Characterization of Complex Proteoform Mixtures by Online Nanoflow Ion-Exchange Chromatography-Native Mass Spectrometry.

The characterization of proteins and complexes in biological systems is essential to establish their critical properties and to understand their unique functions in a plethora of bioprocesses. However, it is highly difficult to analyze low levels of intact proteins in their native states (especially those exceeding 30 kDa) with liquid chromatography (LC)-mass spectrometry (MS). Herein, we describe for the first time the use of nanoflow ion-exchange chromatography directly coupled with native MS to resolve mixtures of intact proteins. Reference proteins and protein complexes with molecular weights between 10 and 150 kDa and a model cell lysate were separated using a salt-mediated pH gradient method with volatile additives. The method allowed for low detection limits (0.22 pmol of monoclonal antibodies), while proteins presented nondenatured MS (low number of charges and limited charge state distributions), and the oligomeric state of the complexes analyzed was mostly kept. Excellent chromatographic separations including the resolution of different proteoforms of large proteins (>140 kDa) and a peak capacity of 82 in a 30 min gradient were obtained. The proposed setup and workflows show great potential for analyzing diverse proteoforms in native top-down proteomics, opening unprecedented opportunities for clinical studies and other sample-limited applications.

Co-assembled supramolecular hydrogels of cell adhesive peptide and alginate for rapid hemostasis and efficacious wound healing.

Injectable hydrogels are promising materials for applications in non-compressive wound management. Yet difficulties remain for the fabrication of mechanically stable hydrogel materials with inherent functionalities in both hemostatic control and wound healing without additional supplements of growth factors. Herein, we reported the co-assembly of a cell adhesive peptide conjugate (Pept-1) and alginate (ALG), to confer supramolecular hydrogels with excellent mechanical properties and high efficacy in both hemostatic control and wound healing requiring no additional growth factors. The co-assembling process of Pept-1 and ALG, which was mediated by electrostatic interactions and metal chelation, afforded a composite hydrogel with denser nanofibrillar structures and better mechanical strength when comparing to the Pept-1 gel alone. As-prepared Pept-1/ALG hydrogels exhibited excellent injectability and thixotropic properties, making them ideal materials for wound dressing. The composite hydrogel induced fast hemostasis when spiked with whole blood in vitro, and reduced the amount of bleeding to ∼18% of the untreated control in a liver puncture mouse model. Meanwhile, it promoted adhesion and migration of fibroblast NIH3T3 cells in vitro, and accelerated the rate of wound healing in a full-thickness skin defect model of mice. In addition, the Pept-1/ALG hydrogel showed excellent biocompatibility with no obvious hemolytic activity. In future, the strategy of utilizing co-assembled nanostructures composed of biofunctional peptides and polysaccharides could be further exploited to construct a broad range of nanocomposite materials for a variety of biomedical applications.

Doxorubicin-reinforced supramolecular hydrogels of RGD-derived peptide conjugates for pH-responsive drug delivery.

Drug incorporation in hydrogels often brings undesirable effects on the stability or mechanical properties of the system. To address this problem, we report the design and synthesis of a RGD-derived peptide conjugate (1-RGDH) for its co-assembly with a commonly used chemotherapeutic drug, doxorubicin (DOX), that formed electrostatic interactions with the 1-RGDH peptide and reinforced the supramolecular network of nanofibers within the matrix of the hydrogel. The hybrid hydrogel demonstrated excellent viscoelastic and shear-thinning properties that greatly facilitated the development of injectable drug delivery systems. Furthermore, it demonstrated a unique pH responsive release of DOX under weakly acidic conditions, paving ways for the controlled release of drug cargos in a typical tumor microenvironment with mild acidity. Finally, the DOX-incorporated hydrogel exhibited a superior anti-tumor efficacy in non-small-cell lung cancer cells A549 compared to the aqueous solution of free DOX, with an integrin receptor-mediated endocytosis pathway revealed for the cellular uptake of DOX-incorporated nanofibers.

Identification of heavily glycated proteoforms by hydrophilic-interaction liquid chromatography and native size-exclusion chromatography - High-resolution mass spectrometry.

BACKGROUND: The non-enzymatic glycation of proteins and their advanced glycation end products (AGEs) are associated with protein transformations such as in the development of diseases and biopharmaceutical storage. The characterization of heavily glycated proteins at the intact level is of high interest as it allows to describe co-occurring protein modifications. However, the high heterogeneity of glycated protein makes this process challenging, and novel methods are required to accomplish this. RESULTS: In this study, we investigated two novel LC-HRMS methods to study glycated reference proteins at the intact protein level: low-flow hydrophilic-interaction liquid chromatography (HILIC) and native size-exclusion chromatography (SEC). Model proteins were exposed to conditions that favored extensive glycation and the formation of AGEs. After glycation, complicated MS spectra were observed, along with a sharply reduced signal response, possibly due to protein denaturation and the formation of aggregates. When using HILIC-MS, the glycated forms of the proteins could be resolved based on the number of reducing monosaccharides. Moreover, some positional glycated isomers were separated. The SEC-MS method under non-denaturing conditions provided insights into glycated aggregates but offered only a limited separation of glycated species based on molar mass. Overall, more than 25 different types of species were observed in both methods, differing in molar mass by 14-162 Da. 19 of these species have not been previously reported. SIGNIFICANCE: The proposed strategies show great potential to characterize highly glycated intact proteins from native and denaturing perspectives and provide new opportunities for fast clinical diagnoses and investigating glycation-related diseases.

Wound microenvironment-responsive dually cross-linked nanofibrillar peptide hydrogels for efficient hemostatic control and multi-faceted wound management.

The wound microenvironment-responsive hydrogel, featuring a dually cross-linked architecture, offers distinct advantages in the realm of drug delivery due to its exceptional mechanical properties and responsiveness to stimuli. In this investigation, a versatile dually cross-linked hydrogel was synthesized. The initial framework was established through non-covalent interactions employing a self-assembling peptide indomethacin-Gly-Phe-Phe-Tyr-Gly-Arg-Gly-Asp (abbreviated as IDM-1), while the second framework underwent chemical cross-linking of chitosan (CS) mediated by genipin. This dually-network arrangement significantly bolstered the structure, proving effective for hemostatic control. In addition, hydrogels can be triggered for degradation by proteases highly expressed in the wound microenvironment, releasing drugs like indomethacin (IDM) and CS. This characteristic introduced efficient multi-faceted wound management in vitro and in vivo, such as anti-inflammatory and antibacterial activities, ultimately augmenting the wound healing process. Thus, the development of a dually cross-linked hydrogel that enables smart drug release triggered by specific wound microenvironment presents considerable potential within the realm of wound management.

Influence of ion-pairing reagents on the separation of intact glycoproteins using hydrophilic-interaction liquid chromatography - high-resolution mass spectrometry.

Hydrophilic-interaction liquid chromatography (HILIC) of intact proteins offers high-resolution separations of glycoforms of glycoproteins differing in the number of (neutral) glycans. However, to obtain efficient separations it is essential that the positively charged sites of the proteins are shielded by acidic (negative) ion-pair reagents (IPRs), so as to enhance the contribution of the hydroxyl groups of the (neutral) sugars in the glycoprotein. Here, we studied the influence of various IPRs that differ in physico-chemical properties, such as hydrophobicity and acidity, on the capillary-scale HILIC separation of intact (glyco)proteins. We evaluated the use of fluoroacetic acid (MFA), difluoroacetic acid (DFA), trifluoroacetic acid (TFA), and heptafluorobutyric acid (HFBA) as diluents for sample preparation, as solvents for sample loading on a reversed-phase trap prior to the HILIC separation, and as mobile-phase components for HILIC and HILIC-MS. To reduce the contribution of ion-exchange interaction with the (silica-based) stationary phase, we used an acrylamide-based monolithic column. We studied the influence of the different IPRs on each step of the separation of a mixture of proteins of different size and hydrophilicity and on the separation of the five glycoforms of ribonuclease B. The content of IPR in the sample was shown not to affect the separation and the MS detection. However, a low content of TFA and DFA in the mobile phase is favourable, as it reduces adduct formation and leads to higher signal intensity. The optimized HILIC conditions successfully resolved nine major glycoforms groups of a ∼40 kDa glycoprotein horseradish peroxidase (HRP), as an example of a complex glycoprotein.

A high-performance electrochemical biosensor using an engineered urate oxidase.

We constructed a high-performance biosensor for detecting uric acid by immobilizing an engineered urate oxidase on gold nanoparticles deposited on a carbon-glass electrode. This biosensor showed a low limit-of-detection (9.16 nM), a high sensitivity (14 µA/µM), a wide range of linearity (50 nM-1 mM), and more than 28 days lifetime.

Dopamine-functionalized hyaluronic acid microspheres for effective capture of CD44-overexpressing circulating tumor cells.

As one of the biomarkers of liquid biopsy, circulating tumor cells (CTCs) provides important clinical information for cancer diagnosis. However, accurate separation and identification of CTCs remains a great deal of challenge. In present work, we developed novel dopamine-functionalized hyaluronic acid microspheres (HA-DA microspheres) to capture CD44-overexpressing CTCs. The dopamine was grafted onto the hyaluronic acid chain, which was polymerized and cross-linked by oxidation of the catechol groups. Afterwards, a facile microfluidic chip was designed and developed to fabricate the HA-DA microspheres with a diameter of about 45 µm. Our results showed that the CD44+ cells (i.e., HeLa, HepG2, A549, MCF-7 and DU-145 cells) could be selectively captured. Then a double-layer microfluidic filter (DLMF) was fabricated for dynamic isolation and detection of CTCs in blood samples. Many slit openings with 15 µm in height were designed to allow white blood cells to clear away, while the microspheres with CTCs were intercepted in the DLMF, which achieved effective separation of CTCs from blood cells. The approach exhibited high capture efficiency even at the cell density as low as 10 cells/mL. We believe the DLMF integrated with HA-DA microspheres could be a promising approach for isolation and detection of CD44-overexpressing CTCs, which is useful for prognosis and early metastasis of cancer patients.

Double-crosslinked nanocomposite hydrogels for temporal control of drug dosing in combination therapy.

Temporal control of drug dosing is indispensable for a successful combination therapy that utilizes cisplatin (CDDP) and irinotecan (IRN), with clinical evidence supporting a higher response rate when CDDP was administered prior to IRN. Herein, a peptide-based nanocomposite hydrogel (CDDP/Pept-AlgNP/IRN) was designed for differential release of CDDP and IRN to maximize synergism of two drugs. First, a double-crosslinking strategy was exploited for structural reinforcement of hydrogel, with integration of coordination interactions between CDDP and hydrogelator (Pept) as well as electrostatic interactions between Pept and alginate nanoparticles (AlgNP/IRN), that afforded nanocomposite hydrogel with 42-fold increase in storage modulus comparing to peptide gel alone. Next, the nanocomposite hydrogel with excellent injectability served as a depot for controlled release of dual drugs, and guaranteed a fast release of CDDP prior to a tunable release of IRN that is dependent on fraction ratios of AlgNP in the composite materials. Comparing to simple mixture of CDDP and IRN solution, CDDP/Pept-AlgNP/IRN hydrogel formulation demonstrated excelling synergism of CDDP and IRN in cell inhibition studies, with efficacious antitumor potency further proved in tumor regression studies in vivo. We believe that the strategy of utilizing co-assembly of multiple pairs of entities (i.e. drug-gelator, nanoparticle-gelator) in composite materials provides a generalized method to design mechanically stable supramolecular hydrogels, and further promises an exact temporal control of drug dosing by packing individual drugs in co-assembled structures/domains to satisfy clinical demands from combination therapy. STATEMENT OF SIGNIFICANCE: This study reports the design of nanocomposite hydrogels with two distinct co-assembling domains for structural reinforcement of hydrogel and differential release of two drugs (CDDP and IRN) in combination therapy. We first investigated the effects of co-assembling processes for the reinforcement of hydrogel. Then we utilized the hydrogel for differential release of CDDP and IRN to achieve better synergistic efficacy of drugs in inhibiting the growth of cancer cell A549 and better anticancer efficacies than single drug formulations or solution mixtures of dual drugs in an A549-xenografted mouse model. We believe that the strategy of packing individual drugs in distinct co-assembling structures promises a paradigm shift for regulating temporal control of drug dosing in combination therapy.

An enzyme-assisted self-delivery system of lonidamine-peptide conjugates for selectively killing cancer cells.

A self-delivery system consisting of lonidamine and a self-assembling peptide was designed for the selective killing of phosphatase-overexpressing cancer cells, which was mediated by both enhanced cellular uptake of LND-peptide and enzyme-triggered intracellular fiber formation, thereby providing a generalized strategy to develop cancer-targeting systems of drug conjugates.

In situ hydrogelation of bicalutamide-peptide conjugates at prostate tissue for smart drug release based on pH and enzymatic activity.

Tissue-specific self-assemblies of supramolecular hydrogels have attracted great interest in material design and biomedical applications, for in situ-formed hydrogels serve as an excellent local depot with tunable release of drug therapeutics. Here we report the design and syntheses of a novel class of histidine-containing hexapeptide derivatives (Nap-1 and ID-1) for in situ hydrogelation at the zinc ion-rich prostate tissue. Thanks to the efficient co-ordination between zinc and histidine, both Nap-1 and ID-1 displayed excellent self-assembly capability with a high sensitivity to zinc ions at ∼0.1 equivalency. To foster a prostate-specific drug delivery system (DDS), ID-1 was chosen for further conjugation with bicalutamide (BLT), a clinically used drug for prostate cancer. The as-synthesized ID-1-BLT retained the self-assembly capability with zinc ions, and conferred supramoelcular hydrogels at the prostate site. Interestingly, ID-1-BLT hydrogels demonstrated tunable drug release profiles in a typical tumor microenvironment, with acidic pH and esterase activity regulating the drug release in a dose dependent manner. Consequently, the hydrogel-based DDS demonstrated enhanced potency and selective cytotoxicity against prostate cancer cell DU145 over normal fibroblast cell NIH3T3, plausibly due to differential cellular uptake of drugs as well as the elevated esterase activities in cancer cells. Finally, the biocompatible hydrogel system demonstrated sustained delivery of drugs at the prostate gland of rats, with a superior in situ drug distribution profile compared to that of aqueous solution of BLT alone.