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1. Effect of polymeric nanoparticles on the stability of a biomimetic model of the lung surfactant Weiam Daear MSc. Student Supervisor: Dr. Elmar Prenner University of Calgary Alberta, Canada 1 2. 1. Introduction: Drug Delivery Routes 2 Mitragotri, S. 2008. The Bridge 38(4): 5-12 Parenteral delivery: Intravenous. Delivery through injections. Transdermal delivery: Delivery through skin patches or lotions. Oral delivery: Liquid or pill delivery through ingestion. Large blood supply but subject to pH degradation. Pulmonary delivery: Delivery through the lungs using aerosols. Close proximity to blood circulation (Heart). Nasal delivery: Delivery through the nasal cavity. Close proximity to the brain. Ocular delivery: Delivery through the eyes e.g. eye drops. There is no one route that is better than the other, it all depends on the nature of the drug and the target location. 3. 1. Introduction: Pulmonary Drug Delivery Route The distance between the air and the blood is around 500-800 nm. Lung surfactant is the first point of interaction of the pulmonary delivery route. Lung surfactant is a monolayer consisting of: 90% lipids 10% neutral lipids (e.g. Cholestrol) 80% phospholipids 10% surfactant proteins A major role of the lung surfactant is to reduce the surface tension to values closer to zero in order to prevent lung collapse. Piknova, B. et al. 2002 Current Opinion in Structural Biology Agassandian M. and Mallampalli R.K. 2013 Biochemica et Biophysica Acta 1831:612-625 3 4. Drug Targeting molecule There is a constantly growing need for an optimal drug delivery with high efficiency, specificity, and stability. Drugs can be either be encapsulated or adsorbed onto the surface of nanoparticles (NPs). Nanoparticles are generally within few hundred nanometers in size. Advantages of using NPs as drug delivery vehicles: Can penetrate tissues/cells of target organ Ability to exhibit controlled release 1. Introduction: Nanoparticles for Drug Delivery 4 Peer, D. et al. 2007 Nature Nanotechnology 5: 751-760 5. 1. Introduction: Nanoparticles for Drug Delivery Examples of Nanoparticles (NPs): Polymeric NPs Gelatin Poly(lactide-co-glycolic acid), PLGA Poly-n-(butyl)cyanoacrylates, PBCA Dendrimers Chain of repeating molecules Liposomes/micelles Lipid based 5 PLGA PBCA Peer, D. et al. 2007 Nature Nanotechnology 5: 751-760 This research focuses on polymeric NPs due to their high stability, biocompatibility and biodegradability. 6. 2. Research Goals Goal 1: Develop a biomimetic model of the lung surfactant. Goal 2: Analyze and understand how the nature of polymeric nanoparticles affect the stability of the lung surfactant monolayer. Gelatin, PLGA, and PBCA nanoparticles Goal 3: Understand the localization of different polymeric nanoparticles with the lung surfactant monolayer. 6 7. Lung surfactant lipid analysis: Postle et al. performed mass spectroscopy analysis on the lipid composition of the lung surfactant. The major lipid classes are phosphatidylcholines (PCs) and phosphatidylglycerols (PGs). Based on the results, the 16:0/16:0 and 16:0/18:1 PCs and PGs were chosen for biomimetic model. 3. Research Methods Postle A.D. et al. 2001Comparative Biochemistry and Physiology Part A 129: 65-73 7 8. 3. Research Methods Lung surfactant lipid biomimetic model: (mol ratios) 45 1,2-dipalmitoylphosphatidyl-choline, DPPC 1 1,2-dipalmitoylphosphatidyl-glycerol, DPPG 8 1-palmitoyl-2-oleoylphosphatidyl-choline, POPC 6 1-palmitoyl-2-oleoylphosphatidyl-glycerol, POPG The double bond introduces fluidity to the lipid. The two major driving factors in the model are : The Overall charge and Fluidity. 8 16:0 /16:0 PC (DPPC) Overall charge: 0PC 16:0/16:0 PG (DPPG) Overall charge: -1PG Overall charge: -1 16:0/18:19 PG (POPG) PG 16:0/18:19 PC (POPC) Overall charge: 0PCDouble bond Postle A.D. et al. 2001Comparative Biochemistry and Physiology Part A 129: 65-73 2 % Cholesterol of the total lipid weight + 9. Studies on the lung surfactant is done using a Langmuir Trough. A movable barrier is used to mimic compression of the lung surfactant during exhalation. Monolayer compression causes an increase in surface pressure recorded by a sensor. 3. Research Methods: Langmuir Trough Pressure Sensor Movable Barrier Aqueous layer Lipids deposited on aqueous layer 10. 3. Research Methods: Langmuir Trough He and Li 2007 Advances in Colloid and Interface Science 131: 91-98 10 The change in surface pressure as the area changes with compression is recorded as a Pressure Area isotherms (-A isotherms). As the monolayer is being compressed, it undergoes different lipid phases. G: Gaseous phase LE: Liquid Expanded LC: Liquid Condensed Area per molecule G phase LE phase LE-LC LC phase Collapse Further compression results in monolayer collapse (multilayer structures) which is known as Collapse Pressure. Surface pressure and surface tension are inversely proportional. A decrease in collapse pressure is a measure of monolayer stability. Direction of compression 11. 3. Research Methods: Brewster Angle Microscopy Brewster Angle Microscopy (BAM) Allows visualization of the lateral domain organization of the monolayer. Plane polarized light is directed at the air-water interface at the Brewster angle where no light is reflected. This angle () is determined through Snells law: tan = n2/n1 For the air-water interface, is equal to 53.1, Image adapted from www.ksvnima.com 11 12. 4. Results: DPPC system 12 As shown by Lai et al., collapse pressure of pure DPPC is around 60 mN/m. This collapse pressure is not reduced significantly with the addition of gelatin NPs. This indicates that gelatin NPs had no effect on the stability of DPPC. DPPC, overall charge: 0 Lai, P. et al. 2009 Journal of Biomedical Technology 6(2): 145-152 This paper shows the effect of gelatin NPs on the major lung surfactant lipid constituent, DPPC. I want to test the effect on other major lipid classes and associated complex mixtures for the development of a biomimetic lung surfactant model. 13. 4. Results: DPPC system DPPC interaction with gelatin NPs: At a surface pressure of ~7 mN/m: Gelatin NPs decreased the size of the DPPC lateral domains. The decrease in the DPPC domain size is correlated to the concentration of NPs being added. 13 DPPC:Nanoparticles (30:1 w/w)DPPC DPPC:Nanoparticles (11:1 w/w) Bar: 50 m DPPC, overall charge: 0 Lai, P. et al. 2009 Journal of Biomedical Technology 6(2): 145-152 14. POPC interaction with gelatin NPs: Collapse pressure of POPC is not reduced by the addition of gelatin NPs. This indicates that gelatin NPs have no effect on the stability of POPC. Small reduction in molecular area due to the addition of gelatin NPs indicate minor lipid loss to the aqueous layer. 4. Results: POPC system 14 0 5 10 15 20 25 30 35 40 45 50 0 50 100 150 200 250 300 SurfacePressure(mN/m) Area (/molecule) POPC POPC + NPs POPC, overall charge: 0 15. POPC interaction with gelatin NPs : At a surface pressure of ~5 mN/m: Gelatin NPs had a minor effect on the POPC monolayer lateral organization. When comparing DPPC to POPC, the major difference is the level of acyl chain saturation (fluidity). This mono-unsaturation prevents the formation of domains. 4. Results: POPC system 15Bar: 50 m POPC POPC:Nanoparticle (10:1 w/w) Bright spots due to addition of NPs POPC, overall charge: 0 16. 16 4. Results: Binary PC system 16 Binary PC (45 DPPC+ 8 POPC) interaction with gelatin NPs: Collapse pressure of Binary PC is not reduced by the addition of gelatin NPs. This indicates that gelatin NPs have no effect on the stability of Binary PC system. A significant shift in the -A isotherm to larger molecular areas is observed. This indicates NP adsorption/insertion into the monolayer film. -10 0 10 20 30 40 50 60 70 0 50 100 150 200 250 SurfacePressure(mN/m) Area (/molecule) Binary PC Binary PC with NPs DPPC, overall charge: 0 POPC, overall charge: -1 17. 1717 Binary PC interaction with gelatin NPs: At surface pressures of ~ 13 mN/m and 25 mN/m: Gelatin NPs decreased the frequency of the lateral domains observed. Bar: 50 m DPPC+POPC Binary PC:Nanoparticle (10:1 w/w) DPPC+POPC Binary PC:Nanoparticle (10:1 w/w) At 13 mN/m At 25 mN/m 4. Results: Binary PC system DPPC, overall charge: 0 POPC, overall charge: -1 18. 4. Results: DPPG system 18 -10 0 10 20 30 40 50 60 0 50 100 150 200SurfacePressure(mN/m) Area (/molecule) DPPG DPPG + NPs DPPG interaction with gelatin NPs: Collapse pressure of DPPG is reduced by ~ 5 mN/m due to the addition of gelatin NPs. DPPG, overall charge: -1 19. 4. Results: DPPG system 191919 Bar: 50 m DPPG DPPG:Nanoparticle (10:1 w/w) DPPG DPPG:Nanoparticle (10:1 w/w) At 10 mN/m At 30 mN/m DPPG interaction with gelatin NPs: At a surface pressures of ~ 10 mN/m and 30 mN/m: Gelatin NPs did not have an effect on the observed domain size but appear to localize at the edges of the domains. (arrows indicate localization) DPPG, overall charge: -1 20. 4. Results: Quaternary system 20 4 lipid system interaction with gelatin NPs: Collapse pressure of 4 lipid system is not reduced by the addition of gelatin NPs. Small reduction in molecular area due to the addition of gelatin NPs indicate change in lipid packing or minor lipid loss to the aqueous layer. -10 0 10 20 30 40 50 60 0 20 40 60 80 100 120 SurfacePressure(mN/m) Area (/molecule) 4 lipid 4 lipid + NPs Complex mixture-1 (48 DPPC+ 8 POPC+ 1 DPPG+ 6 POPG) DPPC, overall charge: 0 POPC, overall charge: -1 DPPG, overall charge: -1 POPG, overall charge: -1 21. 4. Results: Quaternary system 21 4 lipid system interaction with gelatin NPs: At surface pressures of 15 mN/m and 30 mN/m: Gelatin NPs appear to have an effect on domain size and frequency. Bar: 50 m 4 lipid 4 lipid:Nanoparticle (10:1 w/w) 4 lipid 4 lipid:Nanoparticle (10:1 w/w) At 15 mN/m At 30 mN/m Bar: 50 m DPPC, overall charge: 0 POPC, overall