We thank Prostate Cancer UK (CDF-12-002 Fellowship), and the Engineering and Physical Sciences Research Council (EPSRC) (EP/M008657/1) for funding.

1. Equipment setup
<ol>
	<li>Assemble the syringe pumps and SHM as follows.
	<ol>
		<li>Connect the &#34;To Computer&#34; port of the secondary syringe pump (Pump 02, for aqueous solution) to the &#34;To Network&#34; port of the master syringe pump (Pump 01, for ethanol lipid solution) using Pump to Pump network cable (Figure 1, yellow).</li>
		<li>Connect the &#34;To Computer&#34; port of the master pump to the &#34;RS232 Serial&#34; port of the computer using PC to Pump network cable (Figure 1, blue).</li>
		<li>Connect tubing to each of the inlets and outlets of the SHM using a nut and ferrule. Convert the terminal of the tubing for both inlets to female Luer using another nut and ferrule and a union assembly. Longer tubing of the inlets allows easier attachment to the syringes (Figure 2).</li>
	</ol>
	</li>
	<li>Set up the pump control software.
	<ol>
		<li>Assign the address of the master syringe pump and secondary syringe pump to &#34;Ad:01&#34; and &#34;Ad:02&#34;, respectively, using the &#34;Setup&#34; button of the syringe pump. This only needs to be done for the first time.</li>
		<li>Open the pump control software on the computer. The two syringe pumps should be detected automatically, followed by a beeping sound. Otherwise, click Pumps and Search for pumps to update the connection. (Figure 3).</li>
		<li>Assign Diameter to 12.45 (mm) by choosing &#34;HSW Norm-Ject 5 cc (Dia=12.45)&#34;.</li>
		<li>Assign Rate to 0.25 mL/min for Pump 01 (ethanol lipid solution) and 0.75 mL/min for Pump 02 (aqueous solution). The flow rates correspond to a total flow rate (TFR) of 1 mL/min and aqueous-to-ethanol flow rate ratio (FRR) of 3.</li>
		<li>Assign Volume to any values above 5 mL. 
		NOTE: The targeted infusion volume is set greater than the loaded liquid volume considering the void volume of the tubing.</li>
		<li>Select INF (infusion) mode for both pumps.</li>
		<li>Press Set to confirm the settings.</li>
	</ol>
	</li>
</ol>
2. Prepare the LTSLs
<ol>
	<li>Prepare a LTSL10 or LTSL10-ICG lipid mixture (see Table 1).</li>
	<li>Withdraw 1 mL of lipid mixture and at least 3 mL of (NH4)2SO4 solution using two 5 mL Luer lock syringes.</li>
	<li>Install the two syringes onto the syringe pumps in the upright position by sliding the barrel flange of the syringe to the syringe retainer of the pump, and the plunger flange of the syringe to the pusher block of the pump (Figure 4).</li>
	<li>Wrap the end of the heating tape to the syringe with the aqueous solution. Wrap the other end of the heating tape and temperature probe of the thermostat around the syringe with the lipid solution. It is helpful to practice this step with empty syringes in place in order to ease the assembly process (Figure 5A).</li>
	<li>Connect the two syringes to the female Luer adaptors of the corresponding inlets of the SHM. Make sure the syringes containing the lipid mixture and (NH4)2SO4 solutions are connected ethanol inlet and aqueous inlet, respectively. Adjust the plunger position to remove air bubbles from the syringes (Figure 5B). 
	NOTE: Ensure the syringes are still securely positioned onto the syringe retainer of the pumps.</li>
	<li>Heat up the syringes to above 51 &#176;C using the heating tape using a 10 s heating session. Allow the thermostat to update the temperature of the syringes. Repeat this step in the following steps to maintain the temperature during the infusion. 
	CAUTION: Turn off the heating tape after 10 s to prevent temperature overshoot and allow the thermostat to update the actual temperature. The heating tape should also be handled with care as its temperature rises very quickly. Heating continuously may damage the equipment and syringes, due to the time delay of the thermostat for updating the measured temperature.</li>
	<li>Once the temperature is above 51 &#176;C, run the syringe pumps by pressing Run All in the pump control software (Figure 3).</li>
	<li>Ensure the fluid flow is free of air bubbles and any leakage. Dispose the initial volume (around 0.5 mL) of liquid from the outlet as waste. 
	NOTE: This initial waste volume is not definite and depends on the internal volume of the setup, which is the volume for fluid to travel from the syringes through the tubing and SHM to the outlet.</li>
	<li>Collect the rest of the liquid as liposome samples into a microcentrifuge tube or bijou vial.</li>
	<li>Pause/stop the infusion when the liquid in either of the syringes are almost empty. 
	NOTE: The pumps should be stopped manually, since the pumps may not accurately detect the position when the syringes are empty.</li>
	<li>Place the collected liposome solutions in a 60 &#176;C water bath to anneal for 1.5 h. 
	NOTE: This step is essential in enabling drug loading into the liposomes.</li>
	<li>Transfer the solutions to dialysis tubes. Dialyze the solutions against 1 L of 240 mM (NH4)2SO4 at 37 &#176;C for at least 4 h to obtain purified liposomes. 
	NOTE: The protocol can be paused here. Liposomes at this step are at 5 mM of phospholipid. Purified liposomes can be stored at 4 &#176;C.</li>
	<li>To clean the SHM for repeated use, flush the SHM sequentially with deionized water, ethanol and dry with nitrogen gas.</li>
</ol>
3. Remote loading of DOX into LTSLs by transmembrane pH gradient
<ol>
	<li>Exchange external buffer of LTSLs to HEPES-buffered saline (HBS) by using size exclusion chromatography (SEC) to establish a transmembrane pH gradient.
	<ol>
		<li>Add a total of 25 mL of HBS to the top of a SEC column to prepare the column. Allow all eluent to elute through the column and dispose the eluate.</li>
		<li>Add 1 mL of dialyzed liposomes, prepared from step 2.12, to the column and dispose the elute.</li>
		<li>Add 1.5 mL of HBS to the column and dispose the elute.</li>
		<li>Add 3 mL of HBS to the column and collect the 3 mL of elute. 
		NOTE: The protocol can be paused here. Liposomes are collected at this step and are at 1.67 mM of phospholipid. Buffer exchanged liposomes can be stored at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Incubate LTSLs with doxorubicin (DOX) and purify LTSLs.
	<ol>
		<li>Add DOX solution in 1:20 DOX-to-phospholipid molar ratio into 1 mL of buffer-exchanged liposomes solution (1.67 mmol) contained in a bijou vial. This can be achieved by adding 48.4 &#181;L of 1 mg/mL DOX solution (83.4 &#181;mol).</li>
		<li>Place the bijou vial in a 37 &#176;C water bath for 1.5 h to allow DOX loading into the liposomes.</li>
		<li>Mix 10 &#181;L of the liposomes with 170 &#181;L of HBS and 20 &#181;L of 1% (v/v) Triton X-100 solution in a black 96-well plate. Repeat for three wells. These wells correspond to the &#34;before purification&#34; DOX content.</li>
		<li>In case of preparing LTSL10-ICG, mix 40 &#181;L of the liposomes with 160 &#181;L of DMSO in a clear 96-well plate. Repeat for three wells. These wells correspond to the &#34;before purification&#34; ICG content.</li>
		<li>Purify the liposome solution as described in step 3.1. 
		NOTE: To reuse the column for future purification, clean the column from free DOX by first adding 1 mL of diluted 0.5 M NaOH solution before performing step 3.1.1. Free DOX in red will turn violet-blue and elute through the column quickly.</li>
		<li>Mix 30 &#181;L of the purified liposomes solution with 150 &#181;L of HBS and 20 &#181;L of 1% (v/v) Triton X-100 solution in a black 96-well plate. Repeat for three wells. These wells correspond to the &#34;after purification&#34; DOX content.</li>
		<li>In case of LTSL10-ICG, mix 40 &#181;L of the purified liposomes solution with 160 &#181;L of DMSO in a clear 96-well plate. Repeat for three wells. These wells correspond to the &#34;after purification&#34; ICG content.</li>
		<li>Measure the DOX fluorescence intensity of the wells before (step 3.2.3) and after (step 3.2.5) purification, using a microplate reader (&#955;ex = 485 nm, &#955;em = 590 nm).</li>
		<li>Calculate the encapsulation efficiency of DOX (DOX EE) by taking the ratio of the fluorescence intensities before and after purification. 
		<img alt="Equation 1" src="/files/ftp_upload/60907/60907eq1.jpg" /></li>
		<li>Measure the ICG absorbance of the wells before and after purification, using a microplate reader (600 to 1000 nm).</li>
		<li>Calculate the encapsulation efficiency of ICG (ICG EE) by taking the ratio of the absorbance at 792 nm before and after purification, taking into account the dilution factor (3 times) during the purification. 
		<img alt="Equation 2" src="/files/ftp_upload/60907/60907eq2a.jpg" /></li>
	</ol>
	</li>
</ol>
4. Dynamic Light Scattering (DLS)
<ol>
	<li>Add 50 &#181;L of liposomes solution (step 2.12) to 450 &#181;L of deionized water.</li>
	<li>Place the cuvette inside the DLS instrument and perform the measurement according to the manufacturer&#39;s instructions.</li>
	<li>Record the mean Z-average diameter and dispersity of three measurements for each sample.</li>
</ol>
5. Differential scanning calorimetry (DSC)
<ol>
	<li>Concentrate 1 mL of the liposomes samples (step 2.12) with a centrifugal filter unit to 0.5 mL (final lipid concentration of 10 mM). Using a fixed-angle rotor, spin at 7500 x g for approximately 15 min.</li>
	<li>Transfer 20 &#181;L of (NH4)2SO4 solution and liposomes samples to two respective DSC pans. Seal the pans with DSC hermetic lids using the DSC sample press kit.</li>
	<li>Measure the sample from 30 &#176;C to 60 &#176;C at a heating rate of 1 &#176;C/min using a differential scanning calorimeter.</li>
	<li>Analyze the data with appropriate software. Take the phase transition temperature (Tm) as the onset of the phase transition (melting peak), which is measured by the x-intercept of the tangent of the point of maximum slope.</li>
</ol>
6. Doxorubicin release
<ol>
	<li>Preheat HBS at designated temperature (37 or 42 &#176;C) using a water bath. Prepare an ice water bath for quenching the samples.</li>
	<li>Add 100 &#181;L of purified DOX-loaded liposomes (step 3.2.5) into 1.9 mL of HBS in a microcentrifuge tube. Place the tube into the water bath of the designated temperature.</li>
	<li>Withdraw immediately 200 &#181;L of samples from the tube and quickly place it in the ice water bath to quench any subsequent drug release. This sample corresponds to the initial (t = 0) time point.</li>
	<li>Withdraw 200 &#181;L of samples at subsequent time points (t = 5, 10, 15, 30, 60 min) and quickly place it in the ice water bath to quench any drug release.</li>
	<li>Mix 50 &#181;L of sample of each time point with 150 &#181;L of HBS in a black 96-well plate. Measure the DOX fluorescence intensity using a plate reader.</li>
	<li>Add 20 &#181;L of 1% (v/v) Triton X-100 into random selected wells prepared in step 6.5. Measure the DOX fluorescence intensity of these wells using a plate reader. These values correspond to the fully released (t = &#8734;; 100% release) time point.</li>
	<li>Calculate and plot the percentage of DOX released by interpolating the fluorescence intensity of each time points (I(t)), compared to the initial (I(0)), and fully released (I(&#8734;)), value. 
	<img alt="Equation 3" src="/files/ftp_upload/60907/60907eq3.jpg" /></li>
</ol>
7. Laser Heating and Triggered Release
<ol>
	<li>Set water bath temperature to 37 &#176;C and allow the temperature to stabilize.</li>
	<li>Add 200 &#181;L of DOX loaded LTSL10-ICG ([ICG] = 10 &#181;g/mL) to a clear 96-well plate, then place it in the water bath, keep the bottom immersed in water.</li>
	<li>Set the current of the laser system to 2.27 A. Place the collimator of the laser system at 5 cm vertically above the surface of the 96-well plate, which corresponds to an energy flux of 0.5 W/cm2 [Figure 6]. 
	CAUTION: The laser system should be operated in compliance with relevant laser safety measures.</li>
	<li>Switch on the laser and monitor the temperature every minute using a fiber optic temperature probe.</li>
	<li>At 5 and 10 min, withdraw 10 &#181;L of laser-irradiated liposomes from the clear 96-well plate at and mix with 190 &#181;L of HBS for three wells in a black 96-well plate.</li>
	<li>Mix 10 &#181;L of the liposomes with 170 &#181;L of HBS and 20 &#181;L of 1% (v/v) Triton X-100 solution for three wells in a black 96-well plate. These wells correspond to the &#34;100% released&#34; DOX content. Measure the DOX fluorescent intensity and calculate the DOX release as described in step 6.7.</li>
</ol>

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The authors have nothing to disclose.

The presented protocol describes the preparation of low temperature-sensitive liposomes (LTSLs) using a staggered herringbone micromixer (SHM). The LTSL10 formulation enables temperature-triggered burst release of doxorubicin within 5 minutes at a clinically attainable hyperthermic temperature of 42 &#176;C. Indocyanine green (ICG) can also be co-loaded for photothermal heating triggered the release of DOX. The method relies on: (i) self-assembly of phospholipids into liposomes under a homogenized solvent environment pro...

LTSL formulation is a clinically relevant liposomal product that has been developed to deliver the chemotherapeutic drug doxorubicin (DOX) and allows efficient burst drug release at clinically attainable mild hyperthermia (T &#8776; 41 &#176;C)1. The LTSL formulation consists of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), the lysolipid 1-stearoyl-2-hydroxy-sn-glycero-3-phosphatidylcholine (MSPC; M stands for &#34;mono&#34;) and PEGylated lipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). Upon reaching the phase transition temperature (Tm &#8776; 41 &#176;C), the lysolipid and DSPE-PEG2000 together facilitate the formation of membrane pores, resulting in a burst release of the drug2. The preparation of LTSLs primarily uses a bulk top-down approach, namely lipid film hydration and extrusion. It remains challenging to reproducibly prepare large batches with identical properties and in sufficient quantities for clinical applications3.
Microfluidics is an emerging technique for preparing liposomes, offering tunable nanoparticle size, reproducibility, and scalability3. Once the manufacturing parameters are optimized, the throughput could be scaled-up by parallelization, with properties identical to those prepared at bench scale3,4,5. A major advantage of microfluidics over conventional bulk techniques is the ability to handle small liquid volumes with high controllability in space and time through miniaturization, allowing faster optimization, while operating in a continuous and automated manner6. Production of liposomes with microfluidic devices is achieved by a bottom-up nanoprecipitation approach, which is more time and energy efficient because homogenization processes such as extrusion and sonication are unnecessary7. Typically, an organic solution (e.g. ethanol) of lipids (and hydrophobic payload) is mixed with a miscible non-solvent (e.g. water and hydrophilic payload). As the organic solvent mixes with the non-solvent, the solubility for the lipids is reduced. The lipid concentration eventually reaches a critical concentration at which the precipitation process is triggered7. Nanoprecipitates of lipids eventually grow in size and close into a liposome. The main factors governing the size and homogeneity of the liposomes are the ratio between the non-solvent and solvent (i.e. aqueous-to-organic flow rate ratio; FRR) and the homogeneity of the solvent environment during the self-assembly of lipids into liposomes8.
Efficient fluid mixing in microfluidics is therefore essential to the preparation of homogeneous liposomes, and various designs of mixers have been employed in different applications9. Staggered herringbone micromixer (SHM) represents one of the new generations of passive mixers, which enables high throughput (in range of mL/min) with a low dilution factor. This is superior to traditional microfluidic hydrodynamic mixing devices8,10. The SHM has patterned herringbone grooves, which rapidly mix fluids by chaotic advection9,11. The short mixing timescale of SHM (&#60; 5 ms, less than the typical aggregation time scale of 10&#8211;100 ms) allows lipid self-assembly to occur in a homogenous solvent environment, producing nanoparticles with uniform size distribution3,12.
The preparation of LTSLs with microfluidics is, however, not as straightforward compared to conventional liposomal formulations due to the lack of cholesterol8, without which lipid bilayers are susceptible to ethanol-induced interdigitation13,14,15. Until now, the effect of residual ethanol presents during the microfluidic production of liposomes has not been well understood. The majority of the reported formulations are inherently resistant to interdigitation (containing cholesterol or unsaturated lipids)16, which unlike LTSLs are both saturated and cholesterol-free.
The protocol presented herein uses SHM to prepare LTSLs for temperature triggered-release drug delivery. In the presented method, we ensured the microfluidic-prepared LTSLs are nano-sized (100 nm) and uniform (dispersity &#60; 0.2) by dynamic light scattering (DLS). Furthermore, we encapsulated DOX using the transmembrane ammonium sulfate gradient method (also known as remote loading)17 as a validation of the integrity of the LTSL lipid bilayer. Remote loading of DOX requires the liposome to maintain a pH-gradient in order to achieve high encapsulation efficiency (EE), which is unlikely to happen without an intact lipid bilayer. In this presented method, distinctive from typical microfluidic liposome preparation protocols, an annealing step is required before the ethanol is removed to enable the remote loading capability; i.e. to restore the integrity of the lipid bilayer.
As mentioned previously, hydrophilic and hydrophobic payloads can also be introduced to the initial solutions for the simultaneous encapsulation of payloads during the formation of LTSLs. As a proof-of-concept, indocyanine green (ICG), an FDA-approved near-infrared fluorescent dye, which is also a promising photothermal agent, is introduced to the initial lipid mixture and successfully co-loaded into the LTSLs. An 808 nm laser is used to irradiate the DOX/ICG-loaded LTSLs and successfully induce photothermal heating-triggered burst release of DOX within 5 min.
All the instruments and materials are commercially available, ready-to-use, and without the need for customization. Since all the parameters for formulating LTSLs have been optimized, following this protocol, researchers with no prior knowledge of microfluidics could also prepare the LTSLs, which serves as the basis of a thermosensitive drug delivery system.

<table><tbody><tr><td>1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)</td><td>Lipoid</td><td>PC 16:0/16:0 (DPPC)</td><td></td></tr><tr><td>1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000)</td><td>Lipoid</td><td>PE 18:0/18:0-PEG 2000&lt;br/&gt; (MPEG 2000-DSPE)</td><td></td></tr><tr><td>1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (MSPC)</td><td>Avanti Polar Lipid</td><td>855775P-500MG</td><td>Distributed by Sigma-Adrich; also known as Lyso 16:0 PC&lt;br/&gt; (Not to be confused with 14:0/18:0 PC, which is also termed MSPC)</td></tr><tr><td>4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)</td><td>Sigma-Aldrich</td><td>H3375-100G</td><td></td></tr><tr><td>Adapters, Female Luer Lock to 1/4&quot;-28UNF</td><td>IDEX Health &amp;amp; Science</td><td>P-624</td><td>Requires 2 units. For the inlets</td></tr><tr><td>Adapters, Union Assembly, 1/4&quot;-28UNF</td><td>IDEX Health &amp;amp; Science</td><td>P-630</td><td>Requires 2 units. (One unit included 2 nuts and 2 ferrules)</td></tr><tr><td>Ammonium Sulfate ((NH4)2SO4)</td><td>Sigma-Aldrich</td><td>31119-1KG-M</td><td></td></tr><tr><td>Bijou vial</td><td>VWR</td><td>216-0980</td><td>7 mL, clear, polystyrene vial</td></tr><tr><td>Centrifugal Filter Unit</td><td>Sigma-Aldrich</td><td>UFC801008</td><td>10 kDa MWCO, Amicon Ultra-4 Centrifugal Filter Unit</td></tr><tr><td>Centrifuge</td><td>ThermoFisher Scientific</td><td>Heraeus Megafuge 8R</td><td>With HIGHConic III Fixed Angle Rotor</td></tr><tr><td>Cuvette</td><td>Fisher Scientific</td><td>11602609</td><td>Disposable polystyrene cuvette, low volume, for DLS measurement</td></tr><tr><td>Dialysis Kit - Pur-A-Lyzer Maxi</td><td>Sigma-Aldrich</td><td>PURX12015-1KT</td><td>12-14 kDa MWCO</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>34943-1L-M</td><td></td></tr><tr><td>DLS Instrument</td><td>Malvern Panalytical</td><td>Zetasizer Nano ZS90</td><td></td></tr><tr><td>Doxorubicin Hydrochloride (DOX)</td><td>Apollo Scientific</td><td>BID0120</td><td></td></tr><tr><td>DSC Instrument</td><td>TA Instruments</td><td>TA Q200 DSC</td><td></td></tr><tr><td>DSC Tzero Hermetic Lids</td><td>TA Instruments</td><td>901684.901</td><td>For DSC measurement</td></tr><tr><td>DSC Tzero Pans</td><td>TA Instruments</td><td>901683.901</td><td>For DSC measurement</td></tr><tr><td>DSC Tzero Sample Press Kit</td><td>TA Instruments</td><td>901600.901</td><td>For DSC measurement</td></tr><tr><td>Ethanol</td><td>VWR</td><td>20821.330</td><td>Absolute, &amp;ge;99.8%</td></tr><tr><td>FC-808 Fibre Coupled Laser System</td><td>CNI Optoelectronics Tech</td><td>FC-808-8W-181315</td><td>FOC-01-B Fiber Collimator included.</td></tr><tr><td>Ferrule, 1/4&quot;-28UNF to 1/16&quot; OD</td><td>IDEX Health &amp;amp; Science</td><td>P-200</td><td>For the outlet</td></tr><tr><td>Fibre Optic Temperature Probe</td><td>Osensa</td><td>PRB-G40</td><td></td></tr><tr><td>Glass Staggered Herringbone Micromixer (SHM)</td><td>Darwin Microfluidics</td><td>Herringbone Mixer - Glass Chip</td><td></td></tr><tr><td>Heating Tape</td><td>Omega</td><td>DHT052020LD</td><td>Can be replaced by other syringe heater such as &quot;HTC&quot; or &quot;SRT series&quot; for slower heating. Manual wiring to a 3-pin plug required for 240V models</td></tr><tr><td>Indocyanine Green</td><td>Adooq</td><td>A10473-100</td><td>Distributed by Bioquote Limited (U.K.)</td></tr><tr><td>Luer-lock Syringe, 5 mL</td><td>VWR</td><td>613-2043</td><td>Hanke Sass Wolf SOFT-JECT 3-piece syringes, O.D. 12.45 mm</td></tr><tr><td>Microplate Reader</td><td>BMG Labtech</td><td>FLUOstar Omega</td><td>Installed with 485 nm (exictation) and 590 nm (emission) filters</td></tr><tr><td>Microplate, 96-well, Black, Flat-bottom</td><td>ThermoFisher Scientific</td><td>611F96BK</td><td>For fluorescence measurement in microplate reader</td></tr><tr><td>Microplate, 96-well, Clear, Flat-bottom</td><td>Grenier</td><td>655101</td><td>For absorbance measurement microplate reader</td></tr><tr><td>Nut, 1/4&quot;-28UNF to 1/16&quot; OD</td><td>IDEX Health &amp;amp; Science</td><td>P-245</td><td>For the outlet</td></tr><tr><td>PC to Pump Network Cable for Aladdin, 7ft</td><td>World Precision Instruments</td><td>NE-PC7</td><td>Optional: Syringe pumps can be operated manually</td></tr><tr><td>Pump control software - SyringePumpPro Software License for 2</td><td>World Precision Instruments</td><td>SYRINGE-PUMP-PRO-02</td><td>Optional: Syringe pumps can be operated manually</td></tr><tr><td>Pump to Pump Network Cable for Aladdin, 7 ft</td><td>World Precision Instruments</td><td>NE-NET7</td><td>Optional: Syringe pumps can be operated manually</td></tr><tr><td>Size exclusion chromatography (SEC) column</td><td>GE Life Science</td><td>17085101</td><td>Sephadex G-25 resin in PD-10 Desalting Columns</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>31434-1KG-M</td><td></td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>S5881-500G</td><td></td></tr><tr><td>Syringe Pumps &amp;amp; Cable (DUAL-PUMP-NE-1000)</td><td>World Precision Instruments</td><td>ALADDIN2-220/AL1000-220</td><td></td></tr><tr><td>Thermostat Temperature Controller</td><td>Inkbird</td><td>ITC-308</td><td>Can be replaced by other syringe heater kit/thermostat</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-100ML</td><td></td></tr><tr><td>Tubing, ETFE (1/16&quot; OD)</td><td>IDEX Health &amp;amp; Science</td><td>1516</td><td></td></tr><tr><td>USB To RS-232 Converter</td><td>World Precision Instruments</td><td>CBL-USB-232</td><td>Optional: For computer without RS-232 port</td></tr><tr><td>Water Bath</td><td>Grant Instruments Ltd.</td><td>JB Nova 12</td><td></td></tr></tbody></table>

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 preparation of LTSLs by microfluidics requires the lipid composition of DPPC/MSPC/DSPE-PEG2000 (80/10/10, molar ratio; LTSL10). Figure 7A (left) shows the appearance of as-prepared LTSL10 from step 2.9, as a clear and non-viscous liquid. LTSL10 formulation is developed from the conventional formulation, LTSL4 (DPPC/MSPC/DSPE-PEG2000, 86/10/4, molar ratio) since LTSL4 forms a gel-like viscous sample, as indicated by t...

Watch this Scientific Journal Video about Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes at JoVE.com

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

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 ...

microfluidic-production-lysolipid-containing-temperature-sensitive

Research

JoVE Journal

Bioengineering

8.9K Views.  Queen's University Belfast. 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.

Video: Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

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 ...

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug discovery because they are the targets of more than half of all drugs. An obstacle to conducting biochemical, biophysical, and structural studies of membrane proteins as well as antibody development has been the difficulty in producing large amounts of high-quality membrane protein with correct conformation and activity. Here we describe a &#8220;bilayer-dialysis method&#8221; using a wheat germ cell-free system, liposomes, and dialysis cups to efficiently synthesize membrane proteins and prepare purified proteoliposomes in a short time with a high success rate. Membrane proteins can be produced as much as in several milligrams, such as GPCRs, ion channels, transporters, and tetraspanins. This cell-free method contributes to reducing the time, cost and effort for preparing high-quality proteoliposomes, and provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...

Biochemistry

Synthesis of Gold Nanoparticle Integrated Photo-responsive Liposomes and Measurement of Their Microbubble Cavitation upon Pulse Laser Excitation

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control over the drug release. However, most of the relevant technologies are still in the development process and are unprocurable by clinics. Here, we describe a facile fabrication of these photo-responsive NPs with commercially available gold NPs and thermo-responsive liposomes. Calcein is used as a model drug to evaluate the encapsulation efficiency and the release kinetic profile upon heat/light stimulation. Finally, we show that this photo-triggered release is due to the membrane disruption caused by microbubble cavitation, which can be measured with hydrophone.

This protocol describes a simple preparation method for gold nanoparticle integrated photo-responsive liposomes with the commercially available materials. It also shows how to measure the microbubble cavitation process of the synthesized liposomes upon the treatment of pulsed laser.

Photo-responsive nanoparticles (NPs) have received considerable attention because of their potential in providing spatial, temporal, and dosage control ...

Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 &#176;C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of &#60; 1 hr and achieved a BLM formation rate of ~80%. Moreover, ion channel experiments with gramicidin A demonstrated the feasibility of the membrane system.

We demonstrate a storable, transportable lipid bilayer formation system. A lipid bilayer membrane can be formed within 1 hr with over 80% success rate when a frozen membrane precursor is brought to ambient temperature. This system will reduce laborious processes and expertise associated with ion channels.

An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for ...

Production of siRNA-Loaded Lipid Nanoparticles using a Microfluidic Device

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based RNA delivery systems, namely, RNA-loaded LNPs have attracted attention for RNA therapy. In particular, mRNA-loaded LNP vaccines were approved to prevent COVID-19, thereby leading to the paradigm shift toward the development of next-generation nanomedicines. For the LNP-based nanomedicines, the LNP size is a significant factor in controlling the LNP biodistribution and LNP performance. Therefore, a precise LNP size control technique is indispensable for the LNP production process. Here, we report a protocol for size controlled LNP production using a microfluidic device, named iLiNP. siRNA loaded LNPs are also produced using the iLiNP device and evaluated by in vitro experiment. Representative results are shown for the LNP size, including siRNA-loaded LNPs, Z-potential, siRNA encapsulation efficiency, cytotoxicity, and target gene silencing activity.

Microfluidic-based lipid nanoparticle (LNP) production methods have attracted attention in drug delivery systems (DDSs), including RNA delivery. This protocol describes the fabrication, LNP (siRNA-loaded LNP) production, and LNP evaluation processes using our original microfluidic device named iLiNP.

The development of functional lipid nanoparticles (LNPs) is one of the major challenges in the field of drug delivery systems (DDS). Recently, LNP-based ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

This protocol describes the fabrication of liposomes and how these can be immobilized on a surface and imaged individually in a massive parallel manner using fluorescence microscopy. This allows for the quantification of the size and compositional inhomogeneity between single liposomes of the population.

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

Biology

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are simplistic cellular mimics composed of an aqueous interior surrounded by a lipid bilayer; they are valuable in designing synthetic cells and understanding the fundamentals of biological cells in an in vitro fashion and are important for applied sciences, such as cargo delivery for therapeutic applications. This article describes a detailed working protocol for an on-chip microfluidic technique, octanol-assisted liposome assembly (OLA), to produce monodispersed, micron-sized, biocompatible liposomes. OLA functions similarly to bubble blowing, where an inner aqueous (IA) phase and a surrounding lipid-carrying 1-octanol phase are pinched off by surfactant-containing outer fluid streams. This readily generates double-emulsion droplets with protruding octanol pockets. As the lipid bilayer assembles at the droplet interface, the pocket spontaneously detaches to give rise to a unilamellar liposome that is ready for further manipulation and experimentation. OLA provides several advantages, such as steady liposome generation (&#62;10 Hz), efficient encapsulation of biomaterials, and monodispersed liposome populations, and requires very small sample volumes (~50 &#181;L), which can be crucial when working with precious biologicals. The study includes details on microfabrication, soft-lithography, and surface passivation, which are needed to establish OLA technology in the lab. A proof-of-principle synthetic biology application is also shown by inducing the formation of biomolecular condensates inside the liposomes via transmembrane proton flux. It is anticipated that this accompanying video protocol will facilitate the readers to establish and troubleshoot OLA in their labs.

The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.