Introduction

Materials Science and Engineering of the Low Temperature Sensitive Liposome (LTSL): Composition- Structure-Property Relationships That Underlie its Design and

Performance

DAVID NEEDHAM*

^a

AND MARK W. DEWHIRST

^b

aDepartment of Mechanical Engineering and Material Science, Duke University, Durham NC 27705, USA, and DNRF Niels Bohr Professor, and HCA Academy Visiting Professor, University Southern Denmark, DK-5230 Odense M, Denmark;^bGustavo S. Montana Professor, Director of Tumor Microcirculation Laboratory, Department of Radiation Oncology, Duke University Medical Center, Duke University, Durham, NC 27708, USA

*Email: d.needham@duke.edu

solid-liquid phase transition temperature), and then extruded under positive pressure through a 100-nm nanopore filter, a suspension of unilamellar liposomes is produced, with diameters ofB100 nm.^2,3This simple fabrication process has become the basis for a range of drug-delivery systems based on the biologically ubiquitous and compatible liposome, or small unilamellar vesicle (SUV). Even by the late 1970s a huge number of compounds had been encapsulated in liposomes,⁴ and new applications were being sought for this potentially revolutionary drug-delivery system. One of the principal drugs at the time, doxorubicin, was successfully encapsulated into conventional liposomes and testedin vivo.⁵As reviewed by Waterhouseet al.,⁶the liposome formulations were found to maintain the anticancer activity of free doxorubicin in mice, while at the same time decreasing its associated cardiotoxicity. As a result, two main liposomal formulations of doxorubicin, Doxil^s and Myocett, have been developed and evaluated in clinical trials. Doxil is FDA approved for several clinical indications, and Myocet is approved in Europe and Canada for treatment of metastatic breast cancer in combination with cyclophosphamide.

As listed by Maureret al.,⁷such a self-assembled nanocapsule is made up of just 95,000 molecules of lipid and has an encapsulated volume of 3.810^–19 liters. If a drug is encapsulated at 100 mM, there will be 2.410⁴molecules of drug per nanocapsule. While keeping the drug inside the liposome is a favorable consequence that reduces toxicity^8,9 (and references therein), one major obstacle to a more effective use of liposomes as drug-delivery vehicles is actually to get the drug out, perhaps in response to a local biological or other applied trigger. This is particularly important for treating cancer, especially with the more traditional, highly toxic, chemotherapeutics. Lipids can become ‘‘smart materials’’ when they perform a desired function in response to an environ- mental change.

Given that we want to use the encapsulating function of lipids as bilayers to achieve some drug-delivery goal, we might immediately start to ask,

‘‘What makes the self-assembly process (that is also critical to encapsulating every cell on the planet) form such ultrathin (5 nm), two-molecule-thick structures?’’ and ‘‘How can they be made to have encapsulating and release functions?’’ The basis for all materials design and innovation is a deep and detailed understanding of the relationships between a material’s composition,structureand properties(CSP; see Chapter 1 in this volume for general concepts), and how these relationships influence processing and performance of the material in service, in this case in the bloodstream or other tissue-milieu in the body. We will get into much more detail in subsequent sections of this chapter regarding lipids, lipid bilayers and micelles, but to give an initial example of what we mean by CSP rela- tionships, let’s look at one of the most common lipids that we have relied on as a prototypical lipid bilayer material, 1-stearoyl-2-oleoyl-sn-glycero- 3-phosphocholine (SOPC).

As shown in Figure 2.1, SOPC iscompositionallyloaded with carbons and hydrogens with an empirical formula of C₄₄H₈₆NO₈P. Structurally, it is

34 Chapter 2

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amphipathic comprising a hybrid structure of a glycerol backbone, a phos- phocholine head-group and two hydrocarbon chains. While we will later be concerned with thepropertiesof its assembly as bilayers, this molecule also has properties of its own, like its water solubility that is made up of the two amphipathic parts. We see that while the hydrophilic, glycerylphosphoryl- choline has a solubility S¼4.8 M,¹⁰the two hydrophobic hydrocarbon tails are so relatively insoluble in water (stearoyloleoylglycerol S¼B5 nM (ALOGPS predicted value)) that they largely determine the SOPC molecular solubility of onlyB22 nM (ALOGPS predicted value). Thermodynamically, then, it is this high head-group solubility and extremely low hydrocarbon chain solubility that largely govern the self-assembly of the bilayer in water, i.e. an overriding entropic exclusion of the hydrocarbon from water (the so-called hydrophobic effect¹¹) and a protection of the assembled hydrocarbon chains by a double interface of the phosphocholine to form a bilayer.¹²

So now we have the basis for a membrane capsule. Beyond this global hydrophobic effect though, there are subtleties that determine additional composition-structure-propertyrelationships that, in turn, underlie and direct our ‘‘Smart Materials Design’’. Examples here include the response of the bilayer material to one or more environmental cues, like pH^y,¹³enzymes,¹⁴or, in our case, temperature.^15,16This chapter will present and explore the material relationships that went into the discovery, design and performance of the so- called Low Temperature Sensitive Liposome (LTSL). Single Giant Unilamellar Vesicle (GUV) experiments and data that we have carried out and reported over the past 32 years will be presented and described in terms of classical materials science and materials engineering design. These micropipet experiments and analyses are unique to membrane mechanics and were literally instrumental in arriving at the LTSL formulation. This particular LTSL lipid composition has combined the benefits of lipid encapsulation (reduced drug

Composition Structure Property

C₄₄H₈₆NO₈P Solubility = ~10 nM

Figure 2.1 Chemical composition (empirical formula), molecular structure (space filling) and apropertyof solubility for a prototypical lipid bilayer material, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC).

ySee also Chapter 3. pH-sensitive Liposomes in Drug Delivery, Shivani Rai Paliwal, Rishi Paliwal, and Suresh P Vyas.

Materials Science and Engineering of the Low Temperature Sensitive Liposome 35

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toxicity) together with mild hyperthermia (triggered drug release) to provide a clinically attainable way to get a drug like doxorubicin out of the liposome and into all cells of a tumor (neoplastic, stroma, pericytes and endothelia), in unprecedented amounts.¹⁷

2.1.2 Micelles, Bilayers and Inverted Micelles

The best way to start thinking about how to design a ‘‘smart’’ liposome for any application is to understand what makes a particular lipid molecule form a bimolecular leaflet or lipid bilayer, and what makes it form other structures. It would be the transition or transformation to these other structures with different properties that creates the potential for an environment-sensitive response. Israelachviliet al. treated the lipid-packing problem (what it takes to form micelles, bilayers and inverted micelles) in terms of a chain packing parameter.¹⁸The chain packing parameter is defined relative to the area of the lipid head-group at the lipid-water interface (A), the volume of the entire lipid molecule (V) and its length (l). When V/AlB1, lamellar structures are formed, whereas normal micelles and inverted curved structures are obtained for V/Alo1 and V/Al41, respectively.

With this brief introductory background (see also the references section) let’s now look at what these simple rules mean for bilayer composition-structure- propertyrelationships for a single lipid bilayer that, in the case of the LTSL, contains a majority of di-chain phosphatidylcholine lipids (V/AlB1), and in the same bilayer a minority of a mono-chain phosphatidylcholine (V/Alo1), and a third component, also in low concentration, a distearoyl PEG lipid (also V/Alo1). The goal is to understand in as much detail as possible how this compositionwith bilayer, grain domain and other defectstructurescan have all thepropertiesneeded for it to load, encapsulate and retain a drug, and trigger the release of the encapsulated drug in response to just a few degrees rise in temperature.