Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Nanoparticle Encapsulation Strategy: Leveraging Plant Exine Capsules Used as Secondary Capping for Oral Delivery Di Wu,†,§ Xinyi Wang,†,§ Shishuai Wang,‡ Bin Li,†,§,∥ and Hongshan Liang*,†,§ †
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China College of Culinary and Food Engineering, Wuhan Business University, Wuhan 430056, China § Key Laboratory of Environment Correlative Dietology, Huazhong Agricultural University, Ministry of Education, Wuhan, China ∥ Functional Food Engineering & Technology Research Center of Hubei Province, Wuhan, China Downloaded via NOTTINGHAM TRENT UNIV on July 17, 2019 at 12:10:50 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
‡
ABSTRACT: Protein-based nanoparticles (NPs) with favorable properties including enhanced absorptivity and low toxicity still suffer a major challenge for rapid nutraceutical or drug release after oral administration. Hence, we introduced a secondary encapsulation for unstable factor to attain a controlled-release effect in a gastrointestinal environment. In this work, assembled nanoparticles engineered by nobiletin (NOB), zein, and tannin acid (TA) were first reported for drug delivery systems. The TA added was capable of obtaining further assembly to stabilize nobiletin in comparison with NOB-loaded zein NPs only. Sunflower pollens (SPGs) were selected as carriers for further oral delivery, while zein was chosen as a coating material for capping SPGs absolutely. As a result, the NOB/zein/TA NPs (NZT NPs) obtained had a stable size of 100 nm after 48 h. Besides, they could improve the chemical stability of NOB for at least 120 days at 4 °C compared with zein NPs (ZT NPs). Owing to the secondary capping by SPGs, the final system was able to release selectively via an oral route, that is, achieving no release in a gastric environment and slow release in an intestine environment. Generally, our research proposed a secondary protection model to prevent drug-loaded NPs from resolving after oral administration, which provided a new perspective for nutraceutical or drug encapsulation and controlled-release delivery. KEYWORDS: sunflower pollen grains, nanoparticles, nobiletin, secondary capping, oral delivery system sporopollenin biopolymer.12 The sporopollenin-based extine not only has the capability of protecting sensitive drugs from degradation under harsh conditions (gastric fluid, for example) but also is able to interact with intestinal cells directly for enhanced absorption.13,14 Although SPGs have such an expectant nature, each spike on the SPG surfaces covered with pores for the uptake of macromolecules, promoting the hydration process of pollens,15,16 resulted in the encapsulated payload leaking easily. Previous studies had shown that zein is always applied as a coating film to foods.17−19 Herein, we regarded zein film on the surface of SPGs as a secondary capping to further protect NPs from degrading in the stomach directly. In the present work, we fabricated NZT NPs by the synergism of oxidation reaction among TA molecules and hydrogen bonding between zein and TA. The obtained NPs with high loading capacity and long-term stability were stable after 48 h compared to NOB/zein NPs (NZ NPs). Subsequently, we selected SPGs as a novel delivery carrier and zein as a coating material for secondary capping to protect NPs from resolving in the stomach and effective release in the intestine (Figure 1). Consequently, the secondary encapsulation based on zein-capped SPGs for NZT NPs could achieve the purpose of controlled release in a gastrointestinal
1. INTRODUCTION NOB, a citrus polymethoxylated flavone, possesses massive health benefits including antioxidant activity,1 inhibition of tumor angiogenesis,2 antiinflammator activity,3 and so on. Nevertheless, NOB displays poor water solubility and chemical instability because of methylated groups, resulting in low oral bioavailability and restrictions on practical uses of NOB.4 Recently, self-assembled nanoparticles (NPs) based on protein have attracted widespread interest in improving the chemical stability and bioavailability as potential nutraceutical or drug delivery vehicles. Zein, an alcohol-soluble protein from corn, has been extensively investigated in drug delivery systems due to its capability of self-assembly to form NPs and intrinsic favorable biocompatibility and biodegradability.5−7 Although protein-based NPs own such favorable properties including enhanced absorptivity and low toxicity,8,9 they are also presenting a major challenge for low loading capacity and rapid drug release via oral administration. In our previous study, zein NPs (Z NPs) could provide limited loading for the unsoluble bioactive.10 However, synthesizing self-polymers surrounding NZ NPs by oxidative reaction of TA resulted in great improvement in the loading efficiency of the payload. During the oral delivery system, carriers must be able to protect drugs from degradation in a gastric environment and persist in the intestines long enough to ensure drugs adhere to the cell surface and be transcytosed by intestinal cells.11 Sunflower pollens (SPGs), recognized as safe natural resources, have attracted considerable attention for their large enough inner cavity and sturdy extine based on © XXXX American Chemical Society
Received: Revised: Accepted: Published: A
March 31, 2019 June 2, 2019 July 3, 2019 July 3, 2019 DOI: 10.1021/acs.jafc.9b02003 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 1. Schematic diagram of nanoparticle fabrication, secondary capping, and in vitro release. instrument (Jasco Inc., Easton, MO). All samples were freeze-dried and then compressed with KBr to form pellets. The samples above were measured against a blank KBr pellet as the background. The freeze-drying samples were tested by a DSC (NETZSCH Instruments, Bavaria, Germany). Briefly, the samples were heated in the range 20−200 °C with a heating rate of 5 °C/min and a nitrogen gas flow rate of 60 mL/min. All results were analyzed further by NETZSCH software.20 The XRD curves were obtained by a XRD diffractometer (D8-Advance, Bruker, USA) with pattern radiation of Cu Kα and a weighted average λ of 0.15406 nm. The freeze-drying samples were tested from 5 to 60° with a speed of 10°/min under 40 kV and 40 mA. 2.4. Secondary Capping for Oral Administration. 2.4.1. NP Encapsulation. The prepared NZT48 NPs (2 mL) were encapsulated into 5 mg SPGs by ultrasonic technology for 30 min under 40 kHz and 90% power with a temperature of 25 °C (SCIENTZ, China). The NZT48 NP-loaded SPGs (NZTS) were obtained by centrifugation of 14000 rpm for 90 s, and the bottom was collected. 2.4.2. Zein Coated NZTS Formulations. Portions of 1, 3, and 5 mL of zein (75% ethanol solution) were added into the abovementioned NZT48 NP-loaded SPGs and vacuum overnight for drying. The final NZTS was stored in a desiccator for further characterization. 2.5. In Vitro Release Analysis. For in vitro release, the above NZTS was performed in HCl (0.1 M, 2% Tween-80, 0.5% pepsin) for simulated gastric fluid (SGF) and PBS with 2% Tween-80 and 1% pancreatin contained for simulated intestinal fluid (SIF). Briefly, according to our previous study, the samples were put into SGF for 2 h and then transferred in SIF for further release at 37 °C with a shaking of 100 rpm. On each released time we set, 1 mL of released solution was taken out and 1 mL of fresh buffer media was taken in. The released amount of NOB in the release medium was tested by HPLC (LC-2010C, Shimadzu, Japan).21
environment after oral administration. In short, our research put forward a secondary capping model of encapsulation and protected the unstable factor with poor oral availability.
2. MATERIALS AND METHODS 2.1. Materials. Zein was obtained from Tokyo Chemistry Industry, Co., Ltd. (Tokyo, Japan). Nobiletin was procured from Shanxi Huike Botanical Development Co., Ltd. (Xian, China). Natural sunflower pollen grains were purchased from Carl Science Laboratory (China). Coumarin-6 was procured from Invitrogen (USA). Tannin acid was obtained from Aladdin Chemistry Co., Ltd. Nile Blue was procured from Invitrogen (USA). All other chemicals in our experiment were of analytical grade from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All aqueous solutions in the experiments were prepared using a Milli-Q water purification system (Millipore, Milford, MA, USA). 2.2. Preparation of NZT NPs. Zein and NOB were dissolved in 75% aqueous ethanol solutions to acquire a stock solution with 10 mg/mL of zein and 12 mg/mL of NOB. Then, 0.5 mL of the above solution was added into 9.5 mL of MOPS (0.01 M, pH 8.0) under vigorous stirring to obtain NOB-loaded zein NPs (NZ NPs). Then, 100 μL of TA solution (40 mg/mL) was added rapidly into this dispersion. The obtained NPs of NOB, zein, and TA (NZT NPs) were placed into a water bath for another 48 h to acquire steady NPs, termed NZT48 NPs. The product was then purified by successive dialysis (MWCO 3500) against deionized water for 48 h to remove the free TA. 2.3. Characterization of NZT48 NPs. The size, zeta potential, and PDI of NPs were tested by a laser light scattering instrument (Nano-ZS90, Malvern, U.K.). From the experiment, each sample was fresh solution and was tested with 30 runs. All results were performed at 25 °C with an average of three times. UV−vis spectroscopy was measured by a UV−vis spectrophotometer (UV1100, MAPDA) under 329 nm using fresh solution. XPS observations were determined using an axis ultra DLD instrument (Kratos, U.K.) with frozen-dried powders. Morphological characterization of the formulated NPs was carried out by TEM (JEM-2100F, Japan). The fresh solution was dipped on copper grids coated with carbon and dried under ambient conditions. The experiment was conducted at diverse magnifications with an accelerating voltage of 100 kV. FT-IR spectra were carried out by a Jasco 4100 series
3. RESULTS AND DISCUSSION 3.1. Characterization of NZT NPs. NOB-encapsulated self-assembly NPs were fabricated in this study on the basis of zein and TA to establish a high loading capacity and longterm stability delivery system. The schematic of fabricating NPs was illustrated in Figure 1. Briefly, NOB and zein were B
DOI: 10.1021/acs.jafc.9b02003 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 2. (a) Z-Average, zeta potential, and PDI of NZT NPs within 14 days. (b) Turbidity and photograph of NZT NPs within 14 days. (c) Size distribution of NZT NPs after 48 h and 120 days.
first added into the buffer solution simultaneously and formed aggregates by self-assembly. TA was then adsorbed around the aggregates rapidly. After a 48 h oxidation of TA, the stable NPs with yellowish color were obtained for further use. Figure 2a exhibited the size, zeta potential, and PDI of NZT NPs in a period of 14 days. Obviously, NOB, zein, and TA could form NPs immediately with a size of 200 nm. However, the size was decreased to 100 nm and kept stable after 48 h. The zeta potential of NPs had a similar downward trend from −50 to −35 mV. The possible reason might be that there would take a certain amount of time to stabilize the system and make individual components harmonious subsequently, which would be described in more detail in the following characterization. Moreover, the PDI of NZT48 NPs was within 0.3 and the diameter distributions (data not shown) of NPs exhibited a single peak distribution, indicating these samples might have uniform particle sizes. Figure 2b showed the turbidity of NZT NPs from 0 to 14 days. As shown, the NP solution tended to be markedly clear and transparent after 48 h, which is in accord with the results in Figure 2a. On the basis of a previous study, the color of the solution changed from milky white to brown because of the oxidation of TA, making hydroxyl convert to colored quinones.22 Besides, the NZT NPs could be stable for about 120 days at 4 °C, which achieved a long-term storing stability (Figure 2c). In order to further explore the reason for size variation, UV−vis and XPS spectroscopy were performed to compare the variation of NZT NPs before (0 h) and after stabilization (48 h) in Figure 3. As shown in Figure 3a,b, there was no change of absorption peak in NZT NPs but a marked shift of TA due to the oxidation reaction to produce quinones. On the
basis of previous research, TA had a characteristic absorption peak of 276 nm assigned for the CO group,23 while the only peak was transformed into three peaks owing to the formation of benzoquinone from the oxidation reaction.24,25 Moreover, XPS analysis was acquired to affirm the surface chemical information on NZT and NZT48 NPs. The survey scan spectrum confirms that three peak components C 1s were located at 288.2, 286, and 284.7 eV, while two peak components O 1s existed at 533.1 and 531.7 eV, which was consistent with previous research.26,27 Besides, Figure 3c−f displayed that the peak intensity of hydroxyl group diminished significantly while the carbonyl peak enhanced synchronously as a consequence of oxidation reaction of TA. That is, the COH group could be converted to CO by oxidation during the variety, which could match the conclusion from the UV−vis spectrum further. A reported study pointed out that some polyphenol could self-polymerize and then deposit on a variety of substrates and form a uniform layer.28 Hence, we postulated that TA could adsorb on the surface of NZ NPs first and then self-polymerize by oxidation. As a result, the NPs could be stable once the oxidation had finished. Hence, we used steady NZT48 NPs as the optimal samples in our subsequent experiments. Figure 4a and d show typical size distributions of NZ NPs and NZT48 NPs after 48 h. Briefly, zein and NOB could form NPs of 65 nm immediately but they were separated from the solution to precipitate after 2 h (Figure 4a, data could not be detected after 48 h). However, NZT48 NPs were able to stabilize with sizes of 100 nm. Figure 4b and e exhibited the TEM images of NZ and NZT48 NPs. As shown, the NZ NPs were observed as needle-like crystals, which were already C
DOI: 10.1021/acs.jafc.9b02003 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 3. UV−vis spectrum of NZ, TA, and NZT NPs for 0 h (a) and 48 h (b). XPS analysis of NZT NPs for 0 h (c, e) and 48 h (d, f).
cm−1 and shifted to 3318 cm−1 in ZNT48 NPs, illustrating the hydrogen bondings were formed between zein and TA, since the characterization peaks of hydrogen bondings were in a range of 3200−3400 cm−1.31 Comparing all spectra of NOB, TA, and zein with NPs, the main characteristic absorption peaks of the control could be contained in NZT48 NPs totally, illustrating that the final NPs were composed of these three compounds. Besides, we could draw a conclusion that the stability of the NP system not only depended on the oxidation reaction mentioned above but also required a certain hydrogen bond force. To further evaluate the assembly of NPs, DSC and XRD were performed in Figure 5b,c. DSC curves reflected that pure NOB powders had a sharp melting peak at 138.5 °C and a mild crystallization peak at 88.5 °C.32 By contrast, no discernible peaks appear for TA within the measured temperature range. However, when it comes to NZT48 NPs, the two peaks could not be detected compared to the physical mixing of NZT, with similar peaks to NOB, which
reported in a previous study.20 Nevertheless, NZT48 NPs were shown as regular nanospheres with a homogeneous distribution. Polarizing microscope images were displayed in Figure 4c and f. As could be seen, needle-like crystals were observed markedly in NZ NPs, while no crystals appeared in NZT48 NPs. The above results proved that zein could assemble with NOB to form NPs but the force was too weak to inhibit the crystallization of NOB. The addition of TA enhanced the force between zein and TA to gain a better inhibition effect. Figure 5a displayed the FTIR curves of control and NZT48 NPs, respectively. As shown in Figure 5a, the characterization peaks at 1645 cm−1 were assigned to CO stretching and the peaks at 1588 and 1519 cm−1 were due to the CC stretching in aromatic rings of NOB.29 As for zein, The amide I band at 1656 cm−1 and the amide II band at 1537 cm−1 demonstrating CO stretching and CN stretching were two prominent characterization peaks.30 Furthermore, the hydroxyl groups of zein and TA occurred at 3330 and 3363 D
DOI: 10.1021/acs.jafc.9b02003 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 4. DLS patterns, TEM images, and polarizing microscope images of NZ NPs (a−c) and NZT48 NPs (d−f), respectively. ND represented data not detected.
Figure 5. FTIR spectra (a), DSC curves (b), and XRD patterns (c) of NOB, TA, zein, and NPs.
3.2. Secondary Capping for Oral Administration. Although our fabricated NPs could improve the solubility and loading capacity of NOB, the effective release in a gastrointestinal environment was still facing a formidable challenge for NPs tending to release rapidly in the stomach (2h, 60%) and intestine (8h, 100%) after oral administration (Figure 8a). Therefore, it was exceedingly essential for NPs to carry out a secondary-encapsulation protection strategy for further oral delivery. SPGs, demonstrated as a safety potential delivery vehicle, were widely used to prevent various materials from harsh conditions.36 Hence, our study made a novel attempt to encapsulate NPs into SPGs (termed as NZTS) by ultrasound to fabricate a secondary protection system aiming to resist being resolved under an improper environment. As shown in Figure 6a, the ultrasonic condition had no adverse effect on the diameter of NZT48 NPs. Figure 6b displayed the size distribution of SPGs before and after coating and showed that there was no significant change in size after loading. Figure 6c exhibited the CLSM images of SPGs for control and loading completely by coumarin-6 (green fluorescence) labeled NPs. As presented, the control pollens had a completely exine structure with strong both green and red fluorescence while the pollen cavities were filled with green fluorescence
related to the existent state of NOB, in which NOB was amorphous or molecularly dispersed in NPs.4 XRD patterns were presented in Figure 5c to explore the crystallization of NOB in NPs. NOB, a needle-like crystal, showed massive sharp peaks corresponding to its high crystallization.33 Physical mixing of all compounds still exhibited similar strongly peaks of NOB, which implied that the physical mixing of sample did not affect the crystal structure of compounds. However, the addition of zein led to partial disappearance of the crystalline state. As a result, zein could interact with NOB to a certain degree and prevent NOB from crystallization partially. According to previous studies, zein has the ability to form self-assembled NPs.34,35 Hence, we postulated that zein was capable of forming NPs to protect NOB from crystallizing but with lower loading capacity. Contrarily, there were no discernible peaks in NZT48 NPs, illustrating the amorphous property of coating by zein and TA. The results were able to signify the expected coating effect by oxidation reaction between TA molecules and hydrogen bond by zein and TA. In addition, the encapsulation efficiency of NZT48 NPs was 92.37 ± 0.12% and the loading capacity was 48.46 ± 1.62%, respectively (Figure 8a, inset). E
DOI: 10.1021/acs.jafc.9b02003 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 6. DLS patterns of NZT48 NPs before and after the ultrasonic condition (a). Characterization of SPGs before and after loading: (b) size distribution, (c) confocal micrographs. (d) Confocal micrographs with or without Z-stack after zein coating. Scale bar: 30 μm.
Figure 7. SEM images of SPGs before and after loading (a) and coating (b).
absolutely after loading, illustrating the successful encapsulation of NPs. Besides, the CLSM images in Figure 6d proved that the green autofluorescence by SPGs was observed to embed into Nile Blue-labeled zein entirely (red fluorescence),
further providing a confirmation for successful coating of NZTS by zein. Figure 7a showed the SEM images of control and loading samples. As shown, the sample after loading was attached to massive particles compared to the control one F
DOI: 10.1021/acs.jafc.9b02003 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
Figure 8. Cumulative release profiles of NZT48 NPs (a) and NZTS and NZTSZ (b) in SGF and SIF based on different incubation times. (c) SEM images of NOB-loaded SPGs before and after incubating for 2 h in SGF and 48 h in SIF. Inset: (a) Encapsulation efficiency and loading capacity of NZT48 NPs and (b) encapsulation loading of NZTS.
Figure 8b. As shown, the NZTS (gray line) still exhibited an abrupt release similar to the release trend in NPs (Figure 8a), due to the apertures that existed in pollens which led to NOB leaking easily. Herein, we fabricated zein film as a secondary coating on the surface of SPGs in Figure 7b. Obviously, the pollens were surrounded by zein absolutely (named NZTSZ)
with a smooth pollen cavity, which proved the successful loading of NPs. Besides, the loading capacity of NZTS was calculated as 90.49 ± 1.19 mg/g in the inset of Figure 8b. 3.3. In Vitro Release Study. In order to estimate the release property of NPs in a simulated gastrointestinal environment, an in vitro release study was carried out in G
DOI: 10.1021/acs.jafc.9b02003 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Article
Journal of Agricultural and Food Chemistry
(5) Lee, S.; Alwahab, N. S.; Moazzam, Z. M. Zein-based oral drug delivery system targeting activated macrophages. Int. J. Pharm. 2013, 454, 388−393. (6) Liang, H.; Huang, Q.; Zhou, B.; Lei, H.; Lin, L.; An, Y.; Yan, L.; Liu, S.; Chen, Y.; Li, B. Self-assembled zein−sodium carboxymethyl cellulose nanoparticles as an effective drug carrier and transporter. J. Mater. Chem. B 2015, 3, 3242−3253. (7) Shi, C.; He, Y.; Ding, M.; Wang, Y.; Zhong, J. Nanoimaging of food proteins by atomic force microscopy. Part ii: Application for food proteins from different sources. Trends Food Sci. Technol. 2019, 87, 14−25. (8) Chen, L.; Remondetto, G. E.; Subirade, M. Food protein-based materials as nutraceutical delivery systems. Trends Food Sci. Technol. 2006, 17, 272−283. (9) Xie, J.; Wang, H.; Yi, C.; Meng, Q.; Wei, W. Photo-synthesis of protein-based nanoparticles and the application in drug delivery. Ann. Phys. 2015, 358, 225−235. (10) Liang, H.; Zhou, B.; He, L.; An, Y.; Lin, L.; Li, Y.; Liu, S.; Chen, Y.; Li, B. Fabrication of zein/quaternized chitosan nanoparticles for the encapsulation and protection of curcumin. RSC Adv. 2015, 5, 13891−13900. (11) des Rieux, A.; Fievez, V.; Garinot, M.; Schneider, Y.-J.; Preat, V. Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach. J. Controlled Release 2006, 116, 1− 27. (12) Fraser, W. T.; Scott, A. C.; Forbes, A. E. S.; Glasspool, I. J.; Plotnick, R. E.; Kenig, F.; Lomax, B. H. Evolutionary stasis of sporopollenin biochemistry revealed by unaltered Pennsylvanian spores. New Phytol. 2012, 196, 397−401. (13) Diego-Taboada, A.; Beckett, S. T.; Atkin, S. L.; Mackenzie, G. Hollow pollen shells to enhance drug delivery. Pharmaceutics 2014, 6, 80−96. (14) Uddin, M. J.; Gill, H. S. Ragweed pollen as an oral vaccine delivery system: Mechanistic insights. J. Controlled Release 2017, 268, 416−426. (15) Katifori, E.; Alben, S.; Cerda, E.; Nelson, D. R.; Dumais, J. Foldable structures and the natural design of pollen grains. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 7635−7639. (16) Firon, N.; Nepi, M.; Pacini, E. Water status and associated processes mark critical stages in pollen development and functioning. Ann. Bot. 2012, 109, 1201−1214. (17) Janes, M. E.; Kooshesh, S.; Johnson, M. G. Control of Listeria monocytogenes on the Surface of Refrigerated, Ready-to-eat Chicken Coated with Edible Zein Film Coatings Containing Nisin and/or Calcium Propionate. J. Food Sci. 2002, 67, 2754−2757. (18) Herald, T. J.; Hachmeister, K. A.; Huang, S.; Bowers, J. R. Corn Zein Packaging Materials for Cooked Turkey. J. Food Sci. 1996, 61, 415−418. (19) Shi, C.; He, Y.; Ding, M.; Wang, Y.; Zhong, J. Nanoimaging of food proteins by atomic force microscopy. Part I: Components, imaging modes, observation ways, and research types. Trends Food Sci. Technol. 2019, 87, 3−13. (20) Chen, H.; An, Y.; Yan, X.; Mcclements, D. J.; Li, B.; Yan, L. Designing self-nanoemulsifying delivery systems to enhance bioaccessibility of hydrophobic bioactives (nobiletin): Influence ofhydroxypropyl methylcellulose and thermal processing. Food Hydrocolloids 2015, 51, 395−404. (21) Wu, D.; Liang, Y.; Huang, K.; Jing, X.; Li, B.; Liang, H. Leveraging plant exine capsules as pH-responsive delivery vehicles for hydrophobic nutraceutical encapsulation. Food Funct. 2018, 9, 5436− 5442. (22) Vivas, N.; Glories, Y. Role of oak wood ellagitannins in the oxidation process of red wines during aging. Am. J. Enol. Vitic. 1996, 47, 103−107. (23) Albu, M. G.; Ghica, M. V.; Giurginca, M.; Trandafir, V.; Popa, L.; Cotrut, C. Spectral characteristics and antioxidant properties of tannic acid immobilized on collagen drug-delivery systems. Indian Vet. J. 2009, 60, 666−672.
without any visible pores, achieving a complete coating. As for the release curves, NZTSZ could prevent NPs from decomposing in SGF (2 h,
