among the lipid nanoparticles , 
 liposomes are widely studied as 
 drug delivery vehicles . liposomes protect the encapsulated drugs from being 
 metabolized during the circulation prior to reaching the target . the 
 us food and drug administration has approved liposome - based formulations 
 for the treatment of several types of cancer . however , upon targeting , the passive release of the encapsulated 
 drugs from the liposomes is often slow . reorganization of the lipid domains has been used as a trigger to 
 enhance , and to control the rate and the extent of contents released 
 from liposomes . among the various triggers , decreased ph in the lysosomes 
 has been widely used as a successful strategy to efficiently release 
 the encapsulated liposomal contents . however , imparting 
 ph sensitivity to liposomes requires the synthesis of specialized 
 lipids with structures that are substantially modified , either due 
 to hydrolysis or due to changes in the protonation states of the lipid 
 head groups , at reduced ph . stabilized gas bubbles are widely used as contrast - enhancing 
 agents 
 for ultrasound imaging of perfused tissues . there are many reports of ultrasound - mediated drug release from 
 nanoparticles , liposomes , and other carriers . although ultrasound waves in the khz frequency efficiently 
 release drugs from the carriers ( due to cavitation and high local 
 temperatures ) , the harmful biological effects associated with low - frequency 
 ultrasound limit the usefulness of such strategies . to make liposomes responsive to high - frequency ultrasound , 
 they need to be coupled with gas pockets . echogenic liposomes ( elips ) 
 entrap small amounts of air along with the hydrophilic drug in their 
 aqueous interior , and are currently being developed as drug delivery 
 vehicles for ultrasound - triggered drug release and simultaneous imaging . although there is uncertainty about the exact location and size 
 of the entrapped air bubbles in the elips , their acoustic characterization 
 has been reported extensively in the literature . we are developing targeted , 
 multimodal liposomes for triggered 
 release of encapsulated contents , and simultaneous ultrasound imaging . furthermore , we are interested in enhancing the contents released 
 from the liposomes by employing diagnostic frequency ( mhz ) ultrasound . 
 we have recently demonstrated the ultrasound - enhanced , extracellular 
 release of liposomal contents mediated by the cancer - cell - secreted 
 enzyme matrix metalloproteinase-9 ( mmp-9 ) . herein , we report a strategy to render liposomes ph sensitive by 
 encapsulating ammonium bicarbonate which generates gas bubbles in situ in response to acidic ph . our strategy does not require the use of ph - sensitive lipids in 
 the liposomal formulations . we hypothesize that , at a reduced ph , 
 the hydronium ions diffuse into the aqueous interior of the liposomes , 
 and produce carbon dioxide bubbles , thereby  turning on  
 the echogenicity . as more bubbles are generated , the liposomal 
 bilayer is disturbed , leading to the release of encapsulated contents . we observe that the release was further enhanced by applying ultrasound 
 with a frequency of 1 mhz . to the best of our knowledge there are 
 no reports in the literature of ultrasound enhanced triggered release 
 from ph - tunable echogenic liposomes . we have demonstrated the 
 usefulness of this liposomal delivery 
 system using the panc-1 pancreatic cancer cells . pancreatic cancer 
 is one of the leading causes of cancer - related deaths in both men 
 and women in the united states , with a 5-year survival rate of less 
 than 5% . according to the american cancer society , 
 38,460 pancreatic cancer related deaths ( almost equally split between 
 men and women ) occurred in united states in 2013 . to demonstrate 
 tunable echogenicity , we prepared the liposomes from 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ( popc ) , encapsulating 400 mm 
 ammonium bicarbonate along with the self - quenching dye carboxyfluorescein 
 ( 100 mm ) . we reasoned that , for multilamellar liposomes , the outside 
 hydronium ions need to diffuse through several lipid bilayers in order 
 to generate sufficient amounts of co2 gas inside the liposomes . the presence of several lipid bilayers was also expected to decrease 
 the efficiency of the contents released in response to escaping gas 
 bubbles and ultrasonic excitation . hence , we decided to formulate 
 unilamellar liposomes with a narrow size distribution by sonicating 
 and sequentially extruding ( through 800 and 200 nm polycarbonate membrane 
 filters ) the initially formed multilamellar vesicles . we observed 
 ( with dynamic light scattering ) that the average hydrodynamic diameter 
 of the liposomes is 110  15 nm with a polydispersity index of 
 0.05 ( supporting information , figure s1a ) . these results were corroborated with transmission electron microscopic 
 imaging of the liposomes ( supporting information , figure s1b ) . to demonstrate the tunable echogenicity , we 
 added the liposomes to buffers with different phs ( 7.45.0 ) 
 and recorded the images using a terason t3200 high - frequency ( 1214 
 mhz ) diagnostic ultrasound transducer . we expected that the generated 
 gas bubbles would reflect the ultrasound , and that the contrast of 
 the images would be more pronounced as the amount of generated gas 
 is increased at a lower ph . we observed that there was a lag time 
 before the liposomes became echogenic , and that the duration of this 
 lag phase decreased with the reduced ph . for example , liposomes in 
 the ph 7.4 buffer did not show any ultrasound contrast in 10 min . 
 at ph 6 , the liposomes became weakly echogenic in 5 min , but at ph 
 5 , the liposomes were fairly echogenic within 3 min . the ultrasound 
 images for the liposomes in buffers with different ph values after 
 5 min are shown in figure 1 . figure 1a shows that there is no echo at ph 7.4 ; but as 
 the ph is reduced , progressively stronger echo from the entire cell 
 well is observed ( note that the ultrasound probe is placed at the 
 top of the cell well ) . figures 1b and 1c show how the mean and maximum gray scale values 
 quantitatively change with ph . the echo from the homogeneous suspension 
 of liposomes appears as light bands ( figure 1a ) . they arise 
 due to interference between echoes from subresolution scatterers such 
 as liposomes which themselves are far smaller in size than the ultrasound 
 wavelength . ( a ) ph - dependent diagnostic frequency ultrasound imaging of popc 
 liposomes encapsulating 400 mm ammonium bicarbonate . the dotted white 
 lines represent the regions of interest ( roi ) that were used to calculate 
 the gray scale values . ( b ) mean gray scale values and ( c ) maximum 
 gray scale values for the ultrasound images shown in panel a as a 
 function of ph ( n = 3 ) . we anticipated that the concentration of hydronium ions in 
 the 
 external buffer would affect their diffusion rate inside the liposomes 
 as well as the subsequent generation of co2 bubbles . as 
 the encapsulated ammonium bicarbonate was depleted , the generation 
 of co2 gas slowed down and finally stopped . consistent 
 with this hypothesis , we observed that liposomes in the ph 5 buffer 
 are not echogenic after 20 min ( figure 2 ) . however , we noted that the diameters of the gas bubbles inside the 
 liposomes are likely to be small ( in nanometers ) , and that they may 
 not reflect the ultrasound very well . it is likely that the nanobubbles coalesce in the lipid bilayer 
 of the liposomes , generating larger bubbles , and reflect the ultrasound . popc lipid has a gel low transition temperature ( 2 c ) , 
 and the liposomal bilayer is in the fluid phase under the experimental 
 conditions ( 20 c ) . the loose lipid 
 packing and fluidity of the popc bilayer accommodate the coalescence 
 and the size increase of the gas bubbles . diagnostic frequency 
 ultrasound images of popc liposomes encapsulating 
 400 mm ammonium bicabonate as a function of frequency and incubation 
 time in a ph 5 buffer . the images were acquired by employing high - frequency 
 ( 1215 mhz ; a , b ) , medium - frequency ( 812 mhz ; c , d ) , 
 and low - frequency ( 48 mhz ; e , f ) ultrasound transducers . we analyzed the ultrasound images 
 shown in figure 1a using the imagej software 
 ( http://rsbweb.nih.gov ) to calculate the mean and maximum 
 gray scale values for region 
 of interest ( roi ) shown in figure 1a . as expected , 
 the mean and maximum gray scale values increase with a decreasing 
 ph . we observed that the highest gray scale value was observed at 
 ph 5 , and it does not increase any more below this ph ( data not shown ) . we also observed a time - dependent decrease in the echogenicity of 
 these liposomes at ph 5.0 ( figure 2 ) . these 
 results demonstrated that liposomes are programmed to reflect the 
 ultrasound only after reaching the acidic microenvironment of cancer 
 cells . having demonstrated ph - tunable echogenicity , we decided to determine 
 if the escaping gas bubbles sufficiently disturb the lipid bilayer 
 to release the encapsulated contents from the liposomes . for this 
 endeavor , we incubated the popc liposomes ( encapsulating carboxyfluorescein 
 and 400 mm ammonium bicarbonate ) in buffers with different ph values 
 ( 7.45.0 ) , and monitored the emission intensity of carboxyfluorescein . however , the emission intensity of carboxyfluorescein is quenched 
 as the ph is lowered . to correct for 
 this decreased emission intensity , we measured the absorption spectra 
 of carboxyfluorescein as a function of ph , and we determined the isosbestic 
 point to be 460 nm . subsequently , the dye solution was prepared in 
 buffers with ph of 7.4 , 6.0 , and 5.0 ; the solution was excited at 
 460 nm , and the emission spectra were recorded . we then monitored 
 the emission of the dye at 497 nm ( excitation : 460 nm ) for 2 h. the 
 correction factors were calculated at each ph as a function of time , 
 and all emission intensities were appropriately corrected for calculating 
 the percentage released ( supporting information ) . when the liposomes were incubated in acidic buffers , there 
 was a time lag before dye release ( figure 3 ) . however , the liposomes continued to leak the contents for a considerably 
 long time ( 23 h ) . the continued leakage indicates that the 
 disturbances in the lipid bilayers created by the escaping gas bubbles 
 either are not sealed or take a long time to heal . representative release 
 profiles of carboxyfluorescein from popc 
 liposomes encapsulating 400 mm ammonium bicarbonate . the liposomes 
 were incubated in buffers with ph 7.4 ( blue circles ) , ph 6.0 ( purple 
 triangles ) , and ph 5.0 ( green stars ) . the lines are generated by connecting 
 the observed data points . while the liposomes at a ph of 7.4 ( control ) released only 
 15% 
 of the encapsulated dye in 2 h , at a ph of 5 , the release increased 
 to 55% ( figure 3 ) . when we encapsulated sodium 
 bicarbonate in the liposomes ( instead of ammonium bicarbonate ) , the 
 amount of the content release decreased . in 2 h , we observed that 
 the sodium bicarbonate encapsulated liposomes released 40% of the 
 encapsulated dye ( at ph = 5.0 ; supporting information , figures s3 and s4 ) . for both of these liposomal formulations , the 
 rate of contents release decreased considerably after 2 h. in 3 h 
 at ph 5.0 , the ammonium bicarbonate encapsulated liposomes released 
 75% of the contents , and the sodium bicarbonate encapsulated liposomes 
 released 44% of the contents ( figure 4a ) . decreasing 
 the amount of encapsulated ammonium bicarbonate ( from 400 mm to 200 
 mm ) also reduced the amount of contents release from the liposomes 
 ( figure 4b ) . release of encapsulated carboxyfluorescein 
 from popc liposomes 
 as a function of ph encapsulating ( a ) 400 mm ammonium bicarbonate 
 and ( b ) 200 mm ammonium bicarbonate after 3 h ( n = 
 3 ) . the acidic decomposition of ammonium 
 bicarbonate generates nh3 and co2 , while sodium 
 bicarbonate produces the 
 sodium salt of the buffer , h2o , and co2 . the 
 ammonia gas will react with the hydronium ions in the liposome interior , 
 leading to a reduction in proton concentration . the resultant proton 
 gradient will facilitate the diffusion of more hydronium ions into 
 the liposomal lumen and generate more co2 gas and ammonia . 
 amount of generated gas decreases by reducing the concentration of 
 encapsulated ammonium bicarbonate ( from 400 to 200 mm ) , leading to 
 a reduction in contents release from the liposomes ( figure 4b ) . as an additional control , we prepared the popc 
 liposomes without encapsulating any gas precursor and studied the 
 contents release as a function of ph . we observed minimal release 
 ( < 10% ) of the encapsulated carboxyfluorescein at ph 7.4 and 6.0 . however , at ph 5.0 , about 20% of the dye was released in 2 h ( figure 
 s5 , supporting information ) . we do not 
 have an explanation for this observation yet . in these liposome 
 formulations the popc molecules contain the saturated palmitoyl and the 
 unsaturated oleoyl groups . due to the presence of an alkene in the 
 z - configuration , this lipid does not form a tight bilayer , and the 
 gel transition temperature is also low ( 2 c ) . it is possible that the loose packing of the 
 popc lipids will likely allow the escaping co2 bubbles 
 to coalesce inside the hydrophobic bilayer of the liposomes . the resulting 
 larger gas bubbles will disturb the bilayer while escaping , allowing 
 the contents to leak . to determine if the lipid packing in the liposomal 
 bilayer and the gel transition temperature affect the contents released , 
 we prepared two batches of dspc ( 1,2-distearoyl - sn - glycero-3-phosphocholine ) liposomes that encapsulate ammonium and 
 sodium bicarbonate respectively ( 400 mm each ) . the dspc molecules 
 contain two saturated stearoyl groups and form a tight bilayer with 
 a melting temperature of 56 c . we 
 hypothesized that the tightly packed lipid molecules in the bilayer 
 would hinder the coalescence of the escaping co2 bubbles 
 generated inside the aqueous core of the liposomes . in addition , 
 the rate of diffusion of the hydronium ions across the lipid bilayer 
 would be slower compared to the popc bilayer . we observed that both 
 ammonium bicarbonate and sodium bicarbonate encapsulated dspc liposomes 
 released less than 5% of their contents after incubation for 2 h at 
 a ph of 5.0 ( supporting information , table 
 s1 ) . we employed tapping mode atomic force microscopic imaging 
 to determine 
 if the escaping gas bubbles caused any structural changes ( shape and 
 surface morphology ) to the ammonium bicarbonate encapsulated popc 
 liposomes . after preparation , the ammonium bicarbonate encapsulated 
 liposomes ( ph = 7.4 buffer ) were spherical with an average diameter 
 around 100 nm ( figure 5a ) . however , after incubating 
 in a ph 5.0 buffer for an hour , the liposomes fused , and the majority 
 of the structures showed irregular shapes with sizes up to 800 nm 
 ( figure 5b ) . these results demonstrated that 
 the escaping gas bubbles caused permanent changes to the liposomes 
 morphology , leading to leakage of the encapsulated contents . ( b ) liposomes containing 400 mm ammonium bicarbonate after incubation 
 in ph 5 buffer for an hour . ( c ) buffer containing liposomes after 
 incubation in a ph 5 buffer for an hour . we reasoned that the released gas bubbles inside the liposomes 
 would allow an additional control on the contents released when employing 
 high - frequency ultrasound . to test this hypothesis , we incubated the 
 ammonium bicarbonate encapsulated ( 400 mm ) popc liposomes in buffers 
 with ph of 6.0 ( figure 6a ) and 5.0 ( figure 6b ) , and after 5 min , we exposed them to continuous 
 wave ultrasound ( 1 mhz , 2 w / cm ) for 5 min . when incubated 
 in a ph 5.0 buffer , 80% of the encapsulated contents were released 
 from the liposomes in 2 h ( compared to 55% released in the absence 
 of ultrasound ; figure 6b ) . the corresponding 
 content releases were considerably lower in ph 6.0 buffer ( figure 6a ) . decreasing the concentration of encapsulated 
 ammonium bicarbonate ( from 400 mm to 200 mm ) reduced the amount of 
 contents released upon the application of ultrasound , to 45% ( figure 7 ) . we also observed that the applied ultrasound 
 exerted a maximum effect when applied within 515 min of incubating 
 the liposomes with the ph 5 buffer . it is likely that the generated 
 co2 bubbles escape from the liposomes within 15 min , and 
 after that time , liposomes become less responsive to ultrasound . during 
 the imaging studies , we observed a decrease in the echogenicity of 
 the liposomes after 15 min of incubation in the ph 5 buffer ( figure 2 ) . ultrasound - enhanced 
 ( 1 mhz , cw , 2 w / cm , 5 min ) , ph - triggered 
 release from popc liposomes encapsulating 400 mm ammonium bicarbonate 
 at ph = 6.0 ( a ) and ph = 5.0 ( b ) . orange bars : release after 2 h with ultrasound 
 application ( n = 3 ) . ultrasound - enhanced ( 1 mhz , cw , 2 w / cm , 5 min ) , ph - triggered 
 release from popc liposomes encapsulating 200 of mm ammonium bicarbonate 
 at ph = 6.0 ( a ) and ph = 5.0 ( b ) . orange bars : release after 2 h with ultrasound 
 application ( n = 3 ) . when the ammonium bicarbonate encapsulated popc liposomes 
 were 
 incubated in ph 6 buffer , we observed that applying the ultrasound 
 enhanced the release by 1520% . contrary to the ph 5 experimental 
 results , this enhancement in contents release was not strongly dependent 
 on the time when the ultrasound was applied ( figures 6a and 7a ) . at a ph of 6 , the concentration 
 of hydronium ions was 10 times less compared to that at a ph of 5 . the lower hydronium ion concentration at a ph of 5 contributed to 
 a slow generation of gas bubbles inside the liposomes , and it took 
 longer to consume the encapsulated ammonium bicarbonate . these two 
 factors likely contributed to the results observed with ultrasound 
 at a ph of 6.0 . we observed that applying ultrasound increased 
 liposome solutions 
 temperature from 25 to 30 c . it is possible that a thermal effect 
 along with the mechanical effect could be responsible for the content 
 release . to determine if this temperature change influenced the contents 
 released from liposomes , we repeated the studies ( in a ph 5 buffer ) 
 in a large ice bath . the results from these 
 two experiments were identical , indicating that the temperature increase 
 did not influence the contents released from our ph - sensitive liposomes . however , we note that one can not rule out the 
 possibility of local hot spots being generated in the liposomes themselves 
 even when they are in the ice bath . such temperature hotspot can only 
 be created by the mechanical compression of the air cavity entrapped 
 in the liposomes . having optimized the ultrasound - enhanced 
 release from the ph - sensitive 
 liposomes , we proceeded to demonstrate the effectiveness of the strategy 
 in cellular studies . to demonstrate efficient cellular internalization , 
 we prepared liposomes incorporating 1 mol % 1,2-distearoyl - sn - glycero-3-phosphoethanolamine - n-[folate(polyethylene 
 glycol)-5000 ] ( ammonium salt , commercially available from avanti polar 
 lipids ) and popc encapsulating 100 mm carboxyfluorescein . we selected 
 the folate receptor overexpressing pancreatic ductal carcinoma cells 
 ( panc-1 ) for our cellular studies . after incubating with the liposomes , we imaged the cells by employing 
 a confocal fluorescence microscope . we noticed that liposomes incorporating 
 1 mol % of the folate lipid were taken up more effectively by the 
 panc-1 cells compared to the liposomes without the folate lipid ( figure 8) . if the cells had a higher expression of the folate 
 receptor , the internalization rate was faster . for example , the breast 
 cancer cell line mcf-7 internalized the folate lipid containing liposomes 
 faster compared to the panc-1 cells ( supporting 
 information , figure s6 ) . fluorescence microscopic images for the 
 uptake of ph - tunable , echogenic 
 popc liposomes encapsulating carboxyfluorescein by the folate receptor 
 overexpressing panc-1 cancer cells . images were obtained using different 
 filters : brightfield ( bf ) , fluorescein isothiocyanate ( fitc , green 
 fluorescence ) , and 4,6-diamino-2-phenylindole ( dapi , blue 
 fluorescence ) . ( a ) nontargeted liposomes after 3 h of incubation ( magnification : 
 20 ) . ( b ) nontargeted liposomes after 6 h of incubation ( magnification : 
 20 ) . ( c ) folate - targeted liposomes after 3 h of incubation ( magnification : 
 20 ) . ( d ) folate - targeted liposomes after 6 h of incubation ( magnification : 
 20 ) . after confirming cellular 
 internalization , we encapsulated the anticancer drug doxorubicin in 
 the popc liposomes and studied its release in the cytosol of the panc-1 
 cells ( in the absence and presence of applied diagnostic frequency 
 ultrasound ) . although gemcitabine is the standard chemotherapeutic 
 drug for pancreatic cancer , doxorubicin is currently being tested 
 as a possible adjuvant therapy . we noted , a priori , that some literature reports question the safety of ultrasound 
 for healthy tissues surrounding a tumor . to determine if the ultrasound has any deleterious effects for the 
 normal cells , we seeded the panc-1 cells onto transwell inserts consisting 
 of two chambers . diagnostic frequency ultrasound is reported to pass 
 through the insert and reach the lower chamber . in this experimental design , the panc-1 cells in the upper chamber 
 represented the tumor tissue , receiving direct exposure to the liposomes 
 as well as the applied ultrasound . the cells in the lower chamber 
 represented the neighboring tissue , which may be indirectly exposed 
 to the treatment ( figure 9a ) . the pore size 
 for the transwell insert was 400 nm , and the average diameter for 
 the liposomes was 110 nm . hence , we expected that some liposomes and 
 ultrasound waves would pass 
 through the membrane to reach the lower chamber . ( a ) 
 panc-1 cell viability studies using live ( green ) and dead ( red ) 
 cell staining of different treatment groups ( n = 
 3 ) . the upper chamber cells received direct exposure , whereas the 
 lower chamber cells received indirect exposure to popc liposomes and 
 ultrasound . ( 1 ) folate - targeted doxorubicin liposomes ( encapsulating 
 ammonium bicarbonate ) + ultrasound . ( 2 ) nontargeted doxorubicin liposomes 
 ( encapsulating ammonium bicarbonate ) + ultrasound . ( 4 ) folate - targeted liposomes ( encapsulating ammonium 
 bicarbonate but no doxorubicin ) + ultrasound . ( 5 ) folate - targeted 
 doxorubicin liposomes ( no ammonium bicarbonate encapsulation ) + ultrasound . 
 ( 6 ) ultrasound only . ( 7 ) no treatment ( control ) . the final doxorubicin concentration used 
 was 25 g / ml . ( b ) cell viability of the upper chamber ( orange 
 bars ) and lower chamber ( violet bars ) . upon reaching confluency , we exposed the upper chamber s 
 cells to various combinations of targeted / nontargeted doxorubicin - encapsulated 
 liposomes and ultrasound ( applied between 15 and 20 min of incubation ; 
 figure 9a ) . subsequently , we placed the cells 
 in an incubator for 6 h and stained to visualize the live and dead 
 cells . we observed that indirect exposure to any of the treatments 
 did not cause cell death in the lower chamber ( figures 9a and 9b ) . on the other hand , direct 
 exposure to folate - targeted or nontargeted , ph - tunable , doxorubicin - encapsulated 
 liposomes and ultrasound led to significant cell death in the upper 
 chamber ( figures 9a and 9b ) . we observed that the folate - targeted , doxorubicin and ammonium 
 bicarbonate encapsulated popc liposomes ( figure 9a-1 ) were more toxic ( 14% cell viability ) compared to the corresponding 
 liposomes without bicarbonate encapsulation ( cell viability 25% ; figure 9a-5 ) . it was reported that the cavitation force 
 of exploding co2 bubbles in the lysosomes mechanically 
 disrupts the membranes , leading to the release of lysosomal proteolytic 
 enzymes in the cytosol and to cell death . contrary to this report , we observed less than 5% cell death in 
 the presence of liposomes that only encapsulate ammonium bicarbonate 
 ( i.e. , without doxorubicin ; figure 9a-4 ) . these 
 results indicated that , in our experiments , cavitation induced by 
 co2 bubbles enhanced the toxicity of the liposomal formulations . clearly , the folate - targeted doxorubicin liposomes ( encapsulating 
 ammonium bicarbonate ) in the presence of applied ultrasound were most 
 effective in killing the panc-1 cells ( figures 9a-1 and 9b , group 1 ) . this combination reduced 
 the cell viability to 14% ( figure 9b , group 
 1 ) . interestingly , the free doxorubicin in the presence of applied 
 ultrasound was more effective compared to liposomal doxorubicin ( without 
 folate ) in inducing cell death ( figures 9a-2 
 and 9a-3 ) . it is likely that sonoporation by 
 the ultrasound is contributing to this effect . in the absence of any 
 microbubbles , the pores formed in the cell membranes by the applied 
 ultrasound are likely to be small and transient . possibly , the sizes 
 of these transient pores are large enough to allow doxorubicin molecules 
 to cross the cell membranes . these observations 
 are consistent with literature reports demonstrating a higher uptake 
 of smaller particles compared to larger ones upon sonoporation . we observed that , under our experimental conditions , 
 direct or indirect exposure to the ultrasound does not induce cell 
 death ( figure 9a-6 ) . our objective was 
 not to compare the efficacy of free doxorubicin 
 in the absence and presence of applied diagnostic - frequency ultrasound . our goal was to determine the effectiveness of the ammonium bicarbonate 
 and doxorubicin coencapsulated liposomes when ultrasound is applied . numerous literature reports demonstrate the efficacy of doxorubicin 
 for panc-1 cells ( without the applied ultrasound ) ; hence , we did 
 not include this control experiment . panc-1 is a metastatic 
 pancreatic cancer cell line known to secrete 
 matrix metalloproteinase ( mmp ) -2 and -9 enzymes in the extracellular 
 matrix . these two proteolytic enzymes 
 are responsible for the hydrolysis of the extracellular matrix , leading 
 to the migration and metastasis of cancer cells . ultrasound treatment can loosen the extracellular material 
 surrounding a tumor , resulting in the dissemination of cancer cells 
 into the bloodstream . this action leads to increased migration and 
 metastasis of the cancer cells when exposed to ultrasound . to determine if our experimental conditions 
 contribute to such effects , we conducted migration assays of the panc-1 
 cells in the presence of applied ultrasound . for this endeavor after 
 6 h , we exposed the cells to ultrasound ( 1 mhz , 5 min ) , incubated 
 them overnight , and determined their migration . we observed that there 
 was no significant difference ( p > 0.01 , n = 5 ) in the migration ability of the ultrasound - exposed 
 cells compared to the control samples ( no ultrasound exposure ) . these 
 results suggested that , within our experimental parameters , the migration 
 of the panc-1 cells remained unaffected by the applied ultrasound . 
 further in vivo validation studies with the ammonium 
 bicarbonate encapsulated liposomes are in progress , and the results 
 will be reported in the future . we 
 have successfully demonstrated the proof - of - concept for a new 
 strategy to release liposomal contents in response to reduced ph . 
 with our design , the liposomes encapsulate the gas precursor , ammonium 
 bicarbonate , and do not incorporate ph - sensitive lipids in the bilayer . when incubated in buffers of acidic ph , co2 gas bubbles 
 are generated , thus , inducing echogenicity to the liposomes . the escaping 
 gas bubbles cause structural changes to the liposomes , and release 
 the encapsulated contents ( up to 56% ) . the content release is further 
 enhanced by the simultaneous application of diagnostic - frequency ultrasound 
 ( 1 mhz , 5 min ; 80% release ) . the fluidity of the liposomal membranes 
 plays a crucial role in the contents released . by incorporating a 
 folate lipid in the bilayer , we have successfully targeted the liposomes 
 to pancreatic cancer cells that overexpress the folate receptor on 
 the surface . liposome - encapsulated doxorubicin is efficiently released 
 in the cancer cells , and the release is enhanced by the simultaneous 
 application of diagnostic frequency ultrasound . while the ultrasound 
 was innocuous , the combination of doxorubicin released from the liposomes 
 and ultrasound reduced the viability of pancreatic cancer cells to 
 14% . with further developments , these liposomes have the potential 
 to be an excellent option for ultrasound image guided , targeted drug 
 delivery at tumor sites .