Precise control of liposome size using characteristic time depends on solvent type and membrane properties.

Controlling the sizes of liposomes is critical in drug delivery systems because it directly influences their cellular uptake, transportation, and accumulation behavior. Although hydrodynamic focusing has frequently been employed when synthesizing nano-sized liposomes, little is known regarding how flow characteristics determine liposome formation. Here, various sizes of homogeneous liposomes (50-400 nm) were prepared according to flow rate ratios in two solvents, ethanol, and isopropyl alcohol (IPA). Relatively small liposomes formed in ethanol due to its low viscosity and high diffusivity, whereas larger, more poly-dispersed liposomes formed when using IPA as a solvent. This difference was investigated via numerical simulations using the characteristic time factor to predict the liposome size; this approach was also used to examine the flow characteristics inside the microfluidic channel. In case of the liposomes, the membrane rigidity also has a critical role in determining their size. The increased viscosity and packing density of the membrane by addition of cholesterol confirmed by fluorescence anisotropy and polarity lead to increase in liposome size (40-530 nm). However, the interposition of short-chain lipids de-aligned the bilayer membrane, leading to its degradation; this decreased the liposome size. Adding short-chain lipids linearly decreased the liposome size (130-230 nm), but at a shallower gradient than that of cholesterol. This analytical study expands the understanding of microfluidic environment in the liposome synthesis by offering design parameters and their relation to the size of liposomes.

Preparation and Characterization of pH-Sensitive Capsosomes for Oral Delivery of Therapeutic Proteins.

Oral administration of therapeutic proteins is very challenging because of gastrointestinal instability and decomposition. In this study, we developed a system for oral delivery of superoxide dismutase (SOD) as one of the therapeutic proteins. SOD-loaded capsosomes (SOD-C) were formed by the assembly of chitosan-coated solid lipid nanoparticles and SOD-loaded liposomes (SOD-L). Unlike raw SOD activity decreases to 19.41% in SGF and 13.70% in SIF, the SOD-C in SGF (89.30%) condition retained its initial catalytic activity and decreased but exhibited a three-fold higher raw SOD activity even after incubation in SIF (41.63%). TEM analysis indicated that after intestinal digestion, the residual amount of intact liposomes affected the higher catalytic activity of SOD-C compared to raw SOD and SOD-L. Based on these results, significantly higher cellular uptake of SOD-C was observed compared to raw SOD. Also, SOD-C remarkably suppressed the cellular malondialdehyde (MDA) concentration by maintaining the antioxidative capacity of SOD to remove MDA produced in the oxidative stress-induced cells, thereby contributing to a significant five-fold difference with SOD-R (p < 0.05). This delivery system can facilitate the oral application of other therapeutic proteins, improving gastrointestinal stability.

Controlled rate slow freezing with lyoprotective agent to retain the integrity of lipid nanovesicles during lyophilization.

We designed a novel lyophilization method using controlled rate slow freezing (CSF) with lyoprotective agent (LPA) to achieve intact lipid nanovesicles after lyophilization. During the freezing step, LPA prevented water supercooling, and the freezing rate was controlled by CSF. Regulating the freezing rate by various liquid media was a crucial determinant of membrane disruption, and isopropanol (freezing rate of 0.933 °C/min) was the optimal medium for the CSF system. Lyophilized lipid nanovesicle using both CSF and LPA retained 92.9% of the core material and had uniform size distributions (Z-average diameter = 133.4 nm, polydispersity index = 0.144), similar to intact vesicles (120.7 nm and 0.159, respectively), after rehydration. Only lyophilized lipid nanovesicle using both CSF and LPA showed no changes in membrane fluidity and polarity. This lyophilization method can be applied to improve storage stability of lipid nanocarriers encapsulating drugs while retaining their original activity.

A Simple Method for Continuous Synthesis of Bicelles in Microfluidic Systems.

Bicelle has great potential for drug delivery systems due to its small size and biocompatibility. The conventional method of bicelle preparation contains a long process and harsh conditions, which limit its feasibility and damage the biological substances. For these reasons, a continuous manufacturing method in mild conditions has been demanded. Here, we propose a novel method for DMPC/DHPC bicelle synthesis based on a microfluidic device without heating and freezing processes. Bicelles were successfully prepared using this continuous method, which was identified by the physicochemical properties and morphologies of the synthesized assemblies. Experimental and analytical studies confirm that there is critical lipid concentration and critical mixing time for bicelle synthesis in this microfluidic system. Furthermore, a linear relation between the actual composition of bicelle and initial lipid ratio is deduced, and this enables the size of bicelles to be controlled.

Precise Microfluidic Luminescent Sensor Platform with Controlled Injection System.

Efforts have been devoted to screening various prevalent diseases, such as severe acute respiratory syndrome (SARS) and coronavirus disease 2019 (COVID-19). Real-time polymerase chain reaction (RT-PCR), which is currently the most widely used, has high accuracy, but it requires several facilities and takes a relatively long time to check; so, new testing technology is necessary for a higher test efficiency. A chemiluminescence (CL) sensor is a relatively simple device and suitable as an alternative because it can detect very precise specimens. However, in measurements via CL, the quantitative formulation of reagents that cause color development is important. In the case of mixing using micropipettes, precise analysis is possible, but this technique is limited by uncontrollable errors or deviations in detection amounts. In addition, in using a microfluidic chip to increase field applicability, a syringe pump or other quantification injection tools are required, so problems must be overcome for practical use. Therefore, in this study, a microchip was designed and manufactured to supply a sample of a certain volume by simply blowing air and injecting a sample into the chamber. By utilizing the luminescence reaction of luminol, CuSO4 and H2O2 the performance of the prepared chip was confirmed, and the desired amount of the sample could be injected with a simple device with an error rate of 2% or less. For feasible applications, an experiment was performed to quantitatively analyze thrombin, a biomarker of heart disease. Results demonstrated that biomarkers could be more precisely detected using the proposed microchips than using micropipettes.

Hybrid Nanofiber Scaffold-Based Direct Conversion of Neural Precursor Cells/Dopamine Neurons.

The concept of cellular reprogramming was developed to generate induced neural precursor cells (iNPCs)/dopaminergic (iDA) neurons using diverse approaches. Here, we investigated the effects of various nanoscale scaffolds (fiber, dot, and line) on iNPC/iDA differentiation by direct reprogramming. The generation and maturation of iDA neurons (microtubule-associated protein 2-positive and tyrosine hydroxylase-positive) and iNPCs (NESTIN-positive and SOX2-positive) increased on fiber and dot scaffolds as compared to that of the flat (control) scaffold. This study demonstrates that nanotopographical environments are suitable for direct differentiation methods and may improve the differentiation efficiency.

Lipid-Hydrogel-Nanostructure Hybrids as Robust Biofilm-Resistant Polymeric Materials.

Despite extensive efforts toward developing antibiofilm materials, efficient prevention of biofilm formation remains challenging. Approaches based on a single strategy using either bactericidal material, antifouling coatings, or nanopatterning have shown limited performance in the prevention of biofilm formation. This study presents a hybrid strategy based on a lipid-hydrogel-nanotopography hybrid for the development of a highly efficient and durable biofilm-resistant material. The hybrid material consists of nanostructured antifouling, biocompatible polyethylene glycol-based polymer grafted with an antifouling zwitterionic polymer of 2-methacryloyloxyethyl phosphorylcholine. Based on the unique composite nanostructures, the lipid-hydrogel-nanostructure hybrid exhibits superior dual functionalities of antifouling and bactericidal activities against Gram-negative and Gram-positive bacteria, compared with those of surfaces with simple nanostructures or antifouling coatings. Additionally, it preserves the robust antibiofilm activity even when the material is damaged under external mechanical stimuli thanks to the polymeric composite nanostructure.

Catalytic characteristics of a sn-1(3) regioselective lipase from Cordyceps militaris.

A total of 39 agricultural products were screened for natural sources of lipases with distinctive positional specificity. Based on this, Cordyceps militaris lipase (CML) was selected and subsequently purified by sequential chromatography involving anion-exchange, hydrophobic-interaction, and gel-permeation columns. As a result of the overall purification procedure, a remarkable increase in the specific activity of the CML (4.733 U/mg protein) was achieved, with a yield of 2.47% (purification fold of 94.54). The purified CML has a monomeric structure with a molecular mass of approximately 62 kDa. It was further identified as a putative extracellular lipase from C. militaris by the partial sequence analysis using ESI-Q-TOF MS. In a kinetic study of the CML-catalyzed hydrolysis, the values of Vmax , Km , and kcat were determined to be 4.86 µmol·min-1 ·mg-1 , 0.07 mM, and 0.29 min-1 , respectively. In particular, the relatively low Km value indicated that CML has a high affinity for its substrate. With regard to positional specificity, CML selectively cleaved triolein at the sn-1 or 3 positions of glycerol backbone, releasing 1,2(2,3)-diolein as the major products. Therefore, CML can be considered a distinctive biocatalyst with sn-1(3) regioselectivity. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2744, 2019.

Hydrophilic and lipophilic characteristics of non-fatty acid moieties: significant factors affecting antibacterial activity of lauric acid esters.

It has been proposed that the hydrophilic and/or lipophilic characteristics of fatty acid derivatives affect their antibacterial activities according to their ability to incorporate into the bacterial cell membrane. To verify this hypothesis, six kinds of lauric acid derivatives esterified with different non-fatty acid moieties were selected to confirm whether antibacterial activity from their precursor (i.e., lauric acid) is retained or lost. Three compounds, monolaurin, sucrose laurate, and erythorbyl laurate, exerted bacteriostatic and bactericidal effects against Gram-positive bacteria, while the others showed no inhibitory activity. Interestingly, the calculated log P (octanol-water partition coefficient) values of monolaurin, sucrose laurate, and erythorbyl laurate were - 4.122, - 0.686, and 3.670, respectively, relatively lower than those of the other compounds without antibacterial activity. Moreover, the hydrophilic-lipophilic balance values of the three compounds with antibacterial activity were higher than those of the other compounds, corresponding to the log P result.

Design of a simple paper-based colorimetric biosensor using polydiacetylene liposomes for neomycin detection.

We developed a paper-based analytical device (µPAD) combined with self-signaling polydiacetylene (PDA) liposomes for convenient visual neomycin detection. The simple dot array type of µPAD was fabricated by the wax printing technique, and the PDA liposomes in the aqueous solution were facilely immobilized onto the hydrophilic dot region of the paper substrate. We found that, when the PDA liposomes were inserted to the paper matrix, the stability of the PDA liposomes can be significantly enhanced by adding a hydrophilic reagent such as polyvinyl alcohol and glycerol to the liposome solution. In particular, polyvinyl alcohol (PVA) provides the best stabilization among the various hydrophilic reagents tested in this contribution, and the enhanced stability sharply increased the sensitivity of the PDA liposomes in the paper matrix. Based on the above results, we successfully detected neomycin through both naked-eye observation and fluorescence measurement of PDA signals. The detection limit was 1 ppm and was selective to non-aminoglycoside antibiotics.

Amperometric Detection of Conformational Change of Proteins Using Immobilized-Liposome Sensor System.

An immobilized liposome electrode (ILE)-based sensor was developed to quantify conformational changes of the proteins under various stress conditions. The ILE surface was characterized by using a tapping-mode atomic force microscopy (TM-AFM) to confirm surface immobilization of liposome. The uniform layer of liposome was formed on the electrode. The current deviations generated based on the status of the proteins under different stress were then measured. Bovine carbonic anhydrase (CAB) and lysozyme were tested with three different conditions: native, reduced and partially denatured. For both proteins, a linear dynamic range formed between denatured concentrations and output electric current signals was able to quantify conformational changes of the proteins. The pattern recognition (PARC) technique was integrated with ILE-based sensor to perform data analysis and provided an effective method to improve the prediction of protein structural changes. The ILE-based stress sensor showed potential of leveraging the amperometric technique to manifest activity of proteins based on various external conditions.

Mussel-Inspired Universal Bioconjugation of Polydiacetylene Liposome for Droplet-Array Biosensors.

Most solid-state biosensor platforms require a specific immobilization chemistry and a bioconjugation strategy separately to tether sensory molecules to a substrate and attach specific receptors to the sensory unit, respectively. We developed a mussel-inspired universal conjugation method that enables both surface immobilization and bioconjugation at the same time. By incorporating dopamine or catechol moiety into self-signaling polydiacetylene (PDA) liposomes, we demonstrated efficient immobilization of the PDA liposomes to a wide range of substrates, without any substrate modification. Moreover, receptor molecules having a specificity toward a target molecule can also be attached to the immobilized PDA liposome layer without any chemical modification. We applied our mussel-inspired conjugation method to a droplet-array biosensor by exploiting the hydrophilic nature of PDA liposomes coated on a hydrophobic polytetrafluoroethylene surface and demonstrated selective and sensitive detection of vascular endothelial growth factor down to 10 nM.

Ultra-sensitive detection of kanamycin for food safety using a reduced graphene oxide-based fluorescent aptasensor.

Overuse of antibiotics has caused serious problems, such as appearance of super bacteria, whose accumulation in the human body through the food chain is a concern. Kanamycin is a common antibiotic used to treat diverse infections; however, residual kanamycin can cause many side effects in humans. Thus, development of an ultra-sensitive, precise, and simple detection system for residual kanamycin in food products is urgently needed for food safety. In this study, we identified kanamycin-binding aptamers via a new screening method, and truncated variants were analyzed for optimization of the minimal sequence required for target binding. We found various aptamers with high binding affinity from 34.7 to 669 nanomolar Kdapp values with good specificity against kanamycin. Furthermore, we developed a reduced graphene oxide (RGO)-based fluorescent aptasensor for kanamycin detection. In this system, kanamycin was detected at a concentration as low as 1 pM (582.6 fg/mL). In addition, this method could detect kanamycin accurately in kanamycin-spiked blood serum and milk samples. Consequently, this simple, rapid, and sensitive kanamycin detection system with newly structural and functional analysis aptamer exhibits outstanding detection compared to previous methods and provides a new possibility for point of care testing and food safety.

Characterization of the physicochemical properties of phospholipid vesicles prepared in CO2/water systems at high pressure.

Phospholipid vesicles were prepared by the nonsolvent method using high-pressure CO2/water systems. The membrane properties of vesicles prepared at different pressures and temperatures were mainly characterized based on analysis of the membrane fluidity and membrane polarity, using the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 6-dodecanoyl-N,N-dimethyl-2-naphthylamine, respectively. The CO2(liquid)/water(liquid) and the CO2(supercritical)/water(liquid) two-phase (heterogeneous) systems resulted in the formation of vesicles with high yield (ca. 85%-88%). The membrane fluidity and polarity of the vesicles were similar to those of liposomes prepared by the conventional method. It is suggested that high-pressure CO2 can be used to form an appropriate hydrophobic-hydrophilic interface where phospholipid molecules as a self-assembled membrane.

AOT/isooctane reverse micelles with a microaqueous core act as protective shells for enhancing the thermal stability of Chromobacterium viscosum lipase.

According to the different environmental systems for lipase reactions, changes in thermal stability were investigated by employing the Chromobacterium viscosum lipase and a two-step series-type deactivation model. The half-life (6.81 h) of the lipase entrapped in reverse micelles at 70 °C was 9.87- and 14.80-fold longer than that in glycerol pool or in aqueous buffer. The deactivation constants for the first and second step (k1 and k2) at all temperatures drastically decreased when the lipase was entrapped in reverse micelles. In particular, k1 (3.84 h(-1)) at 70 °C in reverse micelles was 1.57-fold lower than that in aqueous buffer (6.03 h(-1)). Based on the fluorescence spectrometry, the amount of excited forms of tryptophan and tyrosine increased markedly during the thermal-treatment in aqueous buffer, whereas no significant fluctuation was noted in the reversed micellar system. These results indicated that the encapsulation in reverse micelles could be favorable for preventing the enzyme from heat-induced denaturation.

Microfluidic platforms with monolithically integrated hierarchical apertures for the facile and rapid formation of cargo-carrying vesicles.

We fabricated a simple yet robust microfluidic platform with monolithically integrated hierarchical apertures. This platform showed efficient diffusive mixing of the introduced lipids through approximately 8000 divisions with tiny pores (~5 µm in diameter), resulting in massive, real-time production of various cargo-carrying particles via multi-hydrodynamic focusing.

Janus-compartmental alginate microbeads having polydiacetylene liposomes and magnetic nanoparticles for visual lead(II) detection.

Janus-compartmental alginate microbeads having two divided phases of sensory polydiacetylene (PDA) liposomes and magnetic nanoparticles were fabricated for facile sensory applications. The sensory liposomes are composed of PDA for label-free signal generation and 1,2-dipalmitoyl-sn-glycero-3-galloyl (DPGG) lipids whose galloyl headgroup has specific interactions with lead(II). The second phase having magnetic nanoparticles is designed for convenient handling of the microbeads, such as washing, solvent exchange, stirring, and detection, by applying magnetic field. Selective and convenient colorimetric detection of lead(II) and efficient removal of lead(II) by alginate matrix at the same time are demonstrated.

Effect of surface tension and coefficient of thermal expansion in 30 nm scale nanoimprinting with two flexible polymer molds.

We report on nanoimprinting of polymer thin films at 30 nm scale resolution using two types of ultraviolet (UV)-curable, flexible polymer molds: perfluoropolyether (PFPE) and polyurethane acrylate (PUA). It was found that the quality of nanopatterning at the 30 nm scale is largely determined by the combined effects of surface tension and the coefficient of thermal expansion of the polymer mold. In particular, the polar component of surface tension may play a critical role in clean release of the mold, as evidenced by much reduced delamination or broken structures for the less polarized PFPE mold when patterning a relatively hydrophilic PMMA film. In contrast, such problems were not notably observed with a relatively hydrophobic PS film for both polymer molds. In addition, the demolding characteristic was also influenced by the coefficient of thermal expansion so that no delamination or uniformity problems were observed when patterning a UV-curable polymer film at room temperature. These results suggest that a proper polymeric mold material needs to be chosen for patterning polymer films under different surface properties and processing conditions, providing insights into how a clean demolding characteristic can be obtained at 30 nm scale nanopatterning.

Biomimetic detection of aminoglycosidic antibiotics using polydiacetylene-phospholipids supramolecules.

We rationally designed highly sensitive and selective polydiacetylene (PDA)-phospholipids liposomes for the facile detection of aminoglycosidic antibiotics. The detecting mechanism mimics the cellular membrane interactions between neomycin and phosphatidylinositol-4,5-bisphosphate (PIP(2)) phospholipids. The developed PDA-PIP(2) sensory system showed a detection limit of 61 ppb for neomycin and was very specific to aminoglycosidic antibodies only.