Glass flow focusing microfluidic device for liposomes production
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1 Glass flow focusing microfluidic device for liposomes production J. N. Schianti 1 ; J. E. Trevisan 2 ; M. R. Gongora-Rubio 3 ; A.C. Seabra 1, M.H.A. Santana 2, and L. G. de la Torre 2 1. Polytechnical School, University of São Paulo, Brazil 2. School of Chemical Engineering, State University of Campinas, Brazil 3. Institute of Technological Research of São Paulo, IPT, Brazil juliana.schianti@usp.br Abstract Flow focusing microfluidic devices have been applied in different pharmaceutical and chemical processes involving emulsion and nanoparcticle production. The possibility to produce particles in micro and nanoscale with a continuum process presents many advantages such as stability, enhanced control of chemical reactions and of particle size. In this work a study to obtain glass microfluidic device with a flow focusing geometry is presented. This approach involves conventional photolithography, etching process with hydrofluoric acid solution and sealing process with UV glue to obtain microfluidic devices with depths of up to 60 µm. Fabricated devices were used for continuous liposome production, rendering particles down to 150 nm. Key words: microfluidic device, glass, flow focusing, liposomes Introduction Microfluidic devices have been successfully applied to obtain micro and nanoparticles. Advantages such as low energy and reagents consumption, high particle size control and reproducibility allow for these devices to be used in a great numbers of applications in pharmaceutical and chemical processes. Because of the low Reynolds number, the mixtures of the substances are ruled by convective-difusion phenomenon and can produce nanoparticles such as liposomes in a very fast way (1,2,3). Liposomes are phospholipids aggregates structured in bilayers with an aqueous nucleus surrounded by one or multiple concentric lamella. They are uni or multilamellar particles with a diameter that varies from the order of nanometers to micrometers, according to the production protocol and lipid composition. These colloidal structures have been attracting the attention for its applicability as a drug delivery system and as a membrane model. Nowadays, a variety of lipidbased drug formulations is being commercialized or under clinical studies (4,5,6).The classical methods for liposome production are based on the formation of a dried lipid film, ethanol injection among others. In order to obtain liposomes with specific desired size, additional steps are required, such as: sonication, extrusion or high pressure homogenization. The search for new strategies that control the lipid aggregation and particle size are a challenge in the field do liposome technology. In this context, nanoliposomes can be produced in one step only with a microfluidic continuous process with many advantages over classical methods (7,8,9,10). In this work we have used the latter approach to produce liposomes using glass microfluidic devices (11). The glass microfluidic devices were fabricated using a low cost and simple method that involves wet etching and UV glue with high viscosity to seal the microchannels. To seal the system different dilutions of UV glue in pure acetone were studied. This kind of sealing process is cheap, reversible and produces microfluidic systems without any leakage. The liposomes were produced using a flow focusing geometry, controlling the flow rates of water and lipid by a syringe pump. The flow focusing happens when a central fluid flow is restricted by two lateral fluid flows. This containment can break the fluid flow in droplets when the fluids are immiscible, or can be used to obtain particles in diffusive process at the interface between the two fluids. The results
2 showed us that microfluidic offer an important way to obtain liposomes and in futures studies may also encapsulate drugs in the same system. Materials and Experimental In this paper, commercially soda lime microscope glass slides (76 x 26 x 1 mm, Knittel, Germany) were used to obtain a flow focusing microfluidic device. The first step involves a careful cleaning process using sulfuric acid solution H 2 SO 4 + H 2 O 2 (4:1), rinsing in DI water and Isopropyl Alcohol and drying with N 2. On sequence a 50nm chromium film (LMM-IFUSP, BRAZIL) was deposited on the glass substrate by PVD process, using a Sputtering AJA with conditions related in Table I. The chromium film was treated in a muffle furnace at 500 C by two hours with a heating rate of 10 ºC/min. In order to seal the glass layers we studied the use of UV Glue (UV Glue 1201, Pizzani, Brazil). The glue has a high viscosity ( cps), forming a thick film between the glasses and blocking the microchannels. To improve the results it ways studied two dilutions in acetone (30 and 50%).The glue was applied by spin coating, and were studied different velocities rates ( rpm) to obtain the smallest glue thickness. The cure was processed in an UV camera (Philips, UV Germicid, 15W) in 30 seconds. The complete fabrication process is presented in figure 2. a) b) c) d) Table I. Sputtering Conditions Pressure N 2 Flux Power Deposition rate 5 mtorr 20 sccm 20 W 0,566 A/s e) f) The photolithographic process was realized using the TSMRV90 deposited by spin coating and the glass slides were etched HF solution, and it was studied two solutions: HF: HCl: H 2 O (1:1:3 and 1:2:3). In Figure 1 is showed the cross section geometry. With this geometry is possible to promote the hidrodynamic focalization. At the center inlet is introduced the lipid dispersed in ethanol and the two inlets is introduced deionized water. The microchannel has a diffusion width of 5 cm. Figure 2. a) cleaning; b) sputtering process; c) photolithography; d) etching; e) UV sealing and f) the final system. The liposomes were obtained after the cross flow injection of Egg-phosphatidylcholine (Egg-PC) dispersion in ethanol (400, 200 and 100 mm were tested) in the central microchannel. Simultaneously, water was injected in both lateral microchannels. The three streams were fed to the microchannels at controlled flow rate, through a syringe pump. The liposomes were collected in a final recipient as showed in Figure 3. The ethanol and water flow rates were and ml/min, respectively in the best results, however were tested different values to choose the best condition. The liposome average hydrodynamic diameter and size distribution were determined by dynamic light scattering (Malvern Zetasizer). Figure 1. Cross section geometry for hidrodynamic focalization.
3 (a) (b) Figure 3: Experimental apparatus for liposome production. (1) Microfluidic device; (2) Syringe pump for water supply; (3) Syringe pump for lipid supply in ethanol dispersion; (4) Recipient for liposome collection; (5) Stereo microscope; (6)Computer system for on line image acquisition. Results Microfabrication Process The results obtained for the microfabrication process permit us to choose the best conditions to produce the microfluidic devices. With the intent to improve the chromium mask performance it was realized a heat treatment at 500 º C. In Figure 4 are showed two glass wafers with and without the heat treatment. The heat treatment promotes an oxidation varying the color film and increasing the thickness in approximately 3 nm. Figure 5. Glass surface before an etch of 15 min using chromium film as a mask: (a) without heat treatment and (b) with heat treatment. In Figure 6 is shown the graphic of etched depths versus time of etch. This result give us the etch rate for this type of glass and with this parameter is possible define the correct time to obtain a desired depth. The solution with less quantity of HCl (1:1:3) was obtained a etch rate of µm/min and, with the solution (1:2:3) the etch rate is , 02 µm/min. We choose the first solution to produce the microchannels. The use of HCl allows a homogenous etching process and reduces the roughness in the channels surfaces. It is important to note that depending on the glass the etch rates can vary, as we can see at the literature. The glasses have different concentrations of some cations such as Na + and Ca 2+ that interfere in etching process. To study the liposome formation was utilized channels with 50µm of depth. Figure 4. Chromium film with and without heat treatment. The glass on the left with blue coloring was treating in a furnace at 500ºC. In Figure 5(a) and 5(b) are shown the final results for etching times of 15 min for chromium films with and without heat treatment. In both cases there are some imperfections in the glass surface, but in the first image we can note a wrinkled surface. Figure 6. Graphic of etched depths in a period of time.
4 Ibersensor 2010, 9-11 November 2010, Lisbon, Portugal In the Figure 7 are presented the microchannels observed by SEM (LSI-POLI). The interaction reaction is presented in (a), the magnitude was of 200x and resolution of 200 µm; and (b), is possible to observe the microchannels profile, with magnitude of 632 and resolution of 50 µm. It is possible to observe some defects in the glass surfaces and in the sides of microchannels; however these defects did not affect the final results in the liposome production. (a) Figure 8. Graphic of spin coating rate versus thickness film (b) Figure 9. Glass flow focusing system used to produce liposomes. Figure 7. Microchannels observed in detail by SEM. (a) the fluid flow interaction region and (b) the cross section profile. The commercial UV glue has a high viscosity, blocking the channels if was used without any dilution. In Figure 8 are show the graphic relating the glue thickness obtained for each rotation velocity in a spin coat, for different glue dilutions. The best results were obtained with a high velocity and 50% of dilution in acetone and a rotation velocity of 4000 rpm. In this condition is possible to obtain glue films with 2 µm of thickness. The devices were cured in UV chamber before 30 seconds of exposure. The UV glue has a good chemical resistance for a great number of acids solutions and can stand temperature up to 180ºC. In Figure 9 is showed the complete microfluidic system used to obtain the liposomes. The fluids accesses were fixed with commercial epoxy glue. Liposome production The mean hydrodynamic diameter of liposomes was evaluated as function of the lipid concentration in the ethanol stream (400, 200 and 100 mm), the ethanol flow rate (0.005 to 0.01 ml/min) and the ratio between ethanol and water volumetric flows (Table II).According Table II, we can observe that the liposome average diameter is dependent on the initial lipid concentration, the ethanol flow rate and the ratio between water/ethanol volumetric flows (Rwater/ethanol). It was
5 observed that decreasing the lipid concentration, the liposome mean hydrodynamic diameter decreases. When the lipid concentration is 100 mm in the ethanol stream, we obtained the smallest diameter. In this case, the reduction of ethanol flow rate and the increase of R water/ethanol produced liposomes with approximately 270 nm. In order to investigate the difusional process, we used the Continuity Equation for mass transfer to estimate the effect of lipid concentration on the ethanol diffusion lenght (Figure 10). The phenomenon we considered was the diffusion of ethanol from the central stream to the aqueous strain, taking the lipid as a barrier. According the simulation, higher lipid concentration or higher ethanol flow rate requires higher microchannel length to complete the process of ethanol diffusion. It is important to note that we produced the microfluidic device with length of 5 cm for ethanol diffusion. The comparison between Table II and Figure 10, indicates that the process conditions that produced liposomes with the lowest diameters (270 nm) allowed the complete ethanol diffusion trough the 5 cm microchannel length. Higher lipid concentrations and/or ethanol flow rate require higher microchannel length to complete the diffusional process to control the liposome size. It is also important to note that the complete diffusion of ethanol in water improves the liposomes production and higher lipid content promotes an additional barrier to ethanol diffusion. We believe that this could be one of the reasons for higher liposome diameters at higher lipid concentration. Table II. Mean hydrodynamic diameter for liposomes produced at different lipid concentration and ethanol flow rate. Lipid Concentration In ethanol strain Ethanol Flow Rate R (i) Liposome mean hydrodynamic diameter (ii) (mm) (ml/min) (nm±sd) 0, ,3 ± 836, , ,2 ± 522,6 0, ,9 ± 898,8 0, ,7 ± 384, , ,6 ± 118,4 0, ,4 ± 533, , ,8 ± 134,5 0, ,6 ± 37,5 (i) (ii) R: Ratio between Water/Ethanol flows; Z-average diameter. (iii) SD: Standard deviation of 3 independent experiments.
6 Figure 10. Simulation of ethanol diffusion length (x) for different ethanol/lipid flow rates (Q E) in the microfluidic device.( ) Ethanol; Lipid at (_._)100, (.) 200 and (- - -)400 mm in ethanol. Ratio between Water/Ethanol flows (R) of 20. Considering the above process investigation, we selected the lipid concentration of 100 mm in the ethanol stream and we produced liposomes at different R (water/ethanol) (using the ethanol flow rate of 0.007) (Figure 11). (c) R=20 Figure 11. Hydrodynamic focus obtained for the Egg-PC liposomes production in the cross flow microfluidic device at different flow ratio R(water/ethanol) (using the ethanol flow rate of 0.007). (a) R=7,5 The best conditions for our device were obtained at 100mM lipid concentration, ethanol flow rate of ml/min and R water/etanol of 7.5. At this conditions the liposomes diameter was in the range of 110 nm as presented in Figure 12. (b) R=10,57 Figure 12: Intensity-weighted liposomes size distribution obtained after microfluidic processing using the cross flow injection. The liposomes were obtained using 100 mm of lipid dispersion in ethanol, ethanol flow rate of 0.007mL/min and water/ethanol flow ratio of 7.5. The different curves correspond to three independent experiments.
7 These results show that the microfluidic devices offer a great tool to produce liposomes with low size and polidispersivity. In the best results the polidispersivities are in the range of 0.15 to Conclusion The implemented fabrication process allows us to manufacture flow focusing devices with a fast turnaround and low fabrication costs. The cross flow microfluidic device is a promising strategy for the production of homogeneous liposome in nanoscale range and low polidispersivity, useful for applications as a drug delivery system. Some improvements in geometry will allow a better performance with lipids in a higher concentration. Besides, with the geometry improvement, the next steps in this work it will be the introduction of drugs within the liposome in only one microfluidic device. More studies about the physics of fluid flow and liposome formation are still development. References 1 Janh, A. et. al., ACSNANO, Vol. 4, n.º 4 (2010), Castaño-Álvarez, M. et al., Sensors and Actuators B 130 (2008) Zhang S., et al., Chemical Engineering Science 63 (2008) Lasic, D. (1993). Liposomes: From Physics To Applications. Amsterdam: Elsevier Science Publishers B.V. 5 Barnadas-Rodríguez, R., & Sabés, M. International Journal Of Pharmaceutics, 213, (2001), Gregoriadis, G. (1993). Liposome Technology. CRC Press. 7 Wagner, A. et al. Journal of Liposome Research. 12 (2002) Janh, A. et al., J. Am. Chem. Soc., 126, (2004) Jahn, A. et al., Langmuir 23, (2007) Zhang S., et al., Chemical Engineering Journal 144 (2008) C. Iliescu, et. al., Sensors and Actuators A: Physical, 143 [1] (2008)
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