Liposome Nanoparticles

## Introduction

Liposomes are spherical lipid vesicles with a bilayered membrane structure composed of natural or synthetic amphiphilic lipid molecules. Liposomes have been widely used as pharmaceutical carriers in the past decade because of their unique abilities to

1. encapsulate both hydrophilic and hydrophobic therapeutic agents with high efficiency,
2. protect the encapsulated drugs from undesired effects of external conditions,
3. be functionalized with specific ligands that can target specific cells, tissues, and organs of interest,
4. be coated with inert and biocompatible polymers such as polyethylene glycol, in turn prolonging the liposome circulation half-life *in vivo*, and
5. form desired formulations with needed composition, size, surface charge, and other properties.

In this project, we aim to understand the self-assembly mechanism of lipid molecules to form liposomes and to investigate the preparation of liposomes through an extrusion method. Specifically, the goals are to

1. prepare liposomes using an extrusion method,
2. control the size and size distribution of liposomes,
3. evaluate the quality of the synthesized liposomes, and
4. understand the self-assembly mechanism of the formation of liposomes.

### Pre-Lab Questions

You can download the Pre-Lab questions for this experiment here; the Gilson document for Prob. 4 can be downloaded here.

## Background

Liposome nanoparticle synthesis techniques and applications are quite varied. A few relevant references are as follows:

* How to stabilize phospholipid liposome using nanoparticles.
* Nanoparticles in medicine: therapeutic applications and developments.
* Recent advances with liposome as pharmaceutical carriers.
* Liposome application: problems and prospects.
* Vesicles of variable sizes produced by a rapid extrusion procedure.
* The antimicrobial activity of liposomal lauric acids against Propionicbacterium acnes.

## Theory

### Liposome Formation

Lipids are characterized by hydrophobic and hydrophilic domains on a single molecule which can self-assemble to form lipid bilayers in order to minimize unfavorable interactions between the various domains and the surrounding liquids (**Figure 1**).

Many different synthesis routes have been established to produce nanometer-sized liposomes; here, we use an extrusion technique to produce large, unilamellar vesicles (LUVs) by hydration of a lipid film. This synthesis proceeds through five steps:

1. A dilute lipid solution containing phospholipid(s) and cholesterol is prepared in an organic solvent such as chloroform.
2. The lipid solution is dried to yield a multi-layer lipid film on the container surface.
3. Water is added to swell the film.
4. Agitation forms large, multilamellar vesicles (LMVs) from the swollen lipid sheets.
5. Extrusion through small pores produces vesicles with diameter proportional to the pore size. Here, we use 100 nm pores to produce large, unilamellar vesicles (LUVs) with diameters of approximately 120 nm. Small, unilamellar vesicles (SUVs) can be produced by sonication or extrusion through smaller pores.

### Dynamic Light Scattering

As the name implies, liposome nanoparticles should have characteristic lengths on the order of hundreds of nanometers which prohibits visualization by optical microscopy. A common technique for characterizing the size distribution of small particles is dynamic light scattering (DLS). Theoretical development of the correlations used in DLS analysis are beyond the scope of this lab but you should be familiar with the basic operating principles. Fortunately, several useful tutorials are available online and include

* a video to introduce the Principles of Dynamic Light Scattering,
* operational guidelines from Malvern, a DLS instrument manufacturer, which also describes data interpretation,
* theoretical overview from the Weitz Lab at Harvard University, and
* other particle characterization techniques from Malvern, which also describes relevant data interpretation methods.

It's recommended that you review these documents before attempting to interpret your DLS data. Generally, we're interested in the *z*-average diameter, polydispersity index (PDI), and number- and volume-weighted size distributions.

## Standard Operating Procedure

### Safety

* Lab coats and protective eyewear should be worn at all times. All experiments should be performed in the hood with nitrile gloves.
* Dispose of broken glassware in the appropriately labeled receptacles; used filters and spacers can be disposed of in the trash.
* Do not dispose of waste down the drain! A labeled waste bottle should be in the hood for all waste; if you can't find it then notify an instructor or TA and you'll be provided one.
* Be careful when handling syringes and needles for the extrusion procedure and make a note of nearby first aid kits in case of needle puncture.
* Relevant chemical information has been listed in **Table 1**.

### Tutorial Video

Check out the video below for a brief overview of this experiment, then carefully read the instructions below. Keep in mind that **there may be variations** in the equipment or protocol when you're in the lab compared to when this video was created. Ask a TA or instructor if you're not clear on something.

### Preparation

The TA will provide you with several stock solutions which you should dilute for further use as described below. Table 2 summarizes the "baseline" solution volumes and concentrations. Use a pipetter and glass vials for all solutions, and the vortex mixer or sonicator for mixing.

1. **Phospholipid solution:** The TA or instructors will provide you about 5 mL of a stock solution of phospholipid/chloroform solution at 10 mg phospholipid / mL chloroform.
2. **Cholesterol solution:** The TA or instructors will provide you a stock solution of about 1 mL of a cholesterol/chloroform solution at 10 mg cholesterol / mL chloroform.
3. **Lipid solution:** Typical cell membranes contain about 10% cholesterol and 90% phospholipid; this combination will be referred to simply as the "lipid." Prepare 2 mL of a lipid/chloroform solution at 2.25 mg lipid / mL chloroform by pipetting 450 uL phospholipid solution, 50 uL cholesterol solution, and 1.5 mL chloroform into a glass vial. A common procedure in this lab is to vary the cholesterol/phospholipid ratio and examine its effects on the nanoparticles.

**Table 2.** Baseline solutions for liposome synthesis.
 Cholesterol (stock)
 Lipid

#### Gravimetric Verification Procedure for Pipettes

Everyone who uses a pipetter will use it slightly differently from everyone else, and the pipettes themselves have an intrinsic error. You should quantify this variance so that you can accurately report the uncertainty in your solution concentrations by following the gravimetric verification procedure that you practiced in your Pre-lab questions. This procedure can be performed *after* preparing the solutions noted above; a particularly convenient time to perform such a verification is during the chloroform evaporation process noted below.

### Basic Operation

#### Lipid Film Hydration

The first step to produce liposome nanoparticles is to produce a lipid film which is then re-hydrated to form large, multi-lamellar vesicles (LMVs).

1. Use an air stream to gently evaporate *all* chloroform from the lipid solution to yield a lipid film on the vial walls. (Reminder: now is a good time to perform the pipette verification procedure.
2. Hydrate the lipid film by adding 2 mL deionized (DI) water.
3. Agitate in the sonicator (5-10 min) then with the vortex mixer (~3 min) or until the mixture looks cloudy.

**STOP: Before extrusions, ask a TA to receive proper training on luer-lok syringes!**

#### Extrusion

Lipid extrusion is a technique in which a solution of LMVs is forced through a small pore, typically 0.2 - 1.0 um in diameter, to produce small unilamellar vesicles (SUVs, diameter < 10-7 m) or large unilamellar vesicles (LUVs, diamater ~10-7-10-6 m).

1. Disassemble the extruder, then add membrane spacers on the O-rings and a 100 nm polycarbonate membrane between the spacers. Reassemble the extruder, taking care to tighten the nuts enough to prevent leaks but not enough to rupture the membrane.
2. Aspirate the sonicated LMV solution into one of the 1 mL glass syringes.
3. While holding the metal tip, carefully twist the loaded syringe onto the luer-lok connection on one of the teflon parts. Connect the empty syringe onto the opposite luer-lok connection.
4. Push the plunger to perform an extrusion while pulling the opposite plunger to collect the sample in the opposite needle. Push **slowly** to avoid breaking the membrane.
5. Repeat Steps 2-4 as many times as desired (solutions are typically extruded 5-15 times).
6. To improve consistency,
	* always use the same syringes for extruding and collecting the sample,
	* check the membrane occasionally to ensure clogging has not occurred due to lipid aggregation, using acetone to clean any organic deposits on the needles, and
	* have a single person perform all extrusions for an individual sample.

#### Dynamic Light Scattering

A TA or Technical Staff will train one person from each group to operate the dynamic light scattering instrument (DLS). It is **required** to receive training **before** proceeding to measure your samples. Once completed, keep in mind:

* The laser is pointing to the right, therefore make sure the cuvette is positioned correctly.
* If the amount of sample produced after extrusion isn't enough to reach the laser, dilute it with DI water.
* Instructions are located in near the computer

### Shutdown

1. Disassemble, clean with acetone, and dry the extruder and syringes.
2. Collect all waste--including any produced during cleaning--in the provided waste bottle in the hood.
3. Dispose of membranes and spacers in the lab trash.
4. Notify the TA or instructor of any unused stock solution, which must be stored in the refrigerator.

## Common Report Mistakes and Suggestions

The evaluator(s) for this experiment was asked to list three or more common mistakes often made on the reports for this experiment, or to provide three or more suggestions that could improve the quality of reports for this experiment. The responses were as follows:

1. The method section should include all experimental details that others can repeat exactly the experiments after reading this section. This means, we need to provide quantitative concentration, volume, etc.
2. We always need to conduct an odd number of extrusions, because an even number of extrusions will leave the final liposomes in the initial syringe, which may be contaminated.
3. The key results (liposome size and PDI) should be plotted as final figures and presented in the main report (not in the appendix).

## References

1. ↑ Zhang, L.; Granick, S. *Nano Lett.*, **2006,** *6*, 694-8.
2. ↑ Zhang, L. *et al*. *Clin. Pharmacol. Ther.,* **2008,** *5*, 761-9.
3. ↑ Torchilin, V.P. *Nat. Rev. Drug Discov.,* **2005,** *4*, 145-160.
4. ↑ Barenholz, Y. *Curr. Opin. Coll. Int. Sci.* **2001,** *6*, 66-77.
5. ↑ Mayer, L.D.; Hope, M.J.; Cullis, P.R. *Biochim. Biophys. Acta,* **1986,** *858*, 161-168.
6. ↑ Yang, D.R. *et al*. *Biomaterials,* **2009,** *30*, 6035-6040.