Hydrophobic Modification of Poly(γ-glutamic acid) by Grafting 4-Phenyl-butyl Side Groups for the Encapsulation and Release of Doxorubicin.

The delivery of drugs is a great challenge, since most of active pharmaceutical ingredients developed today are hydrophobic and poorly water soluble. From this perspective, drug encapsulation on biodegradable and biocompatible polymers can surpass this problem. Poly(γ-glutamic acid) (PGGA), a bioedible and biocompatible polymer has been chosen for this purpose. Carboxylic side groups of PGGA have been partially esterified with 4-phenyl-butyl bromide, producing a series of aliphatic-aromatic ester derivatives with different hydrophilic-lipophilic balances. Using nanoprecipitation or emulsion/evaporation methods, these copolymers were self-assembled in a water solution, forming nanoparticles with average diameters between 89 and 374 nm and zeta potential values between -13.1 and -49.5 mV. The hydrophobic core containing 4-phenyl-butyl side groups was used for the encapsulation of an anticancer drug, such as Doxorubicin (DOX). The highest encapsulation efficiency was reached for a copolymer derived from PGGA, with a 46 mol% degree of esterification. Drug release studies carried out for 5 days at different pHs (4.2 and 7.4) indicated that DOX was released faster at pH 4.2, revealing the potential of these nanoparticles as chemotherapy agents.

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.

Modification of Microbial Polymalic Acid With Hydrophobic Amino Acids for Drug-Releasing Nanoparticles.

Microbial poly(ß, l-malic acid) was modified with either l-leucine ethyl ester (L) or l-phenylalanine methyl ester (F) to produce amphiphylic copolymers. The degradation of these copolymers in aqueous buffer took place under physiological conditions in a few weeks by hydrolysis of the side chain ester group followed by cleavage of the main chain. Spherical nanoparticles with diameters ranging between 70 and 230 nm were prepared from these copolymers by the dialysis-precipitation method. No alteration of the cell viability was observed after incubation of these nanoparticles in different cell lines. Anticancer drugs temozolomide and doxorubicin were encapsulated in the nanoparticles. Temozolomide was released within several hours whereas doxorubicin took several weeks to be completely liberated.

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(ß,L-Malic Acid) Methyl Esters for Tumor Drug Delivery.

New copolyesters derived from poly(ß,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(ß,L-malic acid) copolyesters are suitable as nanoconjugate platforms for drug delivery.

Nanoparticles made of microbial poly(gamma-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(gamma-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 alpha-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(beta-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(beta,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.

Ionic complexes of biosynthetic poly(malic acid) and poly(glutamic acid) as prospective drug-delivery systems.

The hydrolytic degradability and erythromycin release from stoichiometric ionic complexes of biotechnological poly(beta,L-malic acid)s and poly(gamma,D-glutamic acid)s with alkyltrimethylammonium surfactants were investigated. The influence of pH, temperature and antibiotic load on hydrolysis rate was examined. It was found that poly(malic acid) complexes degraded by a surface erosion mechanism at a higher rate than poly(glutamic acid) complexes, which eroded in bulk. Erythromycin was lodged in the paraffinic subphase of the complexes and upon aging it was delivered according to a sigmoidal profile that appeared to be independent on the antibiotic load.

Thermal decomposition of fungal poly(beta,L-malic acid) and Poly(beta,L-malate)s.

The thermal decomposition of poly(beta,l-malic acid), poly(alpha-methyl beta,l-malate), and ionic complexes of the polyacid with alkyltrimethylammonium salts was studied by TGA, GPC, and FTIR and NMR spectroscopy. It was found that poly(beta,l-malic acid) depolymerized above 200 degrees C by an unzipping mechanism with generation of fumaric acid which is then partially converted in a mixture of maleic acid and anhydride. On the contrary, random scission of the main chain was found to happen in the thermal decomposition of poly(alpha-methyl beta,l-malate). On the other hand, ionic poly(beta,l-malate)s degraded through a well defined three-stage process, the first one being depolymerization of the poly(malate) main chain along with decomposition of the ionic complex. Decomposition of the previously generated alkyltrimethylammonium salts followed by unspecific cracking of the resulting nitrogenated compounds happened at higher temperatures. Mechanisms partially explaining the decomposition processes of the three studied systems were proposed according to collected data.

Nanostructurated complexes of poly(beta,L-malate) and cationic surfactants: synthesis, characterization and structural aspects.

Ionic complexes of microbially produced poly(beta,L-malic acid) and alkyltrimethylammonium surfactants with linear alkyl chains containing even numbers of carbon atoms from 14 up to 22, were investigated. Complexes with a stoichiometric or nearly stoichiometric composition were prepared by precipitation from equimolar mixtures of aqueous solutions of the two components. All complexes were found to adopt supramolecular stratified structures made of alternating layers of poly(beta,L-malate) and surfactant with a periodicity on the length scale of 3-5 nm, which increased proportionally to the length of the polymethylene chain. In these complexes, alkyl side chains with more than 16 carbon atoms were partially crystallized showing reversible melting at temperatures between 40 and 70 degrees C. After melting, a smectic LC phase that isotropicized at approximately 100 degrees C was observed for all of the complexes. Conformational and dimensional changes taking place in the complexes by effect of heating were analyzed by (13)C CP-MAS NMR and powder X-ray diffraction.

Comblike complexes of bacterial poly(gamma,d-glutamic acid) and cationic surfactants.

The ability of microbally produced poly(gamma,d-glutamic acid) to form stable polyelectrolyte-opposite charged surfactant complexes was investigated. A sonicated sample of polyacid with a molecular weight about 10(5) Da and a content of d enantiomer higher than 90% was used in this study. Nearly stoichiometric complexes of poly(gamma,d-glutamate) anions and alkyltrimethylammonium cations bearing linear alkyl chains with even numbers of carbon atoms from 12 up to 22 were "synthesized" by precipitation from equimolar mixtures of aqueous solutions of the two components. All complexes were found to adopt stratified supramolecular structures made of alternating layers of poly(gamma,d-glutamate) and surfactant with a periodicity increasing from 3.2 up to 4.3 nm according to the length of the alkyl side chain. No definite evidence indicative of the conformation adopted by the main chain in these complexes could be afforded. In all cases, the alkyl chains are in an extended conformation and oriented normal or nearly normal to the layer planes. Polymethylene chains with more than 16 carbon atoms were partially crystallized in the complexes in a separated paraffinic phase, whereas no crystallinity was detected for shorter lengths. The crystallized paraffinic phases were found to melt reversibly at temperatures between 40 and 70 degrees C. This process was found to happen with a concomitant expansion-contraction that amounts between 2 and 8% of the long period of the structure but without significant alteration of the layered arrangement.