1. PLGA for drug delivery Huang Juan Huang Junlian Saskia Huijser Rob Duchateau

2. Introduction Aliphatic polyesters have been extensively used as important biodegradable biomaterials for a wide variety of drug delivery carriers and biomedical devices. They have biodegradability, versatile mechanical properties and proven biocompatibility. Poly(L-lactide)(PLA) and poly(glycolide)(PGA) and poly(lactide- co-glycolide)(PLGA) are the most commonly used biodegradable and biocompatible polymers.

3. Synthesis of PLGA 1.Melt polycondensation: step growth Lactic acid Glycolic acid PLGA: poly(lactic acid-co-glycolic acid)

4. Synthesis of PLGA 2. Ring opening polymerization: chain growth Lactide Glycolide PLGA: poly(lactide-co-glycolide ) a) Enzyme catalyst b) Metal catalyst

5. Enzymatic polymerization of PLGA An increase in interest in enzyme-catalyzed organic reactions Several advantages:  Catalysis under mild reaction conditions (Temperature, pH, Pressure)  Nontoxic natural catalyst  Have the ability to be used in bulk reaction media avoiding organic solvents Several disadvantages:  Long reaction time  Low molecular weight

6. Enzyme of lipase PS Red site: Histidine Yellow site: Aspartic acid Green site: Serine Serine

7. Postulated Mechanism

8. Results and discussion SampleEnzyme Time (d) Conversion (%) M w (kDa) PDI PLLAPS79215.01.7 PLLAPS-DI7979.22.2 PLLA-710-- PGAPS29613.02.4 PGAPS-DI21009.11.7 PGA-20-- The results of PLLA and PGA with/without lipase Reaction in bulk in 100 0 C and using 8 wt % lipase

9. PLGA prepared by lipase at 100 0 C for 7 d EntrySamples Feed ratio (L/G) Polymer ratio (L/G) Enzyme L Conv.(%) G Conv. (%) Mw (kDa) PDI T g ( 0 C) Δ T g ( 0 C) 1a1a PLLGA 90/10 88/12PS89100 13.91.950.23.6 2PLLGA 80/20 77/23PS94100 11.71.847.53.4 3PLLGA 70/30 PS96100 14.61.946.43.8 4PLLGA 90/10 82/18PS-DI74100 6.41.445.74.2 5PLLGA 80/20 69/31PS-DI76100 8.71.544.74.0 6PLLGA 70/30 64/36PS-DI86100 7.91.644.03.9 7PDLLGA 80/20 75/25PS85100 10.52.436.35.4 8PDLLGA 80/20 58/42PS-DI78100 7.41.635.75.0 9b9b PLLGA 80/20 79/21PS97100 17.82.248.33.9 10 c PLLGA 80/20 58/42-1957 1.91.3n.d.

10. PLLGA L/G=80/20 using 8 wt% lipase PS at 100 0 C The decrease in polymerization rate may be due to the low concentration of monomers and the high viscosity of the system.

11. PLLGA L/G=80/20 using 8 wt% lipase PS-DI at 100 0 C Both M w and polydispersity increase during the reaction time. The reaction rate of glycolide is faster than lactide.

12. 1 H NMR spectrum (400 MHz, DMSO-d6) of PLLGA (with 8 wt% lipase PS at 100 0 C for 7 d)

13. 13 C{ 1 H} NMR spectra (125MHz, CDCl 3 ) of PLLGA (carbonyl region), a) PLLGA with lipase PS-DI at 100 0 C for 7 d. b) PLLGA with lipase PS at 100 0 C for 7 d

14. L L = (I LL +IL G )/I LG L G = (I GG +I GL )/I GL (n L and n G are lactide and glycolide molar fraction in copolymers respectively ) Entry Glycolide (%) in polymer [n L /(n L +n G )]L LGLG L G /(L G +L L ) (%) a317.73.430.6 b2310.13.224.1 13 C{ 1 H} NMR sequence analysis of PLLGA copolymers A random copolymer would have an average glycolyl sequence length, L G equal to 2.

15. Mass (m/z) = M end group + mM la + nM ga + M K+ (where M end group = 18 or 0, M la = 72, M ga = 58, M K+ = 39) MALDI-ToF MS spectra of PLLGA with lipase PS at 100 0 C.

16. Mass (m/z) Series A Series B Series C mn m nmn 1783-- 17 8214 1785240 13 179 1797030 18 7223 1799-- 14 12188 1811129 19 6232 1813-- 15 11197 18211018 - -327 1825228 20 5241 1827-- 16 10206 Main ion series determined by MALDI-ToF spectrum of PLLGA using lipase Series ASeries BSeries C

17. PLGA prepared by lipase at 130 0 C for 7 d EntrySamples Feed ratio (L/G) Polymer ratio (L/G) Enzyme L Conv. (%) G Conv. (%) Mw (kDa) PDI T g ( 0 C) Δ T g ( 0 C) 1a1a PLLGA 90/10 PS9810019.82.346.94.0 2PLLGA 80/20 79/21PS9610020.22.745.63.5 3PLLGA 70/30 69/31PS9710018.74.636.26.2 4PLLGA 90/10 89/11PS-DI9910011.91.543.34.9 5PLLGA 80/20 79/21PS-DI9710011.21.645.63.5 6PLLGA 70/30 69/31PS-DI9710010.21.642.74.0 7PDLLGA 80/20 79/21PS9710011.53.143.73.8 8PDLLGA 80/20 79/21PS-DI9710011.61.739.24.8 9b9b PLLGA 80/20 72/28-741007.71.342.94.4

18. PLLGA L/G=80/20 using 8 wt% lipase PS at 130 0 C M w decreases after the second day. High temperature increases chain depolymerization. Lipase PS may be denatured at this temperature. PLLGA L/G=80/20 without catalyst at 130 0 C

19. PLLGA L/G=80/20 using 8 wt% lipase PS at 130 0 C PLLGA L/G=80/20 without catalyst at 130 0 C High temperature at 130 0 C increases the polymerization rate.

20. 4.Conclusion  Lipase PS works as catalyst to synthesize of PLGA and the conversion gets to 96%.  Transesterfication has occurred during the reaction.  PLGA copolymers obtained by lipase PS and lipase PS-DI at 100 0 C are block copolymers.  A higher temperature increases the polymerization rate but also increases the depolymerization rate.  The PLGA copolymers from lipase might contain both linear and cyclic chains.

21. Acknowledgement Prof. J.L. Huang Ir. S. Huijser Dr. R. Sablong Dr. R. Duchateau Prof. C.E. Koning Dr. F.G. Karssenberg Prof. P. J. Lemstra Everybody who contributed to my project

22. Thank you for your attention !