Matrix-assisted laser desorption/ionization time-of-flight vs. fast-atom bombardment and electrospray ionization mass spectrometry in the structural characterization of bacterial poly(3-hydroxyalkanoates).

RATIONALE: Bacterial poly(3-hydroxyalkanoates) (PHAs) are an emergent class of plastic materials available from renewable resources. Their properties are strictly correlated with the comonomeric composition and sequence, which may be determined by various mass spectrometry approaches. In this paper we compare fast-atom bombardment (FAB) and electrospray ionization (ESI) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of partially pyrolyzed samples. METHODS: We determined the compositions and sequences of the medium-chain-length PHAs (mcl-PHAs) prepared by bacterial fermentation of Pseudomonas aeruginosa ATCC 27853 cultured in media containing fatty acids with 8, 12, 14, 18, and 20 carbon atoms as carbon sources by means of MALDI-TOFMS of pyrolyzates, and compared the results with those obtained by FAB- and ESI-MS in previous studies. MALDI matrices used were 9-aminoacridine (9-AA) and indoleacrylic acid (IAA). RESULTS: MALDI-TOFMS was carried out in negative ion mode when using 9-AA as a matrix, giving a semi-quantitative estimation of the 3-hydroxyacids constituting the PHAs, and in positive mode when using IAA, allowing us, through statistical analysis of the relative intensity of the oligomers generated by pyrolysis, to establish that the polymers obtained are true random copolyesters and not a mixture of homopolymers or copolymers. CONCLUSIONS: MALDI-TOFMS in 9-AA and IAA of partial pyrolyzates of mcl-PHAs represents a powerful method for the structural analysis of these materials. In comparison with FAB and ESI, MALDI provided an extended mass range with better sensitivity at higher mass and a faster method of analysis.

Synthesis and characterization of poly(3-hydroxyalkanoates) from Brassica carinata oil with high content of erucic acid and from very long chain fatty acids.

Pseudomonas aeruginosa produced medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) when grown on substrates containing very long chain fatty acids (VLCFA, C>20). Looking for low cost carbon sources, we tested Brassica carinata oil (erucic acid content 35-48%) as an intact triglyceride containing VLCFA. Oleic (C18:1), erucic (C22:1), and nervonic (C24:1) acids were also employed for mcl-PHA production as model substrates. The polymers obtained were analyzed by GC of methanolyzed samples, GPC, 1H and 13C NMR, ESI MS of partially pyrolyzed samples, and DSC. The repeating units of such polymers were saturated and unsaturated, with a higher content of the latter in the case of the PHA obtained from B. carinata oil. Statistical analysis of the ion intensity in the ESI mass spectra showed that the PHAs from pure fatty acids are random copolymers, while the PHA from B. carinata oil is either a pure polymer or a mixture of polymers. Weight-average molecular weight varied from ca. 56,000 g/mol for the PHA from B. carinata oil and oleic acid, to about 120,000 g/mol for those from erucic and nervonic acids. The PHAs from erucic and nervonic acids were partially crystalline, with rubbery characteristics and a melting point (Tm) of 50°C, while the PHAs from oleic acid and from B. carinata oil afforded totally amorphous materials, with glass transition temperatures (Tg) of -52°C and -47°C, respectively.

Poly(3-hydroxybutyrate-co-epsilon-caprolactone) copolymers and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-epsilon-caprolactone) terpolymers as novel materials for colloidal drug delivery systems.

Poly(3-hydroxybutyrate-co-epsilon-caprolactone) copolymers and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-epsilon-caprolactone) terpolymers were used by a solvent deposition technique to prepare either micro- or nanoparticles. In particular, the synthesis and analytical characterization of the terpolymers were described.On the basis of copolymer composition and properties, either micro- or nanoparticles were obtained; nanoparticle size was below 500 nm for the suspensions obtained from P(HB-co-CL) copolymers, and even smaller (200-300 nm) for those obtained using terpolymers. Particle size showed only a limited tendency to increase during storage, suggesting a good chemical and physical stability in the short-term storage at room temperature. Some copolymers produced hetero-dispersed microparticles under the same conditions, with a mean size between 10 and 30 microm. These systems showed a tendency to aggregate upon storage at room temperature.The nanoparticles showed a negative surface charge (around -20 mV for those prepared using an UltraTurrax and about -5 mV for those prepared by magnetic stirring). After storage at 4 degrees C the surface charge tend to decrease and these changes have been explained in terms of a partial hydrolysis of the polymeric matrix in aqueous suspension, which led to a change of chemical composition at the surface of the particles.The fluorescent probes calcein and Oil Red O were encapsulated in these systems as models of a hydrophilic and lipophilic drug molecule, respectively. Encapsulation efficiency and in vitro release profiles were studied to evaluate the effect of copolymer properties, such as molecular weight and composition, on their behaviour as potential materials to prepare controlled drug delivery carriers. Calcein was generally better encapsulated (up to 100%) than Oil Red O (10-30%); however, the zeta-potential measurement and in vitro release experiments suggested that a large amount of calcein was adsorbed onto the particle surface and was rapidly released within the first minutes of the test. Conversely, the lipophilic probe was dispersed within the polymeric matrix and its release profile from the nanoparticles was characterized by a considerable lag time (up to 8 h), followed by a slow and almost linear release.As a general trend, we observed that the composition and crystallinity of the tested polymers affected the type and size of obtained systems (micro- or nanoparticles), whereas the molecular weight mainly influenced the probes encapsulation and release.