274093 Ultrasound Induced Calcein Release From Eliposomes

Tuesday, October 30, 2012: 10:05 AM
Allegheny III (Westin )
William G. Pitt, Department of Chemical Engineering, Brigham Young University, Provo, UT, James R. Lattin, Chemical Engineering Dept, Brigham Young University, Provo, UT, Ghaleb A. Husseini, Chemical Engineering Department, American University of Sharjah, Sharjah, United Arab Emirates and David Belnap, Department of chemistry and Biochemistry, Brigham Young University, Provo, UT

Ultrasound Induced Calcein Release from eLiposomes

J.R. Lattin1, W.G. Pitt, Ph.D.1, D.M. Belnap, Ph.D.2, G.A. Husseini, Ph.D.3

1 Chemical Engineering Department, Brigham Young University, 2 Chemistry and Biochemistry Department, Brigham Young University, 3 Chemical Engineering Department, American University of Sharjah, Sharjah,


Liposomes - bilayer lipid vesicles - have proven to be effective and versatile drug carriers. Targeted drug delivery from liposomes could be used to further increase their delivery efficiency by more specifically controlling the site of drug delivery. One such method of targeting involves using ultrasound to release sequestered drugs at a specific target site from carriers such as microbubbles. However, liposomes are not inherently responsive to ultrasound. The goal of the current work is to develop a sub-micron sized liposomal drug carrier that is responsive to low intensity ultrasound. The proposed drug carrier is called an eLiposome, defined as a liposome containing vaporizable emulsion droplets. The nano-sized emulsion droplets are formed from perfluorocarbon liquids with high vapor pressures. During the low pressure phase of an ultrasound wave, the local pressure may drop below this high vapor pressure, allowing the formation and expansion of a vapor phase [1]. As this vapor phase forms and expands, it stretches and disrupts the liposomal membrane, causing local drug release. Emulsions were formed from perfluorohexane (PFC6) and perfluoropentane (PFC5) because of their relatively high vapor pressures and non-toxicity. Additionally, both of these perfluorocarbons have very low solubility in water, enabling nanodroplets to persist in an aqueous environment.

Emulsions were formed by inserting a 20 kHz ultrasound probe into a cuvette containing 0.2 g of PFC and 5 mg of dipalmitoyl phosphatidyl choline (DPPC) in 1.5 mL of water. Large (400 nm) and small (100 nm) emulsion droplets were prepared by varying sonication time and intensity and/or by extrusion. Sizes were verified by dynamic light scattering. eLiposomes were formed using a lipid sheet refolding technique [2]. Interdigitated DPPC sheets were formed from small DPPC vesicles by adding ethanol. After sheet formation, excess ethanol was removed from the sheet suspension by two cycles of centrifugation and washing. 0.2 mL of emulsion was added to 10 mg of interdigitated sheets along with 0.2 mL of water (for TEM imaging) or a 30 mM calcein solution (for release experiments). The solution was heated to 50C and stirred with a magnetic stir bar for 30 minutes, allowing the sheets to fold back into vesicles and trapping nanoemulsion droplets inside [3]. The size distribution of the resulting eLiposomes was controlled by extrusion through an 800-nm polycarbonate filter at 50C. Empty eLiposomes, defined as liposomes refolded from interdigitated sheets in the absence of emulsion, were prepared as a negative control.

The eLiposomes were imaged using cryogenic transmission electron microscopy (cryoTEM). The microscope stage was rotated and the sample was viewed at -45, 0, and +45 to verify encapsulation of the emulsion droplets [3]. The cryoTEM images verify that PFC6 emulsion droplets have been encapsulated inside the lipid vesicles using the sheet refolding technique and that this method was able to encapsulate both large (400 nm) and small (100 nm) emulsion droplets. Figure 1A shows an example of an 800-nm eLiposome with 3 distinct 100-150 nm emulsion droplets. Figure 1B shows an example of an 800-nm eLiposome with one 475-nm emulsion droplet as well as two smaller droplets.

Figure 1. CryoTEM images of 800-nm eLiposomes containing A) three 100-150 nm PFC6 emulsion droplets or B) one 475-nm droplet and two smaller droplets. Scale bars represent 200 nm. The straight (B, top left) and mottled (A, bottom) structures are supporting entities of the holey carbon support film

Calcein containing samples were prepared for quantification of release. During vesicle formation, calcein was encapsulated inside of the eLiposomes at a concentration of approximately 15 mM. At this concentration, the calcein was self-quenched. The external calcein concentration was reduced by allowing the sample to settle at the bottom of the microcentrifuge tube for a few hours to form a gel-like pellet. The top phase was removed and replaced with an NaCl solution. Samples were further diluted by adding 20 L of sample to a cuvette along with 2 mL of NaCl solution in order to leave an external calcein concentration of 1 to 5 M so as to be within the linear region of the concentration curve for calcein. Sample fluorescence was measured using a QuantaMaster fluorometer (Photon Technology International, Birmingham, New Jersey) with excitation and emission wavelengths of 488 nm and 525 nm, respectively. Baseline fluorescence data was collected for 10 seconds at 4 data points per seconds. The cuvette was then removed from the fluorometer and 20-kHz ultrasound was applied using a 3 mm probe (Sonics and Materials, Newton, CT) inserted directly into the cuvette. As concentrated (self-quenched) calcein was released from the interior of the eLiposomes into the surrounding solution, it was diluted below its self-quenching concentration. Fluorescence was again measured after sonication. Finally 25 L of 5% Triton X-100 was added to lyse any remaining liposomes and fluorescence was measured

Figure 2 shows the results as the time of ultrasound exposure was varied from 100 ms to 10 seconds at 1 W/cm2. The eLiposome samples demonstrated increased calcein release compared to conventional liposomes (containing only calcein) and compared to conventional liposomes with external emulsions, suggesting that the encapsulated emulsion droplets add an ultrasound sensitive element to the eLiposomes. The eLiposomes were less sensitive to ultrasound when small emulsion droplets were encapsulated. This is most likely due to the greater Laplace pressure imposed on the droplets as their size decreases [4]. The increased pressure on the droplet adds to the ultrasound intensity required to vaporize the droplet. Because Laplace pressure is inversely proportional to radius, the amount of pressure on the droplet increases as droplets diameter decreases. In this study, the effect of droplet size can be best observed with PFC5 eLiposomes: eLiposomes with large emulsion droplets released approximately twice as much calcein as those with small droplets after 10 seconds of ultrasound exposure. There was also a noticeable difference between eLiposomes formed with PFC5 and PFC6. This difference is most likely due to the difference in vapor pressures; it is easier to promote ultrasonically induced gas formation with the higher vapor pressure of PFC5.

Figure 2. Calcein release from PFC5 eLiposomes (A) and PFC6 eLiposomes (B) when exposed to 20-kHz ultrasound at 1 W/cm2 for varying times. Data is presented for eLiposomes with large (n) and small (o) emulsion droplets, empty control vesicles (l), and empty vesicles with Large (p) or small (r) emulsion droplets added to the exterior solution. Sham experiments were also performed using eLiposomes with large droplets (u). Error bars represent 1 standard deviation.

eLiposomes formed with PFC5 and PFC6 both released significantly more calcein than controls, suggesting that the internal emulsions do indeed add an ultrasound sensitive component to liposomes.



1. Rapoport, N.Y., et al., Controlled and targeted tumor chemotherapy by ultrasound-activated nanoemulsions/microbubbles. Journal of Controlled Release, 2009. 138(3): p. 268-276.

2. Kisak, E.T., et al., The vesosome - A multicompartment drug delivery vehicle. Current Medicinal Chemistry, 2004. 11(2): p. 199-219.

3. Lattin, J.R., D.M. Belnap, and W.G. Pitt, Formation of eLiposomes as a drug delivery vehicle. Colloids and Surfaces B-Biointerfaces, 2012. 89: p. 93-100.

4. Sheeran, P.S., et al., Formulation and Acoustic Studies of a New Phase-Shift Agent for Diagnostic and Therapeutic Ultrasound. Langmuir, 2011. 27(17): p. 10412-10420.


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