Effects Of Multifunctional Small Molecules On Fatty Acid Vesicles

Fatty acid vesicles(FAV)are the aggregates with a closed bilayer or multilayer structure self-assembled from"fatty acid dimers"which is formed by a fatty acid molecule and an ion through hydrogen bonding near their p K_a,they have multiple responses such as pH response and dynamic characteristics of self-replication,growth and division.The researches on FAV have been widely concerned.The hollow closed bilayer or multi-layer structure of FAV endows it with important properties such as compartmentalization and selective permeability,therefore study on the formation of FAV and the construction of stable FAV has an important theoretical significance for the potential application of FAV as a protocell membrane model and microreactor to explore the origin of life and biochemical processes,as well as an application value for the hollow closed structure and selective permeable membrane of FAV as the carrier of drugs,proteins,nutrients and other aspects.FAV composed of pure fatty acid is environmentally sensitive,and its pH range for formation is narrow and mostly alkaline,which makes FAV limited in theoretical study of protocell membrane model and practical application as a carrier.The methods of constructing stable FAV by modification of fatty acid and co-assembly of additional amphiphilic substance and fatty acid have been studied thoroughly and systematically.However,the effects of non-amphiphilic small molecules on stable FAV construction,especially the influence of its pH window and the structure-activity relationship of its own structure on FAV properties,have not been systematically investigated.Fatty acids and fatty acid salts constituting FAV belong to the surface active substance,their self-assembly behaviour in aqueous phase is similar to that of surfactants,thus the theory of critical packing parameter based on surfactant self-assembly behaviour in aqueous phase plays an important role in explain the formation of both pure and hybrid FAV,however the direction and extent of the pH window migration or widening of FAV could not be well explained or predicted only with the variation of packing parameter.Studies on the relationship between the pH window migration or broaden of hybrid FAV or FAV stabilized by water-soluble small molecules through surface interaction with FAV and the variation of packing parameter are still of insufficient.Studies on the construction of FAV between various small molecules and fatty acids and the impact on the stability of FAV are extensive,however the intermolecular interaction information between small molecules and fatty acids or FAV obtained only based on molecular structure is not clear enough,and there are various speculations about the mechanism of interaction.The effects of various small molecules on the pH window of FAV are different,and the comparative study of the ability to influence the pH window of FAV among the small molecules with different functional groups is not sufficient.Focusing on the above problems to be studied,the main research contents and results of this paper are as follows:(1)The effects of multifunctional small molecules on the pH window,morphology and stability of conjugated linoleic acid vesicles were studied by means of laser Tyndall effect,transmission electron microscope(TEM),optical microscope,isothermal titration microcalorimetry(ITC)and binding energy calculation,the similarities and differences of their effects on the pH window of FAV were analyzed from the perspective of different molecular structures of multifunctional small molecules with mainly hydroxyl group as single functional group.The results show that these small molecules can extend the pH window of FAV in both sides through weak non-covalent binding,such as hydrogen bond or ion-dipole interaction,on the FAV surface,and the near neutral-side pH window extends in the following order:disaccharide<monosaccharide≈disaccharide/monosaccharide mixtures≤monosaccharide/monosaccharide mixtures,linear polyalcohols<cyclitol,it indicates that the change of hydrophilicity/hydrophobicity on FAV surface through the binding of these small molecules to FAV and the site effect jointly affect the change of pH window.Compared with the existing results and principles of the effects of long-chain alcohols,alkyl glycosides(APG)and Tween 80 on the pH window FAV,it is further proved that the number of free hydroxyl groups and site effect jointly affect the pH window change of the FAV,and although the ability of polyol small molecule to moderate the pH window of the FAV by surface interaction is weaker than that of the hydrophobic co-assembly of surfactants and fatty acids combined with the effect of hydrogen bonds,such surfactants with hydroxyl groups or ethylene oxide(EO)chain have only the ability to adjust the pH window of FAV in one side.(2)The pH window of oleic acid vesicle(OAV)was defined by laser Tyntall effect and turbidity observation,and effects of amine structure on the pH window were investigated involving in spacer lengths of diamines,numbers of amino groups in polyamines,alkyl chain lengths of primary fatty amines and quaternization.It is found that diamines and polyamines could mostly extend the pH window of OAV into the alkaline region,while primary fatty amines and dodecyltrimethylammonium bromide(DTAB)could result in a new metastable to stable vesicular phase at the low pH region besides the above window extension.Combining the titration curves of typical diethylenetriamine and oleic acid systems and the molecular structures of different amines and the quaternary ammonium salt,it is found that regulation of the pH window of OAV driven by amines is generally relying on transformation between protonation and deprotonation of amino groups under different pH,while cooperating with hydrophobic interaction of fatty amines or electrostatic attraction as a special driven force of DTAB.The results of the thermodynamic parameters for molecular interactions obtained by ITC combined with binding energy calculation show that the non-covalent bonding of diethylenetriamine,as a representative of diamines or polyamines,with the OAV surface is dominated by hydrogen bond or ion-dipole interaction,which has weaker effect on the pH window of OAV than the hydrophobic co-assembly with primary fatty amines or electrostatic attraction with DTAB.(3)The effects of multifunctional small molecules containing different types of functional groups on the pH window and morphology of conjugated linoleic acid vesicles were studied by methods such as appearance changes combined with titration curves and TEM,and the results show that at most concentrations the six amino acids studied have the ability to extend the pH window of FAV to near-neutral region,while have little effect on the alkaline-side pH window of FAV,the three neutral amino acids can extend the pH window of FAV in both sides at lower concentration,and the ability of alcoholamine to extend the pH window to near-neutral region is enhanced with the increase of the number of alcohol hydroxyl groups in the molecular structure,while glycerophosphoric acid and choline glycerophosphate as the building block of phospholipid can extend the pH window of FAV in both sides.The coexistence of a certain concentration of sodium chloride can help improve the ability of amino acids and alcoholamines to extend the pH window of FAV to near-neutral region.Compared with the multifunctional small molecules containing a single functional group,the pH window regulation effect of such multifunctional small molecules on the FAV is also related to the change of hydrophilicity/hydrophobicity on FAV surface,and there may be a selectivity of functional group binding during their binding to the FAV surface.Compared with the pH windows of lysophosphatidylcholine hybrid FAV,polyconjugated linoleic acid vesicle and polyionic vesicles containing fatty acids,it shows that the ability of extend the pH window of FAV to near-neutral or acidic region by co-assembly or multi-pedal anchoring is stronger than that of simple surface interaction.However,the alkaline-side pH windows of the lysophosphatidylcholine hybrid FAV and polyionic vesicles containing fatty acids shrink.(4)Based on the mechanism of the effects monosaccharides on the pH window of FAV through the interaction between monosaccharides and conjugated linoleic acid and conjugated linoleic acid vesicles,and the theoretical analysis of the effects of polyols,polyamines,the multifunctional small molecules with non-monofunctional groups and some surfactants on the pH window of FAV,the criterion for the direction and degree of migration or extending of the pH window of FAV by the microenvironmental polarity changes of FAV surface was obtained.If a small molecular as the second component made the FAV surface more hydrophobic on the alkaline-side pH window while more hydrophilic on the acidic-side via molecular interactions,it could drive the corresponding-side pH window extension,otherwise the corresponding-side pH window shrinkage.This empirical criterion can be used as a supplement to the packing parameters theory,and the analysis results combined with the existing literature and experimental results prove that it has a wide applicability for predicting the direction and degree of pH window migration of FAV.