Self-assembled Lipid Research Articles

Construction of a Synthetic Colostrum Substitute and Its Protection of Intestinal Cells against Inflammation in an In Vitro Model of Necrotizing Enterocolitis.

Colostrum provides bioactive components that are essential for the colonization of microbiota in the infant gut, while preventing infectious diseases such as necrotizing enterocolitis. As colostrum is not always available from the mother, particularly for premature infants, effective and safe substitutes are keenly sought after by neonatologists. The benefits of bioactive factors in colostrum are recognized; however, there have been no accounts of human colostrum being studied during digestion of the lipid components or their self-assembly in gastrointestinal environments. Due to the weaker bile pool in infants than adults, evaluating the lipid composition of human colostrum and linking it to structural self-assembly behavior is important in these settings and thus enabling the formulation of substitutes for colostrum. This study is aimed at the rational design of an appropriate lipid component for a colostrum substitute and determining the ability of this formulation to reduce inflammation in intestinal cells. Gas chromatography was utilized to map lipid composition. The self-assembly of lipid components occurring during digestion of colostrum was monitored using small-angle X-ray scattering for comparison with substitute mixtures containing pure triglyceride lipids based on their abundance in colostrum. The digestion profiles of human colostrum and the substitute mixtures were similar. Subtle differences in lipid self-assembly were evident, with the substitute mixtures exhibiting additional non-lamellar phases, which were not seen for human colostrum. The difference is attributable to the distribution of free fatty acids released during digestion. The biological markers of necrotizing enterocolitis were modulated in cells that were treated with bifidobacteria cultured on colostrum substitute mixtures, compared to those treated with infant formula. These findings provide an insight into a colostrum substitute mixture that resembles human colostrum in terms of composition and structural behavior during digestion and potentially reduces some of the characteristics associated with necrotizing enterocolitis.

Photoinduced bidirectional switching in lipid membranes containing azobenzene glycolipids

Following the reaction of biological membranes to external stimuli reveals fundamental insights into cellular function. Here, self-assembled lipid monolayers act as model membranes containing photoswitchable azobenzene glycolipids for investigating structural response during isomerization by combining Langmuir isotherms with X-ray scattering. Controlled in-situ trans/cis photoswitching of the azobenzene N = N double bond alters the DPPC monolayer structure, causing reproducible changes in surface pressure and layer thickness, indicating monolayer reorientation. Interestingly, for monolayers containing azobenzene glycolipids, along with the expected DPPC phase transitions an additional discontinuity is observed. The associated reorintation represents a crossover point, with the surface pressure and layer thickness changing in opposite directions above and below. This is evidence that the azobenzene glycolipids themselves change orientation within the monolayer. Such behaviour suggests that azobenzene glycolipids can act as a bidirectional switch in DPPC monolayers providing a tool to investigate membrane structure–function relationships in depth.

Control of Enzyme Reactivity in Response to Osmotic Pressure Modulation Mimicking Dynamic Assembly of Intracellular Organelles.

In response to variations in osmotic stress, in particular to hypertonicity associated with biological dysregulations, cells have developed complex mechanisms to release their excess water, thus avoiding their bursting and death. When water is expelled, cells shrink and concentrate their internal bio(macro)molecular content, inducing the formation of membraneless organelles following a liquid-liquid phase separation (LLPS) mechanism. To mimic this intrinsic property of cells, functional thermo-responsive elastin-like polypeptide (ELP) biomacromolecular conjugates are herein encapsulated into self-assembled lipid vesicles using a microfluidic system, together with polyethylene glycol (PEG) to mimic cells' interior crowded microenvironment. By inducing a hypertonic shock onto the vesicles, expelled water induces a local increase in concentration and a concomitant decrease in the cloud point temperature (Tcp ) of ELP bioconjugates that phase separate and form coacervates mimicking cellular stress-induced membraneless organelle assemblies. Horseradish peroxidase (HRP), as a model enzyme, is bioconjugated to ELPs and is locally confined in coacervates as a response to osmotic stress. This consequently increases local HRP and substrate concentrations and accelerates the kinetics of the enzymatic reaction. These results illustrate a unique way to fine-tune enzymatic reactions dynamically as a response to a physiological change in isothermal conditions.

Self-assembled lipid–prodrug nanoparticles

Self-assembled lipid–prodrug nanoparticles

Effect of Glycosylation on Self-Assembly of Lipid A Lipopolysaccharides in Aqueous Solutions.

Lipopolysaccharides (LPSs) based on lipid A produced by bacteria are of interest due to their bioactivity in stimulating immune responses, as are simpler synthetic components or analogues. Here, the self-assembly in water of two monodisperse lipid A derivatives based on simplified bacterial LPS structures is examined and compared to that of a native Escherichia coli LPS using small-angle X-ray scattering and cryogenic transmission electron microscopy. The critical aggregation concentration is obtained from fluorescence probe experiments, and conformation is probed using circular dichroism spectroscopy. The E. coli LPS is found to form wormlike micelles, whereas the synthetic analogues bearing six lipid chains and with four or two saccharide head groups (Kdo2-lipid A and monophosphoryl lipid A) self-assemble into nanosheets or vesicles, respectively. These observations are rationalized by considering the surfactant packing parameter.

Preparation and cytotoxicity evaluation of folic acid-modified YF8-OA self-assembled lipid prodrug nanoparticles

This study aimed to improve the use of YF8, a matrine derivative obtained through chemical transformation of matrine extracted from Sophora alopecuroides. YF8 has demonstrated improved cytotoxicity compared to matrine, but its hydrophobic nature hinders its application. To overcome this, the lipid prodrug YF8-OA was synthesized by linking oleic acid (OA) to YF8 through an ester bond. Although YF8-OA could self-assemble into unique nanostructures in water, it was not sufficiently stable. To enhance the stability of YF8-OA lipid prodrug nanoparticles (LPs), we employed the strategy of PEGylation using DSPE-mPEG2000 or DSPE-mPEG2000 conjugated with folic acid (FA). This resulted in the formation of uniform spherical nanoparticles with greatly improved stability and a maximum drug load capacity upto 58.63%. Cytotoxicity was evaluated in A549, HeLa, and HepG2 cell lines. The results showed that in HeLa cells, the IC50 value of YF8-OA/LPs with FA-modified PEGylation was significantly lower than that of YF8-OA/LPs modified by PEGylation alone. However, no significant enhancement was observed in A549 and HepG2 cells. In conclusion, the lipid prodrug YF8-OA can form nanoparticles in aqueous solution to address its poor water solubility. Modification with FA resulted in further enhanced cytotoxicity, providing a potential avenue for exerting the antitumor activity of matrine analogs.

Lipid-Polymer Hybrid Nanoparticles for Topical Drug Delivery System

Human skin not only functions as a permeation barrier (mainly due to the stratum corneum layer), but also provides a unique delivery pathway for therapeutic and other active agents. These compounds penetrate via intercellular, intracellular and transappendageal routes, resulting in topical delivery (into skin strata) and transdermal delivery (to subcutaneous tissues and into the systemic circulation). Lipid–polymer hybrid nanoparticles (LPHNPs) are next-generation core–shell nanostructures, conceptually derived from both liposome and polymeric nanoparticles (NPs), where a polymer core remains enveloped by a lipid layer. Although they have garnered significant interest, they remain not yet widely exploited or ubiquitous. Recently, a fundamental transformation has occurred in the preparation of LPHNPs, characterized by a transition from a two-step to a one-step strategy, involving synchronous self-assembly of polymers and lipids. Owing to its two-in-one structure, this approach is of particular interest as a combinatorial drug delivery platform in oncology. In particular, the outer surface can be decorated in multifarious ways for active targeting of anticancer therapy, delivery of DNA or RNA materials, and use as a diagnostic imaging agent.
 Keywords: Lipid–polymer hybrid nanoparticle, Topical delivery, Drug delivery, Gene delivery.

Photoresponsive liposomes and LipoParticles by incorporating a photosensitizer agent in their lipid membrane

Liposomes, as self-assembled lipid layer vesicles, have emerged as a promising platform for various biomedical applications such as photodynamic therapy (PDT). LipoParticles are core–shell lipid assemblies with a solid polymer core coated by a lipid membrane, which gather the advantages of both their counterparts. Photosensitizers (PS), used in PDT, are photosensitive molecules than can be activated by an exposure to a light source, leading to the death of pathogenic surrounding cells. In this context, the aim of this study is to elaborate liposomes and LipoParticles incorporating a PS, and to examine their resulting photoresponsive properties by following the release, triggered by an UV irradiation, of incorporated fluorescent (macro)molecules. Al(III) phthalocyanine chloride disulfonic acid (AlPcS2) was chosen as PS, and the release of 5(6)-carboxyfluorescein (CF) or bovine serum albumin conjugated with fluorescein isothiocyanate (BSA-FITC) from liposomes and poly (lactic-co-glycolic acid) (PLGA) core of LipoParticles respectively, was investigated by fluorescence spectroscopy. The results have shown that UV irradiation led to the release of CF molecules depending on the membrane lipid formulation, and that repeated irradiations could increase the amount of CF molecules released from liposomes. The main parameters governing the release of BSA-FITC macromolecules from LipoParticles were the temperature of their elaboration (corresponding to a reorganization of several liposomes on the surface of PLGA particles), the initial PS concentration, and the duration of UV irradiation. Best results were found with a temperature of the LipoParticles elaboration of 20 °C, a PS concentration of 0.1 mM, and a duration of irradiation of 5 min. These results support the fact that similarly to liposomes, LipoParticles can also be endowed with photoresponsive properties, which positions them as interesting nanovectors for PDT.

Structural complexity and physical mechanism of self-assembled lipid as nanocarriers: A review

Lipids such as glyceryl monooleate, phosphatidylcholine, and monoglyceride (CITREM) possess an amphipathic property that allows them to self-assemble into a complex internal structure when interacting with an aqueous solution. Since amphiphilic molecules possess hydrophilic heads and lipophilic tails, hydrophobic effects cause the spontaneous activity of the molecular rearrangement. The self-organization of the molecules often results in the phases of lipid polymorphism, for example microemulsion, inverse bicontinuous cubic (Q2), discontinuous hexagonal (H2), and micellar cubic (I2) Fd3m. Interestingly, these lamellar and non-lamellar phases have been applied in the development of nanocarriers for drug delivery due to their ability to provide a sustained drug release system, better drug bioavailability, and improved overall treatment. However, the attention that they are receiving from their application is not comparable to our understanding of the mechanisms involved in their synthesis. Elucidation of the spontaneous process helps in predicting and tuning the internal structure of an amphiphilic molecule to suit its application. Therefore, this review discusses the formation of lipid polymorphism from the thermodynamic point of view, critical packing parameter, and modified stalk theory.

Controlled lipid self-assembly for scalable manufacturing of next-generation immune stimulating complexes

Immune stimulating complexes (ISCOMs) are safe and effective saponin-based adjuvants formed by the self-assembly of saponin, cholesterol, and phospholipids in water to form cage-like 30–40 nm diameter particles. Inclusion of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) in ISCOM particles yields a promising next-generation adjuvant termed Saponin-MPLA NanoParticles (SMNP). In this work, we detail protocols to produce ISCOMs or SMNP via a tangential flow filtration (TFF) process suitable for scalable synthesis and Good Manufacturing Practice (GMP) production of clinical-grade adjuvants. SMNP or ISCOM components were solubilized in micelles of the surfactant MEGA-10, then diluted below the critical micelle concentration (CMC) of the surfactant to drive ISCOM self-assembly. Assembly of ISCOM/SMNP particles using the purified saponin QS-21 used in clinical-grade saponin adjuvants was found to require controlled stepwise dilution of the initial micellar solution, to prevent formation of undesirable kinetically-trapped aggregate species. An optimized protocol gave yields of ∼77% based on the initial feed of QS-21 and the final SMNP particle composition mirrored the feed ratios of the components. Further, samples were highly homogeneous with comparable quality to that of material prepared at lab scale by dialysis and purified via size-exclusion chromatography. This protocol may be useful for clinical preparation of ISCOM-based vaccine adjuvants and therapeutics.

Discerning perturbed assembly of lipids in a model membrane in presence of violacein: Effects of membrane hydrophobicity

For the lack of effective antibiotics towards antibiotic resisting bacteria, it is required to discover new antibiotics and to understand their antimicrobial mechanism. Violacein is a violet pigment found in several gram-negative bacteria possessing antimicrobial properties to gram-positive bacteria. This present article investigates the insertion ability of this molecule into a model membrane composed of zwitterionic phospholipids. Thermodynamic characterization of lipid monolayers in the presence of violacein was carried out using a single lipid layer formed at air-water interface. The molecule inserts into the layer altering the area occupied by each lipid and the in-plane compressibility of the film. This insertion increases with the hydrophobic chain length of the lipid. The perturbed self-assembly of lipids in a bilayer is quantified using a lipid multilayer system applying the X-ray reflectivity technique. The electron density profile from the reflectivity data shows that the molecule inserts into the fluid phase creating a relatively ordered chain conformation. Further, the insertion into the gel phase is observed to increase with the increased thickness of the hydrophobic core of a bilayer.

Investigating the mechanochemistry of membrane area asymmetry generation: A combined mass-action and micromechanical study using methyl-beta-cyclodextrin.

Membrane asymmetry is important for cell signaling and is coupled with membrane morphological remodeling protein conformational regulation, sorting, and function. With respect to the lipids composing a bilayer, membrane asymmetry is divided into two contributions including 1) area asymmetry- that is, unequal monolayer surface areas that two apposed leaflets would tend to favor were they uncoupled and 2) compositional asymmetry- that is, different lipid compositions between the leaflets. For in vitro research on membrane asymmetry, methyl-β-cyclodextrin (mbCD) has been previously applied as a lipid-exchange catalyst to tune the compositional asymmetry of synthetic membranes. Inspired by this approach, we opted to study the physical chemistry of 1-component vesicle systems where mbCD was present not as a catalyst but as a reactant. In reaction mixtures at ambient conditions containing nanoscale vesicles, we performed a fluoresence-quenching assay to see that mbCD extracts phospholipids from only the outer leaflets of vesicles and, thereby, must generate area asymmetry in such mixtures. For certain molar ratios, mbCD can fully solubilize all phospholipid and we used 90o light scattering to map this behavior on a two-dimensional phase diagram at ambient temperature. Through temperature-cycling experiments, we showed an irreversibility in mbCD-phospholipid phase behavior. This granted access to stable solutions containing no self-assembled lipid structures with larger molar ratios of phospholipid to mbCD than could be achieved by isothermal reaction conditions. By adjusting the molar ratios of these solutions, we generated both negative and positive curvature in microscopic vesicles (i.e., inward and outward bending, respectively). Tunability of the curvature is consistent with mass-action considerations relating to net flow of phospholipid out of/into vesicles. We believe our approach using mbCD pre-complexed with phospholipid will enhance experimental research on the biophysical consequences of area asymmetry.

Biomimetic Stratum Corneum Liposome Models: Lamellar Organization and Permeability Studies

The stratum corneum (SC), the outer layer of the skin, plays a crucial role as a barrier protecting the underlying cells from external stress. The SC comprises three key components: ceramide (CER), free fatty acid (FFA), and cholesterol, along with small fractions of cholesterol sulfate and cholesterol ester. In order to gain a deeper understanding about the interdependence of the two major components, CER and FFA, on the organizational, structural, and functional properties of the SC layer, a library of SC lipid liposome (SCLL) models was developed by mixing CER (phytosphingosine or sphingosine), FFA (oleic acid, palmitic acid, or stearic acid), cholesterol, and cholesterol sulfate. Self-assembly of the SC lipids into lamellar phases was first confirmed by small-angle X-ray scattering. Short periodicity and long periodicity phases were identified for SCLLs containing phytosphingosines and sphingosine CERs, respectively. Furthermore, unsaturation in the CER acyl and FFA chains reduced the lipid conformational ordering and packing density of the liposomal bilayer, which were measured by differential scanning calorimetry and Fourier transform infrared spectroscopy. The introduction of unsaturation in the CER and/or FFA chains also impacted the lamellar integrity and permeability. This extensive library of SCLL models exhibiting physiologically relevant lamellar phases with defined structural and functional properties may potentially be used as a model system for screening pharmaceuticals or cosmetic agents.

Unveiling growth and dynamics of liposomes by graphene liquid cell-transmission electron microscopy.

Liposome is a model system for biotechnological and biomedical purposes spanning from targeted drug delivery to modern vaccine research. Yet, the growth mechanism of liposomes is largely unknown. In this work, the formation and evolution of phosphatidylcholine-based liposomes are studied in real-time by graphene liquid cell-transmission electron microscopy (GLC-TEM). We reveal important steps in the growth, fusion and denaturation of phosphatidylcholine (PC) liposomes. We show that initially complex lipid aggregates resembling micelles start to form. These aggregates randomly merge while capturing water and forming small proto-liposomes. The nanoscopic containers continue sucking water until their membrane becomes convex and free of redundant phospholipids, giving stabilized PC liposomes of different sizes. In the initial stage, proto-liposomes grow at a rate of 10-15 nm s-1, which is followed by their growth rate of 2-5 nm s-1, limited by the lipid availability in the solution. Molecular dynamics (MD) simulations are used to understand the structure of micellar clusters, their evolution, and merging. The liposomes are also found to fuse through lipid bilayers docking followed by the formation of a hemifusion diaphragm and fusion pore opening. The liposomes denaturation can be described by initial structural destabilization and deformation of the membrane followed by the leakage of the encapsulated liquid. This study offers new insights on the formation and growth of lipid-based molecular assemblies which is applicable to a wide range of amphiphilic molecules.

Understanding the rod-to-tube transformation of self-assembled ascorbyl dipalmitate lipid nanoparticles stabilized with PEGylated lipids.

We previously established a nanoparticle-based drug delivery system (DDS) for high-dose ascorbic acid therapy by self-assembly of a lipid-modified ascorbic acid derivative, L-ascorbyl 2,6-dipalmitate (ASC-DP). The particles' morphology should be modified for effective DDSs. Here, we modulated the morphology of self-assembled ASC-DP nanoparticles using two different PEGylated lipids, distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) and cholesterol-polyethylene glycol (Chol-PEG), with various PEG molecular weights. At the preparation molar ratio of 10 : 1 (ASC-DP/PEGylated lipid), rod-like nanoparticles emerged in the ASC-DP/DSPE-PEG system, whereas the ASC-DP/Chol-PEG system yielded tube-like nanoparticles. The internal structures of both rod-like ASC-DP/DSPE-PEG and tube-like ASC-DP/Chol-PEG nanoparticles were similar to that of repeated ASC-DP bilayers. The particles' surfaces featured PEGylated lipids, which stabilized the structure and dispersion of the nanoparticles. For both systems, the particle size increased slightly with increasing the PEGylated lipid's PEG molecular weight. Increasing the PEG molecular weight decreased the inner tunnel size of tube-like ASC-DP/Chol-PEG nanoparticles. A mechanism has been proposed for the rod-to-tube transformation. Surface-layer free-energy changes owing to the mixing of multiple lipids and PEG chain repulsion are thought to underlie the inner tunnels' formation. The rod-to-tube morphology of self-assembled ASC-DP nanoparticles can be modulated by controlling the PEGylated lipids' structure, including the lipid species and the PEG chain length.

Phytantriol phase behaviour in choline chloride urea and water mixtures.

Deep eutectic solvents (DES) are tailorable non-aqueous solvents with promising properties for a range of applications, from industrial dissolution of plant products to biomedicine. They are mixtures of hydrogen bond donors and acceptors with low melting points that can be tailored to specific applications, and many support the self-assembly of amphiphilic molecules into lyotropic liquid crystal phases. Self-assembled lipid structures have potential for numerous applications, including drug delivery. These ordered structures can act as carriers, slow-release vehicles, or microreactors. Lipid self-assembly in non-aqueous solvents, such as deep eutectic solvents, is important for applications at extreme temperatures, or involving water-insoluble or water sensitive components. However, lipid self-assembly in these solvents remains largely unexplored. In this paper, we have examined the self-assembly of phytantriol, a non-ionic lipid, at 10 and 30 wt% in the deep eutectic solvent choline chloride:urea, with and without water. Self-assembly was assessed using small angle X-ray scattering and cross polarised optical microscopy at temperatures from 25-66 °C. We found that pure choline chloride:urea supports a Pn3m cubic phase similar to that formed in water. However, mixtures of the DES with water resulted in phytantriol forming an inverse hexagonal phase and influenced the phase transition temperatures. These results demonstrate that choline chloride:urea can support diverse phase behaviour, and also provides a mechanism for tailoring the phase for particular applications simply by controlling the amount of water in the solvent. In the future this could lead to methods of triggered release of drugs and biomolecules by the simple addition of water which could be critical for drug delivery applications.

Enhancing the spectral range of plant and bacterial light-harvesting pigment-protein complexes with various synthetic chromophores incorporated into lipid vesicles.

The Light-Harvesting (LH) pigment-protein complexes found in photosynthetic organisms have the role of absorbing solar energy with high efficiency and transferring it to reaction centre complexes. LH complexes contain a suite of pigments that each absorb light at specific wavelengths, however, the natural combinations of pigments within any one protein complex do not cover the full range of solar radiation. Here, we provide an in-depth comparison of the relative effectiveness of five different organic "dye" molecules (Texas Red, ATTO, Cy7, DiI, DiR) for enhancing the absorption range of two different LH membrane protein complexes (the major LHCII from plants and LH2 from purple phototrophic bacteria). Proteoliposomes were self-assembled from defined mixtures of lipids, proteins and dye molecules and their optical properties were quantified by absorption and fluorescence spectroscopy. Both lipid-linked dyes and alternative lipophilic dyes were found to be effective excitation energy donors to LH protein complexes, without the need for direct chemical or generic modification of the proteins. The Förster theory parameters (e.g., spectral overlap) were compared between each donor-acceptor combination and found to be good predictors of an effective dye-protein combination. At the highest dye-to-protein ratios tested (over 20:1), the effective absorption strength integrated over the full spectral range was increased to ∼180% of its natural level for both LH complexes. Lipophilic dyes could be inserted into pre-formed membranes although their effectiveness was found to depend upon favourable physicochemical interactions. Finally, we demonstrated that these dyes can also be effective at increasing the spectral range of surface-supported models of photosynthetic membranes, using fluorescence microscopy. The results of this work provide insight into the utility of self-assembled lipid membranes and the great flexibility of LH complexes for interacting with different dyes.

Cell-Mimic Directional Cargo Transportation in a Visible-Light-Activated Colloidal Motor/Lipid Tube System.

Active tether and transportation of cargoes on cytoskeletal highway enabled by molecular motors is key for accurate delivery of vesicles and organelles in the complex intracellular environment. Here, a hybrid system composed of colloidal motors and self-assembled lipid tubes is designed to mimic the subcellular traffic system in living cells. The colloidal motors, composed of gold-coated hematite, display light-activated self-propulsion tunable by the light intensity and the concentration of hydrogen peroxide fuel. Importantly, the motors show light-switchable binding with lipid cargoes and attachment to the lipid tubes, whereby the latter act as the motor highways. Upon assembly, the colloidal motor/lipid tube system demonstrates directional delivery of lipid vesicles, emulating intracellular transportation. The assembly and function of the hybrid system are rationalized by a cooperative action of light-triggered electrophoretic and hydrodynamic effects, supported by finite element analysis. A synthetic analog of the biological protein motor/cytoskeletal filament system is realized for the manipulation and delivery of different matter at the microscale, which is expected to be a promising platform for various applications in materials science, nanotechnology, microfluidics, and synthetic biology.

The future of non-viral gene delivery for the treatment of inherited retinal diseases

In a recent article in Molecular Therapy – Nucleic Acids,1 Sun et al. describe a promising therapy for ABCA4-mediated Stargardt disease (STGD). The therapy is based on a non-viral gene delivery platform designed for the treatment of inherited retinal diseases (IRDs) caused by mutations in large genes. This platform allows for the formulation of self-assembling multifunctional lipid nanoparticles loaded with plasmid DNA containing the gene of interest. The authors’ preliminary study using subretinal injections showed that the therapy was safe and effective for short-term gene expression in STGD1 mice.

Controlled Shrinkage of Microfluidically Generated Microbubbles by Tuning Lipid Concentration.

Monodisperse microbubbles with diameters less than 10 μm are desirable in several ultrasound imaging and therapeutic delivery applications. However, conventional approaches to synthesize microbubbles, which are usually agitation-based, produce polydisperse bubbles that are less desirable because of their heterogeneous response when exposed to an ultrasound field. Microfluidics technology has the unique advantage of generating size-controlled monodisperse microbubbles, and it is now well established that the diameter of microfluidically made microbubbles can be tuned by varying the liquid flow rate, gas pressure, and dimensions of the microfluidic channel. It is also observed that once the microbubbles form, the bubbles shrink and eventually stabilize to a quasi-equilibrium diameter, and that the rate of stabilization is related to the lipid solution. However, how the lipid solution concentration affects the degree of bubble shrinkage, and the stable size of microbubbles, has not been thoroughly examined. Here, we investigate whether and how the lipid concentration affects the degree of microbubble shrinkage. Namely, we utilize a flow-focusing microfluidic geometry to generate monodisperse bubbles, and observe the effect of gas composition (2.5, 1.42, and 0.17 wt % octafluoropropane in nitrogen) and lipid concentration (1-16 mg/mL) on the degree of microbubble shrinkage. For the lipid system and gas utilized in these experiments, we observe a monotonic increase in the degree of microbubble shrinkage with decreasing lipid concentration, and no dependency on the gas composition. We hypothesize that the degree of shrinkage is related to lipid concentration by the self-assembly of lipids on the gas-liquid interface during bubble generation and subsequent lipid packing on the interface during shrinkage, which is arrested when a maximum packing density is achieved. We anticipate that this approach for creating and tuning the size of monodisperse microbubbles will find utility in biomedical applications, such as contrast-enhanced ultrasound imaging and ultrasound-triggered gene delivery.