新型碱木质素微/亚微米颗粒的绿色合成、表征、封装和释放潜力的测量
Summary
我们描述了生物相容性木质素微米和亚微米颗粒的合成和表征的新颖、简单的方法。这些配方为杂质聚合物的利用提供了一种简单的方法，也为合理设计多功能载体基质提供了一种替代方案，在生物医学、制药技术和食品工业中具有潜在的适用性。
Abstract
由于生物聚合物基颗粒作为有效载体系统的巨大潜力，生物聚合物微/纳米技术在人类、兽医、制药和食品技术中的适用性正在迅速增长。使用木质素作为碱性杂聚物生物基质，用于设计创新的微/亚微米配方，可以提高生物相容性，并提供各种活性官能团，为定制配方的理化性质和生物活性提供机会，适用于各种应用。本研究的目的是开发一种简单且环保的方法，用于合成微米和亚微米尺寸的木质素颗粒;评估其理化、光谱和结构特性;并检查它们包封生物活性分子的能力以及在模拟胃肠道培养基中体 外释放生物 类黄酮的潜力。所提出的方法适用于廉价和绿色的溶剂;简单、直接、快速和灵敏的过程，只需很少的设备、无毒物质和简单的表征方法，即可测定对水溶性差的生物活性化合物桑酸和槲皮素的包封能力，以及木质素基质的 体外 释放潜力。
Introduction
如今，由于纤维素、壳聚糖、胶原蛋白、葡聚糖、明胶和木质素等生物聚合物作为设计具有可定制尺寸、物理化学特性和生物功能性的微/亚微米载体的前体，由于它们在组织工程、3D 生物打印、体外的适用性，在生物医学、制药和食品技术行业中有所增加疾病建模平台、包装行业、乳液制备和营养物质输送等 1,2,3.
新颖的研究强调了木质素基水凝胶以及微纳米制剂4 作为用于食品包装材料5、储能6、化妆品7、热/光稳定剂、增强材料和药物载体基质的有利载体8 用于输送疏水分子、改善紫外线阻隔剂9，作为纳米复合材料中的增强剂，以及由于最近的一些安全问题而作为无机纳米颗粒的替代品10,11,12。这种趋势背后的原因是天然生物聚合物的生物相容性、生物降解性和无毒性，以及其已证实的木质素抗氧化潜力和自由基清除、抗增殖和抗菌活性的生物活性 13,14,15,16,17。
科学文献报道了各种合成方法（自组装、抗溶剂沉淀、酸沉淀和溶剂转移）18 以及基于木质素的微/纳米级制剂的表征，包括使用昂贵或有害的溶剂，如四氢呋喃 （THF）、二甲基亚砜 （DMSO）、N，N-二甲基甲酰胺 （DMF） 和丙酮，以及使用大量设备和有毒物质的复杂、间接和繁琐的过程12，19,20.
为了克服后一种缺点，以下协议提出了使用廉价和绿色溶剂合成木质素基微/亚微米颗粒的新方法;简单、直接、快速和灵敏的过程，只需很少的设备、无毒物质和简单的方法来表征和测定对水溶性差的生物活性化合物的包封能力和木质素基质的 体外 释放潜力。所提出的实验室规模生产方法有利于制造功能性木质素载体，这些载体具有可调的尺寸、高封装容量和可持续的 体外 释放行为，利用简单的表征程序和环保化学品，可以在生物医学科学和食品技术的各个领域找到应用。将两种黄酮类化合物作为靶分子封装在木质素颗粒中：桑苷封装在微粒中，槲皮素封装在亚微米颗粒中。两种黄酮类化合物结构的区别只是第二个-OH基团在B-芳环中的位置：-OH基团在桑苷中位于2’位置，在槲皮素中位于3’位置，因此两种有机化合物都是位置异构体。后一个事实假定两种生物活性天然化合物在包封和/或释放过程中具有相似的行为。
Protocol
1.木质素微粒的合成 通过在磁力搅拌器上将 2.5 g 碱木质素溶解在 50 mL 超纯水中，制备 50 mg/mL 碱木质素水溶液。 通过将 1 mL 吐温 80 溶解在 100 mL 超纯水中来制备 1% 吐温 80 溶液。 用超纯水稀释 6.65 mL 的 67% HNO3（密度 = 1.413 g/mL）至最终体积为 50 mL 来制备 2 M 的 HNO3 溶液。 缓慢加入 15 mL 的 1% 吐温 80 溶液到 50 mL 的 50 mg/mL 碱木质素溶液中。<…
Representative Results
执行反溶剂沉淀技术以产生碱木质素微米/亚微米颗粒。将稀释的无机酸-硝酸/有机酸-柠檬酸水溶液分散到碱木质素水溶液中，并富集环保表面活性剂/乙醇，导致生物聚合物溶质逐渐沉淀，超声处理后，最终产生致密的微米/亚微米颗粒悬浮液（图1）。 <strong …
Discussion
用于设计基于生物聚合物的药物载体制剂的现代合成方法的主要关键问题之一是使用有害的有机试剂 – 挥发性和易燃溶剂，如四氢呋喃、丙酮、甲醇，甚至高浓度的DMSO – 由于可能具有毒性作用的表现，这限制了它们在生物医学、制药工业和食品技术中的适用性20， 21,22,23,24.?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究得到了保加利亚科学基金的支持，合同编号为 KΠ-06 H59/3，并得到了特拉基亚大学科学项目编号 07/2023 FVM 的支持。
Materials
| automatic-cell counter | EVE, NanoEnTek | ||
| Citric acid | Sigma | 251275 | ACS reagent, ≥99.5% |
| digital water bath | Memmert | ||
| Eppendorf tubes, 1.5-2 mL | |||
| Ethanol | Sigma | 34852-M | absolute, suitable for HPLC, ≥99.8% |
| Folin–Ciocalteu’s phenol reagent | Sigma | F9252 | |
| freeze dryer | Biobase | ||
| gallic acid | Sigma- | BCBW7577 | monohydrate |
| HCl | Sigma | 258148 | ACS reagent, 37% |
| HNO3 | Sigma | 438073 | ACS reagent, 70% |
| lignin, alkali | Sigma | 370959 | |
| morin | Sigma | PHL82601 | |
| NaCl | Sigma | S9888 | ACS reagent, ≥99.0% |
| Na2CO3 | Sigma | 223530 | powder, ≥99.5%, ACS reagent |
| NaOH | Sigma | 655104 | reagent grade, 97%, powder |
| orbital shaker | IKA | KS 130 basic | |
| pH-meter | Consort | ||
| phosphate-buffered saline (PBS) | Sigma | RNBH7571 | |
| Quercetin hydrate | Sigma | STBG3815V | |
| statistical software for Excel | Microsoft Corporation | XLSTAT Version 2022.4.5. | |
| Tween 80 | Sigma | P8074 | BioXtra, viscous liquid |
| ultracentrifuge | Hermle | Z 326 K | |
| Ultrapure water system | Adrona | INTEGRITY+ | |
| ultrasound homogenizer | Bandelin Sonopuls | HD 2070 | |
| UV/Vis spectrophotometer | Hach-Lange | DR 5000 |
References
- Yu, X., et al. Lignin nanoparticles with high phenolic content as efficient antioxidant and sun-blocker for food and cosmetics. ACS Sustainable Chem. Eng. 11 (10), 4082-4092 (2023).
- Boarino, A., Klok, H. -. A. Opportunities and challenges for lignin valorization in food packaging, antimicrobial, and agricultural applications. Biomacromolecules. 24 (3), 1065-1077 (2023).
- Aadil, K., Barapatre, A., Jha, H. Synthesis and characterization of Acacia lignin-gelatin film for its possible application in food packaging. Bioresour. Bioprocess. 3 (27), 1-11 (2016).
- Sharma, S., et al. Valorization of lignin into nanoparticles and nanogel: characterization and application. Bioresour. Technol. Reports. 18, 101041 (2022).
- Zadeh, E. M., O’Keefe, S. F., Kim, Y. -. T. Utilization of lignin in biopolymeric packaging films. ACS Omega. 3 (7), 7388-7398 (2018).
- Beaucamp, A., et al. Lignin for energy applications – state of the art, life cycle, technoeconomic analysis and future trends (Critical Review). Green Chem. 24, 8193-8226 (2022).
- Antunes, F., et al. From sugarcane to skin: Lignin as a multifunctional ingredient for cosmetic application. Int J Biol Macromol. 234, 123592 (2023).
- Garg, J., et al. Applications of lignin nanoparticles for cancer drug delivery: An update. Materials Letters. 311, 131573 (2022).
- Anushikha, K. K. Lignin as a UV blocking, antioxidant, and antimicrobial agent for food packaging applications. Biomass Conv. Bioref. , 1-14 (2023).
- Freitas, F. M. C., et al. synthesis of lignin nano- and micro-particles: Physicochemical characterization, bioactive properties and cytotoxicity assessment. Int J Biol Macromol. 163, 1798-1809 (2020).
- Rismawati, R., Nurdin, I. A., Pradiptha, M. N., Maulidiyah, A., Mubarakati, N. J. Preparation and characterization of lignin nanoparticles from rice straw after biosynthesis using Lactobacillus bulgaricus. Journal of Physics: Conference Series. 9th International Seminar on New Paradigm and Innovation of Natural Sciences and its Application. 1524, 012070 (2020).
- Worku, L. A., et al. Synthesis of lignin nanoparticles from Oxytenanthera abyssinica by nanoprecipitation method followed by ultrasonication for the nanocomposite application. Journal of King Saud University – Science. 35 (7), 102793 (2023).
- Gala Morena, A., Tzanov, T. z. Antibacterial lignin-based nanoparticles and their use in composite materials. Nanoscale Adv. 4, 4447-4469 (2022).
- Ivanova, D., Nikolova, G., Karamalakova, Y., Marutsova, V., Yaneva, Z. Water-soluble alkali lignin as a natural radical scavenger and anticancer alternative. Int J Mol Sci. 24 (16), 12705 (2023).
- Ivanova, D., Toneva, M., Simeonov, E., Antov, G., Yaneva, Z. Newly synthesized lignin microparticles as bioinspired oral drug-delivery vehicles: Flavonoid-carrier potential and in vitro radical-scavenging activity. Pharmaceutics. 15 (4), 1067 (2023).
- Yaneva, Z., et al. Antimicrobial potential of conjugated lignin/morin/chitosan combinations as a function of system complexity. Antibiotics. 11, 650 (2022).
- Handral, H. K., Wyrobnik, T. A., Lam, A. T. -. L. Emerging trends in biodegradable microcarriers for therapeutic applications. Polymers. 15 (6), 1487 (2023).
- Figueiredo, P., Lintinen, K., Hirvonen, J. T., Kostiainen, M. A., Santos, H. A. Properties and chemical modifications of lignin: Towards lignin-based nanomaterials for biomedical applications. Prog. Mater. Sci. 93, 233-269 (2018).
- Tang, Q., et al. Lignin-based nanoparticles: a review on their preparations and applications. Polymers. 12 (11), 2471 (2020).
- Zhao, W., Simmons, B., Singh, S., Ragauskas, A., Cheng, G. From lignin association to nano-/micro-particle preparation: extracting higher value of lignin. Green Chemistry. 18 (21), 5693-5700 (2016).
- Stewart, H., Golding, M., Matia-Merino, L., Archer, R., Davies, C. Manufacture of lignin microparticles by anti-solvent precipitation: Effect of preparation temperature and presence of sodium dodecyl sulfate. Food Res Int. 66, 93-99 (2014).
- Beisl, S., Friedl, A., Miltner, A. Lignin from micro- to nanosize: Applications. Int. J. Mol. Sci. 18, 2367 (2017).
- Mishra, P. K., Ekielski, A. A simple method to synthesize lignin nanoparticles. Colloids Interfaces. 3, 52 (2019).
- Qian, Y., Deng, Y., Qiu, X., Li, H., Yang, D. Formation of uniform colloidal spheres from lignin, a renewable resource recovered from pulping spent liquor. Green Chem. 16, 2156-2163 (2014).
- Tardy, B. L., et al. Lignin nano- and microparticles as template for nanostructured materials: formation of hollow metal-phenolic capsules. Green Chem. 20, 1335-1344 (2018).
- Silva, M., et al. Paraquat-loaded alginate/chitosan nanoparticles: preparation, characterization and soil sorption studies. J Haz Mat. 190 (1-3), 366-374 (2011).
- Georgieva, N., Yaneva, Z. Comparative evaluation of natural and acid-modified layered mineral materials as rimifon-carriers using UV/VIS, FTIR, and equilibrium sorption study. Cogent Chem. 1 (1), 1-16 (2015).
- Zhang, P., Chen, D., Li, L., Sun, K. Charge reversal nano-systems for tumor therapy. J Nanobiotechnol. 20, 31 (2022).
- Yaneva, Z. L., Georgieva, N. V. Removal of diazo dye from the aqueous phase by biosorption onto ball-milled maize cob (BMMC) biomass of Zea mays. Maced. J. Chem. Chem. Eng. 32 (1), 133-149 (2013).
- Zatorska, M., et al. Drug-loading capacity of polylactide-based micro- and nanoparticles – Experimental and molecular modeling study. Int J Pharmaceutics. 591, 120031 (2020).
- Yaneva, Z., Georgieva, N., Grumezescu, A. M. Chapter 5 – Physicochemical and morphological characterization of pharmaceutical nanocarriers and mathematical modeling of drug encapsulation/release mass transfer processes. Nanoscale Fabrication, Optimization, Scale-Up and Biological Aspects of Pharmaceutical Nanotechnology. , 173-218 (2018).
- Yaneva, Z., Georgieva, N., Staleva, M. Development of d,l-α-tocopherol acetate/zeolite carrier system: equilibrium study. Monatshefte fur Chemie Chemical Monthly. 147 (7), 1167-1175 (2016).
- Yaneva, Z., Georgieva, N. Study on the physical chemistry, equilibrium, and kinetic mechanism of Azure A biosorption by Zea mays biomass. Journal of Dispersion Science and Technology. 35 (2), 193-204 (2014).
Cite This Article
Yaneva, Z., Ivanova, D., Toneva, M. Green Synthesis, Characterization, Encapsulation, and Measurement of the Release Potential of Novel Alkali Lignin Micro-/Submicron Particles. J. Vis. Exp. (205), e66216, doi:10.3791/66216 (2024).