chondroitin-sulfates and pyrene

Topics: Anticoagulants; Biosensing Techniques; Chondroitin Sulfates; Drug Contamination; Fluorescent Dyes; Heparin; Heparin Lyase; Humans; Limit of Detection; Peptides; Polysaccharide-Lyases; Pyrenes; Spectrometry, Fluorescence

Heparin is extensively used as an anticoagulant drug during surgery. Two fluorophore-functionalized cationic oligopeptides HS 1 and HS 2 were developed to monitor heparin ratiometrically in aqueous media. Upon binding to heparin, HS 1 and HS 2 undergo a conformational change from an open form to a folded form, which leads to a distinct change in the fluorescence properties. HS 1 switches from pyrene monomer emission to an excimer emission. For HS 2, a fluorescence resonance energy transfer (FRET) process is enabled between a naphthalene donor and a dansyl acceptor. This method is highly selective for heparin relative to other similar biological analytes such as hyaluronic acid or chondroitin sulfate. HS 1 and HS 2 could also detect heparin ratiometrically in diluted bovine serum. The strong ratiometric emission color change can also be observed by the naked eye. Addition of the polycationic protein protamine releases both HS 1 and HS 2 from their heparin complex, which simultaneously restores pyrene monomer emission for the first case and decreases the FRET process for the latter case, respectively. Dynamic light scattering (DLS) and AFM studies confirm aggregate formation of heparin with HS 1 and HS 2.

Topics: Animals; Cattle; Chondroitin Sulfates; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Heparin; Hyaluronic Acid; Limit of Detection; Naphthalenes; Oligopeptides; Polyamines; Polyelectrolytes; Protein Binding; Protein Conformation; Pyrenes

A ratiometric fluorescence sensor based on pyrene was designed for selective detection of heparin in HEPES (N-(2-hydroxyethyl)piperazine-N'-ethanesulfonic acid) buffer and serum sample. Pyrene and long-chain alkanes were linked through bisquaternary functionality in the sensor which could interact with heparin via supramolecular assembly. A ratiometric fluorescent signal change of the sensor can be observed because of the specific monomer-excimer conversion of pyrene which is modulated by the supramolecular self-assembly of sensor and heparin. Upon addition of heparin, the excimer emission of the sensor at 489 nm is observed and the monomer emission intensity at 395 nm decreases concomitantly. Addition of heparin derivatives with very similar structure such as chondroitin 4-sulfate or hyaluronic acid to the same sensor solution only leads to very smaller changes in intensity ratios probably because of lower charge density and more distant spatial distribution of anions (or disadvantageous spatial orientation of anions) as compared to those of heparin. The novel sensor can effectively differentiate heparin from its derivatives with relatively low background interference and wide linear response in HEPES and serum. A linear calibration curve is obtained from 0 to 3.4 μM for heparin quantification in serum.

Topics: Animals; Cattle; Chondroitin Sulfates; Fluorescent Dyes; Heparin; HEPES; Hyaluronic Acid; Hydrogen-Ion Concentration; Pyrenes; Water

A water-soluble pyrene-based butterfly shaped conjugated oligoelectrolyte (TFP) is synthesized and integrated with graphene oxide (GO) to form a label-free assay for heparin detection. Efficient fluorescence quenching occurs between TFP and GO because of strong electrostatic and π-π interactions, leading to nearly dark emission in the absence of analytes. Addition of heparin into TFP solution significantly minimizes the fluorescence quenching of GO toward TFP, which is less effective for the heparin analogues, such as hyaluronic acid and chondroitin 4-sulfate. As a consequence, the solution emits strong yellow fluorescence only in the presence of heparin, which allows for light-up visual discrimination of heparin from its analogues. Moreover, the linear light-up response of the TFP/GO integrated assay enables heparin quantification in the range of 0-1.76 U/mL with a limit of detection of 0.046 U/mL, which is practical for heparin monitoring during postoperative and long-term care. This study thus demonstrates a new synthetic strategy to develop GO-based chemical and biological sensing without the employment of dye-labeled biomolecules.

Topics: Chondroitin Sulfates; Electrolytes; Graphite; Heparin; Hyaluronic Acid; Oxides; Pyrenes; Spectrometry, Fluorescence; Static Electricity

Pluronic F127 (PF127), one of the polymers which can inhibit drug efflux transporters in cancer therapy, was used to produce amphiphilic nanocarriers for doxorubicin (DOX). In order to stabilize the nanocarriers, the hydroxyl groups on both termini of PF127 were acrylated and reacted with methacrylated chondroitin sulfate (CSMA) to form CS-PF127 nanogel. The introducing CSMA has carboxylic acid groups which can be used to react with a folic acid-polyethylene glycol (FA-PEG). Folic acid, having high binding affinity to tumor-associated folate receptors (FR), provides a selective delivery of doxorubicin (DOX) to FR-positive tumor cells. DOX was loaded either in a cationic DOX.HCl form through the electrostatic interactions with the negative charges of chondroitin sulfate, or in a free DOX form by solubilization into the PPO core compartment of PF127. The loading efficiency and release behavior of DOX prepared from two different formulations are compared. The synthesis of CS-PF127 and FA-PEG grafted CS-PF127 (FA-CS-PF127) was characterized by nuclear magnetic resonance spectrometry (NMR), ultraviolet/visible spectroscopy (UV), and X-ray photoelectron spectroscopy (XPS). With a fluorescent probe technique, the critical aggregation concentrations (CAC) are 7.5 x 10(-2)mg/mL for CS-PF127 and 7.9 x 10(-2)mg/mL for FA-CS-PF127, respectively. The spherical images of nanogels were visualized with the use of the transmission electron microscope (TEM). The particle diameters measured by dynamic light scattering (DLS) are 299.6+/-8.2nm for CS-PF127 and 138.3+/-12.3 for FA-CS-PF127, neither aggregation nor change in sizes in double deionized (DD) water after 20 days. The better cellular uptake of FA-CS-PF127 in KB cells was evidenced by confocal laser scanning microscopy (CLSM) and flow cytometry upon loading Rhodamine123 as a probe.

Topics: Acrylates; Binding, Competitive; Biological Availability; Cell Line, Tumor; Cell Survival; Chondroitin Sulfates; Doxorubicin; Drug Carriers; Endocytosis; Fluorescent Dyes; Folic Acid; Humans; Magnetic Resonance Spectroscopy; Methacrylates; Microscopy, Electron, Transmission; Molecular Structure; Nanostructures; Particle Size; Poloxamer; Polyethylene Glycols; Pyrenes; Spectrometry, Fluorescence; Spectrometry, X-Ray Emission; Spectrophotometry, Ultraviolet

Novel polymeric amphiphilic copolymers were synthesized using chondroitin sulfate (CS) as a hydrophilic segment and poly(L-lactide) (PLLA) as a hydrophobic segment. Micelles of those copolymers were formed in an aqueous phase and were characterized by 1H NMR spectra, fluorescence techniques, dynamic light scattering (DLS), atomic force microscopy (AFM), and confocal microscopy. Their critical aggregation concentrations (CAC) are in the range of 0.0043-0.0091 mg/mL at 25 degrees C. The partition equilibrium constants, Kv, of the pyrene probe in the aqueous solution were from 3.65 x 10(5) to 1.41 x 10(6) at 25 degrees C. The mean diameters of the micelles were below 200 nm, and their sizes were narrowly distributed. The AFM images revealed that the self-aggregates were spherical. Additionally, the CSn-PLLA micelles can efficiently transport within the cells via endocytosis as observed from confocal microscopy.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Chondroitin Sulfates; Fibroblasts; Humans; Hydrophobic and Hydrophilic Interactions; Light; Micelles; Molecular Sequence Data; Particle Size; Polyesters; Pyrenes; Scattering, Radiation; Sensitivity and Specificity; Surface-Active Agents