APPLICATION NOTE

Flexible nanoparticle development: processoptimization & scale-up of a wide range of synthetic
nanomedicines with Sunshine
In contrast, by using microfluidic technology to combine reactants in a precise mixing geometry using
stable, highly controlled, laminar flows, the reaction
chemistry is highly controlled which results in theformation of NPs with significantly improved consistency. Through the use of a hydrodynamic flow
focusing approach, the reaction time for nanoparticle
synthesis (and therefore particle size/morphology)
can be precisely and reproducibly tuned simply byvarying the flow rates of the reactants into the Sunny(microfluidic chip).

Introduction
Nanomedicine is one of the most promising fields indrug delivery, with the potential to help overcome theconstraints of conventional formulation approaches
and find widespread applications in the developmentof novel drug products and therapeutics. Synthetic
nanomaterials such as liposomes, lipid nanoparticles(LNPs), and polymer nanoparticles can be used invarious ways to improve the solubility, stability, andbioavailability of various Active Pharmaceutical Ingredients (APIs), which in turn can increase API transport
across cell membranes, as well as extending the invivo circulation time for enhancing the efficacy andsafety of the APIs.

Recent years have seen a rapid increase in theinvestigation and development of nanomedicines
using particles such as LNPs for delivering vaccines
and advanced therapies, with microfluidics being
widely adopted as a core technique for their synthesis(Figure 1). However, previous commercially available microfluidic technologies have required users
to devote significant hands-on time to conduct theDue to the wide range of complex physiochemical properties of APIs, from small molecule drug
compounds to nucleic acids and proteins, a flexible
nanoparticle generation process is beneficial to enable the formation of different particle types depending on the API in question and the desired properties.
Sunshine from Unchained Labs offers an automatedmicrofluidic approach to developing and optimizing
milliliter-scale nanoparticle synthesis protocols for arange of material types and particle structures, aswell as having the capability to continuously manufacture them in multi-liter quantities for pre-clinicaltesting.
Over the past few decades, lipid-based nanoparticles (including liposomes, lipoplexes and LNPs) and
nanoparticles (NPs) of various polymers, metals
and oxides have been produced by batch methods
such as thin film hydration, sonication, and nanoprecipitation; however, these traditional techniques
are laborious and can involve large batch volumes.
Furthermore, these methods lack batch-to-batch
consistency in terms of particle size, morphology, andencapsulation efficiency, which are important factors
in the efficacy of the final drug formulation.

Figure 1: An example Sunny setup for the synthesis of nanoparticles, e.g. LNPs. At the first Sunny junction, in the case of LNPs, lipids in ethanol and mRNA in aqueous buffer are brought together
in a laminar manner to generate a hydrodynamically focused flowof reagents. The reagents diffuse into one another reducing thesolubility of the lipids which drives the self-assembly process. Thismode of mixing provides highly controlled reaction conditions andresults in very consistent and tunable LNP formation. An optional
second Sunny is included for the in-line addition of diluent for pH
adjustment and reduction of solvent concentration in collected
samples.

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FLEXIBLE NANOPARTICLE DEVELOPMENT: PROCESS OPTIMIZATION & SCALE-UP OF A WIDE RANGE OFSYNTHETIC NANOMEDICINES WITH SUNSHINE

syntheses, often involving larger reagent volumes andthe use of high-cost consumables.

Synthesis of PLGA nanoparticles
with Sunshine

In order to significantly accelerate the developmentof nanomedicines, researchers need reliable, lowcost, and efficient nanoparticle production methods
that can work flexibly with a wide range of materials.
Unchained Labs’ pioneering microfluidic technology
is utilized in Sunshine to produce homogeneous andreproducible nanoparticles (Figure 2). With Sunshine,users can control flow rate ratio (FRR), total flow rate
(TFR), precursor volume, sample collection volume
and optional in-line dilution to enable the controlled,
reproducible, and monodispersed formation ofvarious nanoparticles. Sunnies not only reduces
consumable cost but also provides a system which iscompatible with a wide array of reagents and solvents.

Poly (lactic-co-glycolic acid) (PLGA) nanoparticles
are commonly used in the field of drug delivery andcontrolled release, owing to their biocompatibility andbiodegradability. An excellent example of the use of
PLGA NPs is in the delivery of ocular drugs, such as
the anti-inflammatory drug triamcinolone acetonide.
Encapsulation of these drugs in PLGA results in an
elongated release profile which reduces the required
dosage and the frequency of unpleasant drug administration processes, increasing patient compliance.
Additionally, PLGA NPs exhibit unique properties, i.e.
specific surface area and functionality, which enable
PLGA encapsulated drugs to overcome the challengeof the ocular barrier (Jiang et al).1
Precise control of the nanoparticle’s size is crucial as
size is crucial in determining the ultimate efficacy of
API delivery. Traditional batch methods of PLGA NP
synthesis lack reproducibility due to the inability to
precisely control the reaction conditions. The resultof this is typically generating particles of variable size
but can also lead to uncontrolled particle aggregation, either of which will have a detrimental effect on
experimental results and hamper drug discovery &development.

Parameter

Figure 2: Sunshine

This application note illustrates how Sunshine canwork flexibly with multiple material types to produce
nanomaterials for use in nanomedicine development.
Various nanoparticles were synthesized using PLGA,
two distinct liposomal formulations, and two mRNA-LNP formulations. The particles produced were
all within the size range of 20 nm to 150 nm, with apolydispersity index (PDI) <0.2 and yielding encapsulation efficiencies greater than 90%. This work
demonstrates that being able to precisely control
and vary the FRR, TFR and degree of dilution of thereactants is central to being able to fully characterize
and optimize nanoparticle synthesis and Sunshine is
the ideal tool to achieve this.

Description

Aqueous Phase (A)Deionized (DI) water

Organic Phase (O)0.5% (w/v) PLGA in
acetoneFlow Rate Ratio(FRR)

Variable (A:O)

Total Flow Rate
(TFR)

3 mL/minSunny

Sunny 275 XT

Table 1: Parameters for the synthesis of PLGA nanoparticles using
Sunshine.

1 doi: 10.2147/IJN.S272750

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Sunshine enables the rapid development of PLGA
nanoparticles. In this example, a Sunny 275 XT, whichmixes reagents using hydrodynamic flow focusing(HFF), was used to synthesize the nanoparticles.
The size of PLGA nanoparticles was precisely andreproducibly controlled between 83 nm and 57 nm
by varying the FRR from 1:1 to 8:1 (aqueous to organic
phase correspondingly) while maintaining a constant
TFR of 3 mL/min (Figure 3). Notably, the polydispersity index (PDI) for the produced nanoparticles
averages below 0.1, indicating the monodispersity of
the PLGA nanoparticles formed. This is critical for aconsistent release profile for an encapsulated API.

Synthesis of liposomes withSunshine
Liposomes are lipid-based vesicles that are formedof one or more phospholipid bilayers. They can act
as biomimetic compartments with a membrane thathas a significant similarity to natural cell membranes.
Due to the relative simplicity of their formulation,
together with excellent biocompatibility, high bioavailability, and flexibility in delivering both hydrophilic andhydrophobic APIs, liposomes have garnered growing
and significant interest as a delivery system for a wide
range of cargos. For these reasons, liposomes are
currently the most common class of FDA-approved
nanomedicines.

Such PLGA particles can be further optimized using
variables such as TFR, Sunny type, and by introducing
stabilizing materials to the aqueous phase.

Parameter

Description

Aqueous Phase (A)1x PBS, pH 7.4

Organic Phase (O)Phospholipon 90G (1 mg/mL), DDAB (0.1 mg/mL) in
ethanol

Dilution

1x PBS pH 7.4

Flow Rate Ratio(FRR)

Variable (A:O:D)

Total Flow Rate
(TFR)

3 mL/minSunny
Figure 3: PLGA nanoparticles produced by varying aqueous
to organic FRR at a TFR of 3 mL/min. Particle size distribution
and polydispersity index (PDI) were determined by dynamic lightscattering (DLS). The error bars represent standard deviation of
the mean, n=3..

Sunny 100 XT, with Sunny
190 XT for dilution

Table 2: Parameters for the synthesis of phospholipon-based
liposomes using Sunshine.

In this first example, the results of a liposomal synthesis using a formulation based on phospholipon
90G and a cationic lipid, dimethyl-dioctadecyl-ammonium bromide (DDAB) is reported. Unlike PLGA
NP synthesis where acetone is required as a solvent,the lipid components for this liposome formulation
were suspended in ethanol to form the organic input
while phosphate buffered saline (1 x PBS, pH 7.4) was
employed for both the aqueous and dilution inputs.
When evaluating a new formulation, a rapid screenof FRR can be used effectively to explore parameters
and identify both ideal and non-ideal conditions forparticle formation (Figure 4).

Using the Sunny Suite Software, users can optimize
the FRR, TFR, NP precursor volume, sample collection
volume and allow optional in-line dilution to synthesize controlled, reproducible, and monodispersed
nanoparticles. For example, a series of 10 low-volume
experiments with a fixed pair of reagents testing at
various conditions of FRR, TFR, and dilution ratio
could be executed automatically in 15 minutes inprotocol mode.

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FLEXIBLE NANOPARTICLE DEVELOPMENT: PROCESS OPTIMIZATION & SCALE-UP OF A WIDE RANGE OFSYNTHETIC NANOMEDICINES WITH SUNSHINE

Parameter

Description

Organic Phase (O)DMPC: cholesterol: DMGPEG 2000 (2:1:0.05
molar) in ethanol

Aqueous Phase (A)PBS (Phosphate Buffered
Saline) (1X, pH 7.4)

Dilution Phase (D)PBS (Phosphate Buffered
Saline) (1X, pH 7.4)

Flow Rate Ratio(FRR)

3:1:1 (A:O:D)

Total Flow Rate (TFR)
Sunny
Figure 4: Phospholipon-based liposomes produced by varying
aqueous to organic FRR at TFR of 3 mL/min. Liposome sizes
ranged from 20 nm to 150 nm, in which particle size distribution
and polydispersity index (PDI) were determined by dynamic lightscattering (DLS). The error bars represent standard deviation of
the mean n=3.

Variable
Sunny 100 XT, with Sunny
190 XT for dilution

Table 3: Conditions to produce liposome-based DMPC: cholesterol: DMG-PEG 2000 using Sunshine with FRR at 3:1:1 (A:O:D).

The data presented in Figure 5 shows that increasing
the TFR results in an 18% reduction in the hydrodynamic size of the liposomes produced, from 133
nm to 109 nm. Increasing the TFR also resulted inparticles being produced with an improved, reduced
PDI further highlighting the importance of assessing
the response of a formulation to a range of process
conditions.

The results presented above show the size of theliposomes produced varying between 20 nm and 150
nm in direct response to the FRR being used. In thisexperiment the ratio of aqueous:organic:diluent wasvaried from 1:0.5:1 to 1:3.5:1, keeping the aqueous anddiluent components fixed and increasing the organic
component. The TFR was maintained at 3 mL/min for
all experiments.
At lower organic ratios, PDIs were undesirably high,whereas a ratio of 1:3:1 gave the lowest PDI, albeit ata larger than desired particle size. A ratio of 1:2:1 gavea suitable particle size of around 80 nm, with a PDI
reproducibly below 0.2. Further investigation of otherconditions such as TFR, Sunny geometry, and so on,
can then be investigated at this optimized flow rate
ratio to further optimize PDI and other performance
criteria.
A second example of liposome synthesis is shown
below using a formulation of 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), cholesterol, and1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000 (DSPE-PEG 2000) in a molar ratioof 2:1:0.05, respectively. For this study, the effect of
TFR on liposome size was examined. Sunshine was
employed in Protocol Mode to execute a batch ofexperiments in which the TFR was increased from 2
mL/min to 5 mL/min while maintaining a FRR of 3:1:1(aqueous:organic:dilution).

Total Flow Rate (mL/min)Figure 5: Conditions to produce liposomes based on DMPC,
cholesterol and DMG-PEG 2000 using Sunshine with various
TFRs and a fixed FRR of 3:1:1 (A:O:D). Particle size distribution
and polydispersity index (PDI) were determined by dynamic lightscattering (DLS). The error bars represent standard deviation of
the mean, n=3.

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Figure 6 shows that the size of mRNA-LNPs formed
reduced by 14.7%, from 68 nm to 58 nm, as the FRR
increased from 2:1 to 6:1 (aqueous:organic). Particle
size and PDI were assessed by DLS post-dialysis anda consistently low PDI was recorded for all conditions
tested.

Synthesis of lipid nanoparticles
with Sunshine
Over recent years, lipid nanoparticles (LNPs) havearguably become the most significant subset of
lipid-based nanocarriers. LNPs were initially derived
from well-established liposome technology, withthe addition of cationic or ionizable cationic lipids forencapsulation of negatively charged oligonucleotide
materials, such as siRNA, mRNA, miRNA, and DNA
via electrostatic interactions. The field of study has
grown exponentially, and work is being conducted
to fully understand the implications of formulation,
composition, size, surface chemistry and a host ofother parameters to enable the effective and targeted delivery of a wide range of payloads, from CRISPR
therapies to mRNA vaccines.

Parameter
Aqueous Phase (A)Luciferase mRNA
(4basebio PLC) in acetate
buffer, pH 4

Organic Phase (O)SM-102: DSPC:
cholesterol: DMG-PEG
2000 (50:10:38.5:1.5
molar ratio) in ethanol

Dilution (D)Flow Rate Ratio(FRR)
Total Flow Rate (TFR)

Figure 6: mRNA-LNP produced by various FFR (aqueous:organic)
at a fixed TFR of 5 mL/min with no dilution. LNPs ranged from 58
nm to 68 nm post-dialysis, with an average PDI of 0.12. Particle
size distribution and polydispersity index (PDI) were determinedby dynamic light scattering (DLS). The error bars represent thestandard deviation of the mean, n=3.

Description

Using a Quant-it™ RiboGreen RNA Assay Kit (Thermo
Fisher Scientific), hereafter ‘RiboGreen’, the mRNA encapsulation efficiency (EE%) was examined. While the
TFR was maintained at 5 mL/min and the N/P ratio
was kept constant at 6, the data in Figure 7 showsthe encapsulation efficiency (EE) was above 90% for
all FRRs tested with a FRR of 6:1 (aqueous:organic)
providing the highest encapsulation efficiency of 96%.

No dilution
Variable (A:O)
5 mL/min

N/P

6

Sunny

Sunny 190 XT

Table 4: Parameters for the synthesis of mRNA-LNP utilizing
Sunshine.

Here, a formulation of the ionizable cationic lipid SM102 with DSPC, cholesterol and DMG-PEG 2000, in a
molar ratio of 50:10:38.5:1.5, was prepared. Luciferase mRNA (4basebio PLC) was encapsulated with aratio of cationic lipid charge to the mRNA’s phosphate
negative charge (the N/P ratio) of 6. LNPs were
produced using Sunshine to automatically perform aseries of small volume experiments each at different
FRRs while maintaining a TFR of 5 mL/min.

Figure 7: The mRNA encapsulation efficiency (EE%) in the LNPs
produced was obtained by the RiboGreen assay. mRNA-LNPs
were produced using Sunshine at a fixed TFR of 5 mL/min andvarious FRR (aqueous:organic) with no dilution. The N/P ratio wasfixed at 6. The error bars represent standard deviation of themean, n=3.

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FLEXIBLE NANOPARTICLE DEVELOPMENT: PROCESS OPTIMIZATION & SCALE-UP OF A WIDE RANGE OFSYNTHETIC NANOMEDICINES WITH SUNSHINE

A second LNP formulation based on the cationic lipid
DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) was prepared as detailed in Table 5 below. For
this work, the payload is Polyadenylic acid (Poly(A),Merck, Cat# GE27-4110-01) which is a single-stranded RNA homopolymer of the nucleotide Adenine withan average chain length of >200 nucleotides. Poly(A)has been demonstrated to be a suitable analogue formRNA for the purpose of particle synthesis experiments (data available on request).

Parameter

Description

Aqueous Phase (A)Poly(A) in citrate, pH 6

Organic Phase (O)DOTAP: DSPC:
cholesterol: DMG-PEG
2000 (40:10:47.5:2.5
molar) in ethanol

Dilution (D)The Poly(A) was loaded into DOTAP-based LNPs
which were made using Sunshine in combination
with a Sunny 490 Trident T. A FRR of 3:1 (aqueous:organic) was maintained throughout a seriesof experiments in which the TFR was varied from 5
mL/min to 12 mL/min to explore the effect of TFR
on the size and polydispersity of the nanoparticles
produced. As shown in Figure 9 the Poly(A)-loaded DOTAP-based LNPs produced could be consistently and reproducibly synthesized with sizes
ranging between 128 nm to 66 nm with the PDI ofaround 0.13.
Three replicates were performed for each TFR. All
LNPs produced exhibited highly consistent sizes
with PDIs below 0.2 and with a relative standard
deviation (RSD) that was less than 4%, which isregarded as repeatable.

1x PBS, pH 7.4

Flow Rate Ratio(FRR)

3:1 (A:O)

Total Flow Rate (TFR)

Variable

N/P

8

Sunny

Sunny 490 Trident T

Table 5: Parameters for the synthesis of Poly(A)-LNP based
DOTAP utilizing Sunshine..

Figure 9: Poly(A) encapsulated in DOTAP-based LNPs produced
at various TFRs at a fixed FRR (3:1) (aqueous:organic). LNPs
ranged from 66 nm to 128 nm, with polydispersity index (PDI)
values around 0.13. Particle size distribution and PDI were determined by dynamic light scattering (DLS) The error bars represent
standard deviation of the mean, n=3. .

Figure 8: Sunny 490 Trident T - entry of DOTAP lipids in organic
solvent and payload Poly(A) in aqueous buffer, hydrodynamic
flow focusing, and subsequent mixing assembles Poly (A)-LNPs,
with the option of in-line dilution. The linear connector affixes tothe top surface of the Sunny, enabling the mRNA input to be split
on-chip into 2 fluid streams and recombined with the lipid/ethanol input at the trident junction..

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to other synthesis methods and can allow for theparticle size to be readily manipulated.

Conclusions
Due to the distinct formulation properties of thedifferent materials used in nanoparticle synthesis, it is challenging to determine an optimization
process which would be generally applicable.
This has led to high demand for an all-in-one
system capable of rapidly synthesizing and effectively screening process parameters to optimize
nanoparticle production from a variety of nanomaterials. In addition, conventional methods ofbulk synthesis typically produce nanoparticles
that are not homogeneous or predictable. In fact,
effective pharmacological delivery requires extremely precise and reproducible control of theparticles produced, therefore particles producedby batch methods are not a particularly attractive
option for the pharmaceutical industry.

Sunshine can be utilized to rapidly optimize theformation of a wide variety of nanoparticles, fromPLGA nanoparticles, liposomes, and ionizable or
cationic-LNPs. By allowing users to define andautomatically execute batches of experiments(each with different TFRs and FRRs), the optimal
set of conditions for nanoparticle synthesis can be
determined. Once determined these conditions
can be used by Sunshine to produce volumes ofnanoparticles up to 10s of liters per day in continuous mode.

Materials and methods
For the production of PLGA nanoparticles, poly(lactic-co-glycolic acid) (Sigma Aldrich, UK) was
dissolved in acetone (Sigma Aldrich, UK) at aconcentration of 0.5% (w/v). Deionized water wasused for the aqueous phase. All formulated solutions underwent filtration using a 0.2 µm filter. A
Sunny 275 XT was used for all experiments.

Microfluidics, unlike conventional batch synthesis
methods, provides the ability to manipulate particle sizes by adjusting the TFR, FRR and/or chipgeometry. For example, increasing the flow rate
ratio of the aqueous to organic phase, for mostformulations, results in a reduction in nanoparticle
size.

To produce the liposomes based on phospholipon and DDAB, phospholipon 90G (Lipoid, Switzerland) and DDAB (Fisher Scientific, UK) weredissolved in ethanol (reagent grade 99%, Sigma
Aldrich, UK) at concentration of 1 mg/mL and 0.1
mg/mL respectively. For the aqueous and dilution
phase, 1x PBS at pH 7.4 was used. All prepared
solutions were filtered with a 0.2 µm filter prior
to use. Particles were formed on a Sunny 100 XT
with a second, Sunny 190 XT included in series forin-line addition of diluent.

During the nanoparticle syntheses described here
it can be seen that adjusting the FRR to increase
the organic component typically results in an
increase in the size of the particles produced. The
formation of larger particles is driven by the higher
concentration of lipids/polymer per unit volume
and longer diffusion/mixing time.
Similarly, TFR also plays a critical role in determining the size of particles formed, as increasing TFR(and therefore fluid velocity & Reynolds number)through a mixing geometry will reduce mixing time,
and therefore the size of the resulting nanoparticles.

The second liposome formulation was preparedusing DMPC (Avanti, USA), cholesterol (Sigma
Aldrich, UK), and DMG PEG 2000 (Avanti, USA)
which were dissolved in anhydrous ethanol (Sigma Aldrich, UK) with the molar ratio of 2:1:0.05,
respectively. 1x PBS at pH 7.4 was used for theaqueous phase and dilution buffer. For these liposomes, a Sunny 190 XT was used for both synthesis and in-line dilution.

Sunshine provides a unique platform for the development of nanoparticles by combining precision
microfluidics and automation to rapidly screen andidentify the optimal process conditions for a given
formulation and nanoparticle type. With microfluidics, the resulting nanoparticles typically exhibit amuch narrower size distribution when compared

SM-102-based mRNA-LNPs were synthesized using DSPC (Avanti, USA), DMG PEG-2000 (Avanti,USA), cholesterol (Sigma Aldrich, UK) was dis-

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solved in ethanol to a final concentration of 10 mg/
mL. SM-102 (BroadPharm, San Diego, US) was
supplied as a 100 mg/mL solution in ethanol andcombined with the initial lipid solution to prepare
a mixture with an overall concentration of 10 mM
with a relative molar ratio of 50:10:38.5:1.5 of
SM-102, DSPC, cholesterol and DMG PEG-2000
respectively. For the aqueous phase, 89.44 µg/mL
of mRNA (Luciferase) was dissolved in 50 mM ofacetate buffer, pH 4 (nuclease-free). A Sunny 100
XT was used for LNP synthesis.

Particle size distribution and polydispersity index(PDI) were determined by DLS. All samples wereanalyzed in triplicate with the mean size and standard deviation (SD) of the replicates reported.
The nanoparticles formed were dialyzed using a
dialysis kit (Pur-A-lyzerTM Midi Dialysis Kit, Sigma,UK) and placed in a beaker containing 1 L of PBS 1x
pH 7.4. The dialysis was run for 3 hours to remove
the ethanol and exchange the buffer medium. The
post-dialysis solution of nanoparticles was stored
in the fridge for further investigation.

DOTAP-based LNPs were produced using DOTAP(Avanti, USA), DSPC (Avanti, USA), DMG PEG2000 (Avanti, USA), cholesterol (Sigma Aldrich,
USA). The lipid components were dissolved in ethanol to form a 10 mM solution with the molar ratioof 40:10:47.5:2.5 of DOTAP, DSPC, cholesterol and
DMG PEG-2000 respectively. For the aqueous
phase, 64 µg/mL of Poly(A) was prepared in 50
mM of citrate buffer, pH 6.0 (nuclease-free). A
Sunny 490 Trident T was used for LNP synthesis.

The encapsulation efficiency (EE%) of mRNA-LNP
was quantified by RiboGreen assay by using
Triton X-100 to break open the LNP to determine
concentration of total mRNA in the sample andcomparing this value to the concentration of freemRNA, determined in the absence of Triton. The
fluorescence intensity of RiboGreen was measure on a Microplate reader (FLUOROstar, BMG,
Germany).

For all nanoparticles prepared, the organic (ethanol or acetone) and aqueous solutions were mixed
microfluidically and automatically using Sunshine
to perform batches of experiments at various total
flow rates and flow rate ratios.

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.

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