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luteolin microparticles formation using a supercritical fluids assisted technique
Powder Technology 356 (2019) 899e908
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Zein/luteolin microparticles formation using a supercritical fluids assisted technique I. Palazzo a, R. Campardelli b, *, M. Scognamiglio a, E. Reverchon a a b
Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II, 132 e 84084 Fisciano (SA), Italy Department of Civil, Chemical and Environmental Engineering (DICCA), University of Genoa, Via Opera Pia 15, 16145 Genova (GE), Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 February 2019 Received in revised form 5 September 2019 Accepted 12 September 2019 Available online 13 September 2019
In this work, a supercritical-based process, named Supercritical Assisted Injection in a Liquid Antisolvent (SAILA), has been proposed for the entrapment of an antioxidant, luteolin, in zein microparticles. Zein submicro and micrometric particles, with an average size between 0.26 and 2 mm, were obtained using ethanol-water mixtures as solvent with composition ranging from 70/30 to 90/10 volume ratio. Then, the experiments for the coprecipitation of zein/luteolin were performed. Using SAILA, zein/luteolin microparticles with a mean diameter of 1.20 ± 0.20 ÷ 1.26 ± 0.17 mm and good entrapment efficiencies, up to 82%, were obtained. The coprecipitates were characterized by a homogeneous drug dispersion in the polymer matrix. The antioxidant activity of coprecipitated particles was comparable to the one of luteolin in native state. Coprecipitates showed faster dissolution rate (2 ÷ 3 h) with respect to the physical mixture zein/ luteolin (20h), with a consequent increase in luteolin bioavailability. © 2019 Elsevier B.V. All rights reserved.
Keywords: Luteolin Zein Microparticles Supercritical fluids
1. Introduction Encapsulation is largely applied to stabilize nutraceutical products. Some natural compounds (e.g. proteins, vitamins, minerals, antioxidants) have a beneficial effect and they are used to create functional foods. Flavonoids are polyphenols that are abundant in food; e.g., vegetables, fruits, and medicinal herbs. They play an important role in defending plant cells against microorganisms, insects, and UV irradiation [1,2]. Investigation and use of flavonoids in functional foods have been object of great interest especially because they demonstrate a significant beneficial to human and animal health for their anti-inflammatory, anti-oxidant, cytostatic, apoptotic and estrogenic actions [3]. It has been noticed that flavonoids may be a cancer preventive [4]. Among the flavonoids, luteolin is present in a wide range of plants such as in celery, green pepper, perilla leaf, chamomile tea, broccoli, and carrots [5]. The activity and potential health benefits of luteolin are limited to instability under acidic conditions encountered in the stomach, low permeability within the gut, processing and storage that result in small quantity of the molecules available by oral administration [6,7]. Therefore, luteolin delivery requires formulations to maintain
* Corresponding author. E-mail address: [email protected] (R. Campardelli). https://doi.org/10.1016/j.powtec.2019.09.034 0032-5910/© 2019 Elsevier B.V. All rights reserved.
the active molecular form up to the time of consumption and preserve the stability, bioactivity and bioavailability. Several encapsulation techniques of food ingredients and additives have been proposed such as emulsions, micelles, liposomes and biopolymer matrices [8e10]. Considering entrapment in biopolymer matrix for in the food industry, the materials used must be granted GRAS (generally regarded as safe) and relatively cheap. Furthermore, techniques used need to be economical, reproducible and robust. A variety of natural and/or synthetic polymers [11e13] have been intensively investigated for formulate intelligent and selective delivery systems in order to maximize action of the active compounds in food applications. Among natural polymers, proteinbased polymers are attracting the attention of scientists and industry as possible encapsulation materials for nutraceutical compounds [14]. Most of the protein polymers that have been used for nutraceutical delivery are water-soluble proteins with limited sustained-release characteristics [15]. Zein is a protein extracted from corn gluten meal and it is one of the few cereal proteins extracted in a relatively pure form [16,17]. It is a hydrophobic compound and for this reason it has been successfully applied as a promising carrier for encapsulation and controlled release of water-insoluble compounds in the pharmaceutical and food industries [18e25]. To produce zein micro and nanoparticles for encapsulation and delivery application, several
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industrial scalable approaches have been recently reported. Zein nanoparticles using a liquid-liquid dispersion process were produced for the potential development of food-grade delivery systems. Zein nanoparticles of 100e200 nm were produced, and smaller particles were formed at a higher shear rate, at higher ethanol concentration or at lower zein concentration in stock solutions [26]. Using this process, two essential oils, thymol and carvacrol, were encapsulated in the nanoparticles of zein using the liquid-liquid dispersion method [23]. Zein colloidal particles below 350 nm, without or with sodium caseinate (NaCas) as the stabilizer, were obtained using a continuous technique termed Flash NanoPrecipitation (FNP) [27]. GomezEstaca et al. produced zein nanoparticles with a compact spherical structure and a narrow size distribution by electrohydrodynamic atomization [28]. Using a built-in ultrasonic dialysis process (BUDP), Jiang et al. studied the effects of dialysate properties on zein self-assembly. In their works, they obtained zein microspheres with a size around 1e2 mm [29] and a zein-based microcarrier system was proposed for targeted oral delivery using indomethacin and hydroxycamptothecin as model drugs [30e32]. However, these technologies show some limitations and need to be further evaluated before industrialization. Several problems are connected to these processes for example, difficulties controlling of the zein particles size [33], large use of toxic organic solvent, with consequently expenses to reduce the solvent residues, possible degradation of the active product due to thermal stress, dimension of zein particles caused by process parameters [28]. Supercritical carbon dioxide (SC-CO2) based processes have been proposed as a promising alternative to traditional techniques to overcome their limitations. They are frequently used in several fields such as extraction, micronization, coprecipitation and entrapment of polymers and drugs [34e38]. In particular, using Supercritical Assisted Atomization (SAA) luteolin was micronized and encapsulated of using polyvinylpyrrolidone (PVP) and dextran [34,39]. Zein micronization was also attempted using supercritical anti-solvent precipitation (SAS) [40]. In particular, zein microparticles were produced using 2% w/w of zein in a 90% aqueous ethanol mixture. However, the particles obtained were characterized by an irregular shape and wide distribution of the dimensions. Better results were obtained using methanol as solvent; in particular, nanometric particles of zein were obtained varying the percentage of methanol from 95% to 100% [40]. Therefore, the production of zein particles at nanoscale with uniform size unless 100% methanol used as solvent is difficult and it is necessary a large flow rate of supercritical CO2 to remove the toxic methanol residue [41]. Liu et al. developed a drug nanocrystal delivery system by coupling SAS process with BUDP. In detail, 10-Hydroxycamptothecin nanocrystals (HCPT) were successfully incorporated into zein microspheres by combining the SAS process with BUDP: the co-precipitation of HCPT and zein prepared using the SAS process was dispersed into ethanolewater as the dialysis solution for BUDP [42]. Recently, Franco et al. have proposed the coprecipitation of zein/ diclofenac using SAS technique: zein micronization was studied and microparticles with a mean diameter of 4.19 mm were obtained, at 90 bar, 40 C using DMSO as solvent. The coprecipitation of zein/ diclofenac was also investigated at various process conditions: in particular, higher polymer/drug ratio (20/1 and 30/1) assured an efficient coprecipitation of the two compounds [43]. Hu et al. have produced coprecipitated particles of zein/lutein 24/1 by supercritical antisolvent using a mixture of acetone/DMSO (7/3 v/v) as solvent with an entrapment efficiency varying between 67% and 83% using a range of temperature between 32 C and 45 C. However, decreasing the polymer/drug ratio (12/1), entrapment efficiencies were drastically reduced (34%) and the produced particles were
aggregated, with the presence of external drug crystals not encapsulated in the polymer matrix [44]. Supercritical Assisted Injection in a Liquid Antisolvent (SAILA) is a recent proposed process based on the continuous injection of an expanded liquid (formed by an organic solvent, a solid solute and SC-CO2) in an antisolvent solution [45]. SC-CO2 acts as a co-solute being miscible with the solution to be treated and favors the atomization in the antisolvent. In this process the solute has to be soluble in the solvent, but not in the antisolvent and at the same time the solvent and the antisolvent have to be completely miscible. A saturator filled with packing elements is used and a near-equilibrium solution between solute, solvent and CO2 is formed. This solution is sprayed through a thin wall injector into the precipitation vessel containing water, used as antisolvent. The mixing, between the two fluids, produces a rapid supersaturation and the precipitation of the particles. This process has numerous advantages, such as no thermal degradation, the possibility of using nontoxic solvents (for example acetone and ethanol), producing directly a stabilized suspension with a good control on particle size distribution. This process was successfully used to micronize pharmaceutical compounds [45] and polymers [46,47] obtaining micrometric and nanometric particle suspensions of controlled size and distribution. Recently, composite drug-polymer of piroxicam/ PLGA particles were produced [48]. Therefore, the aim of this work is to explore the application of SAILA as an alternative technique for the production of composite zein/luteolin microparticle suspensions to be used in nutraceutical fields. For this reason, first feasibility tests for the micronization of zein proteins using the SAILA process will be conducted; then, the optimization of the operative parameters for the coprecipitation of zein/luteolin will be tested. In particular, the effect of process parameters on particle diameter, size distribution and entrapment efficiency will be systematically studied. Morphological, granulometric, solid state and drug release analyses will be performed to characterize coprecipitates and to investigate the distribution of the active compound inside the particles. Moreover, the antioxidant activity of the composite compound will be evaluated and compared with the pure antioxidant. 2. Materials Zein was purchased from Sigma (SigmaeAldrich, St. Louis, USA), micronized luteolin was supplied from Epitech Group Research Laboratories. Carbon dioxide, CO2, purity 99.5% was provided by Morlando Group (Naples, Italy). Polysorbate (Tween 80, Sigma Aldrich Chemical Co., Milan, Italy), acetone (AC, purity 99.9%, Sigma Aldrich Chemical Co., Milan, Italy), ethanol (EtOH, purity 99.8%, Sigma Aldrich Chemical Co., Milan, Italy), 2,2-diphenyl-1picrylhydrazyl (DPPH, Sigma Aldrich Chemical Co., Milan, Italy), 2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, Sigma Aldrich Chemical Co., Milan, Italy) were used as received. Distilled water was produced in the laboratory. 3. Apparatus The SAILA apparatus (Fig. 1) consists of two feed lines, used to deliver compressed CO2 and liquid solvent to a saturator. Carbon dioxide (R1) is cooled using a refrigerating bath (RB), pumped (P1, Lewa Eco, model LDC-M-2), preheated and delivered to the mixing vessel, saturator (S). The solvent liquid solution (R2), in which a solid solute is dissolved, is pumped using a membrane pump (P2, Gilson, model 305). The saturator is a high-pressure static mixer with an internal volume of 0.15 dm3. It is packed with stainless steel perforated saddles and thermally heated by thin band heaters. The liquid solution is prepared dissolving a known quantity of solute (polymer and drug) in an organic solvent. SC-CO2 is continuously
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4.3. Entrapment efficiency and dissolution tests The entrapment efficiency was measured dissolving 5 mg of washed microspheres in 3 mL of a solvent mixture water/ethanol 20/80 and measuring the absorbance of the solution at 350 nm. The entrapment efficiency was calculated as the ratio between the measured and theoretical drug contents. Dissolution tests were performed suspending microspheres (3 mg) containing luteolin in 2 mL of phosphate-buffered saline (PBS) and loading the suspension in dialysis sack. Then, luteolin release profiles were determined in 250 mL of PBS at pH 7.4 continuously agitated at 200 rpm in a 37 C incubator to maintain adequate sink conditions. Released luteolin was determined continuously measuring the absorbance at 350 nm (UVevis mod. Cary 50, Varian). All analysis performed in triplicate. 4.4. Solid state characterization
Fig. 1. SAILA process layout. R1: CO2 reservoir; R2: Liquid solution reservoir; RB: refrigerating bath; P1: CO2 pump; P2: solvent pump; P3: antisolvent peristaltic pump; S: saturator; TC: temperature controller; PC: pressure controller.
solubilized in the liquid solution, forming an expanded liquid, whose composition determines the position in the vapor-liquid equilibrium diagram (VLE) of the system CO2-organic solvent, and depends on the operative conditions of temperature, pressure and on gas to liquid ratio (GLR), expressed as weight ratio. The operative conditions are monitored with a temperature controller (TC) and a pressure controller (PC). At the exit of the mixer, the expanded solution is injected through a micrometric nozzle (nozzles of 80 and 100 mm diameter were used in this work) in an antisolvent phase, water. A surfactant, Tween 80 at 0.2% w/w, was added to the antisolvent phase to stabilize the formed particles. The water phase is continuously pumped using a peristaltic pump (P3) from a reservoir to the receiving vessel; a regulation valve is used to recover continuously the suspension and to maintain constant the liquid level in the vessel. Experiments were performed in triplicates.
4. Methods 4.1. Particles diameter The suspensions were characterized with Dynamic Light Scattering (DLS) (Zetasizer, mod.5000, Malvern Instruments Ltd., UK) to obtain Particles Size Distribution (PDS). Mean diameter (MD), standard deviation (SD) and polydispersity index (PDI) of the particles were measured for each sample. The sample was analyzed 3 times in order to calculate the mean value of the measurements.
4.2. Particles morphology Particle morphology was analyzed using a Field EmissionScanning Electron Microscope (FESEM, LEO 1525, Carl Zeiss SMT AG). Particles were centrifuged at 6500 rpm for 20 min at 4 C (Thermo Scientific, mod. IEC CL30R) and filtered using a membrane (Millipore MF membrane filter, Filter type 0.2 mm HA). Membrane were dried at air in order to recover microparticles. To perform FESEM analysis, powders were coated with a gold layer, using a sputter coater (thickness 250 Å, model B7341; Agar Scientific, Stansted, UK).
Solid-state analysis of the precipitates was performed by powder X-ray diffraction (PXRD; model D8 Advance; Bruker AXS, Madison, WI) using a copper-sealed tube source. Samples were placed in the holder and flattened with a glass slide to assure a good surface texture. The measuring conditions were nickel-filtered Cu Ka radiation, l ¼ 1.54 A, and a 2q angle ranging from 5 to 90 with a scanning rate of 3 s/step and a step size of 0.592 . Microparticle thermal characteristics were determined by differential scanning calorimetry (DSC; model TC11, Mettler Toledo, Inc., Columbus, OH). An accurately weighed amount of powder (±5 mg) was crimped in a standard aluminum pan that was pierced and heated from 25 to 350 C at a scanning rate of 10 C/min in a nitrogen atmosphere at a flow rate of 50 mL/min. Fourier transform infrared (FT-IR) spectra was performed with a FTIR spectrophotometer (IR-Tracer100, Shimadzu). The samples were combined with a small amount of potassium bromide and were prepared by compressing the powders in a hydraulic press. Analyses were performed at 25 C, in the range 400e4000 cm1 at a resolution of 1 cm1 as the mean of 16 measurements to reduce the noise. 4.5. Antioxidant activity 4.5.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay. The evaluation of radical scavenging activity (antioxidant activity) was performed adapting the method of Blois (1958) with modifications. An ethanolic solution of DPPH 1 104 M was prepared. To obtain a calibration curve, 100 mL of different luteolin concentrations (100 mg/mL, 50 mg/mL, 20 mg/mL, 10 mg/mL and 5 mg/mL dissolved in H2O/Ethanol 20/80 v/v solution) were added in 2900 mL of DPPH solution. Each samples were left at room temperature for 30 min under light protection. The absorbance was measured at 517 nm. The lower the absorbance of the reaction mixture, the higher is free radical scavenging activity. A fixed quantity of zein/luteolin coprecipitates was then dissolved in H20/ Ethanol 20/80 v/v and DPPH method was applied. All determinations were carried out in triplicate. The scavenging ability of DPPH radical was calculated by using the following equation:
A Scavenging effect ð%Þ ¼ 1 A 100 AB
(1)
where AA is the absorbance at 517 nm of the sample treated with DPPH in ethanol solution, and AB is the control absorbance at 517 nm of DPPH in ethanol. The inhibition percentage obtained in this way was compared with the pure unprocessed samples percentage, to calculate the decrease of DPPH inhibition capacity between unprocessed and processed samples.
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Table 1 Zein precipitation SAILA tests performed at different water/ethanol and water/ acetone solvent mixture. Mean diameters (MD), standard deviation (SD) and polydispersity index (PDI) are also reported. Test
Solvent mixture
Mixture composition [v/v]
MD ± SD [mm]
PDI
Z_01 Z_02 Z_03 Z_04 Z_05
H2O/EtOH H2O/EtOH H2O/EtOH H2O/AC H2O/AC
30/70 20/80 10/90 30/70 20/80
0.31 ± 0.09 0.41 ± 0.14 1.33 ± 0.20 0.38 ± 0.08 0.44 ± 0.18
0.24 0.35 0.30 0.40 0.41
4.5.2. 2,20 -Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assay ABTS method was used to reinforce the results obtained from the DPPH radical scavenging activity assay. ABTS was dissolved in deionized water to a 7 mM concentration. ABTS radical cation (ABTSþ) was produced by reacting ABTS solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 16 h before use. For the study, 1 mL of ABTSþ solution was diluted in PBS (25 mL) until to obtain an absorbance of 0.7 (±0.02) at 734 nm. A calibration curve was prepared as DPPH assay. The coprecipitates were dissolved in in H20/Ethanol 20/80 v/v and after, 100 mL of each samples was mixed with 2900 mL of ABTSþ solution. The absorbance reading was taken at 30 C for 10 min after initial mixing (AA) and an appropriate solvent blank reading was taken (AB). All determinations were carried out in triplicate. The percentage of inhibition of ABTSþ was calculated using above formula (Eq. (1)).
4.6. Statistical analysis The results are expressed as mean ± SD. Normal distribution of the variables was analyzed by the ShapiroeWilk test followed by
the WilcoxoneManneWhitney U test, ANOVA or Student’s t-test (StataSE 12 statistical software, StataCorp LP, USA). Statistically significant differences among groups were considered when P .05. 5. Experimental results The principle at the basis of all the liquid antisolvent processes is that to obtain the solute precipitation, the solute has to be soluble in the solvent and not soluble in the antisolvent. At the same time the solvent and the antisolvent have to be completely miscible. SAILA adds to traditional processes the peculiar properties of supercritical fluids, namely of SC-CO2, to reduce the surface tension of the liquid solution. Operating with SC CO2 smaller droplets can be produced when the injection is performed, due to the reduction of the cohesive forces. Zein is not soluble in water, ethanol or acetone but, it is soluble in aqueous ethanol and aqueous acetone mixtures. For this reason, zein micronization and coprecipitation were tested using the SAILA process using 90e70% (w/w) aqueous ethanol (H2O/EtOH) and 90e70% (w/w) aqueous acetone (H2O/AC) solutions as solvent and water as antisolvent. 5.1. Zein precipitation feasibility experiments Ternary diagrams CO2-ethanolewater are reported in the literature [49]. They have been used in this work, to located the SAILA operative point in the miscibility hole of the ternary system. During the feasibility experiments, zein was processed setting SAILA process conditions selected in a previous study [48]: saturator temperature of 60 C, gas liquid ratio (GLR) 1.5 w/w, CO2 flow rate 12 g/ min, nozzle diameter 80 mm and the polymer was dissolved in the solvent mixture at a concentration of 5 mg/mL. The operating pressure was set at 100 bar for all the experiments. The antisolvent was maintained at room temperature. The composition of the
Fig. 2. FESEM of zein particles precipitated using different solvent mixtures: (a) 20/80 H2O/EtOH, (b) 20/80 H2O/AC.
Table 2 SAILA experiments performed on zein/luteolin mixtures using different solvent mixture and injector diameter with polymer/drug ratio at 20/1 and saturator temperature of 60 C. Test
Solvent mixture composition [v/v]
ZL_01 ZL_02 ZL_03 ZL_04 ZL_05 ZL_06 ZL_07 ZL_08
30/70 20/80 10/90 30/70 20/80 10/90 30/70 20/80
H2O/EtOH H2O/EtOH H2O/EtOH H2O/EtOH H2O/EtOH H2O/EtOH H2O/AC H2O/AC
Injector diameter [mm]
Pressure [bar]
MD ± SD [mm]
PDI
EE [%]
80 80 80 100 100 100 80 80
100 100 100 98 98 98 82 82
0.27 ± 0.05 1.26 ± 0.17 1.35 ± 0.43 0.38 ± 0.13 1.20 ± 0.22 1.43 ± 0.33 0.51 ± 0.20 0.59 ± 0.19
0.53 0.25 0.64 0.71 0.37 0.46 0.39 0.32
43.42 68.52 e 23.37 81.79 e 32.52 27.93
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solvent mixture was changed to understand its effect on PSD and morphology of zein particles. H2O/EtOH and H2O/AC mixtures were used at different composition (30/70, 20/80, 10/90 v/v). The composition 10/90 H2O/AC was not used because the polymer was not soluble in this mixture. Stable and homogeneous suspensions were obtained for all the tests, and the used solvent mixture are summarized in Table 1. Submicro and microparticles were successfully obtained in all
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the experiments, as reported in Table 1. Stable aqueous suspensions were produced, with a stability over three mounts of observation, without the presence of sedimenting particles. In Fig. 2, FESEM image of the tests performed at mixture composition of 20/80 of H2O/EtOH and H2O/AC are reported for comparison. In all cases tested, spherical and well separated particles were obtained. The data reported in Table 1 show that, decreasing water content in the solvent mixture, an increase of particles diameter
Fig. 3. FESEM images of zein/luteolin microparticles produced using different injector diameter (80 mm on the left and 100 mm on the right) and different solvent mixture ratio H2O/ Ethanol at 60 C, GLR 1.5, polymer concentration 5 mg/mL, polymer/drug ratio 20/1.
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can be observed for both solvent mixture studied. This result could be explained considering that, as the amount of water decreases in the starting solution, zein solutions become gradually closer to the saturation. Since in the SAILA process, the formation of the particles is based on the precipitation of the solute due to an antisolvent effect, precipitation rate is directly proportional to the degree of supersaturation [47,50]. For this reason, for solutions in which solute concentration is closer to saturation, the precipitation, due to the anti-solvent effect, produces larger particles and a wider particle size distribution. 5.2. Coprecipitation feasibility experiments SAILA coprecipitation feasibility tests were carried out using different solvent mixtures H2O/EtOH and H2O/AC. Process parameters were left unchanged with respect to zein precipitation experiments. Polymer concentration in solvent mixtures was set at 5 mg/mL and polymer/antioxidant ratio at 20/1. Experimental results are reported in Table 2. It can be observed that, using ethanol in the solvent mixture, when the amount of water was reduced from 30/70 to 10/90, a progressive increase in the size of the obtained particles was obtained, passing from 0.27 ± 0.05 mm to 1.35 ± 0.43, respectively. Considering the FESEM images of Fig. 3, it is possible to note that operating at 30/70 H2O/EtOH, mainly spherical particles of submicrometric dimensions (0.27 ± 0.05 mm) have obtained (Fig. 3a); whereas, when the quantity of water decreased, particles produced were spherical with a micrometric diameter of 1.26 ± 0.17 mm (Fig. 3b). In the case of a solvent mixture of 10/90 H2O/EtOH, the precipitated material was irregular showing two morphologies: crystals of drug and spherical particles of zein. Thus, in this case the coprecipitation was not successful, resulting in a visible separate precipitation of polymer and drug (Fig. 3c). Entrapment efficiencies ranged between 43 and 68% for aqueous ethanol. In particular, in Table 2 it is possible to observe that luteolin was entrapped in the polymer matrix with a lower entrapment efficiency in the case of the tests carried out with the ratio H2O/ EtOH 30/70; in this case the efficiency was 43.44%. Entrapment efficiency of 68.52% was obtained using the 20/80 ratio. The same set of experiments was performed using a 100 mm injector. Data reported in Table 2 show that, also in this case, coprecipitates were obtained only from mixtures with 70e80% ethanol (Fig. 3d and e); therefore, the mixture 10/90 H2O/ETOH is not useful to produce composite particles because, as reported in Fig. 3f, polymer particles separated from crystal of luteolin were obtained. The increase of the injector diameter from 80 to 100 mm, resulted in a slight increase of the average size of coprecipitated particles (see Table 2 and Fig. 3). Entrapment efficiencies of 23.37% and 81.79% were obtained starting form mixtures containing 70% and 80% ethanol respectively. Moreover, as in the previous case, the best mixture for the production of zein/luteolin coprecipitates is 20/80 H2O/EtOH; indeed, a larger value of entrapment efficiency was obtained. Coprecipitation experiments were also performed using the H2O/AC mixtures in the 30/70 and 20/80 ratios. The composition 10/ 90 H2O/AC ratio was not used because the polymer is not soluble in this mixture. Data reported in Table 2, show that the obtained particles were characterized by sub-micrometric dimensions for both mixtures tested, in particular, in the range 0.51 ± 0.20 and 0.59 ± 0.19 mm respectively. Particles were spherical also in this case, but, in the case of acetone, the solvent mixture did not significantly influence the particle size distribution. However, entrapment efficiencies were lower than those obtained using the ethanol mixture. Indeed, entrapment efficiencies between 27% and 32% were obtained. For this reason, only the ethanol water solvent mixture was used for the further experiments reported in this work.
Some experiments were performed changing the polymer/drug ratio and saturation temperature using the solvent mixture H2O/ EtOH at the volumetric ratio 20/80. The experiments performed are summarized in Table 3. Zein/luteolin polymer/drug ratio was varied from 20/1 to 5/1 w/w, fixing the overall solution concentration at 5 mg/mL. Example of FESEM images of the precipitated powders are shown in Fig. 4. The 20/1 ratio FESEM image is not reported because it was already shown in Fig. 3b. When a mixture zein/luteolin 5/1 w/w was processed, the precipitated powder was irregular and it is possible to note that crystals precipitated separately from spherical particles (Fig. 4a) and are organized as flower-like structures. Increasing polymer/ drug ratio at 10/1, the phenomenon of crystals precipitation was eliminated, only spherical particles with a mean diameter of 0.25 ± 0.07 nm are visible in the corresponding FESEM image (Fig. 4b). At the ratio zein/luteolin 20/1, regular microparticles were
Table 3 SAILA tests performed for zein/luteolin at different polymer/drug ratios, temperatures and at pressure of 100 bar. Test
Polymer/drug ratio
Temperature [ C]
MD ± SD [mm]
PDI
EE [%]
ZL_09 ZL_10 ZL_02 ZL_11 ZL_12 ZL_13 ZL_14
5/1 10/1 20/1 10/1 10/1 20/1 20/1
60 60 60 80 40 80 40
0.26 ± 0.08 0.25 ± 0.07 1.26 ± 0.17 0.20 ± 0.04 0.25 ± 0.05 0.23 ± 0.03 1.26 ± 0.24
0.64 0.59 0.25 0.44 0.41 0.26 0.19
e 46.45 68.52 3.6 63.96 15.77 80.15
Fig. 4. FESEM images of zein/luteolin particles produced using different polymer/drug ratio at 60 C, 92 bar, GLR 1.4, polymer concentration 5 mg/mL and solvent mixture H2O/EtOH 20/80.
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also obtained, as discussed in the previuos set of experiments (Fig. 3b). Luteolin entrapment efficiency of the coprecipitated particles obtained at 10/1 of polymer drug ratio was 46.4%; whereas, an efficiency of 68.5% was obtained at 20/1 ratio. Using 10/1 and 20/1 polymer/drug ratio, also the effect of higher and lower temperatures was studied, in particular the same set of experiments was performed at temperature of 80 C and 40 C. These temperatures have been chosen because in the SAILA process, an increase in temperature generally produced a reduction in the size of the particles produced [45]; whereas, operating at temperatures of about 40 C, good entrapment efficiencies were observed for composite systems zein/lutein [44]. Suspensions were successfully produced at 80 C and 40 C for the tested polymer/drug ratio see Table 3 (# 4e7). From the data reported in Table 3 and from the FESEM images of Fig. 5, it is possible to note a reduction in the mean diameter when the temperature increased and the polymer/drug ratio decreased. In the tests carried out at 80 C the formation of aggregates was observed. Better results were obtained when the saturator temperature was set at 40 C; indeed, being natural compounds, lower temperatures can favor conservation and processability of the material. The trends of the entrapment efficiencies with the variation of temperature and polymer/drug ratio are summarized Fig. 6, where the data obtained from the tests carried out at 60 C is also reported. Fig. 6, shows a clear trend: the entrapment efficiency increases with decreasing the temperature and with the increasing of polymer/drug ratio in all the studied cases. The increase of the entrapment efficiency at lower temperatures could be due to a slower diffusion of the drug in the anti-solvent phase. During the precipitation, part of the luteolin tends to spread towards the antisolvent phase, in which it has a reduced solubility. For this reason, when the temperature is low, this diffusion effect is reduced. Regarding the influence of the polymer/drug on the entrapment efficiency, a larger quantity of polymer favors the coprecipitation between the two compounds. The presence of a larger quantity of
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polymer molecules favors luteolin wrapping during zein precipitation, resulting in larger entrapment efficiencies. 5.3. Characterization of coprecipitates FT-IR analyses were carried out for unprocessed luteolin, pure zein, and SAILA processed zein/luteolin in the case of coprecipitation and in the case of separate precipitation of polymer and antioxidant, to analyze the chemical bonds of native and processed structures. The comparison of FT-IR spectra (Fig. 7a) shows that the characteristic peaks of processed compounds did not shown displacements with respect to the spectra of unprocessed compounds, confirming that the process does not modify compounds structure. It is also evident that, in coprecipitates (ZL_05), the spectrum is much more similar to polymer spectrum even if there are peaks (between 1000 and 2000 cm-1) due to the presence of the drug; whereas, when the luteolin is not coprecipitated (ZL_11) the spectrum is closer to that of unprocessed luteolin, because drug crystals are not encapsulated in the polymeric matrix. Unprocessed polymer, antioxidant and a coprecipitated powder were also characterized by X-ray diffraction (XRD) analyses. These analyses revealed that unprocessed drug shows the typical crystalline structure of luteolin: whereas, when a coprecipitation was obtained, zein/luteolin samples showed an amorphous structure similar to that native Zein (Fig. 7b). This result can be explained considering that luteolin is distributed in the polymeric matrix, and the drug has a low mobility among polymer chains; therefore, and the sample is predominantly in the amorphous state. DSC analysis was also carried out (Fig. 7c): zein does not present particular variations with temperature, whereas, luteolin shows an endothermic peak around 350 C due to crystal fusion. It is possible to note that the composite particles of zein/luteolin show a behavior very similar to that of polymer: in none of the analyzed tests the peak of crystallinity of the drug is detected. Therefore, the microdispersion of luteolin in the polymeric matrix, led to the
Fig. 5. FESEM images of Zein/Luteolin particles produced using different temperature and polymer/drug ratio, with solvent mixture H2O/EtOH at 20/80 composition.
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Fig. 6. Trend of the entrapment efficiency at different temperatures and zein/luteolin ratios.
formation of an amorphous compound. Drug release tests were also performed to compare the dissolution kinetics of different samples. Indeed, if the coprecipitation has properly occurred, luteolin is dispersed inside zein and its
release rate should be influenced by the presence of the polymer. Release kinetics of the drug in a phosphate buffer (PBS) was studied, at a temperature of 37 C, and the results obtained were compared with the release kinetics of the physical mixture zein/ luteolin (Fig. 8a). It has been found that the composite microparticles release the drug faster than the physical mixture. The physical mixture completes the dissolution of the drug in about 20 h. Zein/ luteolin coprecipitates, instead, produce a complete dissolution of the antioxidant in the time interval of 2 h and 30 min/3 h. When the coprecipitation is successful, luteolin should be uniformly dispersed at nanometric level in the polymer matrix and it is, therefore, released rapidly by a mechanism of diffusion through the polymer in consequence of degradation/swelling of zein particles. It has to be considered that, with respect to the physical mixture obtained using zein and luteolin separately precipitated by SAILA, coprecipitates allow to increase the area exposed to the release medium, thus increasing the dissolution rate [34]. In Fig. 8b, as further confirmation, of these considerations, release test performed for the powder obtained at the process conditions in which the polymer and the drug precipitated separately is reported. A dissolution test of the drug released from a physical mixture formed at the same polymer/antioxidant ratio was performed and it is also reported for comparison purpose. It can be seen that, when coprecipitation was not successful, release times similar to physical
Fig. 7. FT-IR analyses of unprocessed and processed powders; (b) XRD analyses of unprocessed and processed powders.
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Fig. 8. Luteolin release from zein microparticles produced at different temperature and injector diameter with the polymer/drug ratio ratio 20/1.
Table 4 Radical scavenging activity of coprecipitates with DPPH and ABTS assay. Tests
ZL_02 ZL_05 ZL_07 ZL_08
Theoretical loading [mg/mL]
EE
50.0 61.0 52.0 58.5
68.5 81.8 32.5 27.9
DPPH
ABTS
Scavenging effect [%]
Scavenging effect [%]
16.6 26.9 9.5 10.8
24.8 29.4 28.7 27.8
mixture are obtained. The previous characterizations confirmed the presence of drug in the microparticles produced, the good entrapment efficiencies in the polymer matrix and the increase of the luteolin bioavailability. However, antioxidants are very delicate substances and their antioxidant power is greatly reduced due to environmental degradation agents such as light or heat; therefore, they need to be protected and encapsulated in biocompatible polymers, zein in this case. The entrapment process conditions could lead to a degradation and a reduction of the intrinsic properties of luteolin. This aspect is fundamental to determine the success of the process. Therefore, spectrophotometric assays (DPPH and ABTS as explained in Material and Methods section) have been used for the determination of the radical scavenging activity of the obtained coprecipitates. The test was first performed on pure luteolin. Samples were prepared dissolving luteolin at different concentrations in H2O/ETOH 20/80 and adding at each sample the ethanolic solution of DPPH and ABTS. The absorbance values obtained were converted in terms of % inhibition. Hence, with the same technique, the coprecipitated samples were analyzed. The scavenging activity of each sample analyzed is reported in Table 4, where it is also reported the theoretical loading of the luteolin inside the particles and the entrapment efficiency. The analyzed samples preserved their antioxidant activity from comparison with native antioxidant. Therefore, it is possible to confirm that the process has not largely changed the antioxidant properties of luteolin. 6. Conclusions SAILA technique was successfully used in the precipitation of zein, and in the production of composite particles of zein/luteolin. Zein submicro and micrometric particles, with an average size between 0.26 and 2 mm, were obtained. The coprecipitation process was successful only at selected, optimized operative conditions, in
the other cases separated precipitation of zein and luteolin was observed. Large polymer/drug ratios (20/1) and low temperatures (40 C) allowed the simultaneous precipitation of the two compounds with high luteolin entrapment efficiencies (up to 82%). The entrapment efficiency of the antioxidant in the polymer matrix was also affected by temperature because both luteolin and zein are natural compounds and sensitive to the process temperature used. SAILA coprecipitates enhanced the dissolution rate of the antioxidant compound and should be improve its bioavailability. References [1] J.B. Harborne, C.A. Williams, Advances in flavonoid research since 1992, Phytochemistry. 55 (6) (2000) 481e504, 2000/11/01/. [2] D.I. Tsimogiannis, V. Oreopoulou, Free radical scavenging and antioxidant activity of 5,7,30 ,40 -hydroxy-substituted flavonoids, Innovative Food Sci. Emerg. Technol. 5 (4) (2004) 523e528, 2004/12/01/. [3] J.A. Ross, C.M. Kasum, Dietary flavonoids: bioavailability, metabolic effects, and safety, Annu. Rev. Nutr. 22 (1) (2002) 19e34. PubMed PMID: 12055336. [4] M.L. Neuhouser, Review: dietary flavonoids and cancer risk: evidence from human population studies, Nutr. Cancer 50 (1) (2004) 1e7, 2004/09/01. [5] L.-L. Miguel, Distribution and biological activities of the flavonoid luteolin, Mini-Rev. Med. Chem. 9 (1) (2009) 31e59. [6] P. Zhou, L.-P. Li, S.-Q. Luo, H.-D. Jiang, S. Zeng, Intestinal absorption of luteolin from peanut hull extract is more efficient than that from individual pure luteolin, J. Agric. Food Chem. 56 (1) (2008) 296e300, 2008/01/01. [7] Z. Chen, M. Tu, S. Sun, S. Kong, Y. Wang, J. Ye, et al., The exposure of luteolin is much lower than that of apigenin in oral administration of flos chrysanthemi extract to rats, Drug Metabol. Pharmacokinet. 27 (1) (2012) 162e168. [8] M. Reza Mozafari, C. Johnson, S. Hatziantoniou, C. Demetzos, Nanoliposomes and their applications in food nanotechnology, J. Liposome Res. 18 (4) (2008) 309e327, 2008/01/01. [9] H. Yu, Q. Huang, Enhanced in vitro anti-cancer activity of curcumin encapsulated in hydrophobically modified starch, Food Chem. 119 (2) (2010) 669e674, 2010/03/15/. [10] K. Pathakoti, M. Manubolu, H.-M. Hwang, Nanostructures: current uses and future applications in food science, J. Food Drug Anal. 25 (2) (2017) 245e253, 2017/04/01/. [11] L.S. Nair, C.T. Laurencin, Biodegradable polymers as biomaterials, Prog. Polym. Sci. 32 (8) (2007) 762e798, 2007/08/01/. [12] V.R. Sinha, A. Trehan, Biodegradable microspheres for protein delivery, J. Control. Release 90 (3) (2003) 261e280, 2003/07/31/. [13] J.M. Dang, K.W. Leong, Natural polymers for gene delivery and tissue engineering, Adv. Drug Deliv. Rev. 58 (4) (2006) 487e499, 2006/07/07/. [14] A.O. Elzoghby, W.M. Samy, N.A. Elgindy, Protein-based nanocarriers as promising drug and gene delivery systems, J. Control. Release 161 (1) (2012) 38e49, 2012/07/10/. [15] G. Wang, H. Uludag, Recent developments in nanoparticle-based drug delivery and targeting systems with emphasis on protein-based nanoparticles, Expert Opin. Drug Deliv. 5 (5) (2008) 499e515, 2008/05/01. [16] W.L.J. Zein, A history of processing and use, Cereal Chem. 79 (1) (2002) 1e18. [17] L.C. Swallen, A. Zein, New industrial protein, Ind. Eng. Chem. 33 (3) (1941) 394e398, 1941/03/01. [18] X. Liu, Q. Sun, H. Wang, L. Zhang, J.-Y. Wang, Microspheres of corn protein, zein, for an ivermectin drug delivery system, Biomaterials. 26 (1) (2005)
908
I. Palazzo et al. / Powder Technology 356 (2019) 899e908
109e115, 2005/01/01/. [19] L.F. Lai, H.X. Guo, Preparation of new 5-fluorouracil-loaded zein nanoparticles for liver targeting, Int. J. Pharm. 404 (1) (2011) 317e323, 2011/02/14/. [20] Y. Luo, B. Zhang, M. Whent, L. Yu, Q. Wang, Preparation and characterization of zein/chitosan complex for encapsulation of a-tocopherol, and its in vitro controlled release study, Colloids Surf. B: Biointerfaces 85 (2) (2011) 145e152, 2011/07/01. [21] Y. Luo, Z. Teng, Q. Wang, Development of Zein nanoparticles coated with carboxymethyl chitosan for encapsulation and controlled release of vitamin D3, J. Agric. Food Chem. 60 (3) (2012) 836e843, 2012/01/25. [22] L. Chen, M. Subirade, Elaboration and characterization of soy/zein protein microspheres for controlled nutraceutical delivery, Biomacromolecules. 10 (12) (2009) 3327e3334, 2009/12/14. [23] Y. Wu, Y. Luo, Q. Wang, Antioxidant and antimicrobial properties of essential oils encapsulated in zein nanoparticles prepared by liquideliquid dispersion method, LWT Food Sci. Technol. 48 (2) (2012) 283e290, 2012/10/01/. [24] B. Zhang, Y. Luo, Q. Wang, Development of silverzein composites as a promising antimicrobial agent, Biomacromolecules. (9) (2010) 2366e2375, 2010/09/13;11. _ Ünalan, I. Arcan, F. Korel, A. Yemeniciog lu, Application of active zein-based [25] I.U. films with controlled release properties to control listeria monocytogenes growth and lipid oxidation in fresh Kashar cheese, Innovative Food Sci. Emerg. Technol. 20 (2013) 208e214, 2013/10/01. [26] Q. Zhong, M. Jin, Zein nanoparticles produced by liquideliquid dispersion, Food Hydrocoll. 23 (8) (2009) 2380e2387, 2009/12/01/. [27] K.-K. Li, X. Zhang, Q. Huang, S.-W. Yin, X.-Q. Yang, Q.-B. Wen, et al., Continuous preparation of zein colloidal particles by Flash NanoPrecipitation (FNP), J. Food Eng. 127 (2014) 103e110, 2014/04/01/. [28] J. Gomez-Estaca, M.P. Balaguer, R. Gavara, P. Hernandez-Munoz, Formation of zein nanoparticles by electrohydrodynamic atomization: effect of the main processing variables and suitability for encapsulating the food coloring and active ingredient curcumin, Food Hydrocoll. 28 (1) (2012) 82e91, 2012/07/01/ . [29] G. Liu, J. Feng, W. Zhu, Y. Jiang, Zein self-assembly using the built-in ultrasonic dialysis process: microphase behavior and the effect of dialysate properties, Colloid Polym. Sci. 296 (1) (2018 January 01) 173e181. [30] G. Liu, J. Pang, Y. Huang, Q. Xie, G. Guan, Y. Jiang, Self-assembled nanospheres of folate-decorated zein for the targeted delivery of 10-hydroxycamptothecin, Ind. Eng. Chem. Res. 56 (30) (2017) 8517e8527, 2017/08/02. [31] H. Wang, X. Zhang, W. Zhu, Y. Jiang, Z. Zhang, Self-assembly of Zein-based microcarrier system for colon-targeted oral drug delivery, Ind. Eng. Chem. Res. 57 (38) (2018) 12689e12699, 2018/09/26. [32] H. Wang, W. Zhu, Y. Huang, Z. Li, Y. Jiang, Q. Xie, Facile encapsulation of hydroxycamptothecin nanocrystals into zein-based nanocomplexes for active targeting in drug delivery and cell imaging, Acta Biomater. 61 (2017) 88e100, 2017/10//. PubMed PMID: 28433787. eng. [33] Y. Wang, G.W. Padua, Formation of Zein microphases in ethanolwater, Langmuir. 26 (15) (2010) 12897e12901, 2010/08/03. [34] A. Di Capua, R. Adami, E. Reverchon, Production of luteolin/biopolymer microspheres by supercritical assisted atomization, Ind. Eng. Chem. Res. 56 (15)
(2017) 4334e4340, 2017/04/19. [35] P. Chattopadhyay, R. Huff, B.Y. Shekunov, Drug encapsulation using supercritical fluid extraction of emulsions, J. Pharm. Sci. 95 (3) (2006) 667e679, 2006/03/01/. [36] P. Trucillo, R. Campardelli, E. Reverchon, Production of liposomes loaded with antioxidants using a supercritical CO2 assisted process, Powder Technol. 323 (2018) 155e162, 2018/01/01/. [37] L. Baldino, M. Scognamiglio, E. Reverchon, Extraction of rotenoids from Derris elliptica using supercritical CO2, J. Chem. Technol. Biotechnol. 93 (12) (2018) 3656e3660. [38] C. Gimenez-Rota, I. Palazzo, M.R. Scognamiglio, A. Mainar, E. Reverchon, Della porta G. b-carotene, a-tocoferol and rosmarinic acid encapsulated within PLA/ PLGA microcarriers by supercritical emulsion extraction: encapsulation efficiency, drugs shelf-life and antioxidant activity, J. Supercrit. Fluids 146 (2019) 199e207, 2019/04/01/. [39] A. Di Capua, R. Adami, L. Izzo, E. Reverchon, Luteolin/dextran-FITC fluorescent microspheres produced by supercritical assisted atomization, J. Supercrit. Fluids 130 (2017) 97e104, 2017/12/01/. [40] Q. Zhong, M. Jin, D. Xiao, H. Tian, W. Zhang, Application of supercritical antisolvent technologies for the synthesis of delivery systems of bioactive food components, Food Biophys. 3 (2) (2008 June 01) 186e190. [41] L. Yangchao, W. Qin, Zein-based micro- and nano-particles for drug and nutrient delivery: a review, J. Appl. Polym. Sci. 131 (2014) 16. [42] G. Liu, S. Li, Y. Huang, H. Wang, Y. Jiang, Incorporation of 10hydroxycamptothecin nanocrystals into zein microspheres, Chem. Eng. Sci. 155 (2016) 405e414, 2016/11/22. [43] P. Franco, E. Reverchon, I. De Marco, Zein/diclofenac sodium coprecipitation at micrometric and nanometric range by supercritical antisolvent processing, J. CO2 Utilization 27 (2018) 366e373, 2018/10/01/. [44] D. Hu, C. Lin, L. Liu, S. Li, Y. Zhao, Preparation, characterization, and in vitro release investigation of lutein/zein nanoparticles via solution enhanced dispersion by supercritical fluids, J. Food Eng. 109 (3) (2012) 545e552, 2012/ 04/01/. [45] R. Campardelli, E. Reverchon, a-Tocopherol nanosuspensions produced using a supercritical assisted process, J. Food Eng. 149 (2015) 131e136. [46] R. Campardelli, E. Oleandro, R. Adami, E. Reverchon, Polymethylmethacrylate (PMMA) sub-microparticles produced by supercritical assisted injection in a liquid antisolvent, J. Supercrit. Fluids 92 (2014) 93e99, 2014/08/01/. [47] R. Campardelli, E. Oleandro, E. Reverchon, Supercritical assisted injection in a liquid antisolvent for PLGA and PLA microparticle production, Powder Technol. 287 (2016) 12e19, 2016/01/01/. [48] R. Campardelli, E. Reverchon, Instantaneous coprecipitation of polymer/drug microparticles using the supercritical assisted injection in a liquid antisolvent, J. Supercrit. Fluids 120 (2017) 151e160, 2017/02/01/. [49] M.A. Rodrigues, J. Li, A.J. Almeida, H.A. Matos, Azevedo EGd, Efficiency of water removal from water/ethanol mixtures using supercritical carbon dioxide, Braz. J. Chem. Eng. 23 (2006) 205e212. [50] I.J. Joye, D.J. McClements, Production of nanoparticles by anti-solvent precipitation for use in food systems, Trends Food Sci. Technol. 34 (2) (2013) 109e123, 2013/12/01/.
Contents lists available at ScienceDirect
Powder Technology journal homepage: www.elsevier.com/locate/powtec
Zein/luteolin microparticles formation using a supercritical fluids assisted technique I. Palazzo a, R. Campardelli b, *, M. Scognamiglio a, E. Reverchon a a b
Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II, 132 e 84084 Fisciano (SA), Italy Department of Civil, Chemical and Environmental Engineering (DICCA), University of Genoa, Via Opera Pia 15, 16145 Genova (GE), Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 February 2019 Received in revised form 5 September 2019 Accepted 12 September 2019 Available online 13 September 2019
In this work, a supercritical-based process, named Supercritical Assisted Injection in a Liquid Antisolvent (SAILA), has been proposed for the entrapment of an antioxidant, luteolin, in zein microparticles. Zein submicro and micrometric particles, with an average size between 0.26 and 2 mm, were obtained using ethanol-water mixtures as solvent with composition ranging from 70/30 to 90/10 volume ratio. Then, the experiments for the coprecipitation of zein/luteolin were performed. Using SAILA, zein/luteolin microparticles with a mean diameter of 1.20 ± 0.20 ÷ 1.26 ± 0.17 mm and good entrapment efficiencies, up to 82%, were obtained. The coprecipitates were characterized by a homogeneous drug dispersion in the polymer matrix. The antioxidant activity of coprecipitated particles was comparable to the one of luteolin in native state. Coprecipitates showed faster dissolution rate (2 ÷ 3 h) with respect to the physical mixture zein/ luteolin (20h), with a consequent increase in luteolin bioavailability. © 2019 Elsevier B.V. All rights reserved.
Keywords: Luteolin Zein Microparticles Supercritical fluids
1. Introduction Encapsulation is largely applied to stabilize nutraceutical products. Some natural compounds (e.g. proteins, vitamins, minerals, antioxidants) have a beneficial effect and they are used to create functional foods. Flavonoids are polyphenols that are abundant in food; e.g., vegetables, fruits, and medicinal herbs. They play an important role in defending plant cells against microorganisms, insects, and UV irradiation [1,2]. Investigation and use of flavonoids in functional foods have been object of great interest especially because they demonstrate a significant beneficial to human and animal health for their anti-inflammatory, anti-oxidant, cytostatic, apoptotic and estrogenic actions [3]. It has been noticed that flavonoids may be a cancer preventive [4]. Among the flavonoids, luteolin is present in a wide range of plants such as in celery, green pepper, perilla leaf, chamomile tea, broccoli, and carrots [5]. The activity and potential health benefits of luteolin are limited to instability under acidic conditions encountered in the stomach, low permeability within the gut, processing and storage that result in small quantity of the molecules available by oral administration [6,7]. Therefore, luteolin delivery requires formulations to maintain
* Corresponding author. E-mail address: [email protected] (R. Campardelli). https://doi.org/10.1016/j.powtec.2019.09.034 0032-5910/© 2019 Elsevier B.V. All rights reserved.
the active molecular form up to the time of consumption and preserve the stability, bioactivity and bioavailability. Several encapsulation techniques of food ingredients and additives have been proposed such as emulsions, micelles, liposomes and biopolymer matrices [8e10]. Considering entrapment in biopolymer matrix for in the food industry, the materials used must be granted GRAS (generally regarded as safe) and relatively cheap. Furthermore, techniques used need to be economical, reproducible and robust. A variety of natural and/or synthetic polymers [11e13] have been intensively investigated for formulate intelligent and selective delivery systems in order to maximize action of the active compounds in food applications. Among natural polymers, proteinbased polymers are attracting the attention of scientists and industry as possible encapsulation materials for nutraceutical compounds [14]. Most of the protein polymers that have been used for nutraceutical delivery are water-soluble proteins with limited sustained-release characteristics [15]. Zein is a protein extracted from corn gluten meal and it is one of the few cereal proteins extracted in a relatively pure form [16,17]. It is a hydrophobic compound and for this reason it has been successfully applied as a promising carrier for encapsulation and controlled release of water-insoluble compounds in the pharmaceutical and food industries [18e25]. To produce zein micro and nanoparticles for encapsulation and delivery application, several
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industrial scalable approaches have been recently reported. Zein nanoparticles using a liquid-liquid dispersion process were produced for the potential development of food-grade delivery systems. Zein nanoparticles of 100e200 nm were produced, and smaller particles were formed at a higher shear rate, at higher ethanol concentration or at lower zein concentration in stock solutions [26]. Using this process, two essential oils, thymol and carvacrol, were encapsulated in the nanoparticles of zein using the liquid-liquid dispersion method [23]. Zein colloidal particles below 350 nm, without or with sodium caseinate (NaCas) as the stabilizer, were obtained using a continuous technique termed Flash NanoPrecipitation (FNP) [27]. GomezEstaca et al. produced zein nanoparticles with a compact spherical structure and a narrow size distribution by electrohydrodynamic atomization [28]. Using a built-in ultrasonic dialysis process (BUDP), Jiang et al. studied the effects of dialysate properties on zein self-assembly. In their works, they obtained zein microspheres with a size around 1e2 mm [29] and a zein-based microcarrier system was proposed for targeted oral delivery using indomethacin and hydroxycamptothecin as model drugs [30e32]. However, these technologies show some limitations and need to be further evaluated before industrialization. Several problems are connected to these processes for example, difficulties controlling of the zein particles size [33], large use of toxic organic solvent, with consequently expenses to reduce the solvent residues, possible degradation of the active product due to thermal stress, dimension of zein particles caused by process parameters [28]. Supercritical carbon dioxide (SC-CO2) based processes have been proposed as a promising alternative to traditional techniques to overcome their limitations. They are frequently used in several fields such as extraction, micronization, coprecipitation and entrapment of polymers and drugs [34e38]. In particular, using Supercritical Assisted Atomization (SAA) luteolin was micronized and encapsulated of using polyvinylpyrrolidone (PVP) and dextran [34,39]. Zein micronization was also attempted using supercritical anti-solvent precipitation (SAS) [40]. In particular, zein microparticles were produced using 2% w/w of zein in a 90% aqueous ethanol mixture. However, the particles obtained were characterized by an irregular shape and wide distribution of the dimensions. Better results were obtained using methanol as solvent; in particular, nanometric particles of zein were obtained varying the percentage of methanol from 95% to 100% [40]. Therefore, the production of zein particles at nanoscale with uniform size unless 100% methanol used as solvent is difficult and it is necessary a large flow rate of supercritical CO2 to remove the toxic methanol residue [41]. Liu et al. developed a drug nanocrystal delivery system by coupling SAS process with BUDP. In detail, 10-Hydroxycamptothecin nanocrystals (HCPT) were successfully incorporated into zein microspheres by combining the SAS process with BUDP: the co-precipitation of HCPT and zein prepared using the SAS process was dispersed into ethanolewater as the dialysis solution for BUDP [42]. Recently, Franco et al. have proposed the coprecipitation of zein/ diclofenac using SAS technique: zein micronization was studied and microparticles with a mean diameter of 4.19 mm were obtained, at 90 bar, 40 C using DMSO as solvent. The coprecipitation of zein/ diclofenac was also investigated at various process conditions: in particular, higher polymer/drug ratio (20/1 and 30/1) assured an efficient coprecipitation of the two compounds [43]. Hu et al. have produced coprecipitated particles of zein/lutein 24/1 by supercritical antisolvent using a mixture of acetone/DMSO (7/3 v/v) as solvent with an entrapment efficiency varying between 67% and 83% using a range of temperature between 32 C and 45 C. However, decreasing the polymer/drug ratio (12/1), entrapment efficiencies were drastically reduced (34%) and the produced particles were
aggregated, with the presence of external drug crystals not encapsulated in the polymer matrix [44]. Supercritical Assisted Injection in a Liquid Antisolvent (SAILA) is a recent proposed process based on the continuous injection of an expanded liquid (formed by an organic solvent, a solid solute and SC-CO2) in an antisolvent solution [45]. SC-CO2 acts as a co-solute being miscible with the solution to be treated and favors the atomization in the antisolvent. In this process the solute has to be soluble in the solvent, but not in the antisolvent and at the same time the solvent and the antisolvent have to be completely miscible. A saturator filled with packing elements is used and a near-equilibrium solution between solute, solvent and CO2 is formed. This solution is sprayed through a thin wall injector into the precipitation vessel containing water, used as antisolvent. The mixing, between the two fluids, produces a rapid supersaturation and the precipitation of the particles. This process has numerous advantages, such as no thermal degradation, the possibility of using nontoxic solvents (for example acetone and ethanol), producing directly a stabilized suspension with a good control on particle size distribution. This process was successfully used to micronize pharmaceutical compounds [45] and polymers [46,47] obtaining micrometric and nanometric particle suspensions of controlled size and distribution. Recently, composite drug-polymer of piroxicam/ PLGA particles were produced [48]. Therefore, the aim of this work is to explore the application of SAILA as an alternative technique for the production of composite zein/luteolin microparticle suspensions to be used in nutraceutical fields. For this reason, first feasibility tests for the micronization of zein proteins using the SAILA process will be conducted; then, the optimization of the operative parameters for the coprecipitation of zein/luteolin will be tested. In particular, the effect of process parameters on particle diameter, size distribution and entrapment efficiency will be systematically studied. Morphological, granulometric, solid state and drug release analyses will be performed to characterize coprecipitates and to investigate the distribution of the active compound inside the particles. Moreover, the antioxidant activity of the composite compound will be evaluated and compared with the pure antioxidant. 2. Materials Zein was purchased from Sigma (SigmaeAldrich, St. Louis, USA), micronized luteolin was supplied from Epitech Group Research Laboratories. Carbon dioxide, CO2, purity 99.5% was provided by Morlando Group (Naples, Italy). Polysorbate (Tween 80, Sigma Aldrich Chemical Co., Milan, Italy), acetone (AC, purity 99.9%, Sigma Aldrich Chemical Co., Milan, Italy), ethanol (EtOH, purity 99.8%, Sigma Aldrich Chemical Co., Milan, Italy), 2,2-diphenyl-1picrylhydrazyl (DPPH, Sigma Aldrich Chemical Co., Milan, Italy), 2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, Sigma Aldrich Chemical Co., Milan, Italy) were used as received. Distilled water was produced in the laboratory. 3. Apparatus The SAILA apparatus (Fig. 1) consists of two feed lines, used to deliver compressed CO2 and liquid solvent to a saturator. Carbon dioxide (R1) is cooled using a refrigerating bath (RB), pumped (P1, Lewa Eco, model LDC-M-2), preheated and delivered to the mixing vessel, saturator (S). The solvent liquid solution (R2), in which a solid solute is dissolved, is pumped using a membrane pump (P2, Gilson, model 305). The saturator is a high-pressure static mixer with an internal volume of 0.15 dm3. It is packed with stainless steel perforated saddles and thermally heated by thin band heaters. The liquid solution is prepared dissolving a known quantity of solute (polymer and drug) in an organic solvent. SC-CO2 is continuously
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4.3. Entrapment efficiency and dissolution tests The entrapment efficiency was measured dissolving 5 mg of washed microspheres in 3 mL of a solvent mixture water/ethanol 20/80 and measuring the absorbance of the solution at 350 nm. The entrapment efficiency was calculated as the ratio between the measured and theoretical drug contents. Dissolution tests were performed suspending microspheres (3 mg) containing luteolin in 2 mL of phosphate-buffered saline (PBS) and loading the suspension in dialysis sack. Then, luteolin release profiles were determined in 250 mL of PBS at pH 7.4 continuously agitated at 200 rpm in a 37 C incubator to maintain adequate sink conditions. Released luteolin was determined continuously measuring the absorbance at 350 nm (UVevis mod. Cary 50, Varian). All analysis performed in triplicate. 4.4. Solid state characterization
Fig. 1. SAILA process layout. R1: CO2 reservoir; R2: Liquid solution reservoir; RB: refrigerating bath; P1: CO2 pump; P2: solvent pump; P3: antisolvent peristaltic pump; S: saturator; TC: temperature controller; PC: pressure controller.
solubilized in the liquid solution, forming an expanded liquid, whose composition determines the position in the vapor-liquid equilibrium diagram (VLE) of the system CO2-organic solvent, and depends on the operative conditions of temperature, pressure and on gas to liquid ratio (GLR), expressed as weight ratio. The operative conditions are monitored with a temperature controller (TC) and a pressure controller (PC). At the exit of the mixer, the expanded solution is injected through a micrometric nozzle (nozzles of 80 and 100 mm diameter were used in this work) in an antisolvent phase, water. A surfactant, Tween 80 at 0.2% w/w, was added to the antisolvent phase to stabilize the formed particles. The water phase is continuously pumped using a peristaltic pump (P3) from a reservoir to the receiving vessel; a regulation valve is used to recover continuously the suspension and to maintain constant the liquid level in the vessel. Experiments were performed in triplicates.
4. Methods 4.1. Particles diameter The suspensions were characterized with Dynamic Light Scattering (DLS) (Zetasizer, mod.5000, Malvern Instruments Ltd., UK) to obtain Particles Size Distribution (PDS). Mean diameter (MD), standard deviation (SD) and polydispersity index (PDI) of the particles were measured for each sample. The sample was analyzed 3 times in order to calculate the mean value of the measurements.
4.2. Particles morphology Particle morphology was analyzed using a Field EmissionScanning Electron Microscope (FESEM, LEO 1525, Carl Zeiss SMT AG). Particles were centrifuged at 6500 rpm for 20 min at 4 C (Thermo Scientific, mod. IEC CL30R) and filtered using a membrane (Millipore MF membrane filter, Filter type 0.2 mm HA). Membrane were dried at air in order to recover microparticles. To perform FESEM analysis, powders were coated with a gold layer, using a sputter coater (thickness 250 Å, model B7341; Agar Scientific, Stansted, UK).
Solid-state analysis of the precipitates was performed by powder X-ray diffraction (PXRD; model D8 Advance; Bruker AXS, Madison, WI) using a copper-sealed tube source. Samples were placed in the holder and flattened with a glass slide to assure a good surface texture. The measuring conditions were nickel-filtered Cu Ka radiation, l ¼ 1.54 A, and a 2q angle ranging from 5 to 90 with a scanning rate of 3 s/step and a step size of 0.592 . Microparticle thermal characteristics were determined by differential scanning calorimetry (DSC; model TC11, Mettler Toledo, Inc., Columbus, OH). An accurately weighed amount of powder (±5 mg) was crimped in a standard aluminum pan that was pierced and heated from 25 to 350 C at a scanning rate of 10 C/min in a nitrogen atmosphere at a flow rate of 50 mL/min. Fourier transform infrared (FT-IR) spectra was performed with a FTIR spectrophotometer (IR-Tracer100, Shimadzu). The samples were combined with a small amount of potassium bromide and were prepared by compressing the powders in a hydraulic press. Analyses were performed at 25 C, in the range 400e4000 cm1 at a resolution of 1 cm1 as the mean of 16 measurements to reduce the noise. 4.5. Antioxidant activity 4.5.1. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) assay. The evaluation of radical scavenging activity (antioxidant activity) was performed adapting the method of Blois (1958) with modifications. An ethanolic solution of DPPH 1 104 M was prepared. To obtain a calibration curve, 100 mL of different luteolin concentrations (100 mg/mL, 50 mg/mL, 20 mg/mL, 10 mg/mL and 5 mg/mL dissolved in H2O/Ethanol 20/80 v/v solution) were added in 2900 mL of DPPH solution. Each samples were left at room temperature for 30 min under light protection. The absorbance was measured at 517 nm. The lower the absorbance of the reaction mixture, the higher is free radical scavenging activity. A fixed quantity of zein/luteolin coprecipitates was then dissolved in H20/ Ethanol 20/80 v/v and DPPH method was applied. All determinations were carried out in triplicate. The scavenging ability of DPPH radical was calculated by using the following equation:
A Scavenging effect ð%Þ ¼ 1 A 100 AB
(1)
where AA is the absorbance at 517 nm of the sample treated with DPPH in ethanol solution, and AB is the control absorbance at 517 nm of DPPH in ethanol. The inhibition percentage obtained in this way was compared with the pure unprocessed samples percentage, to calculate the decrease of DPPH inhibition capacity between unprocessed and processed samples.
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Table 1 Zein precipitation SAILA tests performed at different water/ethanol and water/ acetone solvent mixture. Mean diameters (MD), standard deviation (SD) and polydispersity index (PDI) are also reported. Test
Solvent mixture
Mixture composition [v/v]
MD ± SD [mm]
PDI
Z_01 Z_02 Z_03 Z_04 Z_05
H2O/EtOH H2O/EtOH H2O/EtOH H2O/AC H2O/AC
30/70 20/80 10/90 30/70 20/80
0.31 ± 0.09 0.41 ± 0.14 1.33 ± 0.20 0.38 ± 0.08 0.44 ± 0.18
0.24 0.35 0.30 0.40 0.41
4.5.2. 2,20 -Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assay ABTS method was used to reinforce the results obtained from the DPPH radical scavenging activity assay. ABTS was dissolved in deionized water to a 7 mM concentration. ABTS radical cation (ABTSþ) was produced by reacting ABTS solution with 2.45 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 16 h before use. For the study, 1 mL of ABTSþ solution was diluted in PBS (25 mL) until to obtain an absorbance of 0.7 (±0.02) at 734 nm. A calibration curve was prepared as DPPH assay. The coprecipitates were dissolved in in H20/Ethanol 20/80 v/v and after, 100 mL of each samples was mixed with 2900 mL of ABTSþ solution. The absorbance reading was taken at 30 C for 10 min after initial mixing (AA) and an appropriate solvent blank reading was taken (AB). All determinations were carried out in triplicate. The percentage of inhibition of ABTSþ was calculated using above formula (Eq. (1)).
4.6. Statistical analysis The results are expressed as mean ± SD. Normal distribution of the variables was analyzed by the ShapiroeWilk test followed by
the WilcoxoneManneWhitney U test, ANOVA or Student’s t-test (StataSE 12 statistical software, StataCorp LP, USA). Statistically significant differences among groups were considered when P .05. 5. Experimental results The principle at the basis of all the liquid antisolvent processes is that to obtain the solute precipitation, the solute has to be soluble in the solvent and not soluble in the antisolvent. At the same time the solvent and the antisolvent have to be completely miscible. SAILA adds to traditional processes the peculiar properties of supercritical fluids, namely of SC-CO2, to reduce the surface tension of the liquid solution. Operating with SC CO2 smaller droplets can be produced when the injection is performed, due to the reduction of the cohesive forces. Zein is not soluble in water, ethanol or acetone but, it is soluble in aqueous ethanol and aqueous acetone mixtures. For this reason, zein micronization and coprecipitation were tested using the SAILA process using 90e70% (w/w) aqueous ethanol (H2O/EtOH) and 90e70% (w/w) aqueous acetone (H2O/AC) solutions as solvent and water as antisolvent. 5.1. Zein precipitation feasibility experiments Ternary diagrams CO2-ethanolewater are reported in the literature [49]. They have been used in this work, to located the SAILA operative point in the miscibility hole of the ternary system. During the feasibility experiments, zein was processed setting SAILA process conditions selected in a previous study [48]: saturator temperature of 60 C, gas liquid ratio (GLR) 1.5 w/w, CO2 flow rate 12 g/ min, nozzle diameter 80 mm and the polymer was dissolved in the solvent mixture at a concentration of 5 mg/mL. The operating pressure was set at 100 bar for all the experiments. The antisolvent was maintained at room temperature. The composition of the
Fig. 2. FESEM of zein particles precipitated using different solvent mixtures: (a) 20/80 H2O/EtOH, (b) 20/80 H2O/AC.
Table 2 SAILA experiments performed on zein/luteolin mixtures using different solvent mixture and injector diameter with polymer/drug ratio at 20/1 and saturator temperature of 60 C. Test
Solvent mixture composition [v/v]
ZL_01 ZL_02 ZL_03 ZL_04 ZL_05 ZL_06 ZL_07 ZL_08
30/70 20/80 10/90 30/70 20/80 10/90 30/70 20/80
H2O/EtOH H2O/EtOH H2O/EtOH H2O/EtOH H2O/EtOH H2O/EtOH H2O/AC H2O/AC
Injector diameter [mm]
Pressure [bar]
MD ± SD [mm]
PDI
EE [%]
80 80 80 100 100 100 80 80
100 100 100 98 98 98 82 82
0.27 ± 0.05 1.26 ± 0.17 1.35 ± 0.43 0.38 ± 0.13 1.20 ± 0.22 1.43 ± 0.33 0.51 ± 0.20 0.59 ± 0.19
0.53 0.25 0.64 0.71 0.37 0.46 0.39 0.32
43.42 68.52 e 23.37 81.79 e 32.52 27.93
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solvent mixture was changed to understand its effect on PSD and morphology of zein particles. H2O/EtOH and H2O/AC mixtures were used at different composition (30/70, 20/80, 10/90 v/v). The composition 10/90 H2O/AC was not used because the polymer was not soluble in this mixture. Stable and homogeneous suspensions were obtained for all the tests, and the used solvent mixture are summarized in Table 1. Submicro and microparticles were successfully obtained in all
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the experiments, as reported in Table 1. Stable aqueous suspensions were produced, with a stability over three mounts of observation, without the presence of sedimenting particles. In Fig. 2, FESEM image of the tests performed at mixture composition of 20/80 of H2O/EtOH and H2O/AC are reported for comparison. In all cases tested, spherical and well separated particles were obtained. The data reported in Table 1 show that, decreasing water content in the solvent mixture, an increase of particles diameter
Fig. 3. FESEM images of zein/luteolin microparticles produced using different injector diameter (80 mm on the left and 100 mm on the right) and different solvent mixture ratio H2O/ Ethanol at 60 C, GLR 1.5, polymer concentration 5 mg/mL, polymer/drug ratio 20/1.
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can be observed for both solvent mixture studied. This result could be explained considering that, as the amount of water decreases in the starting solution, zein solutions become gradually closer to the saturation. Since in the SAILA process, the formation of the particles is based on the precipitation of the solute due to an antisolvent effect, precipitation rate is directly proportional to the degree of supersaturation [47,50]. For this reason, for solutions in which solute concentration is closer to saturation, the precipitation, due to the anti-solvent effect, produces larger particles and a wider particle size distribution. 5.2. Coprecipitation feasibility experiments SAILA coprecipitation feasibility tests were carried out using different solvent mixtures H2O/EtOH and H2O/AC. Process parameters were left unchanged with respect to zein precipitation experiments. Polymer concentration in solvent mixtures was set at 5 mg/mL and polymer/antioxidant ratio at 20/1. Experimental results are reported in Table 2. It can be observed that, using ethanol in the solvent mixture, when the amount of water was reduced from 30/70 to 10/90, a progressive increase in the size of the obtained particles was obtained, passing from 0.27 ± 0.05 mm to 1.35 ± 0.43, respectively. Considering the FESEM images of Fig. 3, it is possible to note that operating at 30/70 H2O/EtOH, mainly spherical particles of submicrometric dimensions (0.27 ± 0.05 mm) have obtained (Fig. 3a); whereas, when the quantity of water decreased, particles produced were spherical with a micrometric diameter of 1.26 ± 0.17 mm (Fig. 3b). In the case of a solvent mixture of 10/90 H2O/EtOH, the precipitated material was irregular showing two morphologies: crystals of drug and spherical particles of zein. Thus, in this case the coprecipitation was not successful, resulting in a visible separate precipitation of polymer and drug (Fig. 3c). Entrapment efficiencies ranged between 43 and 68% for aqueous ethanol. In particular, in Table 2 it is possible to observe that luteolin was entrapped in the polymer matrix with a lower entrapment efficiency in the case of the tests carried out with the ratio H2O/ EtOH 30/70; in this case the efficiency was 43.44%. Entrapment efficiency of 68.52% was obtained using the 20/80 ratio. The same set of experiments was performed using a 100 mm injector. Data reported in Table 2 show that, also in this case, coprecipitates were obtained only from mixtures with 70e80% ethanol (Fig. 3d and e); therefore, the mixture 10/90 H2O/ETOH is not useful to produce composite particles because, as reported in Fig. 3f, polymer particles separated from crystal of luteolin were obtained. The increase of the injector diameter from 80 to 100 mm, resulted in a slight increase of the average size of coprecipitated particles (see Table 2 and Fig. 3). Entrapment efficiencies of 23.37% and 81.79% were obtained starting form mixtures containing 70% and 80% ethanol respectively. Moreover, as in the previous case, the best mixture for the production of zein/luteolin coprecipitates is 20/80 H2O/EtOH; indeed, a larger value of entrapment efficiency was obtained. Coprecipitation experiments were also performed using the H2O/AC mixtures in the 30/70 and 20/80 ratios. The composition 10/ 90 H2O/AC ratio was not used because the polymer is not soluble in this mixture. Data reported in Table 2, show that the obtained particles were characterized by sub-micrometric dimensions for both mixtures tested, in particular, in the range 0.51 ± 0.20 and 0.59 ± 0.19 mm respectively. Particles were spherical also in this case, but, in the case of acetone, the solvent mixture did not significantly influence the particle size distribution. However, entrapment efficiencies were lower than those obtained using the ethanol mixture. Indeed, entrapment efficiencies between 27% and 32% were obtained. For this reason, only the ethanol water solvent mixture was used for the further experiments reported in this work.
Some experiments were performed changing the polymer/drug ratio and saturation temperature using the solvent mixture H2O/ EtOH at the volumetric ratio 20/80. The experiments performed are summarized in Table 3. Zein/luteolin polymer/drug ratio was varied from 20/1 to 5/1 w/w, fixing the overall solution concentration at 5 mg/mL. Example of FESEM images of the precipitated powders are shown in Fig. 4. The 20/1 ratio FESEM image is not reported because it was already shown in Fig. 3b. When a mixture zein/luteolin 5/1 w/w was processed, the precipitated powder was irregular and it is possible to note that crystals precipitated separately from spherical particles (Fig. 4a) and are organized as flower-like structures. Increasing polymer/ drug ratio at 10/1, the phenomenon of crystals precipitation was eliminated, only spherical particles with a mean diameter of 0.25 ± 0.07 nm are visible in the corresponding FESEM image (Fig. 4b). At the ratio zein/luteolin 20/1, regular microparticles were
Table 3 SAILA tests performed for zein/luteolin at different polymer/drug ratios, temperatures and at pressure of 100 bar. Test
Polymer/drug ratio
Temperature [ C]
MD ± SD [mm]
PDI
EE [%]
ZL_09 ZL_10 ZL_02 ZL_11 ZL_12 ZL_13 ZL_14
5/1 10/1 20/1 10/1 10/1 20/1 20/1
60 60 60 80 40 80 40
0.26 ± 0.08 0.25 ± 0.07 1.26 ± 0.17 0.20 ± 0.04 0.25 ± 0.05 0.23 ± 0.03 1.26 ± 0.24
0.64 0.59 0.25 0.44 0.41 0.26 0.19
e 46.45 68.52 3.6 63.96 15.77 80.15
Fig. 4. FESEM images of zein/luteolin particles produced using different polymer/drug ratio at 60 C, 92 bar, GLR 1.4, polymer concentration 5 mg/mL and solvent mixture H2O/EtOH 20/80.
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also obtained, as discussed in the previuos set of experiments (Fig. 3b). Luteolin entrapment efficiency of the coprecipitated particles obtained at 10/1 of polymer drug ratio was 46.4%; whereas, an efficiency of 68.5% was obtained at 20/1 ratio. Using 10/1 and 20/1 polymer/drug ratio, also the effect of higher and lower temperatures was studied, in particular the same set of experiments was performed at temperature of 80 C and 40 C. These temperatures have been chosen because in the SAILA process, an increase in temperature generally produced a reduction in the size of the particles produced [45]; whereas, operating at temperatures of about 40 C, good entrapment efficiencies were observed for composite systems zein/lutein [44]. Suspensions were successfully produced at 80 C and 40 C for the tested polymer/drug ratio see Table 3 (# 4e7). From the data reported in Table 3 and from the FESEM images of Fig. 5, it is possible to note a reduction in the mean diameter when the temperature increased and the polymer/drug ratio decreased. In the tests carried out at 80 C the formation of aggregates was observed. Better results were obtained when the saturator temperature was set at 40 C; indeed, being natural compounds, lower temperatures can favor conservation and processability of the material. The trends of the entrapment efficiencies with the variation of temperature and polymer/drug ratio are summarized Fig. 6, where the data obtained from the tests carried out at 60 C is also reported. Fig. 6, shows a clear trend: the entrapment efficiency increases with decreasing the temperature and with the increasing of polymer/drug ratio in all the studied cases. The increase of the entrapment efficiency at lower temperatures could be due to a slower diffusion of the drug in the anti-solvent phase. During the precipitation, part of the luteolin tends to spread towards the antisolvent phase, in which it has a reduced solubility. For this reason, when the temperature is low, this diffusion effect is reduced. Regarding the influence of the polymer/drug on the entrapment efficiency, a larger quantity of polymer favors the coprecipitation between the two compounds. The presence of a larger quantity of
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polymer molecules favors luteolin wrapping during zein precipitation, resulting in larger entrapment efficiencies. 5.3. Characterization of coprecipitates FT-IR analyses were carried out for unprocessed luteolin, pure zein, and SAILA processed zein/luteolin in the case of coprecipitation and in the case of separate precipitation of polymer and antioxidant, to analyze the chemical bonds of native and processed structures. The comparison of FT-IR spectra (Fig. 7a) shows that the characteristic peaks of processed compounds did not shown displacements with respect to the spectra of unprocessed compounds, confirming that the process does not modify compounds structure. It is also evident that, in coprecipitates (ZL_05), the spectrum is much more similar to polymer spectrum even if there are peaks (between 1000 and 2000 cm-1) due to the presence of the drug; whereas, when the luteolin is not coprecipitated (ZL_11) the spectrum is closer to that of unprocessed luteolin, because drug crystals are not encapsulated in the polymeric matrix. Unprocessed polymer, antioxidant and a coprecipitated powder were also characterized by X-ray diffraction (XRD) analyses. These analyses revealed that unprocessed drug shows the typical crystalline structure of luteolin: whereas, when a coprecipitation was obtained, zein/luteolin samples showed an amorphous structure similar to that native Zein (Fig. 7b). This result can be explained considering that luteolin is distributed in the polymeric matrix, and the drug has a low mobility among polymer chains; therefore, and the sample is predominantly in the amorphous state. DSC analysis was also carried out (Fig. 7c): zein does not present particular variations with temperature, whereas, luteolin shows an endothermic peak around 350 C due to crystal fusion. It is possible to note that the composite particles of zein/luteolin show a behavior very similar to that of polymer: in none of the analyzed tests the peak of crystallinity of the drug is detected. Therefore, the microdispersion of luteolin in the polymeric matrix, led to the
Fig. 5. FESEM images of Zein/Luteolin particles produced using different temperature and polymer/drug ratio, with solvent mixture H2O/EtOH at 20/80 composition.
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Fig. 6. Trend of the entrapment efficiency at different temperatures and zein/luteolin ratios.
formation of an amorphous compound. Drug release tests were also performed to compare the dissolution kinetics of different samples. Indeed, if the coprecipitation has properly occurred, luteolin is dispersed inside zein and its
release rate should be influenced by the presence of the polymer. Release kinetics of the drug in a phosphate buffer (PBS) was studied, at a temperature of 37 C, and the results obtained were compared with the release kinetics of the physical mixture zein/ luteolin (Fig. 8a). It has been found that the composite microparticles release the drug faster than the physical mixture. The physical mixture completes the dissolution of the drug in about 20 h. Zein/ luteolin coprecipitates, instead, produce a complete dissolution of the antioxidant in the time interval of 2 h and 30 min/3 h. When the coprecipitation is successful, luteolin should be uniformly dispersed at nanometric level in the polymer matrix and it is, therefore, released rapidly by a mechanism of diffusion through the polymer in consequence of degradation/swelling of zein particles. It has to be considered that, with respect to the physical mixture obtained using zein and luteolin separately precipitated by SAILA, coprecipitates allow to increase the area exposed to the release medium, thus increasing the dissolution rate [34]. In Fig. 8b, as further confirmation, of these considerations, release test performed for the powder obtained at the process conditions in which the polymer and the drug precipitated separately is reported. A dissolution test of the drug released from a physical mixture formed at the same polymer/antioxidant ratio was performed and it is also reported for comparison purpose. It can be seen that, when coprecipitation was not successful, release times similar to physical
Fig. 7. FT-IR analyses of unprocessed and processed powders; (b) XRD analyses of unprocessed and processed powders.
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Fig. 8. Luteolin release from zein microparticles produced at different temperature and injector diameter with the polymer/drug ratio ratio 20/1.
Table 4 Radical scavenging activity of coprecipitates with DPPH and ABTS assay. Tests
ZL_02 ZL_05 ZL_07 ZL_08
Theoretical loading [mg/mL]
EE
50.0 61.0 52.0 58.5
68.5 81.8 32.5 27.9
DPPH
ABTS
Scavenging effect [%]
Scavenging effect [%]
16.6 26.9 9.5 10.8
24.8 29.4 28.7 27.8
mixture are obtained. The previous characterizations confirmed the presence of drug in the microparticles produced, the good entrapment efficiencies in the polymer matrix and the increase of the luteolin bioavailability. However, antioxidants are very delicate substances and their antioxidant power is greatly reduced due to environmental degradation agents such as light or heat; therefore, they need to be protected and encapsulated in biocompatible polymers, zein in this case. The entrapment process conditions could lead to a degradation and a reduction of the intrinsic properties of luteolin. This aspect is fundamental to determine the success of the process. Therefore, spectrophotometric assays (DPPH and ABTS as explained in Material and Methods section) have been used for the determination of the radical scavenging activity of the obtained coprecipitates. The test was first performed on pure luteolin. Samples were prepared dissolving luteolin at different concentrations in H2O/ETOH 20/80 and adding at each sample the ethanolic solution of DPPH and ABTS. The absorbance values obtained were converted in terms of % inhibition. Hence, with the same technique, the coprecipitated samples were analyzed. The scavenging activity of each sample analyzed is reported in Table 4, where it is also reported the theoretical loading of the luteolin inside the particles and the entrapment efficiency. The analyzed samples preserved their antioxidant activity from comparison with native antioxidant. Therefore, it is possible to confirm that the process has not largely changed the antioxidant properties of luteolin. 6. Conclusions SAILA technique was successfully used in the precipitation of zein, and in the production of composite particles of zein/luteolin. Zein submicro and micrometric particles, with an average size between 0.26 and 2 mm, were obtained. The coprecipitation process was successful only at selected, optimized operative conditions, in
the other cases separated precipitation of zein and luteolin was observed. Large polymer/drug ratios (20/1) and low temperatures (40 C) allowed the simultaneous precipitation of the two compounds with high luteolin entrapment efficiencies (up to 82%). The entrapment efficiency of the antioxidant in the polymer matrix was also affected by temperature because both luteolin and zein are natural compounds and sensitive to the process temperature used. SAILA coprecipitates enhanced the dissolution rate of the antioxidant compound and should be improve its bioavailability. References [1] J.B. Harborne, C.A. Williams, Advances in flavonoid research since 1992, Phytochemistry. 55 (6) (2000) 481e504, 2000/11/01/. [2] D.I. Tsimogiannis, V. Oreopoulou, Free radical scavenging and antioxidant activity of 5,7,30 ,40 -hydroxy-substituted flavonoids, Innovative Food Sci. Emerg. Technol. 5 (4) (2004) 523e528, 2004/12/01/. [3] J.A. Ross, C.M. Kasum, Dietary flavonoids: bioavailability, metabolic effects, and safety, Annu. Rev. Nutr. 22 (1) (2002) 19e34. PubMed PMID: 12055336. [4] M.L. Neuhouser, Review: dietary flavonoids and cancer risk: evidence from human population studies, Nutr. Cancer 50 (1) (2004) 1e7, 2004/09/01. [5] L.-L. Miguel, Distribution and biological activities of the flavonoid luteolin, Mini-Rev. Med. Chem. 9 (1) (2009) 31e59. [6] P. Zhou, L.-P. Li, S.-Q. Luo, H.-D. Jiang, S. Zeng, Intestinal absorption of luteolin from peanut hull extract is more efficient than that from individual pure luteolin, J. Agric. Food Chem. 56 (1) (2008) 296e300, 2008/01/01. [7] Z. Chen, M. Tu, S. Sun, S. Kong, Y. Wang, J. Ye, et al., The exposure of luteolin is much lower than that of apigenin in oral administration of flos chrysanthemi extract to rats, Drug Metabol. Pharmacokinet. 27 (1) (2012) 162e168. [8] M. Reza Mozafari, C. Johnson, S. Hatziantoniou, C. Demetzos, Nanoliposomes and their applications in food nanotechnology, J. Liposome Res. 18 (4) (2008) 309e327, 2008/01/01. [9] H. Yu, Q. Huang, Enhanced in vitro anti-cancer activity of curcumin encapsulated in hydrophobically modified starch, Food Chem. 119 (2) (2010) 669e674, 2010/03/15/. [10] K. Pathakoti, M. Manubolu, H.-M. Hwang, Nanostructures: current uses and future applications in food science, J. Food Drug Anal. 25 (2) (2017) 245e253, 2017/04/01/. [11] L.S. Nair, C.T. Laurencin, Biodegradable polymers as biomaterials, Prog. Polym. Sci. 32 (8) (2007) 762e798, 2007/08/01/. [12] V.R. Sinha, A. Trehan, Biodegradable microspheres for protein delivery, J. Control. Release 90 (3) (2003) 261e280, 2003/07/31/. [13] J.M. Dang, K.W. Leong, Natural polymers for gene delivery and tissue engineering, Adv. Drug Deliv. Rev. 58 (4) (2006) 487e499, 2006/07/07/. [14] A.O. Elzoghby, W.M. Samy, N.A. Elgindy, Protein-based nanocarriers as promising drug and gene delivery systems, J. Control. Release 161 (1) (2012) 38e49, 2012/07/10/. [15] G. Wang, H. Uludag, Recent developments in nanoparticle-based drug delivery and targeting systems with emphasis on protein-based nanoparticles, Expert Opin. Drug Deliv. 5 (5) (2008) 499e515, 2008/05/01. [16] W.L.J. Zein, A history of processing and use, Cereal Chem. 79 (1) (2002) 1e18. [17] L.C. Swallen, A. Zein, New industrial protein, Ind. Eng. Chem. 33 (3) (1941) 394e398, 1941/03/01. [18] X. Liu, Q. Sun, H. Wang, L. Zhang, J.-Y. Wang, Microspheres of corn protein, zein, for an ivermectin drug delivery system, Biomaterials. 26 (1) (2005)
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I. Palazzo et al. / Powder Technology 356 (2019) 899e908
109e115, 2005/01/01/. [19] L.F. Lai, H.X. Guo, Preparation of new 5-fluorouracil-loaded zein nanoparticles for liver targeting, Int. J. Pharm. 404 (1) (2011) 317e323, 2011/02/14/. [20] Y. Luo, B. Zhang, M. Whent, L. Yu, Q. Wang, Preparation and characterization of zein/chitosan complex for encapsulation of a-tocopherol, and its in vitro controlled release study, Colloids Surf. B: Biointerfaces 85 (2) (2011) 145e152, 2011/07/01. [21] Y. Luo, Z. Teng, Q. Wang, Development of Zein nanoparticles coated with carboxymethyl chitosan for encapsulation and controlled release of vitamin D3, J. Agric. Food Chem. 60 (3) (2012) 836e843, 2012/01/25. [22] L. Chen, M. Subirade, Elaboration and characterization of soy/zein protein microspheres for controlled nutraceutical delivery, Biomacromolecules. 10 (12) (2009) 3327e3334, 2009/12/14. [23] Y. Wu, Y. Luo, Q. Wang, Antioxidant and antimicrobial properties of essential oils encapsulated in zein nanoparticles prepared by liquideliquid dispersion method, LWT Food Sci. Technol. 48 (2) (2012) 283e290, 2012/10/01/. [24] B. Zhang, Y. Luo, Q. Wang, Development of silverzein composites as a promising antimicrobial agent, Biomacromolecules. (9) (2010) 2366e2375, 2010/09/13;11. _ Ünalan, I. Arcan, F. Korel, A. Yemeniciog lu, Application of active zein-based [25] I.U. films with controlled release properties to control listeria monocytogenes growth and lipid oxidation in fresh Kashar cheese, Innovative Food Sci. Emerg. Technol. 20 (2013) 208e214, 2013/10/01. [26] Q. Zhong, M. Jin, Zein nanoparticles produced by liquideliquid dispersion, Food Hydrocoll. 23 (8) (2009) 2380e2387, 2009/12/01/. [27] K.-K. Li, X. Zhang, Q. Huang, S.-W. Yin, X.-Q. Yang, Q.-B. Wen, et al., Continuous preparation of zein colloidal particles by Flash NanoPrecipitation (FNP), J. Food Eng. 127 (2014) 103e110, 2014/04/01/. [28] J. Gomez-Estaca, M.P. Balaguer, R. Gavara, P. Hernandez-Munoz, Formation of zein nanoparticles by electrohydrodynamic atomization: effect of the main processing variables and suitability for encapsulating the food coloring and active ingredient curcumin, Food Hydrocoll. 28 (1) (2012) 82e91, 2012/07/01/ . [29] G. Liu, J. Feng, W. Zhu, Y. Jiang, Zein self-assembly using the built-in ultrasonic dialysis process: microphase behavior and the effect of dialysate properties, Colloid Polym. Sci. 296 (1) (2018 January 01) 173e181. [30] G. Liu, J. Pang, Y. Huang, Q. Xie, G. Guan, Y. Jiang, Self-assembled nanospheres of folate-decorated zein for the targeted delivery of 10-hydroxycamptothecin, Ind. Eng. Chem. Res. 56 (30) (2017) 8517e8527, 2017/08/02. [31] H. Wang, X. Zhang, W. Zhu, Y. Jiang, Z. Zhang, Self-assembly of Zein-based microcarrier system for colon-targeted oral drug delivery, Ind. Eng. Chem. Res. 57 (38) (2018) 12689e12699, 2018/09/26. [32] H. Wang, W. Zhu, Y. Huang, Z. Li, Y. Jiang, Q. Xie, Facile encapsulation of hydroxycamptothecin nanocrystals into zein-based nanocomplexes for active targeting in drug delivery and cell imaging, Acta Biomater. 61 (2017) 88e100, 2017/10//. PubMed PMID: 28433787. eng. [33] Y. Wang, G.W. Padua, Formation of Zein microphases in ethanolwater, Langmuir. 26 (15) (2010) 12897e12901, 2010/08/03. [34] A. Di Capua, R. Adami, E. Reverchon, Production of luteolin/biopolymer microspheres by supercritical assisted atomization, Ind. Eng. Chem. Res. 56 (15)
(2017) 4334e4340, 2017/04/19. [35] P. Chattopadhyay, R. Huff, B.Y. Shekunov, Drug encapsulation using supercritical fluid extraction of emulsions, J. Pharm. Sci. 95 (3) (2006) 667e679, 2006/03/01/. [36] P. Trucillo, R. Campardelli, E. Reverchon, Production of liposomes loaded with antioxidants using a supercritical CO2 assisted process, Powder Technol. 323 (2018) 155e162, 2018/01/01/. [37] L. Baldino, M. Scognamiglio, E. Reverchon, Extraction of rotenoids from Derris elliptica using supercritical CO2, J. Chem. Technol. Biotechnol. 93 (12) (2018) 3656e3660. [38] C. Gimenez-Rota, I. Palazzo, M.R. Scognamiglio, A. Mainar, E. Reverchon, Della porta G. b-carotene, a-tocoferol and rosmarinic acid encapsulated within PLA/ PLGA microcarriers by supercritical emulsion extraction: encapsulation efficiency, drugs shelf-life and antioxidant activity, J. Supercrit. Fluids 146 (2019) 199e207, 2019/04/01/. [39] A. Di Capua, R. Adami, L. Izzo, E. Reverchon, Luteolin/dextran-FITC fluorescent microspheres produced by supercritical assisted atomization, J. Supercrit. Fluids 130 (2017) 97e104, 2017/12/01/. [40] Q. Zhong, M. Jin, D. Xiao, H. Tian, W. Zhang, Application of supercritical antisolvent technologies for the synthesis of delivery systems of bioactive food components, Food Biophys. 3 (2) (2008 June 01) 186e190. [41] L. Yangchao, W. Qin, Zein-based micro- and nano-particles for drug and nutrient delivery: a review, J. Appl. Polym. Sci. 131 (2014) 16. [42] G. Liu, S. Li, Y. Huang, H. Wang, Y. Jiang, Incorporation of 10hydroxycamptothecin nanocrystals into zein microspheres, Chem. Eng. Sci. 155 (2016) 405e414, 2016/11/22. [43] P. Franco, E. Reverchon, I. De Marco, Zein/diclofenac sodium coprecipitation at micrometric and nanometric range by supercritical antisolvent processing, J. CO2 Utilization 27 (2018) 366e373, 2018/10/01/. [44] D. Hu, C. Lin, L. Liu, S. Li, Y. Zhao, Preparation, characterization, and in vitro release investigation of lutein/zein nanoparticles via solution enhanced dispersion by supercritical fluids, J. Food Eng. 109 (3) (2012) 545e552, 2012/ 04/01/. [45] R. Campardelli, E. Reverchon, a-Tocopherol nanosuspensions produced using a supercritical assisted process, J. Food Eng. 149 (2015) 131e136. [46] R. Campardelli, E. Oleandro, R. Adami, E. Reverchon, Polymethylmethacrylate (PMMA) sub-microparticles produced by supercritical assisted injection in a liquid antisolvent, J. Supercrit. Fluids 92 (2014) 93e99, 2014/08/01/. [47] R. Campardelli, E. Oleandro, E. Reverchon, Supercritical assisted injection in a liquid antisolvent for PLGA and PLA microparticle production, Powder Technol. 287 (2016) 12e19, 2016/01/01/. [48] R. Campardelli, E. Reverchon, Instantaneous coprecipitation of polymer/drug microparticles using the supercritical assisted injection in a liquid antisolvent, J. Supercrit. Fluids 120 (2017) 151e160, 2017/02/01/. [49] M.A. Rodrigues, J. Li, A.J. Almeida, H.A. Matos, Azevedo EGd, Efficiency of water removal from water/ethanol mixtures using supercritical carbon dioxide, Braz. J. Chem. Eng. 23 (2006) 205e212. [50] I.J. Joye, D.J. McClements, Production of nanoparticles by anti-solvent precipitation for use in food systems, Trends Food Sci. Technol. 34 (2) (2013) 109e123, 2013/12/01/.