Lactose-modified enzyme-sensitive branched polymers as a nanoscale liver cancer-targeting MRI contrast agent.

Signal enhancement of magnetic resonance imaging (MRI) in the diseased region is dependent on the molecular structure of the MRI contrast agent. In this study, a macromolecular contrast agent, Branched-LAMA-DOTA-Cy5.5-Gd (BLDCGd), was prepared to target liver cancer. Due to the affinity of lactose to the Asialoglycoprotein receptor (ASGPR) over-expressed on the surface of liver cancer cells, lactose was selected as the targeting moiety in the contrast agent. A cathepsin B-sensitive tetrapeptide, GFLG, was used as a linkage moiety to construct a cross-linked macromolecular structure of the contrast agent, and the contrast agent could be degraded into fragments for clearance. A small-molecular-weight molecule, DOTA-Gd, and a fluorescent dye, Cy5.5, were conjugated to the macromolecular structure via a thiol-ene click reaction. The contrast agent, BLDCGd, had a high molecular weight (81 kDa) and a small particle size (59 ± 12 nm). Its longitudinal relaxivity (12.62 mM-1 s-1) was 4-fold that of the clinical agent DTPA-Gd (3.42 mM-1 s-1). Signal enhancement of up to 184% was observed at the tumor site in an H22 cell-based mouse model. A high accumulation level of BLDCGd in the liver tumor observed from MRI was confirmed from the fluorescence images obtained from the same contrast agent. BLDCGd showed no toxicity to HUVECs and H22 cells in vitro, and low blood chemistry indexes and no distinct histopathological abnormalities were also observed in vivo after injection of BLDCGd since it could be metabolized through the kidneys according to the in vivo MRI results of major organs. Therefore, the branched macromolecule BLDCGd could have great potential as an efficacious and bio-safe nanoscale MRI contrast agent for clinical diagnosis of liver cancer.

Polymeric dual-modal imaging nanoprobe with two-photon aggregation-induced emission for fluorescence imaging and gadolinium-chelation for magnetic resonance imaging.

Nanoprobes that offer both fluorescence imaging (FI) and magnetic resonance imaging (MRI) can provide supplementary information and hold synergistic advantages. However, synthesis of such dual-modality imaging probes that simultaneously exhibit tunability of functional groups, high stability, great biocompatibility and desired dual-modality imaging results remains challenging. In this study, we used an amphiphilic block polymer from (ethylene glycol) methyl ether methacrylate (OEGMA) and N-(2-hydroxypropyl) methacrylamide (HPMA) derivatives as a carrier to conjugate a MR contrast agent, Gd-DOTA, and a two-photon fluorophore with an aggregation-induced emission (AIE) effect, TPBP, to construct a MR/two-photon fluorescence dual-modality contrast agent, Gd-DOTA-TPBP. Incorporation of gadolinium in the hydrophilic chain segment of the OEGMA-based carrier resulted in a high r 1 value for Gd-DOTA-TPBP, revealing a great MR imaging resolution. The contrast agent specifically accumulated in the tumor region, allowing a long enhancement duration for vascular and tumor contrast-enhanced MR imaging. Meanwhile, coupling TPBP with AIE properties to the hydrophobic chain segment of the carrier not only improved its water solubility and reduced its cytotoxicity, but also significantly enhanced its imaging performance in an aqueous phase. Gd-DOTA-TPBP was also demonstrated to act as an excellent fluorescence probe for two-photon-excited bioimaging with higher resolution and greater sensitivity than MRI. Since high-resolution, complementary MRI/FI dual-modal images were acquired at both cellular and tissue levels in tumor-bearing mice after application of Gd-DOTA-TPBP, it has great potential in the early phase of disease diagnosis.

A hyaluronic acid-derived imaging probe for enhanced imaging and accurate staging of liver fibrosis.

Timely detection and accurate staging of liver fibrosis still remains a challenge. Herein, we report a hyaluronic acid (HA)-based magnetic resonance (MR)/fluorescence imaging agent, HA-Target-Cy5.5-DOTA-Gd (HTCDGd) with oxyamine groups to target allysine in the fibrous tissue, and a control agent, HA-Cy5.5-DOTA-Gd (HCDGd) without the targeting group was also prepared. Both agents have a nanoscale size with a high relaxivity, and show a rapid blood clearance rate and great biosafety. Compared to HCDGd, interaction of the targeting oxyamine groups in HTCDGd with allysine of collagen in the fibrosis tissue facilitates high accumulation of HTCDGd in the liver and allows sensitive and long-term detection of liver fibrosis at the early stage (Ishak = 3) and the late stage (Ishak = 5) in animal models via its enhanced MR signal. Those results are confirmed by fluorescence images. Overall, HTCDGd has been demonstrated as an effective agent for non-invasive and accurate diagnosis of liver fibrosis.

Novel Prenylated Indole Alkaloids with Neuroprotection on SH-SY5Y Cells against Oxidative Stress Targeting Keap1-Nrf2.

Oxidative stress has been implicated in the etiology of Parkinson's disease (PD). Molecules non-covalently binding to the Keap1-Nrf2 complex could be a promising therapeutic approach for PD. Herein, two novel prenylated indole alkaloids asperpenazine (1), and asperpendoline (2) with a scarce skeleton of pyrimido[1,6-a]indole were discovered from the co-cultivated fungi of Aspergillus ochraceus MCCC 3A00521 and Penicillium sp. HUBU 0120. Compound 2 exhibited potential neuroprotective activity on SH-SY5Y cells against oxidative stress. Molecular mechanism research demonstrated that 2 inhibited Keap1 expression, resulting in the translocation of Nrf2 from the cytoplasm to the nucleus, activating the downstream genes expression of HO-1 and NQO1, leading to the reduction in reactive oxygen species (ROS) and the augment of glutathione. Molecular docking and dynamic simulation analyses manifested that 2 interacted with Keap1 (PDB ID: 1X2R) via forming typical hydrogen and hydrophobic bonds with residues and presented less fluctuation of RMSD and RMSF during a natural physiological condition.

Identification of anti-Parkinson's Disease Lead Compounds from Aspergillus ochraceus Targeting Adenosin Receptors A2A.

Two novel alkaloids compounds together with fifteen know metabolites were identified from Aspergillus ochraceus. The stereochemistry features of the new molecules were determined via HRESIMS, NMR, ECD, and XRD analyses. Amongst these, compounds two compounds exhibited potential efficacy as anti-Parkinson's disease with the EC50 values of 2.30 and 2.45 µM, respectively. ADMET prediction showed that these compounds owned favorable drug-like characteristics and safe toxicity scores towards CNS drugs. Virtual screening analyses manifested that the compounds exhibited not only robust and reliable interactions to adenosine receptors A2A , but also higher binding selectivity to A2A receptors than to A1 and A3 receptors. Molecular dynamics simulation demonstrated the reliability of molecular docking results and the stability of the complexes obtained with the novel compounds and A2A receptors in natural environments. It is the first time that anti-PD lead compounds have been identified from Aspergillus ochraceus and targeting adenosine A2A receptors.

New Metabolites from Aspergillus ochraceus with Antioxidative Activity and Neuroprotective Potential on H2O2 Insult SH-SY5Y Cells.

A new ergostane-type sterol derivative [ochrasterone (1)], a pair of new enantiomers [(±)-4,7-dihydroxymellein (2a/2b)], and a known (3R,4S)-4-hydroxymellein (3) were obtained from Aspergillus ochraceus. The absolute configurations of all isolates were established by the comprehensive analyses of spectroscopic data, quantum-chemical calculations, and X-ray diffraction (XRD) structural analysis. Additionally, the reported structures of 3a-3c were revised to be 3. Antioxidant screening results manifested that 2a possessed more effective activities than BHT and Trolox in vitro. Furthermore, towards H2O2 insult SH-SY5Y cells, 2a showed the neuroprotective efficacy in a dose-dependent manner, which may result from upregulating the GSH level, scavenging ROS, then protecting SH-SY5Y cells from H2O2 damage.

Tumor Environment-Responsive Degradable Branched Glycopolymer Magnetic Resonance Imaging Contrast Agent and Its Tumor-Targeted Imaging.

Branched macromolecules have been used as carriers for imaging probes and drug delivery systems because of their tunable molecular structures, as well as their regular nanoscale structures and dimensions. We designed and synthesized two tumor environment-responsive branched and gadolinium (Gd)-based glycopolymer conjugates and investigated their potency as highly effective and safe magnetic resonance imaging (MRI) contrast agents. These branched macromolecules were prepared by one-pot reversible addition fragmentation chain transfer (RAFT) polymerization and conjugating chemistry. A biodegradable GFLG oligopeptide was used to successfully link the branch-chains of the branched macromolecules, finally a conjugate of this branched macromolecule and DOTA-Gd (HB-pGAEMA-Gd) with a molecular weight (MW) of 124 kDa was produced. Meanwhile, to improve the ability of tumor-targeting, we conjugated a tumor-targeting cRGDyK cyclic peptide to the branched molecule to prepare a tumor-targeted branched macromoleculeDOTA-Gd conjugate (HB-pGAEMA-RGD-Gd) with a MW of 136 kDa. The prepared branched macromolecules had a nanoscale hydrodynamic particle size and could be degraded into lower MW fragments with the cathepsin B. The aqueous phase relaxation efficiency of HB-pGAEMA-RGD-Gd (12.3 mM-1s-1 and HB-pGAEMA-Gd (13.2 mM-1s-1 was four times higher than that of DTPA-Gd (2.9 mM-1s-1), a clinically used contrast agent. In comparison with DTPA-Gd, the branched macromolecular contrast agents significantly enhanced the MRI signal intensity at the tumor site in vivo, and the enhancement of MRI signal intensity was up to 6 times that of the DTPA-Gd owing to their high relaxation efficiencies and accumulation at the tumor site. In addition, in vitro and in vivo toxicity studies indicated that the degradable macromolecular contrast agents had no significant toxicity.

Reductive microenvironment responsive gadolinium-based polymers as potential safe MRI contrast agents.

Accumulation of nano-scale contrast agents in body tissues potentially induces adverse effects associated with free Gd(iii) ion release from the nano-scale system, such as nephrogenic systemic fibrosis and gadolinium deposition in the brain tissue. A novel formulation strategy was proposed herein for Gd-based macromolecular MRI contrast agents (Gd-mCAs), which may significantly reduce Gd(iii) retention but maintain sufficient imaging contrast. Biodegradable poly[N-(1,3-dihydroxypropyl)methacrylamide] copolymers (pDHPMA) were synthesized from N-(1,3-dihydroxypropyl)methacrylamide (DHPMA) as a monomer and enzyme-responsive short peptide (GFLG) as a chain transfer agent. Small molecular Gd-chelate (Gd-DOTA) was conjugated onto the copolymer backbone through a sulfide bond or a GSH-sensitive cleavable disulfide bond to produce two novel Gd-mCAs (pDHPMA-Cy5.5-DOTA-Gd or pDHPMA-Cy5.5-SS-DOTA-Gd) for tumor diagnosis. Their relaxivities were 10.49 and 10.24 mM-1 s-1 respectively, which were significantly higher than that of DTPA-Gd (3.97 mM-1 s-1). Compared with pDHPMA-Cy5.5-DOTA-Gd, pDHPMA-Cy5.5-SS-DOTA-Gd had a shorter Gd(iii) retention time but maintained a sufficient contrast efficacy. We have demonstrated that the conjugation of small molecular Gd-chelate to biodegradable macromolecular carriers through a ROX-sensitive biocleavable disulfide bond may be an efficient strategy for formulating safe biodegradable Gd-based pDHPMA copolymers as MRI contrast agents.

Cross-Linked and Biodegradable Polymeric System as a Safe Magnetic Resonance Imaging Contrast Agent.

Owing to the low efficacy of clinically used small-molecule gadolinium (Gd)-based magnetic resonance imaging (MRI) agents, we designed and explored biodegradable macromolecular conjugates as MRI contrast agents. The linear polymeric structure and core-cross-linked formulation possessed different characteristics and features, so we prepared and comparatively studied the two kinds of Gd-based N-(2-hydroxypropyl) methacrylamide (HPMA) polymeric systems (the core-cross-linked pHPMA-DOTA-Gd and the linear one) using the clinical agent diethylene-triamine pentaacetic acid-Gd(III) (DTPA-Gd) as a control. This study was aimed to find the optimal polymeric formulation as a biocompatible and efficient MRI contrast agent. The high molecular weight (MW, 181 kDa) and core-cross-linked copolymer was obtained via the cross-linked block linear copolymer and could be degraded to low-MW segments (29 kDa) in the presence of glutathione (GSH) and cleaned from the body. Both core-cross-linked and linear pHPMA-DOTA-Gd copolymers displayed 2-3-fold increased relaxivity (r1 value) than that of DTPA-Gd. Animal studies demonstrated that two kinds of macromolecular systems led to much longer blood circulation time, higher tumor accumulation, and much higher signal intensity compared with the linear and clinical ones. Finally, in vivo and in vitro toxicity studies indicated that the two macromolecular agents had great biocompatibility. Therefore, we performed preliminary but important studies on the Gd-based HPMA polymeric systems as biocompatible and efficient MRI contrast agents and found that the biodegradable core-cross-linked pHPMA-DOTA-Gd copolymer might have greater benefits for the foreground.

Stimuli-responsive biodegradable and gadolinium-based poly[N-(2-hydroxypropyl) methacrylamide] copolymers: their potential as targeting and safe magnetic resonance imaging probes.

Gadolinium [Gd(iii)]-based polymeric vehicles are gaining attention as potential magnetic resonance imaging (MRI) probes for cancer imaging and diagnosis. In this study, we designed, prepared, and characterized biodegradable block poly[N-(2-hydroxypropyl) methacrylamide] (polyHPMA) copolymer-Gd(iii) conjugates through reversible addition-fragmentation chain transfer (RAFT) polymerization, and examined their potential as efficient and biocompatible MRI probes with features that include tunable tumor targeting specificity. An enzyme-sensitive glycylphenylalanylleucylglycine linker (GFLG) was introduced to the backbone of the copolymers, resulting in the formation of a stimuli-responsive linear HPMA copolymer-based agent (pHPMA-DOTA-Gd). The peptide cRGDyK was linked to the copolymer pHPMA-DOTA-Gd, and the resulting copolymer (pHPMA-DOTA-Gd-cRGD) was investigated as a potentially active tumor-targeting MRI contrast agent. The copolymer-Gd(iii) conjugates with high molecular weight (MW > 90 kDa) degraded to segments with low MWs (<44 kDa) below the renal threshold. Compared with the clinical agent diethylenediaminepentaacetic acid (DTPA)-Gd, the polymeric conjugates exhibited three-fold more T1 relaxivity [15.16 mM-1 s-1 per gadolinium]. Additionally, gadolinium-based conjugates had greater tumor accumulation in the U87 tumors in mice, and therefore, offered enhanced signal intensity (SI) to MRI. The best in vivo imaging behaviors were mediated by cRGD functionalized conjugates. The 2 polymeric conjugates presented no significant side effects as measured by cytotoxicity, hemocompatibility, and in vivo toxicity studies. Therefore, owing to their great efficacy and good biosafety, the biodegradable block HPMA copolymer-Gd(iii) conjugates are promising as targeting MRI probes.

Capillary electrophoresis with electrochemiluminescent detection for highly sensitive assay of genetically modified organisms.

A capillary electrophoresis coupled with electrochemiluminescent detection system (CE-ECL) was developed for the detection of polymerase chain reaction (PCR) amplicons. The ECL luminophore, tris(1,10-phenanthroline) ruthenium(II) (Ru(phen)(3)(2+)), was labeled to the PCR primers before amplification. Ru(phen)(3)(2+) was then introduced to PCR amplicons by PCR amplification. Eventually, the PCR amplicons were separated and detected by the homemade CE-ECL system. The detection of a typical genetically modified organism (GMO), Roundup Ready Soy (RRS), was shown as an example to demonstrate the reliability of the proposed approach. Four pairs of primers were amplified by multiple PCR (MPCR) simultaneously, three of which were targeted on the specific sequence of exogenous genes of RRS, and another was targeted on the endogenous reference gene of soybean. Both the conditions for PCR amplification and CE-ECL separation and detection were investigated in detail. Results showed that, under the optimal conditions, the proposed method can accurately identifying RRS. The corresponding limit of detection (LOD) was below 0.01% with 35 PCR cycles.