Polyamidoamine dendrimer-mediated hydrogel for solubility enhancement and anti-cancer drug delivery.

The application of hydrogels for anti-cancer drug delivery has garnered considerable interest in the medical field. Current cancer treatment approaches, such as chemotherapy and radiation therapy, often induce severe side effects, causing significant distress and substantial health complications to patients. Hydrogels present an appealing solution as they can be precisely injected into specific sites within the body, facilitating the sustainable release of encapsulated drugs. This localized treatment approach holds great potential for reducing toxicity levels and improving drug delivery efficacy. In this study we developed a hydrogel delivery system containing polyamidoamine (PAMAM) dendrimer and polyethylene glycol (PEG) for solubility enhancement and sustained delivery of hydrophobic anti-cancer drugs. The three selected model drugs, e.g. silibinin, camptothecin, and methotrexate, possess limited aqueous solubility and thus face restricted application. In the presence of vinyl sulfone functionalized PAMAM dendrimer at 45 mg/mL concentration, drug solubility is increased by 37-fold, 4-fold, and 10-fold for silibinin, camptothecin, and methotrexate, respectively. By further crosslinking of the functionalized PAMAM dendrimer and thiolated PEG, we successfully developed a fast-crosslinking hydrogel capable of encapsulating a significant payload of solubilized cancer drugs for sustained release. In water, the drug encapsulated hydrogels release 30%-80% of their loads in 1-4 days. MTT assays of J82 and MCF7 cells with various doses of drug encapsulated hydrogels reveal that cytotoxicity is observed for all three drugs on both J82 and MCF7 cell lines after 48 h. Notably, camptothecin exhibits higher cytotoxicity to both cell lines than silibinin and methotrexate, achieving up to 95% cell death at experimental conditions, despite its lower solubility. Our experiments provide evidence that the PAMAM dendrimer-mediated hydrogel system significantly improves the solubility of hydrophobic drugs and facilitates their sustained release. These findings position the system as a promising platform for controlled delivery of hydrophobic drugs for intratumoral cancer treatment.

Advances in Studying Glycosaminoglycan-Protein Interactions Using Capillary Electrophoresis.

Methods for studying interactions between glycosaminoglycans (GAGs) and proteins have assumed considerable significance as their biological importance increases. Capillary electrophoresis (CE) is a powerful method to study these interactions due to its speed, high efficiency, and low sample/reagent consumption. In addition, CE works effectively under a wide range of physiologically relevant conditions. This chapter presents the state of the art on CE methods for studying GAG-protein interactions including affinity capillary electrophoresis (ACE), capillary zone electrophoresis (CZE), frontal analysis (FA)/frontal analysis continuous capillary electrophoresis (FACCE), and capillary electrokinetic chromatography (CEC) with detailed experimental protocols for ACE and CZE methods.

Plasmonic resonance energy transfer from a Au nanosphere to quantum dots at a single particle level and its homogenous immunoassay.

Plasmonic resonance energy transfer (PRET) from a Au nanosphere (AuNS) to a quantum dot (QD) is discovered at the single particle level. A homogenous immunoassay based on this PRET is verified using a prostate specific antigen (PSA) as an example. The limit of detection of the PSA is determined to be 0.2 fM.

Polyamidoamine dendrimer-PEG hydrogel and its mechanical property on differentiation of mesenchymal stem cells.

BACKGROUND: Biocompatible hydrogel systems with tunable mechanical properties have been reported to influence the behavior and differentiation of mesenchymal stem cells (MSCs). OBJECTIVE: To develop a functionalized hydrogel system with well-defined chemical structures and tunable mechanical property for regulation of stem cell differentiation. METHODS: An in situ-forming hydrogel system is developed by crosslinking vinyl sulfone functionalized polyamidoamine (PAMAM) dendrimer and multi-armed thiolated polyethylene glycol (PEG) through a thiol-ene Michael addition in aqueous conditions. The viability and differentiation of MSCs in hydrogels of different stiffness are conducted for 21 days under corresponding induction media. RESULTS: MSCs are viable in synthesized hydrogels after 48 hours of culture. By varying the concentrations of PAMAM dendrimer and PEG, hydrogels of different gelation time and stiffness are achieved. The MSC differentiation indicates that more osteogenic differentiation is observed in hard gel (5,663 Pa) and more adipogenic differentiation is observed in soft gel (77 Pa) in addition to the differentiation caused by each individual induction media during the process of culture. CONCLUSIONS: A biocompatible in situ-forming hydrogel system is successfully synthesized. This hydrogel system has shown influences on differentiation of MSCs and may potentially be important in developing therapeutic strategies in medical applications.

Asynchrony of spectral blue-shifts of quantum dot based digital homogeneous immunoassay.

A femtomolar digital homogenous immunoassay is developed based on sensitively distinguishing the immunocomplexes labeled with quantum dot (QD) aggregates from the excessive free monodisperse single QDs. The success in quantifying the carcino-embryonic antigen and alpha-fetoprotein in plasma validated the feasibility of our approach for clinical tests.

A Single-Molecule Homogeneous Immunoassay by Counting Spatially "Overlapping" Two-Color Quantum Dots with Wide-Field Fluorescence Microscopy.

We developed a single-molecule homogeneous immunoassay by counting spatially "overlapping" two-color quantum dots (QD) under a wide-field fluorescence microscope. QD 655 with red fluorescence and QD 565 with green fluorescence were modified with capture and detection antibodies, respectively. A capture antibody-modified QD 655 and a detection antibody-modified QD 565 were conjugated by a corresponding antigen molecule to form a "sandwich" immunocomplex. The conjugated QD 655 could not be distinguished from the conjugated QD 565 by fluorescent microscopy because the distance between them was smaller than the resolution of an optical microscope (approximately 200 nm). The immunocomplex color became yellow because of the spatial "overlap" of the red and green fluorescence. The number of the yellow spots was equal to the number of immunocomplex molecules, while the concentration of the antigen was related to the ratio of the yellow dots to the red dots. The successful quantification of two model proteins in the human plasma, namely, alpha-fetoprotein and carcinoembryonic antigen, demonstrated the accuracy and reliability of our approach.

Sensing Active Heparin by Counting Aggregated Quantum Dots at Single-Particle Level.

Developing highly sensitive and highly selective assays for monitoring heparin levels in blood is required during and after surgery. In previous studies, electrostatic interactions are exploited to recognize heparin and changes in light signal intensity are used to sense heparin. In the present study, we developed a quantum dot (QD) aggregation-based detection strategy to quantify heparin. When cationic micelles and fluorescence QDs modified with anti-thrombin III (AT III) are added into heparin sample solution, the AT III-QDs, which specifically bind with heparin, aggregate around the micelles. The aggregated QDs are recorded by spectral imaging fluorescence microscopy and differentiated from single QDs based on the asynchronous process of blue shift and photobleaching. The ratio of aggregated QD spots to all counted QD spots is linearly related to the amount of heparin in the range of 4.65 × 10 -4 U/mL to 0.023 U/mL. The limit of detection is 9.3 × 10 -5 U/mL (∼0.1 nM), and the recovery of the spiked heparin at 0.00465 U/mL (∼5 nM) in 0.1% human plasma is acceptable.

Thiol-ene crosslinking polyamidoamine dendrimer-hyaluronic acid hydrogel system for biomedical applications.

A series of alkene functionalized polyamidoamine (PAMAM) dendrimers were synthesized to prepare in situ forming hydrogels with varied gelation time and mechanical properties through crosslinking with thiolated hyaluronic acid (HS-HA). By varying the alkenyl groups on the dendrimers, the gelation time displayed a large range from 8 seconds to 18 hours, and the modulus of the hydrogels ranged from 36 to 183 Pa under experimental conditions. Investigation by (1)H-NMR spectroscopy revealed that the gelation time and the stiffness of the hydrogels were governed by the degree of electron deficiency of alkenyl groups on the dendrimers. This research provided a systematic study on the relationship between chemical structures versus gelation time and mechanical properties of hydrogels, which could guide the way to synthesize in situ forming hydrogels with designated gelation time and stiffness for biomedical applications. Further, a RGD peptide was attached to the PAMAM dendrimers to enhance cell attachment and proliferation. Viability assays of Human Umbilical Vein Endothelial Cells (HUVEC) in the synthesized hydrogels demonstrated the biocompatibility of the hydrogels after 48 hours of culturing, and the RGD peptide improved the viability of HUVEC cells in hydrogels. We believe the PAMAM/HA hydrogel system is a tuneable and biocompatible system for diverse biomedical applications.

Studying glycosaminoglycan-protein interactions using capillary electrophoresis.

Methods for studying interactions between glycosaminoglycans (GAGs) and proteins have assumed considerable significance as their biological importance increases. Capillary electrophoresis (CE) is a powerful method to study these interactions due to its speed, high efficiency, and low sample/reagent consumption. In addition, CE works effectively under a wide range of physiologically relevant conditions. This chapter presents state-of-the-art on CE methods for studying GAG-protein interactions including affinity capillary electrophoresis (ACE), capillary zone electrophoresis (CZE), frontal analysis (FA)/frontal analysis continuous capillary electrophoresis (FACCE), and capillary electrokinetic chromatography (CEC) with detailed experimental protocols for ACE and CZE methods.

Determination of binding constants between one protein and multiple carbohydrates by affinity chromatography on a microchip.

Development of rapid, reliable and high throughput methods for evaluating the interactions between different carbohydrates and a same protein is critical to carbohydrate drug development. In this study, we develop a novel strategy based on an affinity chromatography for quickly determining the binding constants of different carbohydrates to a same protein. The core of our method is the inversely proportional relationship between the binding constant and a new termed parameter, critical elution concentration (CMC). CMC is defined as the lowest concentration of displacing reagent, a series of carbohydrates herein, at which the protein specifically bond to the affinity column can be eluted off as an intact peak by the carbohydrate solution in a certain time. The interactions between a series of sulfate polysaccharides and granulocyte colony-stimulating factor (G-CSF) are selected as model. Through a 200 µm long heparin affinity column microfabricated inside a channel of 50 µm width and 20 µm height, the binding constant of each G-CSF-polysaccharide binding pair can be obtained within 1h, around one sixth of time needed by traditional capillary electrophoresis based method.

Dynamic affinity chromatography in the separation of sulfated lignins binding to thrombin.

Sulfated low molecular weight lignins (LMWLs), a mixture of chemo-enzymatically prepared oligomers, have been found to be potent antagonists of coagulation. However, structures that induce anticoagulation remain unidentified. The highly polar sulfate groups on these molecules and the thousands of different structures present in these mixtures make traditional chromatographic resolution of sulfated LMWLs difficult. We performed dynamic thrombin affinity chromatography monitored using chromogenic substrate hydrolysis assay to isolate sulfated LMWL fractions that differed significantly in their biophysical and biochemical properties. Three fractions, I(35), I(55) and Peak II, were isolated from the starting complex mixture. Independent plasma clotting assays suggested that I(35) possessed good anticoagulation potential (APTT=4.2µM; PT=6.8µM), while I(55) and Peak II were approximately 10- and 100-fold less potent. The ESI-MS spectrum of this oligomeric fraction showed multiple peaks at 684.8, 610.6, 557.4, 541.4, 536.5, and 519.4m/z, which most probably arise from variably functionalized ß-O4ß-ß-linked trimers and/or a ß-O4ß-O4-linked dimers. The first direct observation of these structures in sulfated LMWLs will greatly assist in the discovery of more potent sulfated LMWL-based anticoagulants.

Designing allosteric regulators of thrombin. Monosulfated benzofuran dimers selectively interact with Arg173 of exosite 2 to induce inhibition.

Earlier, we reported on the design of sulfated benzofuran dimers (SBDs) as allosteric inhibitors of thrombin (Sidhu et al. J. Med. Chem.201154 5522-5531). To identify the site of binding of SBDs, we studied thrombin inhibition in the presence of exosite 1 and 2 ligands. Whereas hirudin peptide and heparin octasaccharide did not affect the IC(50) of thrombin inhibition by a high affinity SBD, the presence of full-length heparin reduced inhibition potency by 4-fold. The presence of γ' fibrinogen peptide, which recognizes Arg93, Arg97, Arg173, Arg175, and other residues, resulted in a loss of affinity that correlated with the ideal Dixon-Webb competitive profile. Replacement of several arginines and lysines of exosite 2 with alanine did not affect thrombin inhibition potency, except for Arg173, which displayed a 22-fold reduction in IC(50). Docking studies suggested a hydrophobic patch around Arg173 as a plausible site of SBD binding to thrombin. The absence of the Arg173-like residue in factor Xa supported the observed selectivity of inhibition by SBDs. Cellular toxicity studies indicated that SBDs are essentially nontoxic to cells at concentrations as high as 250 mg/kg. Overall, the work presents the localization of the SBD binding site, which could lead to allosteric modulators of thrombin that are completely different from all clinically used anticoagulants.

Sulfated, low molecular weight lignins inhibit a select group of heparin-binding serine proteases.

Sulfated low molecular weight lignins (LMWLs), designed as oligomeric mimetics of low molecular weight heparins (LMWHs), have been found to bind in exosite II of thrombin. To assess whether sulfated LMWLs recognize other heparin-binding proteins, we studied their effect on serine proteases of the coagulation, inflammatory and digestive systems. Using chromogenic substrate hydrolysis assay, sulfated LMWLs were found to potently inhibit coagulation factor XIa and human leukocyte elastase, moderately inhibit cathepsin G and not inhibit coagulation factors VIIa, IXa, and XIIa, plasma kallikrein, activated protein C, trypsin, and chymotrypsin. Competition studies show that UFH competes with sulfated LMWLs for binding to factors Xa and XIa. These results further advance the concept of sulfated LMWLs as heparin mimics and will aid the design of anticoagulants based on their novel scaffold.

Designing nonsaccharide, allosteric activators of antithrombin for accelerated inhibition of factor Xa.

Antithrombin is a key regulator of coagulation and prime target of heparins, clinically used anticoagulants. Heparins induce a two-step conformational activation of antithrombin, a process that has remained challenging to target with molecules devoid of the antithrombin-binding pentasaccharide DEFGH. Computational screening of a focused library led to the design of two tetra-sulfated N-arylacyl tetrahydroisoquinoline variants as potential nonsaccharide activators of antithrombin. A high yielding synthetic scheme based on Horner-Wadsworth-Emmons or Pictet-Spengler reactions was developed to facilitate the functionalization of the tetrahydoisoquinoline ring, which upon further amidation, deprotection, and sulfation gave the targeted nonsaccharide activators. Spectrofluorometric measurement of affinity displayed antithrombin binding affinities in the low to high micromolar range at pH 6.0, I 0.05, 25 °C. Measurement of second-order rate constants of antithrombin inhibition of factor Xa in the presence and absence of the designed activators showed antithrombin activation in the range of 8-80-fold in the pH 6.0 buffer. This work puts forward 20c, a novel tetra-sulfated N-arylacyl tetrahydroisoquinoline-based molecule, that activates AT only 3.8-fold less than that achieved with DEFGH, suggesting a strong possibility of rationally designing sulfated organic molecules as clinically relevant AT activators.

Rational design of potent, small, synthetic allosteric inhibitors of thrombin.

Thrombin is a key enzyme targeted by the majority of current anticoagulants that are direct inhibitors. Allosteric inhibition of thrombin may offer a major advantage of finely tuned regulation. We present here sulfated benzofurans as the first examples of potent, small allosteric inhibitors of thrombin. A sulfated benzofuran library of 15 sulfated monomers and 13 sulfated dimers with different charged, polar, and hydrophobic substituents was studied in this work. Synthesis of the sulfated benzofurans was achieved through a multiple step, highly branched strategy, which culminated with microwave-assisted chemical sulfation. Of the 28 potential inhibitors, 11 exhibited reasonable inhibition of human α-thrombin at pH 7.4. Structure-activity relationship analysis indicated that sulfation at the 5-position of the benzofuran scaffold was essential for targeting thrombin. A tert-butyl 5-sulfated benzofuran derivative was found to be the most potent thrombin inhibitor with an IC(50) of 7.3 µM under physiologically relevant conditions. Michaelis-Menten studies showed an allosteric inhibition phenomenon. Plasma clotting assays indicate that the sulfated benzofurans prolong both the activated partial thromboplastin time and prothrombin time. Overall, this work puts forward sulfated benzofurans as the first small, synthetic molecules as powerful lead compounds for the design of a new class of allosteric inhibitors of thrombin.

Study of physico-chemical properties of novel highly sulfated, aromatic, mimetics of heparin and heparan sulfate.

Heparin (H) and heparan sulfate (HS) play major roles in a number of biological processes. Yet, H/HS-based pharmaceutical agents are also associated with multiple adverse effects. This has led to the concept of designing noncarbohydrate, aromatic mimetics that modulate H/HS function. In this work, we study a library of synthetic, aromatic H/HS mimetics for their capillary electrophoretic profiles, the acid and base stability, and aqueous-organic partitioning property. The nonsugar H/HS mimetics exhibit electrophoretic properties similar to sulfated oligosaccharides suggesting that the mimetics can be rapidly and quantitatively analyzed. Stability studies show that the mimetics are essentially stable under neutral and basic conditions in a manner similar to the heparins, but are considerably unstable under acidic conditions in contrast to heparins. The measurement of partition coefficients show major differences within the sulfated mimetics as well as between the measured and calculated log P values. Understanding these physico-chemical properties is expected to have significant implications in the pharmaceutical development of this growing class of molecules.

Interaction of antithrombin with sulfated, low molecular weight lignins: opportunities for potent, selective modulation of antithrombin function.

Antithrombin, a major regulator of coagulation and angiogenesis, is known to interact with several natural sulfated polysaccharides. Previously, we prepared sulfated low molecular weight variants of natural lignins, called sulfated dehydrogenation polymers (DHPs) (Henry, B. L., Monien, B. H., Bock, P. E., and Desai, U. R. (2007) J. Biol. Chem. 282, 31891-31899), which have now been found to exhibit interesting antithrombin binding properties. Sulfated DHPs represent a library of diverse noncarbohydrate aromatic scaffolds that possess structures completely different from heparin and heparan sulfate. Fluorescence binding studies indicate that sulfated DHPs bind to antithrombin with micromolar affinity under physiological conditions. Salt dependence of binding affinity indicates that the antithrombin-sulfated DHP interaction involves a massive 80-87% non-ionic component to the free energy of binding. Competitive binding studies with heparin pentasaccharide, epicatechin sulfate, and full-length heparin indicate that sulfated DHPs bind to both the pentasaccharide-binding site and extended heparin-binding site of antithrombin. Affinity capillary electrophoresis resolves a limited number of peaks of antithrombin co-complexes suggesting preferential binding of selected DHP structures to the serpin. Computational genetic algorithm-based virtual screening study shows that only one sulfated DHP structure, out of the 11 present in a library of plausible sequences, bound in the heparin-binding site with a high calculated score supporting selectivity of recognition. Enzyme inhibition studies indicate that only one of the three sulfated DHPs studied is a potent inhibitor of free factor VIIa in the presence of antithrombin. Overall, the chemo-enzymatic origin and antithrombin binding properties of sulfated DHPs present novel opportunities for potent and selective modulation of the serpin function, especially for inhibiting the initiation phase of hemostasis.

First steps in the direction of synthetic, allosteric, direct inhibitors of thrombin and factor Xa.

Designing non-saccharide functional mimics of heparin is a major challenge. In this work, a library of small, aromatic molecules based on the sulfated DHP scaffold was synthesized and screened against thrombin and factor Xa. The results reveal that (i) selected monomeric benzofuran derivatives inhibit the two enzymes, albeit weakly; (ii) the two enzymes recognize different structural features in the benzofurans studied suggesting significant selectivity of recognition; and (iii) the mechanism of inhibition is allosteric. The molecules represent the first allosteric small molecule inhibitors of the two enzymes.

Capillary electrophoretic study of small, highly sulfated, non-sugar molecules interacting with antithrombin.

Affinity CE (ACE) was used to study interactions of small, highly sulfated, aromatic molecules with antithrombin (AT). The high charge density of the small molecules induces differential migration of the complex resulting in a versatile method of assessing binding affinities, nature of interactions and site of binding on the inhibitor. Scatchard analysis of the interaction of three tetrahydroisoquinoline-based polysulfated molecules with AT results in monophasic profiles with affinities in the range of 40-60 microM in 20 mM sodium phosphate buffer, pH 7.4. For a pentasulfated molecule, a biphasic profile with affinities of 4.7 and 30 microM was observed. Measurement of K(D) as a function of ionic strength of the medium indicated that ionic and non-ionic forces contribute 2.4 and 1.9 kcal/mol, respectively, at pH 7.4 and 100 mM NaCl. Competitive binding studies showed that the tetrahydroisoquinoline-based molecules do not compete with a high-affinity heparin pentasaccharide. In contrast, the affinity of these tetrahydroisoquinoline derivatives decreases dramatically in the presence of an extended heparin-binding site ligand. Overall, ACE analysis of small, sulfated aromatic molecules interacting with AT is relatively easy and obviates the need for an external signal, e.g. fluorescence, for monitoring the interaction. In addition to affording biochemical knowledge, the small sample requirement and fast analysis time of ACE could be particularly advantageous for high-throughput screening of potential anticoagulants.

On designing non-saccharide, allosteric activators of antithrombin.

Antithrombin, a plasma glycoprotein serpin, requires conformational activation by heparin to induce an anticoagulant effect, which is mediated through accelerated factor Xa inhibition. Heparin, a highly charged polymer and an allosteric activator of the serpin, is associated with major adverse effects. To design better, but radically different activators of antithrombin from heparin, we utilized a pharmacophore-based approach. A tetrahydroisoquinoline-based scaffold was designed to mimic four critical anionic groups of the key trisaccharide DEF constituting the sequence-specific pentasaccharide DEFGH in heparin. Activator IAS(5) containing 5,6-disulfated tetrahydroisoquinoline and 3,4,5-trisulfated phenyl rings was found to bind antithrombin at pH 7.4 with an affinity comparable to the reference trisaccharide DEF. IAS(5) activated the inhibitor nearly 30-fold, nearly 2- to 3-fold higher than our first generation flavanoid-based designs. This work advances the concept of antithrombin activation through non-saccharide, organic molecules and pinpoints a direction for the design of more potent molecules.