Patent Application
20180000745
The present invention relates to chitosan nanoparticles coated with an edible canola protein, and a process for coating and protection of chitosan particles with an edible canola protein. The coated chitosan particles can be used for encapsulation and delivery of bioactive material and drugs.
Functional foods and nutraceuticals have potential to improve human health and wellness; however the low bioavailability of many bioactive food compounds makes this potential health benefit difficult to achieve. Encapsulation of bioactive compounds is recognized as an effective approach to improve bioavailability by providing protection against food processing and digestion in the gastrointestinal (GI) tract, and improving their solubility and/or permeability in the GI tract. Chitosan has been explored as a wall material for preparing particles due to its unique mucoadhesive and permeation enhancing properties. However, chitosan is degraded at low pH. To overcome this hurdle, chitosan particles are often coated with synthetic co-polymers such as Eudragit™, which is commercially available in many different forms. However, in food uses, natural polymers such as food proteins, are more desirable compared to the synthetic ones.
In one aspect, the invention comprises a nanoparticle comprising cruciferin, chitosan and a bioactive material. The bioactive material may be a water soluble or insoluble compound or a probiotic, and may be incorporated into the nanoparticle during formation of the nanoparticle.
In another aspect, the invention may comprise a method of forming a nanoparticle for delivery of a bioactive material, comprising the steps of:
Preferably, the pH of the combined solution is adjusted to between about 5.5 to about 6.5, and the ratio of cruciferin to chitosan is in the range of about 3:2 to about 5:2 by weight.
The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.
In one embodiment, the invention comprises the use of an edible canola protein as a functional coating to produce a nanoparticle delivery system. Most food proteins form heat-induced aggregations at temperatures above about 70° C., in which different compounds can be entrapped and slowly be released. Since heat-set hydrogels may not be suitable for encapsulation of heat-sensitive compounds, an alternative method, cold gelation, in which proteins form a gel using multivalent ions or in combination with other polymers, may be applied. In one specific embodiment, cruciferin is used to coat chitosan particles, resulting in particles with improved stability at low pH.
As used herein, a nanoparticle is an agglomeration comprising cruciferin and chitosan molecules, having at least one dimension less than about 1000 nm, and preferably less than about 500 nm. Preferably, the nanoparticle is roughly spherical, with cruciferin forming a complete or partial shell around a core of chitosan. A bioactive material may be incorporated into the nanoparticle, and may be associated with the chitosan, wholly or partially encapsulated by cruciferin. A bioactive material may be any compound or combination of compounds which is intended to be consumed by a patient or subject and which may have a therapeutic or biological effect. A bioactive material may include probiotic bacteria or viruses. The bioactive compound may be a pharmaceutical or nutraceutical compound.
Cruciferin is the major canola protein (11S or 12S globulin) accounting for ˜65% of total canola proteins. The meal remaining after oil extraction contains 35-40% proteins, and canola proteins are considered potential food proteins as they contain a well-balanced amino acid composition, high contents of lysine (6.0%) and sulfur-containing amino acids (3 to 4%), and a high protein efficiency ratio. Furthermore, canola proteins have been shown to exhibit emulsifying and gelling properties, and may be broken down into bioactive peptides. Cruciferin, with an isoelectric point (IEP) of around 7.2 and a molecular weight of about 300 kDa, is composed of six subunits and is resistant to gastric digestion. As used herein, cruciferin may comprise all isoforms and variants thereof, including recombinant variants, which form nanoparticles with chitosan in a cold-gelation type method.
Chitosan is a water-soluble, linear polysaccharide derived from chitin of fungi cell walls or exoskeleton of crabs and shrimp, comprising a copolymer of acetylated and deacetylated glucosamine. Chitosan may be produced by the deacetylation of chitin, where the degree of deacetylation may vary from about 50% to about 100%. Chitosan may have an average molecular weight (number or weight average) in the range of about 3800 to about 20,000 Daltons. As used herein, chitosan includes any of its derivatives or variants which form nanoparticles with cruciferin in a cold-gelation type method.
In one preferred embodiment, cruciferin-coated chitosan nanoparticles are prepared. Such particles may have an average size of 50 to 500 nm, and preferably in the range of about 100 to 200 nm. The nanoparticles may be prepared in solution at a pH of between about 4.5 to about 6.5, preferably about 5.5, and a cruciferin:chitosan ratio of between about 3:2 to about 5:2 (by weight). The cruciferin shell surrounds the chitosan core in the particles. The particles may encapsulate a bioactive material. The cruciferin-coated chitosan nanoparticles particles may protect the bioactive material against pH and enzymatic degradation in the stomach and intestine, thus, they may serve as carriers for compounds sensitive to stomach and intestinal conditions.
In addition to the controlled release of the compounds in the intestine, the nanoparticles may also be absorbed directly by cells, thereby improving bioavailability due to the mucoadhesive and paracellular permeation enhancing properties of chitosan. Moreover, in another embodiment, the nanoparticles might also act as colon targeting delivery systems since chitosan is degraded in the colon and release the encapsulated bioactive material in the colon. Since cruciferin is a GRAS natural polymer, bioactive-loaded particles might be used for food fortification; this is the advantage of the particles compared to synthetic polymers-coated chitosan particles.
Exemplary embodiments of the present invention are described in the following Examples, which are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter. As will be apparent to those skilled in the art, various modifications, adaptations and variations of the specific disclosure herein can be made without departing from the scope of the invention claimed herein.
Commercial canola meal, obtained from Richardson Oilseed Company (Lethbridge, AB, Canada) was ground, passed through a 35-mesh screen and stored at −20° C. for further use. Caco-2 cells (HTB37) were obtained at passage 19 from the American type culture collection (Manassas, Va.). Dulbecco's modified eagle medium (DMEM), 0.25% (w/v) trypsin-0.53 mM EDTA, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), fetal bovine serum, 1% nonessential amino acids, and 1% antibiotics were all procured from Gibco Invitrogen (Burlington, ON, Canada). (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT), dimethyl sulfoxide (DMSO), coomassie brilliant blue G-250, β-carotene, chitosan (molecular weight of 140-220 kDa and degree of acetylation ≦40%), urea, sodium dodecyl sulfate (SDS), dithiothreitol (DTT), pepsin, pancreatin, Coumarin 6 and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma (Oakville, ON, Canada).
Cruciferin was extracted using the method previously developed and described (Akbari et al. 2015) A slurry of canola meal:water (1:10, W:V) was acidified to pH 4, stirred for 2 h and centrifuged at 10,000 rpm for 20 min at 4° C. The resultant pellet was mixed with 10× volume of water, and the pH was adjusted to 12 while stirring at room temperature for 1 h. After centrifugation, the supernatant was adjusted to pH 4 to precipitate proteins. The collected precipitate was washed twice with acidified water (pH 4), centrifuged and freeze-dried as cruciferin isolate.
To prepare the particles, cruciferin and chitosan were solubilized at different concentrations at alkaline (pH 12) and acidic (pH 3) conditions, respectively, and then the cruciferin solution was dropwise added to the chitosan solution to achieve a pH ˜4.5. The pH of the mixture was adjusted to different pHs and the formed particles suspensions were studied. 18 mL of cruciferin solution in water at different protein concentrations (2, 3, 5, 7.5, and 10 mg/mL) and pH 12 was dropwise added to 18 mL chitosan solution (2 mg/mL in 0.1 M acetic acid) containing 0.04% w/v sodium azide while stirring. The pH of the suspensions was adjusted to 3.5, 4.5, 5.5, 6.5, and 7.5, and the suspensions were stirred for 1 h. The stability and turbidity of the particle suspensions were used to assess conditions for particles formation. The stability of the particles was studied for a period of 3 weeks stored at 4° C. The turbidity was measured at 600 nm using a UV/VIS spectrophotometer (V-530 Jasco, Japan).
Since the formation of particles would increase the turbidity of the suspensions, an indicator of 50% increase in the turbidity compared to initial protein solution was used to determine an appropriate particles formation. Our results showed that the mixtures did not form particles at pHs 3.5 and 4.5 (low turbidity) likely due to high repulsive forces between positively-charged cruciferin and chitosan, formed at pHs 5.5 and 6.5 at certain ratios of cruciferin:chitosan, and precipitated at pH 7.5. At pHs 5.5 and 6.5, in general, the appearance of the particles suspensions changed from clear to opaque at increasing cruciferin concentrations. Whereas the mixtures did not form highly-turbid suspension at the ratio of 2:2 (by weight) and the particles prepared at the ratios of 7.5:2 and 10:2 were not stable and precipitated; the particles prepared at the ratios of 3:2 and 5:2 were stable and therefore used for further studies.
Size of the particles was determined by dynamic light scattering using Malvern Nanosizer ZS (Malvern, Worcestershire, UK). Zeta potential of the particles was also measured by laser doppler velocimetry using the Nanosizer. Prior to measurement, samples were diluted in 10 mM phosphate buffers (the same pH of particle suspensions) to obtain a slight opalescent dispersion to prevent multiple scattering effects. Morphology of the prepared particles was studied using a transmission electron microscopy (TEM, Philips Morgagni 268, FEI Company, The Netherlands) at 80 kV. The particles were freshly prepared at the cruciferin:chitosan ratio of 5:2 and pH 5.5, diluted 10 times, put on Formvar-covered copper grids, negatively stained with 2% phosphotungstic acid for 20 S, and then air dried.
The zeta potential and size of chitosan and cruciferin before forming the particles were ˜+43 mV and 25 nm, and −41 mV and 105 nm at pH 3 and 12, respectively. The surface charge of cruciferin-coated chitosan nanoparticles formed at pH 5.5 with 165 nm size was ˜+18 mV. In general, the particle size increased at increasing pHs from 5.5 to 6.5, likely due to decreased protonation of chitosan (pKa˜6.4) and cruciferin (IEP ˜7.2) (Table 1), which would decrease repulsion forces between chitosan and cruciferin, leading to particle aggregation and even precipitation at increasing cruciferin and chitosan ratio above 5:2 (data not shown). The zeta potential of particles was not affected. Similar trend was reported for chitosan/tripolyphosphate and chitosan/hydroxypropyl methylcellulose phthalate by Makhlof et al, (2011).3 Polydispersity index (PDI) of the particles sizes was ˜0.35, revealing satisfactory particles uniformity. Therefore, the particles prepared at the cruciferin:chitosan ratios of 3:2 and 5:2 at pH 5.5 were appropriate, and the ratio of 5:2 was chosen to prepare particles for further studies. Morphology of cruciferin and chitosan solutions and the particles were examined using TEM, No specific structure was observed in the cruciferin and chitosan solution (
The surface hydrophobicity of cruciferin and the particles was determined using ANS fluorescent probe. Briefly, the samples were diluted to ten concentrations ranging from 0.0025 to 0.1 mg/mL using 0.1 M citrate buffer at pH 5. Then, 20 μL of 8 mM ANS solution prepared in the same buffer was added to 4 mL of the diluted samples and the fluorescence intensity was measured at emission and excitation wavelengths of 470 nm and 390 nm, respectively, at a slit width of 5 nm using a 1-cm path length quartz cell on Shimadzu RF-5301PC spectrofluorophotometer (Kyoto, Japan). The buffer containing ANS and diluted samples (in the absence of ANS), as blanks, was subtracted from the measured fluorescence values. The slope of the linear plot of net fluorescence values versus protein concentrations was used as an index of the protein surface hydrophobicity.
The surface hydrophobicity (S0) of cruciferin was not affected before and after particle formation (8613±3 and 8421±34, respectively), which indicated that the particle formation process didn't affect protein folding/unfolding and/or the availability of hydrophobic groups on the protein surface, Unlike Boeris et al. (2011) who reported chitosan decreased the S0 of the pepsin/chitosan complex due to blocking the surface hydrophobic groups; the unchanged S0 during particle formation suggested that either S0 of cruciferin in the coating layer was not affected by chitosan, or the coating layer was just composed of cruciferin.
The intrinsic fluorescence emissions of cruciferin and the particles were measured using a Shimadzu RF-5301PC spectrofluorophotometer (Kyoto, Japan) equipped with a 1-cm path length quartz cell. The protein concentration and pH of the samples were adjusted to 0.005 mg/mL and pH 5.5 using 10 mM citrate buffer. The fluorescence measurements were performed using the excitation wavelength of 295 nm and the emission wavelengths of 300 to 650 nm, both at a slit width of 5 nm. The interactions between encapsulated β-carotene and the particles were also studied using the intrinsic fluorescence property. In brief, different concentrations of β-carotene, soluble in ethanol, were encapsulated in the particles as will be explained in 2.7.2 section. The intrinsic fluorescence of the β-carotene-loaded particles, at final β-carotene concentrations of 0.002-0.06 mg/mL, was also measured using 295 nm excitation wavelength. The emission spectra were recorded from of 300 to 650 nm wavelength.
The intrinsic fluorescence of a protein strongly depends on the conformation that the protein adopts in response to bulk solution conditions. Intrinsic fluorescence property of a protein is due to the presence of aromatic amino acids such as tryptophan and tyrosine. The intensity of intrinsic fluorescence property of the particles at 594 nm was lower than that of cruciferin (
The importance of hydrophobic, electrostatic, hydrogen and disulfide interactions in forming the particles were evaluated using SDS, NaCl, urea and DTT, respectively. The particles suspension was mixed with the same volume of each dissociating agent at different concentrations. The mixtures were adjusted to pH 5.5 and vortexed 10 s and after 6 h, their turbidity were measured at 600 nm using the UV/VIS spectrophotometer. The turbidity of the mixtures was compared to that of initial particles suspension. Decrease in the turbidity of the mixtures was considered as an indicator of the degree of particles dissociation.
As described above, adding alkaline cruciferin solution (negatively-charged) into acidic chitosan solution (positively-charged) led to a pH value of ˜4.5, which was lower than IEP of cruciferin (˜7.2) and pKa of chitosan (˜6.4); therefore, both cruciferin and chitosan polymers were positively-charged which would prevent the particle formation due to the presence of repulsive forces. At increasing pHs to 5.5-6, the decreased protonation of the polymers also decreased their repulsive forces, enabling them to come closer to form different interactions.
The formation of particles could be affected by a number of interactions such as electrostatic, disulfide, hydrophobic and hydrogen bonding. To understand the responsible force for the particle formation, effects of dissociating agents, namely SDS, DTT, urea and NaCl, on the suspensions turbidity were determined. The suspension turbidity was decreased by 89 and 14% in the presence of urea and NaCl, respectively, but was not affected by DTT (
Freeze-dried samples were dried in a vacuum desiccator using phosphorous pentoxide for 48 h. The dried samples (˜5 mg) were weighed in hermetically sealed aluminium pans, sealed and loaded into a differential scanning calorimeter (Q2000-DSC, TA Instruments, New Castle, Del., USA). The samples were heated at a scanning rate of 10° C./min from 15 to 300° C. under inert nitrogen atmosphere. An empty aluminium pan was used as a reference.
The denaturation temperature of cruciferin powder was recorded at 174° C. (
Freeze-dried samples were dried in the vacuum desiccator before FTIR analysis. The milled samples in KBr (˜10 mg) were loaded on a Thermo Nicolet 8700 FTIR spectrometer (Madison, Wis., USA). A background spectrum in air was obtained before running the samples. The infrared spectra were collected from the wavenumber of 400 to 4000 cm−1 at a resolution of 4 cm−1. Each sample was subjected to 64 repeated scans. The spectra were smoothed and standardized, and their baselines were calibrated using Omnic 8.1 software (Madison, Wis., USA). The recorded spectra were also deconvoluted in amide I band region (1700-1600 cm−1) using the software at a bandwidth of 25 cm−1 and an enhancement factor of 2.5. The detected amide I bands in the spectra were assigned to protein secondary structures using previously established wavenumber ranges.
In FTIR spectra, amide I (1600-1700 cm−1) and amide II (1480-1545 cm−1), corresponding respectively to C═O stretching and N—H bending, provide useful information about structural changes in polymers. In comparison with amide I, amide II has much less sensitivity to conformational changes but it is more sensitive to hydrogen-bonding changes in the environment of N—H groups. In our study, while amide I band in the spectra of cruciferin, chitosan and the particles shifted in a small range of 1654-1656 cm−1, amide II peak shifted from 1537 cm−1 wavenumber in cruciferin spectrum to 1558 cm−1 in the particles (
Toxicity of the prepared particles on Caco-2 cells was assessed using MTT method developed by Mosmann (1983) with some modifications.23 The MTT test measures the activity of dehydrogenase enzyme, which can convert MTT to insoluble purple formazan in Caco-2 cells. The formazan is dissolved in DMSO and the intensity of formed purple color determines the enzyme activity and cell viability. Caco-2 cells were seeded (50,000 cell/mL) in high-glucose and L-glutamine DMEM medium supplemented with 10% FBS, 1% non-essential amino acids, 1% penicillin and streptomycin, and 2.5% HEPS in a 96-well plate (200 μl/cell) and incubated at 37° C. in a humidified incubator with 5% CO2 for 24 h. After incubation, the media in the cells was replaced with freshly prepared particles suspended in the DMEM medium at different concentrations of 0.5, 1.5, and 2.5 mg/mL. DMEM without nanoparticles was used as control. After 24 h incubation, the wells were gently washed three times with PBS, and then 15 μl MTT (5 mg/mL in PBS) and 185 μl medium were added to the cells and incubated for 4 h. Medium was removed and the formed formazan crystals were dissolved in 150 μl DMSO. Absorbance was measured at 570 nm using a microplate reader (GENios, Tecan, Mannedorf, Switzerland). Relative cell viability (%) was calculated by comparing the absorbance of the particle samples with that of the control.
Incubation of the particles at three concentrations of 0.5, 1.5, and 2.5 mg/mL for 24 h didn't show any significant change in the cellular viability compared to the control (
To study the cell uptake of nanoparticles in Caco-2 cells, coumarin-6, as a fluorescent marker, was encapsulated into the nanoparticles. The cellular uptake of coumarin-6-loaded nanoparticles was compared with free coumarin-6 (not encapsulated). In brief, 1 mL of coumarin-6 solution in ethanol at concentrations ranging from 10 to 400 μg/mL was mixed with 9 mL 2 mg/mL chitosan solution in 0.1 M acetic acid. 9 mL of 5 mg/mL cruciferin solution (solubilized at pH 12) was added dropwise to the solution while stirring. The pH of the suspension was adjusted to 5.5, stirred for 2 h and then centrifuged at 40000 g and 4° C. for 30 min, Loaded particles pellet was collected, washed twice with water and centrifuged to remove free coumarin-6. The labelled particles were re-suspended in DMEM medium at concentrations of 0.1, 0.5 and 1 mg/mL. The size and zeta potential of the re-suspended particles were measured. Caco-2 cells (105 cells/well) were seeded on cover glasses placed in 6-well plates (Costar, Corning, N.Y.), and incubated until cells were about 80% confluent. 2.5 mL of labelled-particles suspensions or free coumarin-6 solution in DMEM medium was added to each well and incubated for 6 h at 37° C. Then, the cells were washed three times with PBS to remove free nanoparticles and coumarin-6. Cell membranes were stained with 10 μg/mL Alexa Fluor 594-Concanavalin A conjugate in PBS for 10 min. After three washings with PBS, the cells were fixed using ice-cold solution of 3.7% formaldehyde for 30 min, Cell nuclei were also stained with 0.3 μg/mL DAPI for 10 min and three washings with PBS. The coverslips covered with fixed cells were removed from the wells, inverted on slides containing 40 μL (90% glycerol in PBS), then dried overnight in dark at room temperature, and kept at 4° C. The cells were observed with a CLSM 510 Meta confocal laser scanning microscope (Carl Zeiss Microscopy, Jena, Germany) using an oil immersion objective (63×) at wavelengths of 405, 561 and 488 nm to visualize cell nuclei, membranes and labelled particles, respectively. Images were processed with ZEN 2011 LE software (Carl Zeiss, AG, Oberkochen, Germany).
Confocal microscopy images of Caco-2 cells after 6 h incubation with coumarin-6-loaded particles and free coumarin-6 were shown in
Brilliant blue-loaded particles were prepared by adding dropwise 18 mL of cruciferin solution (pH 12) to 18 mL chitosan solution (2 mg/mL in 0.1 M acetic acid) containing brilliant blue (1 mg/mL) and 0.04% (w/v) sodium azide, and then the pH was adjusted to 5.5. After stirring for 30 min, the particle suspension was centrifuged at 40000 g, 4° C. for 30 min, and the particle pellet was collected, washed twice with water (the same pH as suspension), and centrifuged to remove the adsorbed brilliant blue on the particle surface. The content of free brilliant blue in the combined supernatants was measured using the spectrophotometer at 590 nm. Encapsulation efficiency (EE), loading capacity (LC), and particle preparation yield (PPY) were calculated using the following equations:
EE (%)=100×(A−B)/A
LC (%)=100×(A−B)/C
PPY (%)=100×C/D
Encapsulating β-carotene in particles was performed by slowly adding 2 mL β-carotene ethanol solution (0.5 mg/mL) to 18 mL chitosan (2 mg/mL in 0.1 M acetic acid), and then 18 mL of cruciferin solution (solubilized at pH 12) was added dropwise, the pH of the suspension was adjusted to 5.5, stirred for 30 min, and then concentrated to 30 mL using a rotary vacuum evaporator (Heidolph 2-Collegiate, Germany) at 50° C. 1 mL of the concentrated suspension containing the loaded particles was mixed with 5 mL hexane, vortexed for 10 sec and then centrifuged at 10,000 rpm for 20 min at 4° C. The β-carotene extracted in the organic phase represented both unencapsulated and loosely-adsorbed β-carotene on the surface of particles. Absorbance of the organic phases was measured at 450 nm using the UV-VIS spectrometer and EE, LC and PPY were calculated.
Release experiments were performed in simulated gastric fluid (SGF) followed by simulated intestinal fluid (SIF). Brilliant blue- and β-carotene-loaded particles were prepared using the previously described method. To simulate gastric condition, 20 mL of above suspensions was adjusted to pH 1.4 using 10 mL of 0.05 M HCl containing 1 mg/mL pepsin and was stirred at 100 rpm and 37° C. in a water bath. Release studies were also performed at pH 1.4 and in the absence of pepsin. 0.5 mL samples were withdrawn from the release media at predetermined intervals. 0.25 mL of 1 M sodium bicarbonate was added to the samples to increase pH to 7.4 and inactivate pepsin. Brilliant blue-containing samples were centrifuged (40000 g) and released brilliant blue in supernatant was measured. β-carotene was extracted from the withdrawn samples using 2.5 mL hexane and quantified using the method previously described. After 2 h incubation in SGF, pepsin was inactivated by raising pH to 7.4 using 10 mL of 1 M sodium bicarbonate and release studies were followed in SIF medium by adding 5 ml of 200 mM phosphate buffer pH 7.4 in the absence and presence of 2 mg/mL pancreatin. 0.5 mL samples were withdrawn at predetermined intervals. To stop pancreatin activity after sampling, brilliant blue-containing samples were heated at 90° C. for 5 min while for β-carotene samples, free β-carotene was immediately extracted using hexane. The concentrations of released brilliant blue and β-carotene in SGF and following SIF media were measured and their cumulative percentages were calculated based on the total amount of the initial loaded compounds and the released compounds in recovery studies.
Encapsulation property and release behaviour, two important factors affecting the efficiency of a delivery system, were studied for the particles. Brilliant blue and β-carotene, representing hydrophilic and hydrophobic model compounds, were encapsulated in the particles. While the encapsulation efficiency (EE), loading capacity (LC) and particle preparation yield (PPY) of brilliant blue were 72.4%±0.7, 16.3%±0.8 and 55.5%±2.6, those for β-carotene were 98.8%±0.2, 7.7%±0.2 and 44.5%±1.1, respectively. Previously the EE of β-carotene was reported to be 39.5%±0.3 in chitosan-β-lactoglobulin nanoparticles, 54.7% and 87.6%, respectively, in chitosan-alginate beads and sodium caseinate sub-micelles. The higher EE of the particles suggested that the particles are appropriate carriers for both water-soluble and -insoluble compounds.
In vitro release of brilliant blue and β-carotene from the particles was studied using 2 h incubation in SGF followed by 6 h in SIF with and without pepsin and pancreatin, respectively (
In SIF (pH 7.4) and in the absence of pancreatin, the particles stayed resistant and only less than 5-10% of encapsulated brilliant blue and B-carotene were released (
However, in the presence of pancreatin, the previously released brilliant blue (during in SGF) was re-adsorbed by the particles and precipitated along with particles after centrifugation. The reason might be due to the degradation of the cruciferin layer by pancreatin which released positively-charged amine groups of chitosan and then bound to the previously released negatively-charged brilliant blue. Similar results were reported by Chen and Subirade (2005).
However, unlike brilliant blue-loaded particles, degradation of the cruciferin layer by pancreatin released ˜15% of β-carotene in SIF. The released β-carotene from enzymatic-hydrolyzed particles might be related to the β-carotene which was entrapped in the cruciferin coating layer. Afterward, the remaining encapsulated β-carotene, entrapped in the chitosan-based core, was not released since chitosan was resistant to the pH and enzymes in SIP. Similar results were reported in a chitosan dispersed system coated with aminoalkyl methacrylate copolymer RS (Eudragit RS) and lactose-sodium alginate-chitosan composite particles. Therefore, the particles are promising carriers for delivery of sensitive compounds to both gastric and intestinal conditions such as protein-based bioactive compounds.
The encapsulated β-carotene might affect the thermal property and structure of the particles. In the study of the DSC thermograms, pure crystalline β-carotene showed two melting peaks at 168 and 176° C. (
At the temperature range of 15-160° C. (prior to the melting points), endothermic water evaporation peaks were presented at 146° C. and 140° C. for empty and β-carotene-loaded particles, respectively (
To study the effect of the encapsulation on the structure of the particles, the FTIR spectra of β-carotene, particles and β-carotene-loaded particles were compared in
Binding of β-carotene to the particles can also be characterized by determination of intrinsic fluorescence of tryptophan residues in cruciferin structure. The intensity of particles was initially decreased after encapsulation of β-carotene (
All Experiments were carried out in triplicates and results were statistically analyzed using ANOVA and Duncan tests (version 9.2, SAS Institute Inc., Cary, N.C., USA).
The description of the present invention has been presented for purposes of illustration and description, but it is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. Embodiments were chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.
References in the specification to “one embodiment”, “an embodiment”, etc., indicate that the embodiment described may include a particular aspect, feature, structure, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, or characteristic with other embodiments, whether or not explicitly described. In other words, any element or feature may be combined with any other element or feature in different embodiments, unless there is an obvious or inherent incompatibility between the two, or it is specifically excluded.
It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as “solely,” “only,” and the like, in connection with the recitation of claim elements or use of a “negative” limitation. The terms “preferably,” “preferred,” “prefer,” “optionally,” “may,” and similar terms are used to indicate that an item, condition or step being referred to is an optional (not required) feature of the invention.
The singular forms “a,” “an,” and “the” include the plural reference unless the context clearly dictates otherwise. The term “and/or” means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrase “one or more” is readily understood by one of skill in the art, particularly when read in context of its usage.
As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percents or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as “up to”, “at least”, “greater than”, “less than”, “more than”, “or more”, and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into sub-ranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio.
The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% of the value specified. For example, “about 50” percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term “about” can include one or two integers greater than and/or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term “about” is intended to include values and ranges proximate to the recited range that are equivalent in terms of the functionality of the composition, or the embodiment.
The following references are incorporated herein by reference (where permitted) as if reproduced in their entirety. All references are indicative of the level of skill of those skilled in the art to which this invention pertains.
