APPLICATION NOTE

Accelerate your LNP formulation screening with
Sunscreen’s automated microfluidic technology
used for LNP screening, the process requires the user
to individually load reagents, a time consuming andpotentially wasteful process, and rely on disposable,
high-cost, microfluidic mixing chips, or rely on pipettemixing, which is uncontrolled, hard to reproduce andnot scalable.

Introduction
Lipid Nanoparticles (LNPs) are the leading methodology for the clinical delivery of nucleic acids (NA)applications. The potential for hugely efficient translation from concept to clinic – the first doses of SpikeVax
for clinical testing were rolled out 41 days after theCOVID-19 sequence was published – means that
LNPs stand to revolutionise the drug development
process. Sunscreen is here to boost your formulation
screening and accelerate future discoveries with 96
automated experiments in under 6 hours.
The proved efficacy and outstanding safety profile ofLNP technology have led to an explosion of products
in development, with over 18 products currently inclinical trials. However, LNPs are anything but a simple platform technology. Each formulation must carefully select an ionisable/cationic lipid for RNA binding
and delivery, a structural lipid, cholesterol derivative
and a pegylated lipid, and the relative amounts ofeach. Newer developments are increasing the ranges
of materials used and increasing the number andcomplexity of components, with both passive andactive targeting giving the option of targeting specificorgans and cell types.

Figure 1: LNP formulation effect on biodistribution in-vivo.

Sunscreen is the first product on the market tocombine automated, high-throughput testing, withthe essential precision, scalability and reproducibility of microfluidics, allowing for up to 96 separate experiments to be run in one experimental
set, with no intervention from the operator. Additionally, the Sunnies (microfluidic chips) used arecomposed of ultra-smooth, inert glass; with theautomated wash step built into the experimental
process, these Sunnies are indefinitely reusable.

The mixing of the lipid components, commonly inethanol, and nucleic acids, in aqueous buffer, is alsoa critical step – the total flow rate (TFR) of materials,
the ratio of materials (flow rate ratio, FRR) and thetype of flow (laminar/chaotic/turbulent) affects theloading efficiency, particle structure, and particle size.
All these factors change the pKa, membrane fluidity
and stability of the LNPs, adjusting how they interact
with the complex biomolecular environment foundin-vivo, which can affect biodistribution, expression
and efficacy of the NA.
The resulting design space for LNPs is huge – testing
all these factors can rapidly lead to 100s or 1000s ofexperiments needing to be run. When many of the
microfluidic systems currently on the market are

Figure 2: Sunscreen.

1

ACCELERATE YOUR LNP FORMULATION SCREENING TO LIGHTSPEED WITH SUNSCREEN’S FULLY AUTOMATED
MICROFLUIDICS(error in tables and graphs is the standard deviationof the triplicate measurements unless otherwise
specified).

Sunscreen can use a range of Sunnies to affectdifferent mixing speeds and types, giving full flexibility in how the device is run. The standard Sunny
490 Trident T utilizes hydrodynamic flow focussing(HFF), compressing a central, ethanol/lipid flow withaqueous buffer to reduce the time taken for diffusive
mixing. The flows are very controlled and laminar,
allowing for exquisite control of particle properties.
Alternatively, the Sunny 50 Micromixer intentionallyinduces chaotic flow, leading to reduced mixing times
at lower flow rates (and therefore smaller particlesizes).

EE% was carried out by Invitrogen Quant-it Ribogreen assay following manufacturer’s instructions.
Samples were measured intact and after lysing
with 1% Triton X, and Fluorescence was measured by
FLUOstar Omega plate reader.

Results
Sunscreen’s total of 96 experiments is drawn fromthe fact that it runs from a standard well-plate
format. Robotic arms automate the aspiration of there-agents from the input well plates – as little as 105µL per input – after which the samples are held insample loops.

All Sunnies are compatible with both Sunscreen andSunshine (our process development and scale upsystem) - which are designed to provide a seamless
transition between the stages of your LNP development pipeline.

The system is then primed with driving fluid to establish steady-state flow and pressure in the system,
driven by precision syringe pumps. The reagents arethen released from the sample loops into the mainsystem, where the calibrated tubing and valve timings
allow the two reagent packets to meet on the Sunnysimultaneously, giving excellent mixing control andfidelity. The particles formed are then held in a final
sample loop before being dispensed into the collection well plate.

Methods
Stock solutions in ethanol of 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP),
Dimethyldioctadecylammonium Bromide (DDAB),
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- methoxypolyethylene
glycol-2000 (DSPE-PEG2000), 1,2-Dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000(DMG-PEG2000) (Avanti Polar Lipids) and cholesterol(Chol) (Sigma-Aldrich), were made up. The lipids weremixed in various ratios, with total concentrations of 6mg/mL.

All the flow paths, including the Sunny and fluid handling needles are automatically cleaned when not inuse during each experiment, eliminating sample crosscontamination.
Collected sample sizes range from 200 µL up to 2 mL,with total flow rates from 100 µL/min up to 30 mL/
min and flow rate ratios of between 1:1 and 5:1, giving
a huge range of flexibility to the screening process.
Each sample has a completely customizable headand tail cut to ensure the centre of the sample packet,
with the highest mixing quality and consistency, iscollected – or the entire sample packet. Anti-dispersion technology (ADT) is used to protect the sample
packets form the driving fluids and preserve highconcentrations of reagents.

PolyA (Cytiva) was dissolved in 50 mM citrate buffer,pH 6.00, to give a Nitrogen/Phosphorus (N/P) ratio of
8 for each formulation.
Unless otherwise specified, the TFR was set to 9 mL/min, with a FRR of 3:1 (Aqueuous:Organic). 50 mM citrate buffer, pH 6.00 was used as a driver fluid for theaqueous phase and ethanol was used as a driver fluidfor the organic phase. Total sample size was 1000 µL,
with a 100 µL head and tail cut, giving a total collectedvolume for each sample of 800 µL.

Sunscreen was run through a series of tests toexplore its performance. The first of these looks at
reproducibility across 96 experiments, essential forAll particle size and PDI measurements were carriedout via DLS measurements. All samples were dilutedby 10x in 1x PBS. Samples were measured in triplicate

2ACCELERATE YOUR LNP FORMULATION SCREENING TO LIGHTSPEED WITH SUNSCREEN’S FULLY AUTOMATED
MICROFLUIDICS

Figure 3: Reproducibility and Recoverability of Sunscreen running a DOTAP cLNP formulation over 96 experiments.

Parameter

Description

Z-Average

94.59 nm ± 0.71 nm

PDI

0.142 ± 0.01

RSD0.76%

Total Timewith 90 repeats of a single formulation (DOTAP:DSPC:Chol:DMG-PEG2000, 40:10:37.5:2.5 molar ratio)
and 6 ‘poor’ formulations, with no PEGylated lipid,
included to show system recovery after catastrophic
aggregation of a sample.

05 hours 30 minutes

Reproducibility across the 90 replicates was excellent, with an average particle size of 94.59±0.71, andan overall relative standard deviation (RSD) of lessthan 1% (Figure 3). PDIs were also very consistent, at
0.142±0.01 overall. Samples without PEG were highly
aggregated and polydisperse but had no effect on
the following samples produced, indicating no crosscontamination between samples.

Table 1: Overall results of reproducibility testing. ± is given as thestandard deviation of all results combined. Relative Standard
Deviation (RSD) is given for Z-Average.

establishing that differences in particle properties aresolely down to the formulations and conditions selected and not through random variation in the process
as is often seen with, for example, pipette mixing.

Formulation screen

The second, the formulation screen, uses a fixed set ofconditions – now established to give consistent results
in a stable formulation – but varies the formulation
components to assess how different formulations
affect particle properties, free of other variables andwith low random error.

A large screen of 32 cationic LNP (cLNP) formulations
was carried out in triplicate, investigating the effectsof cationic lipid identity (DOTAP vs. DDAB), structural lipid identity (DSPC vs DOPE), PEG lipid identity(DSPE-PEG2000 vs DMG-PEG2000) and PEG lipid
molar % (0.5, 2.5, 5 and 10 mol%). All experiments
were run from the same 96-well plate, in a single
experimental run lasting 5.5 hours without any
operator intervention or interruption. The particles
were analyzed for Z-average, PDI, and encapsulation
efficiency (EE%).

Finally, a single formulation was run with a screenof the TFR, from 5 to 12 mL/min to assess the effecton particle size and PDI afforded by adjusting flow
conditions.

Reproducibility
To establish the baseline performance and reproducibility of Sunscreen, a 96 experiment series was run,3

ACCELERATE YOUR LNP FORMULATION SCREENING TO LIGHTSPEED WITH SUNSCREEN’S FULLY AUTOMATED
MICROFLUIDICS

Figure 4: Results of cLNP formulation screen. Z-Average and PDIs are the average of each result (n=3), with the error bars given by thestandard deviation.

increases, particle size drops (Figure 5). Less expected is the higher responsiveness of particle size toDSPE-PEG2000 compared to DMG-PEG2000, andthe lower response to DMG-PEG2000 level on DDAB
cLNP particle sizes.

The resulting wall of data is presented above, informulation groups (Figure 4). This single experimental set, run over 5.5 hours, generated more data
than a single graph can accurately convey. Someobvious trends are quickly identified – as PEG level

Figure 5: Effect of DMG-PEG2000 lipid mol% on particle size of cLNPs B – effect of DSPE-PEG2000 on particle size of cLNPs.

identified, including the effect of the structural lipid
on PDI which was higher for DOPE in each case, andthe lower encapsulation efficiency of DDAB/DSPEPEG2000 formulations (Figure 6).

Excluding formulations with 0.5% PEG lipid, which
tended to have low PDIs but high standard deviations
in size measurement, indicating some level of unpredictability/instability, several other relationships were4ACCELERATE YOUR LNP FORMULATION SCREENING TO LIGHTSPEED WITH SUNSCREEN’S FULLY AUTOMATED
MICROFLUIDICSBA

EE% of formulation combinations

Effect of Structural Lipid on PDI (2.5%, 5%, 10% PEG)

Figure 6: A- Effect of structural lipid on cLNP PDI. Significance level calculated via 2 tailed Students T-Test. B – encapsulation efficiency
of cLNP formulations. Significance level calculated via Dunnet’s Test against DDAB/DSPC/Chol/DSPE-PEG2000 formulations.

Flow rate screen
The last test intentionally varied flow conditions toadjust particle size with a set formulation (DOTAP:DSPC:Chol:DMG-PEG2000, 40:10:37.5:2.5 molar ratio).
Particles were formed at 5, 7, 9 and 12 mL/min. As
expected, the particle size drops as the flow rateincreases, as the focusing of the central ethanol flow
occurs faster and mixing times are decreased.
This is clearly evident in the data, with a statisticallysignificant difference in particle size between each
set of flow rates, demonstrating the excellent control
and reproducibility of LNPs produced on Sunscreen(Figure 7).

Figure 7: Effect of TFR on DOTAP cLNP size and PDI. Error bars
represent the SD of 3 measurements. Significance level calculated via 2 tailed Students T-Test. Significance levels are indicated by* p = 0.05, ** p = 0.01.

TFR(mL/min)

Z-Average (nm)PDI

5137.9 ±1.11

0.11 ±0.01

7110.6 ±1.960.13 ±0.00

988.24 ±0.52

0.13 ±0.00

1269.48 ±0.38

0.14 ±0.00

Table 2: Effect of TFR on DOTAP cLNP average size and PDI. Error
is given by the SD (n=3).

5ACCELERATE YOUR LNP FORMULATION SCREENING TO LIGHTSPEED WITH SUNSCREEN’S FULLY AUTOMATED
MICROFLUIDICS

Conclusion
Sunscreen’s winning combination of automation andmicrofluidics mean it is an essential tool in any LNP
development laboratory. When paired up with aStunner for 96-well plate format particle size analysis, a full plate of 96 formulations can be synthesized
and characterized within a working day – all using justa single Sunny.
The consistency provided by precision microfluidic
mixing guarantees high quality, reproducible particles,
and the automation allows integration into complex,
parallel work flows to supercharge your throughput. Whether you are screening cargos, novel lipids,
formulation compostions, or production parameters,
Sunscreen leaves everything else in the shade!

Unchained Labs
4747 Willow Rd,
Pleasanton, CA 94588
Phone: 1.925.587.9800
Toll-free: 1.800.815.6384
Email: info@unchainedlabs.com© 2023 Unchained Labs. All rights reserved. The UnchainedLabs logo, Sunscreen, and Sunshine are trademarks and/or registered trademarks of Unchained Labs. All otherbrands or product names mentioned are trademarks
owned by their respective organizations.

Rev A6