1. Molecular & Cell Biology
Recitation
Spring, 2011
Recitation Instructors:
Prof. Eric Brenner eb50@nyu.edu
Erik Duboue eduboue@gmail.com
Nathan Poslusny njp243@nyu.edu

2. How can the recitation help you?
Recitation Participation: 5%
(Attendance, Discussion, Group Work)
Recitation Presentation: 5%
Class Paper: 10%
=20% Final Grade

3. Recitation 1:
Lipids and Cell Membrane Fluidity

4. Part 1: Using lipids to help fight cancer
Background: What are the major problems with
current chemotherapeutic techniques?
•Toxicity to non-target cells
•Drug stability
•Target cell specificity

5. Hard tumors are particularly difficult to treat
with chemotherapy. Why?
• They have poorly dispersed
vascular systems, which
reduces exposure to blood-
borne drugs.
• They are densely fibrous,
which serves as a physical
barrier.
• They have high internal
pressures, which means
soluble drugs are not easily
delivered into the tumor

6. Is there a “magic bullet” that can target drugs
right into cancers that are difficult to treat?
If so, what is this new technology called?

7. Nanomedicine
(The medical application of
nanotechnology)
Nano drugs = 10 to 100 nanometers

8. Nanomedicine uses nanoparticles
Nanoparticles preferentially
accumulate at tumor sites because
tumors lack an effective lymphatic
drainage system.
Hence, poor vascular structure, such
as that found in hard tumors, is
advantageous for nanomedicine

9. So, how can membranes be used in nanomedicine?
Liposomes are artificially prepared
vesicles made from a lipid bilayer.
Liposomes can be filled with
drugs, and used to deliver
these drugs to cancer cells and
other diseased cells or tissues
To understand how liposomes are made, we must first
review membranes and their characteristic
components…

10. Part 1I: What are key
Features of
Membranes?
• Sheet-like Structures
• Asymmetric Bilipid Leaflets
• Closed Boundaries
• Lipids and Proteins (but also carbohydrates, and
other molecules…)
• Non-covalent Assemblies - disassociates and easily
reconstituted
• Fluid (not random)
How do the properties of lipids confer
the characteristics of cell membranes?

11. Basic Structure of a Phospholipid:
What does amphipathic mean?
*Having both hydrophilic & hydrophobic properties
How are membrane
components oriented?
*It is energetically favorable for amphipathic lipids to form
membranes, as a lipid bilayer allows for the hydrophobic tails to be
buried & the hydrophilic head groups to interact with either the
cytosol or the extracellular space.

12. Side Chains:
Can be Saturated or Unsaturated
Saturated fatty acids are
“Saturated” with H’s
all single bonds form a
straight chain
Unsaturated fatty acids
are missing some H’s
double bonds form kinks

13. What consequence do these different forms
have on membrane properties?
• Saturated side chains are straight and don’t take
up much room, meaning that they can pack
tightly together, increasing density and making
membranes less-fluid
What is healthy vs. unhealthy?
Ex. butter (saturated fats) are solid at room temp

14. Sphingolipids are defined by
their sphingosine group, are
(mostly) uncharged, and
commonly found in neuronal
cell membranes
What kind of lipid is this?
(what’s the distinguishing feature?)
*sphingolipid, defined by the sphingosine (red)
What are the features of the side chains?
*saturated
What is the overall charge on this lipid?
*uncharged

15. Unsaturated chains
“kink”, can’t pack in as
densely made make the
membrane more fluid
Ex. oils are liquid at room
temp

16. What kind of lipid is this?
(what’s the distinguishing feature?)
*phospholipid
What are the features of the side chains?
*one saturated, one unsaturated
What is the overall charge on this lipid?
*it has one (-) & one (+), but overall it is uncharged

17. This lipid is made of ceramide &
glucose
What kind of lipid is this? (what is it made
of?)
*glycolipid (it contains glucose)
What are the features of the side chains?
*one saturated, one unsaturated
What is the overall charge on this lipid?
Any +, -, neutral?
*it is uncharged
Unsaturated fats help maintain membrane
fluidity.
What other famous lipid also helps?

18. Cholesterol
What features make it amphipathic?
*the ring structure is hydrophobic, and the -OH is
hydrophilic
What is the overall charge on this lipid?
*uncharged
Is it found mostly inside the bilayer or does it stick out?
*it is found inside the bilayer
What can this structure tell us about how
this molecule may work in the membrane?
*it helps maintain fluidity by preventing interactions between fatty acid side chai

19. Lipid Content of Membranes…
Varies Among Membranes
Examples:
Myelinated Nerve Cells:
Cholesterol and Cerebrosides
Liver Cell Plasma membranes:
Cholesterol and Phospholipids

20. How can we measure the various lipids in a
membrane?
2. Remove Lipids, but
1. Obtain Sample
leave behind
Tissues, Cell
proteins, carbohydrates,
Extracts, etc…)
etc.)
How? Ch
PE
PC
Organic solvent eg.
chloroform/methanol, cyclohexane PS
How detect?
thin-layer chromatography, HPLC and
more advanced methods

21. Part II: Techniques
How do we study membrane lipids?
Prepare "Ghosts"
What’s osmotic lysis?
Place the cell in a hypotonic solution allowing it to burst

22. Ghost can be used to study membrane composition
Intact cell membranes are impermeable to enzymes and SITS;
however, ghosts are leaky, and enzymes and SITS can enter them!
RBC Leaky Ghost

23. How can we assess the asymmetry of the
lipid composition of leaflets?
*Sequentially use enzymes that selectively degrade different components
Sphingomyelinase: degrades sphingomyelin
Sea snake venom: contains phospholipases that
degrade phosphoglycerides
SITS: membrane impermeable compound that binds to -NH3+
(primary amine) groups and then fluoresces

24. Before treatment
Red Blood Cells* Ghosts*
Sphingomyelin 26% 26%
Phosphatidyl Choline 32% 32%
Phosphatidyl
31% 31%
Ethanolamine
10% 10%
Phosphatidyl Serine
1% 1%
Other
* These values are expressed as percentages of
phospholipids alone; cholesterol and glycolipids are not
included.

25. What do these lipids share in common?
*these are all phospholipids

26. Treatment with sphingomyelinase
Sphingomyelin
RBC Leaky Ghost
sphingomyelin: 26%-
sphingomyelin: 26%->0%
>6%

27. Treatment with sphingomyelinase
Sphingomyelin
RBC Leaky Ghost
sphingomyelin: 26%-
sphingomyelin: 26%->0%
>6%

28. Sphingomyelin quantification and location
Red Blood Cells Leaky Ghosts
Sphingomyelin 26% 6% 26% 0%
Phosphatidyl
Choline 32% 32%
Phosphatidyl
Ethanolamine 31% 31%
Phosphatidyl
10% 10%
Serine
Other, including Difference=
degradation 1% 21% 1% 27% sum of
products changes
Which leaflet contains sphingomyelin?
*it is in both the inner (6%) & outer(20%) leaflet

29. Sea snake venom contains phospholipases that degrade
phosphoglycerides
Red Blood Cells Leaky Ghosts
Sphingomyelin 26% 26%
Phosphatidyl
Choline 32% 9% 32% 0%
Phosphatidyl
31% 25% 31% 0%
Ethanolamine
Phosphatidyl
10% 10% 0%
Serine
Other, including
degradation 1% 30% 1% 74%
products
Which leaflets contain phosphoglycerides?
*the phospholipids are in both the inner (44%) and outer (29%) leaflet

30. SITS: membrane impermeable compound that binds to -
NH3+ groups and then fluoresces
Red Blood Cells Leaky Ghosts
Fluorescent Signal /+ +++
Where are the primary amines principally located?
*in the inner leaflet

31. Conclusion:
The lipid content varies between the leaflets within a
membrane
Interior Layer Exterior Layer
Sphingomyelin 6% 20%
Phosphatidyl
9% 23%
Choline
Phosphatidyl
25% 6%
Ethanolamine
Phosphatidyl
10% 0
Serine
Other 1%

32. Red Blood Cells Leaky Ghosts
Sphingomyelin 26% 26%
Phosphatidyl
32% 9% 32% 0%
Choline
Phosphatidyl
31% 25% 31% 0%
Ethanolamine
Phosphatidyl 10% 10% 0%
Serine
Other, including 1% 30% 1% 74%
degradation
products

33. Now, how do we
target…
…toxic drugs with high specificity to solid
tumors, while maintaining drug stability?

34. Liposomes
Preparation
How do you “fill” them with the drug?
*Fill liposomes with the pharmaceutical agent by adding it to the
buffer before sonication
How could you purify the liposomes?
*purify liposomes using gel filtration (you are selecting against free
lipids and giant liposome blobs, so it makes sense to select based on
size)

35. You can also prepare liposomes with planar bilayers
Pinhole
Side view in chamber

36. Prepare Planar Bilayers (cont.)
a. Place a fine-tip paintbrush into membrane-forming solution.
b. Stroke it over a hole (1 mm in diameter)
c. Bilayer forms

37. PEG or
other
compound

38. Her-2 isreceptorin
• Her-2 receptor overexpressed
~25% of breast cancer patients
• “Trastuzumab”, a recombinant HER-2
antibody conjugated to chemo-loaded
lipsomes
• Hard tumors have “enhanced
permeability and retention effect”. That
is liposomes will preferentially
“extravasate” in the abnormal blood
vessels that occur in tumors.

39. Dox = doxorubicin
Clinical Cancer Research (2002) 8: 1172-1181

40. Homework
How would you prepare Anti-Her2
immunoliposomes?
- Show individual steps in a flow chart.
Also, how does the liposome deliver its contents
into a cancer cell once it has arrived at the
cancer cell?
For reference see - Liposome-based drug delivery in breast cancer treatm
Park, J. W. Breast Cancer Research (2002) 4: 95-99