the outer membranes of organisms serve to enclose the functioning cell and separate it from the external environment . this vital protective function is complicated by the need for the cell to permit the entry of nutrients and the egress of waste products . it is unclear what were the earliest barriers that permitted separate cells to evolve and ultimately to combine into higher life forms [ 2 , 3 ] . what is clear is that along with the development of protective or confining membranes , structures and mechanisms had to evolve   that would permit selective passage of ions and molecules through them . today , the proteins that regulate ion balance and transmembrane transport are extremely complex molecules that interact directly with the membranes in which they are embedded , and they exhibit remarkable selectivity ( specificity ) in their chemical functions . during the past two decades , considerable effort has been expended to develop synthetic amphiphiles that will insert into membranes and exhibit at least some of the functions of highly complex protein channels [ 7 , 8 ] . there has been considerable success in this arena , and a number of reviews describe the efforts [ 913 ] . our own effort in this area initially involved the compounds we have called  hydraphiles  . these compounds are typically constructed from three crown ether macrocycles linked by spacer chains of varying lengths . they readily insert into bilayer membranes and conduct ions through them . when studied by planar bilayer conductance methods , these symmetrical molecules are nonrectifying and show toxicity to bacteria , yeast , and even to mammalian cells . the toxic effect results from a rapid disruption of ion homeostasis .   the hydraphile molecules may be viewed as problematic in biological applications owing to their toxic effects . however , at concentrations below those at which they are toxic , they show no effect on bacterial growth , but exhibit a remarkable ability to enhance the efficacy of antibiotics . an example is the recently reported use of hydraphiles in direct injection chemotherapy , as described below . the initial design of the hydraphiles   in many ways paralleled contemporaneous channel designs developed by jullien and lehn   and by carmichael et al . . our approach was based on biophysical data concerning protein channel properties and function because the x - ray structure of a channel protein was unavailable . some guidance was taken from the solid state structure of bacteriorhodopsin , which is an integral membrane protein . estimates were made of the overall length that would be required for the molecule to function within a membrane . an important consideration was what element or module would serve as the amphiphile 's head group and what , if anything , would serve as an energy - lowering element that would reside at the midplane of the bilayer . the various considerations and strategies were described several years ago in a focus article   and will be recounted here only in the general sense . the first structure to be prepared in this effort is shown in figure 1 . the diaza-18-crown-6 macrocycle was chosen to function both as the amphiphilic head group and as what we call the  central relay . previous studies showed that diazacrowns could function effectively as amphiphile head groups [ 25 , 26 ] . the 18-membered ring macrocycles were chosen because they are more readily prepared than other sizes of macrocycles of this type and because we expected them to bind k more strongly than na . in principle , this difference should confer na  >  k selectivity upon the channel because more strongly bound k would be released and transported less readily than weakly bound na . the distance spanned by each carbon - carbon bond in an extended gauche conformation is about 1.25   . two such chains , and the central macrocycle , would give an overall extension to the molecule of approximately 40   . the so - called insulator regime of a phospholipid bilayer is generally thought to have a thickness of about 3035   . the overall distance spanned by the medial macrocycle depends significantly upon whether it is parallel or perpendicular to the membrane 's fatty acid chains . initially , we surmised that the central macrocycle would be perpendicular to the fatty acid chains in the bilayer , but parallel to the two macrocyclic head groups . in this conformation , the macrocycle would span a distance closer to 30   than to 35   . however , it was thought that the central macrocycle would not need to be exactly parallel to the head group surfaces of the membrane ; this would allow for some flexibility in the channel 's overall length . a key reason that the third , central , or medial macrocycle was included in the overall span of the channel was that chemical intuition suggested that some energy lowering element would be required at the midplane of the bilayer . the midplane is the region most remote from the polar head groups and therefore is the least polar portion of the head group - midplane - head group transmembrane gradient . the initial thought was that it would be energetically unfavorable for an ion to traverse a distance as large as 3035   . an encounter between the ion and macrocycle might permit water exchange or a transient complexation interaction . mature consideration , it was apparent that any such complexation interaction between transporter and ion would require additional energy for release of the ion . the overall design required the  distal  macrocycles to function as head groups in the amphiphilic sense [ 27 , 28 ] and as entry portals for ions . hydrocarbon chains containing 12 carbons were used as covalent links . these dodecylene chains would connect the distal macrocycles with the medial macrocycle . each distal crown was then attached to another 12-carbon chain in order to improve interactions with the fatty acid chains in the bilayer , to make the molecule more cylindrical , and to better shield the ion pathway . the chemical structure of the first synthetic hydraphile ( 1 ) is shown in figure 1 , and the biophysical evidence suggests that it is positioned within the bilayer membrane , in figure 2 . one of the macrocycles is linked to two other macrocycles and two of the macrocycles are linked to dodecylene chains . the synthetic challenge was therefore to make a molecule that is overall symmetric and has several symmetrical elements , but in which both macrocycles , and dodecylene chains are connected in slightly different ways . this was accomplished by desymmetrizing the distal macrocycles and then linking them to the central macrocycle by alkylation or by acylation followed by reduction . characterization of the initial channel compound was done by using a dynamic nmr method developed by riddell and coworkers . in this technique , the vesicles in the bulk suspension and the surrounding medium both contain sodium ions . because the vesicles were prepared in the medium , the concentration of na inside and outside of the vesicles is identical . a na - nmr spectrum of this system shows a single resonance because na ions within the liposomes and in the bulk phase experience the same buffer environment . if an nmr shift reagent such as dy is added to the external medium ( the bulk aqueous phase ) , two na resonances will be observed : those within the liposomes and protected from the influence of dy and those in the bulk phase . because there is significantly more sodium in the external solution than is contained within the vesicles , the dy - shifted resonances will be more intense and observed at different positions . if a compound that can insinuate itself into the membrane and lead to sodium exchange is added to the solution , ion transport through the membrane will be observed . this will lead to changes in the respective linewidths . from the change in linewidth , the experiment described , while valuable and reproducible , is both cumbersome and labor intensive . in our experiments , the synthetic channel compounds we prepared were always studied in parallel with the natural peptide channel former , gramicidin ( figure 3 ) . gramicidin is a natural bacterial peptide ( mixture ) that forms robust channels in bilayer membranes . in our studies , whatever rate was observed with gramicidin , that rate was set arbitrarily to a value of 100 . whatever rates were observed for other channel compounds were then normalized to the gramicidin value . this permitted us to obtain reproducible rates at different times on different instruments when run by different operators . compound 1 , shown in figure 1 , transported sodium cation through phospholipid bilayers at a rate equal to approximately 2530% that of gramicidin . this may not seem impressive at first flush . recall that excellent carriers such as valinomycin conduct about 10 ions  sec whereas pore formers typically conduct ions in the 1010 ions  sec range . it should also be noted that a number of the synthetic ion channels were studied in a concentric tube apparatus intended to evaluate carrier transport . there was , however , no correlation between the ability of these compounds to serve as carriers and their channel - like behavior compared to gramicidin 's behavior in liposomal membranes . the dynamic nmr studies described above established that the synthetic ion channel compounds were effective ion transporters . for example a compound that had two macrocyclic rings connected to each other with a dodecylene chain and terminated by two dodecylene chains was found to be inactive . this molecule was too short to span the bilayer , and it lacked the medial macrocycle . n , n -didodecyldiaza-18-crown-6 . these two  partial channel  elements are pictured in figure 4 . other structural fragments of compound 1 first , they have a pore size dictated by the amino acid sequence that leads to a major conductance state . third , a protein channel is typically selective for a particular cation , anion , or molecular species . for example a cation channel is selective for positively charged ions and either potassium or sodium or sometimes proton will be favored over other cationic species and certainly over anions . fourth when the channel is functioning in a bilayer , it typically shows behavior in which the channel undergoes a sequence of open and close events . typically , when a channel is open , the conductance has a unique magnitude . when the pore is closed , ions fail to flow because the phospholipid bilayer is impermeable . if two channels are simultaneously detected in the open state , the peak observed will be twice the height of that observed for a single channel because the pore size is consistent . of course ,  subconductance states  also occur , but these are beyond the scope of this discussion . this so - called  open - close  behavior is detected by an analytical instrument called a planar bilayer conductance voltage clamp apparatus . a liposome or vesicle has a curved surface , but the membrane present in this device is planar and confined to an orifice of approximately 200  m . as a result the trace obtained for a synthetic channel - forming compound with a bilayer clamp instrument is shown in figure 5 along with its structure . when this pattern is observed for a compound , it confirms that its behavior is mimicking that of protein channels . interact directly with other molecules or with the medium to give signals that can be detected at very low concentrations with good accuracy . we prepared a fluorescent synthetic ion channel to use as a probe of structure and activity . the compound is identical in structure to 1 ( figure 1 ) except that the two terminal dodecyl groups have been replaced by fluorescent dansyl residues ( 2 , see figure 6 ) . the fluorescent maximum observed for dansyl channel 2 depends upon the polarity of the medium in which it is present . we measured max for the dansyl channel in a range of solvents from nonpolar to polar . we then measured the fluorescence signal for the dansyl channel embedded in a liposomal membrane . the dansyl groups experienced a polarity approximately equal to that of ethanol (  = 20 ) . we inferred from this that the dansyl channel was not globular nor were its head groups in contact with the highly polar aqueous phase (  = 80 ) .   in another set of experiments , we measured fluorescence resonance energy transfer between the dansyl channel and another channel in which the dansyl groups were replaced by n - methylindolyl residues ( 3 ) . using mixtures of these two channels in molar ratios from 01 and 10 permitted us to plot the fluorescence response as a function of mole fraction . the slope of this line is reported to correspond to the molecularity of channel function . certain phospholipids are available that have on them a radical quenching group called a tetramethylpyrrolidine n - oxide or  tempo  group . these quenching groups are placed along the fatty acid chain at different distances from the head group . by using several of these phospholipids , it is possible to triangulate the distance between a synthetic ion channel 's fluorescent head group , in this case dansyl ( 2 ) , and the midplane of the bilayer . the data obtained in this experiment showed that the dansyl residues were ~15   from the midplane of the bilayer . this also corresponds to the putative thickness of the insulator regime within the bilayer . taken together , these data show that the dansyl groups reside in what we call the  midpolar regime . the midpolar regime is the portion of the phospholipid that contains glycerol and the ester oxygens . if the dansyl residues were in contact with water , the fluorescence response would be a larger red shift than was observed experimentally ( water  = 80 ) . if the dansyl channel was present in the membrane in a globular conformation , the dansyl groups would exhibit a dielectric constant in the range of 25 , similar to that within the membrane 's insulator regime . a breakthrough in the area of protein channel biochemistry occurred when the structure of the kcsa voltage gated potassium - selective protein channel was reported by doyle and coworkers in 1998 . the kcsa channel is a protein , and the conductance pore of which has 8 transmembrane helices . the overall length of the pore is 45   , and its diameter varies along its distance  . from inside the cell ( bottom ) the pore begins as a tunnel 18   in length ( the internal pore ) and then opens into a wide cavity ( ~10   across ) near the middle of the membrane . this latter description can also be applied to the synthetic ion channel family that we have called the benzyl hydraphiles . compound 4 has 12-carbon spacers , each of which has an extended length of about 15   . the benzyl groups extend the molecule farther , and conformational folding would make the overall structure shorter . the twin dodecyl chains comprise the  tunnel  described by mackinnon and the  cavity  near the middle of the membrane  corresponds to the central macrocycle . to our knowledge , the earliest imaging experiment involving any synthetic ion channel was conducted with a hydraphile having fluorescent side arms . in this particular case , it was hypothesized in this experiment that the hydraphile would insert into a cell 's plasma membrane and cause the cell 's periphery to be fluorescent . the images that were obtained showed a fluorescent halo throughout the bacterial periphery , with little infiltration into the cytosol ( figure 8) . the resolution of this fluorescence experiment was not high enough to determine the precise location of the fluorescent residue within the dual external membranes of gram - negative e. coli . of course , the outer membrane of e. coli has channels within it called porins , into which or through which the hydraphile could embed or pass . in the absence of high - resolution data , it appears that the hydraphiles insinuate themselves into both membranes . it is clear that some of the hydraphiles must penetrate both membranes because these synthetic ion channels are toxic to bacteria as well as to yeast at various concentrations . in fact , using a range of hydraphile lengths , we were able to show that the most active channels were most toxic to the bacteria . the toxicity of hydraphiles to bacteria and to yeast was determined initially by using the so - called kirby - bauer test . in this assay a petri dish is filled with growth medium and cellulose disks are placed upon the surface . each of the cellulose disks used was impregnated with a solvent , a control compound , or a compound of interest . if there is no bacteriostatic effect , the bacteria will grow to the edge of the cellulose disk . if there is a bacteriostatic effect , growth will stop at some distance from the disk . since the disk is circular , the surrounding dead space is called a  halo .  when hydraphiles were studied by using this test , toxicity both to bacteria and to yeast was apparent . a hydraphile having relatively short ( octylene ) spacer chains ( 5 ) was used as a control . this compound was known to be functionally impaired in bilayer membranes ( figure 9 ) . hydraphile 5 has all of the polarity and structural elements that the active hydraphile 4 possesses , but it is too short to span the membrane . thus , 5 was the ideal control and showed almost no activity in the kirby - bauer test . when more quantitative studies were undertaken , it was found that hydraphile 5 ( 12-carbon spacers ) was approximately 15-fold more active than control 5 , which contains 8-carbon spacers . detailed studies demonstrated length dependence between hydraphiles and ion transport function in a bilayer membrane . when compounds were too short to span the bilayer , essentially no activity was observed . when compounds were significantly longer than the thickness of a bilayer , function was observed , but it was generally poor . this lower level of activity was attributed to the inability of the longer compounds to achieve an appropriate conductance state conformation . the compounds that had 12- ( 4 ) , 14- ( 7 ) , or 16-carbon ( 8) spacer chains showed good activity and were similar in their ability to conduct sodium ions . the graph of figure 9 shows this relationship in the upper line labeled  na transport . the graph of figure 9 superimposes toxicity results for compounds 410 with their toxicity either to gram - negative e. coli or to gram - positive bacillus subtilis . the left ordinate scale shows the minimum inhibitory concentration ( mic ) of hydraphile required to kill the bacteria . the superimposed transport of sodium cation is presented on the same logarithmic scale as ion release from liposomes of 0100% . the plots show a strong inverse relationship , which is especially satisfying considering that chemical and biological responses are being compared . first , were they toxic to mammalian cells as well as to prokaryotes and to primary eukaryotes ? third , if the hydraphiles were toxic to mammalian cells , could they still function as ion channels at low ( sublethal ) concentrations ? the last question was answered by using the patch clamp technique on human embryonic kidney ( hek 293 ) cells . after a period of observation , the cell was bathed in saline to extract the hydraphile and the electrical activity of the membrane gradually decreased . later studies showed that although the hydraphiles were less toxic to mammalian cells than to bacteria or to yeast , the difference was too small to permit their effective use as a therapeutic agent . although the selectivity between bacteria and mammalian cells was insufficient to use hydraphiles as medicaments , two possible applications were considered . one of these was to take advantage of the toxicity and use hydraphiles as agents for direct injection chemotherapy . the other was to use the ability of hydraphiles to penetrate membranes as a synergistic component with known antibiotic compounds . erythromycin , kanamycin , rifampicin , and tetracycline   are all fda - approved antibiotics ( figure 10 ) . the mode of action of each of these compounds is known , and each is different from the other . experiments were conducted to determine the minimum inhibitory concentrations ( mics ) for each antibiotic against several organisms : e. coli , pseudomonas aeruginosa , and b. subtilis . the toxicity of each antibiotic was then assayed when nontoxic concentrations of hydraphile were present . the first significant finding of this study was that the presence of 1  m 4 or 7 had no effect on the growth of e. coli . in the presence of this same concentration of 7 , when hydraphile 7 was coadministered ( = 1  m ) with erythromycin , the minimum inhibitory concentration ( mic ) observed for the antibiotic changed from 200  m to 25  m . a similar increase was observed for rifampicin : the mic determined in the absence of hydraphile ( 50  m ) improved 16-fold to 3.1  m . tetracycline is relatively active against e. coli ; its mic under the conditions studied was 3.1  m . in the presence of 7 [ 1  m ] , additional , unpublished studies have shown that hydraphile synergy applies to other antibiotics and to a variety of prokaryotes or to the yeast saccharomyces cerevisiae . a possible mechanism for the activity is membrane disruption that permits more facile entry of the antibiotic into and through the bilayer . alternately , the hydraphiles may disrupt the function of efflux pumps that eject the antibiotics . notwithstanding , higher concentrations of hydraphiles do exhibit cellular toxicity , and this property was used to advantage as discussed in the next section . a basic principle of chemotherapy is that the rapid growth of tumor cells can be used against them in therapy . thus , tumor cells are susceptible to the poison as are such rapidly growing cells as hair and gut epithelium . if the tumor is localized , it can be surgically excised , although anticancer drugs are often administered as adjuvant or prophylactic agents . an alternative to surgery is to directly inject a suitable toxin at the tumor site . second , the tumor must be localized ( not metastasized ) as is generally required for a surgical approach . both ethanol ( etoh , ch3ch2oh ) and acetic acid ( ch3cooh ) have been studied as direct injection ( also called percutaneous injection ) therapeutics , and both achieved some success . a key limitation was the fact that these small molecules diffuse rapidly throughout tissue making the desired localization problematic . a potential advantage of hydraphiles in this context is that they insert in bilayers and achieve their toxic effect by disrupting ion homeostasis without rapidly diffusing from the site of injection . advantages of the hydraphiles as direct injection chemotherapeutics include their hydrophobicity , their ability to insert in a membrane and disrupt ion homeostasis , and their low proclivity to diffuse within tissue . a range of hydraphiles has been studied , and in all cases , their octanol - water partition coefficients ( logp ) were at least 10 ( i.e. , logp > 10 ) in favor of octanol . the lack of diffusion was surmised simply by comparing the size and number of polar elements in a hydraphile with a molecule such as ethanol . this was done in concert with a study to confirm that rapid tissue damage would take place at the site of hydraphile injection . imaging agents are critical to locating a tumor or disease site and to following the progress of therapy . compound 11 ( figure 11 ) was prepared as an imaging probe that could detect anionic phospholipids . the two dipicolylamine - zinc(ii ) cationic binding sites ( blue ) give the probe an affinity for phosphatidyl serine ( ps ) , and phosphatidyl glycerol ( pg ) . the latter occurs broadly in bacterial membranes and ps is the most abundant anionic lipid that occurs in mammalian cells . the dipicolylamine - zinc(ii ) affinity group is attached to a carbocyanine fluorophore ( red , cy7 ) that has an excitation wavelength ( exc ) at 794  nm and emits at 810  nm . this emission is in the near infrared region and is readily observed from within a living specimen , whereas the emission from conventional fluorescent dyes with green and red wavelengths is obscured by tissue components . a particular advantage of probe 11 is that apoptosis can be observed because of changes in the internal and external membrane structures that occur upon cell death . typically , when a mammalian cell dies , phosphatidylserine moves from the internal membrane , where it is abundant , to the extracellular side . the anionic phosphatidylserine head group is complexed by the affinity element of 11 , and the site of cell death can readily be identified by the localization of fluorescence . it has recently been demonstrated that probe 11 will image cell death , as it occurs in mouse muscle tissue treated with various agents including etoh , ketamine , and compound 4 ( figure 12 , frames a - e ) . note that the hydrophobicity of hydraphiles requires that they be dissolved in etoh , and thus any solvent - related toxicity from it will provide an important baseline for comparison . of the three agents tested , hydraphile 4 was the most biologically active with a maximum target / nontarget ratio of 6 , compared to a range of 3 - 4 for ethanol and ketamine . histology confirmed the muscle tissue damages elicited by 4 and the uptake of nir probe 11 at the surface of these cells ( figure 12 , frames f and g ) . a subsequent in vivo study was conducted to determine the biological activities of an increased number of hydraphile compounds and structural subunits . the three most active hydraphiles noted during in vitro and antimicrobial studies , 4 , 7 , and 8 , with c12 , c14 , and c16 chains , were thus tested in vivo using probe 11 to detect muscle damage . as is apparent in frames a , b , and c of figure 13 , there was no difference between the three compounds , indicating that additional bioactivity could not be gleaned from refinement of channel length . a final in vivo study was conducted to assess the behavior of two structural subunits of 4 . compound 12 is n , n -didodecyl-4,13-diaza-18-crown-6 and is the central span of the hydraphile , but lacks the two terminal macrocycles . in previous studies in liposomes and mammalian cells , this central relay component showed no tendency to form channels , although it did exhibit toxicity to bacteria and to yeast . compound 13 is monobenzyl-4,13-diaza-18-crown-6 and comprises the distal macrocycles that form the channel opening on each side of the membrane . this compound has not demonstrated measurable activity in assays with liposomes , bacteria , or mammalian cells . panels d and e of figure 13 show that 12 and 13 did not have measurable activity in excess of the etoh control ( frame f ) during in vivo studies of tissue damage . these results confirm that hydraphile components themselves will not elicit maximum tissue damage unless they are covalently bound to yield the structure given as 4 . it has now been just over twenty years since hydraphiles were first conceived and synthesized . considerable synthetic effort has been dedicated to delineating the structural basis for hydraphile ion transport activity . further , an exhaustive experimental effort has been maintained over this time period to establish and definitively confirm a channel mechanism for these molecules . this work has evolved from in vitro and biophysical assays to work in pro- and eukaryotic cells and now to living mice . in the latter case , in vivo imaging has been instrumental in establishing that ( 1 ) hydraphiles are biologically active toxins that elicit acute tissue damage , ( 2 ) structural subunits of hydraphiles will not cause the same level of damage as when covalently bound , and ( 3 ) the channel length optimization derived from in vitro and cell studies is also applicable to in vivo studies . the continued evolution of this work provides exciting new opportunities for hydraphiles as therapeutics in a variety of disease models including cancer .
hydraphiles are a class of synthetic ion channels that now have a twenty - year history of analysis and success . in early studies , 
 these compounds were rigorously validated in a wide range of in vitro assays including liposomal ion flow detected by nmr or ion - selective electrodes , as well as biophysical experiments in planar bilayers . during the past decade , 
 biological activity was observed for these compounds including toxicity to bacteria , yeast , and mammalian cells due to stress caused by the disruption of ion homeostasis . 
 the channel mechanism was verified in cells using membrane polarity sensitive dyes , as well as patch clamping studies . 
 this body of work has provided a solid foundation with which hydraphiles have recently demonstrated acute biological toxicity in the muscle tissue of living mice , as measured by whole animal fluorescence imaging and histological studies . here 
 we review the critical structure - activity relationships in the hydraphile family of compounds and the in vitro and in cellulo experiments that have validated their channel behavior . 
 this report culminates with a description of recently reported efforts in which these molecules have demonstrated activity in living mice .