Article pubs.acs.org/Langmuir
Encapsulating Nanoemulsions Inside eLiposomes for Ultrasonic Drug Delivery Marjan Javadi,† William G. Pitt,*,† David M. Belnap,‡ Naakaii H. Tsosie,† and Jonathan M. Hartley† †
Chemical Engineering Department and ‡Department of Chemistry and Biochemistry, Brigham Young University, Provo, Utah 84602, United States ABSTRACT: An eLiposome is a liposome encapsulating an emulsion nanodroplet and can be used for drug delivery. For example, therapeutic agents are encapsulated inside the eLiposomes, and the application of ultrasound can cause the emulsion droplet to change from liquid to gas, thus increasing the volume inside the vesicle and causing rupture and the release of the drug. In this research, two different methods were used to prepare eLiposomes. In the first method, emulsion droplets were made of perfluorohexane or perfluoropentane and stabilized with 1,2-dipalmitoyl-sn-glycero-3-phosphate. A layer of 1,2-dimyristoyl-snglycero-3-phosphocholine was dried in a round-bottomed flask. Then the emulsion suspension was added to the flask. As the suspension hydrated the phospholipids, they formed liposomes around the emulsions. In the second method, emulsions and liposomes were made separately, and then they were mixed using ultrasound. The advantage of this second method compared to the previous one is that eLiposomes can be made with fewer restrictions because of incompatible combinations of surfactants. Dynamic light scattering and transmission electron microscopy were used to measure the size of the emulsions, liposomes, and eLiposomes. The size of eLiposomes is appropriate for extravasation into tumors with malformed capillary beds. We hypothesize that ultrasound breaks open these eLiposomes. Both types of eLiposomes were constructed with folate attached via a poly(ethylene glycol) tether to induce endocytosis of the eLiposome. The latter eLiposomes were successfully used to deliver calcein as a model drug to HeLa cells.
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INTRODUCTION Passive and active drug targeting are the first steps in the process of delivering a drug or drug carrier beyond the endothelial barrier of capillaries and then attaching it to a target cell. However, these need to be followed by triggered release to control the time and the site of drug delivery more precisely. This article describes a new delivery vehicle that combines ultrasound with phase-changing liquids to trigger the release of therapeutics from a liposome. Liposomes have many advantages as drug-delivery agents. They are biocompatible and biodegradable, and they can be functionalized with surface molecules such as poly(ethylene glycol) (PEG) to increase their circulation time1 or with targeting ligands (e.g., folate or antibodies) to direct their attachment to specific cells.2 Drug-carrying liposomes can be formed by hydrating phospholipids in an aqueous phase containing the therapeutic. As the spherical liposomes form, the drug is encapsulated in the inner aqueous compartment of the liposome. Alternatively, the liposome can be formed first and then the drug can be loaded by pH gradient techniques.3 In addition to liposomes, nanoemulsions are also commonly used in the pharmaceutical industry. The discrete phase of an emulsion is usually a hydrocarbon, but perfluorocarbon (PFC) emulsions are finding increased use in ultrasonic drug delivery and as ultrasound contrast agents. PFC emulsions have traditionally been used as oxygen carriers in blood substitutes.4 Recently there has been much attention paid to perfluor© 2012 American Chemical Society
ocarbon (PFC) emulsions as phase-changing liquid, with particular emphasis on the use of ultrasound to produce the phase change.5−9 Ultrasound has been used for decades in medical imaging and in some therapeutic applications.10,11 Its great advantage is that energy can be transmitted across the skin without surgical intervention. Ultrasound has recently been used in targeted drug delivery in which the ultrasound is focused on a tissue, and the therapeutic may be free or contained in micelles or liposomes.12 For nearly all ultrasonic drug-delivery applications, acoustic pressure waves cause oscillations and the eventual collapse of gas bubbles, called cavitation phenomena; these cavitation events are the foundation of mechanisms leading to the release of therapeutics from carriers13−17 and the disruption of cell membranes12,18,19 that allow more rapid internalization of the locally released agent. A challenge with using ultrasound and bubbles for drugdelivery systems is that the bubbles are too large to extravasate from the circulatory system into the tumor tissue, so the location of drug release is restricted to the circulatory system. However, small liposomes, less than 1 μm in diameter, can escape through the leaky capillaries found in many tumors.20 Usually liposomes do not contain gas, so they are not easily activated by ultrasound to release therapeutics. However, there Received: June 15, 2012 Revised: September 17, 2012 Published: September 18, 2012 14720
dx.doi.org/10.1021/la303464v | Langmuir 2012, 28, 14720−14729
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is a class of liposomes, called echogenic liposomes or bubble liposomes, that are designed to contain a gas phase such that they scatter ultrasound and can be used as both a drug-delivery vehicle and an ultrasound contrast agent.21−27 Our research group is developing a new delivery vehicle that contains nanoemulsions of PFC liquids inside liposomes.28 By using a PFC with a boiling temperature near body temperature, such as perfluorohexane (PFC6, bp = 57 °C) or perfluoropentane (PFC5, bp = 29 °C), we can use ultrasound to cause an expansion of the PFC liquid into gas, which will break open the liposome and release the drug. During the low-pressure phase of the ultrasound wave, the local pressure drops below the vapor pressure of the PFC and may induce a phase transition from liquid to gas. Although the gas may condense back into liquid when the local pressure increases, during the short time of gas formation the increased volume can break open the liposome because the lipid bilayer membrane can sustain only 3% expansion before rupture.29,30 The challenge is to create stable liposomes that sequester therapeutics along with emulsions that are easily activated by ultrasound. Recently, we have formulated such an emulsion-containing liposome, which we call an eLiposome.28 We call our first formulation a two-step eLiposome because liposomes are formed twice. First, small (
