Dominique Domurado

Biocompatibility of Polycations: In Vitro Agglutination and Lysis of Red Blood Cells And In Vivo Toxicity

The effects of six polycations were studied in vitro on red blood cells (RBC) and in vivo after intravenous administration. Hemagglutination and hemolysis depended not only on the molar mass and the concentration of these polycations, but also on their chemical nature. The hemagglutination and hemolysis induced by poly (l -lysine), diethylaminoethyldextran, poly(dimethyldiallylammonium) chloride and poly[2-(dimethylamino)ethyl methacrylate] was low to moderate, whereas a severe hemolysis was induced by a partially quaternized poly[thio-1- (N, N -diethyl-aminoethyl)ethylene]. In the case of poly (ε -lysine), no significant hemagglutination nor hemolysis was observed. The presence of plasma proteins reduced both agglutination and hemolysis. This protective effect was enhanced when the polycations interacted with plasma proteins before contact with RBC. In the presence of albumin, the behavior depended on the polycation and on the order of addition of the three components of the suspension, namely albumin, polycation and RBC. Depending on the polycation, albumin-polycation complexes were either less active or more active on RBC than the same polycation in protein-free medium. In vivo the studied polycations induced an immediate mortality except poly (ε -lysine), which induced a delayed mortality. The minimal dose of polycations inducing immediate mortality paralleled their effect on RBC.

Synthesis, Degradation, and Antimicrobial Properties of Targeted Macromolecular Prodrugs of Norfloxacin

ABSTRACT Long-term antibiotic treatment is required to cure tuberculosis. Targeted antibiotics should improve the efficacy of treatment by concentrating the drugs close to the bacteria. The aim of the present study was to synthesize targeted conjugates. For this purpose, we used mannose as a homing device to direct norfloxacin into macrophages. Dextran was used as the polymer bearing both mannose and norfloxacin. Using different peptide spacer arms to link norfloxacin to dextran, we demonstrated that norfloxacin acts as an antibiotic only when it is released in its native form. Also, targeting by using mannose as a homing device is required to achieve antimycobacterial activity in vivo. Thus, norfloxacin, which is inactive against mycobacteria in its native form in vivo, can be transformed into an active drug by targeting.

Interactions between red blood cells and a lethal, partly quaternized tertiary polyamine.

Partially quaternized poly[thio-1-(N,N-diethyl-aminomethyl) ethylene]s, Q-P(TDAE)(x) with x indicating the percentage of quaternized subunits, have been proposed as potential carriers for drugs insoluble in water. However these cationic polyelectrolytes form emboli upon intravenous administration. In order to study the mechanism, Q-P(TDAE)(11) was incubated in vitro with red blood cells (RBCs) suspended in various aqueous media such as autologous plasma, autologous serum, albumin dissolved in phosphate buffer, plasma-serum mixtures and Tris buffer. The deformability of the RBC membrane studied by viscometry was not affected by the polycation. Q-P(TDAE)(11)-induced hemagglutination was studied by optical microscopy. It depended on the polymer concentration and on the presence of plasma proteins. As ghosts were formed in some cases, hemolysis was investigated by measuring potassium and hemoglobin released from RBCs. Fibrinogen and serum proteins, except albumin, protected RBCs from hemolysis. Moreover the order of addition of the suspension components modulated dramatically the Q-P(TDAE)(11)-induced hemolysis. Addition of Q-P(TDAE)(11) to whole blood caused hemolysis whereas addition of the polymer to plasma prior to contact with RBCs did not affect the cell integrity. In contrast, addition of the polymer to RBCs suspended in albumin solution caused greater hemolysis than the addition to whole blood, and the contact between Q-P(TDAE)(11) and albumin prior to RBC addition still enhanced cell lysis. Two conclusions can be drawn from these observations: (i) Q-P(TDAE)(11) induces both hemagglutination, probably through electrostatic interaction, and hemolysis, because Q-P(TDAE)(11) disrupted the RBC lipid bilayer; (ii) proteins can decrease or increase the deleterious effects of Q-P(TDAE)(11) on RBCs.

Synthesis of polyglutamine and dextran conjugates of streptomycin with an acid-sensitive drug-carrier linkage

Abstract A polymeric prodrug of streptomycin was prepared by coupling the drug via a spacer, glycine hydrazide, onto a polymeric carrier. In a first step, glycine N -Boc-hydrazide was linked to a 4-nitrophenyl chloroformate activated polymer. After removing the Boc group, streptomycin was coupled with the polymeric hydrazide with formation of a hydrazone bond. In order to target the drug-carrier derivative to the macrophages, 6-aminohexyl-α- D -mannopyranoside side groups were introduced. The polymeric streptomycin derivative was shown to be non hemolytic. The hydrolytic stability of the polymer-streptomycin conjugate was studied at physiological and lysosomal pH. Streptomycin release was found to be faster in the lysosomal pH range.

Synthesis and ring-opening polymerisation of a new alkyne-functionalised glycolide towards biocompatible amphiphilic graft copolymers

A new functional dilactone, 3-(2-propynyl)-1,4-dioxane-2,5-dione (4), was synthesized from ethyl glyoxalate and propargyl bromide via a 4-step reaction sequence. Ring-opening (co)polymerisation of the alkyne-functionalised monomer 4 with L-lactide was carried out in dichloromethane at 30 °C using N,N-dimethylaminopyridine as a catalyst and benzyl alcohol as an initiator. The resulting alkyne-functionalised copolyesters were characterized by 1H NMR, size exclusion chromatography (SEC) and MALDI-TOF spectroscopy. Azide end-functionalised PEG was then grafted onto the polyester backbone with multiple pendant alkyne moieties using copper-catalysed azide–alkyne cycloaddition (click chemistry). The graft copolymers were characterized by 1H NMR, SEC and DOSY NMR. The aggregation behavior of the copolymers in water was investigated by fluorescence spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). The critical aggregation concentration was in the range of 10–50 mg L−1. The nano-sized objects were rod-like in shape with a diameter of 100 nm and a length of around 400 nm.

In Vivo Fate of End-Chain Radiolabelled Poly(β-malic acid), a Water-Soluble Biodegradable Drug Carrier

In a first attempt to determine the fate of poly(β-malic acid) after intravenous injection in mice, polymer end-chain 14C-radiolabelling was achieved using 14C-triethylamine as the initiator for the ring-opening polymer ization of benzyl malolactonate. The corresponding poly(β-malic acid) sodium salt (Mw ∼ 30,000) exhibited an activity of 4.2 μCi :g-1. Aliquots of a neutral isoosmotic solution of the latter were given intravenously to mice through a lateral tail vein. Radioactivity was counted in the liver, kidney, intestine, lung, brain, spleen, heart, muscle, urine and blood for various post-injection times up to 24 hours. Fast urinary excretion (70% after 1 hour and 90% after 6 hours) was observed. For all the sites investigated, radioactivity decreased exponen tially except in the liver and kidneys where a small peak was detected after 2 hours. Further investigations with poly(β-malic acid) radiolabelled in repeat ing units will be necessary to overcome the shortcomings of the end-chain radiolabelling method applied to degradable polymers.

Synthesis and in vitro stability of macromolecular prodrugs of norfloxacin

Abstract Oligopeptide derivatives of the antimicrobial drug norfloxacin, having the tetrapeptides gly-phe-ala-leu or gly-phe-leu-gly substituted on the piperazine amino group, were subsequently coupled with chloroformate-activated dextran. In vitro release experiments in aqueous buffer demonstrate the conjugates to be stable in buffers of pH 7.4 and 5.5. However, in the presence of lysosomal enzymes, the derivatives are degraded. The rate of degradation is followed by HPLC-analysis.

Design and Development of Immunomodulatory Antigen Delivery Systems Based on Peptide/PEG–PLA Conjugate for Tuning Immunity

Cancer vaccines are considered to be a promising tool for cancer immunotherapy. However, a well-designed cancer vaccine should combine a tumor-associated antigen (TAA) with the most effective immunomodulatory agents and/or delivery system to provoke intense immune responses against the TAA. In the present study, we introduced a new approach by conjugating the immunomodulatory molecule LD-indolicidin to the hydrophilic chain end of the polymeric emulsifier poly(ethylene glycol)-polylactide (PEG-PLA), allowing the molecule to be located close to the surface of the resulting emulsion. A peptide/polymer conjugate, named LD-indolicidin-PEG-PLA, was synthesized by conjugation of the amine end-group of LD-indolicidin to the N-hydroxysuccinimide-activated carboxyl end-group of PEG. As an adjuvant for cancer immunotherapeutic use, TAA vaccine candidate formulated with the LD-indolicidin-PEG-PLA-stabilized squalene-in-water emulsion could effectively help to elicit a T helper (Th)1-dominant antigen-specific immune response as well as antitumor ability, using ovalbumin (OVA) protein/EG7 cells as a TAA/tumor cell model. Taken together, these results open up a new approach to the development of immunomodulatory antigen delivery systems for vaccine adjuvants and cancer immunotherapy technologies.

In Vivo Fates of Degradable Poly(β-Malic Acid) and of its Precursor, Malic Acid

To determine whether degradation could influence the in vivo elimination pattern of poly(β-malic acid) in mice, radioactive urinary excretion and 14CO2 expiration were studied after intravenous injection of 14C-radiolabeled poly(β-malic acid) and of its precursor, 14C-malate. The precursor administration led to rapid 14CO2 exhalation, and only negligible urinary elimination. The reverse was observed for the polymer. It was concluded that: (i) the in vivo degradation of poly(β-malic acid) chains, if any during the 24-h period of the study, did not release detectable malate molecules, (ii) the large urinary excretion of poly(β-malic acid) was due to the molar masses being less than the renal filtration threshold, (iii) the degradation of the poly(β-malic acid) chains in blood was slow enough to allow the fraction with higher molar masses to enter the interstitial space of the tissues, and possibly cells.

Solubilization of Water-Insoluble Drugs Due to Random Amphiphilic and Degradable Poly(dimethylmalic acid) Derivatives

Amphotericin B (AmB) and clofazimine are potent drugs hindered by their low water solubilities and their toxicities. Carriers able to increase their apparent water solubilities are needed for these drugs and for other molecules with similar properties. Random amphiphilic copolymers derived from poly(dimethylmalic acid) were obtained using different hydrophobization ratios and side group sizes. Apparent water solubilities of pyrene, clofazimine, and AmB were increased up to 10 000, 20 000 and 1000 times, respectively, in aqueous solutions containing these polymers. The presence of sodium chloride in polymer solution increased pyrene solubility but decreased the solubilities of clofazimine and AmB, compared to the salt-free solutions. Synergy between hydrophobic and electrostatic interactions was observed for polar and cationic molecules. Degradation studies showed that the examined polymers were degradable, but none of them were totally degraded in 28 days. These polymers could be used as a new tool for drug solubilization.