Doxorubicin encapsulated blend of sitagliptin-lignin polymeric drug delivery system for effective combination therapy against cancer.

In this research, a sitagliptin-lignin biopolymer (SL) containing zinc selenide quantum dots (ZnSe QDs) and doxorubicin (doxo) was synthesized. The fabricated polymeric drug delivery system was characterized via FTIR, XRD, SEM, TGA, IR, and DSC. SLQD-Doxo exhibited an irregular surface with a 32 nm diameter and well-defined surface chemistry. Drug loading efficiency was assessed at different concentrations, pH levels, time intervals, and temperatures, and drug kinetics were calculated. Maximum drug release was observed at 6 µmol concentration after 24 h, pH of 6.5 and 45 °C. The maximum drug encapsulation efficiency was 81.75 %. SLQD-Doxo demonstrated 24.4 ± 1.04 % anti-inflammatory activity, and the maximum lipoxygenase inhibition in a concentration-dependent manner was 71.45 ± 2.02 %, compared to indomethacin, a standard anticancer drug. The designed system was applied to breast cancer MCF-7 cells to evaluate anticancer activity. Cytotoxicity of SLQD-Doxo resulted in 24.48 ± 1.64 dead cells and 74.39 ± 4.12 viable cells. Lignin's polyphenolic nature resulted in good antioxidant activity of LLQD-Doxo. The combination of SLQD-Doxo was appropriate for drug delivery at high temperatures and acidic pH of tumor cells compared to healthy cells.

Graphene oxide-metal oxide nanocomposites for on-target enrichment and analysis of phosphorylated biomolecules.

The surface of matrix-assisted laser desorption/ionization mass spectrometry target is modified for improved signal strength and detection of analytes. The developed method includes on-target enrichment and detection of phosphopeptides/phospholipids using graphene oxide-lanthanide metal oxides (samarium, gadolinium, dysprosium, and erbium) nanocomposites. Enriched phosphopeptides are detected using material enhanced laser desorption/ionization mass spectrometry and phospholipids by laser desorption/ionization-mass spectrometry. Nanocomposites are prepared using graphene oxide with respective metal salts at high pH. They are characterized for nano-morphology, chemistry, porosity, composition, crystallinity, and thermal stability. Phosphopeptides enrichment protocol is developed and optimized for tryptic ß-casein digest and that of phospholipids by phosphatidylcholine standard. Statistical analyses of phosphopeptides and phospholipids from milk show overlapping results for gadolinium, dysprosium, and erbium oxide nanocomposites. GO-Gd2 O3 has better enrichment efficiency and application as LDI material. Selectivity for GO-Dy2 O3 is 1:2500, for GO-Sm2 O3 is 1:3500, and 1:4000 for GO-Gd2 O3 . GO-Er2 O3 has a sensitivity of 25 fmol, whereas the highest sensitivity is down to 0.5 fmol for GO-Gd2 O3 . On-target enrichment is batch to batch reproducible with a standard deviation of <1, reduced time of enrichment to 10 min, and ease of operation compared to solid-phase batch extraction. The developed method enriches serum phosphopeptides characteristic of cancer-related phosphoproteins.

Tellurium doped zinc imidazole framework (Te@ZIF-8) for quantitative determination of hydrogen peroxide from serum of pancreatic cancer patients.

The tellurium doped zinc imidazole framework (Te@ZIF-8) is prepared by a two-step hydrothermal strategy for the electrochemical sensing of hydrogen peroxide. Material is characterized by transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDS), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The electrochemical characterization of the MOF modified electrode is done by a three-electrode system. Electrochemical sensing of hydrogen peroxide is made by cyclic voltammetry, amperometry, and impedance measurements. Results demonstrate that Te@ZIF-8 shows a detection limit of 60 µM with linearity up to 0.98855. Material is stable to 1000 cycles with no significant change in electrochemical response. Amperometry depicts the recovery of hydrogen peroxide from human serum up to 101%. Impedance curve reveals the surface of Te@ZIF-8-GCE (glassy carbon electrode) as porous and rough and an interface is developed between analyte ions and the sensing material. Finally, the modified electrode is used for the quantitative determination of hydrogen peroxide from serum samples of pancreatic cancer patients, diagnosed with CA 19-9.

Highly porous terpolymer-ZIF8@BA MOF composite for identification of mono- and multi-glycosylated peptides/proteins using MS-based bottom-up approach.

A hydrophilic terpolymer MOF composite is designed with high surface area and porosity to enrich mono- and multi-glycosylated peptides facilitating a bottom-up approach. Terpolymer@ZIF-8 is synthesized using free radical polymerization followed by layer by layer ZIF-8 fabrication. Subsequent surface modification was made by aminophenylboronic acid (AMBA). The enrichment ability of terpolymer@ZIF-8@BA is evaluated by using tryptic digest of IgG and HRP to exemplify mono- and multi-glycosylated protein samples. Improved selectivity of 1:200 for spiked HRP in BSA digest and sensitivity down to 1 fmol µL-1 is achieved. Batch to batch reproducibility is better 1% RSD which favors the adoption of the developed method for routine N-linked glycopeptide/protein determination. Cost-effective nature of given approach is given by regeneration of the material up to four cycles. Total 318 N-linked glycopeptides have been identified from 1 µL human serum digest after subjecting the enriched and PNGase-treated deglycosylated peptides to LC-MS. Thus, terpolymer@ZIF-8@BA holds the potential both for mono- and multi-glycosylated peptides from complex biological sample. Graphical abstract.

Boronic acid functionalized MOFs as HILIC material for N-linked glycopeptide enrichment.

Highly specific enrichment of N-linked glycopeptides from complex biological samples is crucial prior to mass spectrometric analysis. In this work, a hydrophilic metal-organic framework composite is prepared by the growth of UiO-66-NH2 on graphene sheets, followed by its post-synthetic modification to attach boronic acid to form GO@UiO-66-PBA. The fabrication of graphene oxide-MOF composite results in enhanced surface area with improved thermal and chemical stability. The synthesized MOF nanocomposite is characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and BET. A crystalline structure with high porosity offering large surface area and good hydrophilicity of the nanocomposite assists as an enrichment tool in glycoproteomics. The GO@UiO-66-PBA nanocomposite selectively enriches N-linked glycopeptides from tryptic digests of horseradish peroxidase (HRP) and immunoglobulin (IgG). GO@UiO-66-PBA nanoparticles show a low detection limit (1 fmol) and good specificity (1:200), reusability and reproducibility for N-linked glycopeptide enrichment from IgG digest. The binding capacity of GO@UiO-66-PBA is 84 mg/g for protein concentration, with a good recovery of 86.5%. A total of 372 N-linked glycopeptides corresponding to different glycoproteins are identified from only 1 µL of human serum digest. Thus, the presented research work can be an efficient separation platform for N-linked glycopeptide enrichment from complex samples, which can be extended to cost-effective routine analysis. Graphical abstract.

Enrichment of HDL proteome and phospholipidome from human serum via IMAC/MOAC affinity.

High-density lipoproteins (HDLs) have anti-inflammatory and antioxidant properties and are potentially cardio-protective. Defective HDL function is caused by alterations in both the proteome and lipidome of HDL particles. As potential biomarkers, the development of analytical methods is necessary for the enrichment of HDLs. Therefore, a method for selective enrichment of HDLs using immobilized metal ion affinity chromatography (IMAC) and metal oxide affinity chromatography (MOAC) is presented. SPE-based isolation of HDLs from whole serum is adopted as an alternative to traditional ultracentrifugation methods followed by SDS-PAGE. The enrichment mechanism relies on isoelectric points of lipoproteins and metal oxide. Negatively charged lipoprotein particles interact with positively charged metal oxides and IMAC affinity, which acts as a cation. Identified proteins from HDL through MALDI-MS analysis are apo AI, AII, AIV, CI, CIII, E, J, M, H, serum amyloid A and other nonapoproteins that are part of HDL particles and perform cellular functions. This serum-based proteomics approach gives insight into the functional role of HDL. HDL-associated phospholipids have also been analyzed by LDI-MS. Results suggest that the adopted analytical strategy is a feasible idea to extract lipoproteins from serum. A comparative study of healthy and diseased samples using this approach will provide valuable information in future.

Magnetite nanoparticles coated with chitosan and polyethylenimine as anion exchanger for sorptive enrichment of phosphopeptides.

An anion exchange solid-phase sorbent is described. Chitosan coated magnetite nanoparticles were modified with polyethylenimine which is positively charged at pH 3 and therefore can be used for the magnet-supported enrichment of phosphopeptides which are negatively charged at this pH value. A 2-step strategy was used to synthesize the sorbent. The materials were characterized by transmission electron microscopy, scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, thermogravimetry and magnetic moment analysis. The anion exchanger was applied to extract phosphopeptides from a ß-casein digest. Characteristic analytical figures include (a) a loading buffer of pH 3, (b) and elution buffer of pH 11, (c) a loading time of 5 min, (d) good selectivity (the ß-casein to BSA ratio is 1:1000), and (e) excellent sensitivity (1 fmol). The optimized method was applied to egg yolk digest, non-fat milk digest, and diluted human serum. Graphical abstractSchematic representation of synthesis of PEI@chitosan@Fe3O4 nanoparticles, and of the enrichment of phosphopeptides by magnetic solid phase extraction prior to the determination of the peptides by MALDI-MS analysis.

Metal-organic framework-based affinity materials in proteomics.

Metal-organic frameworks (MOFs) are an eminent addition to materials science research because of their versatile properties due to which their applications are wide spread in proteomics. They are used in various fields due to their characteristics like higher surface area, specific symmetry, ease of modification, and availability of a variety of ligands. As affinity sorbents, they have shown higher selectivity, sensitivity, and reproducibility than conventionally used materials. They are applied for the enrichment of phosphopeptides, glycopeptides, low mass peptides, and as laser desorption/ionization (LDI) matrices for small-molecule analysis. This review captures the insight of applying MOFs in the field of mass spectrometry-based proteomics. The specific features are discussed regarding MOFs as affinity sorbents for the selective capture of biological molecules like phosphopeptides and glycopeptides from complex samples. The potential of MOFs as LDI mass spectrometry (LDI-MS) matrices for small-molecule analysis is also evaluated. MOFs have also been used as enzymatic reactors for the digestion of proteins, prior to MS analysis. MOF-based affinity materials and bioreactors reduce proteome complexity and improve detection sensitivity and coverage. Size-exclusion effects of MOFs help in subtracting the abundant proteins in peptidomics. Several limitations of MOFs are addressed, which include stability under varying pH conditions, the unclear interaction mechanism between the MOFs and targeted analytes, and the non-specific binding that interferes during the analysis because of metal centers and ligands in the MOFs. This will open up MOF-based research to overcome the limitations and improve the performance of MOFs as selective and sensitive materials. Graphical abstract ᅟ.

Preparation of Highly Porous Coordination Polymer Coatings on Macroporous Polymer Monoliths for Enhanced Enrichment of Phosphopeptides.

We describe a protocol for the preparation of hybrid materials based on highly porous coordination polymer coatings on the internal surface of macroporous polymer monoliths. The developed approach is based on the preparation of a macroporous polymer containing carboxylic acid functional groups and the subsequent step-by-step solution-based controlled growth of a layer of a porous coordination polymer on the surface of the pores of the polymer monolith. The prepared metal-organic polymer hybrid has a high specific micropore surface area. The amount of iron(III) sites is enhanced through metal-organic coordination on the surface of the pores of the functional polymer support. The increase of metal sites is related to the number of iterations of the coating process. The developed preparation scheme is easily adapted to a capillary column format. The functional porous polymer is prepared as a self-contained single-block porous monolith within the capillary, yielding a flow-through separation device with excellent flow permeability and modest back-pressure. The metal-organic polymer hybrid column showed excellent performance for the enrichment of phosphopeptides from digested proteins and their subsequent detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented experimental protocol is highly versatile, and can be easily implemented to different organic polymer supports and coatings with a plethora of porous coordination polymers and metal-organic frameworks for multiple purification and/or separation applications.

Development of new multifunctional terpolymer sorbent for proteomics applications.

Determination of the availability of phases for specific separations is an important task achieved by a separation chemist. This becomes vital when the complex samples like biofluids are dealt with in proteome science. The work presented here involves the synthesis and application of terpolymeric sorbent with different functionalizations adopted for the selective enrichment of biomolecules of interest from biological fluids. Synthesis of terpolymer was carried out by the radical polymerization of monomers: methyl acrylate, acrylic acid and vinyl acetate with diethylene glycol dimethacrylate as cross-linking agent, benzoyl peroxide as initiator and chloroform as a porogenic solvent. Characterization was done through Fourier transform infrared spectroscopy, scanning electron microscopy and nitrogen adsorption porosimetry. The polymer was further modified to immobilized metal ion affinity chromatographic material, with immobilized Fe(3+)/La(3+) ions that allowed phosphopeptide enrichment from tryptic digests of standard proteins as well as milk, egg yolk and human serum. Sensitivity of enrichment down to 50 fmol was achieved in the presence of complex protein background as bovine serum albumin. Hydrophobicity was introduced through octadecyl amine, which provides comparable results to ZipTip C18/C4 for desalting of complex mixtures of all caseins. Analysis of the enriched content was performed by Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

Newly developed poly(allyl glycidyl ether/divinyl benzene) polymer for phosphopeptides enrichment and desalting of biofluids.

The polymeric materials have contributed significantly in the area of bioanalytical science. The functionalization of polymeric backbone after its development brings unique selectivity towards the target biomolecules. In present work, the functionalities of choice have been introduced through the ring-opening of allyl glycidyl ether. The utility of polymer is widened through derivatizations to immobilized metal ion affinity chromatographic (IMAC) material for the phosphopeptides enrichment and Reversed Phase (C-18) for the desalting prior to MALDI-MS analysis. The polymer-IMAC in addition to Fe(3+) is also immobilized with lanthanide ions like La(3+), Eu(3+), and Er(3+). The amount of Fe(3+) immobilized is determined as 0.7928 mg/g. Spherical morphology with narrow particle size dispersion is revealed by scanning electron microscopy (SEM). The surface area, pore volume and size distribution is determined by nitrogen adsorption porosimetery. The elemental composition and purity level is confirmed by energy dispersive X-ray spectroscopy (EDX) data. The derivatization to IMAC and RP is evaluated by Fourier transform infrared (FT-IR) spectroscopy. The polymer enables the efficient phosphopeptide enrichment to equal degree from casein variants, non-fat milk, egg yolk, human serum, and HeLa cell extract. The identification of phosphorylation sites can lead to the phosphorylation pathways to understand the post-translational modifications. The identification with their sequence coverage is made using Mascot and Phosphosite Plus. It is sensitive to enrich the phosphopeptides down to 2 femtomoles with very high selectivity of 1:2000 with BSA background. These attributes are linked to the higher surface area (173.1554 m(2)/g) of the designed polymer. The non-specific bindings, particularly the Fe(3+) linked acidic residues are also avoided. Four characteristic phosphopeptides (fibrinopeptide A and their hydrolytic products) from fibrinogen α-chain are identified from the human serum after the enrichment, which have link to the hepatocellular carcinoma (HCC). The proportions of fibrinogen and their phosphorylation products enriched by poly(AGE/DVB)-IMAC open new horizons in the biomarker discovery.

High affinity phosphopeptides enrichment and desalting of biological materials on newly engineered poly(glycidyl propargyl ether/divinyl benzene).

The new synthetic polymers have a key role to play in the separation science. The derivatization of these polymers has made them an efficient class of substrate, having unique properties and the selectively tailored surface chemistries for target molecules. The deeper and detailed characterization of complex sample types has become feasible due to the enhanced selectivity and sensitivity offered by these polymer materials. In present work, a bifunctional monomer glycidyl propargyl ether (GPE) is thermally polymerized with divinylbenzene to form poly(GPE/DVB). Some of the physical and chemical properties are characterized by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX) and Fourier transform infrared spectroscopy (FTIR). The synthesized polymer is further derivatized to IMAC (immobilized metal ion affinity chromatography) and is investigated by loading different metal ions (Fe(3+), Ti(4+), Zr(4+), and La(3+)). The trypsin-digested products of phosphoproteins, such as casein, nonfat milk, egg yolk, and human blood serum, are used to explore its phosphopeptide enrichment ability from complex samples followed by the off-line MALDI-MS analysis. Furthermore, polymeric reversed phase (RP) is created by octadecyl amine (ODA) to be employed in the desalting of complex mixtures and the results are compared with commercially available ZipTip C-18 and Aspire RP30 Desalting Tip. Serum profiling of healthy and diseased samples demonstrates the potential of this new polymer to impart in the disease diagnosis. Ovarian carcinoma serum samples are used for the detection of phosphopeptides based biomarkers.

Versatile nanocomposites in phosphoproteomics: a review.

Protein phosphorylation is one of the most important post-translational modifications. Phosphorylated peptides are present in low abundance in blood serum but play a vital role in regulatory mechanisms and may serve as casual factors in diseases. The enrichment and analysis of phosphorylated peptides directly from human serum and mapping the phosphorylation sites is a challenging task. Versatile nanocomposites of different materials have been synthesized using simple but efficient methodologies for their enrichment. The nanocomposites include magnetic, coated, embedded as well as chemically derivatized materials. Different base materials such as polymers, carbon based and metal oxides are used. The comparison of nanocomposites with respective nanoparticles provides sufficient facts about their efficiency in terms of loading capacity and capture efficiency. The cost for preparing them is low and they hold great promise to be used as chromatographic materials for phosphopeptide enrichment. This review gives an overview of different nanocomposites in phosphoproteomics, discussing the improved efficiency than the individual counterparts and highlighting their significance in phosphopeptide enrichment.