Title: Exploring the Solid State Properties of Enzymatic Poly(amine-co-ester) Terpolymers to Expand their Applications in Gene Transfection Voevodina I, Scandola M, Zhang J, Jiang Z Ref: RSC Adv, 4:8953, 2014 : PubMed
Polymers bearing amino functional groups are an important class of materials capable of serving as non-viral carriers for DNA delivery to living cells. In this work biodegradable poly(amine-co-ester) terpolymers were synthesized via ring-opening and polycondensation copolymerization of lactone (sigma-caprolactone (CL), omega-dodecalactone, omega-pentadecalactone (PDL), and omega-hexadecalactone) with diethyl sebacate (DES) and N-methyldiethanolamine (MDEA) in diphenyl ether, catalyzed by Candida antarctica lipase B (CALB). All lactone-DES-MDEA terpolymers had random distributions of lactone, sebacate, MDEA repeat units in the polymer chains. PDL-DES-MDEA terpolymers were studied in the composition range from 21 mol% to 90 mol% PDL whereas the terpolymers with other lactones were investigated at a single composition (80 mol% lactone). DSC and WAXS analyses showed that all investigated terpolymers crystallize in their respective homopolylactone crystal lattice. Terpolymers with large lactones and a high lactone content melt well above room temperature and are hard solids, whereas terpolymers with small lactones (e.g. CL) or with a low lactone content melt below/around ambient temperature and are waxy/gluey materials. Given the importance of hydrophobicity in influencing gene delivery, water contact angle measurements were carried out on lactone-DES-MDEA terpolymers showing that it is possible to tune the hydrophilic-to-hydrophobic balance by varying polymer composition and size of lactone units. To demonstrate the feasibility of using solid terpolymers as nanocarriers for DNA delivery, PDL-DES-MDEA copolymers with 65-90% PDL were successfully transformed into free-standing nanoparticles with average particle size ranging from 163 to 175 nm. Our preliminary results showed that LucDNA-loaded nanoparticles of the terpolymer with 65% PDL were effective for luciferase gene transfection of HEK293 cells.
Title: Lipase-catalyzed copolymerization of omega-pentadecalactone with p-dioxanone and characterization of copolymer thermal and crystalline properties Jiang Z, Azim H, Gross RA, Focarete ML, Scandola M Ref: Biomacromolecules, 8:2262, 2007 : PubMed
Candida antarctica Lipase B (CALB), a metal-free enzyme, was successfully employed as catalyst for ring-opening copolymerization of omega-pentadecalactone (PDL) with p-dioxanone (DO) under mild reaction conditions (<80 degrees C, atmospheric pressure). Poly(PDL-co-DO) with high molecular weight (Mw > 30 000) and a wide range of comonomer contents was synthesized using various PDL/DO feed ratios. During the copolymerization reaction, large ring PDL was found to be more reactive than its smaller counterpart DO, resulting in higher PDL/DO unit ratios in polymer chains than the corresponding PDL/DO monomer ratios in the feed. The copolymers were typically isolated in 50-90 wt % yields as the monomer conversion was limited by the equilibrium between monomers and copolymer. 1H and 13C NMR analysis on poly(PDL-co-DO) formed by CALB showed that the copolymers contain nearly random sequences of PDL and DO units with a slight tendency toward alternating arrangements. Copolymerization with PDL was found to remarkably enhance PDO thermal stability. Differential scanning calorimetry (DSC) and wide-angle X-ray scattering (WAXS) results demonstrate high crystallinity in all copolymers over the whole range of compositions. Depending on copolymer composition, the crystal lattice of either PDO or PPDL hosts units of the other comonomer, a behavior typical of an isodimorphic system. In poly(PDL-co-DO), both melting temperature and melting enthalpy display a minimum at 70 mol % DO, that is, at the pseudoeutectic composition. WAXS diffractograms show one crystal phase (that of either PPDL or PDO) on either side of the pseudoeutectic and coexistence of PPDL and PDO crystals at the pseudoeutectic.
Title: Cocrystallization of random copolymers of omega-pentadecalactone and epsilon-caprolactone synthesized by lipase catalysis Ceccorulli G, Scandola M, Kumar A, Kalra B, Gross RA Ref: Biomacromolecules, 6:902, 2005 : PubMed
Random copolymers were prepared by Candida antarctica lipase B (Novozyme-435) catalyzed copolymerization of omega-pentadecalactone (PDL) with epsilon-caprolactone (CL). Over the whole composition range PDL-CL copolymers are highly crystalline (melting enthalpy by differential scanning calorimetry, above 100 J/g; crystallinity degree by wide-angle X-ray scattering, WAXS, 60-70%). The copolymers melt at temperatures that linearly decrease with composition from that of poly(omega-pentadecalactone) (PPDL; 97 degrees C) to that of poly(epsilon-caprolactone) (PCL; 59 degrees C). The WAXS profiles of PCL and PPDL homopolymers are very similar, except for the presence in PPDL of the (001) reflection at 2theta = 4.58 degrees that corresponds to a 19.3 angstroms periodicity in the chain direction. In PDL-CL copolymers the intensity of this reflection decreases with increasing content of CL units and vanishes at 50 mol % CL, as a result of randomization of the ester group alignment and loss of chain periodicity. PDL-CL copolymers crystallize in a lattice that gradually changes from that of one homopolymer to that of the other, owing to comonomer isomorphous substitution. Cocrystallization of comonomer units is also shown by a random PDL-CL copolymer obtained in a polymerization/transesterification reaction catalyzed by C. antarctica lipase B (Novozyme-435) starting from preformed PCL and PDL monomer.
A novel type of hydrolase was purified from culture fluid of Paucimonas (formerly Pseudomonas) lemoignei. Biochemical characterization revealed an unusual substrate specificity of the purified enzyme for amorphous poly((R)-3-hydroxyalkanoates) (PHA) such as native granules of natural poly((R)-3-hydroxybutyrate) (PHB) or poly((R)-3-hydroxyvalerate) (PHV), artificial cholate-coated granules of natural PHB or PHV, atactic poly((R,S)-3-hydroxybutyrate), and oligomers of (R)-3-hydroxybutyrate (3HB) with six or more 3HB units. The enzyme has the unique property to recognize the physical state of the polymeric substrate by discrimination between amorphous PHA (good substrate) and denatured, partially crystalline PHA (no substrate). The pentamers of 3HB or 3HV were identified as the main products of enzymatic hydrolysis of native PHB or PHV, respectively. No activity was found with any denatured PHA, oligomers of (R)-3HB with five or less 3HB units, poly(6-hydroxyhexanoate), substrates of lipases such as tributyrin or triolein, substrates for amidases/nitrilases, DNA, RNA, casein, N-alpha-benzoyl-l-arginine-4-nitranilide, or starch. The purified enzyme (M(r) 36,209) was remarkably stable and active at high temperature (60 degrees C), high pH (up to 12.0), low ionic strength (distilled water), and in solvents (e.g. n-propyl alcohol). The depolymerase contained no essential SH groups or essential disulfide bridges and was insensitive to high concentrations of ionic (SDS) and nonionic (Triton and Tween) detergents. Characterization of the cloned structural gene (phaZ7) and the DNA-deduced amino acid sequence revealed no homologies to any PHB depolymerase or any other sequence of data banks except for a short sequence related to the active site serine of serine hydrolases. A classification of the enzyme into a new family (family 9) of carboxyesterases (Arpigny, J. L., and Jaeger, K.-E. (1999) Biochem. J. 343, 177-183) is suggested.
