β-Carotene by Supercritical

Nov 3, 2015 - Moreover, to produce powder with the largest BC content, it is possible to state that the best proportion between the two solutes is 10/...
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Coprecipitation of Polyvinylpyrrolidone/β-Carotene by Supercritical Antisolvent Processing Valentina Prosapio, Ernesto Reverchon, and Iolanda De Marco* Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II, 132, 84084, Fisciano (SA), ITALY ABSTRACT: Production of coprecipitated microparticles is an ambitious application of supercritical antisolvent precipitation (SAS). In this work, coprecipitates of β-carotene (BC) and polyvinylpyrrolidone (PVP) have been successfully obtained using this technique. Nanostructured microparticles in the range 1−2.4 μm were obtained, varying the polymer molecular weight (10 000 and 40 000 g/mol), the polymer/drug ratio (from 10/1 to 20/1), the operating pressure (from 85 to 100 bar), and the total concentration in the liquid solution (from 3 to 7 mg/mL). In particular, at 85 bar, 40 °C, 5 mg/mL, with a PVP/BC equal to 10/1, and using the polymer with the lower molecular weight, well-defined coprecipitated microparticles were obtained. Particles produced operating at these conditions showed a vitamin dissolution rate 10 times faster than the one of unprocessed BC, confirming the effective coprecipitation, and the increase of the β-carotene dissolution rate. The precipitation process and the mechanisms involved are also discussed.

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increase of BC dissolution rate. Yi at al.16 used the homogenization−evaporation method to coprecipitate BC using sodium caseinate, whey protein, and soybean protein as carriers; they obtained particles in the range 78−370 nm and compared the dissolution profiles of BC using the different carriers, but no comparison with the dissolution profile of unprocessed BC was proposed to demonstrate the improved bioavailability. Processes based on supercritical carbon dioxide (scCO2) have been proposed as an efficient alternative to conventional processes, thanks to fast mass transfer, high solvent power, high density, near zero surface tension, low viscosity, and high diffusivity, which can be tuned varying pressure and temperature. They have been successfully applied in different fields: micronization of several kind of materials,17−25 extraction of natural matters,26 impregnation of metals or drugs in aerogels,27,28 membranes,29 and scaffold production.30,31 Among these techniques, a supercritical antisolvent (SAS) process has been successfully used to micronize pharmaceuticals, coloring matters, polymers, and biopolymers;32,33 moreover, some papers have been focused on the interpretation of the mechanisms typical of SAS precipitation.34−38 However, in SAS literature, only few papers 39−43 are devoted to coprecipitates formation, due to the difficulty in their production using this technique. In these works, the particles obtained are generally irregular and coalescing,39 with broad particle size distributions,40 low drug loadings41 and, in some cases, the demonstration of the occurred coprecipitation is questionable.42,43 Franceschi et al.44 and Priamo et al.45 proposed the application of the SAS-like solution enhanced dispersion with supercritical fluids (SEDS) technique for the encapsulation of

INTRODUCTION Nutraceuticals are defined as functional ingredients (antioxidants, vitamins, etc.) that, if taken regularly, can prevent or treat some diseases.1 β-Carotene (BC) is a carotenoid, present in sweet potatoes, carrots, spinaches, pumpkins, broccoli, peas, peppers, pink grapefruits, papayas, and peaches.2 It is a precursor of Vitamin A and its intake is fundamental to reduce the risk of cardiovascular diseases,3 osteoporosis,4 and to prevent cancer of the stomach, esophagus, lung, oral cavity, pharynx, pancreas, and colon.2,5 Carotenoids are not produced by human organisms; therefore, they have to be acquired with the diet; however, BC easily undergoes chemical or physical degradation in the presence of light, moisture, oxygen, and high temperature, during storage.6 To avoid these adverse effects and to preserve the vitamin nutritional properties, encapsulation systems have been developed. These composite particles are formed by a drug or a vitamin uniformly dispersed in a polymeric matrix or by drug or vitamin particles coated with a polymeric shell. In these systems, the carrier should be a hydrophilic polymer, since BC shows low water solubility and, therefore, a reduced bioavailability. Polyvinylpyrrolidone (PVP) is a water-soluble polymer, broadly used as carrier in pharmaceutical, biomedical, and food applications, since it possesses the GRAS (generally regarded as safe) status and is included in the FDA (food and drug administration) list. It has the ability to retard crystal growth7 and to enhance the dissolution rate of poorly water-soluble drugs.8 Various conventional processes have been reported for the production of coprecipitates: spray-drying,9 emulsification/ solvent evaporation,10 centrifugal extrusion,11 freeze-drying,12 and coacervation.13 All of these techniques show some drawbacks, such as lack of control over particle size and particle size distribution, possible degradation of the product, and high residual solvent.14 More specifically, Liang et al.15 proposed the spray-drying of nanoemulsions for the encapsulation of BC in starch; they obtained highly irregular particles and did not report the dissolution tests to confirm an © 2015 American Chemical Society

Received: Revised: Accepted: Published: 11568

September 18, 2015 November 3, 2015 November 3, 2015 November 3, 2015 DOI: 10.1021/acs.iecr.5b03504 Ind. Eng. Chem. Res. 2015, 54, 11568−11575

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Industrial & Engineering Chemistry Research BC in poly(3-hydroxybutirate-co-hydroxyvalerate (PHBV) using dichloromethane as the liquid solvent. They obtained powders formed by a large and irregular material, not characterizable in terms of size and morphology and did not report dissolution tests and solvent residue analyses despite the toxicity of the used solvent. He at al.46 used SEDS process for the coprecipitation of BC in poly ethylene glycol (PEG) obtaining irregular flakes with a BC loading lower than 50%; moreover, also in this case, the dissolution tests were missing. Hong et al.47 proposed SEDS technique for the production of BC/proanthocyanidin microspheres; however, they obtained results similar to those previously described. Moreover, none of these authors proposed an explanation of the coprecipitation mechanisms. However, it is possible to successfully obtain coprecipitates using SAS technique. For example, in a recent paper, Prosapio et al.48 proposed the use of SAS technique for the production of PVP-folic acid (FA) microspheres, investigating the effect of different operating parameters to identify the optimal process conditions. They obtained two different morphologies: (a) nanoparticles with a mean size down to 50 nm, working at pressures far above the mixture critical pressure (MCP) of the binary system solvent/antisolvent; (b) well separated spherical microparticles, working near above the MCP. Dissolution tests performed on the powder samples showed that only the powders precipitated slightly above the MCP showed a FA dissolution rate significantly higher than the one of unprocessed FA, confirming the occurred coprecipitation. In the hypothesis that PVP can exert a general positive effect in the formation of SAS coprecipitates, in this work, we applied SAS process to the coprecipitation of PVP/BC microparticles, to extend the applicability of this technique to other solutes of nutraceutical interest and to gain more information about the involved precipitation mechanisms.

Figure 1. Schematic representation of SAS apparatus. V CO2 supply; S liquid solution supply; RB refrigerating bath; P1, P2 pumps; TC thermocouple; M manometer; PC precipitation chamber; MV micrometering valve; LS liquid separator; BPV back pressure valve; R rotameter.

pressure is regulated by a backpressure valve (BPV, Tescom, model 26-1723-44, Italy), is used to recover the liquid solvent (LS). At the exit of the second vessel, CO2 flow rate and the total quantity of antisolvent delivered are measured by a rotameter (R) and a dry test meter, respectively. SAS Procedure. An SAS experiment usually begins delivering CO2 to the SAS vessel until the desired pressure is reached. When antisolvent steady flow is established, the mixture of organic solvents is sent through the nozzle to the chamber for at least 15 min. When a quasi-steady state composition of solvent and antisolvent is realized inside the SAS vessel, the flow of the solvent is stopped and the liquid solution is delivered through the nozzle, producing the precipitation of solute. At the end of the solution delivery, supercritical CO2 continues to flow, to wash the chamber, eliminating the solution formed by the liquid solubilized in the supercritical antisolvent. If the final purge with pure CO2 is not performed, the organic solvents mixture contained in the fluid phase condenses during the depressurization step and can solubilize or modify the precipitates. At the end of the washing step, CO2 flow is stopped and the precipitator is depressurized down to atmospheric pressure. Analytical Methods. Samples of the precipitated material were observed by a Field Emission Scanning Electron Microscope (FESEM, mod. LEO 1525, Carl Zeiss SMT AG, Oberkochen, Germany). Powder was dispersed on a carbon tab previously stuck to an aluminum stub (Agar Scientific, United Kingdom) and, then, was coated with gold−palladium (layer thickness 250 Å) using a sputter coater (mod. 108 A, Agar Scientific, Stansted, United Kingdom). Particle size distributions (PSDs) of the powders were measured from FESEM photomicrographs using the Sigma Scan Pro image analysis software (release 5.0, Aspire Software International Ashburn, VA). Approximately 1000 particles, taken at high enlargements and in various locations inside the

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MATERIALS, METHODS, AND PROCEDURES Materials. Polyvinylpyrrolidone (PVP, average molecular weight 10 000 and 40 000 g/mol), β-carotene (BC, purity ≥93%), acetone (AC, purity 99.8%), and ethanol (EtOH, purity 99.8%) were supplied by Sigma−Aldrich (Italy). CO2 (purity 99%) was purchased from SON (Italy). All materials were used as received. SAS Apparatus. A schematic representation of the apparatus used for SAS experiments is reported in Figure 1. Two high pressure pumps (Milton Roy, model Milroyal B and Milroyal D, France) are used to deliver the supercritical CO2 (P1) and the liquid solution (P2). A cylindrical vessel of 500 cm3 internal volume (I.V.) (i.d. = 5 cm) is used as the precipitation chamber (PC). The liquid solution is delivered to the precipitator through a thin wall stainless steel nozzle (internal diameter equal to 100 μm). Supercritical CO2, after a preheating, is cocurrently delivered to the precipitator through another port located on the top of the vessel. Pressure is measured by a test gauge manometer (M, Salmoiraghi, model SC-3200, Italy) and regulated by a micrometering valve (MV, Hoke, model 1315G4Y, Spartanburg, SC) located at the exit of the precipitator. Temperature is set by a proportional− integral−derivative (PID) controller (Watlow, USA) connected with electrically controlled thin bands. A stainless steel filter (pore diameter of 0.1 μm) located at the bottom of the precipitator is used to collect the produced powder, allowing the CO2-solvent solution to pass through. A second collection vessel located downstream the micrometering valve, whose 11569

DOI: 10.1021/acs.iecr.5b03504 Ind. Eng. Chem. Res. 2015, 54, 11568−11575

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Industrial & Engineering Chemistry Research Table 1. Summary of SAS Experiments Performed on PVP/BC Using a Mixture AC/EtOH 70/30 (v/v)a

a

no.

PVP molecular weight [g/mol]

PVP/BC [w/w]

P [bar]

C [mg/mL]

morphology

m.d. [μm]

s.d. [μm]

1 2 3 4 5 6 7 8 9 10

10000

1/0 0/1 10/1

85

5

0.81

0.23

85

5

85

5

10000

15/1 20/1 10/1

5

10000

10/1

90 100 85

MP C NP MP MP MP MP MP MP

0.25 2.43 1.70 1.63 2.06 1.24 1.15

0.05 0.81 0.51 0.49 0.64 0.44 0.50

40000 10000 10000

3 7

MP: microparticles. NP: nanoparticles. C: crystals. m.d.: mean diameter. s.d.: standard deviation.

precipitator, were analyzed in the elaboration of each particle size distribution. Histograms representing the particle size distributions were fitted using Microcal Origin Software (release 8.0, Microcal Software, Inc., Northampton, MA). Drug loading and powder dissolution studies were performed using an UV/vis spectrophotometer (model Cary 50, Varian, Palo Alto, CA). Accurately weighted samples containing an equivalent amount of BC (1 mg) were suspended in 1.5 mL of phosphate buffered saline solution (PBS) with 0.5% of Tween80 and placed into a dialysis sack; it was then incubated in 300 mL of PBS at pH 7.4, continuously stirred at 250 rpm and 37 °C. Each analysis was carried out in triplicate and the proposed curves are the mean profiles. BC loadings were measured by UV−vis analysis, measuring the absorbance obtained in the release medium at the end of the drug release; i.e., when all BC was released from the microspheres to the outer water phase. The absorbance was, then, converted into BC concentration, using a calibration curve.

Figure 2. FESEM image of BC precipitated from AC/EtOH 70/30 at 85 bar, 40 °C, and 5 mg/mL.

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EXPERIMENTAL RESULTS All SAS experiments were carried out using a CO2 flow rate of 15 NL/min, a solution flow rate of 1 mL/min, at a temperature of 40 °C. Since BC is soluble in acetone (AC), whereas PVP is soluble in ethanol (EtOH), a mixture AC/EtOH 70/30 (v/v) was used as the liquid solvent. A preliminary check on the formation of a homogeneous solution of PVP/BC/EtOH/AC was made, using PVP with both molecular weights, to be sure that one liquid phase without solute precipitation can be formed. Then, the effect of PVP molecular weight, PVP/BC w/ w ratio, operating pressure and total concentration of solutes in the liquid solution (total amount of PVP+BC used) was investigated. A selection of the performed experiments is summarized in Table 1, with the indication of the operating conditions employed, the obtained morphology and, in the case of particles, their mean diameter (m.d.) and standard deviation (s.d.). Each experiment was carried out in duplicate. Two preliminary SAS experiments were performed using the solutes separately, to know their behavior under the same operating conditions. Both the tests were carried out at 85 bar and 40 °C, i.e., in proximity of the MCP of the system. These process conditions were selected because, in a previous work,48 it has been demonstrated that coprecipitation occurred when microparticles were formed. When BC alone was processed (no. 2 in Table 1), the precipitate, from a macroscopic point of view, appeared as an orange powder that, observed at SEM, was formed by large and irregular particles. In Figure 2, a FESEM image of the precipitated powder is reported. The micronization was clearly unsuccessful.

PVP(10) (Mw = 10 000 g/mol) alone, instead, when SAS processed using the same solvent mixture of the previous experiment, precipitated in form of a white powder characterized by microparticles with a mean diameter of 0.8 μm (no. 1 in Table 1). A SEM image of this precipitate is shown in Figure 3, as confirmation of the suitability of the polymer processing by SAS.48 This preliminary test on PVP alone was necessary, because, in a previous work,48 PVP was chosen to produce coprecipitates using dimethyl sulfoxide (DMSO) as liquid solvent; therefore, the precipitation from

Figure 3. FESEM image of PVP precipitated from AC/EtOH 70/30 at 85 bar, 40 °C, and 5 mg/mL. 11570

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Industrial & Engineering Chemistry Research EtOH/AC mixtures of PVP in form of microparticles had to be verified. Summarizing the results of these preliminary tests, it is possible to observe that the two compounds show completely different behavior when SAS processed at the same operating conditions and that BC alone is a bad candidate for SAS micronization. Effect of PVP Molecular Weight. In the first set of experiments the effect of the polymer molecular weight was investigated (nos. 3−4 in Table 1), working at the same conditions used in the previous experiments. The scope of these experiments was to select the most appropriate PVP molecular weight for the coprecipitation experiments. First, a test was performed using a PVP with a molecular weight of 40 000 g/mol (PVP(40), run no. 3 in Table 1), fixing the polymer/vitamin ratio equal to 10/1. Then, the same experiment was carried out using a lower polymer molecular weight (10000 g/mol) (PVP(10), run no. 4 in Table 1). It is very relevant to consider that, despite the two experiments were carried out at the same operating conditions, the obtained powders had a different color. In particular, using the polymer with the higher molecular weight the powder was orange with some white points, suggesting that probably the two solutes precipitated separately (Figure 4a). The powder obtained using

Figure 5. FESEM images of PVP/BC 10:1 processed at 85 bar, 40 °C, 5 mg/mL: (a) PVP molecular weight of 40 000 g/mol; (b) PVP molecular weight of 10 000 g/mol.

coprecipitates are formed by microspheres. Therefore, of the lower molecular weight PVP was used in the experiments that follow. Effect of Polymer/Vitamin Ratio. The second parameter taken into account was the polymer/vitamin ratio, which was increased first at 15/1 and then at 20/1 (nos. 5−6 in Table 1), keeping constant the other process parameters. When PVP/BC 15/1 was processed, a yellow powder was again obtained, but it was characterized by a lighter yellow color: this result was expected and can be attributed to the larger amount of PVP used with respect to the vitamin. The FESEM images (an example is reported in Figure 6a) showed

Figure 6. FESEM images of PVP/BC particles precipitated from AC/ EtOH 70/30 at 85 bar and 40 °C at different polymer/drug ratios: (a) 15/1, (b) 20/1 w/w.

the presence of slightly coalescing microparticles. Processing PVP/BC 20/1, a larger coalescence is visible, as shown in the FESEM image reported in Figure 6b, and the intensity of the yellow color decreases further. These two last experiments showed that, increasing the PVP/ BC ratio from 10/1 (no. 4) to 20/1 (no. 6), a worsening of particle morphology occurred. Moreover, to produce powder with the largest BC content, it is possible to state that the best proportion between the two solutes is 10/1, that was fixed for the subsequent experiments. Effect of the Operating Pressure. The effect of the operating pressure on PVP/BC coprecipitates morphology, particle size, and particle size distribution was, then, investigated, fixing the polymer/vitamin ratio at 10/1. The pressure range explored was between 85 and 100 bar, since in a previous work48 it was observed that good conditions for a successful coprecipitation are located slightly above the mixture critical point (MCP) of the binary system solvent/CO2. An experiment was carried out at 90 bar; in correspondence with these operating conditions, spherical microparticles (Figure 7a) with a mean diameter of about 2 μm (the larger particles have diameter of about 2.8 μm, the smaller ones of 0.8 μm) were obtained, as it is possible to observe from the SEM image and from data in Table 1 (run no. 7). When the process pressure

Figure 4. Photos of the filter of PVP/BC 10:1 processed at 85 bar, 40 °C, 5 mg/mL: (a) PVP molecular weight of 40 000 g/mol; (b) PVP molecular weight of 10 000 g/mol.

the PVP with the lower molecular weight, instead, showed a yellow color that was intermediate between that of BC and PVP alone (Figure 4b). This result is the visual evidence that, in this case, probably the coprecipitation is successful; this hypothesis has been adequately demonstrated in the following parts of the work. The microscopic analysis of the powders of different molecular weight PVP showed that, in the case of the higher molecular weight PVP, slightly coalescing nanoparticles were obtained, as showed in the FESEM image reported in Figure 5a; whereas, the powder obtained using the lower molecular weight PVP is formed by spherical microparticles with a mean diameter of about 2.43 μm (the larger particles have diameter of about 3.1 μm, the smaller ones of 0.7 μm), as shown in Figure 5b. The comparison of the FESEM images of the powders obtained from the two experiments showed that the morphology obtained using the lower molecular weight polymer is more compatible with a coprecipitation. Indeed, on the basis of a previous work,48 it is expected that 11571

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Industrial & Engineering Chemistry Research

containing an equivalent amount of BC (1 mg) were suspended in 1.5 mL of PBS with 0.5% of Tween-80 and placed into a dialysis sack; it was then incubated in 300 mL of PBS at pH 7.4, continuously stirred at 250 rpm and 37 °C. The analyses revealed that the powders of PVP/BC 10/1, processed at 85 bar and 40 °C, obtained using the polymer with the higher molecular weight (PVP(40)) (run no. 3 of Table 1), show a drug content equal to 70% with respect to the initial value; whereas, the powders obtained using the polymer with the lower molecular weight (PVP(10)) (run no. 4 of Table 1) show a drug content near 100% with respect to the initial value. The dissolution rate of each sample was monitored for 80 h by plotting the percentage of dissolved β-carotene as a function of time. The analyses were performed for unprocessed βcarotene, SAS processed PVP/BC 10/1 with a PVP molecular weight of 40 000 g/mol and with a PVP molecular weight of 10000 g/mol; all results are shown in Figure 9. The profile of

Figure 7. FESEM images of PVP/BC particles precipitated from AC/ ETOH 70/30 at 40 °C and different pressures: (a) 90; (b) 100 bar.

was increased to 100 bar, slightly smaller microparticles were produced with a mean diameter of about 1.2 μm (the larger particles have diameter of about 1.7 μm, the smaller ones of 0.45 μm) (see run no. 8 in Table 1), as shown in Figure 7b. Comparing the volumetric particle size distributions of the particles, it is possible to observe that, increasing the operating pressure, the particle size reduced, and the PSD became narrower, as can be seen in Figure 8. This result is in agreement with SAS literature,34 as we will see in the discussion of the results.

Figure 8. Volumetric particle size distributions of PVP/BC 10/1 coprecipitates obtained at different pressures.

Figure 9. Dissolution profiles of BC in PBS at 37 °C and pH 7.4.

Effect of the Total Solute Concentration. The effect of the total concentration of solutes in the liquid solution was studied at 85 bar, 40 °C, fixing PVP/BC at 10/1. Using as a reference the experiment no. 4, two experiments with a higher and a lower concentration were performed. Using an overall concentration equal to 3 mg/mL, well separated microparticles with a mean diameter of 1.15 μm were obtained. Increasing the concentration at 7 mg/mL, highly coalescing particles were produced, which cannot be characterized in terms of dimensions. Comparing the results obtained at 3 and 5 mg/ mL, it is possible to observe that increasing the concentration the mean diameter increased and the particle size distribution become wider. Analysis of Precipitates. To evaluate the quantity of vitamin present in the coprecipitates and its dissolution profiles, UV−vis analyses were performed on the processed samples and on unprocessed BC. Drug loading analyses were performed, determining the ratio between the BC content revealed by the UV−vis and the BC solubilized together with PVP in the mixture EtOH/AC before the SAS test. As previously indicated in the Materials, Methods, and Procedures section, samples

the untreated vitamin shows a complete dissolution time in 80 h. The sample formed by PVP/BC nanoparticles, obtained using the polymer with higher molecular weight, instead, shows a faster dissolution rate, reaching the 100% dissolution in 40 h. However, only the sample formed by PVP/BC microparticles obtained with the lower polymer molecular weight shows a radically different behavior, with a dissolution profile in which the 100% of the vitamin was released in only 8.5 h. The last curve was identical for run nos. 4 and 9, indicating that the total concentration of the liquid solution had no effect on the release curves. Therefore, for PVP/BC microparticles, the dissolution rate of the vitamin was 5 times faster than PVP/BC nanoparticles and 10 times faster than the unprocessed BC.

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DISCUSSION As stated in the Introduction, SAS coprecipitation is difficult to achieve. An explanation of this problem is given by the analysis of the precipitation processes that are activated during this operation. The first is nucleation and growth that is generally activated when the operating point is located far above the 11572

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Industrial & Engineering Chemistry Research MCP of the studied system organic solvent-antisolvent.49 In this case, basically nanoparticles are produced,34 and the two species (polymer and active compound) tend to precipitate separately48 forming a sort of intimate physical mixture: coprecipitation is substantially unsuccessful. The second precipitation process, that is characteristic of SAS, is the one that develops after liquid solution jet breakup, when it is injected in the precipitator. In this case, microdroplets are formed in which the liquid is eliminated by the formation of a supercritical solution with CO2; the dried droplets are the basis of the microparticles (acting as a confined reactor) formed at the end of the precipitation process.49 This precipitation mechanism is favored at process conditions near the MCP and is potentially responsible of the formation of coprecipitates. The further difficulty is that the presence of two solutes, in most cases, can obstacle the droplets formation. In this work, it is evident that β-carotene alone tends to produce crystals; PVP, instead, depending on its molecular weight tends to produce spherical particles of different mean size. The key question was the following: due to its capacity to block and control the morphology of the compound precipitating in its presence, can PVP force the coprecipitation of BC, producing microdroplets according to the second discussed SAS mechanism and obtain a successful coprecipitation? We discovered that PVP molecular weight also plays an important role in successful coprecipitation; indeed, the nanoparticles shown in Figure 5a, obtained using PVP 40 000 g/mol, show a nucleation and growth process that induces separate precipitation of the two solutes, confirmed by the color of coprecipitates, but also by the β-carotene release tests (Figure 9). When PVP 10 000 g/mol is used, microparticles are obtained (Figure 5b), whose color is intermediate between the ones of BC and PVP (Figure 4b); also their morphology (spherical microparticles) indicates that the coprecipitation mechanism could be successful in this case. This result is confirmed by BC release tests in Figure 9: the dissolution process is concluded in only 8.5 h that is about 10 times faster than β-carotene alone and about 5 times faster than the intimate physical mixture formed when nanoparticles were obtained. Moreover, all (100%) BC was recovered in the precipitates; whereas, only 70% was present in the case of nanoparticles formation. The missing BC was probably lost by dissolution in the supercritical mixture solvents + CO2 in the precipitator and, therefore, it could be recovered in the separator put downstream the precipitation chamber. The final consideration is about the dispersion of BC inside the PVP based microparticles. We can expect that a nanodispersion has been obtained. There are some good reasons to hypothesize this morphology: • First, it has been demonstrated50 that this is the usual mode of coprecipitation in droplets containing large PVP quantities. • Second, PVP is a powerful crystal growth inhibitor and no traces of crystalline BC have been formed. • Third, the very fast dissolution of coprecipitates is reasonably due to the very fast dissolution of PVP (that is readily soluble in water based solution), the release of small BC nanoparticles, and their subsequent fast solubilization. Considering all the experimental evidence collected in this work, it is possible to state that looking at the experiments

performed at different operating pressures (nos. 4, 7, and 8 in Table 1), it was observed that, increasing the pressure, the mean diameter of the particles reduced and the PSD became narrower. Indeed, increasing the pressure, the formation of the supercritical mixture solvent/antisolvent is faster and, therefore, the velocity of the dynamic surface tension vanishing increases, leading the precipitation of smaller particles.51 Looking at the experiments performed at different solute concentrations (nos. 4, 9, and 10 in Table 1), it was observed that, up to 5 mg/mL, increasing the liquid solution concentration, the mean diameter increased and the particle size distribution was enlarged. This trend has been frequently observed in SAS experiments and can be explained using the classical theory of crystallization (nucleation and growth) or considering an increase of the viscosity of the solution with a consequent increase of jet breakup time, that led to a disequilibrium between the jet breakup time and the dynamic surface tension vanishing time. The loading analysis confirmed the presence of both solutes in the samples; in particular, they show that using the PVP with the lower molecular weight, the drug content with respect to the initial value remained unchanged with respect to the injected solution. However, these data are not sufficient to demonstrate that PVP and BC were coprecipitated: for this reason, dissolution tests were performed. Dissolution test results allow us to conclude that only in the case of microparticle production was the coprecipitation successful.

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CONCLUSIONS SAS process of PVP based solutions demonstrated to be efficient in producing polymer/drug coprecipitation when accurate process conditions are chosen. Indeed, for the first time, well-separated microparticles PVP/BC were produced, overcoming the limitations observed in SAS literature. The induced precipitation by microdroplets formation and drying is the key step of the successful coprecipitation of the two solutes and of the 10 times increase of BC dissolution rate.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +39-89-964057. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) DeFelice, L. S. The nutraceutical revolution: its impact on food industry R&D. Trends Food Sci. Technol. 1995, 6 (2), 59. (2) Keijer, J.; Bunschoten, A.; Palou, A.; Franssen-van Hal, N. L. W. Beta-carotene and the application of transcriptomics in risk−benefit evaluation of natural dietary components. Biochim. Biophys. Acta, Mol. Basis Dis. 2005, 1740, 139. (3) Stahl, W.; Sies, H. Antioxidant activity of carotenoids. Mol. Aspects Med. 2003, 24, 345. (4) Chen, G.; Zhu, Y.-Y.; Cao, Y.; Liu, J.; Shi, W.; Liu, Z.; Chen, Y. Association of dietary consumption and serum levels of vitamin A and β-carotene with bone mineral density in Chinese adults. Bone 2015, 79, 110. (5) Li, X.; Wang, G.; Chen, D.; Lu, Y. β-Carotene and astaxanthin with human and bovine serum albumins. Food Chem. 2015, 179, 213. (6) Hosseini, S. M. H.; Emam-Djomeh, Z.; Sabatino, P.; Van der Meeren, P. Nanocomplexes arising from protein-polysaccharide electrostatic interaction as a promising carrier for nutraceutical compounds. Food Hydrocolloids 2015, 50, 16.

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DOI: 10.1021/acs.iecr.5b03504 Ind. Eng. Chem. Res. 2015, 54, 11568−11575

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Industrial & Engineering Chemistry Research (7) Ledet, G. A.; Graves, R. A.; Glotser, E. Y.; Mandal, T. K.; Bostanian, L. A. Preparation and in vitro evaluation of hydrophilic fenretinide nanoparticles. Int. J. Pharm. 2015, 479, 329. (8) Oth, M. P.; Moes, A. J. Enhanced in vitro release of indomethacin from non-aqueous suspensions using indomethacin-polyvinylpyrrolidone coprecipitates. Int. J. Pharm. 1985, 24, 275. (9) Vehring, R. Pharmaceutical particle engineering via spray drying. Pharm. Res. 2008, 25, 999. (10) O’Donnell, P. B.; McGinity, J. W. Preparation of microspheres by the solvent evaporation technique. Adv. Drug Delivery Rev. 1997, 28, 25. (11) Oxley, J. D. Coextrusion for food ingredients and nutraceutical encapsulation: principles and technology. Food Res. Int. 2013, 54, 837. (12) Heidebach, T.; Först, P.; Kulozik, U. Influence of casein-based microencapsulation on freeze-drying and storage of probiotic cells. J. Food Eng. 2010, 98, 309. (13) Nihant, N.; Grandfils, C.; Jérôme, R.; Teyssié, P. Microencapsulation by coacervation of poly(lactide-co-glycolide) IV. Effect of the processing parameters on coacervation and encapsulation. J. Controlled Release 1995, 35, 117. (14) Wang, W.; Liu, G.; Wu, J.; Jiang, Y. Co-precipitation of 10hydroxycamptothecin and poly (l-lactic acid) by supercritical CO2 anti-solvent process using dichloromethane/ethanol co-solvent. J. Supercrit. Fluids 2013, 74, 137. (15) Liang, R.; Huang, Q.; Ma, J.; Shoemaker, C. F.; Zhong, F. Effect of relative humidity on the store stability of spray-dried betacarotene nanoemulsions. Food Hydrocolloids 2013, 33, 225. (16) Yi, J.; Lam, T. I.; Yokoyama, W.; Cheng, L. W.; Zhong, F. Betacarotene encapsulated in food protein nanoparticles reduces peroxyl radical oxidation in Caco-2 cells. Food Hydrocolloids 2015, 43, 31. (17) Della Porta, G.; Nguyen, B.-N. B.; Campardelli, R.; Reverchon, E.; Fisher, J. P. Synergistic effect of sustained release of growth factors and dynamic culture on osteoblastic differentiation of mesenchymal stem cells. J. Biomed. Mater. Res., Part A 2015, 103 (6), 2161. (18) Liparoti, S.; Adami, R.; Reverchon, E. Supercritical Assisted Atomization: effect of operative conditions on PVP microparticle size and morphology. J. Supercrit. Fluids 2015, 97, 31. (19) Reverchon, E.; Adami, R. Supercritical assisted atomization to produce nanostructured chitosan-hydroxyapatite microparticles for biomedical application. Powder Technol. 2013, 246, 441. (20) Sampaio de Sousa, A. R.; Simplìcio, A. L.; de Sousa, H. C.; Duarte, C. M. M. Preparation of glyceryl monostearate-based particles by PGSS®Application to caffeine. J. Supercrit. Fluids 2007, 43, 120. (21) Campardelli, R.; Baldino, L.; Reverchon, E. Supercritical fluids applications in nanomedicine. J. Supercrit. Fluids 2015, 101, 193. (22) Campardelli, R.; Della Porta, G.; Gomez, L.; Irusta, S.; Reverchon, E.; Santamaria, J. Au-PLA nanocomposites for photothermally controlled drug delivery. J. Mater. Chem. B 2014, 2 (4), 409. (23) De Marco, I.; Reverchon, E. Supercritical carbon dioxide + ethanol mixtures for the antisolvent micronization of hydrosoluble materials. Chem. Eng. J. 2012, 187, 401. (24) Prosapio, V.; Reverchon, E.; De Marco, I. Antisolvent micronization of BSA using supercritical mixture carbon dioxide +organic solvent. J. Supercrit. Fluids 2014, 94, 189. (25) Prosapio, V.; Reverchon, E.; De Marco, I. Production of lysozyme microparticles to be used in functional foods, using an expanded liquid antisolvent process. J. Supercrit. Fluids 2016, 107, 106. (26) Herrero, M.; Mendiola, J. A.; Cifuentes, A.; Ibáñez, E. Supercritical fluid extraction: Recent advances and application. J. Chromatogr. A 2010, 1217 (16), 2495. (27) Caputo, G.; Scognamiglio, M.; De Marco, I. Nimesulide adsorbed on silica aerogel using supercritical carbon dioxide. Chem. Eng. Res. Des. 2012, 90 (8), 1082. (28) Smirnova, I.; Suttiruengwong, S.; Arlt, W. Feasibility study of hydrophilic and hydrophobic silica aerogels as drug delivery systems. J. Non-Cryst. Solids 2004, 350, 54. (29) Baldino, L.; Cardea, S.; Reverchon, E. Supercritical assisted enzymatic membranes preparation, for active packaging applications. J. Membr. Sci. 2014, 453, 409.

(30) Pisanti, P.; Yeatts, A. B.; Cardea, S.; Fisher, J. P.; Reverchon, E. Tubular perfusion system culture of human mesenchymal stem cells on poly-L-lactic acid scaffolds produced using a supercritical carbon dioxide-assisted process. J. Biomed. Mater. Res., Part A 2012, 100, 2563. (31) Baldino, L.; Cardea, S.; De Marco, I.; Reverchon, E. Chitosan scaffolds formation by a supercritical freeze extraction process. J. Supercrit. Fluids 2014, 90, 27. (32) De Marco, I.; Prosapio, V.; Cice, F.; Reverchon, E. Use of solvent mixtures in supercritical antisolvent process to modify precipitates morphology: Cellulose acetate microparticles. J. Supercrit. Fluids 2013, 83, 153. (33) Wang, Y.; Wang, Y.; Yang, J.; Pfeffer, R.; Dave, R.; Michniak, B. The application of a supercritical antisolvent process for sustained drug delivery. Powder Technol. 2006, 164 (2), 94. (34) Reverchon, E.; De Marco, I. Mechanisms controlling supercritical antisolvent precipitate morphology. Chem. Eng. J. 2011, 169, 358. (35) Rossmann, M.; Braeuer, A.; Dowy, S.; Gallinger, T. G.; Leipertz, A.; Schluecker, E. Solute solubility as criterion for the appearance of amorphous particle precipitation or crystallization in the supercritical antisolvent (SAS) process. J. Supercrit. Fluids 2012, 66, 350. (36) Marra, F.; De Marco, I.; Reverchon, E. Numerical analysis of the characteristic times controlling supercritical antisolvent micronization. Chem. Eng. Sci. 2012, 71, 39. (37) De Marco, I.; Knauer, O.; Cice, F.; Braeuer, A.; Reverchon, E. Interactions of phase equilibria, jet fluid dynamics and mass transfer during supercritical antisolvent micronization: the influence of solvents. Chem. Eng. J. 2012, 203, 71. (38) De Marco, I.; Rossmann, M.; Prosapio, V.; Reverchon, E.; Braeuer, A. Control of particle size, at micrometric and nanometric range, using supercritical antisolvent precipitation from solvent mixtures: application to PVP. Chem. Eng. J. 2015, 273, 344. (39) Montes, A.; Gordillo, M. D.; Pereyra, C.; Martinez de la Ossa, E. J. Co-precipitation of amoxicillin and ethyl cellulose microparticles by supercritical antisolvent process. J. Supercrit. Fluids 2011, 60, 75. (40) Uzun, I. N.; Sipahigil, O.; Dincer, S. Coprecipitation of Cefuroxime Axetil−PVP composite microparticles by batch supercritical antisolvent process. J. Supercrit. Fluids 2011, 55, 1059. (41) Wang, W.; Liu, G.; Wu, J.; Jiang, Y. Co-precipitation of 10hydroxycamptothecin and poly (l-lactic acid) by supercritical CO2 anti-solvent process using dichloromethane/ethanol co-solvent. J. Supercrit. Fluids 2013, 74, 137. (42) Nerome, H.; Machmudah, S.; Fukuzato, R.; Higashiura, T.; Youn, Y.; Lee, Y.; Goto, M. Nanoparticle formation of lycopene/βcyclodextrin inclusion complex using supercritical antisolvent precipitation. J. Supercrit. Fluids 2013, 83, 97. (43) Kalogiannis, C. G.; Michailof, C. M.; Panayiotou, C. G. Microencapsulation of Amoxicillin in Poly(L-lactic acid) by Supercritical Antisolvent Precipitation. Ind. Eng. Chem. Res. 2006, 45, 8738. (44) Franceschi, E.; De Cesaro, A. M.; Feiten, M.; Ferreira, S. R. S.; Dariva, C.; Kunita, M. H.; Rubira, A. F.; Muniz, E. C.; Corazza, M. L.; Oliveira, J. V. Precipitation of β-carotene and PHBV and coprecipitation from SEDS technique using supercritical CO2. J. Supercrit. Fluids 2008, 47, 259. (45) Priamo, W. L.; de Cezaro, A. M.; Ferreira, S. R. S.; Oliveira, J. V. Precipitation and encapsulation of β-carotene in PHBV using carbon dioxide as anti-solvent. J. Supercrit. Fluids 2010, 54, 103. (46) He, W.; Suo, Q.; Shan, A.; Li, C.; Huang, Y.; Li, Y.; Zhu, M. Production of natural carotene-dispersed polymer microparticles by SEDS-PA co-precipitation. J. Mater. Sci. 2007, 42, 3495. (47) Hong, H.; Suo, Q.; He, W.; Li, C. P. Formation of Carotene/ Proanthocyanidin Composite Microparticles via the SolutionEnhanced Dispersion by Supercritical Fluids (SEDS) Process through Prefilming Atomization. Ind. Eng. Chem. Res. 2007, 46, 6630. (48) Prosapio, V.; De Marco, I.; Scognamiglio, M.; Reverchon, E. Folic acid−PVP nanostructured composite microparticles by supercritical antisolvent precipitation. Chem. Eng. J. 2015, 277, 286. (49) Reverchon, E.; Torino, E.; Dowy, S.; Braeuer, A.; Leipertz, A. Interactions of phase equilibria, jet fluid dynamics and mass transfer 11574

DOI: 10.1021/acs.iecr.5b03504 Ind. Eng. Chem. Res. 2015, 54, 11568−11575

Article

Industrial & Engineering Chemistry Research during supercritical antisolvent micronization. Chem. Eng. J. 2010, 156, 446. (50) Shah, N.; Sandhu, H.; Choi, D. S.; Chokshi, H.; Malik, A. W. Amorphous Solid Dispersions: Theory and Practice; Springer, 2014. (51) De Marco, I.; Reverchon, E. Influence of pressure, temperature and concentration on the mechanisms of particle precipitation in supercritical antisolvent micronization. J. Supercrit. Fluids 2011, 58, 295.

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DOI: 10.1021/acs.iecr.5b03504 Ind. Eng. Chem. Res. 2015, 54, 11568−11575