International Journal of Pharmaceutics 640 (2023) 122985

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International Journal of Pharmaceutics

journal homepage: www.elsevier.com/locate/ijpharm

Sequential Flash NanoPrecipitation for the scalable formulation of stable

core-shell nanoparticles with core loadings up to 90%
Nicholas J. Caggiano a, 1, Satya K. Nayagam a, 1, Leon Z. Wang a, Brian K. Wilson a, Parker Lewis a,
Shadman Jahangir a, Rodney D. Priestley a, b, Robert K. Prud’homme a, Kurt D. Ristroph a, *
a
Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ 08544, United States
b
Princeton Materials Institute, Princeton University, Princeton, NJ 08544, United States

A B S T R A C T

Flash NanoPrecipitation (FNP) is a scalable, single-step process that uses rapid mixing to prepare nanoparticles with a hydrophobic core and amphiphilic stabilizing
shell. Because the two steps of particle self-assembly – (1) core nucleation and growth and (2) adsorption of a stabilizing polymer onto the growing core surface –
occur simultaneously during FNP, nanoparticles formulated at core loadings above approximately 70% typically exhibit poor stability or do not form at all.
Additionally, a fundamental limit on the concentration of total solids that can be introduced into the FNP process has been reported previously. These limits are
believed to share a common mechanism: entrainment of the stabilizing polymer into the growing particle core, leading to destabilization and aggregation. Here, we
demonstrate a variation of FNP which separates the nucleation and stabilization steps of particle formation into separate sequential mixers. This scheme allows the
hydrophobic core to nucleate and grow in the first mixing chamber unimpeded by adsorption of the stabilizing polymer, which is later introduced to the growing
nuclei in the second mixer. Using this Sequential Flash NanoPrecipitation (SNaP) technique, we formulate stable nanoparticles with up to 90% core loading by mass
and at 6-fold higher total input solids concentrations than typically reported.

1. Introduction
Nanoparticle (NP) delivery of hydrophobic compounds is of
increasing interest for solubility enhancement since a large proportion
of new small molecule drugs exhibit poor solubility in water, thereby
limiting oral bioavailability (Lipinski, 2000; Savjani et al., 2012; Thayer,
2010). NP delivery is also of interest for parenteral and oral delivery of
hydrophilic compounds, including biomolecules such as proteins, pep­
tides, and nucleic acids (Anselmo et al., 2019; Paunovska et al., 2022).
Several approaches to prepare nanoparticles have been presented and
reviewed;(Khan et al., 2019; Kumar et al., 2020) however, many tech­
niques are limited by low core loading (mass fraction of core material
per mass of total NP) or poor process scalability (Cai et al., 2015; Qin
et al., 2017; Shen et al., 2017; Zhang et al., 2015). Our research has
focused on a kinetically controlled, polymer-directed, rapid precipita­
tion process termed Flash NanoPrecipitation (FNP). FNP relies on
controlled turbulent micromixing in specially designed mixing cham­
bers to produce supersaturations as high as 10,000 in 1.5 ms (Johnson
and Prud’homme, 2003a; Pinkerton et al., 2015). The technique is
scalable and has been demonstrated to produce the same monodisperse
nanoparticles from laboratory scales (milligrams) to industry scales
(kilograms) (Bobbala et al., 2018; Feng et al., 2019a; Li et al., 2014; Yu
et al., 2021; Zeng et al., 2019). Although FNP typically produces NPs
with core loadings of 33–70%, even higher drug loadings are desired to
reduce the mass of excipients relative to the therapeutic compound
(Saad and Prud’homme, 2016; Shen et al., 2017; Tao et al., 2019; Zhu,
2014). Additionally, producing NPs at higher mass concentrations re­
duces solvent waste and improves process mass intensity.
Rapid precipitation processes, including FNP, fail to produce stable
nanoparticles when either (a) the total solids concentration during
precipitation, or (b) the concentration of core material relative to the
concentration of the stabilizer, is too high. Normally, the maximum
achievable total solids concentration of NPs is approximately 0.1 wt% in
the final dispersion (Pagels et al., 2018; Pustulka et al., 2013). At low
total solids concentrations, the ratio of core material to stabilizer (e.g.,
drug loading) for nonionic hydrophobic species is usually limited to
20–50% (Pagels et al., 2018). The limitations in total solids and drug
loading are related to the mechanism of self-assembly. It has been sug­
gested that for formulations with core loadings above 70%, the ability of
stabilizing polymer molecules to adsorb strongly onto a particle nucleus
(so-called ‘anchoring efficiency’) is decreased due to the presence of
stabilizer molecules which have become kinetically trapped within the

* Corresponding author at: Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47909, United States.
E-mail address: ristroph@purdue.edu (K.D. Ristroph).
1
Contributed equally.

https://doi.org/10.1016/j.ijpharm.2023.122985
Received 28 February 2023; Received in revised form 18 April 2023; Accepted 21 April 2023
Available online 29 April 2023
0378-5173/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
N.J. Caggiano et al.
growing particle core (Pustulka et al., 2013). This reduces the effective
amount of available stabilizer and introduces packing inefficiencies in
the drug-loaded NP core. The result is larger, more polydisperse nano­
particles which exhibit poor size stability over time (Fig. 1A).
Temporally separating the precipitation of the core materials from
the adsorption of the stabilizing polymer surface material is a strategy to
overcome the limitations of loading. One method to achieve this sepa­
ration is through the use of mixed solvent systems to effect sequential
separation of components. For example, Liu, Weitz, et al. demonstrated
a two-step batch nanoprecipitation process where two sequential ali­
quots of antisolvent were added to an initial ternary solvent mixture
containing core and polymer stabilizer. The first antisolvent addition
caused precipitation of the core material, while the stabilizer remained
soluble. After the second addition core–shell nanoparticles were pro­
duced with loadings of up to 59% (Han et al., 2020; Liu et al., 2020). A
similar effect can also be produced in a continuous process using
microfluidics to introduce antisolvents sequentially for the core and
stabilizing compounds (Liu et al., 2017). However, both cases require
careful design of solvent systems to achieve sequential precipitation.
Additionally, there are limitations to scaling microfluidics to large-scale
nanoparticle production. In microfluidics, rapid solvent diffusion is
achieved using the micron length scales of the microfluidic device; in
small geometries the flows are laminar. In contrast, during turbulent
mixing in FNP the small length scales for diffusion are achieved by using
turbulence to establish solvent-antisolvent striations at the Kolmogorov
length scale (Johnson and Prud’homme, 2003b). FNP has been applied
to various payloads and stabilizing polymer chemistries and is highly
scalable (Feng et al., 2019a; Pinkerton et al., 2017; Pustulka et al., 2013;
Yan et al., 2021; Yu et al., 2021; Zeng et al., 2019).
Our process, which we term Sequential Flash NanoPrecipitation
(SNaP), is accomplished by the sequential arrangement of two mixing
chambers. In the first chamber, the hydrophobic core material is
precipitated by rapid mixing and antisolvent-induced supersaturation.
Before the hydrophobic cores can aggregate beyond the desired size
scale, they enter a second FNP mixing chamber where the stabilizing
polymer is introduced, which adsorbs onto the NP core surfaces
(Fig. 1B). Conceptually, this is similar to the ability to introduce coating
layers onto nanoparticle surfaces via FNP, as we have demonstrated for
the coating of latex spheres(D’Addio et al., 2012) or inorganic oxide
Fig. 1. Principles of conventional FNP and sequential SNaP processes. (A) FNP utilizes a one-step mixing process where aggregation and stabilization occur
simultaneously. (B) Proposed two-step mixing process where aggregation occurs followed by stabilization.
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International Journal of Pharmaceutics 640 (2023) 122985
colloids (Budijono et al., 2010; Ungun et al., 2009). In SNaP, the delay
time between the initial core precipitation and the subsequent applica­
tion of the stabilizing layer is determined by the residence time in the
tubing connecting the first and second chambers. This delay time is
intentionally kept to a minimum to limit the extent of core aggregation
which occurs between the precipitation and coating steps.
2. Materials and methods
2.1. Reagents
Polymeric core material polycaprolactone diol (PCL, 2 kDa) was
obtained from Sigma-Aldrich (St. Louis, MO) and used without further
purification. Hydroxypropyl methylcellulose acetate succinate
(HPMCAS; AQOAT® AS-HF) was generously provided by Shin-Etsu
Chemical Co. (Tokyo, Japan). Tetrahydrofuran (THF, HPLC grade)
was purchased from Fisher Chemical (Hampton, NH). Sodium hydroxide
and sodium chloride were purchased from Sigma-Aldrich (St. Louis,
MO). Deionized water (18.2 MΩ•cm) was prepared using a Thermo
Scientific Nanopure Diamond water system (Waltham, MA).
2.2. Sequential mixing and nanoparticle characterization
2.2.1. Preparation of FNP control formulations.
Flash NanoPrecipitation was performed using a confined impinging
jet mixer (CIJ) as reported previously (Han et al., 2012; Markwalter
et al., 2019; Saad and Prud’homme, 2016). In all nanoparticle formu­
lations studied here, PCL was the core material and HPMCAS was the
stabilizing material. In brief, a solvent stream was prepared by dis­
solving polycaprolactone (PCL, 2 kDa) in tetrahydrofuran at various
concentrations. An aqueous antisolvent stream was prepared by dis­
solving HPMCAS free acid at various concentrations in water with
15 mM NaCl along with 1.08 equivalents of NaOH to ionize the succinyl
groups on the polymer. The solvent and antisolvent solutions (0.5 mL
each) were loaded into separate Luer-lip syringes, which were attached
to a CIJ and rapidly depressed. The mixer effluent was immediately
collected in a 4 mL water reservoir. Formulation details for conventional
FNP formulations, including stream compositions, NP loadings, size, and
polydispersity index can be found in Table S1.
N.J. Caggiano et al.
2.2.2. Preparation of SNaP formulations.
For sequential nanoprecipitation, two CIJ mixers were used. Two
2.5 mL glass Luer-lock syringes were attached to the first mixer, one
containing 1 mL of a THF solvent stream with dissolved PCL and the
other containing 1 mL of 15 mM NaCl in water. The effluent port from
this mixer was attached to one inlet on the second CIJ mixer by PEEK
tubing (described below). To the remaining port of the second mixer was
attached a 5 mL glass Luer-lock syringe containing 2 mL of HPMCAS
(with 1.08 equivalents of NaOH) at various concentrations in water with
7.5 mM NaCl. All three syringes were depressed simultaneously, and the
effluent from the second mixer was immediately collected in a 6 mL
water reservoir. Formulation details for SNaP formulations, including
stream compositions, NP loadings, size, and polydispersity index can be
found in Table S2.
2.2.3. Size characterization by dynamic light scattering.
Following formulation, aliquots were removed from nanoparticle
dispersions and diluted in water to a NP concentration of 0.1 mg mL− 1
for characterization. Size and polydispersity index (PDI) were measured
by dynamic light scattering (DLS) using a Zetasizer Nano-ZS (Malvern
Instruments, Malvern, UK) (n = 3). Particle size is reported as Z-average
hydrodynamic diameter obtained from intensity-weighted distributions.
Nanoparticle stability was monitored and evaluated using both visual
observation and DLS.
2.3. Equipment and mixing setups
A mixing stand was designed in-house to allow for depression of
multiple syringes for the SNaP process in making small-scale formula­
tions, as shown in Figure S1. The stand base plate was modified to seat
two confined impinging jet (CIJ) mixers and, later, one CIJ and one
multi-inlet vortex mixer (MIVM) (Liu et al., 2008; Markwalter et al.,
2019). Detailed CAD drawings can be found in Figure S2. For SNaP, the
outlet of the first mixer was connected to one of the inlets of the second
mixer with 250 mm of 0.030-inch inner diameter polyether ether ketone
(PEEK) tubing. Syringes were fixed on the remaining inlets of both
mixers. The depression plate was then manually pushed down to initiate
mixing. The time required to depress syringes using the mixing stand
was measured as tinject = 0.35s using video footage of the process and
averaging across multiple trials. This corresponds to a volumetric flow
rate of 2.3 mL s− 1. For SNaP, the total time between when a fluid
element entered the mixing chamber of the first mixer (mixing chamber
volume ~ 0.2 mL) and the second mixer was 0.14 s.
3. Results and discussion
3.1. Mixing setup and rationale for sequential nanoprecipitation
The sequential mixing process can be divided into two steps: pre­
cipitation and stabilization (Fig. 2). Low molecular weight (2 kDa) PCL
Fig. 2. (A) Schematic of SNaP using two connected confined impinging jet (CIJ) mixers, or (B) a CIJ mixer and a multi-inlet vortex mixer. Core material is
precipitated in the first mixer using an antisolvent stream before stabilizer is added in the second mixer.
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International Journal of Pharmaceutics 640 (2023) 122985
was used here as a model core material because it behaves similarly to
nonionic, small-molecule hydrophobic drugs during the precipitation
process. In the first mixing step, the nanoparticle core material (PCL)
was dissolved in a water-miscible organic solvent (tetrahydrofuran) and
then rapidly mixed against an aqueous antisolvent stream using a CIJ
mixer. The antisolvent stream in FNP is typically deionized water; in this
work, a NaCl concentration of 7.5 mM was maintained in all mixing
chambers to prevent self-stabilization of polycaprolactone cores via the
electrostatic surface charge that has been observed on hydrophobic
surfaces in water (Roger and Cabane, 2012; Zhang et al., 2012). Control
experiments performed with NaCl and without stabilizer showed that
PCL did not self-stabilize and instead formed large aggregates.
Following the initial precipitation step, the effluent from the first CIJ
mixer was fed via tubing to the second CIJ mixer, where it was mixed
with a solution containing the nanoparticle surface stabilizing material.
To avoid redissolution of the PCL cores formed in the first mixer, the
solvent concentration in the second mixer must not be increased above
that of the first mixer effluent (50% water / 50% THF, v/v). Since the CIJ
mixer has only two inlets and requires nearly equivalent stream flow
rates to drive momentum annihilation (Saad and Prud’homme, 2016),
the two-CIJ-mixer setup in Fig. 2 requires that the solution introduced in
the second mixer be primarily aqueous.
Due to this requirement, a water-soluble stabilizing material must be
used in a sequential nanoprecipitation scheme using two CIJ mixers. In
this work we used hydroxypropyl methylcellulose acetate succinate
(HPMCAS), a semisynthetic cellulosic polymer reported previously as
appropriately amphiphilic for application as a stabilizer in FNP nano­
particle formulations (Caggiano et al., 2022; Feng et al., 2019b, 2018;
Ristroph et al., 2019). However, many of the polymers studied in the
FNP literature are water-insoluble diblock copolymers that are not
appropriate for this application. To use these in the SNaP process, the
second mixer must have more than two inlets, or else not require equal
stream volumes. The multi-inlet vortex mixer (MIVM)(Liu et al., 2008;
Markwalter et al., 2019) with four inlets, which was developed for FNP
as an alternative to the CIJ geometry, meets these requirements and will
be a focus of future work on SNaP. Initial efforts to use an MIVM as the
second mixer of SNaP (Fig. S1D-E) to enable a diblock copolymer sta­
bilizer were attempted for a formulation with a vitamin E acetate core
and poly(styrene)-b-poly(ethylene glycol) (PS-b-PEG) stabilizer at 83%
core loadings. The resulting nanoparticles had a Z-average diameter of
approx. 200 nm (Figure S3); however, further development for the CIJ-
to-MIVM system will be reserved for future work.
3.2. Nanoparticle formation
3.2.1. Governing variables for precipitation and self-assembly.
Pagels et al. showed that the size of NPs prepared by FNP is inde­
pendent of the core material used, provided that material is sufficiently
hydrophobic to rapidly precipitate in the mixed solvent environment of
an FNP mixing chamber (log Poctanol/water > ~4.5) (Pagels et al., 2018).
N.J. Caggiano et al.
Their work also showed that, for a given core material and given sta­
bilizer, the two variables that control NP size are the core loading and
total mass concentration (TMC) of the formulation. Core loading is
discussed in the introduction: NP size increases as core loading in­
creases, and NPs either rapidly destabilize or do not form altogether at
core loadings above 75% for block copolymer stabilized nanoparticles.
Pagels defined the TMC in terms of the total solids in the organic feed
stream to FNP (Pagels et al., 2018). Since in SNaP there are multiple feed
streams and mixers, we here define the TMC as the sum of the mass of
the core material and the mass of the stabilizer in the system divided by
the final solution volume including the collection reservoir (in practice,
the difference between this definition and that used in Pagels et al. is a
factor of 10). Higher TMC during the formulation process yields a more
concentrated final NP suspension, which translates to a reduction of
post-processing times (e.g., spray drying times or ultrafiltration con­
centration times) and an improvement of the overall process mass in­
tensity. For formulations of equal core loading, increasing the TMC
increases the resulting NP size. Above a critical TMC, visible aggregates
are observed, indicating that not all core material is encapsulated. As
with the upper limit of core loading, the likely mechanism for this
limitation is the entrainment of stabilizer molecules within NP cores. As
TMC increases, the average distance between two adjacent molecules
during NP assembly decreases, which can lead to stabilizer molecules
becoming trapped among rapidly assembling core molecules. Typical
TMCs for FNP formulations are around 1–2 mg mL− 1 (Markwalter et al.,
2019).
3.2.2. Nanoparticle core loading.
The average size and polydispersity index (PDI) of formulations
prepared by FNP or SNaP at a fixed TMC of 1 mg mL− 1 and different core
loadings are given in Fig. 3A. Conventional FNP produced a combination
of NPs and visible aggregates at core loadings of 75% and higher. In
contrast, no visible aggregates were observed in SNaP formulations at
core loadings of 50%, 63%, 75%, 83%, and 90%. At 95% core loading,
some replicates exhibited aggregates while others did not. At 99% core
loading aggregates were consistently observed. Therefore, the maximum
achievable core loading for this system is likely in the range of 90–95%.
It is important to note that DLS measurements did not always detect the
aggregates, which tended to adhere to vial walls or form a film on the
surface of the liquid in a vial. Close visual scrutiny was necessary to
identify aggregates at this small scale (5–10 mL per formulation), and
representative photographs are provided in the SI (Figure S4). DLS
measurements for the conventional FNP formulations produced at
loadings above 75% indicated that the population of non-aggregated
NPs had smaller average diameter and lower PDI than corresponding
SNaP NPs. However, these DLS measurements are not representative of
the true particle size distribution inclusive of the population of aggre­
gates. At pharmaceutical scale, the formation of aggregates would lead
to product losses and reduced reproducibility, hindering scale-up efforts
(Feng et al., 2019a; Li et al., 2014; Zeng et al., 2019). As such, DLS
measurements for visibly aggregated NP formulations are not reported
Fig. 3. (A) Z-average size (circles) and polydispersity index (PDI, crosses) of NPs produced by SNaP (black) or conventional FNP (orange) plotted versus core loading
for a fixed TMC of 1 mg mL− 1, (B) DLS traces for the six SNaP formulations shown in (A).
4
International Journal of Pharmaceutics 640 (2023) 122985
here.
As core loading was increased, the Z-average diameter of the SNaP
NPs decreased slightly and the PDI increased slightly. Fig. 3B shows the
intensity-weighted particle size distributions for the five SNaP formu­
lations from Fig. 3A. All six DLS traces exhibited at least slight deviation
from monomodality which became more prominent with increased core
loading. This was reflected in increased PDI values.
3.2.3. Total mass concentration during mixing.
The average size and polydispersity index (PDI) of formulations
prepared by FNP or SNaP at a fixed core loading of 50% and various
TMCs are shown in Fig. 4. Above a TMC of 2 mg mL− 1, conventional FNP
produced aggregates along with NPs, whereas SNaP produced NPs
without aggregates at TMCs up to 6 mg mL− 1, representing a six-fold
increase in the solids concentration in the effluent from the two-mixer
setup. Again, close visual scrutiny was necessary to identify aggregates
in conventional FNP formulations at this scale; DLS measurements alone
were not sufficient to determine whether aggregation occurred
(Figure S4).
3.2.4. Particle size.
For formulations in which both methods yielded NPs without ag­
gregates in Figs. 3 and 4, NPs made by sequential precipitation were
consistently larger and had higher PDI values than those made by single-
step precipitation. This was in line with expectations, as NP cores in the
sequential precipitation process had more time to aggregate before
growth was arrested by HPMCAS adsorption in the second mixer. As
described below, however, the delay time between mixers does not fully
account for this difference in size.
Pagels modified Smoluchowski’s model for diffusion-limited aggre­
gation to model NP assembly under FNP conditions (Pagels et al., 2018).
One equation from the modified model can be used to back-calculate the
amount of time required for NPs to assemble:
( )13
r=
tkB Tccore
πμρcore
where NP radius r (meters) may be written as a function of time t
(seconds), absolute temperature T, concentration of core material ccore
(kg m− 3) solution viscosity μ (kg m− 1 s− 1), core material density ρcore (kg
m− 3), and the Boltzmann constant kB (m2 kg s− 2 K− 1).
This model was used to calculate the difference in expected assembly
time between the two comparable formulations at 62.5% core loading in
Fig. 3. These calculations estimate that the 196 nm diameter particles
produced by conventional FNP assembled over approximately 520 ms,
compared with 2,700 ms for the 340 nm diameter SNaP NPs. As the
delay time between the entrance from one mixer to the next mixers was
estimated at only 140 ms, this delay alone does not appear to fully ac­
count for the 2,200 ms difference in predicted assembly time.
Importantly, the residence time in a CIJ under standard operation is
only about 90 ms. For NP formulations with elevated core loadings or
high TMCs, then, the model predicts that particle assembly is not
N.J. Caggiano et al.
Fig. 4. (A) Z-average size (circles) and polydispersity index (PDI, crosses) of NPs produced by SNaP (black) or conventional FNP (orange) plotted versus TMC for a
fixed core loading of 50%, (B) DLS traces for the four SNaP formulations shown in (A).
complete by the time a fluid element exits the final mixer of either
conventional FNP or SNaP. Here, the mixer effluent containing still-
assembling NPs was collected in a downstream quench reservoir,
where mixing was not as finely controlled as the rapid turbulent mixing
inside the CIJ. This may have contributed to the larger sizes and higher
PDI values of the final SNaP NPs. Despite the difference in final size
(discussed further below), the ‘pre-templating’ of the SNaP cores ap­
pears to have enabled stable particle formation at high core loadings and
TMCs without the formation of visible aggregates. The 140 ms of time
for PCL cores to nucleate in SNaP prior to HPMCAS exposure in the
second mixer should have been sufficient for nuclei 125 nm in diameter
to form. These cores then began to be coated by HPMCAS in the second
mixer.
If both formulations finished assembling after (poorly-controlled)
dilution into additional water downstream of the good mixing – dilution
which lowered the TMC and affected solvent quality (and therefore
supersaturation) for this final stage of assembly – the NP cores made by
conventional FNP may have been smaller because of longer exposure to
the HPMCAS stabilizer. NP cores formed by conventional FNP were
exposed to HPMCAS for all of the pre-quench assembly time, whereas
cores formed by SNaP were only exposed to HPMCAS for less than half of
the pre-quench assembly time.
The above example is an important point that should be considered
when preparing NPs by FNP using a CIJ mixer. For NPs that are expected
to take much longer than 90 ms to assemble (predictable as a function of
TMC, core loading, and stabilizer), some or most particle assembly takes
place downstream of the mixer. In industrial applications, the MIVM
geometry removes the need for a downstream quench reservoir and is,
therefore, recommended for formulations (whether SNaP or conven­
tional FNP) made at high TMCs and/or high core loadings, which are
expected to take more time to assemble than the residence time in the
mixer.
3.2.5. Nanoparticle size stability
All formulations made by SNaP in Figs. 3 and 4 remained stable in a
90:10 volumetric mixture of water to THF for 24 h (Figure S5). Size was
monitored by DLS, and vials were visually scrutinized for aggregate
formation. Representative photographs from visual inspection of various
formulation outcomes are included as Figure S4.
4. Conclusions
The results presented here offer proof of principle for an extension of
FNP technology that separates the nucleation and stabilization steps of
nanoparticle assembly. Using two sequentially connected FNP mixers,
nanoparticles can be formulated at higher core loadings and TMCs than
in conventional single-step FNP. NPs with core loadings up to 90% were
formulated without the aggregates that form via conventional FNP at the
same mass loading and TMC. This improvement in loading and TMC are
major advantages for this SNaP technique. Additionally, only minimal
modification of existing FNP equipment is required to facilitate the
5
International Journal of Pharmaceutics 640 (2023) 122985
sequential precipitation process.
When two CIJ mixers are used, the choice of amphiphilic stabilizer is
limited to water-soluble species that can be introduced in an aqueous
stream into the second mixer. Moving forward, we recommend that the
second mixer be an MIVM for improved scalability and flexibility, as this
would result in better in-mixer and downstream solvent quality control
and would enable the use of a wider range of amphiphilic stabilizer
chemistries, including those that are only soluble in organic solvents. A
formulation prepared using this CIJ-to-MIVM setup using a vitamin E
core and block copolymer PS-b-PEG stabilizer was developed and is
reported in SI (Figure S3). However, manual operation of a CIJ-to-
MIVM SNaP system is challenging and is limited by available syringe
volumes and geometries. Independent control of stream flowrates (and
thus solvent quality in the MIVM) can only be achieved using pumps to
feed each stream into the mixers. Development of a lab setup for CIJ-to-
MIVM and MIVM-to-MIVM operation is currently being pursued in our
laboratory.
For formulations where NPs can be formed by both SNaP and con­
ventional FNP, SNaP NPs are larger in diameter. We attribute some of
this difference to NP assembly which occurs downstream of the final CIJ
mixer in a less-controlled solvent environment. This would be solved by
the CIJ-to-MIVM setup described above. However, it is conceivable that
increasing the delay time between mixers could lead to tunable particle
size in the 300–1000 nm range, which is currently difficult to achieve by
FNP and which may have applications in depot delivery or microparticle
formulation. This will be another focus of future studies.
Funding sources
Funding support has come from the Helen S Hunt Fund to the
Princeton University School of Engineering and Science. We also
acknowledge support from the Bill and Melinda Gates Foundation under
grants INV-010674, INV-010674, and INV-042629.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijpharm.2023.122985.
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