Synthesis and hemocompatibility of amino (di-)butyldeoxy modified hydroxyethyl starch.

Cationic polymers are of interest as the basis for obtaining various biomaterials. Hydrophilic biopolymers and their modification products are of main interest. Biocompatibility is the prime criterion that makes this material usable for said purposes. In this research, hydroxyethyl starch (HES) was used as a basis for synthesis of aminodeoxy derivatives, containing n-butylamin (BA) and dibutylamin (DBA) fragments. Bromodeoxy HES was an intermediate compound. The structure of synthesized polymers was confirmed with NMR, elemental analysis and FTIR methods. The derivatives with 0.6 and 0.9 degree of substitution were tested for compatibility with blood. The research showed that HES base does not have an anticoagulation activity, does not affect human platelet aggregation and in concentrations up to 10 mg/mL of cell suspension in a buffer solution does not destroy red blood cell membrane, and therefore can be used as a component of drug delivery systems. Addition of aminodeoxy derivatives of HES hindered development of ADP-induced platelet aggregation. Derivatives of HES-DBA and HES-BA0.9 may also be of interest, but their concentration must not exceed 1 * 10-5 mg/mL of blood. Biodegradation of HES cationic derivatives were analyzed through identification of reducing sugars after treatment with amylase and pancreatin.

Hydroxyethyl starch-10-hydroxy camptothecin conjugate: synthesis, pharmacokinetics, cytotoxicity and pharmacodynamics research.

10-hydroxy camptothecin (10-HCPT) is an antitumor agent effective in the treatment of several solid tumors but its use is hampered by poor water solubility, low lactone stability, short plasma half-life and dose-limiting toxicity. In this study, 10-HCPT-hydroxyethyl starch (HES) conjugate was prepared to overcome these limits of 10-HCPT. The solubility of 10-HCPT conjugate was 0.72 mg/ml, about 100 times to free 10-HCPT. The 10-HCPT conjugate showed good sustained release effect in phosphate-buffered saline (PBS), rat plasma and liver homogenate. Meanwhile, 10-HCPT-HES conjugate achieved much lower IC50 and higher cytotoxicity effects than the free 10-HCPT on Hep-3B liver cancer cells. The pharmacokinetics results of 10-HCPT-HES conjugate demonstrated that the biological half-life of 10-HCPT was increased from 10 min to 4.38 h and the bioavailability was 40 times higher than the commercial 10-HCPT injection. The pharmacodynamics results indicated that 10-HCPT-HES conjugate had a better antitumor efficiency against nude mouse with Hep-3B tumor than the commercial 10-HCPT injection, and the inhibition ratio of tumor was 83.9 and 27.8%, respectively, at the same administration dosage. These findings suggest that 10-HCPT-HES conjugate is a promising drug delivery system providing improved long circulating effect, greater stability and better antitumor effect.

Tailoring the stealth properties of biocompatible polysaccharide nanocontainers.

Fundamental development of a biocompatible and degradable nanocarrier platform based on hydroxyethyl starch (HES) is reported. HES is a derivative of starch and possesses both high biocompatibility and improved stability against enzymatic degradation; it is used to prepare nanocapsules via the polyaddition reaction at the interface of water nanodroplets dispersed in an organic miniemulsion. The synthesized hollow nanocapsules can be loaded with hydrophilic guests in its aqueous core, tuned in size, chemically functionalized in various pathways, and show high shelf life stability. The surface of the HES nanocapsules is further functionalized with poly(ethylene glycol) via different chemistries, which substantially enhanced blood half-life time. Importantly, methods for precise and reliable quantification of the degree of functionalization are also introduced, which enable the precise control of the chemistry on the capsules' surface. The stealth properties of these capsules is studied both in-vitro and in-vivo. The functionalized nanocapsules serve as a modular platform for specific cell targeting, as they show no unspecific up-taken by different cell types and show very long circulating time in blood (up to 72 h).

A comparative study of neurotoxic potential of synthesized polysaccharide-coated and native ferritin-based magnetic nanoparticles.

AIM: To analyze the neurotoxic potential of synthesized magnetite nanoparticles coated by dextran, hydroxyethyl starch, oxidized hydroxyethyl starch, and chitosan, and magnetic nanoparticles combined with ferritin as a native protein. METHODS: The size of nanoparticles was analyzed using photon correlation spectroscopy, their effects on the conductance of planar lipid membrane by planar lipid bilayer technique, membrane potential and acidification of synaptic vesicles by spectrofluorimetry, and glutamate uptake and ambient level of glutamate in isolated rat brain nerve terminals (synaptosomes) by radiolabeled assay. RESULTS: Uncoated synthesized magnetite nanoparticles and nanoparticles coated by different polysaccharides had no significant effect on synaptic vesicle acidification, the initial velocity of L-[(14)C]glutamate uptake, ambient level of L-[(14)C]glutamate and the potential of the plasma membrane of synaptosomes, and conductance of planar lipid membrane. Native ferritin-based magnetic nanoparticles had no effect on the membrane potential but significantly reduced L-[(14)C]glutamate transport in synaptosomes and acidification of synaptic vesicles. CONCLUSIONS: Our study indicates that synthesized magnetite nanoparticles in contrast to ferritin have no effects on the functional state and glutamate transport of nerve terminals, and so ferritin cannot be used as a prototype, analogue, or model of polysaccharide-coated magnetic nanoparticle in toxicity risk assessment and manipulation of nerve terminals by external magnetic fields. Still, the ability of ferritin to change the functional state of nerve terminals in combination with its magnetic properties suggests its biotechnological potential.

Protein HESylation for half-life extension: synthesis, characterization and pharmacokinetics of HESylated anakinra.

Half-life extension (HLE) is becoming an essential component of the industrial development of small-sized therapeutic peptides and proteins. HESylation(®) is a HLE technology based on coupling drug molecules to the biodegradable hydroxyethyl starch (HES). In this study, we report on the synthesis, characterization and pharmacokinetics of HESylated anakinra, where anakinra was conjugated to propionaldehyde-HES using reductive amination, leading to a monoHESylated protein. Characterization using size exclusion chromatography and dynamic light scattering confirmed conjugation and the increase in molecular size, while Fourier transform infrared spectroscopy showed that the secondary structure of the conjugate was not affected by coupling. Meanwhile, microcalorimetry and aggregation studies showed a significant increase in protein stability. Surface plasmon resonance and microscale thermophoresis showed that the conjugate retained its nanomolar affinity, and finally, the pharmacokinetics of the HESylated protein exhibited a 6.5-fold increase in the half-life, and a 45-fold increase in the AUC. These results indicate that HESylation(®) is a promising HLE technology.

Synthesis and physicochemical characterization of a series of hemoglobin-based oxygen carriers: objective comparison between cellular and acellular types.

A series of hemoglobin (Hb)-based O(2) carriers, acellular and cellular types, were synthesized and their physicochemical characteristics were compared. The acellular type includes intramolecularly cross-linked Hb (XLHb), polyoxyethylene (POE)-conjugated pyridoxalated Hb (POE-PLP-Hb), hydroxyethylstarch-conjugated Hb (HES-XLHb), and glutaraldehyde-polymerized XLHb (Poly-XLHb). The cellular type is Hb-vesicles (HbV) of which the surface is modified with POE (POE-HbV). Their particle diameters are 7 +/- 2, 22 +/- 2, 47 +/- 17, 68 +/- 24, and 224 +/- 76 nm, respectively, thus all the materials penetrate across membrane filters with 0.4 microm pore size, though only the POE-HbV cannot penetrate across the filter with 0.2 microm pore size. These characteristics of permeability are important to consider an optimal particle size in microcirculation in vivo. POE-PLP-Hb ([Hb] = 5 g/dL) showed viscosity of 6.1 cP at 332 s(-1) and colloid osmotic pressure (COP) of 70.2 Torr, which are beyond the physiological conditions (human blood, viscosity = 3-4 cP, COP = ca. 25 Torr). XLHb and Poly-XLHb showed viscosities of 1.0 and 1.5 cp, respectively, which are significantly lower than that of blood. COP of POE-HbV is regulated to 20 Torr in 5% human serum albumin (HSA). HES-XLHb and POE-HbV/HSA showed comparable viscosity with human blood. Microscopic observation of human red blood cells (RBC) after mixing blood with POE-PLP-Hb or HES-XLHb disclosed aggregates of RBC, a kind of sludge, indicating a strong interaction with RBC, which is anticipated to modify peripheral blood flow in vivo. On the other hand, XLHb and POE-HbV showed no rouleaux or aggregates of RBC. The acellular Hbs (P(50) = 14-32 Torr) have their specific O(2) affinities determined by their structures, while that of the cellular POE-HbV is regulated by coencapsulating an appropriate amount of an allosteric effector (e.g., P(50) = 18, 32 Torr). These differences in physicochemical characteristics between the acellular and cellular types indicate the advantages of the cellular type from the physiological points of view.

Modulation of deferoxamine toxicity and clearance by covalent attachment to biocompatible polymers.

A class of high molecular weight iron chelators has been prepared by covalently attaching deferoxamine (DFO), by its amino group, to a variety of biocompatible polymers such as dextran and hydroxyethyl-starch. The iron-binding properties of DFO are virtually unchanged after the attachment procedure, but the toxicity and circulatory half-life are profoundly altered. Competitive iron-binding experiments indicate that the conjugates retain a high affinity for ferric iron. In addition, the derivatives inhibit iron-driven lipid peroxidation as effectively as the parent drug. However, the LD50 in mice (based on DFO equivalents) is approximately 4000 mg/kg for dextran-DFO as compared to 250 mg/kg for free DFO. Consistent with the greatly decreased LD50, intravenous administration of the conjugates in dogs at a dose of 100 mg/kg (body weight) does not cause the severe hypotension associated with intravenous administration of DFO. The plasma half-lives of these adducts are increased greater than 10-fold for dextran-DFO and hydroxyethyl-starch-DFO compared to the free drug. Finally, and most importantly, the conjugates are effective in mediating in vivo iron mobilization and excretion. Because recent evidence implicates iron as an important component of tissue injury in many disease states, these high molecular weight iron chelators may have potential for improved therapy, allowing higher sustained plasma concentrations of the active drug.

Preparation and evaluation of Tc-99m hydroxyethyl starch as a potential radiopharmaceutical for lymphoscintigraphy: comparison with Tc-99m human serum albumin, Tc-99m dextran, and Tc-99m sulfur microcolloid.

Tc-99m hydroxyethyl starch (Tc-99m HES) prepared with a labeling efficiency greater than 95% was evaluated in rabbits for visualization of lymphatic channels and lymph nodes, and the findings compared with Tc-99m human serum albumin (Tc-99m HSA), Tc-99m dextran (Tc-99m DXT), and Tc-99m sulfur microcolloid (Tc-99m SMC). Tc-99m HES showed good visualization of lymphatic channels and regional nodes and it had the highest clearance rate from the injection site (p less than 0.01). Tc-99m HES showed greater uptake by the nodes than Tc-99m DXT (p less than 0.001) at 90 minutes post-injection. The concentration of Tc-99m HES in the lymphatic channels was higher than that of Tc-99m SMC at 90 minutes post-injection (p less than 0.001). Preliminary clinical studies of Tc-99m HES yielded high quality lymphoscintigrams of the leg, and the pelvic and para-aortic lymph nodes in less than 10 minutes post-injection. In addition, partially and completed obstructed lymphatics could be differentiated from normal lymphatic pathways. In conclusion, Tc-99m HES is a promising radiopharmaceutical for imaging of lymphatic channels and nodes.