www.sciencemag.org/content/349/6251/956/suppl/DC1

Supplementary Materials for

Production of amorphous nanoparticles by supersonic spray-drying with a

microfluidic nebulator

Esther Amstad, Manesh Gopinadhan, Christian Holtze, Chinedum O. Osuji, Michael P. Brenner, Frans Spaepen, David A. Weitz*

*Corresponding author. E-mail: weitz@seas.harvard.edu

Published 28 August 2015, Science 349, 956 (2015) DOI: 10.1126/science.aac9582

This PDF file includes: Materials and Methods

Supplementary Text

Figs. S1 to S8

Caption for movie S1

Other supplementary material for this manuscript includes: Movie S1

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Materials and Methods Production of drug nanoparticles Fenofibrate nanoparticles (NPs) were produced from ethanol solutions containing 5

mg/ml fenofibrate. Ethanol was chosen as a solvent because of its high volatility; it thus rapidly evaporates during the drying process. Moreover, the saturation concentration of fenofibrate in ethanol at room temperature is ~ 50 mg/ml. If NPs are spray dried using solvents that wet the channel walls, such as ethanol, the initial solute concentration cannot exceed 10% of its saturation concentration. Otherwise, NPs start to form inside the channel, adsorbing on the walls, and eventually leading to clogging. However, this limitation is specific to our design, and could be relaxed if the design were modified or if higher air pressures were used.

To initiate the operation of the microfluidic nebulator, we injected compressed air into the air inlets by applying a pressure of 0.28 MPa to all the air inlets. We subsequently injected the ethanol solution through one liquid inlet at 1 ml/h using volume-controlled syringe pumps. We blocked the second inlet for liquids. Spray dried NPs were collected 15 cm away from the nozzle on a substrate suitable for the further characterization.

Clotrimazole, danazole, and estradiol nanoparticles were prepared using the same procedure. As the saturation concentration of these drugs in ethanol is ≥ 50 mg/ml, all drugs were dissolved at a concentration of 5 mg/ml in ethanol.

Production of inorganic nanoparticles CaCO3 nanoparticles were prepared through a precipitation reaction by mixing two

aqueous solutions, one containing 5 mM CaCl2 and the other one containing 5 mM Na2CO3; to prepare solutions, Millipore water (resistivity ≥ MΩ) was used. Compressed air was injected into the nebulator through the air inlets by applying a pressure of 0.28 MPa to all the air inlets. The two aqueous solutions were injected into the nebulator through the two liquid inlets; the flow rate of each of these solutions was 0.5 ml/h. The spray dried nanoparticles were collected 20 cm away from the nozzle.

BaSO4 nanoparticles were prepared following the same procedure. In this case two aqueous solutions were used, one containing 5 mM BaCl2 and the other solution containing 5 mM K2SO4.

Iron oxide nanoparticles were also produced through a precipitation reaction. However, in this case, one aqueous solution contained a mixture of two salts, namely 2 mM FeCl3 and 1 mM FeCl2, the other solution contained 100 mM NaOH.

Amorphous NaCl nanoparticles were prepared by simply drying an aqueous solution containing NaCl. By analogy to the production of the other nanoparticles, compressed air was injected through the air inlets by applying 0.28 MPa to all the air inlets. An aqueous solution containing 4 mM NaCl was injected through one liquid inlet while the second liquid inlet was blocked. Spray dried nanoparticles were collected 20 cm away from the nozzle.

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Characterization of the NPs NPs were deposited on a one side polished Si wafer and coated with a 5 nm thick

PtPd film to make the sample electrically conductive. SEM images were acquired at an acceleration voltage of 2 kV using the secondary electron detector.

High resolution transmission electron microscopy (HRTEM) images were acquired on a JEOL 2100 TEM with an acceleration voltage of 200 kV. Electron dispersive spectroscopy (EDS) was acquired on a Zeiss Libra 200 kV monochromated aberration corrected scanning tunnelling electron microscopy (STEM).

The structure of the NPs was measured with X-ray diffraction (XRD) by depositing them on a one side polished Si wafer. XRD was acquired on a Scintag XDS 2000 diffractometer using a copper-Kα radiation (1.54 Å) at a speed of 2°C/min.

To conduct differential scanning calorimetry (DSC) measurements, particles were sprayed into an Al pan that was subsequently hermetically sealed. DSC spectra were acquired on a TA instrument at a heating rate of 1 °C/min.

To determine the chemical composition of the NPs, they were sprayed onto a one side polished Si wafer. X-ray photoelectron spectroscopy (XPS) analysis was performed on a Kα XPS (Thermoscientific) with a 180° double focussing hemispherical analyzer and a 128-channel detector.

Quantification of the NP size We manually measured the diameter of at least 300 NPs using SEM images acquired

at a magnification of 100 kX.

Supplementary Text 1. Fenofibrate NPs 1.1 Characterization of the NP size Fenofibrate NPs spray dried by applying 0.28 MPa to the air inlets of the nebulator

had an average diameter of 14 nm, as indicated in the SEM image in Fig. 1D in the main text and in the low resolution TEM image in Fig. S1.

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Fig. S1. Transmission electron micrograph of fenofibrate NPs spray dried at a pressure of 0.28 MPa and collected on a carbon coated Cu-TEM grid.

1.2 Characterization of the NP structure As a negative control for the analysis of the structure of fenofibrate NPs, we co-spray dried fenofibrate with Pluronics F127, a block-copolymer that acts as a heterogeneous nucleant. We dissolved 5 mg/ml fenofibrate in ethanol and added 5 mg/ml Pluronics F127 to this solution. To accelerate the dissolution of Pluronics F127, we heated the solution to 40°C. We spray dried the resulting solution under identical conditions as used for the preparation of pure fenofibrate nanoparticles and analyzed the structure of the resulting NPs with XRD. We observed clear diffraction peaks for fenofibrate NPs co-spray dried with Pluronics F127, as shown in trace (1) of Fig. S2. By contrast, pure Pluronics F127, deposited on a Si wafer, did not show any diffraction peak, as shown by trace (2) of Fig. S2. The distinct peak measured for fenofibrate co-spray dried with Pluronics F127 indicates that these fenofibrate NPs are crystalline, in stark contrast to pure spray dried fenofibrate NPs. These results suggest that Pluronics F127 acted as a heterogeneous nucleant as its solubility in ethanol is much lower than that of fenofibrate.

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Fig. S2 XRD spectra of (1) fenofibrate NPs co-spray dried with Pluronics F127 and (2) Pluronics F127.

1.3 Solubility

We qualitatively tested the solubility of fenofibrate by depositing a-NPs on glass slides at high packing densities such that adjacent NPs came in contact with each other and coalesced. Upon addition of large volumes of water, amorphous particles rapidly dissolved in the water. There was no sign for crystallization upon addition of water whatsoever. By contrast, if water was added to a large crystal of fenofibrate, almost no fenofibrate dissolved, demonstrating that the solubility of amorphous fenofibrate is much higher, as predicted by theory (17, 34).

1.4 Stabilization of NPs To ensure fenofibrate nanoparticles remain stability against crystallization even if deposited at a density, they must be physically separated. To accomplish this, we sprayed them into a matrix of Pluronics, whose surface was rough and thus had a high surface to volume ratio. Even though Pluronics does not inhibit crystallization of fenofibrate, NPs sprayed into Pluronic matrices at high densities showed no sign of crystallinity even if stored at room temperature under ambient conditions for at least 7 months.

2 Characterization of clotrimazole, danazol, and estradiol NPs

We determined the structure of spray dried clotrimazole, danazol, and estradiol NPs using XRD, by analogy to the characterization of fenofibrate NPs. None of the spectra acquired on spray dried drug NPs displayed any diffraction peak, as shown in Fig. S3, indicating that these drug NPs were all amorphous.

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Fig. S3 XRD spectra of (1) clotrimazole, (2) danazol, and (3) estradiol NPs spray dried at a pressure of 0.28 MPa. In addition, we characterized the chemical composition of clotrimazole NPs using XPS. To prevent desorption of the NPs from the substrate, which would contaminate the analysis chamber, we sprayed them onto a Pluronics F68 matrix that has previously been deposited on a Si wafer. We analyzed the N 1s and Cl 2p peak of this sample, as shown in Fig. S4. We quantified the atomic ratio of N : Cl by measuring the area of the XPS peaks using CasaXPS and normalizing it by the photoelectron cross section of the respecting element. We obtained an atomic ratio of 2.05, which is in good agreement with the theoretical value of 2. These results confirm that spray dried NPs were indeed composed of clotrimazole.

Fig. S4 XPS traces of (A) N1s and (B) Cl 2p peaks of spray dried clotrimazole NPs.

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3 Characterization of inorganic NPs 3.1 Chemical characterization of spray dried CaCO3 NPs As CaCO3 NPs are produced by mixing an aqueous solution containing CaCl2 with

one containing Na2CO3 salts, we obtain CaCO3 and NaCl. To determine the composition of the resulting nanoparticles and to localize the Na+ and Cl- ions, we performed EDS on them. To obtain a sufficient signal to noise ratio, we produced CaCO3 NPs with a diameter of approximately 50 nm. We deposited these particles on a carbon coated 300 mesh Cu grid and acquired an STEM image using the secondary electron detector (Fig. S5a). In addition, we measured EDS maps of the Ca, Cl, and Na signals. No macroscopic phase separation of CaCO3 and NaCl was observed, as shown in Figs. S5b to S5d.

Fig. S5 Chemical analysis of spray dried CaCO3 NP. (A) STEM image acquired with a secondary electron detector. (B-D) EDS maps from the same particles of (B) Ca, (C) Na, and (D) Cl.

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3.2 Characterization of inorganic amorphous NPs BaSO4 NPs and iron oxide NPs produced through precipitation reactions were

amorphous, as shown in the HRTEM in Figs. S6A and S6B.

Fig. S6 HRTEM image of spray dried (A) BaSO4 and (B) iron oxide NPs with the corresponding Fourier transforms in the insets.

3.3 Chemical characterization of amorphous NaCl NPs We produced amorphous NaCl NPs from aqueous solutions containing NaCl by simply drying water sufficiently fast to kinetically suppress the formation of stable crystal NaCl nuclei, as shown in the main text. In addition to analyzing the binding energy of the Na 1s electrons of the spray dried NaCl NPs, shown in Fig. 4F in the main text, we also analyzed the binding energy of the Cl 1s using electrons using XPS. The Cl 1s peak measured on amorphous, spray dried NaCl NPs is significantly broader than that measured on the crystalline NaCl NPs and the reference crystalline bulk NaCl, as shown in Fig. S7. This indicates that the distance between Cl ions and their next neighbors differed, in contrast to Cl ions contained in crystalline NaCl. However, if we exposed these amorphous nanoparticles to a high dose of electrons, they transformed into a single crystal; this transformation could be followed in situ using HRTEM. These results provide further evidence that these amorphous nanoparticles are indeed NaCl NPs and not contaminants that could have been introduced into the sample during the spray dry process.

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Fig. S7 XPS spectra of the Cl 2p peak of (1) crystalline NaCl produced by slowly evaporating an aqueous solution containing 40 mM NaCl, and (2-3) spray dried NaCl NPs produced from an aqueous solution containing (2) 40 mM and (3) 4 mM NaCl.

4 Spray drying of amorphous nanoparticles All amorphous NPs consisted either of an anisotropic molecule or they were composed of at least two different types of elements. By contrast, gold and silver NPs are composed of a single type of an isotropic element; they were crystalline if spray dried under identical conditions. This difference in the structure and composition of the building blocks of NPs might be a contributing factor for the crystalline structure of spray dried Au NPs and Ag NPs. However, further studies are warranted to determine the reason for the crystalline structure of these NPs.

5 Quantification of the probability for crystal nuclei to form

Spray dried NPs are amorphous if drops dry faster than crystal nuclei can form. To quantify the probability for crystal nuclei to form in an evaporating drop, we must calculate the time required to dry a drop as well as the time-dependent probability for crystal nuclei to form as the drop dries.

5.1 Drying of a drop

The time required to dry a drop depends on the solvent evaporation rate as well as on the drop size.

5.1.1 Estimation of the solvent evaporation rate

The evaporation rate of a liquid is typically limited by the time it takes for molecules to diffuse across the boundary layer. However, in our case, the boundary layer,

  200 nm, was very thin as the air velocity, u, was very high and the

kinematic viscosity of the air, ν, very low; here r is the drop radius. In this case, ethanol

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molecules diffused much faster across this boundary layer than they impinged the drop surface. Hence, the evaporation rate was equal to the impingement rate of the solvent on the drop surface at equilibrium vapor pressure for that temperature. For molecules with

mass m, and a vapor pressure pvap, kinetic gas theory gives this as per unit area

and time; here kB is the Boltzmann constant (36).

5.2 Estimation of the drop size

We estimated the drop size by measuring the size of the spray dried NPs. Assuming no solvent evaporated before the sub-µm sized drops formed, we can estimate the drop size using the known initial solute concentration, as described in the main text.

We also quantified the drop size by balancing the shear stress with the Laplace

pressure,   ; here µ is the viscosity of air, dv is the difference in the velocity of the air and the drop, l is the distance between the drop surface and the channel wall, γ is the

surface tension, and the radius of the drop. To determine dv, we quantified the velocity of the drop using movies acquired with a high-speed camera. We estimated the volumetric air flux by connecting a gas-tight syringe to the outlet of nebulators with different numbers of air inlets. We measured the time required to fill the gas syringe with 50 cm3 of air as a function of the number of air inlets the nebulator had; we kept the pressure at all the air inlets at 0.28 MPa. Assuming that the difference in the air velocity at the outlet of a device with n+1 pairs of inlets to that of a device with n pairs of inlets can be assigned to the air that flows through this additional air inlet, we could estimate the air velocity profile in the entire nebulator. For the calculations on the drop size, we only used the air velocity at the last 2D junction as this is the only place where the shear stress exceeds the Laplace pressure.

5.3 Probability for crystal nuclei to form

The time required to form a crystal nucleus depends on the solute concentration as well as on the temperature. If drops are homogeneous, the solute concentration steadily increases as the drop dries because the number of solute molecules contained in a drop remains unchanged but the drop volume continuously decreases. However, if the solution undergoes spinodal decomposition, a solute poor and a solute rich phase forms; thus, the solute concentration becomes spatially heterogeneous. In this case, the solute concentration in each of the two phases remains constant as the drop dries and only the relative volume fraction of the two phases changes. The probability for crystal nuclei to form in each phase is then determined by their solute concentrations in the two phases, which strongly depend on the interaction parameter of the solute molecules and the temperature. Thus, to quantify the probability for crystal nuclei to form as the drop evaporates, we calculated the solute concentration in each of the two phases.

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5.3.1 Calculations of the difference in the Gibbs free energy

To quantify the interaction parameter, ε, we calculated the difference between the

chemical potential of the pure amorphous phase, , and the pure crystalline phase, ,

  , as shown in Fig. S8; here xc, sat is the molar saturation concentration of the crystal phase, R the gas constant, T the

absolute temperature, ΔHm the heat of melting of fenofibrate, the undercooling, and Tm the melting temperature of fenofibrate. We measured xc, sat in ethanol at T = 20⁰C to be 0.08, and Tm to be 80⁰C. We determined ΔHm of fenofibrate to be 28.88 kJ/mol, by integrating the area of the melting peak measured with DSC. We thus obtained ε = 6.7 kJ/mol. We plotted the difference in the Gibbs free energy between the pure, individual components of the solution and the solution itself

as a function of the molar fraction of the solute, x, as shown in Fig. 2C in the main text of this paper. The chemical potential

of fenofibrate in solution is then   .

Fig. S8 An example of the Gibbs free energy, G, as a function of the solute concentration, x. We used the Gibbs free energy to calculate the interaction parameter, ε, as well as the equilibrium concentration of the amorphous phase, xa, equ.

5.3.2 Calculations of the nucleation rate

We calculated the probability for nuclei to form inside drops as they evaporate. A drop of a total volume V contains n1 moles of fenofibrate molecules, a solute, which constitutes

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the α phase. As the solvent evaporates, the solute concentration steadily increases. We let

the solute concentration, (i = 1, 2; 1:solute), and its mole fraction, , increase in discrete time intervals of Δt, which is the time required to reduce the drop radius by 0.28 nm, the approximate size of one ethanol molecule. We estimated

= 33 ns; here AEtOH is the area of an ethanol molecule, the

impingement rate of ethanol molecules on the drop surface, pV the vapor pressure of ethanol, m the weight of an ethanol molecule. The molar volume of the α phase is

. From this follows .We assumed that the nucleating phase, β, consists

only of fenofibrate; its molar volume is .

The number of critical nuclei per unit volume is

(1)

where is the work to form a spherical critical nucleus. Here Δµ is the

difference between the chemical potential of a fenofibrate molecule in solution, µ, and its chemical potential in the pure crystalline phase, , as shown in Fig. S8, and γ is the solution-crystal interfacial energy; we estimated this to be on the order of 10 mJ/m2. The nucleation rate, in events per unit volume per second, is then:

(2)

Each of the factors is explained below.

A solute molecule adjacent to the critical nucleus can join the nucleus with a diffusional

jump frequency . For a three-dimensional random walk with jump frequency and distance a, the diffusion coefficient is

(3) and hence

(4) where we take a as the average intermolecular distance; we ignore differences between solute and solvent. The Zeldovich factor Z arises from the Becker-Döring steady-state nucleation condition:

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  (5)

where W(i) is the work to assemble a cluster of size i; its second derivative, which arises from a series expansion, is evaluated at the critical size. The Zeldovich factor is usually on the order of 0.01-0.1 (24).

The number of molecules at the surface of the critical nucleus is . The number of

molecules in the critical nucleus is . The volume of the critical nucleus is then .

If we assumed that the nucleus is spherical, its radius and the area is

.

Since the average area of a molecule on the surface is approximately ,

.

The site factor, or accommodation factor, f, is the fraction of the molecules in the solution layer around the nucleus that can be incorporated into the nucleus with one diffusional jump. It includes requirements of surface topology such as ledges or kinks and molecular rotational effects. In addition, it includes the fraction of molecules in the solution layer that are solute atoms. Since the critical nucleus is in unstable equilibrium with the solution, the fraction of solute molecules in the layer next to the nucleus is simply their

mole fraction, . We can therefore separate this factor and obtain   .

In our case f’ is small and the growth of the nuclei is interface-limited. The concentration of the solution at the interfaces is thus the same as in the bulk and is not limited by diffusion, so we can apply the same argument for all the sizes of the nuclei. Thus, the Becker-Döring steady-state calculation remains valid. Thus, the nucleation frequency in events per m3 and per second is

. (6)

The molar volume of the α phase and the average interatomic distance in that phase are

approximately related as resulting in

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. (7)

The factor in parentheses is of order one, so that to a good approximation we can write:

  (8)

and

(9)

where Vi is the drop volume at time i and Δt the time it takes to evaporate a monolayer of ethanol.

5.3.3 Calculation of the temperature inside the drop

The solute concentration in the two phases is temperature-dependent and the very high evaporation rate of the solvent results in strong evaporative cooling of the drop. To compute the solute concentrations, we calculated the temperature in the drop as it evaporates by balancing the cooling caused by evaporating Δm of the solvent and the heat the air transfers to the remaining drop with a mass m and a surface area, A,

; here h is the heat transfer coefficient. We used the latent heat of evaporation ΔHevap and the specific heat capacity cp of ethanol. The temperature of the drop at time t1 is Tdrop 1 and one iteration step before, at time t0, it was Tdrop 0. We used again the time it takes to reduce the radius of the drop

by the length of an ethanol molecule, = 33 ns as the discrete iteration interval. Since the air flow is very high, we assumed Tair to remain constant at room temperature. Also the boundary layer for heat transport is very thin; thus, we estimated the heat transfer coefficient of air to be as high as 60 kJ/mol (37). This very high heat transfer coefficient prevents drops from freezing. Instead, the temperature in the drop falls to -70⁰C, well below Tg of fenofibrate, within 1-3 µs but stabilizes at these low values.

Movie S1 Movie S1: Operation of the microfluidic nebulator using water as a model non-wetting fluid. The movie is slowed down 100×.