Patent Application: US-201515312943-A

Abstract:
the present invention relates to the formation of emulsions using small amphipathic molecules , such as peptides which self - assemble to form an interface between at least two substantially immiscible liquids . the emulsions may find application is a variety of technological fields , such as in food , cosmetics , life style products , coating , catalysis , encapsulation , drug delivery and / or cell assays . there is also provided a method of making such emulsions , as well as methods of tailoring the stability of the emulsions for particular applications .

Description:
the present invention will now be further described by way of example and with reference to the figures which show :— fig1 shows ( a ) cartoon of self - assembly and formation of fibrous network of aromatic short peptide amphiphiles at oil / water interface . ( b ) cartoon of oil - in - water droplets stabilized by peptide fibrous network . ( c ) chemical structure of aromatic peptide derivatives including fmoc - yl , fmoc - ya , fmoc - ys , fmoc - ff , fmoc - fff and pyrene - yl . fig2 shows ( a ) optical photographs of glass vials in which chloroform - in - water emulsions ( white foamy layer ) were prepared by adding 10 mmol · l − 1 fmoc - yl phosphate buffer solution ( ph 8 ) to chloroform with manual agitation . from left to right , the volume ratio of buffer solution to chloroform is altered from 1 : 9 , 3 : 7 , 5 : 5 , 7 : 3 , to 9 : 1 and samples are named as w1c9 , w3c7 , w5c5 , w7c3 and w9c1 . ( b ) fluorescent microscope image of chloroform - in - water emulsion droplets stabilized by fmoc - yl networks containing fitc in water phase ( sample w3c7 ). scale bar is 50 μm . ( c ) fluorescent microscope image of chloroform - in - water emulsion droplets stabilized by tht labelled fmoc - yl networks ( sample w3c7 ). scale bar is 50 μm . ( d ) ftir spectra of self - assembly of fmoc - yl in chloroform ( black ), d 2 o phosphate buffer solution ( ph 8 ) ( red ) and at interfaces stabilizing emulsions ( blue ). ( e ) sem micrographs of fmoc - yl networks at chloroform / water interface stabilizing the chloroform - in - water emulsions . the samples are prepared at freeze - drying condition . the scale bars are 50 μm ( left ) and 2 μm ( right ). ( f ) sem micrograph of fmoc - yl microcapsules at chloroform / water interface . the sample is prepared at air - drying condition and scale bar is 2 μm . fig3 shows ( a ) fluorescent microscope images of chloroform - in - water emulsion droplets stabilized by fmoc - ya ( left ) and fmoc - ys ( right ) networks containing fitc in water phase . scale bar is 50 μm . ( b ) ftir spectra of 10 mmol · l − 1 fmoc - yl , fmoc - ya and fmoc - ys in d 2 o phosphate buffer solution ( ph 8 ). ( c ) table of the calculated partition coefficient ( c log p ) and the measured partitioning of peptides between water , chloroform and accumulated at the interface , the critical emulsion concentration of fmoc - yl , fmoc - ya and fmoc - ys and the average diameters of emulsions droplets . fluorescent microscope images of ( d ) chloroform - in - water emulsion droplets stabilized by fmoc - ff containing fitc in water phase , ( e ) water - in - chloroform emulsion droplets stabilized by fmoc - fff containing fitc in water phase and ( f ) chloroform - in - water emulsion droplets stabilized by pyrene - yl . scale bar is 50 μm . fig4 shows ( a ) optical photographs of glass vials in which chloroform - in - water emulsions ( white foamy layer ) were prepared with 10 mmol · l - 1 sds solution and fmoc - yl phosphate buffer solution by manual agitation . the top images show freshly prepared emulsions and the bottom images show emulsions incubated for 2 weeks . ( b ) optical photographs of glass vials in which chloroform - in - water emulsions ( white foamy layer ) were prepared with 10 mmol · l - 1 sds solution and fmoc - yl phosphate buffer solution by manual agitation . the top images show freshly prepared emulsions and the bottom images show emulsions heated at 60 ° c . for 3 hours . ( c ) optical photographs of glass vials in which chloroform - in - water emulsions ( white foamy layer ) were prepared with 10 mmol · l - 1 sds and fmoc - yl in 100 mm phosphate , chloride and thiocyanate buffer solution by manual agitation . the top images show freshly prepared emulsions and the bottom images show emulsions incubated for 24 hours . fig5 shows ( a ) optical microscope images of adding 1 mg · ml − 1 thermolysin buffer solution into chloroform - in - water emulsion droplets stabilized with 2 mmol · l − 1 fmoc - yl buffer solution after 0 , 20 , 40 , 60 seconds . scale bar is 50 μm . ( b ) histogram of the size distribution of adding 1 mg · ml − 1 thermolysin phosphate buffer solution into chloroform - in - water emulsion droplets stabilized with 2 mmol · l − 1 fmoc - yl buffer solution after 0 , 20 , 40 , 60 seconds . ( c ) optical photographs of vials in which emulsions formed ( left ) and demulsified ( right ) in addition of thermolysin . emulsions were stabilized with 2 mmol · l − 1 fmoc - yl buffer solution in absence ( left ) and presence ( right ) of 1 mg · ml − 1 thermolysin after 10 min . fig6 : schematic for computational stabilized emulsions a ) tripeptides kyf / kff / kyw / dff / ffd b ) aqueous md simulations showing self assembled nanostructures c ) self - assembled stabilized emulsions droplet . simulation time 9 . 6 μs . fig7 : experimental observations of peptide emulsions a ) emulsions formed from each of the tripeptides b ) fluorescent microscope of kyw labeled with i ) sudan ii ii ) thioflavin t iii ) overlay of both , scale bar 10 μm c ) ftir of the samples in the aqueous state d ) ftir of samples in the emulsions state showing amide i region . fig8 : temperature effects on peptide emulsions at a ) 30 ° c . b ) 60 ° c . fig9 ( a ) schematic representation of the behaviour of fmoc - ypl before and after alkaline phosphatase dephosphorylation in a chloroform / water biphasic system , showing the ability of fmoc - yl to stabilize emulsions , contrary to fmoc - ypl which follows a surfactant - type behavior and relaxes back to two - phases after 1 hour . cyan blue represents water , yellow chloroform and green the alkaline phosphatase ; ( b ) chemical structures of aromatic peptide amphiphiles fmoc - ypl and fmoc - yl ; ( c ) alkaline phosphatase structure . fig1 . zoomed - in tem image ( original in figure s1 from esi ) and macroscopic appearance of fmoc - ypl ( a ) and tem image of fmoc - yl ( b ), showing the importance of the alkaline phosphatase to initiate self - assembly into a nanofibrous network and self - supporting hydrogel ( ammonium molybdate 2 % stain ); ( c ) normalised fluorescence emission spectra of fmoc - ypl ( 0 h ) and fmoc - yl achieved 24 h after enzyme addition ( un - normalised data included in figure s3 from esi ) ( excitation 280 nm ); ( d ) representation of the lambda max wavelength at which fluorenyl peaks were observed before and 24 hours after enzyme addition , showing a redshift ( except for the fmoc - ypl control ); ( e ) amide region of ftir absorbance of fmoc - ypl and fmoc - yl ; ( f ) dephosphorylation from fmoc - ypl to fmoc - yl monitored by reversed phase hplc . fig1 a ) optical photographs of glass vials showing the behaviour of fmoc - ypl and fmoc - yl in a chloroform / water biphasic system , immediately after hand shaking for 5 seconds and after 1 hour / 2 weeks , in addition to the ability of fmoc - ypl , completely demulsified after 2 weeks , to form an emulsion when alkaline phosphatase is added ; ( b ) fluorescence microscopy image of chloroform - in - water emulsion stabilized by nanofibrous networks of fmoc - yl containing fitc in water phase . scale bar is 100 μm ; ( c ) sem image of chloroform - in - water emulsion droplet . scale bar is 1 μm . inset presents a zoomed - in chloroform - in - water droplet . scale bar is 10 μm ; ( d ) dephosphorylation monitored by reversed phase hplc in buffer and in the biphasic system , showing that alkaline phosphatase is active in the chloroform / water system ; ( e ) dephosphorylation monitored by reversed phase hplc when adding alkaline phosphatase to the demulsified fmoc - ypl at different timings . fig1 . ( a ) snapshot of fmoc - ypl system after 200 ns . fmoc is represented in blue , tyrosine and leucine in red , phosphate group in black , ions in grey , water in red and octanol in cyan . inset presents one tyr - tyr h - bonding between 2 molecules , and the surfactant - type behaviour , coloured by element ; ( b ) snapshot of fmoc - yl system after 200 ns . fmoc is represented in blue , tyrosine and leucine in red , water in red and octanol in cyan . inset presents fmoc - leu and fmoc - tyr h - bonds between 2 molecules ; c ) hydrogen bonds per molecule between fmoc - ypl molecules throughout the simulation in biphasic system ; ( d ) hydrogen bonds per molecule between fmoc - yl molecules throughout the simulation . in this work , there is disclosed a series of aromatic short peptide amphiphiles which are exemplified by combining 9 - fluorenylmethoxycarbonyl ( fmoc ) or pyrene ( pyr ) with di - or tri - peptides tyrosine - leucine ( yl ), tyrosine - alanine ( ya ), tyrosine - serine ( ys ), di - phenylalanine ( ff ) and tri - phenylalanine ( fff ) with varying hydrophobicity and functional groups ( fig1 ) these peptide amphiphiles self - assemble at organic / aqueous interfaces rapidly forming a highly stable microcapsules fibrous network . unlike absorption of traditional surfactants with hydrophilic head and hydrophobic tail at interfaces , the nanostructures self - assembled by aromatic π - π stacking and hydrogen bonding of peptide sequences to stabilize the organic ( or water ) droplets in aqueous ( or organic ) media ( fig1 b ). initial interest in studying aromatic peptide amphiphiles at the organic / aqueous interface started from the observation that aromatic peptide fmoc - yl forms a gel in both phosphate buffer solution ( 10 mm ) and chloroform ( 25 mm ). at low concentrations fmoc - yl ( 0 . 5 mm ) was shown to transfer between aqueous and organic phases to reach an equilibrium distribution . by adding different volumes of chloroform to 10 mm fmoc - yl buffer solution at 80 ° c . ( the volume ratio of buffer solution to chloroform is altered from 1 : 9 , 3 : 7 , 5 : 5 , 7 : 3 , to 9 : 1 ), after manual agitation for 5 seconds emulsions form in vials , as fig2 a shown . unexpectedly , these remain stable for months , showing that the formed peptide layers provide an excellent barrier to prevent coalescence . in the images shown , the milky layers are emulsions and the transparent layers above and below are the aqueous and chloroform phase , respectively . uv - vis was used to determine the amount of fmoc - yl that remained in water phase and transferred to chloroform . fluorescein isothiocyanate ( fitc ) was used to label the aqueous phase in the emulsion layers for imaging by fluorescence microscopy . fig2 b indicates that chloroform - in - water emulsions form after emulsification stabilized by fmoc - yl . the absorption of fmoc - yl at the chloroform / water interfaces could be quantified by uv - vis spectra by measuring the concentration in each phase and with the remainder absorbed at the interface . based on uv analysis , in a 50 : 50 water / chloroform system , the amount of fmoc - yl absorbed at the chloroform / water interface could be calculated as 1 . 9 mmol · m − 2 . the calculated maximum absorption of a close - packed monolayer of fmoc - yl is 3 . 4 μmol · m − 2 indicating that fmoc - yl absorbed at the interfaces is composed of a film rather than a monolayer . the structure of this peptide interfacial film at the chloroform / water interface was investigated , using a range of microscopy and spectroscopy technologies . thioflavin t ( tht ) was used to label self - assembled peptide structures . after dissolving the fmoc - yl with tht in both solvents , there is low emission in water and almost no emission in chloroform is observed , while upon gelation ( 24 h ) the appearance of stronger emission in both water and chloroform demonstrates that the self - assembled β sheet - like of fibrous structures are formed . tht was subsequently used to label the interfacial film , fig2 c shows that a fmoc - yl shell stabilized the organic droplets suggesting the self - assembly of peptide β sheet - like structures at the interface . infrared spectroscopy was then used to determine the h - bonding interactions that underpin self - assembly of fmoc - yl fibrous structure in water , chloroform and at the interface . fig2 d shows an infrared absorption spectrum in d 2 o typical for peptides in a well - ordered β sheet - like arrangement with peaks at 1623 and 1684 cm − 1 for amide and carbamate moieties , respectively . in chloroform , these peaks were observed at 1632 and 1687 cm − 1 with an additional absorption at 1652 cm − 1 indicating the presence of a less - ordered h - bonding network . additionally , a peak assigned to the carboxylate group of the c - terminus was found at 1588 cm − 1 in water , while a peak was observed at 1708 cm − 1 in chloroform indicating protonation of the c - terminus in the organic solvent . at the interface , fmoc - yl adopts a confirmation that is similar to the chloroform system with two peaks , indicating a less ordered β sheet - like environment ( 1623 and 1641 cm − 1 ). the c termini remain ( in part ) deprotonated as indicated by a peak at ( 1588 cm − 1 ) suggesting that the nanostructured network is predominantly situated in the aqueous phase . a scanning electron microscope ( sem ) was used to visualize the interfacial fmoc - yl film . freeze - drying was used to prepare the samples with retention of structure . fig2 e shows fibrous networks at the interface . upon air - drying condition , fig2 f shows the peptide stabilized emulsion droplets that remain as microcapsules after solvent evaporation and consequent shrinking . the properties of aromatic peptide amphiphiles , such as hydrophobicity and chemical groups which may affect the emulsification , can be altered by changing the aromatic group or peptide sequence . we prepared a series of peptide amphiphiles by changing one amino acid on the peptide sequence with decreasing hydrophobicity from tyrosine - leucine ( yl ), tyrosine - alanine ( ya ) to tyrosine - serine ( ys ). fmoc - ya can form gel in both buffer solution and chloroform ( 30 mm ), while fmoc - ys only forms gel in aqueous media under these conditions . atomic force microscopy ( afm ) images show the formation of fibrous structure of fmoc - yl , fmoc - ya and fmoc - ys gels in buffer solution to demonstrate their propensity for unidirectional assembly . fig3 a shows that fmoc - ya and fmoc - ys can also stabilize chloroform - in - water emulsions . infrared spectra ( fig3 b ) confirm the presence of β sheet - like h - bonding in fmoc - yl and fmoc - ya in aqueous media with a much weaker contribution for fmoc - ys . fig3 c lists the calculated partition coefficient ( c log p ) and the measured partitioning of peptides between water , chloroform and accumulated at the interface . these values correlate with the critical emulsion concentration ( obtained from emulsification experiments ) of fmoc - yl , fmoc - ya and fmoc - ys and the average diameters of emulsions droplets . it demonstrates that more hydrophobic peptide amphiphiles and stronger h - bonding interactions between the peptide backbones lead to stronger absorption at the chloroform / water interfaces resulting in a decrease in size of emulsion droplets and lower critical emulsion concentrations . to further increase the hydrophobicity , di - and tri - phenylalanine were tested as the peptide sequences ( fmoc - ff and fmoc - fff ). fmoc - ff was previously demonstrated to form nanofibrous structures ( ref ). fig3 d shows that self - assembled fibres labelled with fitc stabilize the chloroform droplets at the interface . the fmoc - fff amphiphiles become too hydrophobic to dissolve in the water phase , but they do dissolve in chloroform . upon preparation of 10 mm fmoc - fff chloroform solution and added to buffer solution containing fitc and emulsification , the core of the emulsion droplets are fluorescent which indicates the formation of water - in - chloroform emulsions ( fig3 e ). replacing the fmoc group with pyrene , amphiphiles and the emulsion systems are inherently endowed with fluorescent properties . fig3 f shows the formation of chloroform - in - water emulsion with blue emission stabilized by self - assembly of pyrene - yl . there is the clear expectation , based upon the results presented that it should be possible to change the peptide sequences and aromatic moieties to further to optimize and functionalize the emulsion systems . the attraction of using self - assembled barriers of aromatic peptides amphiphiles to stabilize emulsions rather than traditional surfactants , e . g . sodium dodecyl sulfate ( sds ), dramatically enhances the stability of emulsions . fig4 a shows that after leaving them for two weeks at room temperature , the emulsions stabilized by sds are demulsified and phase - separated , while the emulsions stabilized by fmoc - yl are still stable . fmoc - yl stabilized emulsions are heat stable with no visible change observed after exposure to 60 ° c . for 3 h , which is comparable to the performance of sds ( fig4 b ). in 100 mm phosphate , chloride and thiocyanate solution , 10 mm sds stabilized emulsions are phase - separated after 24 h as fig4 c shows , while fmoc - yl networks are not influenced by the salt effect . the inventors have observed that fmoc - yl can also stabilize both hexadecane - in - water and mineral oil - in - water emulsions instead of chloroform making the approach generally applicable for variety of organic media . another vital advantage of using peptide self - assembly to stabilize the emulsion systems is the ability to digest the stabilizing film using a suitable enzyme . proteases , the enzymes that cleave peptide bonds cause disassembly of amphiphiles . fig5 a and 5 b show that after adding thermolysin to fmoc - yl stabilized emulsions , the emulsion droplets are demulsified and coalesce to bigger droplets in 60 seconds . when the conversion of cleaving reaction , catalyzed by thermolysin in aqueous media , reaches 50 %, detected by high - performance liquid chromatography ( hplc ) after 10 minutes , the complete demulsification and phase separation was observed fig5 c . the computational screening protocol , reported previously , 1 - 2 was applied to identify tripeptides that were able to self - assemble in water . from this initial screen of approximately 8000 , a subset of tripeptides were identified that showed the potential to form fibers and bilayer structures , which was considered as a pre - requisite for the compounds to act as emulsifiers . this process led to the selection of five tripeptides ( fig6 a ) that were simulated , using the martini coarse - grained force field , 3 for a further 9 . 6 μs in both water and water / octane solutions kyw , kff and kyf have been previously identified as tripeptides that are able to form hydrogels . the simulation of these peptides in water results in extended fibril structures ( fig6 b ), which is indicative of self - assembled fibers . however , two additional peptides were identified : ffd and dff , which after further simulation in water , were shown to self - assemble into a bilayer - like structure ( fig6 b ). since this new structure shows potential amphiphilic behavior , this suggests it may be a strong candidate for forming emulsions . the simulation of the tripeptides in a biphasic system was modeled through the use of an octane / water solvent box . each of the tripeptides were then subjected to a new 9 . 6 μs simulation in the biphasic system to determine whether they would be able to stabilize the octane within the aqueous solution . the simulations show the assembly of the organic solvent as droplets with the peptides assembled at the water / octane interface . as expected , the peptides assemble with the hydrophobic groups exposed to the organic core of the droplet thus decreasing the interfacial tension between the two phases . similarly , the hydrophilic groups act as a barrier for the water phase . the arrangement of the peptides in such an assembly indicates the peptides act as amphiphiles stabilizing the interface . the inability of the tripeptides to form nanofibers around the interface is related to the size limitations of the model . the simulation involves 300 tripeptides , which does not provide sufficient coverage , when self - assembled into a fiber , to encapsulate the octane droplet . nonetheless , the ability of the tripeptides to interact with both the organic and aqueous phases is considered as a positive indicator for these molecules to act as emulsifiers and therefore laboratory experiments were carried out to test this prediction . the five tripeptides were purchased at & gt ; 98 % purity . each of the tripeptides were then dissolved in water and the ph was altered to a neutral ph ˜ 7 . 5 . to create the emulsions , 100 μl of sudan ii labeled rapeseed oil was added to each of the systems . rapeseed oil was chosen for comparison with food regulated oils . homogenization was carried out on each sample for 5 secs thereafter ; the samples were stored for 24 hrs to ensure a stable emulsion was formed . visual inspection of the resulting emulsions revealed a variety of stabilities across the five tripeptides . kyf , kff and kyw form more stabilized emulsions than dff and ffd , which suggests that the ability of these tripeptides to form nanofibres , as observed in the aqueous state , may play a pivotal role in the stability of the emulsion . the opacity of the samples ( fig7 a ) differs between the surfactant like ( ffd and dff ) and fibrous ( kyf , kff , kfw ) emulsifiers , with the more opaque emulsion indicating greater stability due to the complete dispersion of oil within the aqueous phase . the inventors have also observed that emulsions can be formed using other oils , such as vegetable oil in combination with the peptides identified . fluorescent microscopy was carried out on each of the samples to identify the size and distribution of the droplets as well as to identify how the peptides interact at the interface . labeling the organic phase with sudan ii revealed a mixture containing stabilized organic droplets . the introduction of thioflavin t , 4 which labels the peptide region ( β - sheet formation ) shows that the kyw is localized to the interface of the droplets . this suggests that , in the case of kyw , the tripeptide is self - assembling into fibrils are at the interface to create a network which is stabilizing the droplet , as observed herein for a range of fmoc - dipeptides . the combination of the two dyes further highlights the localization of the tripeptide to the surface of the droplet , with negligible sensitivity away from the interfacial region . to confirm that the tripeptides form self - assembled nanoscale network structures , rather than aggregating in a surfactant - like manner at the interface , ftir spectroscopy was carried out to identify key interactions that are indicative of self - assembly of the peptides . to characterize the structures , studies were initially performed on the tripeptides in the aqueous phase , where kyf , kff , and kfw are known to self - assemble and ffd and dff do not ( fig7 c ). significant changes are observed in the infrared spectroscopy upon aggregation of the peptides into nanostructures . primarily , intense ir peaks around 1625 cm − 1 and 1650 cm − 1 show strong hydrogen bonding between the amide groups of the peptide chains , which are present in the fiber forming peptides . similarly , a larger broad peak around 1560 cm − 1 is indicative of the deprotonated carboxylate group , coo − . the shift and broadening of this peak from the solution state to the gel state indicates an introduction of a salt bridge between either the corresponding termini or side group . this proposes a head to tail interaction between the peptides with hydrogen bonding between the two the self - assembled structures giving an overall extended stable structure . in the biphasic systems where the emulsions are formed , the samples that form fibers ( kyf , kff , kyw ) have identical ftir spectra to that observed in the aqueous phase . this indicates that similar fibrous networks are forming and therefore , these droplets are most likely stabilized by nanofiber networks . in contrast , comparison of the ftir spectra for ffd and dff between the aqueous and biphasic systems , reveal the emergence of peaks in the emulsion state indicating the formation of nanostructures , which stabilize the droplets . these peaks correspond to the c ═ o stretch of the peptide backbone . since this is not observed in the aqueous state , this suggests that the introduction of the oil induces the self - assembling process for these peptides . although , the peak is relatively weak , the single peak could show the parallel arrangement of the peptides , which are assembling in a similar manner to the traditional surfactant model . the ability to trigger the separation of an emulsion through environmental triggers is useful property within a variety of application areas . 5 in particular , the ability to degrade emulsions at various temperatures is a key property of interest for the application of emulsions in the food industry . 6 therefore , the thermal stability of the emulsions was investigated for each of the five tripeptides . each sample was placed in an oil bath and the emulsions were monitored in 10 ° c . intervals ( fig8 ). as the temperature is increased the emulsion separates into two different layers . this de - emulsification is observed for all samples at 60 ° c . apart from kyf , which remains in the emulsified state . this shows that kyf has a higher tolerance for heat than the other samples and that there are opportunities to tune heat resistance by sequence . across the range in temperatures , it is clear that dff and ffd de - emulsify relatively quickly which correlates with the initial observations that these peptides are not as strong emulsifiers , but also that traditional surfactants have thermal stability issues . kff begins to breakdown at approximately 40 ° c . with kyw breaking down at 50 ° c . suggesting the strength of the hydrogel is directly proportional to the stability of the emulsion . switchable or stimuli - responsive surfactant ability is attractive for emulsion ( de ) activation at specific industrial process stages . by applying a specific trigger , the control of the emulsifying ability is enabled , being this a rapid efficient method of creating / breaking emulsions at a desired stage . we studied the enzymatic conversion , using alkaline phosphatase , of the precursor fmoc - tyrosine phosphate - leucine ( fmoc - ypl , fig9 b ) into fmoc - tyrosine - leucine ( fmoc - yl , fig1 b ) in the aqueous phase . having established the ability of the enzyme to trigger the self - assembly process , the second part of the experiment was to investigate the on - demand formation of amphiphile fmoc - yl fibres at the chloroform / water interface , converting the surfactant - adsorbed biphasic mixture into a network - stabilized emulsion ( fig9 a ). enzymatic conversion of fmoc - ypl to fmoc - yl . the precursor solution , 10 mm fmoc - ypl in 0 . 6 m sodium phosphate buffer ph 8 , is not able to form a gel and does not show any evidence of fibre formation ( fig1 a ) due to electrostatic repulsion between deprotonated phosphate groups . upon addition of alkaline phosphatase , the clear solution of the precursor fmoc - ypl was converted into fmoc - yl , producing a nanofibrous network ( fig1 b ) that resulted in a hydrogel . monitoring of the dephosphorylation reaction by reversed phase hplc ( fig1 f ) revealed that approximately 90 % of fmoc - ypl is converted into fmoc - yl after 2 hours , with complete conversion to fmoc - yl achieved within 24 hours ( fig1 f ). when chloroform is added in a 1 : 1 volume ratio to the 5 mm fmoc - ypl buffer solution and hand - shaken for 5 seconds , an emulsion is formed . fmoc - ypl follows a surfactant - like behaviour , visible by the formed foam , where the amphiphile absorbs to the oil / water interface . however , the structure of fmoc - ypl implies an inability to effectively stabilize the interface and demulsification occurs after one hour ( fig4 a ). on the other hand , when alkaline phosphatase is added to the biphasic system containing fmoc - ypl and hand - shaken , nanostructures of fmoc - yl are self - assembled at the interface between water and chloroform , creating an emulsion that is stable for months ( fig1 a ), as demonstrated previously . enzyme - triggered self - assembly allows temporal control over emulsification , achieved by adding alkaline phosphatase to the fmoc - ypl biphasic system ( fig1 a ). molecular dynamic ( md ) simulations were carried out to investigate the ability of fmoc - ypl and fmoc - yl to form ordered structures in a biphasic environment . the 60 molecules of fmoc - ypl or fmoc - yl were randomly distributed in the water phase of a large box which contained an tip3p water and octanol . the tendency of both fmoc - ypl and fmoc - yl to aggregate towards the interface of the solvents was observed in the simulations ( see final snapshots of the 200 ns simulation in fig1 a and 12 b ). from the final snapshots of the system it is clear that although both systems are able to assemble at the interface of the solvents , fmoc - ypl is evenly distributed along the length of the box , with minimal penetration into the octanol solvent , occurring predominantly for the fmoc residues and the leu residues ( fig6 a ). in contrast , fmoc - yl is able to form a more ordered aggregate which allows partitioning of the resulting fibre - like structure into the octanol phase ( fig1 b ), which is consistent with the results from partitioning experiments ( table 1 ). in conclusion , we demonstrate the use of short peptides , optionally containing aromatic groups , to self - assemble into nanofibrous networks at the organic / aqueous interface to emulsify and stabilize a variety of oil - in - water or water - in - oil emulsions . the formation of peptide microcapsules at interfaces provides long - term and higher stability against temperature and varied salts , compared with traditional surfactants , e . g . sds . the peptide amphiphiles can , in some embodiments , be designed by altering aromatic moieties and peptide sequences to manipulate the emulsion stability , droplet size and / or critical emulsion concentration . certain peptide microcapsules can disassemble in presence of proteolytic enzymes enabling on - demand demulsification under physiological conditions , or in response to elevated temperature . it is expected that through appropriate molecular design to include fully biocompatible analogues , by replacing the aromatic components with biocompatible ligands , such as nucleotides or other suitable groups , or using unmodified self - assembling aromatic peptides , as described . the interfacial networks presented here facilitate encapsulation and compartmentalization with potential applications in for example drug delivery and release , and the food industry . we report the first unprotected tripeptides capable of self - assembling in biphasic systems to stabilize emulsions . these emulsions have shown a range of stabilities at both ambient and elevated temperatures giving a range of properties that are tunable and dependent on sequence . in particular , we show that fibrillar structures form more stable emulsions compared with the traditional surfactant model . we have demonstrated the possibility of using aromatic dipeptide amphiphiles to enzymatically self - assemble at organic / aqueous interfaces to stabilize chloroform - in - water emulsions . we have demonstrated that fmoc - yl is able to self - assemble in water following its enzymatic generation from the non - assembling precursor fmoc - ypl . the self - assembled fmoc - yl was shown to form nanofibres through non - covalent interactions , including π - stacking and h - bonding . when in a biphasic system , enzymatically - triggered fmoc - yl self - assembles into nanofibrous networks at the chloroform / water interface , stabilizing the chloroform - in - water droplets and generating emulsions , which are stable for months . the stability of the emulsions and the possibility of switching on the emulsifier ability by adding the enzyme at different timings provides an extremely promising tool for several applications in chemical and other processes requiring emulsion formation . fmoc - tyr ( 97 %), h - leu - otbu . hcl (≧ 98 . 0 %), ala - otbu . hcl (≧ 98 . 0 %), 0 - tert - butyl - l - ser - tert - butyl ester hydrochloride (≧ 98 . 0 %), n , n - diisopropylethylamine ( dipea ) (& gt ; 99 . 5 %), trifluoroacetic acid ( 99 %), sodium hydroxide , 1 - pyreneacetic acid , fluorescein isothiocyanate isomer i (≧ 90 %), thioflavin t , sodium phosphate monobasic monohydrate ( acs reagent , 98 . 0 %- 102 . 0 %), sodium phosphate dibasic heptahydrate ( acs reagent , 98 . 0 %- 102 . 0 %), hexadecane ( 99 %) and mineral oil were purchased from sigma - aldrich and used as received . fmoc - phe - phe - phe - oh was purchased from bachem . 2 -( 1h - benzotriazole - 1 - yl )- 1 , 1 , 3 , 3 - tetramethyluronium hexafluorophosphate ( hbtu ) was purchased from novabiochem . phosphate buffer solution ( ph 8 ) was prepared by dissolving 94 mg nah 2 po 4 . h 2 o and 2 . 5 g na 2 hpo 4 . 7h 2 o in 100 ml water . fmoc - tyr ( po ( nme 2 ) 2 - oh ( 537 . 55 g · mol − 1 ) was purchased from novabiochem . fmoc - tyr - oh ( 403 . 43 g · mol − 1 ), l - leucine tert - butyl hydrochloride ( 223 . 74 g · mol − 1 ) and alkaline phosphatase from bovine , expressed in pichia pastoris ( 5000 u · mg − 1 protein , 20 mg protein · ml − 1 , 0 . 049 ml , apparent molar weight 160 kda ) were supplied by sigma aldrich . one enzyme unit corresponds to the quantity of alkaline phosphatase hydrolysing 1 μmol of 4 - nitrophenyl phosphate per minute at ph 9 . 8 and 37 ° c . fmoc - tyr - oh ( 1 g , 2 . 48 mmol ), h - leu - otbu . hcl ( 0 . 663 g , 2 . 98 mmol ) and hbtu ( 0 . 941 g , 2 . 98 mmol ) were dissolved in anhydrous dimethylformamide (˜ 15 ml ) with addition of dipea ( 1 . 08 ml , 6 . 2 mmol ). the mixture was stirred for 24 hours . product precipitated by adding saturated sodium bicarbonate solution (˜ 30 ml ) and was extracted into ethyl acetate (˜ 50 ml ). the mixture was washed with equal volume of saturated brine , 1m hydrochloric acid and brine . the resulting organic layer was dried by anhydrous magnesium sulphate and the ethyl acetate was removed by evaporation in vacuum . the resulting solid was then purified by column chromatography by using 2 . 5 % methanol in dichloromethane as eluent . fractions were tested using tlc under uv ( 254 nm ) light to visualize the spots . fractions containing the compound were combined and solvent removed in vacuum . the removal of the t - bu was carried out by dissolving the sample in dichloromethane and adding 10 ml of trifluoroacetic acid . the mixture was stirred for 24 hours . the dichloromethane was removed by evaporation in vacuum and the tfa was removed with toluene (˜ 10 ml ) and thf (˜ 2 ml ). solvent was removed by evaporation in vacuum ( carried out in triplicate ). the resulting solid was washed 6 times with cold diethyl ether and the product dried under vacuum . the synthesis of the other fmoc - dipeptides ( fmoc - ya - oh and fmoc - ys - oh ) follows the same experimental procedure as fmoc - yl . purity by hplc ( 214 nm )= 97 . 00 %. δ h ( dmso , 500 mhz ): 12 . 6 ( 1h , s , oh ), 9 . 2 ( 1h , s , tyr oh ), 8 . 34 - 8 . 32 ( 1h , nh d , j = 8 hz ), 7 . 89 - 7 . 87 ( 2h , d , j = 7 . 5 hz , 2 fluorenyl ar - ch ), 7 . 66 - 7 . 62 ( 2h , m , 2 fluorenyl ar - ch ), 7 . 55 - 7 . 53 ( 1h , d , j = 9 hz , nh ), 7 . 43 - 7 . 39 ( 2h , m , 2 fluorenyl ar - ch ), 7 . 34 - 7 . 27 ( 2h , m , 2 fluorenyl ar - ch ), 7 . 10 - 7 . 09 ( 2h , d , j = 8 . 3 hz , 2 tyr ar - ch ), 6 . 64 - 6 . 62 ( 2h , d , j = 8 . 35 hz , 2 tyr ar - ch ), 4 . 25 - 4 . 10 ( 5h , m , fluorenyl ch , fluorenyl ch 2 and c α h ), 2 . 92 - 2 . 91 ( 1h , m , tyr ch ), 2 . 73 - 2 . 71 ( 1h , m , tyr ch ), 1 . 68 - 1 . 62 ( 1h , m , leu ch ) 1 . 55 - 1 . 52 ( 2h , m leu ch 2 ), 0 . 9 - 0 . 83 ( 6h , m leu 2ch 3 ) ms ( es +): m / z 517 . 2 , [ m + h ] + . purity by hplc ( 214 nm )= 99 . 00 %. δ h ( dmso , 500 mhz ): 12 . 5 ( 1h , s , oh ), 9 . 1 ( 1h , s , tyr oh ), 8 . 28 - 8 . 26 ( 1h , d , j = 8 hz , nh ), 7 . 88 - 7 . 87 ( 2h , d , j = 7 . 5 hz , 2 fluorenyl ar - ch ), 7 . 65 - 7 . 61 ( 2h , m , 2 fluorenyl ar - ch ), 7 . 53 - 7 . 51 ( 1h , d , j = 9 hz , nh ), 7 . 43 - 7 . 39 ( 2h , m , 2 fluorenyl ar - ch ), 7 . 34 - 7 . 28 ( 2h , m , 2 fluorenyl ar - ch ), 7 . 10 - 7 . 09 ( 2h , d , j = 8 . 3 hz , 2 tyr ar - ch ), 6 . 64 - 6 . 62 ( 2h , d , j = 8 . 35 hz , 2 tyr ar - ch ), 4 . 19 - 4 . 10 ( 5h , m , fluorenyl ch , fluorenyl ch 2 and c α h ), 2 . 92 - 2 . 91 ( 1h , m , tyr ch ), 2 . 73 - 2 . 71 ( 1h , m , 1tyr ch ), 1 . 33 - 1 . 29 ( 3h , ala ch 3 ) ms ( es +): m / z 475 . 07 , [ m + h ] + . purity by hplc ( 214 nm )= 97 . 5 . 00 %. δ h ( dmso , 500 mhz ): 12 . 5 ( 1h , s , oh ), 9 . 1 ( 1h , s , tyr oh ), 8 . 28 - 8 . 26 ( 1h , d , j = 8 hz , nh ), 7 . 88 - 7 . 87 ( 2h , d , j = 7 . 5 hz , 2 fluorenyl ar - ch ), 7 . 65 - 7 . 61 ( 2h , m , 2 fluorenyl ar - ch ), 7 . 53 - 7 . 51 ( 1h , d , j = 9 hz , nh ), 7 . 43 - 7 . 39 ( 2h , m , 2 fluorenyl ar - ch ), 7 . 34 - 7 . 28 ( 2h , m , 2 fluorenyl ar - ch ), 7 . 10 - 7 . 09 ( 2h , d , j = 8 . 3 hz , 2 tyr ar - ch ), 6 . 64 - 6 . 62 ( 2h , d , j = 8 . 35 hz , 2 tyr ar - ch ), 4 . 7 - 4 . 6 ( 1h , m c α h ) 4 . 19 - 4 . 10 ( 4h , m , fluorenyl ch , fluorenyl ch 2 and c α h ), 2 . 92 - 2 . 91 ( 3h , m , 1tyr ch , ser ch 2 ), 2 . 73 - 2 . 71 ( 1h , m , tyr ch ). ms ( es +): m / z 491 . 00 [ m + h ] + . 1 - pyreneacetic acid ( 0 . 50 g , 1 . 92 mmol ), l - tyrosine tert - butyl ester ( 0 . 46 g 1 . 93 mmol ) and hbtu ( 0 . 740 , 1 . 95 mmol ) were mixed in 10 ml dry dmf . 0 . 89 ml ( 4 . 8 mmol ) of dipea was added to this solution and the mixture was stirred overnight under nitrogen atmosphere . after reaction , the product was extracted by 50 ml of ethyl acetate after successive wash with 20 ml of 1 n nahco 3 and 20 ml of 1 n hydrochloric acid and then dried over mgso 4 . after evaporation of the solvent , this compound was purified by column chromatography on silica gel using dichloromethane / methanol ( 95 : 5 ) as eluent ( 0 . 7 g , 75 %). then , the tert - butyl group of the compound ( 0 . 6 g , 1 . 25 mmol ) was removed by the reaction with trifluoroacetic acid ( 2 ml ) in dry dichloromethane for 15 hours . the solvent and excess trifluoroacetic acid was removed under vacuum to get pyrene - y acid ( 0 . 53 g , 1 . 25 mmol ). then pyrene - yl - otbu was obtained by the peptide coupling reaction with pyrene - y acid ( 0 . 52 g , 1 . 22 mmol ), l - leucine tert - butyl ester hydrochloride ( 0 . 23 g 1 . 25 mmol ), hbtu ( 0 . 47 , 1 . 25 mmol ) and 0 . 58 ml ( 3 . 1 mmol ) of dipea in 10 ml dry dmf . the pure product was obtained ( 0 . 38 g , 52 %) by column chromatography on silica gel using dichloromethane / methanol ( 95 : 5 ) as eluent . finally the tert - butyl group of pyrene - yl - otbu ( 0 . 3 g , 0 . 506 mmol ) was removed by using trifluoroacetic acid to get pure pyrene - tl ( 0 . 27 mg , 0 . 5 mmol ). 1 h nmr ( cdcl 3 , 400 mhz ) δ 0 . 79 - 0 . 83 ( m , 6h , ch 3 in leucine moiety ), 1 . 48 - 1 . 57 ( m , 3h , ch and ch 2 in leucine moiety ), 2 . 7 - 2 . 95 ( m , 2h , ch 2 in tyrosine moiety ), 4 . 1 ( s , 2h , ch 2 at pyrene peptide linker ), 4 . 22 - 4 . 28 ( m , 1h , chiral ch in leucine moiety ), 4 . 52 - 4 . 58 ( m , 1h , chiral ch in tyrosine moiety ), 6 . 63 ( d , 2h , ch at ortho position of oh group 3j = 8 . 0 hz ), 7 . 0 ( d , 2h , ch at meta position of oh group , 3 j = 8 . 0 hz ), 7 . 86 ( d , 1h , pyrene aromatic ch 3 j = 8 . 0 hz ), 8 . 07 - 8 . 1 ( m , 1h , pyrene aromatic ch ), 8 . 16 - 8 . 16 ( m , 2h , pyrene aromatic ch and amide nh ), 8 . 18 - 8 . 27 ( m , 4h , pyrene aromatic ch and amide nh ), 8 . 41 - 8 . 43 ( m , 3h , pyrene aromatic ch ). esi - ms : m / z : calcd for c 33 h 32 n 2 o 5 : 536 . 23 [ m + ]; found : 559 . 27 [ m + + na ]. 10 mm fmoc - yl solution was prepared by dissolving 5 . 32 mg fmoc - yl in 1 ml phosphate buffer solution . different volumes of chloroform were added to fmoc - yl buffer solution at 80 ° c . ( the volume ratio of buffer solution to chloroform is altered from 1 : 9 , 3 : 7 , 5 : 5 , 7 : 3 , to 9 : 1 , total volume was always 1 ml ), after hand - shaking for 5 seconds emulsions form in vials . for sem , ir , stability , average particle size , critical emulsion concentration and demulsification measurements , the volume ratio of buffer solution to chloroform was fixed at 7 : 3 . the concentration of all aromatic peptide amphiphiles studied ( fmoc - ya , fmoc - ys , pyr - yl , fmoc - ff and fmoc - fff ) was 10 mm , except for in the determination of the critical emulsion concentration ( 0 . 1 - 10 mm ) and demulsification measurements ( 2 mm ). the structures of fmoc - yl microcapsules were determined by hitachi s800 field emission scanning electron microscope ( sem ) at an accelerating voltage of 10 kev . the transfer of fmoc - yl , ya and ys were analyzed by uv - vis spectroscopy ( jas . c . o v - 660 spectrophotometer ). the formation of fmoc - yl gels and labeling of tht were carried out by fluorescence spectroscopy ( jas . c . o fp - 6500 spectrofluorometer ). high resolution mass spectra ( hrms ) were recorded on a thermo electron exactive . 400 . 1 ( 1h ) nmr spectra were recorded on brucker avance 400 spectrometer at room temperature using perdeuterated solvents as internal standards . the emulsion droplets were characterized by fluorescence microscopy . the devices were mounted on an inverted microscope ( axio observer a1 , zeiss ) and images were acquired using a emccd lucar camera ( andor technologies ). images ( using brightfield and fluorescence microscopy ) were acquired using zeiss × 20 dry objective and the appropriate filter set for the fluorophore being imaged . image was analyzed using imagej . the fibrous structures of fmoc - yl , ya , ys were determined by atomic force microscopy ( afm ). the images were obtained by scanning the mica surface in air under ambient conditions using a veeco diinnova scanning probe microscope ( veeco / bruker , santa barbara , calif ., usa ) operated in tapping mode . 20 μl of solutions were placed on a trimmed and freshly cleaved mica sheet ( g250 - 2 mica sheets 1 ″× 1 ″× 0 . 006 ″; agar scientific ltd , essex , uk ) attached to an afm support stub and left to air - dry overnight in a dust - free environment . the afm scans were taken at 512 × 512 pixels resolution . typical scanning parameters were as follows : tapping frequency 308 khz , integral and proportional gains 0 . 3 and 0 . 5 , respectively , set point 0 . 5 - 0 . 8 v and scanning speed 1 . 0 hz . the β sheet - like arrangement was determined by infrared absorption spectra which were recorded on a bruker vertex 70 spectrometer , averaging 25 scans per sample at a resolution of 1 cm − 1 . samples were sandwiched between two 2 mm caf 2 windows separated with a 50 μm polytetrafluoroethylene ( ptfe ) spacer . peptide structures were obtained via the vmd scripting tool and converted to the martini cg representation using the martinize . py script . 300 molecules of the cg peptide were randomly inserted into a box of dimensions 12 . 5 × 12 . 5 × 12 . 5 nm 3 and solvated with cg water . 300 ions ( cl − or na + ) are added to the system to completely neutralise the charge . the same procedure is carried out for the biphasic system with the addition of 1000 molecules of octane . the addition of the octane was carried out into the water / peptide mixture to give a density approximate to the experimental density of water ( 999 kg m − 3 ). this was achieved by combining a minimised water / tripeptide box with a minimised octane box . the minimised box was equilibrated for 500 , 000 steps with a 25 fs time step ( 12 . 5 ns simulation time ˜ 50 ns real time through the scaling of the time due to the softness of the cg potential . 7 using berendsen algorithm to keep temperature ( 300k ) and pressure ( 1 bar ) constant . periodic boundary conditions are in effect . each system was simulated for 9 . 6 ρs ( simulation time ) where afterwards the last frame was used to identify the finished structure . preparation of the peptides were carried out by dissolution of the peptide in water and the ph was altered to a neutral ph ˜ 7 . 5 . to each of the systems 100 μl of sudan ii labelled rapeseed oil was added . homogenisation was carried out on each of the sample for 5 secs thereafter ; the samples were stored for 24 hrs to ensure a stable emulsion was formed . ftir samples were contained within a standard ir harrink cell between two 2 mm caf2 windows . a 50 um polytetrafluoroethylene ( ptfe ) spacer was places between the spacer . spectra were recorded on a bruker vertex70 spectrometer by averaging 25 scans at a spectral resolution of 1 cm − 1 . fluorescence microscope samples were prepared by placing sample on a glass slide with a cover slip placed on top . a drop of silica oil was place on the sample to allow for a lubricated surface . samples were measured on a nikon eclipse e600 upright fluorescent microscope at × 1000 magnification . these initial observations show samples that showed gelation behaviour , tend to form more stable emulsions that samples that formed other structures . this suggests that the formation of fibrils has a large importance on the peptide ability to self - assemble at the interface forming stable emulsions . the fibrils surround the oil droplets inhibiting coalescence of the droplets . as observed the time stability over a course of 168 hrs showed little or no de - emulsification for kyf , kff and kyw whereas visible de - emulsification is observed for dff and ffd peptides , which do not form fibrils . this observation suggests the formation of the fibrils is a major driving force for the stabilisation of emulsions . the importance of the fibrillar formation has shown to be vital for the stabilisation of the emulsions . comparison of the ftir before and after emulsification sows the key interactions that help to stabilise the droplets . the temperature study shows the range of which the emulsion starts to breakdown . it is shown that the kyf is relatively stable through the temperature range . increase in the temperature between 30 - 40 ° c . shows kff break down . kyw breaks down at slightly higher temperatures approx . 40 - 50 ° c . we see that dff and ffd are relatively de - stabilized at the lower temperatures and the increase in temperature causes minimal change . 10 mm of synthesised fmoc - ypl was prepared in 950 μl of 0 . 6 m sodium phosphate buffer ph 8 and immediately added 50 μl of alkaline phosphatase ( 0 . 0555 u · μl − 1 , or 55 . 53 u · ml − 1 enzymatic concentration ), vortexed and subject to ultra sounds at room temperature . all characterisation was done after 24 hours except when stated otherwise . for the fmoc - ypl precursor , the preparation was the same except no enzyme was added . fmoc - ypl was prepared in the same way as stated before but in a 5 mm concentration to avoid formation of hydrogels . after 24 hours from the fmoc - ypl has been prepared in buffer and the alkaline phosphatase added ( to assure full dephosphorylation ), 500 μl chloroform were added to 500 μl samples and hand - shaken for 5 seconds to make a 50 : 50 chloroform - in - water emulsion . to check if the system is switchable on - demand , besides an immediate addition of alkaline phosphatase to the 50 : 50 chloroform - in water fmoc - ypl samples , the enzyme was added into the demulsified biphasic system of fmoc - ypl 1 week , 2 weeks and 1 month after preparation . photographs were taken and the dephosphorylation assessed by reversed phase hplc . molecular dynamic ( md ) simulations were carried out in namd ( nanoscale molecular dynamics ) program using the charmm force field . each system was minimised , at 300k , with the steep descent technique and then gradually heated up from 0 to 300 k for 55 ps before being equilibrated for 445 ps , to reach the system stabilization . finally , the systems were run within an npt ensemble at 1 atm and 300 k for 200 ns . a 2 . 0 fs time step was used to integrate newton &# 39 ; s motion equation along with a 12 å cut - off for the non - bonded interactions . periodic boundary conditions in the three - dimensional coordinates have been used . 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