Structural Elucidation of Ubiquitin via Gas-Phase Ion/Ion Cross-Linking Reactions Using Sodium-Cationized Reagents Coupled with Infrared Multiphoton Dissociation.

Accurate structural determination of proteins is critical to understanding their biological functions and the impact of structural disruption on disease progression. Gas-phase cross-linking mass spectrometry (XL-MS) via ion/ion reactions between multiply charged protein cations and singly charged cross-linker anions has previously been developed to obtain low-resolution structural information on proteins. This method significantly shortens experimental time relative to conventional solution-phase XL-MS but has several technical limitations: (1) the singly deprotonated N-hydroxysulfosuccinimide (sulfo-NHS)-based cross-linker anions are restricted to attachment at neutral amine groups of basic amino acid residues and (2) analyzing terminal cross-linked fragment ions is insufficient to unambiguously localize sites of linker attachment. Herein, we demonstrate enhanced structural information for alcohol-denatured A-state ubiquitin obtained from an alternative gas-phase XL-MS approach. Briefly, singly sodiated ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS) cross-linker anions enable covalent cross-linking at both ammonium and amine groups. Additionally, covalently modified internal fragment ions, along with terminal b-/y-type counterparts, improve the determination of linker attachment sites. Molecular dynamics simulations validate experimentally obtained gas-phase conformations of denatured ubiquitin. This method has identified four cross-linking sites across 8+ ubiquitin, including two new sites in the N-terminal region of the protein that were originally inaccessible in prior gas-phase XL approaches. The two N-terminal cross-linking sites suggest that the N-terminal half of ubiquitin is more compact in gas-phase conformations. By comparison, the two C-terminal linker sites indicate the signature transformation of this region of the protein from a native to a denatured conformation. Overall, the results suggest that the solution-phase secondary structures of the A-state ubiquitin are conserved in the gas phase. This method also provides sufficient sensitivity to differentiate between two gas-phase conformers of the same charge state with subtle structural variations.

PIMT-Mediated Labeling of l-Isoaspartic Acid with Tris Facilitates Identification of Isomerization Sites in Long-Lived Proteins.

Isomerization of individual residues in long-lived proteins (LLPs) is a subject of growing interest in connection with many age-related human diseases. When isomerization occurs in LLPs, it can lead to deleterious changes in protein structure, function, and proteolytic degradation. Herein, we present a novel labeling technique for rapid identification of l-isoAsp using the enzyme protein l-isoaspartyl methyltransferase (PIMT) and Tris. The succinimide intermediate formed during reaction of l-isoAsp-containing peptides with PIMT and S-adenosyl methionine (SAM) is reactive with Tris base and results in a Tris-modified aspartic acid residue with a mass shift of +103 Da. Tris-modified aspartic acid exhibits prominent and repeated neutral loss of water when subjected to collisional activation. In addition, another dissociation pathway regenerates the original peptide following loss of a characteristic mass shift. Furthermore, it is demonstrated that Tris modification can be used to identify sites of isomerization in LLPs from biological samples such as the lens of the eye. This approach simplifies identification by labeling isomerization sites with a tag that causes a mass shift and provides characteristic loss during collisional activation.

Rapidly Self-Deactivating and Injectable Succinyl Ester-Based Bioadhesives for Postoperative Antiadhesion.

Postoperative adhesion not only causes severe complications for patients but also increases their economic burden. Injectable bioadhesives with adhesiveness to tissues can cover irregular wounds and stay stable in situ, which is a promising barrier for antiadhesion. However, the potential tissue adhesion caused by bioadhesives' indiscriminate adhesiveness between normal and wounded tissue is still a problem. Herein, by using poly(ethylene glycol) succinimidyl succinate (PEG-SS) and gelatin, a succinyl ester-based bioadhesive (SEgel) was fabricated with self-deactivating properties for postoperative antiadhesion. Because N-hydroxysuccinimide esters (NHS-esters) were used as the adhesive group, the bioadhesives' side in contact with the tissue built covalent anchors quickly to maintain the stability, but the superficial layer facing outward withstood fast hydrolysis and then lost its adhesion within minutes, avoiding the indiscriminate adhesiveness. In addition, because of the specific degradation behavior of succinyl ester, the SEgel with proper in vivo retention was achieved without the worry of causing foreign body reactions and unexpected tissue adhesion. Both the cecum-sidewall adhesion and hepatic adhesion models showed that the SEgel markedly reduced the severity of tissue adhesion. These results, together with the ease of the preparation process and well-proven biocompatibility of raw materials, revealed that the SEgel might be a promising solution for postoperative antiadhesion.

Near-Infrared Fluorescence Imaging of Carotid Plaques in an Atherosclerotic Murine Model.

Successful imaging of atherosclerosis, one of the leading global causes of death, is crucial for diagnosis and intervention. Near-infrared fluorescence (NIRF) imaging has been widely adopted along with multimodal/hybrid imaging systems for plaque detection. We evaluate two macrophage-targeting fluorescent tracers for NIRF imaging (TLR4-ZW800-1C and Feraheme-Alexa Fluor 750) in an atherosclerotic murine cohort, where the left carotid artery (LCA) is ligated to cause stenosis, and the right carotid artery (RCA) is used as a control. Imaging performed on dissected tissues revealed that both tracers had high uptake in the diseased vessel compared to the control, which was readily visible even at short exposure times. In addition, ZW800-1C's renal clearance ability and Feraheme's FDA approval puts these two tracers in line with other NIRF tracers such as ICG. Continued investigation with these tracers using intravascular NIRF imaging and larger animal models is warranted for clinical translation.

Synthesis and kinetic characterization of hyperbolic inhibitors of human cathepsins K and S based on a succinimide scaffold.

Cathepsins K and S are closely related papain-like cysteine peptidases and potential therapeutic targets for metabolic and inflammatory diseases such as osteoporosis and arthritis. Here we describe the reduction of a previously characterized succinimide (2,5-dioxopyrrolidine)-containing hyperbolic inhibitor of cathepsin K (methyl (RS)-N-[1-(4-methoxyphenyl)-2,5-dioxopyrrolidin-3-yl]glycinate), to obtain a better and more selective compound (compound 4a - methyl (2,5-dioxopyrrolidin-3-yl)glycinate), which acted as a hyperbolic mixed inhibitor/activator similar to already known allosteric effectors of cathepsin K. We then investigated the potential of the succinimide scaffold as inhibitors of cathepsins K and/or S and synthesized a library of such compounds by 1,4-addition of α-amino acid esters and related compounds to N-substituted maleimides. From the generated library, we identified the first small molecule hyperbolic inhibitors of cathepsin S (methyl ((R)-2,5-dioxopyrrolidin-3-yl)-l-threoninate (compound R-4c) and 3-{[(1S,2R,3'S)-2-hydroxycyclohexyl]amino}pyrrolidine-2,5-dione (compound (1S,2R,3'S-10)). The former acted via a similar mechanism to compound 4a, while the latter was a hyperbolic specific inhibitor of cathepsin S. Given the versatility of the scaffold, the identified compounds will be used as the basis for the development of high-affinity hyperbolic inhibitors of the individual peptidases and to explore the potential of hyperbolic inhibitors for the inhibition of cysteine cathepsins in in vitro models.

Label-retention expansion microscopy.

Expansion microscopy (ExM) increases the effective resolving power of any microscope by expanding the sample with swellable hydrogel. Since its invention, ExM has been successfully applied to a wide range of cell, tissue, and animal samples. Still, fluorescence signal loss during polymerization and digestion limits molecular-scale imaging using ExM. Here, we report the development of label-retention ExM (LR-ExM) with a set of trifunctional anchors that not only prevent signal loss but also enable high-efficiency labeling using SNAP and CLIP tags. We have demonstrated multicolor LR-ExM for a variety of subcellular structures. Combining LR-ExM with superresolution stochastic optical reconstruction microscopy (STORM), we have achieved molecular resolution in the visualization of polyhedral lattice of clathrin-coated pits in situ.

Ex vivo evaluation of a multilayered sealant patch for watertight dural closure: cranial and spinal models.

Cerebrospinal fluid leakage is a frequent complication after cranial and spinal surgery. To prevent this complication and seal the dura watertight, we developed Liqoseal, a dural sealant patch comprising a watertight polyesterurethane layer and an adhesive layer consisting of poly(DL-lactide-co-ε-caprolactone) copolymer and multiarmed N-hydroxylsuccinimide functionalized polyethylene glycol. We compared acute burst pressure and resistance to physiological conditions for 72 h of Liqoseal, Adherus, Duraseal, Tachosil, and Tisseel using computer-assisted models and fresh porcine dura. The mean acute burst pressure of Liqoseal in the cranial model (145 ± 39 mmHg) was higher than that of Adherus (87 ± 47 mmHg), Duraseal (51 ± 42 mmHg) and Tachosil (71 ± 16 mmHg). Under physiological conditions, cranial model resistance test results showed that 2 of 3 Liqoseal sealants maintained dural attachment during 72 hours as opposed to 3 of 3 for Adherus and Duraseal and 0 of 3 for Tachosil. The mean burst pressure of Liqoseal in the spinal model (233 ± 81 mmHg) was higher than that of Tachosil (123 ± 63 mmHg) and Tisseel (23 ± 16 mmHg). Under physiological conditions, spinal model resistance test results showed that 2 of 3 Liqoseal sealants maintained dural attachment for 72 hours as opposed to 3 of 3 for Adherus and 0 of 3 for Duraseal and Tachosil. This novel study showed that Liqoseal is capable of achieving a strong watertight seal over a dural defect in ex vivo models.

Increasing salinity of fibrinogen solvent generates stable fibrin hydrogels for cell delivery or tissue engineering.

Fibrin has been used clinically for wound coverings, surgical glues, and cell delivery because of its affordability, cytocompatibility, and ability to modulate angiogenesis and inflammation. However, its rapid degradation rate has limited its usefulness as a scaffold for 3D cell culture and tissue engineering. Previous studies have sought to slow the degradation rate of fibrin with the addition of proteolysis inhibitors or synthetic crosslinkers that require multiple functionalization or polymerization steps. These strategies are difficult to implement in vivo and introduce increased complexity, both of which hinder the use of fibrin in research and medicine. Previously, we demonstrated that additional crosslinking of fibrin gels using bifunctionalized poly(ethylene glycol)-n-hydroxysuccinimide (PEG-NHS) slows the degradation rate of fibrin. In this study, we aimed to further improve the longevity of these PEG-fibrin gels such that they could be used for tissue engineering in vitro or in situ without the need for proteolysis inhibitors. It is well documented that increasing the salinity of fibrin precursor solutions affects the resulting gel morphology. Here, we investigated whether this altered morphology influences the fibrin degradation rate. Increasing the final sodium chloride (NaCl) concentration from 145 mM (physiologic level) to 250 mM resulted in fine, transparent high-salt (HS) fibrin gels that degrade 2-3 times slower than coarse, opaque physiologic-salt (PS) fibrin gels both in vitro (when treated with proteases and when seeded with amniotic fluid stem cells) and in vivo (when injected subcutaneously into mice). Increased salt concentrations did not affect the viability of encapsulated cells, the ability of encapsulated endothelial cells to form rudimentary capillary networks, or the ability of the gels to maintain induced pluripotent stem cells. Finally, when implanted subcutaneously, PS gels degraded completely within one week while HS gels remained stable and maintained viability of seeded dermal fibroblasts. To our knowledge, this is the simplest method reported for the fabrication of fibrin gels with tunable degradation properties and will be useful for implementing fibrin gels in a wide range of research and clinical applications.

RGDS-Modified Superporous Poly(2-Hydroxyethyl Methacrylate)-Based Scaffolds as 3D In Vitro Leukemia Model.

Superporous poly(2-hydroxyethyl methacrylate-co-2-aminoethyl methacrylate) (P(HEMA-AEMA)) hydrogel scaffolds are designed for in vitro 3D culturing of leukemic B cells. Hydrogel porosity, which influences cell functions and growth, is introduced by adding ammonium oxalate needle-like crystals in the polymerization mixture. To improve cell vitality, cell-adhesive Arg-Gly-Asp-Ser (RGDS) peptide is immobilized on the N-(γ-maleimidobutyryloxy)succinimide-activated P(HEMA-AEMA) hydrogels via reaction of SH with maleimide groups. This modification is especially suitable for the survival of primary chronic lymphocytic leukemia cells (B-CLLs) in 3D cell culture. No other tested stimuli (interleukin-4, CD40 ligand, or shaking) can further improve B-CLL survival or metabolic activity. Both unmodified and RGDS-modified P(HEMA-AEMA) scaffolds serve as a long-term (70 days) 3D culture platforms for HS-5 and M2-10B4 bone marrow stromal cell lines and MEC-1 and HG-3 B-CLL cell lines, although the adherent cells retain their physiological morphologies, preferably on RGDS-modified hydrogels. Moreover, the porosity of hydrogels allows direct cell lysis, followed by efficient DNA isolation from the 3D-cultured cells. P(HEMA-AEMA)-RGDS thus serves as a suitable 3D in vitro leukemia model that enables molecular and metabolic assays and allows imaging of cell morphology, interactions, and migration by confocal microscopy. Such applications can prospectively assist in testing of drugs to treat this frequently recurring or refractory cancer.

Electrochemiluminescence immunosensor for cytokeratin fragment antigen 21-1 detection using electrochemically mediated atom transfer radical polymerization.

The cytokeratin fragment antigen 21-1 (CYFRA 21-1) protein is a critical tumor biomarker tightly related to non-small cell lung cancer (NSCLC). Herein, we prepared an effective electrochemiluminescence (ECL) immunosensor for CYFRA 21-1 detection using electrochemically mediated atom transfer radical polymerization (eATRP). The CYFRA 21-1 antigen was fixed on the electrode surface by constructing a sandwich type antibody-antigen-antibody immune system. The sensitivity of ECL was improved by using the eATRP reaction. In this method, eATRP was applied to CYFRA 21-1 detection antibody with N-acryloyloxysuccinimide as functional monomer. This is the first time that ECL and eATRP signal amplification technology had been combined. Under the optimized testing conditions, the immunosensor showed a good linear relation in the range from 1 fg mL-1 to 1 µg mL-1 at a limit of detection of 0.8 fg mL-1 (equivalent to ~ 134 molecules in a 10 µL sample). The ECL immunosensing system based on eATRP signal amplification technology provided a new way for rapid diagnosis of lung cancer by detecting CYFRA 21-1. The paper prepared an electrochemiluminescence biosensor for ultrasensitive detection of CYFRA 21-1 via eATRP signal amplification strategy, which had the advantages of high sensitivity, reproducibility, and held potential prospect for analysis of low-abundance.

First 3D-Structural Data of Full-Length Guanylyl Cyclase 1 in Rod-Outer-Segment Preparations of Bovine Retina by Cross-Linking/Mass Spectrometry.

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.

Potential of three-step pretargeting radioimmunotherapy using biotinylated bevacizumab and succinylated streptavidin in triple-negative breast cancer xenograft.

OBJECTIVE: Pretargeting radioimmunotherapy (PRIT) is a promising approach that can reduce long-time retention of blood radioactivity and consequently reduce hematotoxicity. Among the PRIT strategies, the combination of biotin-conjugated mAb and radiolabeled streptavidin (StAv) is a simple and convenient method because of its ease of preparation. This study performed three-step (3-step) PRIT using the sequential injection of (1) biotinylated bevacizumab (Bt-BV), (2) avidin, and (3) radiolabeled StAv for the treatment of triple-negative breast cancer (TNBC). METHODS: Four biodistribution studies were performed using 111In in tumor-bearing mice to optimize each step of our PRIT methods. Further, a therapeutic study was performed with optimized 3-step PRIT using 90Y-labeled StAv. RESULTS: Based on the biodistribution studies, the protein dose of Bt-BV and avidin was optimized to 100 µg and 10 molar equivalent of BV, respectively. Succinylation of StAv significantly decreased the kidney accumulation level (with succinylation (6.96 ± 0.91) vs without succinylation (20.60 ± 1.47) at 1 h after injection, p < 0.0001) with little effect on the tumor accumulation level. In the therapeutic study, tumor growth was significantly suppressed in treatment groups with optimized 3-step PRIT using 90Y-labeled succinylated StAv compared to that of the no-treatment group (p < 0.05). CONCLUSIONS: The 3-step PRIT strategy of this study achieved fast blood clearance and low kidney uptake with little effect on the tumor accumulation level, and a certain degree of therapeutic effect was consequently observed. These results indicated that the pretargeting treatment of the current study may be effective for human TNBC treatment.

Mannosylated Cationic Copolymers for Gene Delivery to Macrophages.

Macrophages are desirable targets for gene therapy of cancer and other diseases. Cationic diblock copolymers of polyethylene glycol (PEG) and poly-L-lysine (PLL) or poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (pAsp(DET)) are synthesized and used to form polyplexes with a plasmid DNA (pDNA) that are decorated with mannose moieties, serving as the targeting ligands for the C type lectin receptors displayed at the surface of macrophages. The PEG-b-PLL copolymers are known for its cytotoxicity, so PEG-b-PLL-based polyplexes are cross-linked using reducible reagent dithiobis(succinimidyl propionate) (DSP). The cross-linked polyplexes display low toxicity to both mouse embryonic fibroblasts NIH/3T3 cell line and mouse bone marrow-derived macrophages (BMMΦ). In macrophages mannose-decorated polyplexes demonstrate an ≈8 times higher transfection efficiency. The cross-linking of the polyplexes decrease the toxicity, but the transfection enhancement is moderate. The PEG-b-pAsp(DET) copolymers display low toxicity with respect to the IC-21 murine macrophage cell line and are used for the production of non-cross-linked pDNA-contained polyplexes. The obtained mannose modified polyplexes exhibit ca. 500-times greater transfection activity in IC-21 macrophages compared to the mannose-free polyplexes. This result greatly exceeds the targeting gene transfer effects previously described using mannose receptor targeted non-viral gene delivery systems. These results suggest that Man-PEG-b-pAsp(DET)/pDNA polyplex is a potential vector for immune cells-based gene therapy.

Thermosensitive "Smart" Surfaces for Biorecognition Based Cell Adhesion and Controlled Detachment.

The biorecognition-based control of attachment/detachment of MCF-7 cancer cells from polymer-coated surfaces is demonstrated. A glass surface is coated with a thermoresponsive statistical copolymer of poly(N-isopropylacrylamide-co-acrylamide) [p(NIPAm-co-Am)], which is end-capped with the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide, and the hydrophilic polymer poly(ethylene glycol) (PEG). Below the lower critical solution temperature (LCST) of p(NIPAm-co-Am) (38 °C), the copolymers are in the extended conformation, allowing for accessibility of the GRGDS peptides to membrane-associated integrins thus enabling cell attachment. Above the LCST, the p(NIPAm-co-Am) polymers collapse into globular conformations, resulting in the shielding of the GRGDS peptides into the PEG brush with consequent inaccessibility to cell-surface integrins, causing cell detachment. The surface coating is carried out by a multi-step procedure that included: glass surface amination with 3-aminopropyltriethoxysilane; reaction of mPEG5kDa -N-hydroxysuccinimide (NHS) and p(NIPam-co-Am)15.1kDa -bis-NHS with the surface aminopropyl groups and conjugation of GRGDS to the carboxylic acid termini of p(NIPam-co-Am)15.1kDa -COOH. A range of spectrophotometric, surface, and microscopy assays confirmed the identity of the polymer-coated substrates. Competition studies prove that MCF-7 cancer cells are attached via peptide recognition at the coated surfaces according to the mPEG5kDa /p(NIPam-co-Am)15.1kDa -GRGDS molar ratio. These data suggest the system can be exploited to modulate cell integrin/GRGDS binding for controlled cell capture and release.

Functional Integrin Regulation Through Interactions with Tetraspanin CD9.

Integrins are adhesion receptors that mediate many intercellular and cell-extracellular matrix interactions with relevance in physiology and pathology. Unlike other cellular receptors, integrins critically require activation for ligand binding. Through interaction in cis with other molecules and the formation of tetraspanin-enriched membrane microdomains (TEMs), the tetraspanin CD9 regulates integrin activity and avidity. Here we present three techniques used to study CD9-integrin interactions and integrin activation.

Research progress in biological activities of succinimide derivatives.

Succinimides are well recognized heterocyclic compounds in drug discovery which produce diverse therapeutically related applications in pharmacological practices. Researches in medicinal chemistry field have isolated and synthesized succinimide derivatives with multiple medicinal properties including anticonvulsant, anti-inflammatory, antitumor and antimicrobial agents, 5-HT receptor ligands and enzyme inhibitors. Simultaneously, SAR (Structure-Activity Relationship) analysis has been gradually possessed, along with a great deal of derivatives have been derived for potential targets. In this article, we comprehensively summarize the biological activities and SAR for succinimide derivatives, along with the featuring bioactive molecules reported in patents, wishing to provide an overall retrospect and prospect on the succinimide analogues.

Detecting Necroptosis in Virus-Infected Cells.

Necroptosis has been implicated as a critical cell death pathway in cancers, Alzheimer's and other neurodegenerative diseases, and virus-infected cells. Necroptosis occurs when mixed-lineage kinase domain-like protein (MLKL) punctures the cytoplasmic membrane allowing a rapid influx of water leading to a loss of cellular integrity. As its role in human disease becomes apparent, methods identifying necroptosis will need to be further developed and optimized. Here we describe identification of necroptosis through quantifying cell death with pathway inhibitors and using western blots to identify end points of MLKL activation and protein-protein interactions leading to it.

On-Demand Detachment of Succinimides on Cysteine to Facilitate (Semi)Synthesis of Challenging Proteins.

The maleimide group is a widely used reagent for bioconjugation of peptides, proteins, and oligonucleotides employing Michael addition and Diels-Alder cycloaddition reactions. However, the utility of this functionality in chemical synthesis of peptides and proteins remains unexplored. We report, for the first time that PdII complexes can mediate the efficient removal of various succinimide derivatives in aqueous conditions. Succinimide removal by PdII was applied for the synthesis of two ubiquitin activity-based probes (Ub-ABPs) employing solid phase chemical ligation (SPCL). SPCL was achieved through a sequential three segment ligation on a polymer support via a maleimide anchor. The obtained probes successfully formed the expected covalent complexes with deubiquitinating enzymes (DUBs) USP2 and USP7, highlighting the use of our new method for efficient preparation of unique synthetic proteins. Importantly, we demonstrate the advantages of our newly developed method for the protection and deprotection of native cysteine with a succinimide group in a peptide fragment derived from thioredoxin-1 (Trx-1) obtained via intein based expression to enable ligation/desulfurization and subsequent disulfide bond formation in a one-pot process.

Automated Synthesis of Fluorine-18 Labeled CXCR4 Ligand via the Conjugation with Nicotinic Acid N-Hydroxysuccinimide Ester (6-[18F]SFPy).

The C-X-C motif chemokine receptor 4 (CXCR4) is a seven-transmembrane G protein-coupled receptor that is overexpressed in numerous diseases, particularly in various cancers and is a powerful chemokine, attracting cells to the bone marrow niche. Therefore, CXCR4 is an attractive target for imaging and therapeutic purposes. The goal of this study is to develop an efficient, reproducible, and straightforward method to prepare a fluorine-18 labeled CXCR4 ligand. 6-[18F]Fluoronicotinic acid-2,3,5,6-tetrafluorophenyl ester (6-[18F]FPy-TFP) and nicotinic acid N-hydroxysuccinimide ester (6-[18F]SFPy) have been prepared using 'fluorination on the Sep-Pak' method. Conjugation of 6-[18F]SFPy or 6-[18F]FPy-TFP with the alpha-amino group at the N terminus of the protected T140 precursor followed by deprotection, yielded the final product 6-[18F]FPy-T140. The overall radiochemical yields were 6-17% (n = 15, decay-corrected) in a 90-min radiolabeling time with a radiochemical purity >99%. 6-[18F]FPy-T140 exhibited high specific binding and nanomolar affinity for CXCR4 in vitro, indicating that the biological activity of the peptide was preserved. For the first time, [18F]SFPy has been prepared using 'fluorination on the Sep-Pak' method that allows rapid automated synthesis of 6-[18F]FPy-T140. In addition to increased synthetic efficiency, this construct binds with CXCR4 in high affinity and may have potential as an in vivo positron emission tomography (PET) imaging agent. This radiosynthesis method should encourage wider use of this PET agent to quantify CXCR4 in both research and clinical settings.

Oral GLP1 Gene Delivery by an Antibody-Guided Nanomaterial to Treat Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a chronic and progressive hyperglycemic condition. Glucagon-like peptide-1 (GLP1) is an incretin secreted from pancreatic ß-cells and helps to produce insulin to balance the blood glucose level without the risk of hypoglycemia. However, the therapeutic application of GLP1 is limited by its intrinsic short half-life and rapid metabolic clearance in the body. To enhance the antidiabetic effect of GLP1, we designed a human cysteine-modified IgG1-Fc antibody-mediated oral gene delivery vehicle, which helps to produce GLP1 sustainably in the target site with the help of increased half-life of the Fc-conjugated nanocarrier, protects GLP1 from acidic and enzymatic degradation in the gastrointestinal (GI) tract, uptakes and transports the GLP1 formulation through the neonatal Fc receptor (FcRn), and helps to release the GLP1 gene in the intestine. Our formulation could reduce the blood glucose from about an average of 320 mg/dL (hyperglycemic) to 150 mg/dL (normal blood glucose concentration) in diabetic mice, which is about 50% reduction of the total blood glucose concentration. GLP1 (500 µg) complexed with the IgG1-Fc carrier was proven to be the optimal dose for a complete reduction of hyperglycemic conditions in diabetic mice. A significant amount of insulin production and the presence of GLP1 peptide were observed in the pancreatic islets of oral GLP1 formulation-treated diabetic mice in immunohistochemistry analysis compared to nontreated diabetic mice. The orally given formulation was completely nontoxic according to the histopathology analysis of mice organ tissues, and no mice death was observed. Our antibody-mediated oral gene delivery system is a promising tool for various oral therapeutic gene delivery applications to treat diseases like diabetes.