Method for preparation of well-dispersed, discrete nanoparticles by spray drying techniques

This is a utility application incorporating by reference and claiming priority to previously filed provisional application Ser. No. 61/447,504 filed Feb. 28, 2011 entitled “Method for Preparation of Well-Dispersed CaF 2 Nanoparticles by Spray Drying Techniques”.

In a principal aspect, the present invention relates to a spray drying process for preparation of discrete nanosized particles, such as CaF 2 particles, that are not agglomerated.

Calcium fluoride (CaF 2 ) or ‘CaF 2 -like’ materials are of significant interest in caries prevention as they have been suggested to be the major source of labile fluoride (F) in the oral environment after F treatments. The release of F from the labile store of loosely bound F in the oral cavity environment increases the mineral saturation of oral fluid, and can promote the repair of lesions and reduce demineralization during cariogenic attack. However, a conventional sodium fluoride (NaF) rinse can only lead to very small amounts of CaF 2 deposit formation due to the low concentration of free calcium (Ca) ions in the mouth. In contrast, more CaF 2 deposits in the oral environment have been found to significantly increase the remineralization effects of the F treatment without increasing the F levels.

Previously reported is the preparation of a nano CaF 2 that has a theoretical spherical particle size of 41 nm based on BET (Brunauer-Emmett-Teller) surface area measurements. When used as the F source in an oral rise, these nano CaF 2 were shown to produce a greater F deposition than a NaF rinse of the same F dose in both in vitro and in vivo studies. However, electron microscopic observations indicated that the CaF 2 material consisted primarily of CaF 2 agglomerates, about 300 nm to 800 nm in size, but that the agglomerates, were made up of much small primary CaF 2 particles of <50 nm in size. These agglomerates were not readily dispersed into the primary CaF 2 particles by ultrasound processing, indicating that the primary CaF 2 particles in each cluster were tightly held together. However, there is strong evidence that significantly greater F efficacies can be achieved if the nano CaF 2 or agglomerates can be dispersed into the smaller primary particles. Unwanted agglomeration of nanoparticles due either to the preparation process or post synthesis treatments has thus remained a problem.

Briefly, the present invention comprises a method or process for preparation of dispersed nanosized particles, such as CaF 2 particles, by forming the particles in a soluble salt matrix, such as NaCl, using a spray drying apparatus similar in some aspects to that reported in Preparation and properties of nano-sized calcium fluoride for dental applications. Dent Mater 2008;24(1):111-116 incorporated herein by reference. As an example of the invention, the spray drying process leads to co-precipitation of CaF 2 and NaCl in the form of a cluster consisting of CaF 2 primary and discrete particles distributed in a matrix of NaCl. When exposed to an aqueous solution, the dissolution of NaCl will lead to the dispersion of the solid primary CaF 2 particles that may be collected for appropriate therapeutic use. This method can produce well dispersed, desirably sized nano CaF 2 primary particles, which can be used as a more effective anti-caries agent in rinses or dentifrices, for example.

The method also has application in the formation of other nanosized particulates of various compounds. Generally, the process involves solubilizing appropriate precursor compounds in chosen solvents and then simultaneously dry spraying the discrete solutions together to enable the ionic components to interact or bond thereby forming compounds of highly different or distinct solubility. By spray drying the two and possibly more solubilized compounds, agglomerated but discrete, solid nanoparticles are formed in a solid but soluble matrix. The matrix which is formed has a solubility in a chosen solvent significantly greater than the particles, thus enabling collection of the discrete, agglomerated particles by dissolving the matrix and capturing the particles.

Thus, it is an object of the invention to provide a method for the avoidance of agglomerated clusters of particles, such as CaF 2 particles, and thus provide particles generally of less than 50 nm in size (BET measurement) and 300 nm (dynamic light scattering (DLS) measurement).

It is a further object of the invention to provide a method for efficiently making calcium CaF 2 particles less than 300 nanometers (DLS) in size with greatly reduced agglomeration as a result of the processing steps associated with the creation of such particles.

Yet another object of the invention is to provide techniques for the creation of CaF 2 particles less than 300 nanometers (DLS) in size for their use to significantly increase remineralization effects of the fluoride treatment in effecting caries prevention.

Another object of the invention is to provide a method for making discrete, solid particles of various compounds having a size of nanometer dimension.

These and other objects, advantages and features of the invention will be set forth in the detailed description which follows.

In the detailed description which follows, reference will be made to the drawing comprised of the following figures:

FIG. 1 : X-ray diffraction (XRD) patterns of CaF2/NaCl composite nano particles prepared by spray drying techniques. 215×166 mm (600×600 DPI).

FIG. 2 : Scanning electron microscope (SEM) morphology of nano CaF 2 /NaCl composite particles prepared by spray drying techniques wherein patterns a 1 , a 2 , a 3 are respectively as-prepared CaF 2 -1, CaF 2 -2, CaF 2 -3; and patterns b 1 , b 2 , b 3 are respectively CaF 2 -1, CaF 2 -2 and CaF 2 -3 redispersed in ethanol after washing with distilled water. 330×176 mm (72×72 DPI)>

FIG. 3 : SEM and EDS of nano CaF 2 /NaCl composite particles prepared by spray drying techniques: (a) as prepared sample (at ×5k), (b) washed sample (at ×5k), (c) SEM of a single nano cluster in as prepared sample (at ×70k), (d) EDS spectrum of the single nano cluster (area within the rectangle shown in c). 294×128 mm (72×72 DPI).

FIG. 4 : Particle size distribution graphs of a pure nano CaF 2 sample (CaF 2 -0, (a) graph) and a distilled water-washed nano CaF 2+ NaCl composite sample (CaF 2 -3, (b) graph). 267×190 mm (600×600 DPI).

FIG. 5 : Schematic drawings of (a) a composite cluster containing CaF 2 nanoparticles in the matrix of NaCl, and (b) a cluster containing pure CaF 2 nanoparticles. 323×243 mm (600×600 DPI).

FIG. 6 : Photograph of suspension of a blue dye—incorporated nano amorphous HA (left); after centrifugation (right).

The following examples of the process is directed to the manufacture of discrete particles of CaF 2 having a size generally less than 300 nm and includes the steps of (1) providing at least a first fluoride solution and a discrete calcium solution, (2) simultaneously ejecting said solutions by means of a dual spray drying apparatus in a manner wherein the spray is simultaneously ejected from the spray nozzle(s) in a pattern that effectively co-mingles the sprayed solutions (3) collecting the particulates resulting from the spray drying operation which are in view of the solutes chosen comprised of generally separate CaF 2 particles in a soluble, biocompatible matrix, and (4) dissolving the matrix and collecting the CaF 2 particles. Enabling the process requires appropriate choices of solvents and solutes based upon their ionic reactivity and compound solubility.

Preparation of Nano CaF 2 /NaCl Particles

The nano CaF 2 /NaCl particles were prepared using the spray drying system generally described in On the role of calcium fluoride in the cariostatic mechanism of fluoride. Acta Odontol Scan 1988; 46:341-345, incorporated herewith by reference In the preparation of pure nano CaF 2 , a 4 mmol/L Ca(OH) 2 solution and an 8 mmol/L NH 4 F solution were, following preparation, simultaneously fed to the 2-liquid nozzle of a spray drying apparatus. To incorporate NaCl, NaCl of concentrations of (1.4, and 16) mmol/L were added to both the NH 4 F and Ca (OH 2 ) solutions, respectively. This led to the formation of (CaF 2 +NaCl) composites with CaF 2 /NaCl molar equivalent ratios of 4:1, 1:1, and 1:4, or mass ratios 5.34, 1.31 and 0.33 (Table 1). Pure nano CaF 2 particles were also prepared as the control. All solutions were prepared using reagent grade chemicals and mole ratios of CaF 2 /NaCl in the range of 10:1 to 1:10 are considered within the scope of the process.

The reaction of the solutions led to the formation of (CaF 2 +NaCl) composite particles and NH 4 F (Eqn. 1). The (CaF 2 +NaCl) composite of particles and matrix was collected at the end of the process, while the NH 4 OH was removed as NH 3 and H 2 O vapors with the air flow

Ca(OH) 2 +NH 4 F+NaCl→CaF 2 +NaCl+NH 3 ↑+H 2 O↑  (Eqn. 1)

Microstructural Characterization of Nano CaF 2 /NaCl Particles

The obtained nano powders were characterized by powder X-ray diffraction (XRD), DMAX 2200, Rigaku Denki, Woodlands, Tex.) for crystalline phase identification. Scans were performed between 10°<2θ<60°. The estimated standard uncertainty of the 2θ measurement is 0.01°. The morphology and composition of the initially collected particulate material (powder) was examined using scanning electron microscopy (SEM, JSM-5300, JEOL, Peabody, Mass.) and X-ray energy dispersive spectroscopy (XEDS, equipped in Hitachi S-4700 SEM, Pleasanton, Calif.). The SEM samples were prepared by depositing the powder onto aluminum stubs or carbon tapes (to reduce the A1 interference in XEDS spectrum) from a well-sonicated dilute suspension in pure ethanol. The size distribution of the powders was determined by dynamic light scattering (DLS, Zetaszier, Malvern Instrument, Westborough, Mass.) at a wavelength of 532 nm at 25° C. using a well-sonicated dilute suspension in distilled water (0.25 mg/ml CaF 2 ) as the only particulate matter in the suspension. In order to keep the NaCl concentration constant for all suspensions, an appropriate amount of NaCl solid was added to each of the suspensions such that the NaCl concentration was the same as that in sample CaF 2 -3 (12.8 mmol/L).

To investigate if the CaF 2 can be readily dispersed after the NaCl was dissolved, samples of CaF 2 /NaCl composite powders were washed with distilled water first to dissolve the NaCl, and then centrifuged at (48,000 g) for 10 min (J2-21, Beckman, Brea, Calif.). The solid sediments which should consist of pure CaF 2 were then collected for preparing well-sonicated dilute suspension in pure ethanol for SEM examination and DLS measurement. The sediments were also dried in air for BET and DLS characterization. Multipoint BET surface area analyses were done with ultra high purity nitrogen as the adsorbate gas and liquid nitrogen as the cryogen (ASAP 2020 Surface Area & Porosity Analyzer, Micromeritics, Norcross, Ga.). All samples were degassed at 105° C. for 1 hour and then held at 105° C. for 2 hours with ultra high purity nitrogen purge before the measurement. The estimated standard uncertainty of the BET measurement was 0.15% of the reading. The particle size of the primary crystals of CaF 2 was estimated from the BET surface area by calculating equivalent spherical diameter, or BET particle diameter (d BET ), from the fundamental equation: d BET =6/(p S W ), where p is the theoretical density of the CaF 2 (3.14 g/cm 3 ) and S W is the specific surface area (see Ring, TA. Fundamentals of Ceramic Powder Processing and Synthesis. San Diego; Academic Press; 1996).

XRD patterns of the obtained powders ( FIG. 1 ) showed that the powders include both CaF 2 and NaCl phases and their ratios corresponded to the ones in the starting solution. No traces of other phases such as Ca(OH) 2 , NH 4 F, CaCl 2 or NaF were detected. The peaks of the CaF 2 were relatively broad compared to those of NaCl, suggesting a finer crystal size or less perfect crystal structure for the CaF 2 . The distilled water-washed samples contained only CaF 2 as their XRD patterns (not shown) were similar to that of the CaF 2 -0 sample, showing CaF 2 peaks that are much broader when compared to those of the commercial micro-sized CaF 2 .

SEM examinations ( FIG. 2 ) showed that the as-prepared powder ranged from about 50 nm to 800 nm in size. Also, these powders exhibited numerous spherical protuberances on the surfaces, suggesting that they were formed during the spray drying process through aggregation of the much smaller primary particles. In strong contrast, after washing with distilled water, the material dispersed into much small particles of about 100 nm or less ( FIG. 2 ). The particles became better dispersed with fewer and smaller agglomerates when a greater amount of NaCl was incorporated in the composite clusters during the preparation, and subsequently dissolved in the water. However, when the percentage of NaCl incorporation was very high, such as in the case of sample CaF 2 -3, some NaCl crystals could form separately, rather than as the part of the composite clusters ( FIG. 2 (a 3 )). EDS indicated that the nano composite clusters or powders in as-sprayed samples contained CA, F, Na, and Cl, confirming that the samples contained both CaF 2 and NaCl ( FIG. 3 ).

In comparison, the washed samples contained essentially Ca and F only. However, the CaF 2 /NaCl ratio in a single before-wash composite cluster (intensity of Ca/Na is≈2.7) appeared to be higher than the average ratio (intensity of Ca/Na is≈13) in the bulk sample, suggesting that the CaF 2 /NaCl precipitation may not be homogeneous. This may also explain the SEM observation that some NaCl precipitated as a separate phase as described above.

The DLS measurement in 12.8 mmol/L NaCl solution (Table 2) showed that the mean particle size (z-average) of CaF 2 nanoparticles were greatly reduced when the NaCl was co-precipitated during spray drying and subsequently dissolved in water. The size reduction was greater when more NaCl was incorporated in the composite. The DLS measurement of the distilled water-washed samples dispersed in ethanol (Table 2) showed a similar trend, but the average sizes were smaller and closer to the particle sized observed by SEM. FIG. 4 compares the representative particle size distribution of samples CaF 2 -0 and CaF 2 -3 (washed) in ethanol. The pure CaF 2 sample exhibited a particle size distribution of (200 to 1000) nm with the median value at 425 nm, while the washed CaF 2 -3 sample had a distribution of (45 to 200) nm with the median value at 75 nm. These results, which were in good agreement with the SEM observations, showed that the effective median particle size was decreased by nearly 6-fold. The BET specific surface areas of the washed CaF 2 powders ranged between (49 to 63) m 2 /g (Table 2) and the estimated equivalent spherical particle sizes (d BET ) were (30 to 39) nm. These latter values were similar to the size of the primary particles observed in SEM ( FIG. 3 ), suggesting that the BET particle size values corresponded to those of the primary particles, which were similar in all CaF 2 preparations with a mean value of (36±4) nm.

Process Characterization

Due to the instantaneous atomization and drying process, the spray drying technique makes it possible to co-precipitate two discrete phases with very different solubility into composite particles. To fully understand the process, it is hypothesized that the following sequence of events occurred within each droplet of atomized solution during the spray drying process when preparing pure CaF 2 : (1) the mixed solution, i.e., Ca(OH) 2 +NH 4 F in Eqn.1, became progressively more supersaturated with respect to CaF 2 as water and NH 4 OH were rapidly evaporated, (2) numerous CaF 2 nuclei were formed via homogenous nucleation, (3) the nuclei grew larger and closer to each other as the droplet dried out, and (4) formation of a cluster of tightly aggregated primary nano particles. It seems plausible that, as the droplet dries out, the very last portion of the precipitated CaF 2 would form at the contact points of the primary particles, bridging them together to form a stable three dimensional network that would hinder ready dispersion of the cluster into free primary particles. Therefore, if a highly soluble compound, such as NaCl, is co-precipitated with CaF 2 in the spray-drying process, NaCl precipitation will begin to occur only after formation of the CaF 2 primary particles is nearly complete, i.e., after step (3) described above. In this case, NaCl instead of CaF 2 will be formed in the spaces between the CaF 2 primary particles, leading to formation of a cluster consisting of CaF 2 primary particles distributed in a matrix of NaCl ( FIG. 5 a ) rather than a cluster of tightly interlocked CaF 2 particles ( FIG. 5 b ). When exposed to an aqueous solution, dissolution of NaCl should lead to dispersion of the CaF 2 particles.

The results ( FIG. 1-3 ) show that CaF 2 /NaCl composite particles are successfully formed through the spray drying process, which has similar morphology and particle size as the pure CaF 2 particles prepared previously, i.e., clusters of (50 to 800) nm, consisting of smaller primary particles [11]. Once in the aqueous solution, the NaCl dissolved, releasing the primary CaF 2 nanoparticles ( FIG. 2 ). By these means, better dispersed CaF 2 nanoparticles with a narrow size distribution ( FIG. 2 ). By these means, better dispersed CaF 2 nanoparticles with a narrow size distribution can be obtained ( FIG. 2 , 4 ). A potential problem with this process is that multiple phases could form due to the existence of several ions in the solution and the fast precipitation in the spray drying process. XRD pattern of the obtained powders ( FIG. 1 ), however, showed no traces of other phases such as Ca(OH) 2 , NH 4 F, CaCl 2 or NaF This demonstrated that the least soluble phase, i.e., CaF 2 will precipitate first, and the precipitation of the soluble phases, i.e., NaCl, will begin to occur only after formation of the CaF 2 primary particles is nearly complete. These results supported the feasibility of the approach for preparing well-dispersed nano particles by incorporating a more soluble phase that can be dissolved subsequently.

Three characterization methods have been used in this study: SEM provided a direct image of particle morphology, size, and degree of particle dispersion or agglomeration: DLS measured the particle size distribution in a liquid: BET determined the size of the primary particles. As the particle size measured by DLS is hydrodynamic diameter within a liquid, the results are very much affected by the liquid or medium. Two media have been used in this study. The 12.8 mmol/L NaCl solution was used for all as-sprayed samples as sample CaF 2 -3 contained this amount of NaCl when dissolved in water. This medium is close to the one (1) mmol/L NaCl solution) recommended by the international standard for DLS measurement. However, the particle sizes measured this way (Table 2) appeared to be larger when compared to the SEM observations. A separate DLS measurement of CaF 2 -0 powders in distilled water only gave a value of (497±20) nm (n=3), suggesting that the concentration of the salt in the medium played an important role in the measurement. In addition, to simulate the sample preparation for SEM, ethanol was also used as a medium for DLS measurement of all washed samples (no washing needed for sample CaF 2 -0). The particle sizes obtained by this method were much smaller compared to those measured in NaCl solution (Table 2). However, the results still seemed slightly larger than the SEM observation, which may be due to both the nature of the technique and the existence of negligible amounts of less dispersed particles that caused the small tail on the right side of the size distribution ( FIG. 4 ). In sum, the incorporation of NaCl during the preparation of CaF 2 led to a well dispersed nano CaF 2 particles.

As shown in FIGS. 2 and 4 and Table 2, the degree of particle dispersion for the washed samples increased with the percentage of NaCl co-precipitated with CaF 2 . This is reasonable as the primary CaF 2 particles can be better distributed within a larger volume of the NaCl matrix. However, when the NaCl incorporation was too high, some NaCl crystals could precipitate early in the process, i.e., before significant CaF 2 precipitation has occurred. Rapid growth of these NaCl crystals could occur simultaneously with the precipitation of CaF 2 , forming a separate phase rather than as the matrix of the composite clusters ( FIG. 2 (a 3 )). This suggests that there is a limit for the incorporation of NaCl.

When there is particle agglomeration, the particle size estimated by the BET method is usually much smaller than by other methods, because BET methods provide an approximate measure of the size of primary CaF 2 particles rather than the true size of the agglomerated particles. In contrast, particle size values obtained from the DLS measurements are more closely related to the true physical size of the particles that may have various degrees of agglomeration. Thus, the degree of particle dispersions/agglomeration can be assessed by comparing the values obtained by the BET and DLS methods. Assuming both the primary particles and the agglomerated particles have spherical shapes and the primary particles and the agglomerated particles have spherical shapes and the primary particles are closed packed in an agglomerated particle, we can define the degree of agglomeration (DA) as the averaged number of primary particles contained in the agglomerated particles, which can be estimated by.

DA =volume of composite cluster/volume of primary particle=( d DLs2 / d BET ) 3   (Eqn. 2)

By using the mean primary particle size of 36 nm and the DLS Z-average particle size in ethanol, i.e., d DLs2 in Table 2, the DA values ranged from 2020 for CaF 2 -0 and 85 for CaF 2 -3 Alternatively, if using the median particle size in FIG. 4 , i.e., 425 nm for CaF 2 -0 and 75 nm for CaF 2 -3, the DA values for CaF 2 -0 and CaF 2 -3 were respectively 1645 and 9 These results suggest that our approach is very effective in obtaining better-dispersed nanoparticles. Nonetheless, there were still negligible amounts of less dispersed particles, as revealed by both DLS and SEM.

Preparation of Nano Amorphous HA (Hydroxyapatite)

Nano particles of a composite consisting of HA, NaCl and Brilliant blue dye (CAS Registry Number: 3844-45-9) were synthesized by simultaneously feeding to a two liquid nozzle (1) a solution containing 4 mM Ca(OH) 2 and 3.44 mM NaCl, and (2) a solution containing 2.4 mM H 3 PO 4 and 0.032 g/L of blue dye and spray dried as previously described. The product would have a HA:NaCl mass ratio of 2:1, and the blue dye content of 5 mass %. The collected product was twice washed in distilled water (10 mg/mL) to dissolve the NaCl followed by centrifugation. The supernatant was essentially colorless ( FIG. 6 ), indicating that little or no dye was incorporated into the NaCl phase during the spray drying process. This was expected because large dye molecule is likely to be excluded from NaCl, which has a simple and tight crystal structure. In contrast, the nano amorphous HA particles showed a dark blue color indicating that the large dye molecules were trapped in the HA phase by co-precipitation. These results demonstrate using the technique to incorporate drug molecules (analogous to the blue dye) in HA nano particles by the spray drying method.

DLS measured particle size of the this HA was 228±8 nm (n=3), which is significantly smaller than the size (380±49 nm) of a similarly prepared amorphous HA without the NaCl matrix reported by “Sun L M, Chow L C, Frukhtbeyn S A, Bonevich J E (2010): Preparation and Properties of Nanoparticles of Calcium Phosphates with Various Ca/P Ratios. J Res Natl Inst Stand Technol 115: 243-255.”

It should be noted that, in addition to using NaCl as the matrix phase, other soluble compounds such as potassium nitrate, sodium fluoride, etc., can also be used to obtain similar results. In general, this technique can be used to produce better dispersed nanoparticles for any system as long a (1) the nanoparticles to be prepared are significantly less soluble than the matrix material (on the order of at least 20 times more soluble), and (2) none of the constituents of the matrix material would produce undesired effects on the composition of the nanoparticles being prepared.

This approach is, for example, applicable in preparing better-dispersed calcium phosphate nanoparticles for drug delivery and gene therapy. That is, in the present invention, calcium fluoride, the nano particles to be prepared, has a solubility (in water) of about 0.018 g/L, whereas that of the matrix, sodium chloride, is about 359 g/L. Similarly, solubility of other calcium phosphates are approximately: DCPD (0.15 g/L), DCPA (0.074 g/L), ACP (0.021 g/L), and HA (0.00066 g/L). All of the above have solubility values over 1000 times smaller than that of sodium chloride, therefore requirement (1) above is well satisfied. Since sodium chloride would not significantly interact with calcium phosphate formation in the spray drying process, requirement (2) is also fully met.

A large number of other compounds can also satisfy the two requirements and are good candidates for use the matrix. Non-toxic and biocompatible examples may include mannitol, sucrose, when water is used as the solvent in the spray drying process. If the nano particles to be prepared are not for biomedical uses, additional compounds can be involved.

While there has been set forth various embodiments of the invention and the processes practiced, the invention is to be limited only by the following claims and equivalents thereof.