Montserrat García-Alvarez

Nanoparticles Made of Microbial Poly(γ-glutamate)s for Encapsulation and Delivery of Drugs and Proteins

This study focused on the preparation and evaluation of nanoparticles made of alkyl esters of microbial poly(γ-glutamic acid) (PGGA) to be used as drugs and proteins carrier and delivery systems. Racemic PGGA of bacterial origin was fully methylated or partially esterified to render non-water-soluble polymers. A set of co-polymers containing poly(glutamic acid) and ethyl, hexyl, dodecyl and octadecyl glutamate units with alkyl contents of 50 and 75% was prepared. Spherical nanoparticles with a diameter of 200–250 nm and a narrow distribution were generated from the alkylated polymers by the precipitation-dialysis method. These nanoparticles readily degraded hydrolytically upon incubation in simulated physiological medium at a rate dependent on the alkylation degree and the length of the alkyl group. All these nanoparticles were able to encapsulate efficiently erythromycin. Those made of carboxyl containing polyglutamates were also effective to load α-chymotrypsin. The release of such compounds from nanoparticles upon incubation proceeded essentially following the same profile that is followed in the hydrolysis of the corresponding substrate polymers. The loss of enzyme activity of the incubated protein diminished significantly upon encapsulation in these systems.

Biodegradable Nanoparticles of Partially Methylated Fungal Poly(β-L-malic acid) as a Novel Protein Delivery Carrier

The preparation of nanoparticles from 75% methylated poly(beta-L-malic acid) is described. Their degradation in aqueous environments was examined and the influence of pH and lipase on the rate of hydrolysis was evaluated. Six proteins were used to estimate the loading efficiency of the nanoparticles. The amount of protein retained in the nanoparticles was found to depend on the acid/basic character of the protein. Protein release from the loaded nanoparticles upon incubation in water under physiological conditions encompassed polymer hydrolysis and happened steadily within 3-10 d. The activity loss of entrapped alpha-chymotrypsin caused by loading and releasing depended on the method used for loading.

Synthesis, Degradability, and Drug Releasing Properties of Methyl Esters of Fungal Poly(β,L-malic acid)

Methyl esters of microbial poly(beta,L-malic acid) for conversion degrees of 25, 50, 75, and 100% were prepared by treatment of the polyacid with diazomethane. Esterification proceeded with retention of the molecular weight of the parent polyacid and the copolymers displayed a blocky microstructure consisting of short segments of malic and methyl malate sequences. The thermal stability of the copolyesters was lower than those of the parent homopolymers and all of them were fairly crystalline with melting temperatures within the range of 170-175 degrees C. They were degraded rapidly by water, the hydrolysis rate being highly dependent on the methylation degree. Microspheres with mean-average diameters in the range of 1-20 microm were prepared from the 100% methylated product by the emulsion-evaporation solvent method. Encapsulation of erythromycin was efficiently performed in these microparticles and its releasing upon incubation in simulated physiological medium was evaluated for different drug loads. Drug delivery was observed to occur by a releasing mechanism largely determined by the hydrodegradation of the host polymer and independent of the amount of loaded drug.

Poly(methyl malate) Nanoparticles: Formation, Degradation, and Encapsulation of Anticancer Drugs

PMLA nanoparticles with diameters of 150-250 nm are prepared, and their hydrolytic degradation is studied under physiological conditions. Degradation occurs by hydrolysis of the side chain methyl ester followed by cleavage of the main-chain ester group with methanol and L-malic acid as the final degradation products. No alteration of the cell viability is found after 1 h of incubation, but toxicity increases significantly after 3 d, probably due to the noxious effect of the released methanol. Anticancer drugs temozolomide and doxorubicin are encapsulated in the NPs with 20-40% efficiency, and their release is monitored using in vitro essays. Temozolomide is fully liberated within several hours, whereas doxorubicin is steadily released from the particles over a period of 1 month.

Nanoconjugate platforms development based in poly(β, L-malic acid) methyl esters for tumor drug delivery

New copolyesters derived from poly(β,L-malic acid) have been designed to serve as nanoconjugate platforms in drug delivery. 25% and 50% methylated derivatives (coPMLA-Me(25)H(75) and coPMLA-Me(50)H(50)) with absolute molecular weights of 32 600 Da and 33 100 Da, hydrodynamic diameters of 3.0 nm and 5.2 nm and zeta potential of -15mV and -8.25mV, respectively, were found to destabilize membranes of liposomes at pH 5.0 and pH 7.5 at concentrations above 0.05mg/mL. The copolymers were soluble in PBS (half life of 40 hours) and in human plasma (half life of 15 hours) but they showed tendency to aggregate at high levels of methylation. Fluorescence-labeled copolymers were internalized into MDA-MB-231 breast cancer cells with increased efficiency for the higher methylated copolymer. Viability of cultured brain and breast cancer cell lines indicated moderate toxicity that increased with methylation. The conclusion of the present work is that partially methylated poly(β,L-malic acid) copolyesters are suitable as nanoconjugate platforms for drug delivery.

On the Phase B of Comb‐Like Poly(α‐alkyl‐β‐L‐aspartate)s: A Simulation of the Solubility of Small Penetrants

The structure and thermal properties of the phase B of poly(α-alkyl-β-L-aspartate)s abbreviated PAALA-n (n being the number of carbon atoms in the linear alkyl side chain) with n = 14 and 16 were determined using X-ray diffraction and DSC methods, respectively. These data together with those previously reported by us for the dodecyl deriviative were used to perform Monte Carlo simulations of these comb-like polymers. The results allowed to predict the solubility of different gases in the phase B of PAALA-n. The variation of both the unoccupid space and the excess chemical potential with the size of the penetrant were computed. An attractive interaction between the polymer matrix and the penetrants was founs when small gases were considered. The results have been compared with those recently obtained for crystalline poly(α-alkyl-β-L-aspartate)s bearing short linear alkyl side chains.

Theoretical study of the conformational and electrostatic properties of C4-monosubstituted 2-azetidinones

The conformational preferences and electrostatic properties of 2-azetidinone, 4-(S)-methoxycarbonyl-2-azetidinone and 4-(R)-methyl-2-azetidinone have been studied in gas-phase, aqueous solution and CCl4 solution using quantum mechanical methods. Gas-phase calculations were performed at the ab initio HF, MP2, and MP4 levels and solvent effects were investigated using a self-consistent reaction-field procedure adapted to the AM1 Hamiltonian. An almost planar arrangement was adopted by theβ-lactam ring in the three cases, whereas the alkoxycarbonyl side group was found to display a large conformational flexibility. The effects of the different solvents on the electrostatic properties of the three compounds were investigated by following the changes in both molecular electrostatic potentials and induced dipole moments. The resulting electrostatic parameters were used as static reactivity indices to predict the response of the systems to the attack of nucleophilic reagents. Theoretical results were compared with experimental data available on the structure and properties ofβ-lactams. The validity of the method as a predicting tool was critically discussed.

Ultrasonic degradation of polyaspartates and polyglutamates

In both cases molecular weights of severalof unraveling the mechanism responsible for chain hundreds of thousands daltons or even above the mil-breaking, the value of the method for reducing the mo- lion are typically obtained. Such high molecular sizeslecular weight of polymers without increasing poli- represent a serious inconvenience when these polymersdispersity is of prime practical importance. are handled for processing. In such a case moderateThis work is concerned with the ultrasonic degrada- molecular weights samples are highly desired. Becausetion of a certain group of polyamides that have as dis- amide bonds in the main chain are more reluctant tohydrolysis than ester side groups, cleavage of thesetinctive features to be stereoregularly substituted atpolyamides is unfeasible bychemical means. Ultrasonicthe backbone carbon adjacent to the NH and to containirradiation appears therefore to be an attractivea high density of amide groups in the main chain. Themethod to reduce the molecular weight of these polyam-selected polymers are: (a) the polypeptide poly(g-ben-ides without altering their chemical constitution.zyl-a-

Nanoparticles of Esterified Polymalic Acid for Controlled Anticancer Drug Release

Esterification of microbial poly(malic acid) is performed with either ethanol or 1-butanol to obtain polymalate conjugates capable to form nanoparticles (100-350 nm). Degradation under physiological conditions takes place with release of malic acid and the corresponding alcohol as unique degradation products. The anticancer drugs Temozolomide and Doxorubicin are encapsulated in nanoparticles with efficiency of 17 and 37%, respectively. In vitro drug release assays show that Temozolomide is almost completely discharged in a few hours whereas Doxorubicin is steadily released along several days. Drug-loaded nano-particles show remarkable effectiveness against cancer cells. Partially ethylated poly(malic acid) nano-particles are those showing the highest cellular uptake.

Abstract 3686: Engineering nanoparticles of polymalic acid for controlled delivery of anticancer drugs

Objective: In this work we report new esters obtained by partial or total esterification of Poly(β,L-malic acid) of microbial origin (PMLA) with ethanol or 1-butanol, and the nanoparticles made from them, are explored as drug delivery systems for the anticancer drugs temozolomide (TMZ) and doxorubicin (DOX). Experimental procedures: Esterification of PMLA with either ethanol or 1-butanol was performed with the polyacid dissolved in the corresponding alcohol and using controlled amounts of DCC for activation of the carboxylic side group. The conversion degree and purity was ascertained by 1H NMR. Two methods were employed for the formation of nanoparticles depending on the esterification degree of the polymer. For 100% modified polymers, the emulsion-solvent evaporation method and the precipitation-dialysis for partially esterified PMLA. Particle morphology was monitored by SEM and their sizes were determined by DLS. Primary glioma cell line U87MG and invasive breast carcinoma cell line MDA-MB468 (ATCC, USA) were exposed to drug loaded and unloaded nanoparticles and cell viability and uptake was measured using the MTT method and fluorescent microscopy respectively. Results: Ethyl and butyl PMLA esters with esterification degrees of approximately 50% and fully esterified were obtained. Reaction yields were around 50-70% with higher values attained in the esterification with ethanol. Molecular weights were: 33 KDa for coPMLA-Et50H50; 36 KDa for PMLA-Et100; 34 kDa for coPMLA-Bu50H50 and 53kDa for PMLA-Bu100. Nanoparticles diameters oscillated from 100 to 350 nm, depending on the polymer and on the methodology used for particle formation. DOX and TMZ can be encapsulated in these nanoparticles with efficiencies ranging 20 to 40% w/w. Drug-unloaded nanoparticles were not cytotoxic for the tested cell lines, whereas drug loaded nanoparticles were effective showing significant lower EC50 values when compared with free drug. The most remarkable case was observed for MDA-MB468 cell line treated with free TMZ and TMZ-NPs, in which free TMZ was shown to be ineffective at all concentrations while TMZ loaded nanoparticles reached EC50 at concentrations between 1-2×10-4 M. Two reasons can be invoked to explain such differences. A more sustained presence of encapsulated TMZ in the media and/or the nanoparticle internalization by cells that allows TMZ to be released directly in the cytosol. The most efficient polymer nanoparticles were coPMLA-Et50H50 which showed better internalization of DOX by cells than the free drug. Conclusions: Esterification of microbial poly(malic acid) with ethanol or 1-butanol rendered easily hydrolysable polyesters and copolyesters, suitable for building nanoparticles useful for anticancer drug encapsulation and controlled delivery. Citation Format: Jose Antonio Portilla-Arias, Alberto Lanz-Landazuri, Montserrat Garcia-Alvarez, Antxon Martinez de Ilarduia, Rameshwar Patil, Eggehard Holler, Julia Ljubimova, Sebastian Munoz-Guerra. Engineering nanoparticles of polymalic acid for controlled delivery of anticancer drugs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3686. doi:10.1158/1538-7445.AM2015-3686