Controlled, automated synthesis of lipid nanoparticles

Lipid nanoparticles (LNPs) are powerful carriers for the delivery of active pharmaceutical ingredients (APIs) and biotherapeutics. Their biocompatibility and size, which are important factors when determining the efficacy of an encapsulated API, make them attractive.

Dolomite’s leading microfluidic technology provides a unique automated nanoparticle generation platform for the generation of controllable, homogeneous and reproducible LNPs. The ability to control LNP size from as little as 20 nm allows for a wide scope of end-user applications.

Key features of the Automated Nanoparticle (ANP) System include running in continuous mode to generate large samples once the synthesis is optimized and running protocols with importable experiment tables to produce formulation libraries.

The robustness of the ANP System was shown by comparing the data gathered in ‘protocol’ library generation mode to continuous mode, where the LNP size stayed consistent with a polydispersity index (PDI) of <0.2, and values as low as 0.06 were attainable after optimizing the synthesis with the ANP System.

By controlling the total flow rate (TFR) and flow rate ratio (FRR), facile control over LNP size is accomplished. These key findings can be tailored to any formulation to expedite the development of LNP formulations for applications from vaccine development and drug delivery to gene therapy.

Conventional techniques for making LNPs like thin-film hydration, sonication and extrusion can involve large batch volumes and are laborious. They do not have the ability to easily control encapsulation efficiency (EE %), size and PDI, all of which are crucial factors in the efficacy of liposomal APIs.

The ANP System combines these features (reproducibility, low sample volumes, control over size and PDI) with microfluidic technology, using rapid mixing methods and hydrodynamic flow focusing to nucleate and assemble LNPs.

Put simply, lipids dissolved in a solvent (or a mixture of solvents) are mixed with an anti-solvent (aqueous phase), and the subsequent shift in polarity leads to the self-assembly of lipids into unilamellar vesicles.

The ANP System combines this technology with experiment automation to enable the production of sample libraries. Protocols can be defined in a spreadsheet offline and directly imported into the Flow Control Centre (FCC) Software.

Only a single manual step (loading nanoparticle (NP) precursor) is needed at the start of the protocol once it is set up, enabling walk-away operation and multiple sample production from a single precursor loading.

This article will demonstrate that by utilizing the FCC software accompanying the system that TFR, FRR, NP precursor volume, sample collection volume can be controlled and enable optional in-line dilution with head and tail cuts to ensure the sample collected is representative of material produced in continuous mode.

Automating fluid handling and collection ensures consistency between formulations, reproducibility from batch to batch and stops the variability which is associated with manual operation. The glass microfluidic chips are reusable and robust to decrease consumable costs by using expensive precursor reagents efficiently.

The chips are able to withstand rigorous cleaning with numerous chemicals and can also be autoclaved.

The system automatically runs a wash cycle between each experiment to clean the fluidic pathway and avoid cross-contamination between samples. In between protocol runs, the user also has the choice to carry out further wash cycles manually.

It is also established that the system can be controlled in manual mode to continuously produce a larger batch of the formulation once a formulation has been optimized in protocol mode. The quantity gathered is user-defined and can be run continuously for long periods of time without compromising PDI and size.

a/ ANP System setup. b/ microfluidic chip 1- entry of lipids in solvent and aqueous phase, hydrodynamic flow focussing, and subsequent mixing assembles LNPs, optional inline dilution in microfluidic chip 2 further facilitates LNP growth c/depiction of LNP assembly in microfluidic chip 1.

a/ ANP System setup. b/ microfluidic chip 1- entry of lipids in solvent and aqueous phase, hydrodynamic flow focussing, and subsequent mixing assembles LNPs, optional inline dilution in microfluidic chip 2 further facilitates LNP growth c/depiction of LNP assembly in microfluidic chip 1.

Figure 1. a/ ANP System setup. b/ microfluidic chip 1- entry of lipids in solvent and aqueous phase, hydrodynamic flow focusing, and subsequent mixing assembles LNPs, optional inline dilution in microfluidic chip 2 further facilitates LNP growth c/depiction of LNP assembly in microfluidic chip 1. Image Credit: Dolomite Microfluidics

Results

The FRR between the organic and aqueous phase and the TFR were the key parameters investigated to tune the LNPs’ size.

In addition, a comparison between the continuous mode and protocol was made to show the transferability of the process parameters for bulk production of LNPs.

. .
Organic phase (O) Phospholipon 90G (1 mg/ml), DDAB
(0.1 mg/ml) in ethanol
Aqueous phase (A) 1 x PBS pH 7.4
Dilution (D) 1 x PBS pH 7.4
Flow rate ratio (FRR) Variable (O:A:D)
Total flow rate (TFR) 3 ml/min

 

i/ Lipid nanoparticles produced by varying lipid to aqueous flow rate ratio (FRR) at a total flow rate (TFR) of 3 ml/min. LNP size ranged from 20 nm to 150 nm, mean (n=3). The error bars represent standard deviation of the mean. ii/ LNPs produced at FRR of 3:1:1 at a TFR of 3 ml/min .Three samples collected with an average LNP size of 134 nm and PDI below 0.1. Particle size distribution and polydispersity index (PDI) were determined by Malvern dynamic light scattering (DLS), appendix figure 7. The error bars represent standard deviation of the mean.

Figure 2. i/ Lipid nanoparticles produced by varying lipid to aqueous flow rate ratio (FRR) at a total flow rate (TFR) of 3 ml/min. LNP size ranged from 20 nm to 150 nm, mean (n=3). The error bars represent standard deviation of the mean. ii/ LNPs produced at FRR of 3:1:1 at a TFR of 3 ml/min. Three samples were collected with an average LNP size of 134 nm and PDI below 0.1. Particle size distribution and polydispersity index (PDI) were determined by Malvern dynamic light scattering (DLS), appendix figure 7. The error bars represent standard deviation of the mean. Image Credit: Dolomite Microfluidics

By changing the organic phase in relation to the aqueous phase, the FRR was examined. As seen in Figure 2, data demonstrated that by altering FRR, the LNP size could be changed in a reproducible and consistent way.

. .
Organic phase (O) Phospholipon 90G (1 mg/ml), DDAB
(0.1 mg/ml) in ethanol
Aqueous phase (A) 1 x PBS pH 7.4
Dilution (D) 1 x PBS pH 7.4
Flow rate ratio (FRR) 2:1:1 (O:A:D)
Total flow rate (TFR) Variable

 

Lipid nanoparticles produced by varying total flow rate (TFR) at a fixed 2:1:1 (organic: aqueous: dilution) flow rate ratio FRR. LNPs ranged from 66 nm to 78 nm, average PDI of 0.17 with 0.01 standard deviation. Mean (n=3) particle size distribution and polydispersity index (PDI) were determined by Malvern dynamic light scattering (DLS). The error bars represent standard deviation of the mean.

Figure 3. Lipid nanoparticles produced by varying total flow rate (TFR) at a fixed 2:1:1 (organic: aqueous: dilution) flow rate ratio FRR. LNPs ranged from 66 nm to 78 nm, average PDI of 0.17 with 0.01 standard deviation. Mean (n=3) particle size distribution and polydispersity index (PDI) were determined by Malvern dynamic light scattering (DLS). The error bars represent standard deviation of the mean. Image Credit: Dolomite Microfluidics

PDI of 0.06 was obtained for FRR of 3:1:1, whilst overall the PDI was <0.2 during parameter testing and optimization. For experiments, repeatable minimized PDI could be achieved.

. .
Organic phase (O) Phospholipon 90G (1 mg/ml), DDAB
(0.1 mg/ml) in ethanol
Aqueous phase (A) 1 x PBS pH 7.4
Dilution (D) 1 x PBS pH 7.4
Flow rate ratio (FRR) 1.5:1:1 (O:A:D)
Total flow rate (TFR) Variable

 

Lipid nanoparticles produced by varying total flow rate (TFR) at a fixed 1.5:1:1 (organic; aqueous: dilution) flow rate ratio. LNPs ranged from 57 nm to 66 nm, average PDI of 0.19 with 0.01 standard deviation. Mean (n=3) particle size distribution and polydispersity index (PDI) were determined by Malvern dynamic light scattering.

Figure 4. Lipid nanoparticles produced by varying total flow rate (TFR) at a fixed 1.5:1:1 (organic; aqueous: dilution) flow rate ratio. LNPs ranged from 57 nm to 66 nm, average PDI of 0.19 with 0.01 standard deviation. Mean (n=3) particle size distribution and polydispersity index (PDI) were determined by Malvern dynamic light scattering. Image Credit: Dolomite Microfluidics

To see its effect on the LNP size, TFR was also investigated. The data for the formulation investigated showed no significant change in LNP size with increasing TFR, as seen in Figures 3 and 4. A reduction of 10 nm was observed in the higher TFR (>10 ml/min), as seen in Figure 3.

. .
Organic phase (O) Phospholipon 90G (1 mg/ml), DDAB
(0.1 mg/ml) in ethanol
Aqueous phase (A) 1 x PBS pH 7.4
Dilution (D) 1 x PBS pH 7.4
Flow rate ratio (FRR) 0.5:1:1, 1:1:1, 1.5:1:1 (O:A:D)
Total flow rate (TFR) 3 ml/min

 

Lipid nanoparticles produced continuously at a total flow rate (TFR) of 3 ml/min at varying flow rate ratios (FRR). LNPs ranged from 19 nm to 57 nm, PDI remained consistent between runs ranging from 0.15 to 0,2 with a standard deviation of 0.01. Mean (n=3) particle size distribution and polydispersity index (PDI) were determined by Malvern dynamic light scattering (DLS). The error bars represent standard deviation of the mean.

Figure 5. Lipid nanoparticles produced continuously at a total flow rate (TFR) of 3 ml/min at varying flow rate ratios (FRR). LNPs ranged from 19 nm to 57 nm, PDI remained consistent between runs ranging from 0.15 to 0,2 with a standard deviation of 0.01. Mean (n=3) particle size distribution and polydispersity index (PDI) were determined by Malvern dynamic light scattering (DLS). The error bars represent standard deviation of the mean. Image Credit: Dolomite Microfluidics

The same formulation was then run in continuous mode to further assess the robustness of the system.

. .
Organic phase (O) Phospholipon 90G (1 mg/ml), DDAB
(0.1 mg/ml) in ethanol
Aqueous phase (A) 1 x PBS pH 7.4
Dilution (D) 1 x PBS pH 7.4
Flow rate ratio (FRR) 0.5:1:1, 1:1:1, 1.5:1:1 (O:A:D)
Total flow rate (TFR) 10 ml/min

 

Lipid nanoparticles produced continuously at a total flow rate (TFR) of 10 ml/min at varying flow rate ratio (FRR). LNPs range from 15 nm to 55 nm, PDI remained consistent between runs ranging from 0.15 to 0.2 with a standard deviation of 0.01. Mean (n=3) particle size distribution and polydispersity index (PDI) were determined by Malvern dynamic light scattering (DLS). The error bars represent standard deviation of the mean.

Figure 6. Lipid nanoparticles produced continuously at a total flow rate (TFR) of 10 ml/min at varying flow rate ratio (FRR). LNPs range from 15 nm to 55 nm, PDI remained consistent between runs ranging from 0.15 to 0.2 with a standard deviation of 0.01. Mean (n=3) particle size distribution and polydispersity index (PDI) were determined by Malvern dynamic light scattering (DLS). The error bars represent standard deviation of the mean. Image Credit: Dolomite Microfluidics

There are comparable results between protocol and continuous mode, which indicates that once a formulation has been optimized, it can be reliably bulk produced in continuous mode at various TFR, as seen in Figures 5 and 6.

Discussion

LNP homogeneity and size play a key role in establishing the efficacy of the encapsulated API. So, it is critical that the LNPs are reproducible as well as controllable.

The lipid formulation tested in this article shows that by manipulating the FRR between the organic and aqueous phase (Figure 2), the size of LNP and the PDI can be controlled easily.

Hydrodynamic flow focusing mixes the two phases entirely based on molecular diffusion in the direction of fluid flow streams, known as laminar flow mixing. The ANP System can be utilized with a variety of chip types; mixing can also be achieved by utilizing Dolomite’s micromixer chips to enable mixing by chaotic advection.

As the ratio of organic to aqueous phase increases, the LNP size also increases. The increased size is a result of higher local concentrations of lipid during the liposome growth, which means more lipid can be integrated into the particle structure.

In addition, there is less aggressive particle nucleation as the change in solubility of the lipid is less, which could mean that fewer particles form overall, and so, each one grows due to the amount of material present. Similarly, TFR is also a key factor that can help to decrease the LNP generation time overall.

The TFR results seen in Figures 4 and 5 show that the LNP generation time can be shortened to increase throughput. In this regime, the TFR is not a limiting factor up until >10 ml per minute, at which point TFR starts to have more of an influence; usually, FRR effects dominate at lower TFRs.

TFR can influence the LNP size for other formulations and concentrations of lipids. This can occur because the time needed for diffusion between the organic and aqueous phases is shortened. The speed at which the lipids can self-assemble exceeds the rate of diffusion, resulting in smaller LNPs as a result.

Protocol versus continuous production was investigated to further assess scalability. The results shown in Figures 5 and 6 showed that the LNP size was largely unaffected by the TFR and continuous production.

This demonstrates that LNP assembly stays identical to the protocol mode, and the user can opt to bulk produce after library generation optimization without any additional process development.

The parameters examined here indicate that it is vital to control the mixing method and ratio to attain the desired LNP size. LNP homogeneity as measured by PDI (as low as 0.06 for the formulation tested) is also important for all end applications to enhance efficacy.

For any LNP generation process, reproducibility is key. The results presented in this article display minimal variance between each repeat since the automated process conditions were identical, reliably producing LNPs with a low PDI.

FCC controlled automation of process parameters and hardware ensures that changes in operating conditions or human error are minimized.

Conclusions

The ANP System automates the development of LNP formulations, ensuring consistent results from run to run, providing its users with savings in both time and reagent costs because of its ability to work with small sample volumes.

The experiments can be set up in Excel with ease and easily imported into the software. The hardware provides precise control over experimental parameters like TFR and FRR.

The ANP System can readily generate formulation libraries, tune LNP size and uniformity, and provide scalability to the user. The protocol runs importable experiments automatically, with only a single manual step at the beginning of the protocol run.

The ANP System shows excellent reproducibility with consistent sample-to-sample production of formulation libraries. This single platform enables the user to go from producing small volume LNP libraries to bulk production via a single step, with no requirement to re-validate the process.

The system components are reusable, enabling user-directed wash steps between experiments as they have no additional licensing or expensive consumable costs.

Materials and methods

The lipids utilized in this study, Phospholipon 90G (Lipoid, Switzerland) and Dimethyl Dioctadecyl-ammonium bromide (DDAB) (Fischer Scientific, UK) were dissolved in ethanol (reagent grade 99 %, Sigma Aldrich, UK) at 1 mg/ml and 0.1 mg/ml respectively.

Phosphate buffer saline (PBS) x 1 at pH 7.4 was utilized for the aqueous and dilution phase. All prepared solutions were filtered with a 0.2 µm filter prior to employment. The organic and aqueous were automatically mixed using the Automated Nanoparticle System protocol.

The flow rate ratio of (organic: aqueous: dilution), 0.5:1:1, 1:1:1, 1.5:1:1, 2:1:1, 2.5:1:1, 3:1:1, 3.5:1:1 with the total flow rate of 1,3,6,8,10,15 ml/min were assessed. The gathered samples were analyzed straight after collection by utilizing dynamic light scattering (Malvern Zetasizer, U.K.), which accounted for the ethanol ratio in the sample.

All of the samples were produced and analyzed in triplicate. The relative light intensity scattered by particles was reported as intensity number distribution and polydispersity index (PDI), with error bars representing standard deviation (SD).

Appendix

Malvern mastersizer (DLS) graphs of FRR 3:1:1 at a TFR of 3 ml/min. Intensity, number and volume distribution.

Malvern mastersizer (DLS) graphs of FRR 3:1:1 at a TFR of 3 ml/min. Intensity, number and volume distribution.

Malvern mastersizer (DLS) graphs of FRR 3:1:1 at a TFR of 3 ml/min. Intensity, number and volume distribution.

Figure 7. Malvern mastersizer (DLS) graphs of FRR 3:1:1 at a TFR of 3 ml/min. Intensity, number and volume distribution. Image Credit: Dolomite Microfluidics

About Dolomite Microfluidics

Dolomite is a world leader in Productizing Science™ and an innovator in creating microfluidic devices and solutions. We sell the coolest microfluidic products around the world, often working with partner companies to extend the range of technology available to our customers. Productizing Science™ means creating marketable and commercially successful products from scientific discovery, and Dolomite excels in commercialising microfluidic products which exceed expectations.

We offer modular, standard microfluidic systems benefiting a wide range of applications, always adhering to the principles of having multiple functionalities, scalability, user-friendly design and a cost-effective, flexible solution for our customers.

Moreover, we offer Productizing Science™ as a service, which is a product development & manufacturing partnership creating microfluidic solutions for problems which span an extremely wide range of applications. Customers come to Dolomite with their technical challenges, and Dolomite helps solve these problems using its extensive background technology.

Dolomite also designs & manufactures a wide range of world leading standard components such as OEM products, microfluidic connectors & interfaces, chips, pumps, valves, detectors, sensors & accessories. Finally, we offer design consultancy to create customized chips or connectors and/or a prototyping service for the supply of glass, metal or polymer devices, and custom microfluidic connectors.


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Last updated: Oct 25, 2021 at 7:04 AM

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