This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.
Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N’-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This “easy-to-adopt” approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.
The therapeutic efficacy of cell-based therapies is limited by low cell retention and poor survival1,2. In order to improve the outcome of cell therapies, cell surface engineering via enzymatic manipulation, peptide conjugation, bioorthogonal chemistry and physical encapsulation with biomaterials has been exploited3,4,5,6,7,8,9,10. The current protocol aims to achieve surface engineering of living cells using an "easy-to-adopt" method by applying an ultrathin heparin-incorporated starPEG (heparin-PEG) nanocoating to the cell surface. Surface engineering of pancreatic islets was presented here as an example due to the heterogeneous nature of islets of Langerhans and the disparaging outcomes of current clinical islet transplantation.
Indeed, clinical islet transplantation is currently performed by direct injection of isolated islets into the hepatic portal vein and this procedure is only available for selective patients because of the scarcity of donor materials and low therapeutic efficacy11. Conventionally, alginate has been the most commonly used biomaterial for islet encapsulation and surface modification, although it is less than ideal due to the chemical instability of alginate and inflammatory-related fibrosis12,13. Furthermore, compared to the natural size of islets that ranges between 100 to 200 µm, the alginate-islet microcapsules are larger, ranging between 400 and 800 µm, which exceed the physiological diffusing distance of oxygen. Conformal islet encapsulation, i.e., encapsulating islets without significant alteration of islet volume, was then developed. Thus, deposition of nanomembranes composed of PEG, tetrafluoroethylene, silicon membrane or multi-layered nanocoating (also known as the "layer-by-layer" [LBL] technique) has been reported, resulting in improved in vitro islet survival14,15,16,17,18, although the LBL approach often requires extensive islets handing period for deposition of multiple layers, which may compromise islet viability. Moreover, instability of nanomembranes that relies on electrostatic or covalent interactions between biomembrane layers or hydrophobic interactions between nanomembranes and the islet surface also raises concerns9,14,15,16,22,23,24,25,26.
Another limiting factor that encumbers the therapeutic outcome of intraportal islet transplantation is the instant blood-mediated inflammatory reaction (IBMIR) caused by direct contact of implanted islets with blood, resulting in platelet aggregation, coagulation and adverse immune effect or undesired cellular activation9. To address these problems, an ultra-thin nanocoating composed of star-shaped polyethylene glycol (starPEG) was prepared for its established biocompatibility and versatility as islet housing material. Heparin, a highly sulphated glycosaminoglycan, was also incorporated in the starPEG nanocoating for its anti-inflammatory, anti-coagulant properties and ability to facilitate vascularization by recruiting pro-angiogenic growth factors22,23.
1. Fabrication of Heparin-incorporated Starpeg Nanocoating
2. Mouse Islet Surface Engineering with Heparin-PEG Nanocoating
3. Function of Heparin-PEG Coated Mouse Islets Compared to Non-coated Islets
The heparin-PEG nanocoating was synthesized by conjugation of starPEG-(NH2)8 and heparin using EDC and NHS as coupling agents (Figure 1). Chemical structure of the heparin-PEG nanocoating was examined by FT-IR and as shown in Figure 2, characteristic peaks of heparin could be observed at 3,300–3,600 cm-1, corresponding to the hydroxyl groups of heparin (Figure 2 red). The decrease in amplitude of the peak at 3,300–3,600 cm-1 (Figure 2 blue) represents conjugation between the starPEG-(NH2)8 amide groups and heparin carbonyl group. Amplitude of peak 1,650 cm-1 corresponds to the amide carbonyl stretching vibration was also reduced, indicating sufficient reaction between the carboxylate groups of heparin with succinimidyl succinate and amine of starPEG-(NH2)8. Surface structure of the heparin-PEG nanocoating was examined by atomic force microscopy and it is shown from Lou et al., the nanocoating was approximately 30 nm in height and 2 µm in width with small porous features (dark spots) ranging between 100 to 200 nm in diameter10. Data obtained from scanned electronic microscopy also confirm the highly interconnected porous structure of the heparin-PEG (Figure 3), suggesting that it could be suitable for cell survival during in vivo delivery.
Surface coating of isolated mouse islets was examined and as presented in Figure 4, thin layer of nanocoating, shown by green fluorescence was evenly deposited across the surface of coated islets without causing evident changes on islet volume/size. During coating, it is recommended to keep the islets on ice to maintain their viability. Similarly, the coating period, 10 min in this case, was also optimized to allow maintenance of islets viability. It is worth noting that for better observation of the nanocoating, electron microscopy that examines the cross-sections of coated islets as previously reported9,16 would be more appropriate, although the data would be generated from fixed and embedded islets instead of living islets in culture.
Regarding survival and function of coated islets, islet revascularization and functions in have been assessed in vitro. Considering the beneficial properties of heparin, heparin functionalization onto the islet surface could facilitate islet revascularization in culture and consequently its survival. We have observed that the heparin-PEG coated mouse islets exhibited robust islet viability in culture (Figure 5). Significantly more advanced vascular formation was also evident from islet endothelial cells (MS1) that were co-cultured with heparin-PEG coated islets, indicated by elongated microvessel-like structures and network-like vascular structures (Figure 6).
The nanocoating process incurred no effect glucose-stimulated insulin secretory ability of the heparin-PEG coated islets. Low level of insulin secretion was observed in all treatment groups when islets were perfused with physiological salt solution supplemented with sub-stimulatory level of glucose (2 mmol/L; Figure 7). When islets were stimulated with a supra-physiological level of glucose (20 mmol/L glucose), increase in insulin secretion was observed in all treatment groups.
Figure 1: Chemical structure of Hep-PEG nanocoating for pancreatic islet surface engineering. Islet coating was achieved by covalent crosslinking between the heparin-NHS and primary amines within the cell membrane and star-PEG-(NH2)8. Each heparin molecule possesses multiple carboxyl groups, which were modified to have an NHS group each. The NHS-activated carboxyl groups will react with primary amines of protein to form amide linkages, via which nanocoating of Hep-PEG and islets is stabilized. Please click here to view a larger version of this figure.
Figure 2: Infrared spectrum of star-PEG-(NH2)8, heparin and Hep-PEG nanocoating in dried state. Please click here to view a larger version of this figure.
Figure 3: Scanned electronic microscopy images of Hep-PEG in dried state. Please click here to view a larger version of this figure.
Figure 4: Representative images of heparin-PEG coated islets under fluorescence microscope.
Heparin was pre-labelled with FAM (shown in green). Images are representative of 100 islets. Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 5: Heparin-PEG coated islets exhibited robust islet viability. (A) Data presented as mean± standard error of means, n = 50 islets per group. (B) Living cells were shown in green and dead cells in red. Scale bar = 100 µm. Images are representative of 50 islets. Please click here to view a larger version of this figure.
Figure 6: Heparin-PEG nanocoating facilitates intra-islet revascularisation. Matrigel tube formation of MS1 cells co-cultured with heparin-PEG coated islets and noncoated control islets. Images were taken at 4 and 24 h. Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 7: Heparin-PEG nanocoating incurs no change on islet insulin secretion function. Heparin-PEG coated islets and noncoated control islets (30 each) were exposed to 2 mmol/L (white bar) or 20 mmol/L (black bar) glucose for 30 min. Insulin secretion in response to 20 mmol/L glucose was comparable between the coated and control islets. Data are shown as mean ± standard error of means, n = 10. Please click here to view a larger version of this figure.
In this article, we demonstrate an "easy-to-adopt" approach for living cell surface engineering a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of pancreatic islet surface membrane. Indeed, the amino groups within cell membranes are highly reactive, and as a result, earlier studies have reported interactions between primary amino groups with activated N-hydroxysuccinimidyl (NHS) ester under physiological conditions14,16,21. Furthermore, extensive research has reported that incorporation of heparin, a highly sulphated glycosaminoglycan and important component of the extracellular matrix, during islet encapsulation, could lead to enhanced post-transplantation revascularization and reduced IBMIR22,23. Considering the biocompatibility of PEG and multivalent properties of heparin, we used 8-armed PEG for maximal heparin loading during fabrication of the nanocoating. Heparin was modified with -NHS, which would subsequently react with the -NH2 groups on islet cell membrane. By enabling the covalent bond formation between -NH2 (of cell membrane) and -NHS of the Hep-PEG, the islets would be readily "coated" by the heparin-incorporated PEG, thus forming a nano-thin layer (nanocoating) on the outer surface of the pancreatic islets.
The present approach is different from previously published methods that also selected PEG as the major polymer for islet microencapsulation in that pseudo-bioorthogonal chemistry between the -NHS (of the nanocoating) and -NH2 of the islet cell membrane was used. Considering that stability of islet/cell coating, especially in a complex environment such as the plasma, is crucial to post-transplantation revascularization and survival, the formation between -NHS and -NH2 would be more stable compared to hydrophobic interaction between PEG and cell membrane24, electrostatic interactions9,15,24,25,26 or biological linkage between biotin streptavidin14.
In addition, in contrast to islet coating approach that relies on the LBL approach with extended islet handling period for multi-layer deposition14,16,25, the present technique also requires minimal processing and very short coating period of the isolated islets. Both of these factors are essential for post-transplantation islet survival since islets viability is often already compromised following islet isolation due to damaged ECM during enzymatic digestion. However, one limitation of the present approach is that, unlike LBL, via which thickness of the outer coating could be controlled by increasing or reducing the number of layer deposition, thickness of the Hep-PEG nanocoating cannot be tailored for the time being.
Furthermore, due to the mild condition where chemical reaction between -NHS and -NH2 takes place, the present approach is applicable for living cell surface engineering not limited to pancreatic islets, but most cell therapy. Additionally, considering that heparin is known to interact with a range of cytokines and biologically active molecules, the Hep-PEG nanocoating also presents an open platform that has the potential for incorporation of unlimited biological mediators as well as interfaces for more complex cell surface engineering.
The authors have nothing to disclose.
We are grateful for the financial support of the National Natural Science Funds of China (31770968) and the Tianjin Research Program of Application Foundation and Advanced Technology (17JCZDJC33400).
Reagent | |||
PBS | Hyclone | AAJ207798 | |
Streptozototin | Sigma | S0130 | |
Histopaque | Sigma | 10831 | |
RPMI 1640 | GIBCO, by Life Technologies | 31800022 | |
Fetal Bovine Serum | GIBCO, by Life Technologies | 16000-044 | |
Penicillin Streptomycin | GIBCO, by Life Technologies | 15140 | |
Cell Dissociation Solution | GIBCO, by Life Technologies | 13150-016 | |
DMEM | GIBCO, by Life Technologies | 12800017 | |
D-(+)-Glucose solution | Sigma | G8644 | |
488 phalloidin | Sigma | A12379 | |
CFSE | Sigma | 21888-25mg-F | |
Annexin V/PI apoptosis kit | Dojindo | AD10 | |
DAPI Fluoromount-G | SouthernBiotech | 0100-20 | |
Collagenase from Clostridium, Type XI | Sigma | C7657 | |
Heparin | Sigma-Aldrich | H3149 | |
NHS | Sigma-Aldrich | 56480 | |
EDC | Sigma-Aldrich | 3449 | |
8-armed PEG | J&K Scientific Ltd | 1685176 | |
FAM | Sigma-Aldrich | M041100 | |
5(6)-carboxyfluorescein N-succinimidyl ester | Sigma-Aldrich | 21888 | |
KBr | J&K Scientific Ltd | 32036 | |
3-aminopropyl-triethoxysilane | Sigma-Aldrich | A3648 | |
toluene | J&K Scientific Ltd | S-15497-20X | |
Live/dead staining kit | Biovision, US | K501 | |
BD MatrigelTM, basement membrane matrix, growth factor reduced | BD Bioscience | 354230 | |
Sodium chloride, 99.5% | J&K Scientific Ltd | 105864 | |
Potassium chloride, 99%, extra pure | J&K Scientific Ltd | 991468 | |
Sodium bicarbonate, 99.7%, ACS reagent | J&K Scientific Ltd | 988639 | |
Magnesium chloride hexahydrate, 99%, ACS reagent | J&K Scientific Ltd | 182158 | |
Potassium dihydrogen phosphate, 99%, extra pure | J&K Scientific Ltd | 128839 | |
Magnesium sulfate heptahydrate, 99%, for analysis | J&K Scientific Ltd | 119370 | |
Calcium chloride solution volumetric, 1.0 M CaCl2 | J&K Scientific Ltd | 21114 | |
Bovine Serum Albumin | Sigma-Aldrich | V900933 | |
Rat/Mouse Insulin ELISA kit | Millipore-linco | EZRMI-13K |