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
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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
16:0/18:1Δ9 PG (POPG)
PG
16:0/18:1Δ9 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 (π-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 (θ) is determined through Snell’s law:
tanθ = n2/n1
– For the air-water interface, θ is equal to 53.1º,
Image adapted from www.ksvnima.com
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12. 4. Results: DPPC system
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• 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.
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DPPC:Nanoparticles
(30:1 w/w)DPPC
DPPC:Nanoparticles
(11:1 w/w)
Bar: 50 µm
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
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0
5
10
15
20
25
30
35
40
45
50
0 50 100 150 200 250 300
SurfacePressure(mN/m)
Area (Ų/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 µm
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
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• 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 π-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 (Ų/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 µm
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 (Ų/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 µm
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
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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 (Ų/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
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• 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 µm
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 µm
DPPC, overall
charge: 0
POPC, overall
charge: -1
DPPG, overall
charge: -1
POPG, overall
charge: -1

22. 4. Results: Quinary system
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-10
0
10
20
30
40
50
60
0 20 40 60 80 100 120
SurfacePressure(mN/m)
Area (Ų/molecule)
4 lipid + Chol
4 lipid + Chol + NPs
4 lipid system + Cholesterol
interaction with gelatin NPs:
• Collapse pressure of 4 lipid+Cholesterol
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.
• The plateau indicated by the arrow ( )
resembles that seen with BLES (Bovine
Lipid Extract Surfactant).
DPPC, overall
charge: 0
POPC, overall
charge: -1
DPPG, overall
charge: -1
POPG, overall
charge: -1
Complex mixture-2 (48 DPPC+ 8 POPC+ 1 DPPG+ 6 POPG + 2%w/w Cholesterol )

23. • 4 lipid system + Cholesterol interaction with gelatin NPs:
• At surface pressures of 25 mN/m
– When comparing this system to the 4 lipid system, it can be concluded that
Cholesterol appears to have a significant effect on domain formation.
– Gelatin NPs had a minor effect on the POPC monolayer lateral organization.
4. Results: Quinary system
2323
Bar: 50 µm
4 lipid+Chol
4 lipid+Chol:Nanoparticle
(10:1 w/w)
DPPC, overall
charge: 0
POPC, overall
charge: -1
DPPG, overall
charge: -1
POPG, overall
charge: -1

24. 5. Conclusions
• Isotherms indicate there is no destabilization with the addition of gelatin NPs.
• BAM allowed the visualization of lateral domain organization.
• Increasing the fluidity of the lipid system through a single double bond (DPPC 
POPC) appears to prevent lateral domain formation.
• Binary PC system pressure-area isotherm shift to the larger with the addition of NPs
indicating an insertion/adsorption mode of gelatin NP localization.
• With other measured systems, the addition of gelatin NPs slightly increased the area
indicating an effect on lipid packing or lipid loss to the aqueous layer.
• The addition of Cholesterol to the 4 lipid system affects lipid packing and prevents
the formation of the observable lateral domains.
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25. 6. Future Directions
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• Test how the different chemical nature of polymeric NPs affect the
observed results.
– PLGA and PBCA NPs
• Use fluorescence imaging of NPs to test the localization of NPs
within the lung surfactant.

26. 7. Acknowledgement
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o Funding by Queen Elizabeth II Graduate Scholarship
o Special Thanks to:
• Dr. Elmar Prenner, University of Calgary (Supervisor),
• Sigma Xi for providing such an opportunity, and
• Mohamed Hassanin for help with creating the presentation site.