel hydrogel into diabetic mouse recipients, a big enhancement in post-transplantation blood glucose management and diabetes reversal charge was evident in comparison with islet-only transplantation (Fig. 5B–D). Extra importantly, when donor islets have been transplanted with Gel 2, the blood glucose ranges of the recipient diabetic mice decreased quickly and remained inside the regular vary post-transplantation (Fig. 5B). As compared with the SupraGel group, the Gel 2 group exhibited superior glycemic management and achieved a 100% diabetes reversal charge extra quickly, inside 43 days post-transplantation (Fig. 5C, D). After islet transplantation, the load of recipient mice progressively elevated over time (Fig. 5E). These outcomes recommend that the appliance of SupraGel hydrogel can enhance the result of islet transplantation in treating diabetes, and the utilization of the Gel 2 can additional enhance this efficacy.
The SupraGel + GLP-1-FFG (Gel 2) improves the efficacy of islet transplantation. A Schematic of the experiment design and the teams. B Diabetes reversal charge after islet transplantation in recipient mice (n = 5–6); C Fasting blood glucose ranges after islet transplantation in diabetic recipient mice (n = 5–6); D Space below curve (AUC) calculated based mostly on the fasting blood glucose ranges (n = 5–6); E Physique weight after islet transplantation in recipient mice (n = 5–6); F Intraperitoneal glucose tolerance take a look at (IPGTT) at 60 days post-transplantation (n = 4). Blue asterisks: SupraGel group vs Management group. Orange asterisks: Gel 2 group vs Management group; G Space below curve of the IPGTT curve (n = 4); H Serum insulin degree of mice at 30 min after glucose injection throughout IPGTT experiment (n = 3). All knowledge have been expressed as imply ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001
To additional consider the impact of the Gel 2 on graft operate, a glucose tolerance take a look at (IPGTT) was carried out at 60 days post-transplantation in recipient mice. The outcomes exhibit that in comparison with the management group receiving islet-alone transplantation, mice within the SupraGel group exhibited a extra fast glucose clearance functionality. Notably, mice within the Gel 2 group manifested superior intraperitoneal glucose tolerance in comparison with these within the SupraGel group (Fig. 5F). The end result was additional validated by the realm below the curve (AUC) of the glucose tolerance curve (Fig. 5G). Additional evaluation of serum insulin ranges in mice additionally revealed that, in comparison with the opposite two teams, the Gel 2 group exhibited the next insulin secretion 30 min after glucose injection (Fig. 5H).
SupraGel + GLP-1-FFG improves islet graft survival after transplantation
At 104 days after transplantation, the islet graft was eliminated, and blood glucose within the recipient mice rose quickly again to pre-transplantation ranges (Fig. 5B). Insulin staining of the pancreas in situ additionally confirmed that the native islet β-cells had been destroyed (Determine S5). These end result demonstrates that blood glucose management through the commentary interval is as a result of impact of the islet graft reasonably than the potential affect of residual islets in situ. Immunofluorescence staining of the grafts revealed minimal insulin detection on the graft website within the islet-alone transplantation group. In distinction, the SupraGel group exhibited a bigger insulin-positive space. Extra notably, the Gel 2 group demonstrated even bigger insulin-positive areas in comparison with the opposite two teams (Fig. 6A, B). Within the insulin-positive area, CD31 expression was detected in all teams, indicating that the survived grafts have been nicely vascularized (Fig. 6A, C). These outcomes recommend that Gel 2 promotes islet graft survival after transplantation in vivo.
SupraGel + GLP-1-FFG hydrogel improves islet graft survival. A Consultant pictures of immunofluorescence staining (insulin, CD31, DAPI) of islet graft at 104 days post-transplantation. Scale bar, 100 μm. B Quantitative evaluation of insulin immunofluorescence staining (n = 5). C Quantitative evaluation of CD31 immunofluorescence staining (n = 5). All knowledge have been expressed as imply ± SEM. *P < 0.05, ***P < 0.001
Based mostly on the above outcomes, SupraGel has been demonstrated to boost the efficacy of pancreatic islet transplantation, with GLP-1-FFG additional augmenting this impact. The mechanisms concerned might embrace SupraGel optimizing the microenvironment of the transplantation website to facilitate islet implantation, together with GLP-1-FFG selling islet survival, in the end synergistically enhancing the therapeutic outcomes for diabetes therapy. As talked about above, the YIGSR and RGD purposeful motifs of SupraGel possess the potential to imitate the operate of laminin and activation of the integrin receptors [23, 32, 33], respectively. Biomedical scaffolds or hydrogels based mostly on extracellular matrix (ECM) parts have been extensively investigated in islet transplantation. Previous to transplantation, donor islets bear an isolation process, throughout which their native ECM is disrupted [40]. And the shortage of ECM atmosphere on the transplantation website within the early post-transplantation interval might lead to islet anoikis [41, 42], thus limiting islet engraftment and survival. Using ECM-based biomaterials is conducive to reconstructing the islet microenvironment, selling islet engraftment, and thereby enhancing transplantation efficacy [37, 43, 44]. On this context, the localized motion of supplemented GLP-1-FFG on the transplantation website exerts the anti-apoptotic and pro-survival results of GLP-1 on the transplanted islets [16,17,18], lowering the injury sometimes confronted by islets within the early post-transplantation interval, reminiscent of irritation, hypoxia, and oxidative stress [8, 45, 46].
Whereas this research gives beneficial insights, sure limitations ought to be thought of. This research utilized the kidney capsule because the transplantation website, which, whereas technically easy and reproducible in preclinical fashions, is just not clinically related for human islet transplantation. To boost the translational potential of this hydrogel-based technique, future research ought to consider its efficiency in clinically relevant websites, such because the omentum and subcutaneous areas, which have proven promise as potential scientific islet transplantation websites. These places would enable for a localized, sustained launch of GLP-1 whereas offering a protecting microenvironment for the islets. Advances in supply strategies for in vivo utility of hydrogels, reminiscent of catheter-based administration or injectable formulations, may pave the best way for his or her scientific use in transplantation at extrahepatic websites. Moreover, this research centered on the hydrogel’s skill to boost islet survival and performance in a managed syngeneic mannequin, minimizing variability launched by immune rejection. Nevertheless, to comprehensively assess the hydrogel’s immunomodulatory properties and its potential to mitigate immune-mediated graft rejection, additional investigations incorporating allogeneic or xenogeneic islets, in addition to immune-competent fashions, are warranted.
