| Phospholipids are a kind of amphiphilic molecules with polar heads(hydrophilic)and nonpolar tails(hydrophobic).Phospholipids can form different structures,such as micelles,vesicles,or bilayer sheets,by self-assembling processes when exposed to an aqueous environment.Liposomes are a kind of vesicles consisting of unilamellar or multilamellar lipid bilayers.They have attracted increasing attentions of researchers and been applied to fields of biology,medicine,etc.for their good biodegradability,biocompatibility,and non-immunogenicity since they were found by Bangham in 1965.Giant unilaminar vesicles(GUVs)with diameters larger than 1 ?m have similar membrane structures as cells,and thus,can be used as cell models to study the biochemical properties of plasma membrane,or drug delivery system and biochemical reactor,etc.Significant progress has been made on both preparation methods and applications of GUVs in recent years.However,the mechanism of GUV formation and the effects of various factors are still unclear.This paper designed a microchip first,and based on the chip the mechanism of the factors influencing the lipid hydration was studied,as well as the mechanism of liposome formation.Then,several effective methods to improve the efficiency of the liposome preparation were proposed.Specifically,the work of this paper mainly includes:(1)Experimental verification of the hypothesis that "electrostatic repulsion dominates the formation of liposomes"The hypothesis that "electrostatic repulsion dominates the formation of liposomes"is one of the most commonly used explanations for the mechanism of liposome formation.However,there were still some problems when analyzing many experimental phenomena by it.Therefore,in this paper,the reliability of this hypothesis was verified experimentally first.When explaining the effect of NaCl concentration and lipid type on the lipid hydration,it was found that the hypothesis did explain some experimental phenomena,but there were still many phenomena inconsistent with the hypothesis.Therefore,this statement may not be completely right.Subsequently,the mechanism of lipid hydration and liposome formation were further studied with respect to the influence of aqueous solution and lipid type.(2)The ion specificity in the process of lipid hydrationThe ion species were extended including some monovalent ions and divalent ions to study the ion specificity on lipid hydration.Compared with those in pure water,the influence of different ions can be ordered by CO32->SO42->SCN->NO3->Br->Cl-and Na+>K+.However,the effect of divalence cations was more complicated.When the concentration was very low(<1 mM),the addition of these ions promoted the separation of the supported phospholipid layers(SPL)on the substrate and the formation of liposomes.This effect follows:Zn2+>Mg2+>Ca2+.With the increase of salt concentration,the promoting effect of ions on liposome formation was weakened,and finally suppressed liposome formation.With the help of researches on Hofmeister series,a mechanism of lipid hydration in a salt solution was proposed.Water molecules and ions in salt solution can bind with the polar head and polarize the nonpolar tails producing dispersion forces between the acyl chains and making them bond together,resulting in hydrophobic effect.Therefore,the influence of monovalent ions and divalence anions on lipid hydration can be characterized by their polarization on the acyl chains.The stronger the ions can polarize the acyl chains,the greater the ions would influence lipid hydration compared with pure water.For the specificity of the divalence cations,it was suggested that the ions with a small electronegativity difference with respect to the phosphate group can result in a lower charge density after binding with the polar head.The complexity with lower charge density can thus interact with the part with low polarity or even nonpolar part reducing the hydrophobicity of the lipids at the same ionic strength.The nonpolar part of the phospholipid was usually large and cannot be broken completely.With the increase of ionic strength,ions would always polarize the remaining tails.When polarization was dominant,phospholipids showed an inceasing hydrophobic effect with increasing ion concentration.(3)The specific effect of nonelectrolytes on lipid hydrationThe influence of nonelectrolytes was different from that of ions since the polarization of the nonelectrolytes on the tail was usually weaker.It was found that ethanol molecules could bind to both the lipid head and tail.When the ethanol concentration was relatively low,the SPL separation and liposome formation were improved.However,with high concentration,the hydrophobicity of the lipid was destroyed largely,which was unfavorable to the formation of regular SPL and liposome.The other nonelectrolytes(sucrose,sorbitol,glucose)can only bind with the polar heads since their carbon chains were covered by hydroxyl groups.They not only guaranteed the phospholipid amphipathicity,but also contribute to the swelling of SPL and the formation of liposomes.(4)The specific effect of lipid species on its hydrationSeveral kinds of phospholipids were used to study the specificity of the lipid type on its hydration.It was found that the hydration behavior of lipids,many physical properties of the lipid membrane and even whether it was easy to form liposome for the phospholipid actually did not depend on the overall charge of the phospholipids but the hydrophilicity of the polar head and the hydrophobicity of the nonpolar tail.The polar head would always polarize the nonpolar tail whether in hydrated state or nonhydrated state.The difference was that the former can form regular membrane structure,while the latter can only produce irregular phospholipid aggregates.The more hydrophilic the head was,the easier they can bind with the water molecules and ions,and the stronger the tails would be polarized.The lipids with a hydrophobic effect can rearranged sufficiently in aqueous solution to form regular membrane structures.The hydrophilicity of the lipid head depended on its interaction with the aqueous solution through charge pairing and hydrogen bonding.The hydrophobicity of lipid tail depended on the length and saturation of the acyl chain.In addition,it was found that the effect of ions on the hydrophobic effect of the lipids depended strongly on the properties of the phosphate groups closed to the acyl chains.(5)The improvement of the methods for liposome preparationBased on the above results,it was found that the hydration behavior of phospholipids depended not only on the aqueous solution,but also on their own properties.The aqueous solution influences the phospholipid hydration mainly through two ways.One was binding with the polar head and polarizing the nonpolar tail to make them bond more tightly.The other was binding with the nonpolar tail directly and reduces the nonpolar part and the hydrophobic effect.The effects of phospholipids on their hydration also mainly depend on the hydrophilicity of the polar head and the hydrophobicity of the nonpolar tail.Overall,based on the study of the mechanism of the lipid hydration,several methods to improve the efficiency of liposome preparation have been proposed,and the corresponding experiments have been carried out.? Removing the hydrophobic shelter for the lipids by oxygen plasma treatmentIt was found that the lipids were always difficult to be detached from the substrate to form liposomes since the glass substrate provided a hydrophobic shelter for the lipid tail,which may be one of the important reasons for the low yield of liposomes.Therefore,oxygen plasma surface treatment was used to reduce the hydrophobicity of the glass surface.When the concentration of NaCl was higher than 20 mM,the lipids exhibiting a stronge hydrophobic effect but losing the hydrophobic shelter tended to aggregate in situ to form lipid clusters and then swelled and fused to form dispersed liposomes.With the increase of NaCl concentration,the size of liposomes decreased and the yield increased.This method not only increased the yield of the liposome but also solved the problem of liposome preparation under high ionic strength.? Prehydrating phospholipids with glucose solutionThe results of nonelectrolytes' effect on lipid hydration showed that the binding of carbohydrate molecules can effectively reduce the interaction between lipid heads and suppress fusion of the lipid membranes.Therefore,glucose solution with the most significant effect was chosen to prehyedrate the lipids.Some glucose molecules and water molecules have bound with the lipid heads before adding salt solution and effectively reduced the binding of ions in the salt solution.A good preparation effect for liposome was obtained by this method when NaCl concentration was between 30?70 mM.? preparing liposomes by electroformation on microchipsElectroformation of liposomes was carried out on two kinds of microchips.One was the traditional“sandwished" chip and the other was the microchip with coplanar microelectrode arrays.The liposome preparation was improved after introduction of an exteral electric field.An optimal frequency was found for both of them(10 kHz for the 3D chip and 1 kHz for the 2D chip),at which the preparation efficiency of the liposomes can be maxmized.Compared with the 3D chip,both the yield and size distribution of the formed liposomes were improved on the 2D chip.The electroosmotic flow(EOF)on the lipids was calculated by using COMSOL Multiphysics software,which verified the existence of the characteristic frequencies.? Depositing the lipids on the top substrateAn external force was critical for liposome formation.In experiments,when lipids were deposited on the top substrate,a large number of lipids were detached from the substrate in the form of lipid aggregates and SPL,and continued to swell in aqueous solution to form liposomes.When depositing lipids on the top substrate,the NaCl concentration in which the liposomes can be prepared successfully was extended to 200 mM compared with 20 mM when depositing the lipids on the bottom substrate.This method not only improved the preparation efficiency of liposomes,but also solved the problem of liposome preparation under high ionic strength.The yield was further improved within a low range of ion concentration after introducing an external electric field of 12 V and 10 kHz.In summary,based on the experimental verification of the statement that electrostatic repulsion dominates liposome formation,the specific effect of different ions,non electrolyte and lipid species were studied in their effect on lipid hydration.It was found that the lipid hydration depended not only on the aqueous solution but also on the lipids themselves.Based on this,several attempts for improving the method of liposome preparation were carried out,including removing the hydrophobic shelter of the lipids by oxygen plasma treatment,depositing lipids on the top substrate,and introducing the methods of prehydration and electroformation.In this paper,the mechanism of liposome formation was discussed,which was helpful for establishing a more effective preparation method. |
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