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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 
			(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 
			(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&#34;-28UNF</td>
			<td>IDEX Health &#38; Science</td>
			<td>P-624</td>
			<td>Requires 2 units. For the inlets</td>
		</tr>
		<tr>
			<td>Adapters, Union Assembly, 1/4&#34;-28UNF</td>
			<td>IDEX Health &#38; 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, &#8805;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&#34;-28UNF to 1/16&#34; OD</td>
			<td>IDEX Health &#38; 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 &#34;HTC&#34; or &#34;SRT series&#34; 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&#34;-28UNF to 1/16&#34; OD</td>
			<td>IDEX Health &#38; 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 &#38; 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&#34; OD)</td>
			<td>IDEX Health &#38; 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

A Microfluidic Technique to Probe Cell Deformability

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an efficient manner. Typically, data for ~102 cells can be acquired within a 1 hr experiment. An automated image analysis program enables efficient post-experiment analysis of image data, enabling processing to be complete within a few hours. Our device geometry is unique in that cells must deform through a series of micron-scale constrictions, thereby enabling the initial deformation and time-dependent relaxation of individual cells to be assayed. The applicability of this method to human promyelocytic leukemia (HL-60) cells is demonstrated. Driving cells to deform through micron-scale constrictions using pressure-driven flow, we observe that human promyelocytic (HL-60) cells momentarily occlude the first constriction for a median time of 9.3 msec before passaging more quickly through the subsequent constrictions with a median transit time of 4.0 msec per constriction. By contrast, all-trans retinoic acid-treated (neutrophil-type) HL-60 cells occlude the first constriction for only 4.3 msec before passaging through the subsequent constrictions with a median transit time of 3.3 msec. This method can provide insight into the viscoelastic nature of cells, and ultimately reveal the molecular origins of this behavior.

We demonstrate a microfluidics-based assay to measure the timescale for cells to transit through a sequence of micron-scale constrictions.

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

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 ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

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 ...

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 ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...

Preparation, Administration, and Assessment of In Vivo Tissue-Specific Cellular Uptake of Fluorescent Dye-Labeled Liposomes

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome formulation, liposomes may be preferentially taken up by different cell types in the body. This may influence the efficacy of the therapeutic particle as progression of different diseases is tissue- and cell-type-specific. In this protocol, we present one method for synthesizing and fluorescently labeling liposomes using DSPC, cholesterol, and PEG-2000 DSPE and the lipid dye DiD as a fluorescent label. This protocol also presents an approach for delivering liposomes in vivo and assessing cell-specific uptake of liposomes using flow cytometry. This approach can be used to determine the types of cells that take up liposomes and quantify the distribution and proportion of liposome-uptake across cell types and tissues. While not mentioned in this protocol, additional assays such as immunofluorescence and single-cell fluorescence imaging on a cytometer will strengthen any findings or conclusions made as they permit assessment of intracellular staining. Protocols may also need to be adapted depending on the tissue(s) of interest.

The goal of this protocol is to synthesize fluorescently-labeled liposomes and use flow cytometry to identify in vivo localization of liposomes at a cellular level.

There is a growing interest in using liposomes to deliver compounds in vivo particularly for targeted treatment approaches. Depending on the liposome ...

Microfluidic Model to Mimic Initial Event of Neovascularization

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the initial stage varies dramatically from the following process of neovascularization. Although there are plenty of models to simulate different stages of neovascularization, an in vitro 3D model that capitulates the initial process of neovascularization under the corresponding stimulations of normal vasculature microenvironments is still lacking. Here, we reconstructed an in vitro 3D model that mimics the initial event of neovascularization (MIEN). The MIEN model contains a microfluidic sprouting chip and an automatic control, highly efficient circulation system. A functional, perfusable microchannel coated with endothelium was formed and the process of sprouting was simulated in the microfluidic sprouting chip. The initially physiological microenvironment of neovascularization was recapitulated with the microfluidic control system, by which ECs would be exposed to high luminal shear stress, physiological transendothelial flow, and various vascular endothelial growth factor (VEGF) distributions simultaneously. The MIEN model can be readily applied to the study of neovascularization mechanism and holds a potential promise as a low-cost platform for drug screening and toxicology applications.

Here, we provide a microfluidic chip and an automatically controlled, highly efficient circulation microfluidic system that recapitulates the initial microenvironment of neovascularization, allowing endothelial cells (ECs) to be stimulated by high luminal shear stress, physiological level of transendothelial flow, and various vascular endothelial growth factor (VEGF) distribution simultaneously.

Neovascularization is usually initialized from an existing normal vasculature and the biomechanical microenvironment of endothelial cells (ECs) in the ...