Authors acknowledges the Department of Pharmacy, Quaid-i-Azam University, Islamabad, Pakistan for providing the necessary facilities to complete this study.

1. Screening of the oils, surfactants, and cosurfactants
<ol>
	<li>Screening of the oils, surfactants, and co-surfactants for drug solubility
	<ol>
		<li>Mix 100 mg of rosuvastatin separately in 1 mL of different omega-3 fatty acids-rich oils (fish oil, olive oil, sesame oil, and linseed oil) and 1 mL of surfactants and cosurfactants (Tween 80, Capryol PGMC, PEG 400, and ethanol) by vortexing for 5 min at a fixed-speed of 2,500 rpm. Then, place in a shaking water bath for 48 h at 50 &#176;C.</li>
		<li>After shaking, let the mixture settle for at least 6 h at room temperature so that the undissolved drug is precipitated. Take 0.1 mL of supernatant by using a micropipette and dilute up to 1 mL with methanol.</li>
		<li>Analyze by using a UV-visible spectrophotometer at 242 nm and calculate the concentration by adding the absorption to the straight-line equation of the calibration curve19,20. 
		NOTE: Establish the calibration curve by preparing a stock solution of 100 &#181;g/mL and making serial dilutions up to 0.5 &#181;g/mL. Absorbance is taken for all dilutions and a graph is plotted between absorbance and concentration in a spreadsheet. The straight-line equation (y = mx + b) of the graph is rearranged so that value of absorbance (y) can be used to calculate the unknown concentration (x)19,20.</li>
	</ol>
	</li>
	<li>Screening of the surfactant and co-surfactants for miscibility with oil
	<ol>
		<li>Mix the surfactant and the co-surfactant (Smix) in a 3:1 ratio. Add Smix and oil in different ratios; mix and heat up to 50 &#176;C to ensure homogenization.</li>
		<li>Take 0.1 mL from each mixture and dilute with 25 mL of distilled water in a glass test tube.</li>
		<li>Invert the test tube; the number of inversions at which an emulsion is formed represents the emulsification efficacy and ease of emulsification. Measure the transparency (T%) by measuring the emulsion at 650 nm with a UV-visible spectrophotometer using distilled water as a blank21.</li>
	</ol>
	</li>
</ol>
2. Construction of the pseudo ternary phase diagram
<ol>
	<li>Mix the surfactants and the co-surfactants in various volume ratios (1:0, 1:1, 1:2, 1:3,1:4, and 1:5) to form surfactant mixtures (Smix). Add oil to the Smix in separate vials at various volume ratios of 1:1,1:2,1:3,1:4,1:5,1:6,1:7, 1:8, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, and mix by vortexing.</li>
	<li>Add oil and Smix mixtures in a 10 mL glass vial and heat up to 50 &#176;C with constant stirring at 300 rpm for optimal mixing.</li>
	<li>Cool the mixture to 37 &#176;C and transfer 1 mL from it to a 250 mL water beaker and add dropwise the preheated distilled water (37 &#176;C) under gentle stirring at 50 rpm. Examine the dispersion visually and the mixture that forms a bluish transparent nanoemulsion is regarded as SNEDDS21.</li>
	<li>Construct the pseudoternary plot at different Smix ratios (Supplementary Table 1) through software (e.g., Triplot).</li>
</ol>
3. Optimization via software using a response surface methodology (RSM)
<ol>
	<li>Select three independent variables as oil (A), surfactant (B) and co-surfactant, and run the software for optimization and screening. Then, select the flexible design by choosing &#39;NO&#39; to a hard-to-change selection.
	<ol>
		<li>Observe the effect of these factors critically on dependent variables such as particle size (Y1, nm), zeta potential (Y2, mV), emulsification time (Y3, s) as well as entrapment efficiency (Y4, %) as mentioned in Table 2. 
		NOTE: The runs were increased until the warning signs disappeared and the software itself selected the Box-Benhken design for optimization.</li>
	</ol>
	</li>
	<li>Select the higher and lower values as -1 identifying the lowest variable value, whereas +1 depicts the highest value. The mid-value depicted the middle value. Box-Benhken design suggested a total of 17 runs with five center points as a measure to reduce the error.</li>
	<li>Record the responses to individual runs and fit to linear, 2F1, and quadratic models to ensure the best-fitted model. Generate polynomial equations and utilize to make the inference on the basis of the magnitude of the coefficient corresponding numerical signs.</li>
	<li>Display data of polynomial regression as 3-D plots. Evaluate the best fitted data model by comparing adjusted R2 and predicted R2 value 22. 
	NOTE: The required selection criteria of the formulation was based on grounds of maximum entrapment efficiency, less particle size, higher zeta potential, and minimum emulsification time frame. 
	Y = &#946;0 + &#946;1A + &#946;2B + &#946;3C + &#946;12AB + &#946;13AC + &#946;23BC + &#946;11A2 + &#946;22B2 + &#946;33C2 Eq. (1) 
	Here, &#946;0 appeared as intercept, Y as a response, ad &#946;1, &#946;2, and &#946;3 as linear coefficients. &#914;11, &#946;22, and &#946;33 as quadratic terms and squared coefficients while &#946;12, &#946;13, and &#946;23 as interaction coefficients. A, B, and C employed as independent variables that were selected from results of preliminary study.</li>
</ol>
4. Characterization
<ol>
	<li>Thermodynamic stability studies
	<ol>
		<li>Store the diluted SNEDDS at 4 &#176;C in a refrigerator, and then transfer it to a 50 &#176;C incubator . Perform six such cycles, each cycle being equal to or greater than 48 h.</li>
		<li>Examine the formulation for phase separation23.</li>
	</ol>
	</li>
	<li>Centrifugation test
	<ol>
		<li>Centrifuge the diluted SNEDDS for 30 min at 3,500 rpm at -4 &#176;C.</li>
		<li>Examine the SNEDDS at room temperature for phase separation or sedimentation of drug24.</li>
	</ol>
	</li>
	<li>Dispersibility test for self-emulsification efficacy
	<ol>
		<li>Add 1 mL of the formulation dropwise to 500 mL of double distilled water maintained at 37 &#176;C and 50 rpm. Note the time in which a clear homogenous emulsion is formed by visual inspection.</li>
		<li>Perform a visual assessment according to the following grading system25. 
		Grade 1: a clear bluish emulsion with emulsification time &#60;1 min 
		Grade 2: a bluish-white emulsion with emulsification time &#60;1 min 
		Grade 3: milky appearance with emulsification time &#60;2 min 
		Grade 4: gray, white appearance oil on top with emulsification time &#62;2 min 
		Grade 5: emulsification failure with larger oil droplet on top. 
		NOTE: The time required for emulsification or rate of emulsification is vital to emulsification efficacy. The test is performed in a USP type II dissolution apparatus.</li>
	</ol>
	</li>
	<li>Robustness to dilutions
	<ol>
		<li>Dilute the optimized SNEDDS formulation 50, 100, and 1,000 times with different carriers such as distilled water, 0.1 N HCl (pH 1.0) to mimic gastric pH and phosphate buffer (pH 6.8) to mimic intestinal pH.</li>
		<li>Thoroughly mix the diluted mixtures and set them aside for 12 h.</li>
		<li>Visually examine the formulations for drug precipitation, phase separation, and any other stability issue26,27.</li>
	</ol>
	</li>
</ol>
5. In vitro dissolution studies
<ol>
	<li>Perform the release of drug from SNEDDS and pure drug suspension by using a water shaking bath maintained at 37 &#176;C and 50 rpm.</li>
	<li>Soak the dialysis membranes in the respective media solution for 24 h before the drug dissolution assay to attain good integrity and activation. Fill the drug suspension (in water) and SNEDDS equivalent to 10 mg of rosuvastatin in the dialysis membrane; tie and place in separate beakers (50 mL).</li>
	<li>Take 1 mL of the sample at specific intervals and replenish the beaker with fresh media (1 mL) after each sample.</li>
	<li>Filter the drawn samples and analyze by using a UV-visible spectrophotometer at 242 nm21,28,29,30.</li>
</ol>

The authors have nothing to disclose.

This study was designed to explore the potential of omega-3 fatty acids-rich oil, such as fish oil, sesame oil, olive oil, and linseed oil to act as drug carriers. Self-nanoemulsification was selected as a preferred technique for fabricating the delivery system that lacks water, making it more stable than classical emulsion systems32. Omega-3 fatty acids-rich oils are known for their beneficial health effects18. They have been used as a supplement for various diseases such ...

Lipids have been used for centuries to increase gastrointestinal absorption of water insoluble components of food and medicines1. Emulsions are the formulations used most widely for oral, intravenous (nutritional supplementation), and topical use2. A variety of lipids (fats and oils) are used in the manufacturing of pharmaceutical emulsions and lipid-based self-nanoemulsifying drug delivery systems (SNEDDS). Self-emulsification techniques are widely adopted in pharmaceutical sciences for transmucosal drug delivery. Unlike emulsions, SNEDDS consist of an oil and a surfactant mixture that self-emulsifies in an aqueous medium of the stomach to form emulsion droplets3. They can load lipophilic drugs in the oil phase and prevent them from degrading in the stomach environment4. SNEDDS have been shown to effectively enhance the bioavailable fraction of lipophilic drugs (four to six folds) by enhancing solubility and permeability5,6. The absence of an aqueous phase in SNEDDS offers significant advantages in terms of ease of manufacture and stability as compared to emulsions that are metastable dispersions prone to chemical degradation7. Many lipid excipient combinations are commercially available due to their desirable characteristics8,9.
Cardiovascular disorders are a leading cause of mortality worldwide10 and, hyperlipidemia causes the blood vessels to obstruct blood flow due to thickening of the blood vessels11. Increased dietary lipid uptake and a sedentary lifestyle are the major risk factors for development of hyperlipidemia. In addition to this, lipids have also been shown to directly damage the myocardium of the heart leading to non-ischemic heart failure12. Rosuvastatin is a potent hypolipidemic drug that belongs to the statin class and inhibits cholesterol synthesis leading to the lowering of lipid levels for the treatment of hyperlipidemia/dyslipidemia13. Rosuvastatin is a biopharmaceutical classification system (BCS) class II with poor aqueous solubility (0.01796 mg/mL)14. Recent advances in pharmaceutical research have recognized that lipids used in drug delivery can disturb the lipid profile of patients. The role of emulsions to increase low- and high-density lipoproteins and free cholesterol was demonstrated in the late twentieth century15. In addition to this, lipid-based drug delivery systems have shown to increase triglycerides16 and other lipid metabolites in the blood17. Therefore, there is a dire need to develop pharmaceutical formulations of oils that are unable to disturb the lipid profile of cardiovascular and hyperlipidemic patients.
Fish oil is a rich source of omega-3 fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. Fish oil has shown many health effects with substantial evidence of its beneficial role in cardiovascular and nervous systems18. The aim of the study was to utilize fish oil as an alternative to the conventional oils to formulate SNEDDS for the delivery of a lipophilic drug, rosuvastatin. No previous study has employed fish oil as a carrier to formulate drug delivery systems. Appropriate formulation and processing parameters were selected, and optimization was performed using design expert software.

<table><tbody><tr><td>Ammonium acetate</td><td>Sigma-Aldrich, Germany</td><td>A1542</td><td>Analytical grade</td></tr><tr><td>Capryol PGMC</td><td>Gattefoss&amp;eacute;, France</td><td>RT9P9S09QI</td><td>Analytical grade</td></tr><tr><td>Design Expert Software</td><td>StatEase, United States</td><td>Version 12.0.3.0</td><td>Analytical software (freely available for subscription)</td></tr><tr><td>Dialysis tubing (12,000 Daltons MWCO)</td><td>Visking, UK</td><td>12000.02.30</td><td>Pure regenerated natural cellulose membranes with 12,000 Daltons MWCO</td></tr><tr><td>Dissolution apparatus</td><td>Memmert, Germany</td><td>SV 1422</td><td>USP type II dissolution apparatus</td></tr><tr><td>Ethanol</td><td>Honeywell, Germany</td><td>24194</td><td>Analytical grade</td></tr><tr><td>Fish oil</td><td>Wilshire Labs Pvt(Ltd), Pakistan</td><td>not applicable</td><td>Received as gift sample.</td></tr><tr><td>Hydrochloric acid</td><td>BDH Laboratories Ltd, UK</td><td>BDH3036-54L</td><td>Analytical grade</td></tr><tr><td>Methanol</td><td>Honeywell, Germany</td><td>34966</td><td>Analytical grade</td></tr><tr><td>Refrigerator (Pharmaceutical)</td><td>Panasonic, Pakistan</td><td>MPR-161 DH-PE</td><td>Refrigerator for storage at 4 &amp;deg;C</td></tr><tr><td>Rosuvastatin calcium</td><td>Searle Pharmaceuticals Pvt(Ltd) Pakistan</td><td>not applicable</td><td>Received as gift sample.</td></tr><tr><td>Sodium Hydroxide</td><td>Honeywell, Germany</td><td>38215</td><td>Analytical grade</td></tr><tr><td>Span 80</td><td>BDH Laboratories Ltd, UK</td><td>MFCD00082107</td><td>Analytical grade</td></tr><tr><td>Triplot Software</td><td>MS Excel spreadsheet developed by Tod Thompson</td><td>Triplot Ver. 4.1.2</td><td>Analytical software (freely available)</td></tr><tr><td>Tween-80</td><td>Sigma-Aldrich, Germany</td><td>P1754-500ML</td><td>Analytical grade</td></tr><tr><td>UV-Vis spectrophotometer</td><td>Dynamica, UK</td><td>Halo DB-20</td><td>Double beam spectrophotometer</td></tr><tr><td>Water Bath</td><td>Memmert, Germany</td><td>WNB 7</td><td>Water batch for heating up to 70 &amp;deg;C</td></tr></tbody></table>

self-nanoemulsification of healthy oils to enhance the solubility of lipophilic drugs

The low aqueous solubility of many drugs reduces their bioavailability in the blood. Oils have been used for centuries to enhance the solubility of drugs; however, they can disturb the lipid profile of the patients. In this study, self-nanoemulsifying drug delivery systems of omega-3 fatty acids-rich oils are prepared and optimized for the delivery of lipophilic drugs. Rosuvastatin, a potent hypolipidemic drug, was used as a model lipophilic drug. Fish oil showed more than 7-fold higher solubility of rosuvastatin than other oils and therefore it was selected for the development of self-nanoemulsifying drug delivery systems (SNEDDS). Different combinations of surfactants and co-surfactants were screened and a surfactant mixture of Tween 80 (surfactant) and Capryol PGMC (cosurfactant) were selected for compatibility with fish oil and rosuvastatin. A pseudoternary phase diagram of oil, surfactant, and co-surfactant was designed to identify the emulsion region. The pseudoternary phase diagram predicted a 1:3 oil and surfactant mixture as the most stable ratio for the emulsion system. Then, a response-surface methodology (Box-Behnken design) was applied to calculate the optimal composition. After 17 runs, fish oil, Tween 80, and Capryol PGMC in proportions of 0.399, 0.67, and 0.17, respectively, were selected as the optimized formulation. The self-nanoemulsifying drug delivery systems showed excellent emulsification potential, robustness, stability, and drug release characteristics. In the drug release studies, SNEDDS released 100% of the payload in around 6 h whereas, release of the plain drug was less than 70% even after 12 h. Therefore, omega-3 fatty acids-rich healthy lipids have enormous potential to enhance the solubility of lipophilic drugs whereas, self-emulsification can be used as a simple and feasible approach to exploit this potential.

Herein, nanoformulation of omega-3 fatty acids-rich fish oil are prepared and optimized by self-emulsification with different surfactants and co-surfactants. Figure 1 shows the solubility of rosuvastatin in different oils, surfactants, and co-surfactants. Based on solubility, fish oil was selected as the oil, Tween 80 as the surfactant, and Capryol PGMC as the co-surfactant in the following studies. Table 1 shows the screening of Smix at different ratios to emulsify fish oil...

Watch this Scientific Journal Video about Self-Nanoemulsification of Healthy Oils to Enhance the Solubility of Lipophilic Drugs at JoVE.com

Self-Nanoemulsification of Healthy Oils to Enhance the Solubility of Lipophilic Drugs

Oils used for drug delivery applications can disrupt the lipid profile of patients, which is undesirable in cardiovascular diseases. Omega-3 fatty acids-rich oils are a healthy alternative to conventional oils and have enormous potential for self-emulsified drug delivery systems.

The low aqueous solubility of many drugs reduces their bioavailability in the blood. Oils have been used for centuries to enhance the solubility of drugs; ...

self-nanoemulsification-healthy-oils-to-enhance-solubility-lipophilic

Research

JoVE Journal

Biology

1.0K Views.  Quaid-i-Azam University. Omega-3-vetzuren die rijk zijn aan vetzuren kunnen de oplosbaarheid van lipofiele geneesmiddelen verbeteren zonder het lipidenprofiel te laten ontsporen. Zorgvuldige optimalisatie van olie en hulpstoffen is de sleutel tot het voorbereiden van zelf-nano-emulgerende medicijnafgiftesystemen. Visolie ontspoort het lipidenprofiel niet en heeft zelf gunstige effecten.Optimalisatie versnelt het ontwikkelingsproces van de formulering, bespaart middelen en zorgt voor stabiliteit voor de beoogde...

Video: Self-Nanoemulsification of Healthy Oils to Enhance the Solubility of Lipophilic Drugs

Synthesis of Phase-shift Nanoemulsions with Narrow Size Distributions for Acoustic Droplet Vaporization and Bubble-enhanced Ultrasound-mediated Ablation

High-intensity focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To enhance localized heating and improve thermal ablation in tumors, lipid-coated perfluorocarbon droplets have been developed which can be vaporized by HIFU. The vasculature in many tumors is abnormally leaky due to their rapid growth, and nanoparticles are able to penetrate the fenestrations and passively accumulate within tumors. Thus, controlling the size of the droplets can result in better accumulation within tumors. In this report, the preparation of stable droplets in a phase-shift nanoemulsion (PSNE) with a narrow size distribution is described. PSNE were synthesized by sonicating a lipid solution in the presence of liquid perfluorocarbon. A narrow size distribution was obtained by extruding the PSNE multiple times using filters with pore sizes of 100 or 200 nm. The size distribution was measured over a 7-day period using dynamic light scattering. Polyacrylamide hydrogels containing PSNE were prepared for in vitro experiments. PSNE droplets in the hydrogels were vaporized with ultrasound and the resulting bubbles enhanced localized heating. Vaporized PSNE enables more rapid heating and also reduces the ultrasound intensity needed for thermal ablation. Thus, PSNE is expected to enhance thermal ablation in tumors, potentially improving therapeutic outcomes of HIFU-mediated thermal ablation treatments.

Phase-shift nanoemulsions (PSNE) can be vaporized using high intensity focused ultrasound to enhance localized heating and improve thermal ablation in tumors. In this report, the preparation of stable PSNE with a narrow size distribution is described. Furthermore, the impact of vaporized PSNE on ultrasound-mediated ablation is demonstrated in tissue-mimicking phantoms.

High-intensity  focused ultrasound (HIFU) is used clinically to thermally ablate tumors. To  enhance localized heating and improve thermal ablation in ...

Engineering

Facile Preparation of Internally Self-assembled Lipid Particles Stabilized by Carbon Nanotubes

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled (functionalized) CNTs are used as stabilizers to produce Pickering type oil-in-water (O/W) emulsions. Lipids namely, Dimodan U and Phytantriol are used as emulsifiers, which in excess water self-assemble into the bicontinuous cubic Pn3m phase. This highly viscous phase is fragmented into smaller particles using a probe ultrasonicator in presence of conventional surfactant stabilizers or CNTs as done here. Initially, the CNTs (powder form) are dispersed in water followed by further ultrasonication with the molten lipid to form the final emulsion. During this process the CNTs get coated with lipid molecules, which in turn are presumed to surround the lipid droplets to form a particulate emulsion that is stable for months. The average size of CNT-stabilized nanostructured lipid particles is in the submicron range, which compares well with the particles stabilized using conventional surfactants. Small angle X-ray scattering data confirms the retention of the original Pn3m cubic phase in the CNT-stabilized lipid dispersions as compared to the pure lipid phase (bulk state). Blue shift and lowering of the intensities in characteristic G and G&#39; bands of CNTs observed in Raman spectroscopy characterize the interaction between CNT surface and lipid molecules. These results suggest that the interactions between the CNTs and lipids are responsible for their mutual stabilization in aqueous solutions. As the concentrations of CNTs employed for stabilization are very low and lipid molecules are able to functionalize the CNTs, the toxicity of CNTs is expected to be insignificant while their biocompatibility is greatly enhanced. Hence the present approach finds a great potential in various biomedical applications, for instance, for developing hybrid nanocarrier systems for the delivery of multiple functional molecules as in combination therapy or polytherapy.

We report on a smart application of carbon nanotubes for kinetic stabilization of lipid particles that contain self-assembled nanostructures in their cores. The preparation of lipid particles requires rather low concentrations of carbon nanotubes permitting their use in biomedical applications such as drug delivery.

We present a facile method to prepare nanostructured lipid particles stabilized by carbon nanotubes (CNTs). Single-walled (pristine) and multi-walled ...

Chemistry

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

Bioengineering

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

Emulsions are currently being used to encapsulate and deliver nutrients and drugs to tackle different gastrointestinal conditions such as obesity, nutrient fortification, food allergies, and digestive diseases. The ability of an emulsion to provide the desired functionality, namely, reaching a specific site within the gastrointestinal tract, inhibiting/retarding lipolysis, or facilitating digestibility, ultimately depends on its susceptibility to enzymatic degradation in the gastrointestinal tract. In oil-in-water emulsions, lipid droplets are surrounded by interfacial layers, where the emulsifiers stabilize the emulsion and protect the encapsulated compound. Achieving a tailored digestibility of emulsions depends on their initial composition but also requires monitoring the evolution of those interfacial layers as they are subjected to different phases of gastrointestinal digestion. A pendant drop surface film balance implemented with a multi-subphase exchange allows for simulating the in vitro digestion of emulsions in a single aqueous droplet immersed in oil by applying a customized static digestion model. The transit through the gastrointestinal tract is mimicked by the subphase exchange of the original droplet bulk solution with artificial media, mimicking the physiological conditions of each compartment/step of the gastrointestinal tract. The dynamic evolution of the interfacial tension is recorded in situ throughout the whole simulated gastrointestinal digestion. The mechanical properties of digested interfaces, such as interfacial dilatational elasticity and viscosity, are measured after each digestion phase (oral, gastric, small intestine). The composition of each digestive media can be tuned to account for the particularities of the digestive conditions, including gastrointestinal pathologies and infant digestive media. The specific interfacial mechanisms affecting proteolysis and lipolysis are identified, providing tools to modulate digestion by the interfacial engineering of emulsions. The obtained results can be manipulated for designing novel food matrices with tailored functionalities such as low allergenicity, controlled energy intake, and decreased digestibility.

In vitro Digestion of Emulsions in a Single Droplet via Multi Subphase Exchange of Simulated Gastrointestinal Fluids

A pendant drop surface film balance implemented with a multi-subphase exchange, nicknamed the OCTOPUS, allows for mimicking digestive conditions by the sequential subphase exchange of the original bulk solution with simulated gastrointestinal fluids. The simulated in vitro digestion is monitored by recording in situ the interfacial tension of the digested interfacial layer.

Emulsions are currently being used to encapsulate and deliver nutrients and drugs to tackle different gastrointestinal conditions such as obesity, ...

Biochemistry

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Preparation and Characterization of Nanoliposomes for the Entrapment of Bioactive Hydrophilic Globular Proteins

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their biocompatibility, biodegradability, non-immunogenicity, non-toxicity, and ability to entrap both hydrophilic and hydrophobic compounds. The classical hydration of thin lipid films in an organic solvent is applied herein as a technique to encapsulate tarin, a plant lectin, in nanoliposomes. Nanoliposome size, stability, entrapment efficiency, and morphological characterization are described in detail. The nanoliposomes are prepared using 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (ammonium salt; DSPE-MPEG 2000), and cholesterylhemisuccinate (CHEMS) as the main constituents. Lipids are first dissolved in chloroform to obtain a thin lipid film that is subsequently rehydrated in ammonium sulfate solution containing the protein to be entrapped and incubated overnight. Then, sonication and extrusion techniques are applied to generate nanosized unilamellar vesicles. The size and polydispersity index of the nanovesicles are determined by dynamic light scattering, while nanovesicle morphology is assessed by scanning electron microscopy. Entrapment efficiency is determined by the ratio of the amount of unencapsulated protein to original amount of initially loaded protein. Homogeneous liposomes are obtained with an average size of 155 nm and polydispersity index value of 0.168. A high entrapment efficiency of 83% is achieved.

This study describes classical hydration using the thin lipid film method for nanoliposome preparation followed by nanoparticle characterization. A 47 kDa-hydrophilic and globular protein, tarin, is successfully encapsulated as a strategy to improve stability, avoid fast clearance, and promote controlled release. The method can be adapted to hydrophobic molecules encapsulation.

Liposome nanocapsules have been applied for many purposes in the pharmaceutical, cosmetic, and food industries. Attributes of liposomes include their ...

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading chemotherapeutic drugs, such as doxorubicin (DOX). To achieve this, an ethanolic lipid mixture and ammonium sulfate solution are injected into a staggered herringbone micromixer (SHM) microfluidic device. The solutions are rapidly mixed by the SHM, providing a homogeneous solvent environment for liposomes self-assembly. Collected liposomes are first annealed, then dialyzed to remove residual ethanol. An ammonium sulfate pH-gradient is established through buffer exchange of the external solution by using size exclusion chromatography. DOX is then remotely loaded into the liposomes with high encapsulation efficiency (&#62; 80%). The liposomes obtained are homogenous in size with Z-average diameter of 100 nm. They are capable of temperature-triggered burst release of encapsulated DOX in the presence of mild hyperthermia (42 &#176;C). Indocyanine green (ICG) can also be co-loaded into the liposomes for near-infrared laser-triggered DOX release. The microfluidic approach ensures high-throughput, reproducible and scalable preparation of LTSLs.

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

The presented protocol enables a high-throughput continuous preparation of low temperature-sensitive liposomes (LTSLs), which are capable of loading ...

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

Self-Nanoemulsification of Healthy Oils to Enhance the Solubility of Lipophilic Drugs

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