Sigma xi slideshare final

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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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1. Introduction: Drug Delivery Routes

Mitragotri, S. 2008. The Bridge 38(4): 5-12

Parenteral delivery:Intravenous. Delivery through injections.

Transdermal delivery:Delivery through skin patchesor 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.

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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

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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

Peer, D. et al. 2007 Nature Nanotechnology 5: 751-760

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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

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.

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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.

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• 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

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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.

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

+

• 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

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3. Research Methods: Langmuir Trough

He and Li 2007 Advances in Colloid and Interface Science 131: 91-98

• 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

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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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4. Results: DPPC system

• 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.

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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.

DPPC:Nanoparticles(30:1 w/w)DPPC

DPPC:Nanoparticles(11:1 w/w)

Bar: 50 µm


Lai, P. et al. 2009 Journal of Biomedical Technology 6(2): 145-152

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• 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

0 50 100 150 200 250 3000

5

10

15

20

25

30

35

40

45

50

POPC

POPC + NPs

Area (Å²/molecule)Su

rfac

e Pr

essu

re (m

N/m

)

POPC, overall charge: 0

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• 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

Bar: 50 µm

POPC POPC:Nanoparticle

(10:1 w/w)

Bright spots due to addition of NPs

POPC, overall charge: 0

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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.

0 50 100 150 200 2500

10

20

30

40

50

60

70

Binary PC

Binary PC with NPs

Area (Å²/molecule)Su

rfac

e Pr

essu

re (m

N/m

)


POPC, overall charge: -1

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• 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


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4. Results: DPPG system

0 20 40 60 80 100 120 140 160 180

-10

0

10

20

30

40

50

60

DPPG

DPPG + NPs

Area (Å²/molecule)

Surf

ace

Pres

sure

(mN

/m)

• 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

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4. Results: DPPG system

1919Bar: 50 µm

DPPGDPPG:Nanoparticle

(10:1 w/w) DPPGDPPG:Nanoparticle

(10:1 w/w)


• 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)


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4. Results: Quaternary system

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 20 30 40 50 60 70 80 90 100 110

-10

0

10

20

30

40

50

60

4 lipid 4 lipid + NPs


Surf

ace

Pres

sure

(mN

/m)

Complex mixture-1 (48 DPPC+ 8 POPC+ 1 DPPG+ 6 POPG)




POPG, overall charge: -1

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4. Results: Quaternary system

• 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 lipid4 lipid:Nanoparticle

(10:1 w/w) 4 lipid4 lipid:Nanoparticle

(10:1 w/w)


Bar: 50 µm





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4. Results: Quinary system

20 30 40 50 60 70 80 90 100 110

-10

0

10

20

30

40

50

60

4 lipid + Chol

4 lipid + Chol + NPs


Surf

ace

Pres

sure

(mN

/m)

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).





Complex mixture-2 (48 DPPC+ 8 POPC+ 1 DPPG+ 6 POPG + 2%w/w Cholesterol )

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• 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

23Bar: 50 µm

4 lipid+Chol4 lipid+Chol:Nanoparticle

(10:1 w/w)





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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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6. Future Directions

• 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.

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7. Acknowledgement

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.

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