Kamil Kamiński

pH-Sensitive Genipin-Cross-Linked Chitosan Microspheres For Heparin Removal

Chitosan hydrogel microspheres were obtained by cross-linking chitosan in its inverse emulsion using genipin as cross-linker. The genipin-cross-linked chitosan microspheres (ChGp) swell significantly in water at pH values below 6.5 and shrink to a smaller extent at pH values above 6.5. ChGp microspheres bind heparin in water. The kinetics of heparin binding was found to be pH dependent and was faster and more efficient at a lower pH. That can be also controlled by the weight of ChGp microspheres used. Rate and efficiency of heparin adsorption at pH 7.4, which is typical of blood, could be increased by quaternization of ChGp microspheres using glycidyltrimethylammonium chloride (GTMAC). The polymeric material obtained thus can be potentially useful for heparin removal in biomedical applications.

Chitosan Derivatives as Novel Potential Heparin Reversal Agents

In emergency cases anticoagulant action of heparin needs to be stopped instantaneously, which is usually achieved by intravenous administration of protamine sulfate (PS). However, PS shows many adverse effects. The objective of the present work was to find out if chitosan (Ch) and a cationically modified chitosan, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), may be applied for heparin reversal. For chitosan the efficiency of unfractionated heparin (UFH) binding decreases with increasing pH while for cationically modified chitosan heparin binding is efficient even for high pH values. Complexation of UFH and low-molecular-weight heparin (LMWH) by cationically modified chitosan in the aqueous solution at pH = 7.4 was studied. Complexes of the modified chitosan with UFH are smaller and of lower dispersity than those with PS. Cationically modified chitosan was found to bind both UFH and LMWH. The complex formation capability of cationically modified chitosan is comparable to that of PS.

Cationic derivatives of dextran and hydroxypropylcellulose as novel potential heparin antagonists.

Cationic derivatives of dextran (Dex) and hydroxypropylcellulose (HPC) were studied as potential alternatives of protamine sulfate (PS) used in the reversal of anticoagulant activity of heparin. The modification was performed by the attachment of cationic groups to the Dex main chain or by grafting short side chains of a polycation onto HPC. The cationic derivatives of these polysaccharides were found to bind heparin with the efficiency increasing with growing degree of cationic modification. The degree of cationic modification and consequently the ζ potential of the polymers do not have to be high to achieve effective heparin binding. The size of the complexes of cationic Dex with unfractionated heparin (UFH) is a few micrometers. For complexes of cationic HPC and UFH the size is much below 1 μm, both below and above the lower critical solution temperature of HPC. None of the cationic polysaccharides studied caused hemolysis. The concentrations of the polymers inducing the aggregation of human erythrocytes in vitro were determined.

Cationic derivative of dextran reverses anticoagulant activity of unfractionated heparin in animal models of arterial and venous thrombosis.

Heparin is a natural polymer widely used in medicine especially during the treatment of cardiovascular diseases since it is a potent blood anticoagulant. In case of emergency, e.g., massive hemorrhage, the anticoagulant activity of heparin has to be quickly stopped by the administration of a heparin reversing agent. Currently protamine sulfate, an allergenic protein, is used for this purpose. We are reporting the studies on a new polymeric substance, a cationic dextran derivative, which is able to form complexes with heparin. Dextran is a blood compatible polymer which is also frequently applied in medicine. By substituting dextran with glycidyltrimethylammonium chloride a cationic polymer was obtained that in vitro binds to heparin with an efficiency similar to that of protamine. To investigate the influence of modified dextran on the reversal of conventional heparin we used the models of experimental arterial thrombosis induced by electrical stimulation and chemically induced venous thrombosis. A decrease in bleeding time and activated partial thromboplastin time after administration of the cationic dextran to heparinized rats was found. Moreover, other routinely measured blood parameters are significantly affected. Modified dextran, in contrast to protamine sulfate, significantly increases red blood cell counts, hemoglobin level, and hematocrit value. The data we obtained show that the modified dextran may reduce anticoagulative heparin activity both under in vivo and in vitro conditions. Further clinical studies are needed to estimate whether modified dextran could replace protamine sulfate, especially in dialyzed patients with the end-stage renal disease associated with anemia.

Novel polymeric inhibitors of HCoV-NL63

Abstract The human coronavirus NL63 is generally classified as a common cold pathogen, though the infection may also result in severe lower respiratory tract diseases, especially in children, patients with underlying disease, and elderly. It has been previously shown that HCoV-NL63 is also one of the most important causes of croup in children. In the current manuscript we developed a set of polymer-based compounds showing prominent anticoronaviral activity. Polymers have been recently considered as promising alternatives to small molecule inhibitors, due to their intrinsic antimicrobial properties and ability to serve as matrices for antimicrobial compounds. Most of the antimicrobial polymers show antibacterial properties, while those with antiviral activity are much less frequent. A cationically modified chitosan derivative, N-(2-hydroxypropyl)-3-trimethylammonium chitosan chloride (HTCC), and hydrophobically-modified HTCC were shown to be potent inhibitors of HCoV-NL63 replication. Furthermore, both compounds showed prominent activity against murine hepatitis virus, suggesting broader anticoronaviral activity.

Silicone-stabilized liposomes as a possible novel nanostructural drug carrier.

Development of silicone stabilized liposomes which can serve as novel drug nanocarriers is presented. Silicone precursor 1,3,5,7-tetramethylcyclotetrasiloxane (D4(H)) was introduced into the bilayer of the cationic liposomes prepared from egg yolk phosphatidylocholine (PC) and double-tailed dimethyldioctadecylammonium bromide (DODAB). The silicone material was created inside of the liposomal bilayer in the base-catalyzed polycondensation process of the D4(H) what was confirmed employing (29)Si solid-state MAS NMR and FTIR measurements. Surfactant lysis experiments revealed that resulted systems can be effectively stabilized. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements demonstrated that the silicone-stabilized liposomes have typical lipid vesicles morphology and mean hydrodynamic diameters in the range of about 110nm. They have considerably lower tendency for aggregation than the pristine liposomes. The permeability of vesicles can be tuned by introducing various amounts of silicone precursor into the liposome bilayer, as confirmed in calcein-release studies. The effect of fetal bovine serum (FBS) on the stability of liposomes was also tested in in vitro studies. Biological studies revealed that resulted liposomes can be considered as possible drug nanocarriers because they are not toxic to human skin fibroblasts (HSFs) and mouse embryonic fibroblasts (MEFs).

Nonclinical Evaluation of Novel Cationically Modified Polysaccharide Antidotes for Unfractionated Heparin

Protamine, the only registered antidote of unfractionated heparin (UFH), may produce a number of adverse effects, such as anaphylactic shock or serious hypotension. We aimed to develop an alternative UFH antidote as efficient as protamine, but safer and easier to produce. As a starting material, we have chosen generally non-toxic, biocompatible, widely available, inexpensive, and easy to functionalize polysaccharides. Our approach was to synthesize, purify and characterize cationic derivatives of dextran, hydroxypropylcellulose, pullulan and γ-cyclodextrin, then to screen them for potential heparin-reversal activity using an in vitro assay and finally examine efficacy and safety of the most active polymers in Wistar rat and BALB/c mouse models of experimentally induced arterial and venous thrombosis. Efficacy studies included the measurement of thrombus formation, activated partial thromboplastin time, bleeding time, and anti-factor Xa activity; safety studies included the measurement of hemodynamic, hematologic and immunologic parameters. Linear, high molecular weight dextran substituted with glycidyltrimethylammonium chloride groups at a ratio of 0.65 per glucose unit (Dex40-GTMAC3) is the most potent and the safest UFH inhibitor showing activity comparable to that of protamine while possessing lower immunogenicity. Cationic polysaccharides of various structures neutralize UFH. Dex40-GTMAC3 is a promising and potentially better UFH antidote than protamine.

Biopolymeric hydrogels - nanostructured TiO2 hybrid materials as potential injectable scaffolds for bone regeneration

The present work aims at development of novel hybrid materials from genipin crosslinked collagen or collagen/chitosan hydrogels containing various types of TiO2 nanoparticles characterized with different anatase/rutile ratios. Collagen and chitosan were selected as hydrogel components since they are biopolymers being, like collagen, the major compound present in extracellular matrix or exhibit structural similarity to glycosaminoglycans, like chitosan. TiO2 nanoparticles were introduced to the hydrogel matrices to improve their mechanical properties as well as bioactivity. A series of twelve novel hybrid materials were prepared and their physicochemical, mechanical and biological properties were evaluated. It was found that TiO2 nanostructures introduced to the hydrogels have significant influence on the swelling properties of the synthesized hybrids and their impact is strongly dependent on the type of matrices. The surfaces of hybrid materials were found to be more hydrophilic than these of corresponding hydrogel matrix. It was also observed that, the storage modulus values of the hybrids based on collagen-chitosan hydrogel are comparable to these for plain hydrogels what indicates that the mechanical properties of the materials obtained are satisfactory for possible biomedical application. The in vitro cell culture studies have shown that prepared materials are biocompatible as they can support mitochondrial activity of MEFs as well as MG-63 cells. In vitro experiments performed under simulated body fluid (SBF) conditions have revealed that all studied TiO2 nanoparticles present in hydrogel matrices, regardless of anatase/rutile ratio, successfully induced formation of apatite-like structures. The hybrid materials developed here are promising candidates for preparation of bioactive, injectable scaffolds for tissue engineering.

New arginine substituted derivative of poly(allylamine hydrochloride) for heparin reversal

New derivatives of polyallylamine containing arginine moieties (PAH-ARG) were synthesized. The in vitro tests performed in heparinized blood plasma showed that the complexation of heparin by PAH-ARG polymers allowed the reduction of the activated partial thromboplastin time (aPTT) values to the normal level. The dose of PAH-ARG required for complete reversal of aPTT (prolonged by 1 U of heparin) was half of that required for protamine sulfate, the currently used heparin antagonist. The efficacy of these polymers in the neutralization of heparin was confirmed by in vivo tests using a rat model. PAH-ARG polymers were nontoxic to the fibroblast cells.

The Toxicokinetic Profile of Dex40-GTMAC3—a Novel Polysaccharide Candidate for Reversal of Unfractionated Heparin

Though protamine sulfate is the only approved antidote of unfractionated heparin (UFH), yet may produce life threatening side effects such as systemic hypotension, catastrophic pulmonary vasoconstriction or allergic reactions. We have described 40 kDa dextrans (Dex40) substituted with glycidyltrimethylammonium chloride (GTMAC) as effective, immunogenically and hemodynamically neutral inhibitors of UFH. The aim of the present study was to evaluate in mice and rats toxicokinetic profile of the most promising polymer—Dex40-GTMAC3. Polymer was rapidly eliminated with a half-time of 12.5 ± 3.0 min in Wistar rats, and was mainly distributed to the kidneys and liver in mice. The safety studies included the measurement of blood count and blood biochemistry, erythrocyte osmotic fragility and the evaluation of the histological alterations in kidneys, liver and lungs of mice and rats in acute and chronic experiments. We found that Dex40-GTMAC3 is not only effective but also very well tolerated. Additionally, we found that protamine may cause overt hemolysis with appearance of permanent changes in the liver and kidneys. In summary, fast renal clearance behavior and generally low tissue accumulation of Dex40-GTMAC3 is likely to contribute to its superior to protamine biocompatibility. Intravenous administration of therapeutic doses to living animals does not result in the immunogenic, hemodynamic, blood, and organ toxicity. Dex40-GTMAC3 seems to be a promising effective and safe candidate for further clinical development as new UFH reversal agent.