Chemical Cross-linking Reagents Research Articles

Examining NF-κB genomic interactions by ChIP-seq and CUT&Tag.

An understanding of the mechanisms and logic by which transcription factors coordinate gene regulation requires delineation of their genomic interactions at a genome-wide scale. Chromatin immunoprecipitation-sequencing (ChIP-seq) and more recent techniques, including CUT&Tag, typically reveal thousands of genomic interactions by transcription factors, but without insight into their functional roles. Due to cost and time considerations, optimization of ChIP experimental conditions is typically carried out only with representative interaction sites rather than through genome-wide analyses. Here, we describe insights gained from the titration of two chemical crosslinking reagents in genome-wide ChIP-seq experiments examining two members of the NF-κB family of transcription factors: RelA and c-Rel. We also describe a comparison of ChIP-seq and CUT&Tag. Our results highlight the large impact of ChIP-seq experimental conditions on the number of interactions detected, on the enrichment of consensus and non-consensus DNA motifs for the factor, and on the frequency with which the genomic interactions detected are located near potential target genes. We also found considerable consistency between ChIP-seq and CUT&Tag results, but with a substantial fraction of genomic interactions detected with only one of the two techniques. Together, the results demonstrate the dramatic impact of experimental conditions on the results obtained in a genome-wide analysis of transcription factor binding, highlighting the need for further scrutiny of the functional significance of these condition-dependent differences.

All-natural and high-performance structural material based on lignin-reinforced cellulose

Sustainable materials derived from lignocellulosic resources are attractive alternatives to ubiquitous petrochemical plastics. However, it remains a huge challenge to improve the mechanical properties of lignocellulosic materials through efficient and low-energy processing strategy. Herein, we report a facile lignin-reinforced cellulose strategy to manufacture an all-natural and high-performance structural material from bamboo particles via partial delignification followed by hot-pressing. Partial delignification can regulate the amount of lignin in the bamboo particles and release internal cellulose microfibers. The subsequent hot-pressing drives residual lignin into the cellulose matrix to form a dense structure, which acts as a polyphenolic binder to improve the performance of the structural material. The obtained lignin-reinforced cellulose plate (LRCP) demonstrates high isotropic flexural strength (∼162 MPa), which is higher than that of natural bamboo and typical petrochemical plastics. Moreover, LRCP shows outstanding toughness (∼2.58 MPa m1/2), excellent compressive strength (∼178 MPa), improved wet stability and superior flame resistance. LRCP is all-natural and eco-friendly without the need for additional chemical cross-linking reagents. We believe that this facile and cost-effective strategy provides a promising way to process lignocellulosic resources into all-natural and high-performance structural materials that can serve as powerful plastic alternatives in the future.

Evaluation of photochemically cross-linked collagen/gold nanoparticle composites as potential skin tissue scaffolds.

Collagen type I is the main structural unit in skin tissue and is therefore used preferentially in skin tissue scaffolds. However, collagen-based 3D scaffolds have weak aqueous stability and degradation profiles in their uncross-linked states and chemical cross-linking reagents arise toxicity concerns, which generally restrict the spectrum of their biomedical applicability. Here, the research goal is to photochemically cross-link collagen type I with rose bengal (RB) when subjected to green laser light and to investigate the effect of silk sericin-capped gold nanoparticles (S-AuNP) when incorporated into scaffolds on the cross-linking process and thus on the scaffold properties. All the collagen scaffolds, that is plain collagen (C), collagen/S-AuNP (C-Au), cross-linked collagen (C-RBL), and cross-linked collagen/S-AuNP (C-AuRBL) were characterized for their potential as skin tissue scaffolds. C-AuRBL group had the best thermal stability, resistance to enzymatic degradation, and more uniform pore size distribution. None of the groups had cytotoxicity (cell viability > 70%) regarding the microscopic observations and MTT cell viability assays for L929 fibroblasts. L929 fibroblasts and primary adult human epidermal keratinocytes (HEKa) were also separately seeded on C-AuRBL scaffolds and according to microscopy results, they could support the stimulation of adhesion, morphological changes, and spreading of both cells, thereby encouraging the usage of this fabrication strategy for prospective skin tissue scaffolds.

A Streamlined Method for Detecting Inflammasome-Induced ASC Oligomerization Using Chemical Crosslinking.

The apoptosis-associated speck-like protein containing a caspaseactivationand recruitment domain (ASC) functions as the integral adaptor protein between inflammasome sensors such as NOD-like receptor protein 3 (NLRP3) and the inflammatory caspase, caspase-1. Inflammasome sensor triggering allows recruitment of ASC and the formation of long amyloid-like ASC oligomers that enable binding and proximity-induced activation of caspase-1. The detection of ASC oligomerization thus constitutes a highly specific and direct test for inflammasome complex formation and activation. Here, we describe a simplified and streamlined method for the detection of ASC oligomers via Western blotting, using the chemical crosslinking reagent disuccinimidyl suberate.

Mirror cutting-assisted orthogonal digestion enabling large-scale and accurate protein complex characterization

蛋白质主要以复合物的形式参与各项生命活动。化学交联质谱(CXMS)技术作为近年来新兴的蛋白质复合物解析技术,不仅可实现蛋白质复合物规模化解析,而且普遍适用于任意相对分子质量和纯度的蛋白质复合物样品,因此已成为X-射线晶体衍射技术、冷冻电镜技术等蛋白质复合物解析经典技术的重要补充。目前,CXMS主要采用胰蛋白酶将交联后的蛋白质复合物进行酶切,并进行质谱鉴定。然而鉴定到的交联肽段谱图中b/y特征碎片离子的鉴定数目不足,且因肽段序列匹配连续性较差导致谱图可信度相对较低,影响了蛋白质复合物交联位点的鉴定精准度。该文基于镜像酶正交切割的互补特性,采用胰蛋白酶镜像酶与胰蛋白酶联合酶切的方法,对交联后的蛋白质复合物分别进行酶切,再利用液相色谱-质谱联用技术对酶解产生的交联肽段进行鉴定,进而实现蛋白质复合物的组成、相互作用和结构位点距离约束的解析。对牛血清白蛋白和大肠杆菌全蛋白的交联肽段的分析结果表明,该方法通过增加交联肽段谱图中特征碎裂离子的数目和连续性,鉴定到的大肠杆菌全蛋白的交联位点,较单一胰蛋白酶酶切,提高了16%。因此,镜像酶正交酶切策略能有效提高交联肽段的鉴定准确度和覆盖度,有望为实现规模化的蛋白质复合物精准解析提供新思路。

Apolipoprotein A1 Forms 5/5 and 5/4 Antiparallel Dimers in Human High-density Lipoprotein

Apolipoprotein A1 (APOA1), the major protein of high-density lipoprotein (HDL), contains 10 helical repeats that play key roles in protein-protein and protein-lipid interactions. The current structural model for HDL proposes that APOA1 forms an antiparallel dimer in which helix 5 in monomer 1 associates with helix 5 in monomer 2 along a left-left (LL5/5) interface, forming a protein complex with a 2-fold axis of symmetry centered on helix 5. However, computational studies suggest that other orientations are possible. To test this idea, we used a zero-length chemical cross-linking reagent that forms covalent bonds between closely apposed basic and acidic residues. Using proteolytic digestion and tandem mass spectrometry, we identified amino acids in the central region of the antiparallel APOA1 dimer of HDL that were in close contact. As predicted by the current model, we found six intermolecular cross-links that were consistent with the antiparallel LL5/5 registry. However, we also identified three intermolecular cross-links that were consistent with the antiparallel LL5/4 registry. The LL5/5 is the major structural conformation of the two complexes in both reconstituted discoidal HDL particles and in spherical HDL from human plasma. Molecular dynamic simulations suggest that that LL5/5 and LL5/4 APOA1 dimers possess similar free energies of dimerization, with LL5/5 having the lowest free energy. Our observations indicate that phospholipidated APOA1 in HDL forms different antiparallel dimers that could play distinct roles in enzyme regulation, assembly of specific protein complexes, and the functional properties of HDL in humans.

Chemical cross-linking in the structural analysis of protein assemblies.

For decades, chemical cross-linking of proteins has been an established method to study protein interaction partners. The chemical cross-linking approach has recently been revived by mass spectrometric analysis of the cross-linking reaction products. Chemical cross-linking and mass spectrometric analysis (CXMS) enables the identification of residues that are close in three-dimensional (3D) space but not necessarily close in primary sequence. Therefore, this approach provides medium resolution information to guide de novo structure prediction, protein interface mapping and protein complex model building. The robustness and compatibility of the CXMS approach with multiple biochemical methods have made it especially appealing for challenging systems with multiple biochemical compositions and conformation states. This review provides an overview of the CXMS approach, describing general procedures in sample processing, data acquisition and analysis. Selection of proper chemical cross-linking reagents, strategies for cross-linked peptide identification, and successful application of CXMS in structural characterization of proteins and protein complexes are discussed.

Programmable and Bidirectional Bending of Soft Actuators Based on Janus Structure with Sticky Tough PAA-Clay Hydrogel.

Facile preparation, rapid actuating, and versatile actions are great challenges in exploring new kinds of hydrogel actuators. In this paper, we presented a facile sticking method to prepare Janus bilayer and multilayer hydrogel actuators that benefited from a special tough and adhesive PAA-clay hydrogel. Combining physical and chemical cross-linking reagents, we endowed the PAA gel with both toughness and adhesion. This PAA gel was reinforced by further cross-linking with Fe3+. These two hydrogels with different cross-linking densities exhibited different swelling capabilities and moduli in the media manipulated by pH and ionic strength, thus acting as promising candidates for soft actuators. On the basis of these gels, we designed hydrogel actuators of rapid response in several minutes and precisely controlled actuating direction by sticking two hydrogel layers together. Elaborate soft actuators such as bidirectional bending flytrap, gel hand with grasp, open, and gesturing actions as well as word-writing actuator were prepared. This method could be generalized by using other stimuli-responsive hydrogels combined with the adhesive PAA gel, which would open a new way to programmable and versatile soft actuators.

Omega-3 Fatty Acids Used as Cross-Linkers to Reduce Antigenicity of Wheat Flour

Alpha-linolenic, docosahexaenoic and eicosapentaenoic acids were shown to cross-link hydrolyzed gluten in digested flour, thus reducing immunoreactivity of flour and bread. Residual immunoreactivity in cross-linked flour samples was determined with human coeliac immunoglobulin G (IgG) at 35–45% and 10–30%, respectively, for subtilisin and collagenase treatment. Analogous transglutaminase-cross-linked flour samples exhibited residual immunoreactivity at 34 and 15%. Chemical cross-linking reagents – polyethylenimine and diethanolamine – decreased it below 20%. Similar results were obtained when rabbit anti-QQQPP (Gln-Gln-Gln-Pro-Pro) IgG were used as detection antibodies. Flour samples underwent fermentation, thermal processing and simulated digestion. Residual immunoreactivity in bread made of flour cross-linked with fatty acids was determined with human IgG at 10–70% and 3–60%, respectively, for subtilisin and collagenase treatment. At the same time, transglutaminase cross-linking yielded residual immunoreactivity of bread at 10–20%. Rabbit antibodies did not indicate significant differences between samples of collagenase-treated bread, whether cross-linked with transglutaminase or fatty acids. Practical Applications Given the popularity of cereal-based products in our diet, gluten intolerance is a significant issue. The antigenicity of flour proteins can, to a great extent, be neutralized through hydrolysis and cross-linking with polyunsaturated omega-3 fatty acids. Antigenic amino acid structures are bound deep into the complex surface of the cross-linked protein and are rendered unrecognizable to the consumer's immune system. Cross-linked proteins in flour and bread provoke milder allergy symptoms, allowing dietary restrictions to be reduced. These natural reagents could potentially be a valuable tool in efforts to produce safer bakery products. We propose that they could be used in combination with other known cross-linkers to improve the overall antigenic effect of food processing.

Ionically cross-linkable hyaluronate-based hydrogels for injectable cell delivery

Although hyaluronate is an attractive biomaterial for many biomedical applications, hyaluronate hydrogels are generally formed using chemical cross-linking reagents that may cause unwanted side effects, including toxicity. We thus propose to design and prepare ionically cross-linkable hyaluronate compounds that can form gels in the presence of counter-ions. This study is based on the hypothesis that introduction of alginate to hyaluronate backbones (hyaluronate-g-alginate) could allow for gel formation in the presence of calcium ions. Here, we demonstrated ease of formation of cross-linked structures with calcium ions without additional chemical cross-linking reagents in hyaluronate-g-alginate (HGA) gels. The mechanical properties of HGA gels were regulated through changes in polymer composition and calcium concentration. We also confirmed that HGA gels could be useful in regenerating cartilage in a mouse model following subcutaneous injection into the dorsal region with primary chondrocytes. This finding was supported by histological and immunohistochemical analyses, glycosaminoglycan quantification and chondrogenic marker gene expression. This approach to the design and tailoring of ionically cross-linkable biomedical polymers may be broadly applicable to the development of biomaterials, especially in the drug delivery and tissue engineering fields.

Progress in various crosslinking modification for acellular matrix

Objective To review the current crosslinking strategies for acelluar matrix scaffold, laying the foundation for subsequent experiment. Data sources Data were mainly obtained from recent papers published in PubMed or indexed by Web of Science, with keyword like crosslinking. Results Various crosslinking strategies, including chemical, physical and biological methods, have been introduced to facilitate the performance of fresh acellular matrix. Chemical crosslinking reagents, involved in synthetic and naturally derived agents, need to be eliminated before implantation in case of their potential biotoxicity, although several crosslinking agents with less toxicity and specific characteristics have been developed. Physical crosslinking methods present to be safe, additive-free and relatively controllable for rapid surface functionalization with no consideration of remaining radioactivity. Biological crosslinking strategies have attracted great interest, and have been demonstrated to enhance collagen-based crosslinking since their preparations do not need toxic or potentially biologically contaminated substances and can be carried out under physiological conditions. Conclusions Kinds of crosslinking methods with its potential advantages have been developed to modify raw acelluar matrix, of which the performance are promising after being crosslinked by several crosslinking treatments. Further preclinical and clinical evaluations should be taken to vertify their safety and efficacy for the tissues and organs substitutes in tissue and regenerative medicine.

Effects of Cd2+ on cis-dimer structure of E-cadherin in living cells

E-cadherin, a calcium (Ca2+)-dependent cell–cell adhesion molecule, plays a key role in the maintenance of tissue integrity. We have previously demonstrated that E-cadherin functions in vivo as a cis-dimer through chemical cross-linking reagents. Ca2+ plays an important role in the cis-dimer formation of cadherin. However, the molecular mechanisms by which Ca2+ interacts with the binding sites that regulate cis-dimer structures have not been completely elucidated.As expected for a Ca2+ antagonist, cadmium (Cd2+) disrupts cadherin function by displacing Ca2+ from its binding sites on the cadherin molecules. We used Cd2+ as a probe for investigating the role of Ca2+ in the dynamics of the E-cadherin extracellular region that involve cis-dimer formation and adhesion. While cell–cell adhesion assembly was completely disrupted in the presence of Cd2+, the amount of cis-dimers of E-cadherin that formed at the cell surface was not affected. In our “Cd2+-switch” experiments, we did not find that Cd2+-induced E-cadherin cis-dimer formation in EL cells when they were incubated in low-Ca2+ medium.In the present study, we demonstrated for the first time the effects of Cd2+ on the cis-dimer structure of E-cadherin in living cells using a chemical cross-link analysis.

Formation of thermo‐sensitive polyelectrolyte complex micelles from two biocompatible graft copolymers for drug delivery

Thermo-sensitive polyelectrolyte complex (PEC) micelles assembled from two biocompatible graft copolymers chitosan-g-poly(N-isopropylacrylamide) (CS-g-PNIPAM) and carboxymethyl cellulose-g-poly(N-isopropylacrylamide) (CMC-g-PNIPAM) were prepared for delivery of 5-fluorouracil (5-FU). The PEC micelles showed a narrow size distribution with core-shell structure, in which the core formed from positively charged CS and negatively charged CMC by electrostatic interactions and the shell formed from thermo-sensitive PNIPAM. The synthesized PEC micelles have lower critical solution temperatures (LCST) in the region of 37°C, which is favorable for smart drug delivery applications. The hydrogen bondings between PEC micelles and 5-FU increased the drug loading. Changing temperature, pH or ionic strength, a sustained and controlled release was observed due to the deformation of PEC micelles. Adding glutaraldehyde, a chemical crosslinking reagent, was an efficient way to reinforce the micelles structure and decrease the initial burst release. Cytotoxicity assays showed that drug-loaded PEC micelles retained higher cell inhibition efficiency in HeLa cells.

Collagen crosslinking of porcine sclera using genipin

To evaluate the effect of collagen cross-linking induced by genipin in porcine sclera. Porcine cadaver eyes were treated with genipin at concentrations (by w/v) of 0.01%, 0.03%, 0.1%, 0.3%, 1.0% for 15 and 30 min. Riboflavin/ultraviolet A(UVA)-treated and untreated samples were used as controls. After treatment, scleral strips of 4.0 × 10.0 mm were cut. Twenty-four hours later, the stress-strain parameters of the strips were measured using a biomaterial microtester. The stress and Young's modulus at 8% strain were evaluated. Compared with untreated groups, after treatment with genipin for 15 min, the stress was increased by 66-246%, depending on the concentration of genipin. As for the 30-min groups, the stress was 171-444% higher than that of the control. The difference of the Young's modulus between genipin 15-min groups, except the 0.01% groups (p = 0.095), also had statistical significance (p < 0.05). The Young's modulus had significant difference between the untreated group and the genipin 30-min groups (all p < 0.05). Of 0.3% genipin for 15 min or 0.01% genipin for 30 min had a similar stress-strain curve with those of eyes treated with the riboflavin/UVA group. The sclera exhibited a bluish colour which became deeper with increase concentration and cross-linking time. Collagen cross-linking induced by genipin could increase the biomechanical strength in porcine sclera. The effect depends on the concentration and treatment time of genipin.

1P1-D05 真空遠心インプリントによるコラーゲンビトリゲル薄膜微細流路の開発(バイオアセンブラ)

This paper reports on a novel micro imprinting process of collagen membrane by vacuum evaporation of hydrogel on a template under centrifugal force 〜500G. This process realizes 3D membrane micro/nano structures of extra cellular matrix polymer (e.g. collagen, elastin) with natural self-assembled fibrous structures kept as same as in vivo. The fabricated structure is highly suitable for cell culture scaffold in tissue engineering since no chemical cross-linking reagent is necessary in this process.

Negative ion fragmentations of disulfide-containing cross-linking reagents are competitive with aspartic acid side-chain-induced cleavages

It has been shown that the disulfide moiety in the chemical cross-linking reagent dithiobis(succinimidyl)propionate (DSP), which is similar in structure to the natural cystine disulfide, cleaves preferentially to the peptide backbone in the negative ion mode. However, the tandem mass (MS/MS) spectra of peptides in the negative ion mode are often dominated by products arising from low-energy, side-chain-induced processes, which may compete with any facile cross-linker fragmentations and complicate identification of chemical cross-links in a complex mixture. Two disulfide-containing crosslinking reagents similar to DSP, but with varying spacer arm lengths, were synthesized and the MS/MS spectra of several model peptides cross-linked with these reagents were investigated. Theoretical calculations were used to describe the energetics of the cross-linker fragmentations as well as several low-energy side-chain-induced fragmentations which compete with disulfide cleavages. Altering the spacer arm length of the cross-linker, such that there is one methylene group less than in DSP, results in a more facile cleavage process, whilst the opposite is true when a methylene group is added. Of the low-energy side-chain-induced fragmentations studied, only those from aspartic acid compete significantly with those of the cross-linker disulfide. Low-energy cleavage processes from aspartic acid that compete with cross-linker fragmentations occur in the negative ion MS/MS spectra of the cross-linked peptides, irrespective of the spacer arm length. Other fragmentation pathways do not significantly interfere with low-energy disulfide cleavage, making the presence of additional product ions in the MS/MS spectrum diagnostic for the presence of aspartic acid.

Hydrogel membranes based on gum tragacanth with tunable structure and properties. I. Preparation method using Taguchi experimental design

AbstractGum tragacanth (GT) was exploited to create a series of novel hydrogel membranes with diverse swelling properties. The films of this biopolymer were crosslinked with applying heat only, as well as application of four types of chemical crosslinking reagents, including glycerin, ethylene glycol, triethylene glycol, and glutaraldehyde. The effects of the crosslinking agent concentration, crosslinking reaction temperature (57–97°C) and reaction time (2–24 h) on the equilibrium swelling of each series of the synthesized hydrogels were investigated by the aid of Taguchi method of experimental design. This approach facilitated the study of interaction of a large number of variables spanned by factors and their levels with a small number of experimental runs. In all cases, it was found that by increasing the concentration of each type of the crosslinking reagent and extension of the crosslinking reaction time, the swelling capacity of the prepared hydrogel films decreased. However, by increasing the crosslinking temperature, the equilibrium swelling capacity changed by passing through a minimum. As a whole, the equilibrium swelling values of the hydrogels proved that a broad range of hydrogel membranes having the crosslinking densities of 0.83–23.87 × 10−6 mol/cm3 and swelling capacities of 120–1580 g/g was achieved successfully from GT. Thus, GT was assigned as a promising biopolymer for preparing membranes with diverse structural and swelling characteristics. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

Facile control of RGD-alginate/hyaluronate hydrogel formation for cartilage regeneration

Hydrogels are attractive carriers for delivery of cells in tissue engineering as cell/polymer constructs can be injected into the body in a minimally invasive manner. In this study, we prepared hydrogels using only RGD-alginate and hyaluronate in the presence of primary chondrocytes without additional chemical cross-linking reagents. RGD peptides were introduced to alginate chains to increase the specific interactions with cells, and hyaluronate was used to enhance the cellular interactions and resultant gel formation. The gelation of RGD-alginate/hyaluronate hydrogels was confirmed by rheological measurements. These gels were useful to regenerate cartilage tissues in vivo, which was confirmed by analysis of glycosaminoglycan secretion and chondrogenic gene expression in both qualitative and quantitative manner. This approach to controlling the gelation behavior using only cells and cell-interactive polymers could be useful for delivery of cells and therapeutics in biomedical applications.

Development of a Novel Cross-linking Strategy for Fast and Accurate Identification of Cross-linked Peptides of Protein Complexes

Knowledge of elaborate structures of protein complexes is fundamental for understanding their functions and regulations. Although cross-linking coupled with mass spectrometry (MS) has been presented as a feasible strategy for structural elucidation of large multisubunit protein complexes, this method has proven challenging because of technical difficulties in unambiguous identification of cross-linked peptides and determination of cross-linked sites by MS analysis. In this work, we developed a novel cross-linking strategy using a newly designed MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). DSSO contains two symmetric collision-induced dissociation (CID)-cleavable sites that allow effective identification of DSSO-cross-linked peptides based on their distinct fragmentation patterns unique to cross-linking types (i.e. interlink, intralink, and dead end). The CID-induced separation of interlinked peptides in MS/MS permits MS(3) analysis of single peptide chain fragment ions with defined modifications (due to DSSO remnants) for easy interpretation and unambiguous identification using existing database searching tools. Integration of data analyses from three generated data sets (MS, MS/MS, and MS(3)) allows high confidence identification of DSSO cross-linked peptides. The efficacy of the newly developed DSSO-based cross-linking strategy was demonstrated using model peptides and proteins. In addition, this method was successfully used for structural characterization of the yeast 20 S proteasome complex. In total, 13 non-redundant interlinked peptides of the 20 S proteasome were identified, representing the first application of an MS-cleavable cross-linker for the characterization of a multisubunit protein complex. Given its effectiveness and simplicity, this cross-linking strategy can find a broad range of applications in elucidating the structural topology of proteins and protein complexes.

Potential use of γ-cyclodextrin polypseudorotaxane hydrogels as an injectable sustained release system for insulin

The development of injectable hydrogels for protein delivery is a major challenge. In this study, insulin/α-cyclodextrin (α-CyD) and γ-CyD polypseudorotaxane (PPRX) hydrogels were prepared through inclusion complexation between high molecular weight poly(ethylene glycol) (PEG) and CyDs. The α-CyD and γ-CyD PPRX hydrogels were formed by inserting one PEG chain in the α-CyD cavity and two PEG chains in the γ-CyD cavity. Insulin/CyD PPRX hydrogel formation was based on physical crosslinking induced by self-assembling without chemical crosslinking reagent. The supramolecular structures of insulin/CyD PPRX hydrogels were confirmed with 1H nuclear magnetic resonance ( 1H NMR), X-ray diffraction, differential scanning calorimetry (DSC) and scanning electron microscopy (SEM). The in vitro release study showed that the release rate of insulin from the CyDs PPRX hydrogels decreased in the order of γ-CyD PPRX hydrogel > α-CyD PPRX hydrogel. This decrease was controlled by the addition of CyDs to the medium. The serum insulin level after subcutaneous administration of γ-CyD PPRX hydrogel to rats was significantly prolonged, accompanying with an increase in the area under serum concentration–time curve, which was clearly reflected in the prolonged hypoglycemic effect. In conclusion, these results suggest the potential use of γ-CyD PPRX hydrogel as an injectable sustained release system for insulin.