Mcbii recitation 1 membranes 2011 2

Molecular & Cell Biology Recitation

Spring, 2011

Recitation Instructors:

Prof. Eric Brenner [email protected] Duboue [email protected] Poslusny [email protected]

How can the recitation help you?

Recitation Participation: 5%

(Attendance, Discussion, Group Work)

Recitation Presentation: 5%

Class Paper: 10%

=20% Final Grade

Recitation 1:Lipids and Cell Membrane Fluidity

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

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

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?

Nanomedicine(The medical application of

nanotechnology)Nano drugs = 10 to 100 nanometers

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

Nanomedicine uses nanoparticles

Liposomes are artificially prepared vesicles made from a lipid bilayer.

So, how can membranes be used in nanomedicine?

To understand how liposomes are made, we must first review membranes and their characteristic components…

Liposomes can be filled with drugs, and used to deliver these drugs to cancer cells and other diseased cells or tissues

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?

Basic Structure of a Phospholipid:

What does amphipathic mean?

How are membrane components oriented?

*Having both hydrophilic & hydrophobic properties

*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.

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

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

Ex. butter (saturated fats) are solid at room temp

What is healthy vs. unhealthy?

What kind of lipid is this? (what’s the distinguishing feature?)

Sphingolipids are defined bytheir sphingosine group, are(mostly) uncharged, and commonly found in neuronalcell membranes

*sphingolipid, defined by the sphingosine (red)

*saturated

*uncharged

What are the features of the side chains?

What is the overall charge on this lipid?

Unsaturated chains “kink”, can’t pack in as densely made make the membrane more fluid

Ex. oils are liquid at room temp

What kind of lipid is this? (what’s the distinguishing feature?)


*phospholipid

*one saturated, one unsaturated

*it has one (-) & one (+), but overall it is uncharged


What kind of lipid is this? (what is it made of?)

Unsaturated fats help maintain membrane fluidity.

What other famous lipid also helps?

This lipid is made of ceramide & glucose

*glycolipid (it contains glucose)

*one saturated, one unsaturated

*it is uncharged


What is the overall charge on this lipid? Any +, -, neutral?

What can this structure tell us about how this molecule may work in the membrane?

Cholesterol

*the ring structure is hydrophobic, and the -OH is hydrophilic

*uncharged

*it is found inside the bilayer

Is it found mostly inside the bilayer or does it stick out?


What features make it amphipathic?

*it helps maintain fluidity by preventing interactions between fatty acid side chains

Varies Among Membranes

Examples:

Myelinated Nerve Cells:Cholesterol and Cerebrosides

Liver Cell Plasma membranes:Cholesterol and

Phospholipids

Lipid Content of Membranes…

How can we measure the various lipids in a membrane?

1. Obtain Sample Tissues, Cell Extracts, etc…)

2. Remove Lipids, but leave behind proteins, carbohydrates, etc.)

thin-layer chromatography, HPLC and more advanced methods

Ch

PE

PC

PS

How?

Organic solvent eg. chloroform/methanol, cyclohexane

How detect?

Part II: Techniques

What’s osmotic lysis?

Place the cell in a hypotonic solution allowing it to burst

Prepare "Ghosts" How do we study membrane lipids?

RBC Leaky Ghost

Intact cell membranes are impermeable to enzymes and SITS; however, ghosts are leaky, and enzymes and SITS can

enter them!

Ghost can be used to study membrane composition

How can we assess the asymmetry of the lipid composition of leaflets?

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

*Sequentially use enzymes that selectively degrade different components

Red Blood Cells*

Ghosts*

Sphingomyelin

Phosphatidyl Choline

Phosphatidyl Ethanolamine

Phosphatidyl Serine

Other* These values are expressed as percentages of phospholipids alone; cholesterol and glycolipids are not included.

26% 26%

32% 32%

31% 31%

10% 10%

1% 1%

Before treatment

What do these lipids share in common?

*these are all phospholipids

RBC

Sphingomyelin

Leaky Ghost

sphingomyelin: 26%->6%


Treatment with sphingomyelinase

RBC

Sphingomyelin

Leaky Ghost



Treatment with sphingomyelinase

Sphingomyelin quantification and location

Which leaflet contains sphingomyelin?

Difference= sum of changes

Red Blood Cells Leaky Ghosts

Sphingomyelin

PhosphatidylCholine


Phosphatidyl Serine

Other, including degradation products

26% → 6% 26% → 0%

32% 32%

31% 31%

10% 10%

1% → 21% 1% → 27%

*it is in both the inner (6%) & outer(20%) leaflet

Sea snake venom contains phospholipases that degrade phosphoglycerides


Sphingomyelin

PhosphatidylCholine


Phosphatidyl Serine


Which leaflets contain phosphoglycerides?

26% 26%

32% → 9% 32% → 0%

31% → 25% 31% → 0%

10% 10% → 0%

1% → 30% 1% → 74%

*the phospholipids are in both the inner (44%) and outer (29%) leaflet

SITS: membrane impermeable compound that binds to -NH3

+ groups and then fluoresces


Fluorescent Signal

Where are the primary amines principally located?

−/+ +++

*in the inner leaflet

Interior Layer Exterior Layer

Sphingomyelin 6% 20%

PhosphatidylCholine

9% 23%


25% 6%

Phosphatidyl Serine

10% 0

Other 1%

Conclusion: The lipid content varies between the leaflets within a

membrane


Sphingomyelin

PhosphatidylCholine


Phosphatidyl Serine


26% 26%

32% → 9% 32% → 0%

31% → 25% 31% → 0%

10% 10% → 0%

1% → 30% 1% → 74%

Now, how do we target…

…toxic drugs with high specificity to solid tumors, while maintaining drug stability?

Liposomes Preparation

How do you “fill” them with the drug?

*purify liposomes using gel filtration (you are selecting against free lipids and giant liposome blobs, so it makes sense to select based on size)

How could you purify the liposomes?

*Fill liposomes with the pharmaceutical agent by adding it to the buffer before sonication

Pinhole

You can also prepare liposomes with planar bilayers

Side view in chamber

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

PEG or other compound

Her-2 receptor• Her-2 receptor is overexpressed in

~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.

Dox = doxorubicin

Clinical Cancer Research (2002) 8: 1172-1181

HomeworkHow 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 treatment,Park, J. W. Breast Cancer Research (2002) 4: 95-99

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Mcbii recitation 1 membranes 2011 2