Acoustic activation of drug delivery from polymeric micelles: effect of pulsed ultrasound

Journal of Controlled Release 71 (2001) 239–249www.elsevier.com/ locate / jconrel

Acoustic activation of drug delivery from polymeric micelles:effect of pulsed ultrasound

*Alexander Marin, Md. Muniruzzaman, Natalya RapoportDepartment of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA

Received 16 October 2000; accepted 5 January 2001

Abstract

The effect of a continuous wave (CW) and pulsed 20-kHz ultrasound on the Doxorubicin (DOX) uptake by HL-60 cellsfrom the phosphate buffered saline solution (PBS) and Pluronic micellar solutions was studied. Both CW and pulsedultrasound enhanced DOX uptake from PBS and Pluronic micelles. The main factor that effected drug uptake was ultrasoundpower density; however, with increasing power, the enhanced drug uptake was accompanied by the extensive cell sonolysis.For PBS, no significant effect of duration of the ultrasound pulse or inter-pulse interval on the drug uptake was observed. ForPluronic micelles, the uptake increased with increasing pulse duration in the range 0.1–2 s, overall sonication time being thesame. For 2-s pulses, the uptake was close to that under CW ultrasound. There was no significant effect of the duration of theinter-pulse interval on the drug uptake from Pluronic micelles. The data on the effect of pulse duration on drug uptakesuggest that the characteristic times of drug release from micelles and drug uptake by the cells are comparable. The resultspoint to two independent mechanisms controlling acoustic activation of drug uptake from Pluronic micelles. Bothmechanisms work in concert. The first one is related to the acoustically-triggered drug release from micelles that results inhigher concentration of the free drug in the incubation medium. The second mechanism is based on the perturbation of cellmembranes that results in the increased uptake of the micellar-encapsulated drug. The intracellular uptake of Pluronicmicelles was confirmed by fluorescence microscopy. 2001 Published by Elsevier Science B.V.

Keywords: Controlled drug delivery; Pluronic micelles; Ultrasound; Pulsed ultrasound; Drug uptake; DOX; HL-60 cells

1. Introduction drug action to the target site. One of the prospectiveways of drug targeting is drug encapsulation in

Cancer chemotherapy is often limited by severe polymeric micelles followed by drug release at theside effects caused by chemotherapeutic drugs. A target site triggered by the focused ultrasound [1–6].desirable improvement would be to reduce the dose Polymeric micelles of interest are formed by theand frequency of drug administration and to restrict hydrophobic-hydrophilic block copolymers [1–14].

Their amphiphilic character, size (tens of nanome-ters) and surface properties result in high drug

*Corresponding author. 20 S. 2030 E. Room 108, Department loading capacity and long circulation time in theof Bioengineering, University of Utah, Salt Lake City, UT 84112,

vascular system, which makes them attractive drugUSA. Tel.: 11-801-581-8990; fax: 11-801-585-5151.carriers [7,8]. Among polymeric surfactants, PluronicE-mail address: [email protected] (N.

Rapoport). attracted special attention due to the combination of

0168-3659/01/$ – see front matter 2001 Published by Elsevier Science B.V.PI I : S0168-3659( 01 )00216-4

240 A. Marin et al. / Journal of Controlled Release 71 (2001) 239 –249

advantageous properties that culminated in the hy- These findings warrant a systematic study of thepersensitization of drug resistant cells [9–12]. Fun- effect of ultrasound on the intracellular drug uptakedamental relationships between the composition of from Pluronic micelles, with the ultimate goal toPluronic block copolymers and their hypersensitiza- bring this new technique of drug targeting to clinicaltion effect in MDR cancer cells were recently use.established [12]. From the clinical perspective, pulsed ultrasound is

Pluronic P-105 copolymer was used in our lab to preferable to the CW ultrasound by virtue of reduc-study the effect of ultrasonic stimulation on drug ing the chance of tissue burning. Also, pulseduptake from polymeric micelles [1–6]. Pluronic is a ultrasound has a wider range of controllable parame-triblock copolymer of poly(ethylene)oxide (PEO) ters than CW ultrasound. However, for pulsed ultra-and poly(propylene oxide) (PPO) with a PEO–PPO– sound to be effective, the characteristic times of drugPEO structure. Pluronic is relatively non-toxic; clini- release from micelles and drug uptake by the cellscal use of Pluronic F-128 at concentrations up to 1 should be comparable. If the characteristic time ofwt.% has FDA approval. Hydrophobic central PPO the drug uptake by the cells proves much longer thanblocks form the cores of Pluronic micelles that the pulse duration, pulsed ultrasound might proveencapsulate lipophilic drugs, whereas hydrophilic ineffective. To our knowledge, this issue has neverPEO blocks form the shells that protect micelles been discussed.from recognition by the reticulo–endothelial system. The present study is focused on the effect of

Pluronic copolymers at various aggregation states pulsed ultrasound of various duty cycles (pulsehave been tested as drug carries [1–6,9–12]. durations and inter-pulse intervals) on the Doxorubi-Pluronic molecules in the unimeric form (below the cin (DOX) uptake by HL-60 cells from phosphatecritical micelle concentration, CMC) were found to buffered saline solution (PBS) and Pluronic micelles.considerably increase the cytotoxic activity of abroad range of drugs [9–12]. At the concentrationsabove the CMC, Pluronic forms dense micelles that 2. Materials and methodsencapsulate lipophilic drugs and substantially reduceintracellular drug uptake, which is advantageous for 2.1. Materialspreventing unwanted drug interaction with healthytissues. However, drug uptake at the tumor site Pluronic P-105 with an average molecular weightshould be assured; for this, we have used micelle of 6500, the number of monomeric units in PEO andperturbation by ultrasound [1–5]. Enhanced drug PPO blocks being 37 and 56, respectively, wasuptake from Pluronic micelles under the action of kindly supplied by the BASF Corporation (Mountcontinuous wave (CW) ultrasound and increased Olive, New Jersey); 10 wt.% Pluronic solution inDNA damage caused by the combination of micellar- PBS was used for the experiments. Doxorubicinencapsulated drug and ultrasound has been demon- (DOX) was supplied by the University Hospitalstrated in previous publications [1,2,5,15]. It has (University of Utah, Salt Lake City, UT) as a dosagebeen also shown that ultrasound triggered drug form that comprised 50 mg of DOX and 250 mg ofrelease from Pluronic micelles within a fraction of a lactose.second. Ultrasound pulses longer than 0.5 s provided Fluorescently-labeled Pluronic P-105 was synthes-a peak release that was equal to the stationary release ized and kindly provided to us by Dr Yi Luo,under CW ultrasound [6]. If the pulse duration was Department of Medicinal Chemistry, University ofshorter than 0.5 s, maximum possible release was not Utah. The fluorescent label C-368 (Molecularattained, and for the pulses shorter than 0.2 s Probes, OR) was conjugated to the Pluronic’s endpractically no drug release was observed. The degree hydroxyl groups (the details of the synthesis of theof the acoustically-triggered drug release depended fluorescently-labeled Pluronic and the results on theon a number of factors, including ultrasound fre- Pluronic uptake by HL-60 cells from non-micellarquency, power density, drug–micelle interaction, and micellar solutions of various concentrations willlocal drug concentration, and pulse duration [6]. be reported elsewhere).

A. Marin et al. / Journal of Controlled Release 71 (2001) 239 –249 241

Dulbecco’s PBS, pH 7.4 and sodium dodecyl DOX concentration is cited above). The number ofsulfate (SDS) were purchased from Sigma (St. the cells taken for the experiment ranged between

6 6Louis, MO) and used as received. 3310 and 4310 cells /ml; cells were counted witha hemacytometer. The amount of DOX absorbed by

2.2. Cells the cells was measured by drug depletion from theincubation medium and, in parallel, by drug fluores-

HL-60 promyelocytic cells were kindly provided cence in cell lysates using a photon-counting spectro-by Dr B.K. Murrey (Department of Microbiology, fluorimeter (ISS), model PC-1, Champaign, IL); theBrigham Young University, Provo, UT). They were excitation wavelength was 488 nm and the emissioncultured in RPMI-1640 medium supplemented with wavelength was 590 nm. After the experiment, cells20% fetal calf serum, 2 mM L-glutamine, 0.2% were lysed with a 2%wt. SDS solution at 378C forsodium bicarbonate, and 50 mg/ml gentamicin at 2–3 days with periodical stirring, which resulted in378C in humidified air containing 5% CO . the drug transfer from the cells to the SDS micelles.2

Calibration experiments showed a linear dependence2.3. Insonation of DOX fluorescence intensity on DOX concen-

tration in PBS, Pluronic, or SDS at the concentrationA 20-kHz ultrasound was generated by a micro- range used. The mean of five measurements was

probe transducer (tip diameter 3 mm, Sonics and used for the calculations. The amount of the internal-Materials, Newton, CT); the microprobe was inserted ized DOX was normalized to the cells concentration;directly into the polystyrene test tube (diameter 10 the initial cell concentration was used for the mea-mm) containing the cells at 378C. Power density was surements of drug depletion from the incubationmeasured with a hydrophone (Bruel and Kjaer, type medium, but the final cell concentration was used for8103). The latter measures only space-average power the cell lysate measurements. The final cell con-density. A 3 mm probe operating at 20 kHz is likely centration in lysates was measured by protein contentto produce spherical rather than planar waves. In this using BCA assay (see below). The fluorescencecase the pressure amplitude decreases significantly intensity of tested lysate solutions was compared towith distance from the probe. Hence, there may be that of a standard DOX solution of 0.8 mg/ml.substantial pressure difference within the sonicationchamber. In addition, the active element in Bruel and 2.5. Estimation of cell lysisKjaer, 8103 is located 1 cm into hydrophone. Hence,the actual intensity experienced by the cells may be The degree of cell lysis was measured by ahigher than that noted by the hydrophone. The decrease in cell number after the ultrasonic exposure,ultrasound probe was programmed to generate con- using Micro BCA Protein Assay Reagent [16]. The

21 1tinuous wave (CW) or pulsed ultrasound of varying method is based on the reduction of Cu to Cu bypower densities and duty cycles (ultrasound ‘on’ and proteins in the presence of bicinchoninic acid that

1‘off’ durations). In case of pulsed ultrasound, the forms a colored complex with Cu . The aliquots oftotal ultrasound exposure time was the same for all 20 ml of cell lysates were placed into the 96-wellpulse sequences and was equal to that for CW microtiter plate upon which the BCA reagent (200sonication; for shorter pulses, this required a higher ml) was added; the plate was kept in the thermo-number of pulses and a longer drug–cell contact, as stated bath at 378C for 30 min. The absorbance atschematically illustrated in Fig. 1a. 570 nm was measured and corrected for the back-

ground absorbance of a blank sample. The sets of2.4. Measuring drug uptake by the cells albumin and cell lysate standards were used for the

calibration. Each experiment was made in triplicate.An aliquot of the cell suspension in RPMI-1640 For low degrees of cell sonolysis, the drug uptake

medium was spun, washed with PBS, and the data provided by drug depletion from the incubationmedium was replaced with 3 ml of a 5 mg/ml DOX medium were close to those provided by cell lysatessolution in either PBS or 10 wt.% P-105 (the actual (the difference did not exceed 20%). However, in


Fig. 1. Schematic representation of drug release from micelles under pulse ultrasound: (a) effect of pulse duration; (b) effect of the durationof the inter-pulse interval. Ultrasound induces drug release from micelles resulting in the increase of the free drug concentration in theincubation medium. Drug release increases with increasing pulse duration up to 0.5 s but levels off for longer pulses. The plateau level ofthe drug release under pulsed ultrasound corresponds to that under CW ultrasound. During the ‘ultrasound-off’ phase, the re-encapsulation ofthe drug in restored micelles results in the decrease of the free drug concentration. Again, complete drug re-encapsulation takes about 0.5 s.

case of the extensive cell lysis, there was a large overestimation of the drug uptake). Therefore, indiscrepancy between the drug uptake data provided case of the substantial cell lysis, we based drugby the supernatants and lysates (the latter being uptake measurements only on the drug depletionsubstantially larger than the former). This presumab- from the supernatants.ly resulted from soluble proteins being washed outfrom the damaged cells at the cell washing stage, 2.6. Fluorescence microscopy of the cellswhereas the DNA with the intercalated drug stayed incubated with a fluorescently-labeled Pluronicin the precipitate. This resulted in the underestima-tion of the number of the cells that supplied DNA- The fluorescently-labeled Pluronic was mixed withintercalated drug to the lysates (leading to the the non-labeled Pluronic at a ratio of 1:1000; the


Table 1mixture was dissolved in PBS to give a final PluronicEffect of the intervals between ultrasound pulses on DOX uptakeconcentration of 5.0%wt. HL-60 cells were incubated aby HL-60 cells

or sonicated with the fluorescently-labeled PluronicUltrasound Overall incubation DOX uptakemicelles at 378C for 1 h; upon completion of the

6conditions time (min) (mg/10 cells)incubation, cells were separated, washed, fixed withPBS P-1053% formalin, sealed on glass slides and visualized

with fluorescence microscopy (Nikon, Tokyo, No US 10 0.5060.06 0.1260.04CW 10 0.9560.07 0.3160.06Japan). Ultrasound was generated by 20 kHz trans-1 s on/0.2 s off 12 0.9760.08 0.3060.05ducer mentioned above (tip diameter 13 mm); the1 s on/1 s off 20 0.9060.07 0.2260.05microprobe was inserted into the water bath at the1 s on/5 s off 60 0.9160.08 0.2360.05

distance of 3 cm from the samples.a The initial concentration of DOX is 5 mg/ml, the volume is 3

ml and the total duration of ultrasound, 10 min.

3. Results and discussionthe plateau-level of drug uptake was about 4-fold

3.1. Kinetics of drug uptake from PBS and lower when DOX was delivered from PluronicPluronic micelles micelles in comparison to PBS (Fig. 2 and Table 1).

As shown in Fig. 2, DOX uptake by HL-60 cells 3.2. Effect of ultrasound power density on thefrom both PBS and Pluronic micelles proceeded intracellular drug uptakerather fast; about 70% of the maximum possibleuptake was achieved during the first several minutes The intracellular drug uptake from Pluronic mi-of the cell incubation with the drug, followed by the celles increased with increasing ultrasound powertail that leveled off after approximately 15 min of density (Fig. 3a). For 20-kHz ultrasound, at a power

2incubation. For the same initial drug concentration, density of 33 mW/cm , DOX uptake from Pluronic

Fig. 2. Kinetics of DOX uptake by HL-60 cells from PBS and micellar Pluronic solution (P-105, 10% wt). Initial DOX concentration 5mg/ml; incubation temperature 378C.


Fig. 3. Effect of ultrasound power density on the equilibrium DOX uptake by HL-60 cells from P-105, 10%wt (a) and on the cell lysis (b).Initial DOX concentration 5 mg/ml; incubation temperature 378C, sonication time 20 min. For comparison, DOX uptake from PBS without

2the application of ultrasound is shown. The setting of the instrument used: 2% for power density 1.4 mW/cm , 4% for power density 142 2mW/cm and 8% for power density 33 mW/cm .


micelles was higher than that from PBS without lated) drug in the incubation medium (it should beultrasound (though lower than that from PBS under noted that with fluorescence technique, we cannotthe same ultrasound) (Fig. 3a, Table 1). However, as discriminate between a truly free drug molecules andcould be expected, ultrasound at the higher power those associated with Pluronic unimers, as they havelevels resulted in the extensive cell sonolysis (Fig. equal fluorescence [3]; for the sake of simplicity,3b). Yet, a reasonable compromise between the both drug populations are designated here as ‘free’enhanced drug uptake and enhanced sonolysis may drug). With pulses that are longer than 0.5 s, peakbe obtained in a particular window of power den- release of the free drug under pulsed ultrasound issities and duty cycles. Extensive cell lysis should be equal to the stationary release under CW ultrasoundprevented as it can cause a massive release of [6].lysosomal enzymes and acute inflammation. Drug release is followed by the drug re-encapsula-

tion that occurs during the ‘ultrasound off’ phase of3.3. Effect of 20-kHz ultrasound on the drug pulsed ultrasound. The characteristic times of druguptake: comparison of CW and pulsed ultrasound release and re-encapsulation in Pluronic micelles are

comparable [6]. Re-encapsulation also proceeds3.3.1. Effect of the pulse duration within a fraction of a second, and the complete

For DOX solutions both in PBS and in Pluronic re-encapsulation takes about 0.5 s.micelles, 20-kHz ultrasound substantially increased These considerations are schematically illustrateddrug uptake. The drug uptake from PBS did not in Fig. 1. Fig. 1a shows that the concentration of thedepend on the duration of the pulse in the studied free drug in the incubation medium rises when pulserange of 0.2–2 s (Fig. 4a). Even for a pulse duration duration increases from 0.2 to 0.5 s and then remainsas short as 0.2 s, the overall drug uptake almost constant. Fig. 1a also demonstrates that the drugdoubled in comparison with the unsonicated sample re-encapsulation rate does not depend on the pulse(Fig. 4a). The increased drug uptake from PBS was duration.presumably caused by the cell membrane perturba- Similar considerations are applied for the re-en-tion by ultrasound [17,18]. Our data mean that cell capsulation process. Intervals between the pulses thatmembrane perturbation proceeds within 0.2 s, and are shorter than about 0.5 s don’t provide enoughtherefore kinetics of drug uptake is the same for time for the complete re-encapsulation (Fig. 1b); atpulse duration longer than 0.2 s. longer intervals, complete re-encapsulation of the

In contrast, for Pluronic micelles, drug uptake released drug occurs (this is illustrated in Fig. 1a).increased with pulse duration (Fig. 4b). As shown in As shown in Fig. 4b, the intracellular drug uptakeFig. 4b, some enhancement of drug uptake was continued to increase with pulse duration up to 2 s,observed already at a pulse duration of 0.2 s, though the concentration of the released drug in thewhereas at a pulse duration of 2 s drug uptake was incubation medium leveled off at the pulse durationcomparable to that under CW ultrasound (illustrated of 0.5 s (Fig. 1a). Note that the total ultrasoundhere as a 10-s pulse). This indicated that the com- exposure time was the same for all experiments,plete uptake of the released drug proceeded within while the total time of cell contact with the drug wasapproximately 2 s. longer for shorter pulses, as illustrated in Fig. 1a.

The dependence of the drug uptake from micelles Despite that, drug uptake from Pluronic micelles wason the duration of the ultrasonic pulse differed from larger for longer pulses, i.e. for shorter overall timesthat observed for the drug release from micelles [6], of cell–drug contact. This implied that a veryas explained below. insignificant (if any) uptake proceeded during the

Ultrasound-triggered drug release from Pluronic inter-pulse intervals, as further confirmed by directmicelles does not proceed instantaneously; short measurements of the effect of the length of thepulses don’t provide enough time for the effective inter-pulse duration on the drug uptake (see below).drug release. Equilibrium release is not achieved for This also implied that the average rate of the drugpulses that are shorter than 0.5 s, which results in the uptake from Pluronic micelles during the ultrasoundlower concentration of the free (i.e. not encapsu- pulse increased with pulse duration.


Fig. 4. Effect of the ultrasound pulse duration on DOX uptake by the cells from (a) PBS and (b) P-105, 10%wt; the total sonication time is(a) 10 min and (b) 20 min. The inter-pulse duration is 1 s in all experiments. The number next to the experimental point indicates the overallduration of the experiment. CW ultrasound is presented as a 10-s pulse. Initial DOX concentration 5 mg/ml; incubation temperature 378C;

2space-average power density 1.4 mW/cm (instrument setting 2%).


As mentioned above, for Pluronic micelles, the reasonable to assume that the uptake of the Pluronic-concentration of the drug released from micelles was associated drug molecules increases with the pulseconstant for pulse durations longer than 0.5 s [6], duration. The experiments with the fluorescently-which implies that the uptake of a released drug was labeled Pluronic molecules showed that ultrasoundconstant in the pulse range of 0.5–2s (see Fig. 1). If indeed significantly enhanced the intracellular uptakewe assume that for all pulse durations, the uptake of of Pluronic molecules from micellar solutions (Fig.the free drug was equal to that observed at pulse 5, see below for details).duration of 0.5 s, then the additional uptake over this As shown in Fig. 4b, for the Pluronic-encapsulatedlevel at longer pulses was caused by the uptake of DOX, a series of 2-s pulses resulted in the intracellu-the Pluronic-associated drug. The latter includes both lar drug uptake that was close to that seen under CWthe micellar-encapsulated drug and that associated ultrasound. Taking into consideration that drug re-with Pluronic unimers that may be formed upon encapsulation proceeds in a fraction of a secondultrasound-induced degradation of Pluronic micelles. during the ‘ultrasound-off’ phase, these data indicateWe hypothesize that for Pluronic micelles, the de- that the intracellular uptake of the drug released frompendence of drug uptake on pulse duration stems Pluronic micelles proceeds within 2 s. It may alsofrom the accumulation of ultrasound-induced mem- indicate that the concentration of membrane defectsbrane defects during the ultrasound pulse [17,18]. responsible for the uptake of the micellar-encapsu-The accumulation of membrane defects may result in lated drug levels off after about 2 s of sonication duethe formation of transient pores that are large enough to the competition between defect forming andto allow the flow of the drug encapsulated in healing.Pluronic micelles into the cells; it will also increase Summarizing, the acoustically-enhanced drug up-the flow of the unimer-bound drug. As membrane take from Pluronic micelles may result not only fromdefects accumulate during the ultrasound pulse, it is the ultrasound-triggered drug release from micelles

Fig. 5. Fluorescence micrographs of HL-60 cells (A) incubated and (B) sonicated with the fluorescently-labeled micelles of Pluronic P-105;excitation wavelength 527–552 nm; emission wavelength 577–632 nm. The cells were incubated or sonicated for 1 h with 5% Pluronicsolution that comprised fluorescently-labeled Pluronic at a concentration of 2.1 mg/ml. Ultrasound frequency 20 kHz, power density 14

2mW/cm .


that results in the enhanced uptake of free drug but ultrasound, a very low fluorescence was observed inalso from the perturbation of cell membranes that the cells incubated with a micellar Pluronic solution.increases the uptake of the Pluronic-associated (in The application of CW ultrasound substantiallyparticular micellar-encapsulated) drug. enhanced the fluorescence of the cells (Fig. 5),

Based on the results presented above, the charac- which indicated that ultrasound enhanced the in-teristic time of the intracellular drug uptake is tracellular uptake of Pluronic micelles.comparable to that of the ultrasound-triggered drugrelease from micelles. This holds a considerablepromise for the clinical application of pulsed ultra- 4. Conclusionssound in micellar drug delivery.

The data presented above show that the applica-3.3.2. Effect of the duration of the inter-pulse tion of ultrasound could increase the intracellularintervals drug uptake from Pluronic micelles to the values

For DOX dissolved in PBS, drug uptake under comparable or even overcoming those observed forpulsed ultrasound did not depend on the length of the the conventional delivery medium without ultra-interval between the pulses in the range 0.2–5 s sound. The data suggest two independent mecha-(Table 1). This implies that the equilibrium between nisms controlling acoustic activation of drug uptakethe external and internalized drug established during from Pluronic micelles, both working in concert. Thethe pulse did not change during the inter-pulse first mechanism is related to the acoustically-trig-interval. For Pluronic, for the short inter-pulse gered drug release from micelles that results in theinterval of 0.2 s, drug uptake was close to that under higher concentration of the free drug in the incuba-CW ultrasound (Table 1), which presumably resulted tion medium. The second mechanism is based on thefrom the non-complete re-encapsulation of the re- perturbation of cell membranes that results in theleased drug during the ‘ultrasound off’ time (see increased uptake of the micellar-encapsulated drug.scheme, Fig. 1b). Drug uptake slightly dropped for Thus, with the proper design of the ultrasoundinter-pulse intervals of 1.0 and 5.0 s (Table 1). Note, protocol, the polymeric micelle–ultrasound combina-that for the same ‘ultrasound on’ time, the overall tion can effectively deliver drugs to tumors. Thetime of cell contact with the drug increases with great advantage of this technology is a targetableincreasing intervals between the pulses (Table 1). drug delivery, which conventional delivery systemsThe data suggest that drug uptake during the inter- don’t provide.pulse intervals from (or with) Pluronic micelles didnot proceed to any noticeable degree if the completere-encapsulation of the released drug occurred. This Acknowledgementsimplies that membrane ‘healing’ during the ‘ultra-sound off’ phase is a fast process that takes less than This work was supported by the NIH grant R01a second — otherwise the uptake of a micellar- CA76562-01A1. The authors are grateful to Dr. Yiencapsulated drug would increase with the duration Luo for the synthesis of the fluorescently-labeledof the inter-pulse intervals. Pluronic P-105.

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Acoustic activation of drug delivery from polymeric micelles: effect of pulsed ultrasound