YUAN LI A.PROF QIZHI CHEN, PROF WAYNE COOK

Transcription

1 Synthesis, characterization and properties of biocompatible poly(glycerol sebacate) YUAN LI A.PROF QIZHI CHEN, PROF WAYNE COOK

2 Background Demand of biomaterials in the field of soft tissue engineering PGA, PLA and copolymer (rigidity and plastic deformation) No match PGA PLA CH 3 * C O n * H * C 2 O H O O * n Bio-elastomer: recovery from large scale deformation good biocompatibility with the host tissue

3 Background The crosslinked elastomers Poly (glycerol sebacate) (PGS) O O HO + HO 8 * O O 8 O O n * Advantages: Easy and safe for processing, no toxic catalytic chemicals. Crosslink and hydrogen bonding good mechanical properties Break down to natural metabolic products by simple hydrolysis Hydrolyzable ester bond Disadvantages: Irreproducibility of PGS synthesis Fast release of acidic products structure characterization inflammation Li Y, Chen QZ, et al., RSC Advances, 2012, 2: Biodegradable soft elastomers: synthesis/properties of materials and fabrication of scaffolds

4 Research Aim 1) To explore the effect of synthesis conditions on the PGS structure and properties 2) To estimate the reaction percentage and quantitatively analyze the molecular structure 3) To study the relationship between the PGS structure and mechanical properties as well as cytocompatibility.

5 EXPERIMENTAL PROCEDURE Synthesis condition of PGS Prepolymerisation Mix monomor (1:1 mole ratio, excess hydroxyl group) Glycerol +Sebacic acid 150 C- 8h or 130 C 24h Nitrogen gas PGS-prepolymers Solvent Casting Prepolymer was dissolved in THF to cast on glass Curing /48/72/96/168 hours Cast PGS prepolymer vacuum PGS

6 Two methods calculate reaction percentage: (1) Acid base titration ( unreacted carboxylic acid group) Percentage reaction (%) (1 1 mole unreacted carboxylic acid groups ) 100 mole original carboxylic acid groups ( V 2/( M V0 ) C / m0 n M Gly n Dissolved or swollen in an ethanol (25 wt. %)/toluene (75 wt. %) mixture solvent Titrated with 0.1 mol/l K SA Gly SA ) (2) Mass loss ( by product water) Percentage reaction (%) mole of reacted carboxylic group mole of original carboxylic group (%) m 2 m / M H O H2O 2 SA / M SA 100

7 Characterization Nuclear Magnetic Resonance (NMR) analysis Prepolymer dissolved or gel powder swollen in acetone-d6 Tensile testing Dog-bone shape specimens : t mm (length width thickness) Mini-Instron test : 100N load cell 10 mm/min cross-head speed Cytocompatibility assessment in vitro Samples: PGS-48h, PGS-96h, Poly(D,L-lactic acid), blank culture medium Extract media samples culture SNL mouse fibroblasts Tox-7 assay kit to analysis the degree of cell death AlamarBlue TM assay kit Analysis cell proliferation by measure reduction% of AlamarBlue TM

8 Esterification percentage (%) Esterification percentage (%) Results & Discussions - Reaction Percentage Method 1 (acid base titration); Method 2 (mass loss byproduct water) PGS prepolymerized at 150 C/8h mass loss PGS prepolymerized at 150 C/8h titration PGS prepolymerized at 130 C/24h mass loss PGS prepolymerized at 130 C/24h titration mass loss (byproduct water & evaporated glycerol) PGS prepolymerized at 150 C/8h mass loss PGS prepolymerized at 150 C/8h titration PGS prepolymerized at 130 C/24h mass loss PGS prepolymerized at 130 C/24h titration Post curing time (hours) Post curing time (hours) Gel point at postcuring for 20h 83-84% conversion

9 Characterization of PGS using NMR Sebacate methylene carbons glycerol & glyceride methylene/ methine carbons Carboxylic acid & ester carbons 13 C NMR SPECTRUM OF PGS

10 13 C NMR SPECTRUM OF PGS Twelve 13 C-NMR glyceride signals and two 13 C-NMR glycerol signals HO RO HO glycerol acylglyceride acylglyceride RO RO RO ,2-diacylglyceride 1,3-diacylglyceride 1,2,3-triacylglyceride PGS-gel: 1-acylglyceride decrease 1,3- and 1,2,3-acylglyceride increase

11 EXPERIMENTAL AND THEETICAL PERCENTAGE OF GLYCERIDE ESTER UNITS Structure and chemical shift (ppm) of glycerides Percentage of glyceride ester units (%) PGS: 150 C-8 h (prepolymer) PGS: 150 C-8 h then 130 C-48 h (crosslinked gel) Glycerol 1- monoacylglyceride HO RO monoacylglyceride HO ,2- diacylglyceride 1,3- diacylglyceride 1,2,3- triacylglyceride NMR (1-q) 3 NMR 2(1-q) 2 q NMR (1-q) 2 q NMR 2(1-q)q 2 NMR (1-q)q 2 NMR q ~ ~ RO RO RO NMR- integrated area of carbon peaks of the 13 C NMR spectra The statistical distribution of species - assuming the fractional conversion of hydroxyl groups (q) calculated from titration is the same for primary and secondary alcohols. Primary hydroxy groups have higher reactivity than secondary hydroxy groups

12 Stress (MPa) UTS or E (MPa) Influence of synthesis conditions on mechanical properties a 1.2 PGS-144h PGS-168h 1.0 PGS-96h 0.8 PGS-72h 0.6 PGS-48h 0.4 PGS-24h Strain (%) b PGS-150 C/8h, E PGS-130 C/24, E PGS-130 C/24h, UTS PGS-150 C/8h, UTS PGS-150 C/8h, PGS-150 C/8h, Curing time (hours) (%) UTS & E increased, max decreased monotonically with curing time After a certain curing time, UTS & E and max became almost constant and independent of curing time

13 UTS (MPa) Max Strain (%) Mechanical Properties vs strand density E /3RT a UTS vs Strand density PGS 130 C/24h then cured at 130 C PGS 150 C/8h then cured at 130 C Linear Fit of UTS Linear Fit of UTS b Max strain vs Strand density PGS 130 C/24h then cured at 130 C PGS 150 C/8h then cured at 130 C Power Fit of max y= y=1556x Power Fit of max y= x y=1034x Strand density (mole/m 3 ) The observed trend explained by theory of Taylor and Darin: UTS is approximately linear with strand density until the strand density exceeds a certain value. UTS k Strand density (mole/m 3 ) 1 1 k (1 k3 max ) 2 2 Max Strain at break: k break max 1 0.5

14 Percentage of AlamarBlue reduction (%) Percentage of dead cells (%) Assessment of Biocompatibility Free culture medium PGS cured for 48h PDLLA PGS cured for 96h cells only PDLLA PGS-48h PGS-96h Negative control PDLLA PGS 130 C/48h PGS 130 C/96h Samples The biocompatibility of PGS enhanced by increased curing time Tissue culture duration (days)

15 Assessment of Biocompatibility Reasons why longer curing time improves the material s biocompatibility: Reduction of unreacted carboxylic group (94% converted) O C Less change ph of extract media Higher crosslink density reduces the hydrolysis rate of ester group O C O Hydrolysis

16 CONCLUSION Evaporation of glycerol causes low reproducibility of PGS NMR provide qualitative and semi-quantitative PGS structure information Increased curing time of PGS, increases the UTS and E but decreases ε max Biocompatibility can be improved through a longer curing time Li Y, Cook WD Chen QZ et al, Polym Int, 2012, 6:

17 Acknowledgement SUPERVISION: A.PROF QIZHI CHEN, PROF WAYNE COOK THANKS TO: CNELIS MOHOFF, PETER NICHOLS KARLA CONTRERAS, SHULING LIANG, BING XU, WENCHAO HUANG, HANNING SHI

18 THANK YOU! QUESTION?