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A targeting drug-delivery model via interactions among cells and liposomes under ultrasonic

excitation

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2008 Phys. Med. Biol. 53 3251

(http://iopscience.iop.org/0031-9155/53/12/012)

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Phys. Med. Biol. 53 (2008) 3251–3265 doi:10.1088/0031-9155/53/12/012

A targeting drug-delivery model via interactionsamong cells and liposomes under ultrasonic excitation

Xiaoyu Xi1, Fang Yang2, Di Chen3, Yi Luo4, Dong Zhang1, Ning Gu2

and Junru Wu3

1 Institute of Acoustics, Lab of Modern Acoustics, Nanjing University, Nanjing 210093,People’s Republic of China2 State Key Laboratory of Bioelectronics, Jiangsu Laboratory for Biomaterials and Devices,School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096,People’s Republic of China3 Department of Physics, The University of Vermont, Burlington, VT 05405, USA4 State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry,Nanjing University, Nanjing 210093, People’s Republic of China

E-mail: Jun-ru.wu@uvm.edu

Received 11 March 2008, in final form 30 April 2008Published 27 May 2008Online at stacks.iop.org/PMB/53/3251

AbstractIn our previous work, it was found that acoustic cavitation might play a rolein improving the cell permeability to microparticles when liposomes wereused in an in vitro experiment. The purpose of this project is to expand ourstudy and to learn other possible mechanisms by which cells may interact withliposomes under ultrasound (US) excitation and become transiently permeableto microparticles. It is further hypothesized that two possible scenarios maybe involved in in vitro experiments: (1) drug-carrying liposomes transientlyovercome the cell membrane barrier and enter into a cell while the cell is stillviable; (2) the liposomes incorporate with a cell at its membrane through afusing process. To prove this hypothesis, liposomes of two different structureswere synthesized: one has fluorescent molecules encapsulated into liposomesand the other has fluorescent markers incorporated into the shells of liposomes.Liposomes of each kind were mixed with human breast cancer cells (MCF7-cell line) in a suspension at 5 (liposomes) : 1 (cell) ratio and were then exposedto a focused 1 MHz ultrasound beam at its focal region for 40 s. The US signalcontained 20 cycles per tone-burst at a pulse-repetition-frequency of 10 kHz;the spatial peak acoustic pressure amplitude was 0.25 MPa. It was found thatthe possible mechanisms might include the acoustic cavitation, the endocytosisand cell-fusion. Acoustic radiation force might make liposomes collide withcells effectively and facilitate the delivery process.

(Some figures in this article are in colour only in the electronic version)

0031-9155/08/123251+15$30.00 © 2008 Institute of Physics and Engineering in Medicine Printed in the UK 3251

3252 X Xi et al

1. Introduction

Recent development in molecular biology and bioengineering has shown that gene therapy maychange how several presently incurable diseases including Alzheimer’s disease, Parkinson’sdisease and other genetic diseases will be treated in future (Anderson 1992, Mehier-Humbertand Guy 2005). Targeting drug and DNA delivery technique is potentially one of themost important technologies in modern medicine. Transfection is a process of introducingrecombinant DNA into eukaryotic cells (eukaryotic cells have chromosomes with nucleosomalstructure) and subsequently integrating the DNA into the recipient cell’s chromosomal DNA(Anderson 1992). Current transfection techniques may be divided into two categories: viraland non-viral. The former achieves the goal using viruses as vectors, such as retroviruses andadenoviruses, and the latter does not. The shortcomings of viral techniques include the spatiallyrandom nature and lack of site specificity (no control over where the gene becomes transfected)and possible adverse side effects such as resistance to metabolic degradation and attack by theimmune system. A controllable non-viral transfection tool to deliver DNA and drug safely andefficiently into a cell is urgently needed to elucidate gene structure, regulation and functionfor genetic microbiology research. Currently, electroporation (Wong and Neumann 1982) andgene-gun technology (Kuriyama et al 2007) are the two popular non-viral transfection toolsused for in vitro research. Electroporation uses high-voltage electrical pulses to make cellmembranes transiently permeable, permitting the cellular uptake of foreign macromolecules.Gene-gun technology uses a beam of high-speed DNA-coated gold particles colliding withtargeted cells to facilitate gene transfer. However, like viral transfection techniques, they alsosuffer from the lack of site specificity, difficulties in control, optimization and applicationsin vivo.

It was demonstrated (Bao et al 1997, Miller et al 1999, 2003, Miller and Quddus2001, Ward et al 1999, 2000, Wu et al 2002, 2006b) that ultrasound (US) assisted byencapsulated microbubbles (EMB) could make cell membranes temporarily open and delivermacromolecules into cells through ultrasound exposure; this technique is called sonoporation.Although the transfection efficiency of sonoporation used in vitro and in vivo (Greenleafet al 1998, Lawrie et al 2000) is relatively low at the current stage, it remains a promisingand potentially useful technique. The advantages of using this EMB-assisted ultrasoundtransfection method include site specificity (ultrasound can be easily focused into a desiredvolume) and the ease of manipulating parameters of US for applications in vivo.

Acoustically induced transfection via sonoporation in vitro uses ultrasonic waves ofmegahertz frequency to excite EMBs such as Optison R© (GE Healthcare, Princeton, NJ, USA)microbubbles mixed with cells in a medium. Encapsulated microbubbles could oscillatemoderately under the ultrasonic agitation (it is called non-inertial or stable cavitation),generating a shear stress on the cell membrane of a nearby cell. Microstreaming (a dc flow)as a result of non-inertial cavitation may be established around an oscillating EMB (Gormleyand Wu 1998) facilitating the entrance of DNA into a cell (Wu et al 2002). Encapsulatedmicrobubbles may also oscillate violently and collapse, experiencing inertial cavitation ortransient cavitation (NCRP 2002). In both cases, cell membranes of those cells in a mediummay ‘open’ for short time allowing foreign molecules or DNA to enter the cells (Prentice et al2005). The use of non-inertial cavitation is much more controllable than the inertial cavitationand generates less non-reparable sonoporated cells. Particularly, in targeting drug and genedelivery in in vitro and in vivo experiments, it has been demonstrated that EMBs excitedby moderate intensity US can increase the permeability of cell membranes allowing gene,therapeutic drugs and antibodies to enter cells (Unger et al 2001, 2002, Miller 2006, Tachibanaand Tachibana 2006, Wu 2006) with the affected cells remaining viable. This process is now

Drug-delivery via liposomes under ultrasound excitation 3253

called reparable sonoporation. Tran et al (2007) have performed an in vitro experiment usingthe ruptured-patch-clamp whole-cell technique. They demonstrated the hyperpolarization ofthe membrane of a marked cell (mammary breast cancer cell line MDA-MB-231) duringsonoporation (1 MHz, 0.15 MPa negative peak US; Sono Vue microbubbles were used). Thehyperpolarization of a cell means an above-normal increase in the trans-membrane voltage.They concluded that US-activated oscillations of EMBs modify the electro-physiologic cellactivities by their ‘cellular massage’ action and thus enhance the cell’s permeability towardmacroparticle uptake. ‘Cellular massage’ here may mean the actions on a cell through themoderate shear stress generated by nearby oscillating bubbles.

Another emerging non-viral delivery technique is to use a liposome as a carrier of DNA ordrugs. Liposomes or lipid vesicles are usually spherical structures composed of curved closedlipid bilayers that encapsulate liquid or gas. Their size ranges from nanometers to microns andthe thickness of the membrane is in the order of nanometers. The most attractive features ofthe liposome include its ability to dissolve, protect and carry hydrophilic or hydrophobicmolecules, its biocompatibility with cell membranes, its low antigenicity, its nanometersize allowing it to enter organs such as the lung, spleen and liver through circulation andthe relative ease of adding special ligands to their surface. Compared with EMB, liposomesare more stable and can enclose DNA or other agents to be delivered as a part of its structure(the complex is often called lipoplex). Further, liposomes can be made anionic, cationic orelectronically neutral according to the electrical property of the target cell. For example,cationic liposomes were produced to interact with negatively charged cell membranes, andthus were made effectively to penetrate into cell (Zhdanov et al 2002). Liposomes can alsobe attached to EMBs as structured delivery vehicles (Kheirolomoom et al 2007, Lentackeret al 2007) so that acoustic cavitation generated by EMBs under US excitation can be takenadvantage to improve the cell permeability. It was shown that anti-rabbit IgG, one typeof antibody, conjugated with Alexafluor 647 was delivered into Jurkat cells in suspensionusing the liposomes by 10% duty cycle ultrasound tone-bursts of 2.2 MHz (the in situ spatialacoustic peak intensity = 80 W cm−2) with an efficiency of 13% (Wu et al 2006a). In thiswork, it was found that acoustic cavitation might play a role to improve the cell permeabilityto microparticles when liposomes were used in an in vitro experiment.

The purpose of this paper is to further search the possible mechanisms by which cellsmay interact with liposomes under US excitation and become transiently permeable tomicroparticles. In terms of drug-delivery in in vitro experiments, two possible scenariosmay be involved: (1) drug-carrying liposomes overcome the cell membrane-barrier and enterinto a cell and the cell is still viable; (2) drug-carrying liposomes incorporate with a cellat its membrane via a fusing process and endocytosis (Brown and Greene 1991, Mukherjeeet al 1997), a process by which a cell uptakes some of its extracellular fluid including materialdissolved or suspended in it. To show that both scenarios may be possible, four types ofliposomes of two different structures were prepared; they are FITC-DPPE, FITC-lecithin,NBD-lecithin and NBD-DPPE (refer to section 2.1 for the full chemical names). The mainstructure difference among these four types of liposomes is illustrated in figure 1 and may besummarized as: the fluorescent FITC marker was encapsulated into the liposomes of the firsttwo types. The fluorescent NBD marker was incorporated into the shells of the last two typesof liposomes (no fluorescent material was encapsulated into them). It is also hypothesizedthat at high concentration small (much smaller than the cell size) liposomes might acquiremoderate relative speed with respect to cells in a focused US field via the acoustic radiationforce and collide with cells like a mini gene-gun. Multiple collisions of several liposomes on acell might produce an accumulated effect that improves the permeability of the cell membrane.Thus, reparable sonoporation might occur and facilitate the entrance of liposomes into a cell

3254 X Xi et al

(a) (b)

(c)

Figure 1. Three possible liposome structures: (a) trans-bilayer tail-to-tail dimmers of NBD-cholesterol within phospholipids, non-fluorescent aqueous solution was encapsulated; (b) someNBD-cholesterol monomers paired with phospholipid monomers in otherwise phospholipid bilayermembrane, non-fluorescent aqueous solution was encapsulated; (c) bilayer phospholipids lipidsstructure, fluorescent FITC was encapsulated.

through acoustic cavitation and endocytosis. Liposomes might also combine with a cell at itsmembrane through a fusion process.

2. Materials and methods

2.1. Liposomes preparation

2.1.1. Materials. 1,2-Diacyl-sn-glycero-3 phosphocholine (PC) and fluoresceinisothiocyanate-dextran (FITC) were purchased from Sigma–Aldrich (St. Louis, MO, USA). 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](PEG2000-DPPE) and 25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27-nor-cholesterol (25-NBD-cholesterol) were purchased from Avanti Polar Lipids Inc. (Alabaster,AL, USA). Soybean lecithin was purchased from Nanjing Genetime Biotech Co., Ltd

Drug-delivery via liposomes under ultrasound excitation 3255

Table 1. Summary of liposomes’ ingredients.

Liposome with 25-NBD-cholesterolincorporated at its shell Liposome with FITC encapsulated

PC:PEG2000- SoybeanDPPE:25-NBD- lecithin:cholesterol: PC:PEG2000- Soybeancholesterol 25-NBD-cholesterol DPPE lecithin:cholesterol

Components (NBD-DPPE) (NBD-lecithin) (FITC-DPPE) (FITC-lecithin)

Molar ratio 90:5:5 85:10:5 95:5 85:15

(Nanjing, China) and Cholesterol was purchased from Sinopharm Chemical Reagents Co.,Ltd (Shanghai, China). All other reagents were of analytical grade and were used as received.

2.1.2. Liposome syntheses. Four types of liposomes were synthesized; they were NBD-DPPE, NBD-lecithin, FITC-DPPE and FITC-lecithin. Table 1 summarizes all four liposomes’main gradients. Briefly, they were prepared as follows. An appropriate amount of PC,PEG2000-DPPE and 25-NBD-cholesterol were mixed in the mole percent ratio of 90:5:5 anddissolved in chloroform in a round-bottomed flask. The chloroform solvent was removedunder N2 using a rotary evaporator. Lipid film was rehydrated with PBS (pH 7.4) to yield aconcentration of 10 mg lipid ml−1 by continuous vortex mixing of the flask for 3 h in a 70 ◦Cwater bath. During this period, the lipid suspension formed a milky solution of liposome.Liposomes consisting of soybean lecithin, cholesterol and 25-NBD-cholesterol were made bythe same protocol as described above. In this case, the molar ratio of these two ingredients is85:10:5. Liposome with FITC encapsulated was prepared as follows: a lipid film consisting ofPC:PEG2000-DPPE (95:5) was prepared following the same procedure as described above ina round-bottomed flask, while FITC solution (4 mg ml−1) was added to rehydrate the lipid filminstead of PBS. Some of the FITC was encapsulated inside the liposomes during the swellingof the lipid film. The liposome solution was subsequently centrifuged with 1000 rpm for8 min and the supernatant was discarded. Precipitated materials were washed with PBS andcentrifuged again. The centrifuging and washing steps were repeated three times to removeany unencapsulated fluorophores. Solution containing liposomes with FITC encapsulatedinside was thus prepared. Soybean lecithin and cholesterol were mixed in the ratio of 85:15by mole in chloroform, and following the same method as described above, lecithin-basedliposomes with FITC encapsulated inside were obtained in the solution.

The concentration of all the four types of liposomes prepared before sonications wasdetermined from three samples (the mean and standard deviation were always calculated fromthree samples unless specified otherwise in this paper) to be (2.8 ± 0.4) × 107 ml−1 by ahemacytometer. The possible structures of liposomes produced are illustrated in figure 1. Thetop left one has trans-bilayer tail-to-tail dimmers of NBD-cholesterol within phospholipids,non-fluorescent aqueous solution was encapsulated. The top right one has some NBD-cholesterol monomers paired with phospholipid monomers in otherwise phospholipid bilayermembrane, non-fluorescent aqueous solution was encapsulated. The bottom left one consistsbilayer phospholipids lipids structure, fluorescent FITC was encapsulated. The first twostructures have no fluorescent material encapsulated; they all have fluorescent NBD includedinto the shells. But the first structure has two NBD-cholesterol molecules paired by themselvesand the second structure has an NBD-cholesterol paired with a phospholipid molecule. Thus,the first structure is more stable than the second one under the ultrasonic excitation. Thedetailed analysis of these structures can be found in Rukmini et al’s work (2001).

3256 X Xi et al

Liposome size distribution in the nanometer region was determined by using a CoulterN4 PLUS Submicron Particle Size Analyzer (Beckman Coulter, Inc., Fullerton, CA, USA).The size distributions of liposomes consisting of soybean lecithin and cholesterol in micronregion were found by using Malvern Mastersizer 2000 (Malvern Instruments Ltd, UK).

2.2. Cell preparation

Human breast cancer cells (MCF7-cell line) were incubated in suspension in a humidified37 ◦C, 5% CO2 atmosphere inside 75 cm2 tissue culture flasks (Corning Inc., Corning, NY,USA) filled with a solution of RPMI (Medium 1640, liquid tissue culture medium containinga nutrient blend of amino acids, vitamins, carbohydrates, organic and inorganic supplementsand salts purchased from Sigma–Aldrich) supplemented with fresh 10% fetal bovine serum(FBS) (GIBCO, Grand Island, NY, USA). For ultrasound exposure experiments, exponentiallygrowing cells were harvested by trypsinization and re-suspended in fresh RPMI 1640 mediawith 10% FBS. The concentration of cell was determined to be (3 ± 0.2) × 106 ml−1 using ahemacytometer.

2.3. Scanning electron microscope and the sample preparation

To observe the effects of ultrasound exposure on cell membranes, three MCF7 breast cancercells for each case were imaged using the scanning electron microscopy (SEM) (Leo 1550,Germany). The samples were prepared for SEM as follows. Each sample was covered in 2.5%glutaraldehyde solution for 3 h and then washed in phosphate buffered saline (PBS, pH 7.4 ±0.1) twice. Alcohol dehydration was followed in 50%, 60%, 90% and 100% ethanol for 20 min,respectively, each stage being repeated twice. Critical point drying was then performed(Polaron E3100 critical point dryer), after which the samples were chromium sputter-coatedfor 5 min at 125 mA in an argon atmosphere (approximately 50 nm coating). A field emissionSEM was used with a gun acceleration voltage of 5 kV and a working distance of 8 mm. Thesecondary electron detector was used to image the samples.

2.4. Ultrasound exposure system and flow cytometer

An arbitrary waveform generator (Agilent 33250A, USA) was used to produce a sinusoidalradio frequency (RF) signal (figure 2). This input signal contains repeated 1 MHz tone-bursts,20 cycles per tone-burst at a pulse-repetition-frequency (PRF) of 10 kHz. It was amplified bya broadband 50 dB RF power amplifier (ENI 2100L, Rochester, NY, USA), and then used todrive a self-made focusing transducer of radius 9.2 cm. The central frequency of the transduceris 1 MHz and the focal distance was 8 cm. The plastic test tubes of 10 mm diameter and 75 mmlength (Kimble, Owens-Illinois, Toledo, OH, USA) filled with cell and liposome suspensioncapped by a rubber stopper (the rubber stopper was used as an sound absorber to minimize astanding-wave effect; there was no air between the cap and suspension) were rotated at 200rpm by a dc motor throughout the exposure period; the rotation helped us to mix liposomeswith cells evenly.

The transducer and test tube were mounted in a glass tank filled with degassed water.The test tube was aligned axially with the transducer in such a way that the center of the cellsuspension in a test tube was situated at a distance of 8 cm from the surface of the sourcetransducer.

A calibrated needle broadband hydrophone (TNU0001A, NTR, Seattle, WA, USA) withan active diameter of 0.6 mm and an upper frequency limit of 20 MHz and a low-noise 30 dB

Drug-delivery via liposomes under ultrasound excitation 3257

Figure 2. Ultrasound exposure system.

preamplifier (HPA30, NTR) were used to measure the acoustic pressure in situ. The calibrationof hydrophone was done using the combination of the beam scan integration techniqueand the acoustic power measurement using the acoustic radiation force (Beissner 1993).The attenuation of the wall of test tubes was found by measuring the ultrasound amplitudewith/without placing the test tube in situ and before the hydrophone using a short US tone-burst. The in situ spatial peak-pressure amplitude after the attenuation correction, Psp, changedwith the applied voltage; for the cases discussed in the results section, its value was 0.25 MPaunless specified otherwise.

Each sample consisted of liposome 0.35 ml mixed with 0.7 ml of MCF7 (human breastcancer) cell solution and was exposed to US for 40 s at 22 ◦C (no measurable temperature riseoccurred during sonications). Ultrasound exposure was performed quickly after a sample wasprepared and liposome and cell suspension were mixed.

Immediately after the sonication, the cells were washed twice, re-suspended in 1 ml PBSand tested for viability and fluorescence (it is an indication of successful delivery) by using aflow cytometer (BD FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA). Normally, it isnot possible for liposomes to enter into a cell due to the protection of the cell membrane. Whenfluorescent MCF7 cells were detected by a flow cytometer, liposomes carrying fluorescentmakers (either FITC or NBD molecules) were delivered into the cells or incorporated with thecell membranes.

The cells that survived sonications were further tested for their viability by mixing withTrypan Blue dye at 0.25 MPa; the detailed description may be found in our previous publication

3258 X Xi et al

Figure 3. Mean diameter + standard deviation for various cases. After sonication, NBD-DPPEliposomes’ size changed from 321.6 ± 29.3 nm to 264.2 ± 8.0 nm, while NBD-lecithin liposomes’size did not change statistically.

(Ward et al 2000). Cells stained by Trypan Blue indicate that their plasma membranes aredamaged (lysis) and the cells are unable to exclude the Trypan Blue dye. The cells survivalrate after sonication was found to be (95 ± 4)%.

Three trials were performed in the same fashion for each of the three cases: (1) cells,no US, no-liposome controls (+cells, −liposomes, −US), (2) liposomes plus cells sham-exposed (+cells, +liposomes, −US) and (3) liposomes plus cells ultrasound-exposed (+cells,+liposomes, +US). A t-test statistical model (two-sample assuming unequal variances)provided by a software package SigmaStat R© (SPSS Inc., Chicago, USA) was used to comparethe delivery efficiency among the three cases.

3. Results

3.1. Liposomes size distribution

NBD-DPPE-liposomes’ size distribution changed after sonications (1 MHz, 0.25 MPa, 40 s)and results of the change can be summarized as shown in figure 3. The mean diameterchanged from 321.6 ± 29.3 nm (mean ± standard deviation) to 264.2 ± 8.0 nm with p < 0.02.For NBD-lecithin-liposomes, the mean diameter changed from 406.7 ± 69.4 nm to 397.0 ±60.7 nm with p = 0.42; i.e., statistically there was no significant change. For the FITC-encapsulated liposomes, like NBD-lecithin-liposomes, the mean diameter (760 ± 350 nm)was unchanged statistically after sonications.

3.2. Optimization of acoustic pressure amplitude

The spatial peak acoustic pressure amplitude Psp was altered from 0.0 to 0.34 MPa by adjustingthe output of the function generator from 400 mV to 800 mV with a 100 mV increment.The FITC-encapsulated liposomes were used as markers and fluorescent-count results of the

Drug-delivery via liposomes under ultrasound excitation 3259

Figure 4. Optimization of acoustic pressure amplitude was achieved by comparing flow cytometerreadings represented as mean + standard deviation of different spatial peak acoustic pressureamplitude Psp, 0.0 MPa (no US), 0.17 MPa (400 mV), 0.25 MPa (600 mV) and 0.34 MPa(800 mV).

control (no US), 400 mV (US), 600 mV (US) and 800 mV (US) recorded by the flow cytometerwere summarized in figure 4. The corresponding Psp are 0 MPa, 0.17 MPa, 0.25 MPa and0.34 MPa, respectively. The table on the top right side explains the displayed order of the charton its left. The horizontal axis of each top chart is the logarithmic scale of the fluorescent levelgenerated by FITC and their readings are displayed as the mean + the standard deviations onthe bottom chart. It is evident that the 600 mV (bottom left) case has the highest reading. Itis the indication that 0.25 MPa (600 mV) ultrasound generated the highest delivery efficiency.Therefore, the 600 mV was chosen as the optimum voltage applied in all experiments reportedbelow.

3.3. Delivery efficiency comparison

The comparison of delivery efficiencies is summarized in table 2. The delivery efficiencies ofall four cases were calculated as follows: for example, in the case of NBD-DPPE liposomes,

3260 X Xi et al

Table 2. Comparison of delivery efficiencies.

NBD-DPPE FITC-DPPE

+Cells, −(FITC-

+Cells, −(NBD- +Cells, +(NBD- +Cells, +(NBD- DPPE), −US +Cells, +(FITC- +Cells, +(FITC-

Samples DPPE), −US DPPE), −US DPPE), +US (control) DPPE), −US DPPE), +US

Percent of Control 7.7 ± 0.5 9.7 ± 0.4 Control 29.7.0 ± 3.1 45.40.0 ± 4.0

cells showed (baseline) (baseline)

fluorescent

relative to the

control (%)

Efficiency (%) 2.0 15.7

NBD-lecithin FITC-lecithin

+Cells, −(NBD- +Cells, −(FITC-

lecithin), −US +Cells, +(NBD- +Cells, +(NBD- lecithin), −US +Cells, +(FITC- +Cells, +(FITC-

(control) lecithin), −US lecithin), +US (control) lecithin), −US lecithin), +US

Percent of Control 39.8 ± 2.8 48.6 ± 3.5 Control 32.9 ± 3.3 43.5 ± 3.0

cells showed (baseline) (baseline)

fluorescent

relative to the

control (%)

Efficiency (%) 8.8 10.6

we first calculated the difference of the flow cytometer readings between the suspension whichcontains cells and NBD-DPPE liposomes sham-exposed {+Cells, +(NBD-DPPE), −US} andthe reading for pure cell sham-exposed as a control {+cells, −(NBD-DPPE), −US}. Theresultant difference was then divided by the flow cytometer reading corresponding to the{+Cells, +(NBD-DPPE), −US} case to get the fractional change of the reading of the NBD-DPPE no US case relative to that of the control (pure cells). Then, we repeat the procedurefor the cell and NBD-DPPE liposome suspension US-exposed {+Cells, +(NBD-DPPE), +US}case. The difference between the US case and the no US case is the delivery efficiency aswe defined. Apparently, the efficiencies of the four cases can be ranked from high to low as:FITC-DPPE, FITC-lecithin, NBD-lecithin and NBD-DPPE.

4. Summary and discussion

Four types of liposome, FITC-DPPE, FITC-lecithin, NBD-lecithin and NBD-DPPE, weresynthesized. It was found that NBD-DPPE-type liposomes were smaller than NBD-lecithin-type liposomes. Furthermore, the NBD-DPPE-type liposomes were less stable under the USexposure. This result agrees with the finding of Rukmini et al (2001). Rukmini et al found thatthe low-concentration NBD could form liposomes whose possible structure is illustrated infigure 1(b); i.e. some NBD-cholesterol monomers paired with phospholipid monomers in theotherwise phospholipid bilayer membrane structure. Compared with liposomes of the bilayerstructure shown in figure 1(a), it might be less stable subject to the excitation of US.

The measured delivery efficiencies indicated by the flow cytometer readings of the fourliposomes could be ranked from high to low as: FITC-DPPE, FITC-lecithin, NBD-lecithinand NBD-DPPE (p < 0.05). Both FITC-encapsulated liposomes (FITC-DPPE and FITC-

Drug-delivery via liposomes under ultrasound excitation 3261

Figure 5. The figure contains three SEM images of MCF7 breast cancer cells. The magnificationis 20k for all images. Top: control (no US): the cell surface has smooth characteristic membranetopography. Middle (exposed to US without liposomes for 40 s): the cell surface shows lesssmooth regions. Bottom (+FITC-DPPE liposome+US for 40 s): the cell surface shows manyrough regions also reveals few small ‘holes’ at which the arrows point.

lecithin liposomes) were ranked higher than NBD-type liposomes. It is noted that FITC-typeliposomes as shown in figure 1(c) in general could encapsulate more fluorescent material

3262 X Xi et al

Figure 6. Liposomes may enter a cell via endocytosis or fusion processes.

than NBE-type liposomes. Therefore, if FITC-type structured liposomes entered the cell, thefluorescent reading would increase more than if NBD-type liposomes did. From the drugdelivery point of view, the FITC-type drug-encapsulated liposome is favored due to its higherdrug-carrying capacity and delivery efficiency.

As pointed in our earlier publication (Wu et al 2006a), a passive acoustic cavitationdetector (Roy et al 1990) was used to detect the subharmonic component as possible acousticcavitation events. The results seem to suggest acoustic cavitation did occur during the USexposure with the presence of liposomes and liposomes might serve as cavitation nuclei. Inother words, acoustic cavitation might play a role in generating transient mini holes as shownin figure 5.

Additional mechanisms may include endocytosis and fusion (a cell fuses with bio-compatible liposomes). Figure 6 illustrates these two processes. Exposure to an ultrasonicfield might facilitate these two processes. Both liposomes and cells could be accelerated in anultrasonic field by an acoustic radiation force. We may estimate the speed of a liposome and acell when it was passing the focal region as follows. We use r1 and r2 to represent the radii ofa liposome and a cell, respectively, and let r1 = 300 nm and r2 = 10 µm, the acoustic pressureamplitude p = 0.25 MPa, density of the medium ρ = 1000 kg m−3, the speed of sound c inthe medium = 1500 m s−1 and the viscosity coefficient η = 0.001 Pa s. Since both r1 and r2

are much smaller than the wavelength, a focused US might be considered as a plane wave to aliposome and a cell. The acoustic radiation force (Beissner 1993) acting upon the liposome,F, by an acoustic field may be given by

IS

c< F <

2IS

c, (1)

where I (ultrasound intensity) = p2/2ρc and S (the cross-section of a liposome) = πr2. Ifthe liposome could be considered as a perfect absorber, F = IS/c. If it were a perfectreflector, F = 2IS/c. Apparently, liposomes and cells are between these two extremes. Theparticle moving with the velocity v in the medium experiences a viscous drag force = 6πηrv.Neglecting the inertia of the liposome and cell, the terminal velocity could be calculated byletting F = 6πηrv; i.e., p2r

12ηρc2 < v <p2r

6ηρc2 . Thus, v is proportional to the radius r; i.e.

0.7 mm s−1 < v < 1.4 mm s−1 for liposomes and 23.1 mm s−1 < v < 46.2 mm s−1 forcells. The relative speed between a liposome and a cell should be in the range between22.4 mm s−1 and 44.8 mm s−1. The impact may be significant when they collide. Considering

Drug-delivery via liposomes under ultrasound excitation 3263

Figure 7. The top two are for NBD-DPPE liposome cases and the bottom two are for FITC-DPPEliposome cases. The images on the left are taken using regular light mode and the two on the rightare taken using epifluorescent mode. The top-right image shows liposomes fused with a cell at itsedge (see the cell to that the arrow points), while the fact that the cells in the bottom-right imageare uniformly lit up implies that liposomes entered cells.

5 (liposomes):1 (cell) ratio in the suspension and the mixing action due to the rotation ofthe test tube, there could be multiple collisions for each cell occurred during the experiment.The accumulated effect of the multiple collisions could change the permeability of the cellmembrane.

Three typical scanning electron microscopic (SEM) images of MCF7 human breast cancercells shown in figure 5 might be relevant to those mechanisms. The top one is for a non-sonicated cell; the middle one is for a sonicated cell (no liposome was added into the test-tubeduring US exposure); the bottom one is for a sonicated cell (FITC-DPPE liposomes wereadded during US exposure). It is noted that the non-sonicated cell (top) has a clear ‘relativelysmooth’ cell membrane surface topography in many small regions; the sonicated cell (middle)has many less smooth regions and the cell (bottom) which probably had ‘collisions’ withliposomes under US excitation showed some rough surface regions and some regions thatcontain ‘holes’. These regions with the holes might be caused by sonoporation. We speculatethat liposomes might enter the cell through the holes. It is also possible that from those rough

3264 X Xi et al

regions, endocytosis or fusion processes might also take place. When endocytosis or fusionprocess occur, liposomes might appear at the membrane area of a cell. These observationswere further confirmed by optical images taken under the epifluorescent (excitation wavelength440 nm, fluorescence wavelength 530 nm) modes under a microscope. Figure 7 contains fourimages. Two images on the left were taken using the regular light mode and two on the rightwere taken using the epifluorescent mode. The top two are for NBD-DPPE liposome casesand the bottom two are for FITC-DPPE liposome cases. The image on the top-right suggestsliposomes fused with a cell at its edge (see the cell that the arrow points to; the circumferentialarea is brighter than the core), while the bottom-right shows that liposomes entered cells.

Another point needs to be mentioned. Figure 3 shows that DPPE-type liposomes(≈300 nm) are smaller than lecithin-type liposomes (≈400 nm). This fact may be related tothe delivery efficiency. Table 2 indicates that the delivery efficiency of NBD-DPPE liposomes(2.0%) is smaller than NBD-lecithin liposomes (8.8%), while FITC-DPPE (15.7%) is higherthan FITC-lecithin (10.6%). This may be understood in terms of their size difference. Asmentioned earlier, two possible events might happen: (1) liposomes entered into a cell;(2) liposomes fused with a cell at its membrane. The probability for FITC-DPPE-typeliposomes entering a cell was higher than that of FITC-lecithin-type because the former wassmaller. If liposomes were fused with a cell at its membrane, since a NBD-lecithin liposomewas larger and contained more amount of NBD, it would increase the fluorescence readingmore than a smaller NBD-DPPE liposome. If the entrance of drug into a cell is the purpose,the smaller FITC-DPPE-type structure liposomes may be the choice. On the other hand, if theeffect of the drug can be achieved through the diffusion process passing the cell membrane,the larger NBD-lecithin-type structure liposomes are recommended.

Acknowledgments

This work is supported by grants from National Important Science Research Program ofChina (No 2006CB933206 for Yang and Gu), National Natural Science Foundation of China(No 10774071 for Xi and Zhang; No 90406023 for Yang and Gu), National Natural ScienceFoundation of Jiangsu Province (No BK2007518 for Xi and Zhang) and New England GreenChemistry Consortium, EPA, USA (for Chen and Wu). We also want to thank the reviewersfor their constructive suggestions.

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