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 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 (> 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 °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.
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 ≈ 41 °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 "mono") and PEGylated lipid 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000). Upon reaching the phase transition temperature (Tm ≈ 41 °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 (< 5 ms, less than the typical aggregation time scale of 10–100 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 < 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.
1. Equipment setup
2. Prepare the LTSLs
3. Remote loading of DOX into LTSLs by transmembrane pH gradient
4. Dynamic Light Scattering (DLS)
5. Differential scanning calorimetry (DSC)
6. Doxorubicin release
7. Laser Heating and Triggered Release
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 the large amount of air bubbles trapped in the sample (Figure 7A; right).
DLS measurement of LTSL10 (Figure 7B, red) showed that the Z-Average diameter and dispersity of LTSL10 were 95.28 ± 7.32 nm and 0.100 ± 0.022, respectively, indicating the success of the experiment. Figure 7B (gray) also shows a suboptimal sample, which was prepared at 20 °C, where larger and more dispersed liposomes were obtained.
Figure 7C shows that the DOX EE of LTSL10. DOX EE should usually be around 80%. LTSLs prepared by the conventional method of lipid film hydration with extrusion (LF) are included for comparison, prepared as described elsewhere18. DOX EE of LTSL4 (LF) and LTSL10 (LF) showed decent DOX loading of around 70% and 50%, respectively. Annealing of as-prepared LTSL10 (step 2.11) is essential to enable DOX loading. In the absence of the annealing step, low DOX EE (< 20%) was persistent, regardless of incubation temperature (20 °C to 42 °C) and duration (1 to 24 h). This indicated the failure of LTSL10 to maintain a transmembrane pH gradient, where DOX was instead loaded passively or by adsorption. By annealing the as-prepared LTSL10, DOX EE increased significantly to a mean of 85%, indicating the success of the remote loading of DOX and the presence of the transmembrane pH gradient.
Figure 7D shows the DOX release profile of LTSL10. At 37 °C, the release of encapsulated DOX over 60 min was about 10%. In contrast, at 42 °C, all of the encapsulated DOX was released within 5 minutes, demonstrating the temperature-sensitivity of LTSL10. Similar results were observed with LTSL10 (LF) as a control.
Figure 8 shows the phase transition temperature (Tm) of LTSL10 characterized using differential scanning calorimetry (DSC). Dotted lines as tangent of the point of maximum slope, are added as a visual aid of the onset phase transition temperature (x-intercept of the tangent line). LTSL10 has a relatively broad phase transition with onset at 41.6 °C and peak at 42.6 °C. Similar results were observed with LTSL10 (LF), suggesting a minor difference between the preparation techniques. As a comparison, LTSL4 (LF) has a lower and sharper phase transition, in agreement with the literature1.
Figure 9 shows the characterization of LTSL10-ICG. Effect of initial ICG concentration on size (Figure 9A) and loading efficiencies of DOX and ICG (Figure 9B) are categorized into three concentration ranges. At low ICG concentration (ICG-to-lipid molar ratio of 0.003; initial concentration of 60 µM ICG and 20 mM lipid), Z-average, dispersity and DOX EE were similar to LTSL10 without ICG loading; ICG EE was around 75%. The efficient co-loading of DOX and ICG into LTSL10 can be achieved at this ICG concentration. At intermediate ICG concentrations, while the size and dispersity of the samples were satisfactory, both DOX and ICG EE were reduced. In particular, the decrease in DOX EE indicated the disruption of the liposomal membrane and thus, the pH-gradient. At high ICG concentrations, samples were again gelled; DOX and ICG EE were both significantly decreased.
LTSL10-ICG was irradiated with near-infrared laser (section 7) to induce photothermal heating and triggered the release of DOX (Figure 10). Upon laser irradiation, the sample first heated up to 49.7 °C with a gradual reduction of temperature. Subsequent laser irradiation increased the temperature to 36.7 °C. Quantification of the released DOX indicated that a complete burst release of encapsulated DOX was achieved after the first heating cycle. This was as expected since the temperature reached above 42 °C, in agreement with the DOX release profile shown in Figure 7D. In contrast, LTSL10 without ICG cannot provide photothermal heating, and thus did not release DOX upon laser irradiation.
Figure 1: Photograph of the syringe pumps setup. The "To Network" port of the master pump (Pump 01) is attached to the "To Computer" of the secondary pump (Pump 02; yellow); the "To Computer" port of the master pump is attached to the RS-232 port of the computer (blue). Please click here to view a larger version of this figure.
Figure 2: Photograph of the SHM setup. (A) Assembled view of the SHM setup. (B) Exploded view of the SHM setup. Inlets and outlets of the SHM are connected to tubing using a nut and ferrule. The tubing of both inlets is extended by a longer tubing with nut and ferrule on each end, terminated by a female Luer adapter using a union assembly. Please click here to view a larger version of this figure.
Figure 3: Interface of the pump control software. The two syringe pumps should be detected automatically upon initiating the software; otherwise, click Pumps on the top left corner and Search for pumps. Parameters to be configured are highlighted in red boxes. Please click here to view a larger version of this figure.
Figure 4: Photograph of the syringe pumps and installation of a syringe. (A) Syringe retainer bracket and syringe retainer thumbscrew (2, one on each side) of the syringe pump (yellow box). Pusher block, adjustment thumbscrew, and drive-nut button (white button on the left) of the syringe pump (green box). (B) Position of an installed syringe on the syringe pump. Please click here to view a larger version of this figure.
Figure 5: Photograph of the assembly of syringes, heating element and SHM. (A) Assembly of syringes, heating tape, and thermostat. (B) Assembly of syringes, heating tape, thermostat, and SHM. Please click here to view a larger version of this figure.
Figure 6: Photograph of the laser setup. (A) Photograph of the fiber coupled laser system during operation. (B) Position of the collimator 5 cm above the 96-well plate. Please click here to view a larger version of this figure.
Figure 7: Characterization of LTSL10. (A) Photograph of (left) LTSL10 and (right) LTSL4, before dialysis. LTSL10 appeared as clear and non-viscous liquid, while LTSL4 was gel-like and viscous. (B) Z-average diameter and dispersity of 10 mM of LTSL10 prepared at 20 and 51 °C. Solid bars and open circles (○) indicate Z-average diameter and dispersity, respectively. (C) DOX EE of LTSL4 (LF; white), LTSL10 (LF; white), and LTSL10 before (gray) and after annealing (red). (D) DOX release of LTSL10 (circle) and LTSL10 (LF; cross) at 37 °C (black) and 42 °C (red). Data represent mean ± SD of at least three independent experiments. ***p < 0.001; two-tailed unpaired t-tests. Please click here to view a larger version of this figure.
Figure 8: Thermal properties of LTSL10. Thermographs of LTSL4 (LF), LTSL10 (LF), and LTSL10 characterized by DSC. Dotted lines are added as a visual aid of the onset phase transition temperature. Data represent the mean of at least three independent experiments. Please click here to view a larger version of this figure.
Figure 9: Characterization of LTSL10-ICG. (A) Z-average diameter (red) and dispersity (blue) of ICG-loaded LTSL10. (B) DOX EE (red) and ICG EE (green) of ICG-loaded LTSL10. Data represent the mean ± SD of at least three independent experiments. Please click here to view a larger version of this figure.
Figure 10: Laser-induced photothermal heating triggered the release of DOX -loaded LTSL10 and LTSL10-ICG. (A) The temperature of irradiated samples and (B) DOX release of DOX-loaded LTSL10 (blue) and LTSL10-ICG (red) during the laser-induced photothermal heating. Data represent the mean ± SD of at least three independent experiments. Please click here to view a larger version of this figure.
Table 1: Lipid mixtures, buffers, and stock solutions.
20 mM LTSL10 lipid mixture (DPPC/MSPC/DSPE-PEG2000; 80/10/10, molar ratio) |
For 1 mL of 20 mM LTSL10 lipid mixture: Dissolve 11.75 mg of DPPC with 210 µL of MSPC ethanol stock (5 mg/mL), 558 µL DSPE-PEG2000 ethanol stock (10 mg/mL), and 232 µL of ethanol. Alternatively, equivalent amount of MSPC (1.05 mg) and DSPE-PEG2000 (5.58 mg) can be added as powder. |
20 mM LTSL10-ICG lipid mixture (DPPC/MSPC/DSPE-PEG2000; 80/10/10, molar ratio; ICG-to-lipid molar ratio = 0.003) |
For 1 mL of 20 mM ICG-loaded LTSL10 lipid mixture: Dissolve 11.75 mg of DPPC with 210 µL of MSPC ethanol stock (5 mg/mL), 558 µL DSPE-PEG2000 ethanol stock (10 mg/mL), and 46.5 µL of ICG ethanol stock (1 mg/mL) and 185.5 µL of ethanol. Alternatively, equivalent amount of MSPC (1.05 mg) and DSPE-PEG2000 (5.58 mg) can be added as powder. Contains 60 µM of ICG and 20 mM of lipid. |
240 mM Ammonium sulfate solution (NH4)2SO4, pH 5.4 | Dissolve 31.71 g of (NH4)2SO4 per L of deionized water. The pH of the solution is natively 5.4, additional pH adjustment is not required. |
Doxorubicin solution (DOX), 1 mg/mL | Dissolve 1 mg of DOX per mL of deionized water. |
DSPE-PEG2000 ethanol stock, 10 mg/mL | Dissolve 10 mg of DSPE-PEG2000 per mL of ethanol. |
HEPES-buffered saline (HBS), pH 7.4 | Dissolve 8.0 g of NaCl and 4.766 g of HEPES per L of deionized water. Adjust pH to 7.4 with 2.5 M NaOH solution. |
ICG ethanol stock, 1 mg/mL | Dissolve 1 mg of ICG per mL of ethanol. |
MSPC ethanol stock, 5 mg/mL | Dissolve 5 mg of DPPC per mL of ethanol. |
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 °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 provided by the rapid, chaotic mixing of ethanol and ammonium sulfate solution in the SHM11; (ii) annealing of the liposomes to preserve the integrity of the lipid bilayer essential for DOX loading; and (iii) remote loading of DOX into LTSLs by an ammonium sulfate pH-gradient17. Since the equipment utilized in this protocol is commercially available off-the-shelf and the parameters are optimized, this approach is manageable for users without prior knowledge or microfluidics experience.
One of the most critical steps within the protocol is to ensure the whole assembly is properly secured and fluid can be properly infused (step 2.5). Since the reproducibility of the self-assembly of liposomes relies on a homogenized solvent environment, any instability, such as dislodgement of syringes or introduction of air bubbles, will disturb the stability of the fluid flow and result in suboptimal liposome size and dispersity. This is also the rationale behind step 2.8, where the volume, consisting of the fluid initially occupying the channel and before a stable flow is reached, should be disposed of.
A second critical step for a successful experiment is the annealing step, to enable high DOX EE (step 2.11). In cholesterol-free liposomes, micelle-forming membrane components (i.e. MSPC and DSPE-PEG2000) will accumulate at grain boundaries with a high degree of defects to accommodate a high membrane curvature2. These arrangements thermodynamically favor the formation and stabilize membrane pores, opened liposomes, or bilayer discs. The low DOX EE of LTSL10 without annealing suggested that porous structures existed even below Tm, resulting in the absence of the pH gradient required for DOX loading (Figure 7C). The premature formation of pores below Tm was not observed for LTSLs prepared by lipid film (LTSL4 (LF) and LTSL10 (LF)), where annealing is not required. Furthermore, cholesterol-containing formulations prepared by microfluidics also do not require annealing8. It is, therefore, speculated that the premature formation of pores is a combined effect of the presence of ethanol during the preparation and the lack of cholesterol in the lipid bilayer. Structural defects within the bilayer membrane have been reported to be eliminated by annealing the liposomes above Tm, allowing lipid molecules to redistribute homogeneously and defects to be corrected19. In addition, the annealing process is an irreversible process where annealed liposomes returning to a temperature below Tm do not recreate leaky vesicles19, in agreement with the annealed LTSL10.
The nature of microfluidic preparation of liposome is a nanoprecipitation process, which requires two miscible solvents with distinctive solubility for the lipids: typically, ethanol (as a lipid solvent) and aqueous solution (as a lipid non-solvent). Thus, the presence of ethanol is unavoidable. Therefore, formulations that are sensitive or prone to alcohol-induced interdigitation13, such as cholesterol-free liposomes20, may require modification of the formulation or re-optimization of the protocol. As demonstrated with the preparation of LTSL4, the highly viscous gel was obtained, (Figure 7A), which was likely due to the formation of an interdigitated gel phase15. On the other hand, LTSL10, with its higher polymer concentration that prevents interdigitation21, was prepared successfully. Consequently, an ethanol removal procedure also must be performed; here, it was removed simply by dialysis. While on-chip continuous purification techniques such as tangential flow filtration (capable of both ethanol removal and buffer exchange) have been developed22,23, their implementation (as one-chip or modular) are beyond the aim of this protocol. Nonetheless, in the future, we expect these modular or standardized designs to be optimized and increased in availability, streamlining the microfluidic production process.
Another limitation of the protocol is the sample loss due to the travelling distance of the liquids, namely the initial waste volume (step 2.8) as well as the last fraction of solutions that would be injected but wouldn't reach the outlet. These sample losses are almost unavoidable and may contribute to a significant portion of the preparation volume at bench-scale production, especially when a small volume or precious samples are to be prepared. When necessary, the lipid recovery could be quantified by high performance liquid chromatography-evaporative light scattering detector method that enables rapid quantification of lipid concentrations24. However, once the process is optimized and scaled up, such as by using a larger syringe or fluid reservoir, the throughput could be further scaled up and sample losses would be less significant.
The main difference between this method and the existing preparation method is that liposomes are self-assembled in a controllable solvent environment in a high-throughput, continuous manner. The lipid film method is a batch manufacturing process and requires size homogenization. While very feasible at a bench-scale, it remains challenging to scale up for clinical production. Within existing microfluidic techniques, for instance, microfluidic hydrodynamic focusing, SHM offers a shorter mixing timescale11 and a greater throughput (in the range of mL/min) with lower dilution factor; notwithstanding the preparation of LTSLs has not been reported using other microfluidic devices so far. The major advantage of our approach is the high-throughput, scalable production of thermosensitive liposomes.
Thus far, our microfluidic protocol offers continuous production of LTSL10 with drug loading capability. Payloads other than DOX and ICG are also viable. However, ethanol removal by dialysis, drug remote loading, and purification by SEC column remain as batch processes and are the bottlenecks of the overall formulation process. Future development could focus on utilizing microfluidic approaches (such as tangential flow filtration) to enhance the throughput of these downstream processes and increase the scalability of the protocol.
The authors have nothing to disclose.
We thank Prostate Cancer UK (CDF-12-002 Fellowship), and the Engineering and Physical Sciences Research Council (EPSRC) (EP/M008657/1) for funding.
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) | Lipoid | PC 16:0/16:0 (DPPC) | |
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) | Lipoid | PE 18:0/18:0-PEG 2000 (MPEG 2000-DSPE) |
|
1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (MSPC) | Avanti Polar Lipid | 855775P-500MG | 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) |
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) | Sigma-Aldrich | H3375-100G | |
Adapters, Female Luer Lock to 1/4"-28UNF | IDEX Health & Science | P-624 | Requires 2 units. For the inlets |
Adapters, Union Assembly, 1/4"-28UNF | IDEX Health & Science | P-630 | Requires 2 units. (One unit included 2 nuts and 2 ferrules) |
Ammonium Sulfate ((NH4)2SO4) | Sigma-Aldrich | 31119-1KG-M | |
Bijou vial | VWR | 216-0980 | 7 mL, clear, polystyrene vial |
Centrifugal Filter Unit | Sigma-Aldrich | UFC801008 | 10 kDa MWCO, Amicon Ultra-4 Centrifugal Filter Unit |
Centrifuge | ThermoFisher Scientific | Heraeus Megafuge 8R | With HIGHConic III Fixed Angle Rotor |
Cuvette | Fisher Scientific | 11602609 | Disposable polystyrene cuvette, low volume, for DLS measurement |
Dialysis Kit – Pur-A-Lyzer Maxi | Sigma-Aldrich | PURX12015-1KT | 12-14 kDa MWCO |
Dimethyl Sulfoxide (DMSO) | Sigma-Aldrich | 34943-1L-M | |
DLS Instrument | Malvern Panalytical | Zetasizer Nano ZS90 | |
Doxorubicin Hydrochloride (DOX) | Apollo Scientific | BID0120 | |
DSC Instrument | TA Instruments | TA Q200 DSC | |
DSC Tzero Hermetic Lids | TA Instruments | 901684.901 | For DSC measurement |
DSC Tzero Pans | TA Instruments | 901683.901 | For DSC measurement |
DSC Tzero Sample Press Kit | TA Instruments | 901600.901 | For DSC measurement |
Ethanol | VWR | 20821.330 | Absolute, ≥99.8% |
FC-808 Fibre Coupled Laser System | CNI Optoelectronics Tech | FC-808-8W-181315 | FOC-01-B Fiber Collimator included. |
Ferrule, 1/4"-28UNF to 1/16" OD | IDEX Health & Science | P-200 | For the outlet |
Fibre Optic Temperature Probe | Osensa | PRB-G40 | |
Glass Staggered Herringbone Micromixer (SHM) | Darwin Microfluidics | Herringbone Mixer – Glass Chip | |
Heating Tape | Omega | DHT052020LD | Can be replaced by other syringe heater such as "HTC" or "SRT series" for slower heating. Manual wiring to a 3-pin plug required for 240V models |
Indocyanine Green | Adooq | A10473-100 | Distributed by Bioquote Limited (U.K.) |
Luer-lock Syringe, 5 mL | VWR | 613-2043 | Hanke Sass Wolf SOFT-JECT 3-piece syringes, O.D. 12.45 mm |
Microplate Reader | BMG Labtech | FLUOstar Omega | Installed with 485 nm (exictation) and 590 nm (emission) filters |
Microplate, 96-well, Black, Flat-bottom | ThermoFisher Scientific | 611F96BK | For fluorescence measurement in microplate reader |
Microplate, 96-well, Clear, Flat-bottom | Grenier | 655101 | For absorbance measurement microplate reader |
Nut, 1/4"-28UNF to 1/16" OD | IDEX Health & Science | P-245 | For the outlet |
PC to Pump Network Cable for Aladdin, 7ft | World Precision Instruments | NE-PC7 | Optional: Syringe pumps can be operated manually |
Pump control software – SyringePumpPro Software License for 2 | World Precision Instruments | SYRINGE-PUMP-PRO-02 | Optional: Syringe pumps can be operated manually |
Pump to Pump Network Cable for Aladdin, 7 ft | World Precision Instruments | NE-NET7 | Optional: Syringe pumps can be operated manually |
Size exclusion chromatography (SEC) column | GE Life Science | 17085101 | Sephadex G-25 resin in PD-10 Desalting Columns |
Sodium chloride (NaCl) | Sigma-Aldrich | 31434-1KG-M | |
Sodium hydroxide (NaOH) | Sigma-Aldrich | S5881-500G | |
Syringe Pumps & Cable (DUAL-PUMP-NE-1000) | World Precision Instruments | ALADDIN2-220/AL1000-220 | |
Thermostat Temperature Controller | Inkbird | ITC-308 | Can be replaced by other syringe heater kit/thermostat |
Triton X-100 | Sigma-Aldrich | X100-100ML | |
Tubing, ETFE (1/16" OD) | IDEX Health & Science | 1516 | |
USB To RS-232 Converter | World Precision Instruments | CBL-USB-232 | Optional: For computer without RS-232 port |
Water Bath | Grant Instruments Ltd. | JB Nova 12 |