Proteins conjugated to poly(butyl cyanoacrylate) nanoparticles as potential neuroprotective agents.

Poly(butyl cyanoacrylate) (PBCA) nanoparticles (NPs) can penetrate blood-brain barrier providing the means for drug delivery to the central nervous system. Here, we study attachment of superoxide dismutase (SOD) and anti-glutamate N-methyl D-aspartate receptor 1 (NR1) antibody to PBCA NPs with the ultimate goal to design neuroprotective therapeutics for treatment of secondary spinal cord injury. Synthesis of monodispersed, ∼200 nm-diameter PBCA NPs was performed using polymerization at pH 2.0 with Dextran 70,000 as the stabilizer. Sulfo-HSAB spacers were used to covalently attach SOD and NR1 antibodies to the dextran-coated NPs. The prepared protein-NP conjugates possessed SOD activity and were capable of binding to rat cerebellar neurons. Thus, SOD and NR1 antibodies may be simultaneously attached to PBCA NPs while retaining at least a fraction of enzymatic activity and receptor-binding ability. The conjugates showed neuroprotective efficacy in vitro with rat cerebellar cell cultures challenged by superoxide.

A systematic characterization of the factors influencing polymerization and dynamic behavior of n-butyl cyanoacrylate.

INTRODUCTION: Brain arteriovenous malformations are abnormal connections between arteries and veins without an intervening capillary bed. Endovascular glue embolization with N-butyl cyanoacrylate (NBCA) is an accepted form of treatment. The reported complication rates vary widely from 2% to 15%, and timing of polymerization appears to play a major role. Additionally, the interaction between NBCA and vessel surface as well as the presence of biological catalysts are poorly understood. METHODS: Polymerization time was measured for mixtures of Lipiodol/NBCA of 50/50, 70/30, and 60/40. The influence of pH, temperature, and the presence of biological catalysts on polymerization time was investigated. Contact angles were measured on polyvinyl alcohol cryogel (PVA-C), silicone, and endothelial surfaces in a submerged aqueous environment to assess physical surface interactions. High speed video analysis of glue injection through a microcatheter was performed to characterize simulated coaxial flow. RESULTS: NBCA polymerization rate increased with pH and temperature. A hydrophilic surface such as PVA-C was better than silicone at mimicking the physical properties of endothelium. Live endothelium provided a catalytic surface that at least doubled the rate of polymerization. Blood products further increased the polymerization rate in the following order (slowest to fastest): plasma, platelets, red blood cells (RBCs), and lysed RBCs. These factors could explain the discrepancy between in vitro and in vivo results reported in the current literature. High speed video analysis of NBCA injection showed dripping to jetting transition with significant wall effect which deviated from previous ideal assumptions. CONCLUSIONS: The determinants of NBCA polymerization rate are multifactorial and dependent mainly on the presence of biological catalysts coupled with flow related wall interaction.

Enhanced antitumoral activity of doxorubicin against lung cancer cells using biodegradable poly(butylcyanoacrylate) nanoparticles.

Doxorubicin (Dox) is widely used for the combined chemotherapy of solid tumors. However, the use of these drug associations in lung cancer has low antitumor efficacy. To improve its efficacious delivery and activity in lung adenocarcinoma cells, we developed a biodegradable and noncytotoxic nanoplatform based on biodegradable poly(butylcyanoacrylate) (PBCA). The reproducible formulation method was based on an anionic polymerization process of the PBCA monomer, with the antitumor drug being entrapped within the nanoparticle (NP) matrix during its formation. Improved drug-entrapment efficiencies and sustained (biphasic) drug-release properties were made possible by taking advantage of the synthesis conditions (drug, monomer, and surfactant-agent concentrations). Dox-loaded NPs significantly enhanced cellular uptake of the drug in the A549 and LL/2 lung cancer cell lines, leading to a significant improvement of the drug's antitumoral activity. In vivo studies demonstrated that Dox-loaded NPs clearly reduced tumor volumes and increased mouse-survival rates compared to the free drug. These results demonstrated that PBCA NPs may be used to optimize the antitumor activity of Dox, thus exhibiting a potential application in chemotherapy against lung adenocarcinoma.

Study of the effect of polybutylcyanoacrylate nanoparticles and their metabolites on the primary immune response in mice to sheep red blood cells.

Polybutylcyanoacrylate nanoparticles (PBCN) and their metabolites (polycyanoacrylic acid--PCAA, and n-butanol) were compared with respect to their effects on the primary immune response of mice to sheep red blood cells (SRBC). PCAA was synthetized via a Knoevenagel reaction. Antibody production (hemagglutinins) and E-rosette-forming cells (E-RFC) were used to document the induction of antigen-specific immune response to SRBC in all immunized mice. PBCN showed a time- and dose-dependent effect on the immune response, both humoral and cellular. The inductive phase of immune response was affected preferably. The high dose of PBCN (200 mg kg(-1)) tended to suppress the immune response. This was expressed more in mice treated before antigenic stimulation. Lower dose (10 mgkg(-1)) stimulated the immune response. A significant difference was found in the effects of PBCN and their metabolites on the immune response. PCAA and n-butanol administered at doses equivalent (after lysosomal hydrolysis) to the doses applied of intact PBCN did not impair significantly the immune response. A clear time dependence and dose dependence were not observed. The study led to the hypothesis that the greater suppressive effect of PBCN, relative to either PCAA or n-butanol, or a mixture of them, is probably due to the blocking of the immunopresenting function of macrophages instead of some toxicity towards the immunocompetent cells.

[Forms of an in vivo polymerization product, n-butyl-2-cyanoacrylate, within the gastric wall].

The in vivo morphology of sclerosants, n-butyl-2-cyanoacrylate (Histoacryl; referred hereinafter to briefly as HA) polymer formed within the gastric wall was investigated in an animal experiment. The results indicate that HA polymer formed after local injection of HA into the stomach wall with deliberate avoidance of contact with blood got scattered in an arboroid pattern, suggesting thus that when HA is injected into the gastric mucosa at an extravascular site, the resulting polymer is likely to get scattered and miss the target site. Polymerization products that were formed after injection of HA alone were deep purple, dense, firm, rounded in shape and lesser in adverse histologic influence upon the walls of stomach. On the other hand, those formed by injection of HA with Lipiodol were large-sized, light brown and oval-shaped ones with more irregular margins and less distinct boundaries as compared to those seen after injection of HA alone. These results of the study led us to conclude that at the present time it would seem wise to limit the use of HA for this particular therapeutic purpose only to those cases of bleeding gastric varices.

Transcytosis of CRM197-grafted polybutylcyanoacrylate nanoparticles for delivering zidovudine across human brain-microvascular endothelial cells.

This study investigates the capability of CRM197-grafted polybutylcyanoacrylate (PBCA) nanoparticles (NPs) (CRM197/PBCA NPs) to carry zidovudine (AZT) across the blood-brain barrier (BBB). AZT was loaded on CRM197/PBCA NPs to traverse the monolayer of human brain-microvascular endothelial cells (HBMECs) regulated by human astrocytes. The particle size distribution of AZT-loaded CRM197/PBCA NPs was quite uniform. In addition, AZT-loaded CRM197/PBCA NPs displayed a spherical shape with slightly fluffy exterior. The deposited thin film of AZT-loaded CRM197/PBCA NPs exhibited a hexagonal lattice-like geometry. When the diameter of AZT-loaded CRM197/PBCA NPs decreased, the loading efficiency of AZT on the drug carriers and the permeability coefficient of AZT across the BBB enhanced. An increase in the grafting quantity of CRM197 enhanced the permeability coefficient of AZT across the BBB and the uptake quantity of AZT-loaded CRM197/PBCA NPs by HBMECs. CRM197/PBCA NPs can be promising brain-targeting carriers for delivering AZT across the BBB.

Studies on the transport pathway of PBCA nanoparticles in ocular tissues.

The transport pathway of PBCA nanoparticles through the rabbit cornea and conjunctiva was studied using fluorescence microscopy. Nanoparticles were produced by an emulsion-polymerization process, purified by a GPC procedure, and labelled with rhodamine 6G or propidium iodide as fluorescent laser dyes. The stability of the dye label, particle diameter, and zeta potential of the nanoparticles were determined. Freshly excised rabbit cornea and conjunctiva were incubated with a suspension of labelled nanoparticles for about 30 min in standard perfusion cells. After incubation the particles were visualized, due to their fluorescent character, using laser scanning confocal microscopy. The results show a fluorescence signal inside the cells. In particular conjunctival cells showed an uptake of nanoparticles. Fluorescent particles were visually observed inside the cells, in what appeared to be vesicles or granules. Thus, either endocytosis of the nanoparticles by conjunctival tissue or lysis of the cell wall by nanoparticle metabolic degradation products, are possible explanations of the data. A fluorescence signal was also observed within corneal cells. Only a transcellular pathway was observed. A possible penetration through tight junctions was not noticed; moreover, penetration was observed only into the first two cell layers and no full tissue penetration occurred.

Comparative study on the cytostatic effects and the tissue distribution of 5-fluorouracil in a free form and bound to polybutylcyanoacrylate nanoparticles in sarcoma 180-bearing mice.

The binding of 5-fluorouracil to polybutylcyanoacrylate nanoparticles yielded an enhanced efficacy against Crocker sarcoma S 180 and a higher toxicity of the drug measured by induced leukopenia, body weight loss and premature death. The efficacy was further increased by an increase in the polymer-to-drug ratio. The nanoparticles yielded a prolonged persistence of the 5-fluorouracil in all organs examined including the tumor. These particles hold promise as carriers for cytostatics, since their distribution may be favorably altered by coating them with certain proteins or surfactants or by the attachment of monoclonal antibodies.

Tissue reaction to Histoacryl Blue adhesive and histological investigation of its disappearance in various organs.

The effect of the adhesive Histoacryl Blue on the abdominal organs (liver, stomach, intestines, colon, kidneys, bladder, aorta inferior vena cava and skin wounds) of 15 dogs was investigated histologically. The adhesive was applied in thin or thick layers in various ways: applied on intact or injured surfaces, or introduced into the parenchymal organs. The animals were killed 1, 2, 4, 8 weeks and 3 and 6 months after application of the adhesive and the organs containing Histoacryl Blue were processed histologically. The success of adhesion depended, irrespective of the organ, upon the technique of application and on the quantity of the adhesive used.