Application note: Lipid Microbubbles

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Introduction

This application note presents a step-by-step manufacturing guide to generate monodisperse lipid microbubbles (polydispersity index (PDI) values of ≤10%) using the MicroSphere Creator and the BG5 microfluidic chip. In this application note results obtained with Tide’s proprietary formulation will be presented, alongside results and development using a formulation commonly described in literature.

Perfluorocarbon (PFC), Sulphur hexafluoride (SF6) filled microbubbles with lipid shells are widely used as contrast agents in ultrasound imaging. The key to contrast enhancement are microbubbles with a peak in natural resonance at the diagnostic frequencies used by the ultrasound equipment. The difference in the acoustic response of the resonating microbubbles and the surrounding tissue ensures a higher contrast image. This allows clinicians to obtain functional information related to the perfusion of the patient’s organs such as the liver, kidney, heart as well as the peripheral vasculature. A model of such a microbubble can be seen in figure 1.

The size of each microbubble plays a crucial role, since peak resonance is only achieved when the resonance size of the microbubble precisely matches the operating frequency of the ultrasound equipment, something that is not currently commercially available. In fact, accurately defined microbubble sizes are necessary to maximise efficiency and obtain more information, which is needed for the further development of ultrasound towards new applications such as focused ultrasound therapy in combination with microbubbles and targeted drug delivery.

Figure 1: Schematic model of a microbubble with a lipid shell.

Materials and methods

Lipid preparation

To produce these lipid microbubbles a lipid mixture has to be prepared. This is done following a standard procedure similar to those found in literature [Segers et al., Langmuir 2016, 32, 3937−3944] for the manufacture of ultrasound contrast agents. In this formulation, the following lipids are used:

  • DPPC (1,2-dipalmitoyl-snglycero-3-phosphocholine)
  • DPPA (1,2- dipalmitoyl-sn-glycero-3-phosphate)
  • DPPE-MPEG5000 (1,2-dipalmitoylsn- glycero-3-phosphoethanolamine-N-[methoxypoly(ethylene glycol)-5000])

These lipids are combined at an 80 : 10 : 10 ratio of molar percentages, dissolved in a chloroform: methanol (2 : 1 volumetric ratio) solution and exposed to nitrogen to create a homogenous mixture. A light vacuum is applied to remove the solvents and create a lipid film. This thin film is then dissolved in a concentration of 10 mg/mL using a propylene glycol:glycerol: PBS or water solution (10 : 10 : 80 volumetric ratio) and filtered using a 0.45 μm filter to prevent particles entering the chip.

MicroSphere Creator setup

Microbubbles are produced using the MicroSphere Creator in conjunction with the Tide BG5 microfluidic chip. The BG5 is selected as the patented technology in this chip enables high microbubble production rates and ensures robust results. The driving gas for the lipid solution and the gas that will be encapsulated in the bubbles is SF6. In this case the same gas is used for the continuous phase as well as for the dispersed phase. However, the MicroSphere Creator allows two different gases to be used separately if required, this can ensure exotic gases are not wasted as a driving gas and only used for the creation of the microbubbles. The microfluidic setup can be seen in figure 2.

Figure 2: MicroSphere creator setup for microbubble production. The input gas is connected to the gas inlet connectors. The lipid solution can be inserted in the liquid reservoir after filtration.

When working with microfluidics it is of the utmost importance to work in a clean environment in order to prevent clogging of the system. Always filter the fluids that are introduced to the system by using 0.45 μm filter to prevent particles entering the chip. The use of protective clothing, including a lab coat and gloves are recommeneded. Additionally, it is recommended to ensure that no direct contact is made with the tubing that comes in contact with the fluids entering the system.

On chip monitoring of production

When comparing microbubble radius on chip and after production, the shrinking of bubbles has to be taken into account [Segers et al., Langmuir 2016, 32, 3937−3944]. After production in the nozzle on chip, microbubbles dissolve to a stable final radius in the exit channel. This process results in bubbles with a radius 2.55 times smaller than their initial radius, depending on the formulation used. This can be validated by measuring the bubble size during production, and comparing this to size data after collecting the samples as measured e.g. with a Coulter Counter Multisizer.

Results

Lipid microbubbles created using the MicroSphere creator and the Definity-like lipid formulation are shown in figure 3 and 5. These microbubbles were produced using a liquid pressure of 4000 mbar and a gas pressure of 3300 mbar. To reduce coalescence the liquid reservoir containing the lipid formulation was heated in a hot bath to 85 °C. The 15 degrees increase compared to the 70 °C described by Segers [Lab Chip, 2019, 19, 158] was used to account for cooling down through the tubing and microfluidic chip. In figure 3 the lipid microbubbles can be seen on-chip directly after production. In figure 5 the size distribution after collecting the microbubbles can be seen. Size distribution was determined using a Coulter Counter measurement. In figure 4 and 6 lipid microbubbles created with Tide’s proprietary formulation: TM-22, can be seen. Using TM-22 the microbubbles could be produced at room temperature without coalescence.

Figure 3: Microbubbles produced at 70 °C using the literature formulation; liquid and gas pressures were  4000  and 3300 mbar, respectively.
Figure 4: TM-22 microbubbles produced at room temperature; liquid and gas pressures used were 4000 and 2400 mbar, respectively.
Figure 5: Coulter Counter analysis of the lipid microbubbles using the Definity-like lipid formulation produced at 70 °C. Median bubble diameter of the primary distribution is 6.15 μm with coalescence at 10 μm.
Figure 6: Coulter Counter analysis of lipid microbubbles produced at room temperature using TM-22 formulation. Median bubble diameter is 3.17 μm with no coalescence.

Step by step development using Definity-like lipid formulation

The first step in producing lipid microbubbles using microfluidics is finding a pressure regime that results in stable bubble production in the desired bubble size range. An optimum pressure regime between 2 and 4 bar was used during these experiments.

System settings can be found in table 1. Pictures of the microbubbles produced can be seen in figure 7, 9 and 11. Size distributions (on chip) and median sizes resulting from different pressure settings can be seen in figure 8, 10 and 12. Bubble analysis after collecting using the Coulter Counter can be seen in figure 13. Please note that there are numerous factors influencing the production process; pressure settings might be slightly different for your setup.

Table 1: Pressure settings used for creating lipid microbubble samples with the MicroSphere Creator.

Setting Liquid pressure [mbar] Gas pressure [mbar] Average bubble size on chip [μm]
A 3300 2500 19,40
B 3000 2500 19,40
C 4000 3300 14,50

First microbubbles were produced at room temperature and at pressure setting A. Results can be seen in figure 7 and 8.

Figure 7: Lipid microbubbles produced at room temperature at liquid pressure 3300 mbar and gas pressure 2500 mbar. (Setting A)
Figure 8: On-chip size analysis from setting A produced at room temperature at liquid pressure 3300 mbar and gas pressure 2500 mbar. The microbubbles in the primary distribution have a median size (diameter) of 12.1 μm and a PDI of 11.2%. Total coalescence is 89.4%.

In figure 7 and 8 merging of microbubbles (coalescence) is visible. This measurement was performed on chip using the stroboscopic microscope setup. As stated in by Segers et al. [Lab Chip, 2019, 19, 158] “coalescence probability can be dramatically reduced by increasing the on-chip temperature during bubble formation to around 70 degrees Celsius.”. Another possible solution to reduce coalescence is to increase lipid concentration, however, in this application note the focus will be on temperature and pressure settings and not on lipid concentration [Langmuir 2016, 32, 3937−3944].

To reduce coalescence the liquid reservoir containing the lipid formulation was heated in a hot bath to 85 °C. The 15 degrees increase compared to the 70 °C described by Segers  [Lab Chip, 2019, 19, 158] was used to account for cooling down through the tubing and microfluidic chip. Results from bubble production using similar pressure settings at elevated temperature can be seen in figure 9 and 10 below.

Figure 9: Lipid microbubbles produced at elevated temperature at liquid pressure 3000 mbar and gas pressure 2500 mbar (Setting B)
Figure 10: On-chip size analysis from setting B produced at elevated temperature at liquid pressure 3000 mbar and gas pressure 2500 mbar. The microbubbles in te primary distribution have a median size of 19.4 μm and a PDI of 5.2%. Total coalescence is 36.7%.

In figure 9 and 10 a substantial decrease in coalescence can be seen compared to the results from setting A. However coalescence is still significant. For setting C the pressure regime was increased to 3-4 bar. Results can be seen in figure 11 and 12 below.

Figure 11: Lipid microbubbles produced at elevated temperature at liquid pressure 4000 mbar and gas pressure 3300 mbar (Setting C)
Figure 12: On-chip size analysis from setting C produced at elevated temperature at liquid pressure 4000 mbar and gas pressure 3300 mbar. The microbubbles in the primary distribution have a median size of 14.5 μm and a PDI of 6.9%. Total coalescence is 32.2%.

After production the bubbles were collected in a 2.5 mL closed glass vial pre-filled with SF6 gas. The bubbles were analysed using a Coulter Counter and results can be seen in figure 13.

Figure 13: Coulter Counter analysis of setting C. The median diameter of the primary distribution is 6.15 μm with coalescence at 10 μm.

In figure 10 and 11 can be seen that the microbubbles from setting C have shrunk from 14.50 μm to 6.15 μm. Which is 2.35 times smaller and corresponds to results from Segers et al. [Langmuir 2016, 32, 3937−3944].

When comparing results from setting A, B and C it can be concluded that at lower pressure regimes (Setting A) bubbles are larger and coalescence is higher. Increasing the temperature of the mixture reduced coalescence significantly (Setting B & C). During these experiments the coalescence was not completely reduced due to the difference in heating compared to Segers et al. (heating lipid solution vs. on chip heating).

By optimising the lipid formulation or production parameters the microbubbles can be further optimised and tuned to the desired specifications. By making use of Tide’s TM-22 formulation monodisperse microbubbles can be produced at room temperature. Furthermore, using the pressure control of the MicroSphere Creator the bubble diameter can be tuned to meet your requirements.

Once the production process has been optimised for lipid microbubbles, the MicroSphere Creator can be used to ensure stable production. Using the build in optical measurement and inspection setup the production frequency can be determined in real time. Production frequency gives a good estimate of size uniformity during production. In figure 14 the production stability at different production rates can be seen.

Figure 14: Producing microbubbles at different production frequencies using the MicroSphere Creator. The pressure regulators inside the system ensure fast response and stable production of microbubbles.

If you have any further questions regarding lipid microbubble production, Tide’s TM-22 formulation and/or the MicroSphere Creator, do not hesitate to contact us. Our team of experts is available to help.

 About Tide Microfludics

Tide Microfluidics is a high-tech microfluidic company specialised in the development and manufacturing of innovative ultrasound contrast agents. Tide leverages proprietary microfluidic technology for the production of monodisperse microbubbles to deliver solutions for medical, biochemistry and research applications. We were founded in 2011 to offer commercial microfluidic products and expertise to our customers all over the world. This includes a variety of innovative microfluidic products based on the MicroSphere Creator research platform, which our skilled team of experts supports from out our fully equipped laboratory facilities in Enschede, The Netherlands. Our ambition is to provide easy-to-use microfluidic solutions to enhance our customer’s pharmaceutical and lifescience research worldwide.

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