ABSTRACT
The present investigation confirms that initially implemented procedure to produce poly(methylidene malonate 2.1.2) (PMM 2.1.2) nanoparticles (Lescure et al. Pharm Res 1994;11:1270-77) lead to products mostly containing plasticizing oligomers which strongly lowered glass-transition temperature (Tg), dramatically reduced nanoparticle consistency and rendered them too sensitive to solubilization when diluted in an aqueous medium. From MALDI-TOF spectroscopy analysis, performed on intact colloids, emerged some structural information about these oligomeric species which could result from an intramolecular cyclization mechanism occurring soon in the course of the polymerization process. Thus, with the objective of overcoming these drawbacks, this contribution deals with the variations of manufacturing specifications such as pH and magnetic stirring speed to try and modulate molecular weight (MW) of nanoparticle constituents and reduce oligomer concentration. Although the analyses performed on these new nanoparticles were rather encouraging, the colloid formation yield became so low that it required the development of other methodologies, excluding a previous emulsion step, and allowing a controlled production of PMM 2.1.2-made nanoparticles having better physico-chemical characteristics while keeping good pharmaceutical capabilities.
Subject(s)
Biocompatible Materials/chemistry , Malonates/chemistry , Polyethylenes/chemistry , Biocompatible Materials/chemical synthesis , Calorimetry, Differential Scanning , Chemical Phenomena , Chemistry, Physical , Drug Carriers , Malonates/chemical synthesis , Molecular Weight , Particle Size , Polyethylenes/chemical synthesis , Solubility , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , TemperatureABSTRACT
A new method using surface plasmon resonance (SPR) through the BIAcore was used to demonstrate the specific interaction between an anti-CD4 monoclonal antibody (IOT4a), adsorbed on poly(methylidene malonate 2.1.2) (PMM 2.1.2) nanoparticles, and the CD4 molecule. The results obtained were compared with the interaction of the same immunonanoparticles with rabbit anti-mouse Fc antibodies. The molar ratio (Fc)/(Fab) was 1, suggesting that the same number of epitopes on the Fc and the Fab fragments were accessible after IOT4a adsorption onto nanoparticles. Comparing the observed association rates of free antibody and antibody adsorbed on nanoparticles, the number of molecules of IOT4a antibody on PMM 2.1.2 nanoparticles was estimated as between 2.6 and 3 per nanoparticle. The properties of the antibody-coated nanoparticles are compatible with their use as antibody-targeted pharmacophores.
Subject(s)
Antibodies, Monoclonal/immunology , CD4 Antigens/immunology , Drug Compounding , Malonates/metabolism , Polyethylenes/metabolism , Adsorption , Animals , Antigen-Antibody Reactions , Biosensing Techniques , CD4-Positive T-Lymphocytes/immunology , Drug Carriers , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fc Fragments/immunology , Kinetics , Mice , Microscopy, Electron, Scanning , Recombinant Proteins/immunologyABSTRACT
Interaction between CD4 cell surface protein and HIV-bearing gp120 has been described as the initial step for HIV entry into host cells. Some anti-CD4 antibodies were shown to inhibit this interaction. Biosensor studies using the BIAcore were performed to determine kinetic and thermodynamic parameters of the interaction of one of these antibodies (i.e. IOT4a, clone 13B8-2) with immobilized recombinant soluble CD4 (rsCD4). A non-linear regression method was used to analyze the sensorgrams, showing the existence of a double exponential time curve. A KA of 5.2 x 10(7) M-1 was calculated at 25 degrees C. The complex formation was exothermic (-4.5 kcal.mol-1( and entropically positive (+20 cal.mol-1.K-1). The reaction rate (0.234 x 10(5) M-1.s-1 at 25 degrees C) as well as the enthalpy change of the activated complex (+9.7 kcal.mol-1) are not compatible with a diffusion controlled reaction. The thermodynamic values calculated from equilibrium data corresponded to those calculated from kinetic data confirming the validity of the theoretical approach. As for most antigen-antibody interactions, complex formation was enthalpy driven. The overall positive entropy contribution to the stabilization of the complex is in contrast to that observed for the lysozyme-anti-lysozyme model and is probably due to electrostatic interaction between the epitope and the antibody combining site.
