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internal gelation of alginate
Accepted Manuscript Development of a nutraceutical nano-delivery system through emulsification/ internal gelation of alginate Samira Mokhtari, Seid Mahdi Jafari, Elham Assadpour PII: DOI: Reference:
S0308-8146(17)30272-8 http://dx.doi.org/10.1016/j.foodchem.2017.02.071 FOCH 20624
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
21 October 2016 5 January 2017 15 February 2017
Please cite this article as: Mokhtari, S., Jafari, S.M., Assadpour, E., Development of a nutraceutical nano-delivery system through emulsification/internal gelation of alginate, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/ j.foodchem.2017.02.071
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Development of a nutraceutical nano-delivery system through emulsification/internal gelation of alginate
Running title: Alginate nano-carriers for nutraceuticals
Samira Mokhtari, Seid Mahdi Jafari*, Elham Assadpour
Department of Food Materials and Process Design Engineering, University of Agricultural Sciences and Natural Resources, Gorgan, Iran *Corresponding Details: Tel/Fax: +98 17 324 26 432. E-mails: [email protected]
ABSTRACT Alginate nano/microsphers are produced by emulsification/internal gelation of sodium alginate dispersed within vegetable oils containing surfactant, followed by CaCl2 addition resulting in hardened particles. In this work, the impact was evaluated of alginate, CaCl2, oil and surfactant content on the size and encapsulation efficiency of nanocarriers containing peppermint phenolic extract and prepared by a low energy internal gelation technique. The results revealed that size of nanoparticles decreased at higher oil and surfactant contents, higher molarity of CaCl2 and lower alginate concentrations. Also, it was found that the encapsulation efficiency was inversely proportional to the size of nanoparticles, and the impact of alginate concentration and surfactant content was markedly higher than the other two factors. The composition of 0.5% alginate, 400 ml oil, 0.05 M CaCl2 and 100 ml surfactant was recognized as the optimized treatment with a reasonable encapsulation efficiency of 5.6% and a nanoparticle size of 785 nm.
Keywords: Nanoencapsulation; Alginate; Efficiency; Phenolic extract; Nutraceuticals.
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1. Introduction Nanoencapsulation is one of the interesting applications of nanotechnology normally implemented in food, pharmaceutical and cosmeceutical industries, which enables the controlled release of nutrients in the right zone and time. This technique has numerous advantages for targeted delivery and bioavailability through the cells (Katouzian and Jafari, 2016). In general, nanoencapsulated particles are known as nano-capsules when their size is smaller than 1.0 µm. There are numerous examples of naturally occurring nanoencapsulates, such as casein micelles in milk (<100 nm), mitochondria (500-10,000 nm), and viruses (10-300 nm). The term nanoencapsulation describes encapsulation in the nanometer scale using films, layers, or nano-dispersions (Faridi Esfanjani and Jafari, 2016). The final capsule acts as a nanoscale shield for the nutraceutical or drug molecules/ingredients. Often, the active ingredient is in the molecule or nanoscale state. The major benefit is homogeneity, leading to better encapsulation efficiency as well as improved physical and chemical properties (Khare and Vasisht, 2014). Alginate is an anionic polysaccharide of (1–4)-linked b-D-mannuronic acid (M) and a-L-guluronic acid (G) widely used in bioencapsulation of drugs, nutraceuticals, proteins and cells. Ca-alginate beads represent one of the most widely used carriers for the immobilization of enzymes and proteins as well as for the controlled release of drugs (Sarmento et al, 2006; M Leonard et al, 2004; Chen et al, 2004). Experimental evidence shows that alginate nanoparticles have been successfully prepared using the emulsification/internal gelation method by creating the ideal conditions for the formulation and have recently been extended to the field of nanotechnology (Reis et al, 2006; Faridi Esfanjani and Jafari, 2016). In recent years, hydrogel nanoparticles have gained significant attention as one of the most promising nanoscale delivery systems because they have unique potential through combining the characteristics of a hydrogel system (hydrophilicity and extremely high water content) with those of nanotechnology (very small size). Single or mixed-type biopolymer clusters may be developed from hydrogel particles under certain conditions. 2
Among different bio-macromolecules, which can be used for hydrogel formation, polysaccharides are extremely advantageous to physically or chemically form filled hydrogels. Polysaccharidebased hydrogels can be formed whenever an appropriate counter-ion is added to a polysaccharide dispersion of adequate concentration. As an example, sodium alginate dispersion transforms into alginate hydrogel after mixing (titration) with divalent cations (such as calcium ion). At low concentrations (diluted systems), nanoparticle formation is obtained instead of bulk phase gelation. Rolland et al., (2014), studied the formation of a filled thin alginate hydrogel membrane surrounding an aqueous core through a co-extrusion step in air followed by a sol–gel transition of the shell after immersion into a gelling bath. Some formulation and process parameters can be adjusted to produce smaller particles (Reis et al. 2006). By controlling the conditions under which the water-in-oil emulsion is produced, hydrogel capsule size can be easily controlled from a few nanometers potentially, to millimeters in diameter (Poncelet, 2001). The most recent research limitations and challenges is the inability to reduce particle size of calcium alginate gels. Higher levels of energy such as homogenization at 8000 rpm (Balcão et al., 2013), and magnetic stirring at 1600 rpm for 15 minutes (Reis et al., 2008) are used to lower nanoparticle size. In order to reduce the size of nanoparticles, cationic polymers, like chitosan, have been used; although, this method leads to nanoparticle sizes larger than 700 nm (Lertsutthiwong and Rojsitthisak, 2011). Moreover, how to add solution containing crosslinkers of calcium ions into sodium alginate solution is one of important and determinant factors affecting the particle size that has not yet been discussed. The main difference between the current study and previous research is the much smaller nanoparticle size used here. Therefore, one of the aims of this research was to introduce some modifications for the common internal gelation procedure in order to obtain the same calcium alginate nanogels (in size) using lower energy. Additionally, a detailed study on optimization of nanoencapsulation parameters of internal gelation technique has not yet been done. Therefore, 3
another aim was to maximize the nanoencapsulation efficiency of alginate nanodelivery system by internal gelation method and applying optimal alginate and calcium concentrations, oil and surfactant contents, as well as illustrating the correlation between size and efficiency of final nanocarriers and formulation parameters. Peppermint phenolic extract was selected to be nanoencapsulated as the core material. It is one of the most popular and widely used functional ingredients, with applications in the food, pharmaceutical and flavouring industries (Ghayempour and Mortazavi, 2014; Koo et al., 2014). Peppermint extract readily undergoes oxidation when exposed to the air or UV, and it is highly volatile, which limits its applications in the food and pharmaceutical production (Dong et al., 2011; Koo et al., 2014). The utilization of nanoencapsulated extract instead of free bioactives can overcome the drawbacks of its instability as well as improving its bioavailability. 2. Materials and methods Sodium alginate with an average molecular weight of 12,000–40,000 and medium viscosity (3500 mPa.s for a 2% solution at 25˚C) was purchased from Sigma, UK. Tween 80 and calcium chloride (CaCl2) was supplied from Merck Chemicals Co, Germany. Canola oil was purchased from Ladan Company, Iran. Water used in the experiments was double distilled and deionized. All other reagents used in this work were of analytical grade, and purchased from Sigma–Aldrich (St. Louis, USA). 2.1. Preparation of peppermint phenolic extract Peppermint was obtained from educational farm for medicinal plants of Ferdowsi University of Mashhad, Iran. For the preparation of alcoholic extract, peppermint leaves were dried and milled, obtained powder was mixed in a ratio of 1:20 with 80% ethanol, and then through microwaveassisted extraction, its phenolic extract was prepared, and filtered by paper filters (Whatman, No. 42), and alcoholic extract was liquidized. The remained alcohol eliminated by distillation with a vacuum rotary evaporator and also vacuum oven, and finally was stored at -20°C (Rafiei, et al, 2011). 4
2.2. Nanoencapsulation procedure Nanoencapsulation of peppermint phenolic extract using the internal gelation technique was done according to the You and Peng (2005) method, with some modifications. Briefly, 2 ml phenolic extract was dissolved in 8 ml alginate sodium with varying concentrations (0.5, 0.75 and 1%, w/v). The range of biopolymer concentration for this study was selected on the basis of previous reported works such as You and Peng (2005), 0.05 w/w; Sarmento et al, (2006), 0.063 w/w and De and Robinson, (2003), 0.1 % w/v. Then, the mixed solution of phenolic extract-alginate was added dropwise into different volumes (400, 600 and 800 ml) of canola oil containing various levels (100, 200 and 300 µl) of Tween 80 as a surfactant, while mixing speed was fixed at 800 rpm using a screw magnet in order to create the highest collision. Then the mixture was left for 10 minutes prior to the addition of filtered calcium chloride solution of different molarities (0.05, 0.10 and 0.15 molar). According to Reis et al. (2006), the aqueous/oil phases ratio with values of 1:1 demonstrated the ability to produce alginate particles, so the volume of calcium chloride solution in each treatment was modulated proportional to its volume of canola oil. CaCl2 solution was sprayed on the surface of prepared emulsions in order to achieve the highest surface area and maximum collision of Ca+2 ions onto the sodium alginate nanospheres. Broken emulsion was then centrifuged (20,000 ×g) for 45 min at 4˚C (Sarmento et al, 2006), and finally, the prepared nanoparticles were re-suspended with deionized water. Final nanospheres were formulated based on the 27 experimental runs shown in Table 1. 2.3. Determination of total phenolic content Total phenolic content of each extract was determined by the Folin–Ciocalteu micro-method described by Rafiei, et al., (2012). Briefly, 10 µl of ethanolic extract was mixed with 50 µl folin– Ciocalteu and 580 µl distilled water, and after aging for 5-8 min, 150 ml CaCO3 (1 M) was added. Obtained solution then was kept in dark water bath in 40˚C for 30 min and its absorbance was read by a spectrophotometer (WPA S2000, UK) using a wavelength of 765 nm. For the blank sample, an
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80% ethanol solution was used. The total phenolic content of samples was expressed as gallic acid equivalents using the following linear equation based on the calibration curve: Y= 5.606X – 0.039
R2= 0.099
(1)
where Y is the absorbance at 765 nm and X is the concentration of total phenolics based on gallic acid equivalents (µg/ml) (Javanmardi et al., 2003). To obtain the calibration curve, different concentrations of pure gallic acid were used. Standard solutions of 5, 10, 50, 100, 200, 600 and 700 ppm were prepared, and the absorbance coefficient was calculated for each standard. Zero ppm concentration was considered as the blank sample. 2.4. Analysis of nanoencapsulation efficiency To measure the encapsulation efficiency, total phenolic content (TPC) of 2 ml peppermint extract was measured before adding into sodium alginate solution according to Section 2.3. This volume of phenolic extract was encapsulated after adding to the alginate solution, and the entrapped phenolics in the final alginate nanospheres were released and measured as Entrapped Phenolic Content (EPC). To release nanocapsulated peppermint extract from nanospheres, the prepared capsules were added into 90 ml of 1% (w/v) sodium citrate solution with pH = 6.0 at room temperature, and mixed for 10 minutes by a magnetic stirrer at 100 rpm; then the EPC was calculated after reading its absorbance. Encapsulation Efficiency (EE) was obtained with the following formula: EE = (EPC / TPC) × 100
(2)
2.5. Characterization of nanocarriers For size distribution and particle size data, obtained nanoparticles were redispersed in 5 ml ultrapure water. The mean particle size was measured by dynamic light scattering (DLS: Zeta-sizer, Malvern Instruments Ltd, UK). The morphology of prepared Ca-alginate nanoparticles was also examined using a transmission electron microscope (TEM) instrument (200 kV Hitachi H-8100, Tokyo, Japan), and scanning electron microscopy (SEM) (JEOL JSM-840, 10 kV, Japan). For SEM analysis, samples after dispersing in distilled water were dried on the sample metal stub at the room
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temperature. For TEM images, nanoparticles were spread in SDS (sodium dodecyl sulfate) solution (0.1%) in order to achieve dispersion. 2.6. Experimental design and statistical methods Response surface methodology (RSM) was employed to investigate the variation of encapsulation efficiency and particle size with respect to independent parameters, including alginate concentration, calcium concentration, oil volume and surfactant concentration. The composition of these 4 variables was designed using the central composite design (CCD) approach. CCD is a 2k factorial design with star points and central points. The variables and their levels are shown in Table 1. In general, 27 experimental settings and 3 central points were generated with 4 factors and 3 levels by applying RSM using Design-Expert 6.0.8 (StatEase, Inc., Minneapolis, MN). The numerical and graphical optimizations were also performed by the same software. Experiments were randomized in order to minimize the effects of unexplained variability in the observed responses due to extraneous factors. The center point in the design was repeated in triplicate to calculate the repeatability of the method. A second-order polynomial model was constructed to estimate the responses (Eq. 3):
Y = + ∑ + + ∑ ∑ +
(3)
where Y is the estimated response (dependent variable), b 0 the model constant, bi the linear effect coefficient, bii the quadratic effect coefficient, bij the interaction coefficient for two factors, Xi, Xj the independent variables, e the error, k the number of variables considered, and i and j the codified factors of the system (Hosseini et al., 2015). 3. Results and discussion 3.1. Morphology of alginate nanocarriers Depending on the combination of experimental parameters used to prepare nanospheres, a range of particle sizes was observed. With SEM studies shown in Fig. 1 (a), the three-dimensional view of produced alginate nanospheres were seen to be spherical particles, discrete, and distinct with solid dense structure. The results of TEM and DLS of run 27 as the treatment producing the smallest 7
nanosphere size are shown in Fig. 1 (b) and (c), respectively. There appears to be a discrepancy between the size measured by DLS (≈90nm) and TEM (≈60nm) methods that can be explained by the dehydration of the alginate hydrogel nanoparticles during sample preparation for TEM imaging. Also, DLS measures the apparent size (hydrodynamic radius) of a particle, including hydrodynamic layers that form around hydrophilic particles such as those composed of alginate, leading to an overestimation of nanoparticles size (Langridge et al., 2015).
3.2. Influence of independent variables on nanoencapsulation efficiency The assays with ranges determined in Table 1 were performed and responses for the encapsulation yield (EY) are shown in Fig. 2. Statistical model of EY was obtained as the following Equation: EY = + 4.93800 – 6.60194 X1 + 5.51424×10 -3 X4 + 26.16296 X3 – 8.26296×10-3 X2 – 4.88750×10-3 X1 X4 – 46.35000 X1 X3 + 0.011625 X1 X2 + 0.026562 X3 X4 – 1.74688×10-5 X2 X4 + 0.040625 X2 X3 + 12.43852 X12 _ 1.81481×10 -6 X42 _ 47.03704 X32 + 9.74074×10 -6 X22
(4)
where X1 is alginate concentration, X2 is volume of Tween 80, X3 is CaCl2 molarity, and X4 is volume of oil.
3.2.1. Effect of alginate concentration Higher EY values found for higher alginate concentrations could be explained by higher viscosity of alginate which causes a more cohesion property and consequently can lead to entrapment of higher amounts of peppermint extract as core material (bioactive) incorporated into the final rigid Ca-alginate matrix. This behaviour is more pronounced in the formulations containing lower amounts of Tween 80 as a surfactant because of its interference between oil phase and alginate solution which efficiently reduces surface and interfacial tensions, leading to more escaping of bioactive into the oil, and as a result, lower EY. According to Fig. 2 (a), EY is highly proportional to alginate concentration, and as perturbation graph of EY shows in Fig. 3, it has the highest gradient which conveys the most dependency of EY in alginate concentration among all 4 assessed 8
factors. Motwani et al., (2008), found that encapsulation efficiency of nanoparticles made by coacervation method increases with increasing concentrations of alginate. For maintaining the particle size under nanometer, the optimal value of alginate was calculated as 0.5%. Accordingly, with no guarantee of size range, its lower percentages will cause lower EY values, and equally its higher percentages will lead to higher EY values. Alginates have a long history of use in numerous biomedical applications, including drug delivery systems, as they are biodegradable, biocompatible and mucoadhesive polymers (De and Robinson, 2003). Being hemocompatible and emerging no accumulate in any major organs and no evidence of in vivo degradation makes alginate a suitable vehicle for drug delivery. It has been used in a variety of oral and topical pharmaceutical formulations and it has been specifically used for the aqueous microencapsulation of drugs, in contrast to more conventional solvent-based systems (Rajaonarivony et al., 1993).
3.2.2. Role of surfactant For any emulsification process, a surfactant is usually needed for two main functions. One is to lower the interface tension between the water and oil phases and to make the dispersion of viscous alginate solution into the oil easier. The other purpose is to stabilize emulsion droplets against coalescence (Assadpour, et al, 2016; Mehrnia, et al. 2016). Internal gelation as a method of nanosphere preparation involves emulsification, so a surfactant could play an important role in this process (Vandenberg et al., 2001). The emulsion required was a water-in-oil system, therefore surfactants with lower HLB will be optimal (Baek et al., 2016). Tween 80 with HLB of 4.3 is the most common surfactant being used in the emulsification stage of this study. According to Fig. 2 (b), EY was reversely proportional to the amount of surfactant. A slight reduction or increase in the surfactant content can critically change the EY by affecting the surface tension. Firmness loss of sodium alginate nanogranules is a common defect during the first stage of the encapsulation with a high level of surfactant. The tendency of alginate droplets to maintain their 9
rigidity will be reduced by elevating the Tween 80 as surfactant, consequently, they will be diverted to a higher state of dissolution and lose their entrapped peppermint extract, leading to a lower value of EY. The content of Tween 80 should be balanced to control droplet aggregation and growing of the size before adding CaCl2, and at the same time, to prevent bioactive loss. Its optimal value was determined to be 100 µl for 400 ml oil phase (0.25%). It is worthy to mention that for obtaining particles in nanosize, it is better to use combination of high speed/pressure and short time rather than low speed/pressure and long time since shorter times will partially protect particles of losing their bioactive through exposure to the environment. 3.2.3. Influence of oil volume and CaCl2 molarity There is, to our knowkedge, no research investigating the role of oil and CaCl2 contents on outputs of internal gelation method. As shown in Fig. 2 (d), there is an opposite relation between oil volume and EY value, although it is comparatively less dependent to oil content than other three factors. The reason may be that an increase in oil content provides more space for shrinking of the alginate droplets into smaller ones which results in higher surface of alginate particles, consequently more release of bioactive into oil phase, as a result, lower EY values. With the increase in calcium ions when adding calcium chloride in the second stage, dispersed sodium alginate particles begin to aggregate and size increases when the mixer speed is reduced. At this stage, increasing the concentration of calcium ions will accelerate the exposure of carboxyl groups of alginate particles to calcium ions when they are still in desirable size and turns them into solid particles in a steady matrix form before aggregation. Therefore, as shown in Fig. 3, CaCl2 concentration with a slight gradient is directly proportional to EY value, and it is the most effective factor after alginate concentration. 3.3. Influence of independent variable on the size of alginate nanocarriers Perturbation plots for the size of alginate nanoparticle are presented in Fig 4. The changes in particle size with the formulation parameters followed an exponential equation:
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Particle size = -2255.71914+ 4173.77778 X1 + 1.55333 X4 + 11193.14815 X3 + 5.51741 X2 – 2.20500 X1 X4 – 6420 X1 X3- 3.37500 X4 X3 + 3.53125×10-3 X4 X2+ 11.77500 X3 X2 + 4866.37037 X12 – 9.71296×10-4 X42 – 49940.74075 X32 + 6.6148×10 -3 X2
(5)
where X1 is alginate concentration, X2 is volume of Tween 80, X3 is CaCl2 molarity, and X4 is volume of oil.
3.3.1. Effect of alginate concentration on size of nanocarriers Increasing the alginate concentration resulted bigger particle sizes; for some treatments, transition happened from nanoparticles to microparticles. As shown in Table 1, the run 12 with 0.5% alginate concentration, 100ml Tween, 0.15 M CaCl2 and 400 ml oil came with an EY of 5.26% and particle size of 512 nm, whereas in the run 18 by increasing the alginate concentration to 1%, EY and particle size were obtained as 7.62% and 4303 nm, respectively. For a given application, alginate concentration must be controlled in terms of the particle size, shape and distribution (Reis et al., 2006). Alginate particles at higher concentrations are expected to possess larger sizes due to higher viscosity and hardness which results in a resistance to shear rate of stirring and breakdown to smaller droplets as well as further protection of the bioactive and higher EY. Similar results have also been foundd by other researchers, for example Liu et al (2004) reported that when sodium alginate concentration was higher, the viscosity of aqueous phase was increased, resulted in larger droplets with a wide distribution. 3.3.2. Effect of surfactant on size of nanocarriers When surfactant is adsorbed onto the surface of disperse droplets to form a film against coalescence, lower concentrations may not completely cover these (inter) surfaces of the dispersed phase, causing a decline in the stability of droplets, and due to coalescence, results in larger droplets. According to Liu et al. (2004), on the other hand, higher than 2.0% surfactant is of little benefit to the formulation as the oil-soluble ingredients should diffuse through the water and oil interface in order to initiate the gelation process, thus, a high surfactant concentration may cause 11
mass transfer resistance to protons, prolonging gelation, and causing a low production of particles. As shown in Fig. 4 (b), increase in Tween content leads to smaller particle sizes which could be due to lowering surface tension and facilitating breakdown of sodium alginate droplets into smaller units. Liu et al. (2004), also reported that the size of Ca-alginate beads falls remarkably with an increase in surfactant concentration from 0.5 to 2.0%. Zhao et al. (2010), then demonstrated that increasing Tween 80 concentration reduced the droplet size. Similarly, Lertsutthiwong and Rojsitthisak (2011), reported that Tween 80 is required to produce a small capsule size. Lack of Tween 80 as a surfactant can reversely cause aggregation of sodium alginate droplets, as a result, growing the particle size. Similar results have also been found by Gonzalez Ferreiro et al., (2002), who reported that a lower amount of poly-L-lysine in the formulation led to an irregular surface, colloidal system, probably formed by clustered forms.
3.3.3. Effect of oil content and CaCl2 concentration on size of nanocarriers According to Fig. 4 (d), increase in oil volume efficiently reduces the particle size. At higher oil contents, sodium alginate droplets will shrink much easier while a tight environment (lower oil volume) results in collision of droplets through exerting shear stresses, and then, aggregation and growing of particle size happen. Immediately after the first stage of internal gelation procedure, when particles with desired size have been achieved, Ca2+ ions should be added as a crosslinker to form alginate particles in a solid form (calcium alginate). Mixing during gel formation is important, otherwise aggregation of nanoparticles may happen (Mo et al. 2012). Regarding the stirring process, any delay in this step can cause aggregation of droplets and no formation of small droplets. Hence, in this study adding CaCl2 solution in a spray form was attempted, to maximize collision between Ca2+ ions and hydroxyl groups of sodium alginate. According to Fig. 4 (c), increase in molarity of CaCl2 results in smaller particle sizes, even though it is not affected as much as by other factors. Increase in numbers of Ca2+ ions by elevating the CaCl2 molarity accelerates exposure of hydroxyl groups of alginate to them and partially prevents droplet aggregation, leading to a decrease in particle size. 12
In general, it was found that the dependent variables assessed in this study behave oppositely against independent variables since each factor which increases the EY, reduces the particle size. Pistone et al., (2015), studied the possibility of preparing stable alginate nanohydrogels through cross-linking with zinc in the presence of monovalent salts. Their results showed that a critical zinc concentration was needed to obtain nanoparticles, and below this concentration, particles were not formed. An increase in the ionic strength of the solvent resulted in nanoparticles of lower size as well as lower polydispersity. Chan et al., (2009), reported previously that the drug encapsulation efficiency was affected by the size of micropellets and calcium alginate micropellets with larger sizes had a higher acetaminophen content. According to Fig. 5, alginate concentration was the most effective independent variable on particle size and after that, volume of oil with a lower gradient was the second factor. Internal gelation method is routinely carried out by high speed stirring or high pressure homogenizing. All runs of this study were performed at a stirring speed of 800 rpm in the first stage of the procedure, and nanoparticles smaller than 100 nm in run 26 could be produced. This is an interesting result using this low level of energy for the first time, to our knowledge, and revealing that screw magnets are superior to common magnets in low energy procedures. Also, spraying CaCl2 solution instead of common pouring is much better due to higher collision which is the main reason of reducing the size in this procedure.
3.4. Optimization results The optimum formulation of nanoencapsulation parameters was selected based on obtaining the maximum value of nanoencapsulation efficiency and minimum value of the nanosphere size. The optimum point was validated in triplicate. Experiments were performed to confirm the determined conditions. The formulation composition with of 0.5% alginate concentration, 400 ml oil, 0.05 M CaCl2, and 100 ml surfactant was determined to provide a reasonable EY and an average nanosize of 5.6% and 785nm, respectively. 13
Peppermint extract was selected as an ideal material in terms of easiness and precision of the measurement of phenolic compounds by spectrophotometer compared to other methods and compounds to serve targets of this study and nanoencapsulation of peppermint itself was not of concern. By considering that alginate, in nature, indicates different behaviours in touch with various compounds in terms of rigidity and viscosity and finally in different EYs and sizes, the obtained size and EY values were inclusively relevant to peppermint extract but the optimization results can be extended to nanoencapsulation of any other component. These results are in accordance with the wide range of size and EY values being reported by many studies dealing with nanoencapsulation of various components with different combinations of experimental factors (Chang et al., (2012), 4.73% EY and Reis et al., (2008), 85% EY for nanospheres containing reducible and insulin, respectively). In order to being commercially applicable in nutraceutical area, based on the drug and target organ’s specifications, case studies are needed. Firstly, the optimum size and EY are required to be defined, and secondly, by employing our found combination patterns of experimental factors, the right treatment should be designed. 4. Conclusion In this study, the internal gelation procedure for producing alginate nanospheres was innovatively improved. The impact of formulation parameters on size and encapsulation yield of final nanospheres was also investigated, and these parameters were optimized. In the first step of the procedure, instead of routine soft rod-shaped magnets, screw magnets were used and found to be highly efficient in raising the turbulence. Furthermore, by adding CaCl2 solution as spray droplets instead of the routine way of pouring, maximum collision was found between Ca+2 ions and sodium alginate droplets, which facilitated cross-linking as the aim of the second step of the procedure. Both of these innovations are reported for the first time in this study. All four examined factors were recognized effective on the quality of the procedure and dependency on alginate concentration and surfactant content were found in more intensity than other two factors. Lower concentration of alginate, higher amount of surfactant, oil and calcium chloride led to smaller 14
nanospheres. It was also found that independent factors oppositely affect EY and size. The particle size of the optimal point was found to be 785 nm. Overall, the encapsulation efficiency of internal gelation method is comparatively low, but it can still be considered for some particular nanotargets because of its high safety level. Volatility of phenolic extract should also be considered through procedure of nanoparticle preparation and next assessments can show lower efficiency than norm, even though it can result in comparatively optimum contents and conditions. References Assadpour, E., Maghsoudlou, Y., Jafari, S.-M., Ghorbani, M., & Aalami, M. (2016). Optimization of folic acid nano-emulsification and encapsulation by maltodextrin-whey protein double emulsions. International Journal of Biological Macromolecules, 86, 197-207. Baek, S., Min, J., & Lee, J. W. (2016). Equilibria of cyclopentane hydrates with varying HLB numbers of sorbitan monoesters in water-in-oil emulsions. Fluid Phase Equilibria, 413, 41-47. Balcão, V. M., Costa, C. I., Matos, C. M., Moutinho, C. G., Amorim, M., Pintadom, M. E., Gomes, A. P & et al. (2013). Nanoencapsulation of bovine lactoferrin for food and biopharmaceutical applications. Food Hydrocolloids, 32, 425-431. Chan, L. W., Lee, H. Y., & Heng, PW. S. (2009). Mechanisms of external and internal gelation and their impact on the functions of alginate as a coat and delivery system. Carbohydrate Polymers, 63, 176– 187. Chang, D., Lei, J., Cui, H., Lu, N., Sun, Y., Zhang, X., Gao, C., Zheng, H., & Yin, Y. (2012). Disulfide cross-linked nanospheres from sodium alginate derivative for inflammatory bowel disease: Preparation, characterization, and in vitro drug release behaviour. Carbohydrate Polymer, 88, 663– 669. De, S., & Robinson, D. (2003). P olymer relationships during preparation of chitosan–alginate and poly-llysine–alginate nanospheres. Journal of Controlled Release, 89, 101-112. Dong, Z., Ma, Y., Hayat, K., Jia, C., Xia, S., & Zhang, X. (2011). Morphology and release profile of microcapsules encapsulating peppermint oil by complex coacervation. Journal of Food Engineering, 104(3), 455-460.
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Faridi Esfanjani, A., & Jafari, S. M. (2016). Biopolymer nano-particles and natural nano-carriers for nanoencapsulation of phenolic compounds. Colloids and Surfaces B: Biointerfaces, 146, 532-543. Ghayempour, S., & Mortazavi, S M. (2015). Preparation and investigation of sodium alginate nanocapsules by different microemulsification devices. Journal of Applied Polymer, 132(17), 41904. Gonzalez Ferreiro, M., Tillman, L., Hardee, G., & Bodmeier, R. (2002). Characterization of alginate/poly-Llysine particles as antisense oligonucleotide carriers. International Journal of Pharmaceutics, 239, 47-59. Hosseini, A., Jafari, S. M., Mirzaei, H., Asghari, A., & Akhavan, S. (2015). Application of image processing to assess emulsion stability and emulsification properties of Arabic gum. Carbohydrate polymers, 126, 1-8. Javanmardi, J., Stushnoff, C., Locke, E., & Vivanco, J. M. (2003). Antioxidant activity and total phenolic content of Iranian Ocimum accessions. Food Chemistry, 83, 547-550. Katouzian, I., & Jafari, S. M. (2016). Nano-encapsulation as a promising approach for targeted delivery and controlled release of vitamins. Trends in Food Science & Technology, 53, 34-48. Khare, A R., Vasisht, N. (2014). Nanoencapsulation in the food industry: Technology in the future, in: Gaonkar, A. G., Vasisht, N., Khare, A. R., & Sobel, R. (Eds), Microencapsulation in the Food Industry: A Practical Implementation Guide. Elsevier San Diego, pp. 151-155. Koo, S. Y., Cha, K. H., Song, D. G., Chung, D., & Pan, C. H. (2014). Microencapsulation of peppermint oil in an alginate–pectin matrix using a coaxial electrospray system. International Journal of Food Science & Technology, 49(3), 733-739. Langridge, T. D., Tarver, M. J., & Whitten, S. T. (2015). Temperature effects on the hydrodynamic radius of the intrinsically disordered N-terminal region of the p53 protein. Proteins: Structure, Function, and Bioinformatics, 82(4), 668-678. Lertsutthiwong, P., & Rojsitthiska, P. (2011). Chitosan-alginate nanocapsules for encapsulation of turmeric oil. Die Pharmazie, 66(12), 911-915. Liu, X., Ma, Z., Xing, J., & Liu, H. (2004). Preparation and characterization of amino-silane modified superparamagnetic silica nanospheres. Journal of Magnetism and Magnetic Materials, 270, 1-6.
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Mehrnia, M. A., Jafari, S. M., Makhmal-Zadeh, B. S., & Maghsoudlou, Y. (2016). Crocin loaded nanoemulsions: Factors affecting emulsion properties in spontaneous emulsification. International journal of biological macromolecules, 84, 261-267. Mo, Y., Xiao, K., Shen, Y., & Huang, X. (2012). A new perspective on the effect of complexation between calcium and alginate on fouling during nanofiltration. Separation and Purification Technology, 82, 121-127. Motwani, S. K., Chopra, S., Talegaonkar, T., Kohli, K., Ahmad, F J., & Khar, R. K. (2008). Chitosan– sodium alginate nanoparticles as submicroscopic reservoirs for ocular delivery: Formulation, optimization
and
in
vitro
characterization.
European
Journal
of
Pharmaceutics
and
Biopharmaceutics, 68, 513-525. Pistone, S., Qoragllu, D., Smistad, G., & Hiorth, M. (2015). Formulation and preparation of stable crosslinked alginate-zinc nanoparticles in the presence of a monovalent salt. Soft Matter, 11(28), 57655774. Poncelet, D. (2001). Production of alginate beads by emulsification/internal gelation. Annals of the New York Academy of Sciences, 944, 74–82. Rafiee, Z., Jafari, S. M., Alami, M., & Khomeiri, M. (2011). Microwave-assisted extraction of phenolic compounds from olive leaves; a comparison with maceration. Journal of Animal and Plant Sciences, 21(4), 738-745. Rafiee, Z., Jafari, S. M., Alami, M., & Khomeiri, M. (2012). Antioxidant effect of microwave-assisted extracts of olive leaves on sunflower oil. Journal of Agricultural Science and Technology, 14(SUPPL.), 1497-1509. Reis, C. P., Neufeld, R. J., Vilela, S., Ribeiro, A. J., & Veiga, F. (2006). Review and current status of emulsion/dispersion technology using an internal gelation process for the design of alginate particles. Journal of microencapsulation, 23(3), 245-257. Reis C. P, Veiga, F. J., Ribeiro, A. J., Neufeld, R. J., & Damg, C. (2008). Nanoparticulate Biopolymers Deliver Insulin Orally Eliciting Pharmacological Response. Journal of Pharmaceutical Sciences, 97(12), 5290-5305. Rolland, L., Santanach-Carreras, E., Delma, T., Bibette, J., & Bremond, N. (2014). Physicochemical properties of aqueous core hydrogel capsules. Soft Matter, 10(48), 9668-9674. 17
Sarmento, B., Martins, S., Riberio, A., Veiga, F., Neufeld, R., & Ferreira, D. (2006). Development and comparison of different nanoparticulate polyelectrolyte complexes as insulin carriers. International Journal of Peptide Research and Therapeutics, 12(2), 131-138. Vandenberg, G. W., Drolet, C., Scott, S. L., De la & Noue, J. (2001). Factors affecting protein release from alginate-chitosan coacervate microcapsules during production and gastric/intestinal simulation. Journal of Control Release, 77, 297-307. You, J. O., & Peng, C. A. (2005). Calcium‐Alginate Nanoparticles Formed by Reverse Microemulsion as Gene Carriers, Macromolecular Symposia, 219(1), 147-153. Zhao, Y., Wang, C., Chow, A. H. L., Ren, K., Gong, T., Zhang, Z., & Zheng, Y. (2010). Selfnanoemulsifying drug delivery system (SNEDDS) for oral delivery of Zedoary essential oil: Formulation and bioavailability studies. International Journal of Pharmaceutics, 383, 170-177.
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a
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Table 1. Effect of alginate and surfactant content, CaCl2 concentration and oil content on the size and encapsulation yield (EY) of nanoparticles loaded with peppermint phenolic extract
Run 1 2 3 4 5 6a 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23a 24 25 26 27a a
Alginate (%w/v) (X1) 1.00 0.50 0.75 0.50 1.00 0.75 0.50 0.75 0.75 0.50 1.00 0.50 0.75 1.00 1.00 0.50 0.75 1.00 0.50 0.50 1.00 1.00 0.75 1.00 0.75 0.50 0.75
Surfactant (µ l) (X2) 100 100 200 100 100 200 200 100 200 300 300 100 200 200 100 300 300 100 300 100 300 300 200 300 200 300 200
CaCl2 (molarity) (X3) 0.05 0.05 0.10 0.15 0.05 0.10 0.10 0.10 0.15 0.15 0.15 0.15 0.10 0.10 0.15 0.05 0.10 0.15 0.05 0.05 0.05 0.15 0.10 0.05 0.05 0.15 0.10
Central point
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Oil (mL) (X4 ) 400 400 400 800 800 600 600 600 600 400 400 400 800 600 800 400 600 400 800 800 400 800 600 800 600 800 600
EY (%) 10.2 6.15 8.6 8.8 7.58 6.74 6 7.25 7.45 6.66 10.25 5.26 6.95 7.25 5.8 5.1 7.64 7.62 3.7 5.24 9.61 8.38 7.35 9.19 6.01 4.11 6.92
Mean size (nm) 4533 696 1584 478 4122 1112 412 1974 919 140 923 512 936 2792 2985 361 756 4302 153 608 1257 783 987 945 1425 90 1191
Development of a nutraceutical nano-delivery system through emulsification/internal gelation of alginate
Samira Mokhtari, Seid Mahdi Jafari*, Elham Assadpour Graphical abstract:
Schematic presentation of the internal gelation procedure; (a) shrinkage of alginate sodium droplets; (b) crosslinking with calcium ions.
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Research Highlights: •
Alginate nano-carriers prepared by a low energy internal gelation technique
•
Size of nanoparticles decreased at higher oil and surfactant contents
•
Encapsulation efficiency was inversely proportional to the size of nanoparticles
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0.5% alginate, 400 mL oil, 0.05 M CaCl2 and 100 mL surfactant was the optimized point
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S0308-8146(17)30272-8 http://dx.doi.org/10.1016/j.foodchem.2017.02.071 FOCH 20624
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
21 October 2016 5 January 2017 15 February 2017
Please cite this article as: Mokhtari, S., Jafari, S.M., Assadpour, E., Development of a nutraceutical nano-delivery system through emulsification/internal gelation of alginate, Food Chemistry (2017), doi: http://dx.doi.org/10.1016/ j.foodchem.2017.02.071
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Development of a nutraceutical nano-delivery system through emulsification/internal gelation of alginate
Running title: Alginate nano-carriers for nutraceuticals
Samira Mokhtari, Seid Mahdi Jafari*, Elham Assadpour
Department of Food Materials and Process Design Engineering, University of Agricultural Sciences and Natural Resources, Gorgan, Iran *Corresponding Details: Tel/Fax: +98 17 324 26 432. E-mails: [email protected]
ABSTRACT Alginate nano/microsphers are produced by emulsification/internal gelation of sodium alginate dispersed within vegetable oils containing surfactant, followed by CaCl2 addition resulting in hardened particles. In this work, the impact was evaluated of alginate, CaCl2, oil and surfactant content on the size and encapsulation efficiency of nanocarriers containing peppermint phenolic extract and prepared by a low energy internal gelation technique. The results revealed that size of nanoparticles decreased at higher oil and surfactant contents, higher molarity of CaCl2 and lower alginate concentrations. Also, it was found that the encapsulation efficiency was inversely proportional to the size of nanoparticles, and the impact of alginate concentration and surfactant content was markedly higher than the other two factors. The composition of 0.5% alginate, 400 ml oil, 0.05 M CaCl2 and 100 ml surfactant was recognized as the optimized treatment with a reasonable encapsulation efficiency of 5.6% and a nanoparticle size of 785 nm.
Keywords: Nanoencapsulation; Alginate; Efficiency; Phenolic extract; Nutraceuticals.
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1. Introduction Nanoencapsulation is one of the interesting applications of nanotechnology normally implemented in food, pharmaceutical and cosmeceutical industries, which enables the controlled release of nutrients in the right zone and time. This technique has numerous advantages for targeted delivery and bioavailability through the cells (Katouzian and Jafari, 2016). In general, nanoencapsulated particles are known as nano-capsules when their size is smaller than 1.0 µm. There are numerous examples of naturally occurring nanoencapsulates, such as casein micelles in milk (<100 nm), mitochondria (500-10,000 nm), and viruses (10-300 nm). The term nanoencapsulation describes encapsulation in the nanometer scale using films, layers, or nano-dispersions (Faridi Esfanjani and Jafari, 2016). The final capsule acts as a nanoscale shield for the nutraceutical or drug molecules/ingredients. Often, the active ingredient is in the molecule or nanoscale state. The major benefit is homogeneity, leading to better encapsulation efficiency as well as improved physical and chemical properties (Khare and Vasisht, 2014). Alginate is an anionic polysaccharide of (1–4)-linked b-D-mannuronic acid (M) and a-L-guluronic acid (G) widely used in bioencapsulation of drugs, nutraceuticals, proteins and cells. Ca-alginate beads represent one of the most widely used carriers for the immobilization of enzymes and proteins as well as for the controlled release of drugs (Sarmento et al, 2006; M Leonard et al, 2004; Chen et al, 2004). Experimental evidence shows that alginate nanoparticles have been successfully prepared using the emulsification/internal gelation method by creating the ideal conditions for the formulation and have recently been extended to the field of nanotechnology (Reis et al, 2006; Faridi Esfanjani and Jafari, 2016). In recent years, hydrogel nanoparticles have gained significant attention as one of the most promising nanoscale delivery systems because they have unique potential through combining the characteristics of a hydrogel system (hydrophilicity and extremely high water content) with those of nanotechnology (very small size). Single or mixed-type biopolymer clusters may be developed from hydrogel particles under certain conditions. 2
Among different bio-macromolecules, which can be used for hydrogel formation, polysaccharides are extremely advantageous to physically or chemically form filled hydrogels. Polysaccharidebased hydrogels can be formed whenever an appropriate counter-ion is added to a polysaccharide dispersion of adequate concentration. As an example, sodium alginate dispersion transforms into alginate hydrogel after mixing (titration) with divalent cations (such as calcium ion). At low concentrations (diluted systems), nanoparticle formation is obtained instead of bulk phase gelation. Rolland et al., (2014), studied the formation of a filled thin alginate hydrogel membrane surrounding an aqueous core through a co-extrusion step in air followed by a sol–gel transition of the shell after immersion into a gelling bath. Some formulation and process parameters can be adjusted to produce smaller particles (Reis et al. 2006). By controlling the conditions under which the water-in-oil emulsion is produced, hydrogel capsule size can be easily controlled from a few nanometers potentially, to millimeters in diameter (Poncelet, 2001). The most recent research limitations and challenges is the inability to reduce particle size of calcium alginate gels. Higher levels of energy such as homogenization at 8000 rpm (Balcão et al., 2013), and magnetic stirring at 1600 rpm for 15 minutes (Reis et al., 2008) are used to lower nanoparticle size. In order to reduce the size of nanoparticles, cationic polymers, like chitosan, have been used; although, this method leads to nanoparticle sizes larger than 700 nm (Lertsutthiwong and Rojsitthisak, 2011). Moreover, how to add solution containing crosslinkers of calcium ions into sodium alginate solution is one of important and determinant factors affecting the particle size that has not yet been discussed. The main difference between the current study and previous research is the much smaller nanoparticle size used here. Therefore, one of the aims of this research was to introduce some modifications for the common internal gelation procedure in order to obtain the same calcium alginate nanogels (in size) using lower energy. Additionally, a detailed study on optimization of nanoencapsulation parameters of internal gelation technique has not yet been done. Therefore, 3
another aim was to maximize the nanoencapsulation efficiency of alginate nanodelivery system by internal gelation method and applying optimal alginate and calcium concentrations, oil and surfactant contents, as well as illustrating the correlation between size and efficiency of final nanocarriers and formulation parameters. Peppermint phenolic extract was selected to be nanoencapsulated as the core material. It is one of the most popular and widely used functional ingredients, with applications in the food, pharmaceutical and flavouring industries (Ghayempour and Mortazavi, 2014; Koo et al., 2014). Peppermint extract readily undergoes oxidation when exposed to the air or UV, and it is highly volatile, which limits its applications in the food and pharmaceutical production (Dong et al., 2011; Koo et al., 2014). The utilization of nanoencapsulated extract instead of free bioactives can overcome the drawbacks of its instability as well as improving its bioavailability. 2. Materials and methods Sodium alginate with an average molecular weight of 12,000–40,000 and medium viscosity (3500 mPa.s for a 2% solution at 25˚C) was purchased from Sigma, UK. Tween 80 and calcium chloride (CaCl2) was supplied from Merck Chemicals Co, Germany. Canola oil was purchased from Ladan Company, Iran. Water used in the experiments was double distilled and deionized. All other reagents used in this work were of analytical grade, and purchased from Sigma–Aldrich (St. Louis, USA). 2.1. Preparation of peppermint phenolic extract Peppermint was obtained from educational farm for medicinal plants of Ferdowsi University of Mashhad, Iran. For the preparation of alcoholic extract, peppermint leaves were dried and milled, obtained powder was mixed in a ratio of 1:20 with 80% ethanol, and then through microwaveassisted extraction, its phenolic extract was prepared, and filtered by paper filters (Whatman, No. 42), and alcoholic extract was liquidized. The remained alcohol eliminated by distillation with a vacuum rotary evaporator and also vacuum oven, and finally was stored at -20°C (Rafiei, et al, 2011). 4
2.2. Nanoencapsulation procedure Nanoencapsulation of peppermint phenolic extract using the internal gelation technique was done according to the You and Peng (2005) method, with some modifications. Briefly, 2 ml phenolic extract was dissolved in 8 ml alginate sodium with varying concentrations (0.5, 0.75 and 1%, w/v). The range of biopolymer concentration for this study was selected on the basis of previous reported works such as You and Peng (2005), 0.05 w/w; Sarmento et al, (2006), 0.063 w/w and De and Robinson, (2003), 0.1 % w/v. Then, the mixed solution of phenolic extract-alginate was added dropwise into different volumes (400, 600 and 800 ml) of canola oil containing various levels (100, 200 and 300 µl) of Tween 80 as a surfactant, while mixing speed was fixed at 800 rpm using a screw magnet in order to create the highest collision. Then the mixture was left for 10 minutes prior to the addition of filtered calcium chloride solution of different molarities (0.05, 0.10 and 0.15 molar). According to Reis et al. (2006), the aqueous/oil phases ratio with values of 1:1 demonstrated the ability to produce alginate particles, so the volume of calcium chloride solution in each treatment was modulated proportional to its volume of canola oil. CaCl2 solution was sprayed on the surface of prepared emulsions in order to achieve the highest surface area and maximum collision of Ca+2 ions onto the sodium alginate nanospheres. Broken emulsion was then centrifuged (20,000 ×g) for 45 min at 4˚C (Sarmento et al, 2006), and finally, the prepared nanoparticles were re-suspended with deionized water. Final nanospheres were formulated based on the 27 experimental runs shown in Table 1. 2.3. Determination of total phenolic content Total phenolic content of each extract was determined by the Folin–Ciocalteu micro-method described by Rafiei, et al., (2012). Briefly, 10 µl of ethanolic extract was mixed with 50 µl folin– Ciocalteu and 580 µl distilled water, and after aging for 5-8 min, 150 ml CaCO3 (1 M) was added. Obtained solution then was kept in dark water bath in 40˚C for 30 min and its absorbance was read by a spectrophotometer (WPA S2000, UK) using a wavelength of 765 nm. For the blank sample, an
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80% ethanol solution was used. The total phenolic content of samples was expressed as gallic acid equivalents using the following linear equation based on the calibration curve: Y= 5.606X – 0.039
R2= 0.099
(1)
where Y is the absorbance at 765 nm and X is the concentration of total phenolics based on gallic acid equivalents (µg/ml) (Javanmardi et al., 2003). To obtain the calibration curve, different concentrations of pure gallic acid were used. Standard solutions of 5, 10, 50, 100, 200, 600 and 700 ppm were prepared, and the absorbance coefficient was calculated for each standard. Zero ppm concentration was considered as the blank sample. 2.4. Analysis of nanoencapsulation efficiency To measure the encapsulation efficiency, total phenolic content (TPC) of 2 ml peppermint extract was measured before adding into sodium alginate solution according to Section 2.3. This volume of phenolic extract was encapsulated after adding to the alginate solution, and the entrapped phenolics in the final alginate nanospheres were released and measured as Entrapped Phenolic Content (EPC). To release nanocapsulated peppermint extract from nanospheres, the prepared capsules were added into 90 ml of 1% (w/v) sodium citrate solution with pH = 6.0 at room temperature, and mixed for 10 minutes by a magnetic stirrer at 100 rpm; then the EPC was calculated after reading its absorbance. Encapsulation Efficiency (EE) was obtained with the following formula: EE = (EPC / TPC) × 100
(2)
2.5. Characterization of nanocarriers For size distribution and particle size data, obtained nanoparticles were redispersed in 5 ml ultrapure water. The mean particle size was measured by dynamic light scattering (DLS: Zeta-sizer, Malvern Instruments Ltd, UK). The morphology of prepared Ca-alginate nanoparticles was also examined using a transmission electron microscope (TEM) instrument (200 kV Hitachi H-8100, Tokyo, Japan), and scanning electron microscopy (SEM) (JEOL JSM-840, 10 kV, Japan). For SEM analysis, samples after dispersing in distilled water were dried on the sample metal stub at the room
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temperature. For TEM images, nanoparticles were spread in SDS (sodium dodecyl sulfate) solution (0.1%) in order to achieve dispersion. 2.6. Experimental design and statistical methods Response surface methodology (RSM) was employed to investigate the variation of encapsulation efficiency and particle size with respect to independent parameters, including alginate concentration, calcium concentration, oil volume and surfactant concentration. The composition of these 4 variables was designed using the central composite design (CCD) approach. CCD is a 2k factorial design with star points and central points. The variables and their levels are shown in Table 1. In general, 27 experimental settings and 3 central points were generated with 4 factors and 3 levels by applying RSM using Design-Expert 6.0.8 (StatEase, Inc., Minneapolis, MN). The numerical and graphical optimizations were also performed by the same software. Experiments were randomized in order to minimize the effects of unexplained variability in the observed responses due to extraneous factors. The center point in the design was repeated in triplicate to calculate the repeatability of the method. A second-order polynomial model was constructed to estimate the responses (Eq. 3):
Y = + ∑ + + ∑ ∑ +
(3)
where Y is the estimated response (dependent variable), b 0 the model constant, bi the linear effect coefficient, bii the quadratic effect coefficient, bij the interaction coefficient for two factors, Xi, Xj the independent variables, e the error, k the number of variables considered, and i and j the codified factors of the system (Hosseini et al., 2015). 3. Results and discussion 3.1. Morphology of alginate nanocarriers Depending on the combination of experimental parameters used to prepare nanospheres, a range of particle sizes was observed. With SEM studies shown in Fig. 1 (a), the three-dimensional view of produced alginate nanospheres were seen to be spherical particles, discrete, and distinct with solid dense structure. The results of TEM and DLS of run 27 as the treatment producing the smallest 7
nanosphere size are shown in Fig. 1 (b) and (c), respectively. There appears to be a discrepancy between the size measured by DLS (≈90nm) and TEM (≈60nm) methods that can be explained by the dehydration of the alginate hydrogel nanoparticles during sample preparation for TEM imaging. Also, DLS measures the apparent size (hydrodynamic radius) of a particle, including hydrodynamic layers that form around hydrophilic particles such as those composed of alginate, leading to an overestimation of nanoparticles size (Langridge et al., 2015).
3.2. Influence of independent variables on nanoencapsulation efficiency The assays with ranges determined in Table 1 were performed and responses for the encapsulation yield (EY) are shown in Fig. 2. Statistical model of EY was obtained as the following Equation: EY = + 4.93800 – 6.60194 X1 + 5.51424×10 -3 X4 + 26.16296 X3 – 8.26296×10-3 X2 – 4.88750×10-3 X1 X4 – 46.35000 X1 X3 + 0.011625 X1 X2 + 0.026562 X3 X4 – 1.74688×10-5 X2 X4 + 0.040625 X2 X3 + 12.43852 X12 _ 1.81481×10 -6 X42 _ 47.03704 X32 + 9.74074×10 -6 X22
(4)
where X1 is alginate concentration, X2 is volume of Tween 80, X3 is CaCl2 molarity, and X4 is volume of oil.
3.2.1. Effect of alginate concentration Higher EY values found for higher alginate concentrations could be explained by higher viscosity of alginate which causes a more cohesion property and consequently can lead to entrapment of higher amounts of peppermint extract as core material (bioactive) incorporated into the final rigid Ca-alginate matrix. This behaviour is more pronounced in the formulations containing lower amounts of Tween 80 as a surfactant because of its interference between oil phase and alginate solution which efficiently reduces surface and interfacial tensions, leading to more escaping of bioactive into the oil, and as a result, lower EY. According to Fig. 2 (a), EY is highly proportional to alginate concentration, and as perturbation graph of EY shows in Fig. 3, it has the highest gradient which conveys the most dependency of EY in alginate concentration among all 4 assessed 8
factors. Motwani et al., (2008), found that encapsulation efficiency of nanoparticles made by coacervation method increases with increasing concentrations of alginate. For maintaining the particle size under nanometer, the optimal value of alginate was calculated as 0.5%. Accordingly, with no guarantee of size range, its lower percentages will cause lower EY values, and equally its higher percentages will lead to higher EY values. Alginates have a long history of use in numerous biomedical applications, including drug delivery systems, as they are biodegradable, biocompatible and mucoadhesive polymers (De and Robinson, 2003). Being hemocompatible and emerging no accumulate in any major organs and no evidence of in vivo degradation makes alginate a suitable vehicle for drug delivery. It has been used in a variety of oral and topical pharmaceutical formulations and it has been specifically used for the aqueous microencapsulation of drugs, in contrast to more conventional solvent-based systems (Rajaonarivony et al., 1993).
3.2.2. Role of surfactant For any emulsification process, a surfactant is usually needed for two main functions. One is to lower the interface tension between the water and oil phases and to make the dispersion of viscous alginate solution into the oil easier. The other purpose is to stabilize emulsion droplets against coalescence (Assadpour, et al, 2016; Mehrnia, et al. 2016). Internal gelation as a method of nanosphere preparation involves emulsification, so a surfactant could play an important role in this process (Vandenberg et al., 2001). The emulsion required was a water-in-oil system, therefore surfactants with lower HLB will be optimal (Baek et al., 2016). Tween 80 with HLB of 4.3 is the most common surfactant being used in the emulsification stage of this study. According to Fig. 2 (b), EY was reversely proportional to the amount of surfactant. A slight reduction or increase in the surfactant content can critically change the EY by affecting the surface tension. Firmness loss of sodium alginate nanogranules is a common defect during the first stage of the encapsulation with a high level of surfactant. The tendency of alginate droplets to maintain their 9
rigidity will be reduced by elevating the Tween 80 as surfactant, consequently, they will be diverted to a higher state of dissolution and lose their entrapped peppermint extract, leading to a lower value of EY. The content of Tween 80 should be balanced to control droplet aggregation and growing of the size before adding CaCl2, and at the same time, to prevent bioactive loss. Its optimal value was determined to be 100 µl for 400 ml oil phase (0.25%). It is worthy to mention that for obtaining particles in nanosize, it is better to use combination of high speed/pressure and short time rather than low speed/pressure and long time since shorter times will partially protect particles of losing their bioactive through exposure to the environment. 3.2.3. Influence of oil volume and CaCl2 molarity There is, to our knowkedge, no research investigating the role of oil and CaCl2 contents on outputs of internal gelation method. As shown in Fig. 2 (d), there is an opposite relation between oil volume and EY value, although it is comparatively less dependent to oil content than other three factors. The reason may be that an increase in oil content provides more space for shrinking of the alginate droplets into smaller ones which results in higher surface of alginate particles, consequently more release of bioactive into oil phase, as a result, lower EY values. With the increase in calcium ions when adding calcium chloride in the second stage, dispersed sodium alginate particles begin to aggregate and size increases when the mixer speed is reduced. At this stage, increasing the concentration of calcium ions will accelerate the exposure of carboxyl groups of alginate particles to calcium ions when they are still in desirable size and turns them into solid particles in a steady matrix form before aggregation. Therefore, as shown in Fig. 3, CaCl2 concentration with a slight gradient is directly proportional to EY value, and it is the most effective factor after alginate concentration. 3.3. Influence of independent variable on the size of alginate nanocarriers Perturbation plots for the size of alginate nanoparticle are presented in Fig 4. The changes in particle size with the formulation parameters followed an exponential equation:
10
Particle size = -2255.71914+ 4173.77778 X1 + 1.55333 X4 + 11193.14815 X3 + 5.51741 X2 – 2.20500 X1 X4 – 6420 X1 X3- 3.37500 X4 X3 + 3.53125×10-3 X4 X2+ 11.77500 X3 X2 + 4866.37037 X12 – 9.71296×10-4 X42 – 49940.74075 X32 + 6.6148×10 -3 X2
(5)
where X1 is alginate concentration, X2 is volume of Tween 80, X3 is CaCl2 molarity, and X4 is volume of oil.
3.3.1. Effect of alginate concentration on size of nanocarriers Increasing the alginate concentration resulted bigger particle sizes; for some treatments, transition happened from nanoparticles to microparticles. As shown in Table 1, the run 12 with 0.5% alginate concentration, 100ml Tween, 0.15 M CaCl2 and 400 ml oil came with an EY of 5.26% and particle size of 512 nm, whereas in the run 18 by increasing the alginate concentration to 1%, EY and particle size were obtained as 7.62% and 4303 nm, respectively. For a given application, alginate concentration must be controlled in terms of the particle size, shape and distribution (Reis et al., 2006). Alginate particles at higher concentrations are expected to possess larger sizes due to higher viscosity and hardness which results in a resistance to shear rate of stirring and breakdown to smaller droplets as well as further protection of the bioactive and higher EY. Similar results have also been foundd by other researchers, for example Liu et al (2004) reported that when sodium alginate concentration was higher, the viscosity of aqueous phase was increased, resulted in larger droplets with a wide distribution. 3.3.2. Effect of surfactant on size of nanocarriers When surfactant is adsorbed onto the surface of disperse droplets to form a film against coalescence, lower concentrations may not completely cover these (inter) surfaces of the dispersed phase, causing a decline in the stability of droplets, and due to coalescence, results in larger droplets. According to Liu et al. (2004), on the other hand, higher than 2.0% surfactant is of little benefit to the formulation as the oil-soluble ingredients should diffuse through the water and oil interface in order to initiate the gelation process, thus, a high surfactant concentration may cause 11
mass transfer resistance to protons, prolonging gelation, and causing a low production of particles. As shown in Fig. 4 (b), increase in Tween content leads to smaller particle sizes which could be due to lowering surface tension and facilitating breakdown of sodium alginate droplets into smaller units. Liu et al. (2004), also reported that the size of Ca-alginate beads falls remarkably with an increase in surfactant concentration from 0.5 to 2.0%. Zhao et al. (2010), then demonstrated that increasing Tween 80 concentration reduced the droplet size. Similarly, Lertsutthiwong and Rojsitthisak (2011), reported that Tween 80 is required to produce a small capsule size. Lack of Tween 80 as a surfactant can reversely cause aggregation of sodium alginate droplets, as a result, growing the particle size. Similar results have also been found by Gonzalez Ferreiro et al., (2002), who reported that a lower amount of poly-L-lysine in the formulation led to an irregular surface, colloidal system, probably formed by clustered forms.
3.3.3. Effect of oil content and CaCl2 concentration on size of nanocarriers According to Fig. 4 (d), increase in oil volume efficiently reduces the particle size. At higher oil contents, sodium alginate droplets will shrink much easier while a tight environment (lower oil volume) results in collision of droplets through exerting shear stresses, and then, aggregation and growing of particle size happen. Immediately after the first stage of internal gelation procedure, when particles with desired size have been achieved, Ca2+ ions should be added as a crosslinker to form alginate particles in a solid form (calcium alginate). Mixing during gel formation is important, otherwise aggregation of nanoparticles may happen (Mo et al. 2012). Regarding the stirring process, any delay in this step can cause aggregation of droplets and no formation of small droplets. Hence, in this study adding CaCl2 solution in a spray form was attempted, to maximize collision between Ca2+ ions and hydroxyl groups of sodium alginate. According to Fig. 4 (c), increase in molarity of CaCl2 results in smaller particle sizes, even though it is not affected as much as by other factors. Increase in numbers of Ca2+ ions by elevating the CaCl2 molarity accelerates exposure of hydroxyl groups of alginate to them and partially prevents droplet aggregation, leading to a decrease in particle size. 12
In general, it was found that the dependent variables assessed in this study behave oppositely against independent variables since each factor which increases the EY, reduces the particle size. Pistone et al., (2015), studied the possibility of preparing stable alginate nanohydrogels through cross-linking with zinc in the presence of monovalent salts. Their results showed that a critical zinc concentration was needed to obtain nanoparticles, and below this concentration, particles were not formed. An increase in the ionic strength of the solvent resulted in nanoparticles of lower size as well as lower polydispersity. Chan et al., (2009), reported previously that the drug encapsulation efficiency was affected by the size of micropellets and calcium alginate micropellets with larger sizes had a higher acetaminophen content. According to Fig. 5, alginate concentration was the most effective independent variable on particle size and after that, volume of oil with a lower gradient was the second factor. Internal gelation method is routinely carried out by high speed stirring or high pressure homogenizing. All runs of this study were performed at a stirring speed of 800 rpm in the first stage of the procedure, and nanoparticles smaller than 100 nm in run 26 could be produced. This is an interesting result using this low level of energy for the first time, to our knowledge, and revealing that screw magnets are superior to common magnets in low energy procedures. Also, spraying CaCl2 solution instead of common pouring is much better due to higher collision which is the main reason of reducing the size in this procedure.
3.4. Optimization results The optimum formulation of nanoencapsulation parameters was selected based on obtaining the maximum value of nanoencapsulation efficiency and minimum value of the nanosphere size. The optimum point was validated in triplicate. Experiments were performed to confirm the determined conditions. The formulation composition with of 0.5% alginate concentration, 400 ml oil, 0.05 M CaCl2, and 100 ml surfactant was determined to provide a reasonable EY and an average nanosize of 5.6% and 785nm, respectively. 13
Peppermint extract was selected as an ideal material in terms of easiness and precision of the measurement of phenolic compounds by spectrophotometer compared to other methods and compounds to serve targets of this study and nanoencapsulation of peppermint itself was not of concern. By considering that alginate, in nature, indicates different behaviours in touch with various compounds in terms of rigidity and viscosity and finally in different EYs and sizes, the obtained size and EY values were inclusively relevant to peppermint extract but the optimization results can be extended to nanoencapsulation of any other component. These results are in accordance with the wide range of size and EY values being reported by many studies dealing with nanoencapsulation of various components with different combinations of experimental factors (Chang et al., (2012), 4.73% EY and Reis et al., (2008), 85% EY for nanospheres containing reducible and insulin, respectively). In order to being commercially applicable in nutraceutical area, based on the drug and target organ’s specifications, case studies are needed. Firstly, the optimum size and EY are required to be defined, and secondly, by employing our found combination patterns of experimental factors, the right treatment should be designed. 4. Conclusion In this study, the internal gelation procedure for producing alginate nanospheres was innovatively improved. The impact of formulation parameters on size and encapsulation yield of final nanospheres was also investigated, and these parameters were optimized. In the first step of the procedure, instead of routine soft rod-shaped magnets, screw magnets were used and found to be highly efficient in raising the turbulence. Furthermore, by adding CaCl2 solution as spray droplets instead of the routine way of pouring, maximum collision was found between Ca+2 ions and sodium alginate droplets, which facilitated cross-linking as the aim of the second step of the procedure. Both of these innovations are reported for the first time in this study. All four examined factors were recognized effective on the quality of the procedure and dependency on alginate concentration and surfactant content were found in more intensity than other two factors. Lower concentration of alginate, higher amount of surfactant, oil and calcium chloride led to smaller 14
nanospheres. It was also found that independent factors oppositely affect EY and size. The particle size of the optimal point was found to be 785 nm. Overall, the encapsulation efficiency of internal gelation method is comparatively low, but it can still be considered for some particular nanotargets because of its high safety level. Volatility of phenolic extract should also be considered through procedure of nanoparticle preparation and next assessments can show lower efficiency than norm, even though it can result in comparatively optimum contents and conditions. References Assadpour, E., Maghsoudlou, Y., Jafari, S.-M., Ghorbani, M., & Aalami, M. (2016). Optimization of folic acid nano-emulsification and encapsulation by maltodextrin-whey protein double emulsions. International Journal of Biological Macromolecules, 86, 197-207. Baek, S., Min, J., & Lee, J. W. (2016). Equilibria of cyclopentane hydrates with varying HLB numbers of sorbitan monoesters in water-in-oil emulsions. Fluid Phase Equilibria, 413, 41-47. Balcão, V. M., Costa, C. I., Matos, C. M., Moutinho, C. G., Amorim, M., Pintadom, M. E., Gomes, A. P & et al. (2013). Nanoencapsulation of bovine lactoferrin for food and biopharmaceutical applications. Food Hydrocolloids, 32, 425-431. Chan, L. W., Lee, H. Y., & Heng, PW. S. (2009). Mechanisms of external and internal gelation and their impact on the functions of alginate as a coat and delivery system. Carbohydrate Polymers, 63, 176– 187. Chang, D., Lei, J., Cui, H., Lu, N., Sun, Y., Zhang, X., Gao, C., Zheng, H., & Yin, Y. (2012). Disulfide cross-linked nanospheres from sodium alginate derivative for inflammatory bowel disease: Preparation, characterization, and in vitro drug release behaviour. Carbohydrate Polymer, 88, 663– 669. De, S., & Robinson, D. (2003). P olymer relationships during preparation of chitosan–alginate and poly-llysine–alginate nanospheres. Journal of Controlled Release, 89, 101-112. Dong, Z., Ma, Y., Hayat, K., Jia, C., Xia, S., & Zhang, X. (2011). Morphology and release profile of microcapsules encapsulating peppermint oil by complex coacervation. Journal of Food Engineering, 104(3), 455-460.
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Faridi Esfanjani, A., & Jafari, S. M. (2016). Biopolymer nano-particles and natural nano-carriers for nanoencapsulation of phenolic compounds. Colloids and Surfaces B: Biointerfaces, 146, 532-543. Ghayempour, S., & Mortazavi, S M. (2015). Preparation and investigation of sodium alginate nanocapsules by different microemulsification devices. Journal of Applied Polymer, 132(17), 41904. Gonzalez Ferreiro, M., Tillman, L., Hardee, G., & Bodmeier, R. (2002). Characterization of alginate/poly-Llysine particles as antisense oligonucleotide carriers. International Journal of Pharmaceutics, 239, 47-59. Hosseini, A., Jafari, S. M., Mirzaei, H., Asghari, A., & Akhavan, S. (2015). Application of image processing to assess emulsion stability and emulsification properties of Arabic gum. Carbohydrate polymers, 126, 1-8. Javanmardi, J., Stushnoff, C., Locke, E., & Vivanco, J. M. (2003). Antioxidant activity and total phenolic content of Iranian Ocimum accessions. Food Chemistry, 83, 547-550. Katouzian, I., & Jafari, S. M. (2016). Nano-encapsulation as a promising approach for targeted delivery and controlled release of vitamins. Trends in Food Science & Technology, 53, 34-48. Khare, A R., Vasisht, N. (2014). Nanoencapsulation in the food industry: Technology in the future, in: Gaonkar, A. G., Vasisht, N., Khare, A. R., & Sobel, R. (Eds), Microencapsulation in the Food Industry: A Practical Implementation Guide. Elsevier San Diego, pp. 151-155. Koo, S. Y., Cha, K. H., Song, D. G., Chung, D., & Pan, C. H. (2014). Microencapsulation of peppermint oil in an alginate–pectin matrix using a coaxial electrospray system. International Journal of Food Science & Technology, 49(3), 733-739. Langridge, T. D., Tarver, M. J., & Whitten, S. T. (2015). Temperature effects on the hydrodynamic radius of the intrinsically disordered N-terminal region of the p53 protein. Proteins: Structure, Function, and Bioinformatics, 82(4), 668-678. Lertsutthiwong, P., & Rojsitthiska, P. (2011). Chitosan-alginate nanocapsules for encapsulation of turmeric oil. Die Pharmazie, 66(12), 911-915. Liu, X., Ma, Z., Xing, J., & Liu, H. (2004). Preparation and characterization of amino-silane modified superparamagnetic silica nanospheres. Journal of Magnetism and Magnetic Materials, 270, 1-6.
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Mehrnia, M. A., Jafari, S. M., Makhmal-Zadeh, B. S., & Maghsoudlou, Y. (2016). Crocin loaded nanoemulsions: Factors affecting emulsion properties in spontaneous emulsification. International journal of biological macromolecules, 84, 261-267. Mo, Y., Xiao, K., Shen, Y., & Huang, X. (2012). A new perspective on the effect of complexation between calcium and alginate on fouling during nanofiltration. Separation and Purification Technology, 82, 121-127. Motwani, S. K., Chopra, S., Talegaonkar, T., Kohli, K., Ahmad, F J., & Khar, R. K. (2008). Chitosan– sodium alginate nanoparticles as submicroscopic reservoirs for ocular delivery: Formulation, optimization
and
in
vitro
characterization.
European
Journal
of
Pharmaceutics
and
Biopharmaceutics, 68, 513-525. Pistone, S., Qoragllu, D., Smistad, G., & Hiorth, M. (2015). Formulation and preparation of stable crosslinked alginate-zinc nanoparticles in the presence of a monovalent salt. Soft Matter, 11(28), 57655774. Poncelet, D. (2001). Production of alginate beads by emulsification/internal gelation. Annals of the New York Academy of Sciences, 944, 74–82. Rafiee, Z., Jafari, S. M., Alami, M., & Khomeiri, M. (2011). Microwave-assisted extraction of phenolic compounds from olive leaves; a comparison with maceration. Journal of Animal and Plant Sciences, 21(4), 738-745. Rafiee, Z., Jafari, S. M., Alami, M., & Khomeiri, M. (2012). Antioxidant effect of microwave-assisted extracts of olive leaves on sunflower oil. Journal of Agricultural Science and Technology, 14(SUPPL.), 1497-1509. Reis, C. P., Neufeld, R. J., Vilela, S., Ribeiro, A. J., & Veiga, F. (2006). Review and current status of emulsion/dispersion technology using an internal gelation process for the design of alginate particles. Journal of microencapsulation, 23(3), 245-257. Reis C. P, Veiga, F. J., Ribeiro, A. J., Neufeld, R. J., & Damg, C. (2008). Nanoparticulate Biopolymers Deliver Insulin Orally Eliciting Pharmacological Response. Journal of Pharmaceutical Sciences, 97(12), 5290-5305. Rolland, L., Santanach-Carreras, E., Delma, T., Bibette, J., & Bremond, N. (2014). Physicochemical properties of aqueous core hydrogel capsules. Soft Matter, 10(48), 9668-9674. 17
Sarmento, B., Martins, S., Riberio, A., Veiga, F., Neufeld, R., & Ferreira, D. (2006). Development and comparison of different nanoparticulate polyelectrolyte complexes as insulin carriers. International Journal of Peptide Research and Therapeutics, 12(2), 131-138. Vandenberg, G. W., Drolet, C., Scott, S. L., De la & Noue, J. (2001). Factors affecting protein release from alginate-chitosan coacervate microcapsules during production and gastric/intestinal simulation. Journal of Control Release, 77, 297-307. You, J. O., & Peng, C. A. (2005). Calcium‐Alginate Nanoparticles Formed by Reverse Microemulsion as Gene Carriers, Macromolecular Symposia, 219(1), 147-153. Zhao, Y., Wang, C., Chow, A. H. L., Ren, K., Gong, T., Zhang, Z., & Zheng, Y. (2010). Selfnanoemulsifying drug delivery system (SNEDDS) for oral delivery of Zedoary essential oil: Formulation and bioavailability studies. International Journal of Pharmaceutics, 383, 170-177.
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a
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c Fig. 1. Alginate nanospheres of run=27 loaded with peppermint phenolic extract: (a) SEM photomicrograph, (b) TEM photomicrograph, (c) Size distribution graph
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Table 1. Effect of alginate and surfactant content, CaCl2 concentration and oil content on the size and encapsulation yield (EY) of nanoparticles loaded with peppermint phenolic extract
Run 1 2 3 4 5 6a 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23a 24 25 26 27a a
Alginate (%w/v) (X1) 1.00 0.50 0.75 0.50 1.00 0.75 0.50 0.75 0.75 0.50 1.00 0.50 0.75 1.00 1.00 0.50 0.75 1.00 0.50 0.50 1.00 1.00 0.75 1.00 0.75 0.50 0.75
Surfactant (µ l) (X2) 100 100 200 100 100 200 200 100 200 300 300 100 200 200 100 300 300 100 300 100 300 300 200 300 200 300 200
CaCl2 (molarity) (X3) 0.05 0.05 0.10 0.15 0.05 0.10 0.10 0.10 0.15 0.15 0.15 0.15 0.10 0.10 0.15 0.05 0.10 0.15 0.05 0.05 0.05 0.15 0.10 0.05 0.05 0.15 0.10
Central point
1
Oil (mL) (X4 ) 400 400 400 800 800 600 600 600 600 400 400 400 800 600 800 400 600 400 800 800 400 800 600 800 600 800 600
EY (%) 10.2 6.15 8.6 8.8 7.58 6.74 6 7.25 7.45 6.66 10.25 5.26 6.95 7.25 5.8 5.1 7.64 7.62 3.7 5.24 9.61 8.38 7.35 9.19 6.01 4.11 6.92
Mean size (nm) 4533 696 1584 478 4122 1112 412 1974 919 140 923 512 936 2792 2985 361 756 4302 153 608 1257 783 987 945 1425 90 1191
Development of a nutraceutical nano-delivery system through emulsification/internal gelation of alginate
Samira Mokhtari, Seid Mahdi Jafari*, Elham Assadpour Graphical abstract:
Schematic presentation of the internal gelation procedure; (a) shrinkage of alginate sodium droplets; (b) crosslinking with calcium ions.
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Research Highlights: •
Alginate nano-carriers prepared by a low energy internal gelation technique
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Size of nanoparticles decreased at higher oil and surfactant contents
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Encapsulation efficiency was inversely proportional to the size of nanoparticles
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0.5% alginate, 400 mL oil, 0.05 M CaCl2 and 100 mL surfactant was the optimized point
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