Biodegradable polymers are used in many biomedical devices, such as implants, cellular scaffolds and drug delivery systems. To improve drug delivery capability and to reduce systemic toxicity, the interest in polymer-drug conjugates has increased remarkably. With this aim, pranoprofen (PPF) (an effective non-steroidal anti-inflammatory drug used mainly in ophthalmology) was conjugated to poly(epsilon-caprolactone) (PCL) via microwave-assisted transesterification, acid-catalyzed by p-toluenesulfonic acid. The products obtained were characterized by NMR, matrix assisted laser desorption ionization time of flight mass spectrometry, gel permeation chromatography (which gave proof of the covalent link between PPF and PCL) and thermal analyses. The conjugates were found to contain up to 4.3% w/w of PPF and had molecular weights ranging from 5 to 12 kDa. (c) 2020 Society of Industrial Chemistry

Osteoarthritis (OA) is a debilitating disease affecting joints and impairing the ability to perform everyday tasks. Current treatment regimens tend to provide little to no relief. Therefore, there is a huge need for alternative strategies to manage this painful condition. The delivery of anti-inflammatory drugs into an injured joint with the aim of eliminating articular inflammation and modulate cartilage damage could be a useful strategy to treat OA. Accordingly, the aim of this study is to prepare microparticles (MPs) from new biodegradable poly (3-hydroxybutyrate-co-ε-caprolactone) copolymers (PHBCL) for the potential intra-articular injection of Diclofenac sodium for OA treatment. MPs were prepared starting from copolymers having different molecular weights and an HB/CL molar ratio and studied for their morphologies and size distribution by scanning electron microscopy. Drug loading and encapsulation efficiency were also determined. The in vitro release profile was assessed by the dialysis bag technique and the kinetic profile was evaluated by using several mathematical models revealing a diffusion release mechanism. A1 polymer and related MPs, as representative of the group, were selected for further biological investigation. In vitro studies performed on CaCo-2 and Balb/3T3 cells showed no toxic effects at the desired concentrations as revealed by MTT, CFE and Comet assays.

A biodegradable poly(3-R-hydroxyalkanoate) synthesized by Pseudomonas mediterranea was investigated as a biomaterial to obtain colloidal drug delivery systems. Using a nanoprecipitation method, nanoparticles with a mean size of 155 nm and a negative surface charge were formed. They can be freeze-dried by adding hydroxypropyl-β-cyclodextrin as a cryoprotectant, and they have been shown to efficiently load both a hydrophilic (calcein) and a lipophilic (Nile red) model probe. Since this polymer contains terminal double bonds in the side chains, cross-linking conditions were tested. In particular, under the action of UV rays or irradiation with an incandescent yellow lamp, this polymer tended to cross-link.

ABSTRACTNew biodegradable biomaterials are attracting a huge interest as alternative to conventional polymers used in the field of drug delivery. In this work, we evaluated the ability of new biocompatible and biodegradable polyesters to form nanoparticles (NPs), and tested their potential carrier properties for controlled release of hydrophilic or lipophilic compounds. Multiblock copolymers derived from poly(R‐3‐hydroxybutyrate) and poly(1,4‐butylene adipate) by microwave‐assisted transesterification, having different chemical and physicochemical properties were tested. Nanoprecipitation was applied to obtain NPs with a homogenous size distribution. Oil Red O and calcein were encapsulated as lipophilic and hydrophilic probes, evaluating NP mean size and size polydispersity, surface charge, encapsulation efficiency, and release profile. The release curves were fitted into mathematical models to investigate the release mechanism. NPs stability appeared to be strictly related to storage conditions. The NPs were also successfully autoclaved and their mucoadhesive behavior was assessed by a “mucin‐particle method.” © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2019, 136, 47233.

Pseudomonas aeruginosa ATCC 27853 was cultured on media containing long odd-chain fatty acids. Heptadecanoic, nonadecanoic, and heneicosanoic acids sustained cell growth and resulted in polyhydroxyalkanoate (PHA) accumulation when culturing was conducted under nitrogen starvation conditions. No PHA was produced using a complete or magnesium-deprived medium. The isolated polyesters were characterized by gas chromatography and liquid chromatography-electrospray ionization mass spectrometry (ESI-MS) of methanolyzed samples, 1H and 13C NMR spectroscopy, gel permeation chromatography, ESI MS of partially pyrolyzed samples, and differential scanning calorimetry. These PHAs are composed of seven different odd-chain repeating units starting from 3-hydroxyvalerate, with the highest species being the, to date, unreported constituent 3-hydroxyheptadecanoate, and minor amounts of 2 or 3 even-chain comonomers. The PHAs are soft, sticky, rubber-like materials having glass transition temperatures between -45 and -39°C, melting temperatures between 48 and 52°C, enthalpies of melting around 11J/g, and molar masses ranging from 77 to 188kg/mol. Statistical analysis of the ESI mass spectra of the products of their partial pyrolysis showed that they are pure copolymers and not a blend of copolymers or homopolymers.

Pseudomonas strains produce rhamnolipid mixtures (RLs) that generally consist of one or two molecules of rhamnose linked to one or two molecules of 3-hydroxyalkanoic acid. This study evaluates carbon source effects (glycerol, glucose, myristic acid, and Brassica carinata oil) on the synthesis of monorhamnolipids (mono-RLs) versus dirhamnolipids (di-RLs) in a human isolate of Pseudomonas aeruginosa PAL05. Spectrophotometry, an emulsifying index (E24) test, and an orcinol assay confirmed the production of RLs by PAL05. Purified RLs were characterized by 1H NMR analysis. PAL05 primarily produces mono-RLs when provided carbon sources containing long chain fatty acids (FAs) (myristic acid and B. carinata oil) and di-RLs when provided glycerol or glucose. qRT-PCR analysis showed that delayed expression of rhlC occurred when B. carinata oil was used, but not glycerol, glucose, or myristic acid. Our data show that the carbon source influenced the transcriptional expression of the rhlC gene and, consequently, the predominance of mono-RLs or di-RLs in PAL05 cultures.

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.

Within a research directed to developing new polymeric materials, suitable for decorating the surface of colloidal drug carriers, PEG(5000) polymers containing a free carboxyl or amine group at one end were conjugated to an alpha-lipoamino moiety (LAA). The conjugates were characterized by FT-IR,H-1-NMR, and MALDI-TOF mass spectrometry. They showed the same profile of solubility as the parent PEGs in water and in some polar and apolar solvents of pharmaceutical use. Representative terms showed to be well tolerated when incubated with Caco-2 or L929 cell cultures.Dedicated differential scanning calorimetry (DSC) studies were performed to prove the interaction of increasing molar fractions of the PEG(5000)-LAA conjugates with dipalmitoylphosphatidylcholine (DPPC) bilayers, to gain information about their possible incorporation in drug nanocarriers. While the parent PEGs affected only the superficial structure of bilayers, the amphiphilic PEG-LAA conjugates induced a perturbing effect on the thermotropic behavior of DPPC liposomes, according to the structure of the linked LAA residue. A molar concentration of these PEG-LAA between 5 and 10% was individuated as the most suitable to produce stable vesicles. (C) 2014 Elsevier B.V. All rights reserved.

Copolymers containing (R)-3-hydroxybutyric acid (HB), 1,4-butanediol (B), and adipic acid (A) were synthesized by microwave-assisted transesterification of biodegradable poly(R-3-hydroxybutyrate) (PHB) and poly(1,4-butyleneadipate) (PBA) in solution at different reaction times, composition of the starting mixture, and amount of 4-toluenesulfonic acid, used as a catalyst. The copolyesters were characterized with regard to their molecular weights, thermal properties, molar composition, and average block length of repeating units by gel permeation chromatography (GPC), differential scanning calorimetry (DSC), wide angle X-ray diffraction (WAXS), 1H-NMR, and 13C-NMR, respectively. Random and microblock copolymers could be obtained depending on experimental conditions, with weight average molecular weight of up to 17,000. The glass transition temperature (Tg) of all samples lay in the range between the Tgs of PBA and PHB, while their structure varied from partially crystalline to totally amorphous. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectra of copolymers allowed us to ascertain that they were hydroxyl and carboxyl chain-end terminated.

Linking PEG(2,000) polymers ending in 1 or 2 carboxylic groups to lipoamino acids (LAAs) gives mono- and homo-disubstituted PEG-LAA conjugates. They show an identical solubility to parent PEGs in water and organic solvents. By DSC the degree and depth of interaction of these conjugates with a biomembrane model is studied, gaining information about their future incorporation in drug-loaded nanocarriers. The ability of PEG-LAA conjugates to adopt an ordinate arrangement on the surface of particles and efficiently cover them is demonstrated, compared to DSPE-PEG, by measuring the zeta potential values of negatively charged liposomes prepared in their presence.

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 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.

Copolymers of 3-hydroxybutyrate (3HB) and 3-mercaptopropionate (3MP) or 3-mercaptobutyrate (3MB) units and minor amounts of 3-hydroxypropionate (3HP), 3-hydroxyvalerate (3HV), or 3-mercaptovalerate (3MV) were investigated regarding their microstructure by NMR, electrospray ionization mass spectrometry, and size exclusion chromatography NMR. These copolymers were produced by Ralstonia eutropha strain H16 when cells were cultivated in a mineral salts medium with gluconate as a carbon source for growth and 3MP or 3MB as precursor substrates for incorporation of 3-mercaptoalkanoates. Mass spectrometry analysis of partially methanolyzed or pyrolyzed samples proved the presence of true copolymers or terpolymers. (13)C NMR spectroscopy of intact polymer samples, with values of average block length and degree of randomness deviating from a random sequence model, suggested microblock structures; however, composition analysis by (1)H NMR of fractions obtained by size exclusion chromatography showed significant variations with molecular weight, revealing the presence of blends of poly(3HB-co-3MP-co-3HP) or poly(3HB-co-3MB) with poly(3HB). The experimental NMR carbonyl dyad signal intensities were satisfactorily matched by a random sequence model when the presence of poly(3HB) was taken into account.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of narrowly dispersed molecular weight gel permeation chromatography (GPC) fractions was used to characterize random and microblock poly[(R)-3-hydroxybutyrate-co-epsilon-caprolactone] [P(HB-co-CL)] copolymers obtained via the acid-catalyzed transesterification of the corresponding homopolymers, poly(HRO-hydroxybutyrate] (PHB) and poly(e-caprolactone) (PCL). High-quality mass spectra were obtained, which made it possible to establish the nature of the polymer end groups. Besides the carboxylic termination, two other moieties were found: alcoholic and tosyl end groups. MALDI mass spectra of CL-rich samples possessed mostly tosyl end groups, while HB-rich samples possessed mostly alcoholic end groups, showing that the tosyl moiety is linked prevalently to CL terminal units. The higher resolution of electrospray ionization (ESI) mass spectra of lower molecular weight GPC fractions permitted the identification of the different oligomer species hypothesized in the assignment of the corresponding MALDI mass spectra. Partial methanolysis of these copolymers was explored as a method of producing mixed HB-CL oligomers to be utilized as new synthons, possessing a minor number of chiral centers from those obtained from hydrolysis of biotechnologically synthesized poly(hydroxyalkanoates) (PHAs). Copyright (c) 2006 John Wiley & Sons, Ltd.

Pseudomonas aeruginosa ATCC 27853 accumulated poly(3-hydroxyalkanoates) (PHAs) after growth on saturated fatty acids with an odd number of carbon atoms. No nutrient limitation was required to induce PHA synthesis, although better yields were obtained when the medium was magnesium deprived. A comparative study was carried out between PHAs obtained from C-odd and those from C-even carbon sources. Repeating units identification was performed by gas chromatography (GC) and capillary liquid chromatography-electrospray mass spectrometry (LC-ESI MS) of methanolyzed samples. When C-odd n-alkanoic acids from nonanoic to pentadecanoic were used the lowest hydroxyalkanoate unit found was 3-hydroxyvalerate and the highest 3-hydroxypentadecanoate, whereas when C-even acids from octanoic to eicosanoic were used these were 3-hydroxycaproate and 3-hydroxyeicosanoate, respectively. Weight average molecular weights were in the range 187 000-596 000. DSC traces showed T-m and DeltaH(m) which varied from 43 to 58 degreesC and from 5.9 to 24.8 J/g, with the PHAs generated from C-odd carbon sources having lower values. ESI MS of partially pyrolyzed samples allowed the identification of oligomers up to heptamers, and statistical analysis of the ions intensity in the mass spectra showed that these PHAs are random copolyesters.

Copolymers of (R)-3-hydroxybutyric acid (HB) and epsilon-caprolactone (CL) with a composition ranging from 28 to 81 mol % of HB were synthesized by transesterification of the corresponding homopolymers in solution in the presence of 4-toluenesulfonic acid. The copolyesters were characterized with regard to their molecular weights, thermal properties, molar compositions, and average block length of repeating units by gel permeation chromatography (GPC), differential scanning calorimetry, (1)H NMR, and (13)C NMR, respectively. Random and microblock copolymers could be obtained depending on experimental conditions, with weight-average molecular weights of up to 20,000. The glass transition temperature decreased from 2 to -42 degrees C as the CL content was increased from 0 to 72 mol %. The melting temperature (T(m)) of the PCL phase decreased from 70 to 46 degrees C as the HB content changed from 0 to 47 mol %, while the T(m) of the PHB phase decreased from 177 degrees C to 163 degrees C as the CL content changed from 0 to 72 mol %. Matrix-assisted laser desorption ionization time-of-flight mass spectra of GPC fractionated samples allowed us to ascertain that copolymers rich in HB units have mostly hydroxyl and carboxyl end groups, while copolymers rich in CL units have mostly tosyl and carboxyl end groups.