1. Presented by Naila Sajjad 10-arid-1766 PhD Biochemistry Biodegradable nanoparticles for drug and gene delivery to cells and tissues

2. Content Significance of nanotechnology for drug therapy Nanoparticles Significance of particle size Biodegradable polymers PLGA &PLA Intracellular trafficking Therapeutic applications of PLGA nanoparticles

3. Conti…… Sustained gene delivery Protein delivery Vaccine adjuvant Intracellular targeting Tissue targeting conclusion Future prospects References

4. Nanotechnology Nanoscience Research at the scale of 100nm or less Nanomedicice Drug delivery system

5. Significance of nanotechnology for drug therapy Suitable means of delivering small mol wt drugs & macromolecules such as protein peptides or genes (Moghimi et al.,2001) Targeted (cellular/tisue) delivery of drug (Vinagradov et al., 2002)

6. Significance of particle size High intracellular uptake (Desai et al., 1997) Efficiently penetrate throughout submucosal layers Cross the blood-brain barrier

7. Biodegradable polymers PLGA &PLA Synthetic polymer Polylactide (PLA) and poly (D,L-Lactide-co- glycolide)(PLGA) Hydrolysis Polymer biodegradation

8. Conti…..

9. Plasmid DNA loaded nanoparticle

10. Formation of PLGA nanoparticles Emulsion solvent evaporation technique (Jain,2000) Polyvinyl alcohol (PVA) (Sahoo., 2002) i. Commonly used emulsifier ii. Uniform and smaller in size iii. hydrophilic

11. Solvent evaporation technique

12. Intracellular trafficking Delivery of therapeutic agents to specific compartments or organelles within the cell Targeted delivery is the higher bioavailability of therapeutic agent at its site of action

13. Release of drug entrapped in PLGA matrix Diffusion Block coplymer composition and mol wt. Release of encapsulated therapeutic agent (Lin et al., 2000)

14. Uptake of nanoparticles in S.M.C and V.E.C Phagocytosis Fluid phase pinocytosis Receptor-mediated endocytosis

15. Intracellular uptake pathway

16. Conti… Primary endosomes Sorting endosomes Recycling endosomes Secondary endosomes

17. Intracellular trafficking of nanoparticles

18. Transmission electron microscopic picture of PLGA nanoparticles in the cytoplasm of vascular smooth muscle

19. Conti… Uptake of nanoparticles is time dependent Surface charge reversal

20. Mechanism responsible for the endolysosomal escape of nanoparticle Nanoparticles interact with vesicular membrane inside the cell (Panyam et al., 2002) Destabilization of membrane Escape of nanoparticles into cytoplasmic compartment

21. Exocytosis of nanoparticles Protein (albumin)in serum Antiproliferative effect of dexamethasone-loaded nanoparticles in smooth muscle cell (Davda et al., 2002)

22. Therapeutic applications of PLGA nanoparticles Sustained gene delivery Protein delivery Intracellular targeting Tissue targeting

23. Sustained gene delivery Nanoparticles containing encapsulated plasmid DNA Lysosomal enzymes Hedley at al demonstrated protection of DNA from nuclease when encapsulated into PLGA microsphere Rat bone osteotomy model (Labhasetwar et al., 1999)

24. Marker gene ( Fire fly luciferase &heat sensitive human placental alakaline phosphatase) Gene expression in cell culture in the presence of serum Use of PLGA emulsion containing alkaline phosphatase as marker gene for coating gut suture Rat skeletal muscles (Cohen et al., 2000)

25. Protein delivery Encapsulation of therapeutic proteins & peptide into nanoparticles using emulsion solvent technique (Davda et al., 2000) Loss of therapeutic efficiency due to denaturation/degradation of protein

26. Reasons of protein inactivation Exposure to organic solvents leading to protein adsorption at oil-water interface (Lu et al., 2000) Acidic environment generated during degradation of PLGA matrix due to formation of acidic monomers and oligomers (Zhu et al., 2000)

27. Protection of protein Addition of Bovine serum albumin to aqueous phase before emulsification (Weert et al., 2000) By including buffering base such as magnesium hydroxide to PLGA microsphere formation (Zhu et al., 2000)

28. Vaccine adjuvant Nano and microparticles containing antigen (Raghuvanshi et al., 2001) Alternative to currently used alum Provide sustained release of antigen

29. Systemic and mucosal immunity Adjuvant properties of PLGA nanoparticles containing encapsulated staphylococcal enterotoxin B toxoid

30. Immune response through nanoparticles injection following injection of alum Maximum at 7 weeks then gradually decreased with time Secondary immune response at 19 th week Synergistic immune response after co-injection of TT alum along with TT-loaded nanoparticles (Raghuvanshi et al., 2001)

31. Intracellular targeting Surface charge of nanoparticles (Panyam et al., 2002) Surface modification of nanoparticles with cationic agents like didodecyldimethylammonium bromide (DMAB) Variation in physical properties Attachment of nuclear localization signal to nanoparticle surface

32. Tissue targeting Monoclonal antibodies Epoxy-activation method Active and passive targeting

33. Future prospects Sustained dilivery Issues in drug delivery are becoming more important and specific drugs become available with the knowledge about diseases available from the human genome project All therapeutic agents would optimally require drug delivery and targeting mechanisms to deliver them to target tissues without reducing their therapeutic efficacy.

34. Conti… As the pathophysiology of disease conditions and their cellular mechanisms are understood, drug delivery systems customized to achieve optimal therapeutic efficacy will be more effective Nanoparticles, because of their versatility for formulation, sustained release properties, sub-cellular size and biocompatibility with tissue and cells appear to be a promising system to achieve these important objectives

35. Conclusion Use of biodegradable nanoparticles formed from poly (D,L- Lactide-co-glycolide)(PLGA) for target delivery of plasmid DNA, proteins and low molecular wt compound Rapid escape of PLGA nanoparticles from the endo- lysosomal compartment into cytosol

36. References Maghimi, S.M., A.C.Hunter and J.C. Murray. 2001. Long circulating and target specific nanoparticle.theory to practice. Pharmacol. Rev. 283-318. Vinagradov, S.V., T.K. Bronich and A.V.Kabanov. 2002. Nanosized cationic hydrogels for drug delivery preparation, properties and interaction with cells. Adv. Drug. Del. Rev. 223-233. Labhasetwar, V., J.Bonadio, S.A.Goldstein and R.J.Levy.1999.Gene transfection using biodegradable nanosphere: results in tissue culture and rat osteotomy model. Colloids surfaces. B. Biointerfaces. 281-290. Desai,M.P., V.Labhasetwar, E.Walter, R.J.Levy and G.L. Amidon.1997. The mechanism of uptake of biodegradable microparticle in caco-2cells in size dependent. Pharm.Res. 1568-1573. Sahoo, S.K., J.Panyam, S.Prabhe and V. Labhasetwar. 2002. Residual polyvinyl alcohol associated with poly(D,L-Lactide-co-glycolide)nanoparticles affects their physical properties and cellular uptake. J.Control.Release. 105-114.

37. Conti… Weert, M.V.D., J. Hoechstetter, W.E. Hennink, D.J. Crommelin. 2000.The effect of a water/organic solvent interface on the structural stability of lysozyme. J. Control. 351–359 Lu,L., G.N. Stamatas, A.G. Mikos. 2000. Controlled release of transforming growth factor beta1 from biodegradable polymer microparticles. J. Biomed. Mater. Res.440–451 Raghuvanshi,R.J., A. Mistra, G.P. Talwar, R.J. Levy, V. Labhasetwar. 2001.Enhanced immune response with a combination of alum and biodegradable nanoparticles containing tetanus toxoid. J. Microencapsul. 723– 732

38. Conti….. Cohen,H., R.J. Levy, J. Gao, I. Fishbein, V. Kousaev, S. Sosnoski, S. Slomkowski. 2000. Sustained delivery and expression of DNA encapsulated in polymeric nanoparticles. Gene Ther.1896–1905 Davda,J., V. Labhasetwar. 2002.Characterization of nanoparticle uptake by endothelial cells.Int. J. Pharm.(2002), pp. 51–59 Panyam,J., W.Z. Zhou, S. Prabha, S.K. Sahoo, V. Labhasetwar. 2002.Rapid endo-lysosomal escape of poly ( D, L -lactide-co-glycolide) nanoparticles: Implications for drug and gene delivery. FASEB J.1217–1226 Panyam, J., V. Labhesetwar. 2012. Biodegradable nanoparticles for drug and gene delivery to cells and tissues. J. Pharm. Sci. 64: 61-71

39. Conti….. Lin,S.Y., K.S. Chen, H.H. Teng, M.J. Li.2000 In vitro degradation and dissolution behaviours of microspheres prepared by three low molecular weight polyesters.J. Microencapsul., 577–586