This study was supported by the Bulgarian Scientific Fund under Contract &#8470; K&#928;-06 H59/3 and by Scientific Project No. 07/2023 FVM, Trakia University.

1. Synthesis of lignin microparticles
<ol>
	<li>Prepare a 50 mg/mL alkali lignin aqueous solution by dissolving 2.5 g of alkali lignin in 50 mL of ultrapure water on a magnetic stirrer.</li>
	<li>Prepare 1% Tween 80 solution by dissolving 1 mL of Tween 80 in 100 mL of ultrapure water.</li>
	<li>Prepare a 2 M solution of HNO3 by diluting 6.65 mL of 67% HNO3 (density = 1.413 g/mL) with ultrapure water to a final volume of 50 mL.</li>
	<li>Slowly add 15 mL of the 1% Tween 80 solution to 50 mL of the 50 mg/mL alkali lignin solution.</li>
	<li>Agitate the mixture on a magnetic stirrer at 500 rpm for 10 min so that the surfactant becomes well dispersed.</li>
	<li>Add 20 mL of 2 M HNO3 dropwise with a syringe at a flow rate of approximately 150 &#181;L/s to the mixture.</li>
	<li>Continue stirring the mixture for 30 min when the dark brown solution is transformed into a light brown suspension of microparticles.</li>
	<li>Transfer the suspension into 1.5-2 mL test tubes and centrifuge for 30 min at 15,000 &#215; g in an ultracentrifuge at 10 &#176;C.</li>
	<li>Collect the supernatant for further analyses and rinse the microparticles with ultrapure water.</li>
	<li>Repeat the rinsing/ultracentrifugation procedures 3x.</li>
	<li>Dip the container with the microparticles in an ice bath before the ultrasonic homogenization.</li>
	<li>Homogenize the microparticles for 4 min at an intensity of 93% on an ultrasound homogenizer.</li>
	<li>Lyophilize the microparticles at a temperature of -64 &#176;C in a freeze dryer and store them in an exicator for further use.</li>
</ol>
2. Synthesis of lignin submicron particles
<ol>
	<li>Prepare a 5 mg/mL alkali lignin aqueous solution by dissolving 125 mg of alkali lignin in 25 mL of ultrapure water on a magnetic stirrer.</li>
	<li>Slowly add 1 mL of 96% EtOH to the alkali lignin solution.</li>
	<li>Agitate the mixture on a magnetic stirrer at 500 rpm for 3 min.</li>
	<li>Prepare 50 mL of a 1% solution of citric acid by dissolving 0.5 g of citric acid in ultrapure water to a final volume of 50 mL.</li>
	<li>Add 7 mL of 1% citric acid dropwise with a syringe at a flow rate of approximately 4 mL/min to the mixture.</li>
	<li>Continue stirring the mixture for 10 min when the brown clear solution will transform into a cloudy light brown suspension of submicron particles.</li>
	<li>Transfer the suspension into test tubes and centrifuge for 30 min at 15,000 &#215; g in an ultracentrifuge at 10 &#176;C.</li>
	<li>Collect the supernatant for further analyses and rinse the microparticles with ultrapure water.</li>
	<li>Repeat the rinsing/ultracentrifugation procedures 3x.</li>
	<li>Dip the container with the microparticles in an ice bath before the ultrasonic homogenization.</li>
	<li>Homogenize the microparticles ultrasonically for two cycles of 4 min each at an intensity of 96% in an ultrasound homogenizer.</li>
	<li>Cool the containers for 1 min after the first cycle.</li>
	<li>Lyophilize the microparticles at a temperature of -64 &#176;C in a freeze dryer and store them in an exicator for further use.</li>
</ol>
3. Synthesis of natural flavonoid-encapsulated lignin micro-/submicron particles
<ol>
	<li>Repeat steps 1.1-1.5 for the microparticles.</li>
	<li>Weigh 0.08 g of morin, dissolve it in 1 mL of EtOH, and add this ethanolic solution to the mixture.</li>
	<li>Agitate the mixture on a magnetic stirrer at 500 rpm for 20 min.</li>
	<li>Add 20 mL of 2 N HNO3 dropwise with a syringe at a flow rate of approximately 150 &#181;L/s to the mixture.</li>
	<li>Continue stirring the mixture for 60 min.</li>
	<li>Repeat steps 1.8-1.13.</li>
	<li>Repeat step 2.1 for the submicron particles.</li>
	<li>Weight 0.04 g of quercetin, dissolve it in 1 mL EtOH and add this ethanolic solution to the alkali lignin aqueous solution.</li>
	<li>Agitate the mixture on a magnetic stirrer at 500 rpm for 10 min.</li>
	<li>Repeat steps 2.4-2.13.</li>
</ol>
4. Determination of the encapsulation efficiency of lignin micro-/sumicro- particles
<ol>
	<li>Calculate the content of the added bioactive substance during the procedure for the synthesis of both types of flavonoid-encapsulated lignin particles.
	<ol>
		<li>Determine spectrophotometrically the absorption of the flavonoid in the supernatant obtained during steps 1.9 and 2.8 after diluting it with 96% EtOH.</li>
		<li>Calculate the concentration of the non-entrapped morin/quercetin using the calibration curves of the flavonoids.</li>
		<li>Calculate the encapsulation efficiency (EE, %) of the lignin microparticles towards the natural flavonoids by using equation (1): 
		<img alt="Equation 1" src="/files/ftp_upload/66216/66216eq01.jpg" style="margin: auto;" />&#160; &#160; &#160;(1) 
		Where wo is the total quantity of the bioactive substance added (mg) and wf is the quantity of the free non-entrapped flavonoid (mg).</li>
		<li>Calculate the drug loading capacity (DLC, %)-an important parameter representing the amount of drug in the particles per unit weight of the carrier system-by using eq. (2): 
		<img alt="Equation 2" src="/files/ftp_upload/66216/66216eq02.jpg" style="margin: auto;" />&#160; &#160; &#160;(2) 
		Where wp is the total quantity (yield) of lignin micro-/submicron particles obtained after lyophilization (mg).</li>
	</ol>
	</li>
</ol>
5. Characterization of lignin micro- and submicron particles
<ol>
	<li>Determination of particle number, size, and size distribution
	<ol>
		<li>Assess the particle size and particle size distribution of the samples using an automatic-cell counter with the option for bead count. Add with a micropipette 1 &#181;L of the lignin/flavonoid micro-/submicron particles suspension in ultrapure water in the well of the counting slide required for the operation.</li>
		<li>Wait for the number of particles in 1 mL of the suspension, as well as their number and distribution by size to be shown in the display of the automatic cell counter. 
		NOTE: The apparatus allows the storage of the data on a USB flash. The automatic cell counter special software allows further processing of the saved digital and photo files.</li>
	</ol>
	</li>
	<li>Determination of the content of surface acidic/basic groups of lignin particles by potentiometric titration
	<ol>
		<li>Weight 0.04 g of unloaded/flavonoid-encapsulated lignin particles.</li>
		<li>Transfer them to an Erlenmeyer flask, add 10 mL of 0.1 M HCl, and place the flask on a magnetic stirrer at 250 rpm.</li>
		<li>Fill a 50 mL burette with a 0.1 M standard solution of the titrant NaOH.</li>
		<li>Measure the initial pH of the solution in the Erlenmeyer flask with a bench pH meter before starting the titration.</li>
		<li>Start the titration and measure the pH of the analyzed solution after each 0.5 mL added portion of the titrant.</li>
		<li>Store the experimental data in a table containing the volume of the titrant applied and the corresponding value of pH.</li>
		<li>Stop the titration when an approximately constant value of the pH is reached by increasing the volume of the titrant solution.</li>
		<li>Plot the experimental data in the form of zero-, first- and second-derivative differential titration curves.</li>
		<li>Determine the equivalent points and the corresponding equivalent volumes of the titrants used.</li>
		<li>Calculate the contents of the acidic Aa and Ab basic groups on the surface of unloaded and flavonoid-loaded lignin particles by using equations (3) and (4): 
		<img alt="Equation 3" src="/files/ftp_upload/66216/66216eq03.jpg" style="margin: auto;" /> , mgeq/g&#160; &#160; &#160;(3) 
		<img alt="Equation 4" src="/files/ftp_upload/66216/66216eq04.jpg" style="margin: auto;" /> mgeq/g&#160; &#160; &#160;(4) 
		Where Veqi is the equivalent volume (mL); NT the normality of the titrant (mgeqv/mL); VT the volume of the titrant used for the determination procedure (mL); m the weight of the analyzed sample (g).</li>
	</ol>
	</li>
	<li>Determination of the pH point of zero charge (pHPZC) of lignin-based particles by the solid addition method.
	<ol>
		<li>Prepare 60 mL of 0.1 M aqueous solution of NaCl.</li>
		<li>Add 9 mL of the 0.1 M NaCl solution in each of five stoppered conical flasks and adjust the pH to pHi = 2, 4, 7, 10, and 12 (where i = 1-5 denote the number of the corresponding solution), respectively by the addition of either 0.1 M HCl or 0.1 M NaOH. Adjust the total volume of the solution in each flask to 10 mL exactly by adding NaCl solution of the same strength.</li>
		<li>Add 40 mg of dry lignin particles (unloaded, flavonoid-loaded micro-/submicron) to each flask and cap the flasks securely.</li>
		<li>Secure the flasks upright on an orbital shaker and keep them shaking for 24 h.</li>
		<li>Allow equilibration for 30 min and subsequently measure the final pH (pHf) of the supernatants in each flask.</li>
		<li>Plot pHf values against the corresponding initial pH values (pHi).</li>
		<li>The point of zero charge (pHPZC) is defined as the pH value at which the curve &#916;pH versus pHi intersects the straight line with coordinates (pHi; pHi).</li>
	</ol>
	</li>
	<li>Determination of total phenolic content (TPC) of lignin particles 
	NOTE: The total phenolic content (TPC) of the micro-/submicron lignin particles is determined via a modified Folin-Ciocalteu colorimetric method.
	<ol>
		<li>Mix 200 &#181;L of an aqueous suspension of particles with a concentration of 500 &#181;g/mL with 600 &#181;L of ultrapure water and 200 &#181;L of Folin-Ciocalteu reagent (1:1, v/v).</li>
		<li>After 5 min, add 1.0 mL of 8% Na2CO3 and 1.0 mL of Milli-Q water to the mixture and incubate it in the dark at 40 &#176;C for 30 min in a water bath with intermittent agitation.</li>
		<li>Centrifuge the suspension at 5,300 &#215; g for 2 min.</li>
		<li>Prepare a blank containing no particles.</li>
		<li>Transfer 3.5 mL of the supernatant in a 10 mm quartz cuvette and measure the absorbance on a UV/Vis spectrophotometer in the visible region at 760 nm against the blank.</li>
		<li>Prepare a calibration curve of the standard gallic acid following steps 5.3.1-5.3.5; only instead of 200 &#181;L of the lignin particle suspension, use the ethanolic solution of gallic acid with initial concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, and 200 &#181;g/mL.</li>
		<li>Express the experimental data of the microparticles as mg of gallic acid equivalents in milligrams per gram of dry sample (mg GAE/g).</li>
		<li>Calculate TPC using equation (5): 
		<img alt="Equation 5" src="/files/ftp_upload/66216/66216eq05.jpg" style="margin: auto;" /> mg GAE/g&#160; &#160; &#160;(5) 
		Where CGA is the concentration of the sample equivalent to the concentration of the standard gallic acid obtained from the calibration plot of the acid (&#181;g GA/mL); Cs is the concentration of the sample, which is equal to the dry sample mass divided by the volume of the solvent (&#181;g/mL).</li>
	</ol>
	</li>
</ol>
6. Determination of the in vitro release capacity of lignin particles
<ol>
	<li>Prepare 250 mL of simulated enzyme-free gastric medium by adjusting the pH of standard PBS solution with 0.1 M HCl to pH = 1.2.</li>
	<li>Prepare 250 mL of each of the two simulated intestinal fluid solutions by adjusting the pH of the standard PBS solution with 0.1 M NaOH/0.1 M HCl to pH = 6.8 and 7.4, respectively.</li>
	<li>Add 25 mg of flavonoid-encapsulated micro-/submicron particles to 50 mL of the simulated enzyme-free gastric medium in a glass batch reactor supplied with a mechanical stirrer and place it in a thermal water bath at a constant temperature of T = 37 &#177; 0.2 oC.</li>
	<li>Dip the stirrer to a depth of 2/3 of the liquid volume to ensure full mixing of the solid and liquid phases and ensure maximal mass transfer without stagnant zones.</li>
	<li>Take out 1 mL of sample from the reactor every 10 min up to the 90th min and immediately pipette 1 mL of fresh simulated fluid solution into the reactor to prevent change of the total volume and to ensure sink conditions.</li>
	<li>Repeat the same procedure including steps 6.3-6.6 with both simulated intestinal fluid solutions with pH = 6.8 and 7.4, respectively, for 200 min.</li>
	<li>Perform analogous experiments with unloaded lignin particles in the three simulated media and use the samples as blanks for zeroing the spectrophotometer.</li>
	<li>Determine the absorption of the samples spectrophotometrically after filtering the samples and diluting them with 96% EtOH against the blank samples from step 6.7 and calculate the corresponding flavonoid concentration using the corresponding calibration curves of morin obtained at pH = 1.2, 6.8, and 7.4, respectively.</li>
	<li>Calculate the cumulative release (CR) of the bioflavonoids by using equation (6) in &#181;g/mL and the cumulative release percentage (CRP) by equation (7): 
	<img alt="Equation 6" src="/files/ftp_upload/66216/66216eq06.jpg" style="margin: auto;" />&#160; &#160; (6) 
	Where Ci and Ci+1 are the concentrations of morin/quercetin in the ith and (i+1)th samples (&#181;g/mL); Vs the sample volume taken from the batch reactor (mL); V the total volume of the simulated media (mL). 
	<img alt="Equation 7" src="/files/ftp_upload/66216/66216eq07.jpg" style="margin: auto;" />&#160; &#160; (7) 
	Where Cmax is the maximum concentration of the biologically active compound in the carrier (&#181;g/mL).</li>
</ol>
7. Statistical analyses
<ol>
	<li>Express the experimental data as means &#177; standard deviations (SD) of three independent measurements.</li>
	<li>Determine the statistical significance of the experimental results by performing the ANOVA test as the post hoc test. Consider a value of p &#60; 0.05 statistically significant.</li>
</ol>

Lignin Micro-/Submicron Particle Synthesis for Bioflavonoid Release

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignin+nano-+and+microparticles+as+template+for+nanostructured+materials:+formation+of+hollow+metal-phenolic+capsules.">Lignin nano- and microparticles as template for nanostructured materials: formation of hollow metal-phenolic capsules.</a> Green Chem. 20, 1335-1344 (2018).</li><li>Silva, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraquat-loaded+alginate/chitosan+nanoparticles:+preparation,+characterization+and+soil+sorption+studies.">Paraquat-loaded alginate/chitosan nanoparticles: preparation, characterization and soil sorption studies.</a> J Haz Mat. 190 (1-3), 366-374 (2011).</li><li>Georgieva, N., Yaneva, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+evaluation+of+natural+and+acid-modified+layered+mineral+materials+as+rimifon-carriers+using+UV/VIS,+FTIR,+and+equilibrium+sorption+study.">Comparative evaluation of natural and acid-modified layered mineral materials as rimifon-carriers using UV/VIS, FTIR, and equilibrium sorption study.</a> Cogent Chem. 1 (1), 1-16 (2015).</li><li>Zhang, P., Chen, D., Li, L., Sun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Charge+reversal+nano-systems+for+tumor+therapy.">Charge reversal nano-systems for tumor therapy.</a> J Nanobiotechnol. 20, 31 (2022).</li><li>Yaneva, Z. L., Georgieva, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+diazo+dye+from+the+aqueous+phase+by+biosorption+onto+ball-milled+maize+cob+(BMMC)+biomass+of+Zea+mays.">Removal of diazo dye from the aqueous phase by biosorption onto ball-milled maize cob (BMMC) biomass of Zea mays.</a> Maced. J. Chem. Chem. Eng. 32 (1), 133-149 (2013).</li><li>Zatorska, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug-loading+capacity+of+polylactide-based+micro-+and+nanoparticles+-+Experimental+and+molecular+modeling+study.">Drug-loading capacity of polylactide-based micro- and nanoparticles - Experimental and molecular modeling study.</a> Int J Pharmaceutics. 591, 120031 (2020).</li><li>Yaneva, Z., Georgieva, N., Grumezescu, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+5+-+Physicochemical+and+morphological+characterization+of+pharmaceutical+nanocarriers+and+mathematical+modeling+of+drug+encapsulation/release+mass+transfer+processes.">Chapter 5 - Physicochemical and morphological characterization of pharmaceutical nanocarriers and mathematical modeling of drug encapsulation/release mass transfer processes.</a> Nanoscale Fabrication, Optimization, Scale-Up and Biological Aspects of Pharmaceutical Nanotechnology. , 173-218 (2018).</li><li>Yaneva, Z., Georgieva, N., Staleva, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+d,l-&#945;-tocopherol+acetate/zeolite+carrier+system:+equilibrium+study.">Development of d,l-&#945;-tocopherol acetate/zeolite carrier system: equilibrium study.</a> Monatshefte fur Chemie Chemical Monthly. 147 (7), 1167-1175 (2016).</li><li>Yaneva, Z., Georgieva, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+on+the+physical+chemistry,+equilibrium,+and+kinetic+mechanism+of+Azure+A+biosorption+by+Zea+mays+biomass.">Study on the physical chemistry, equilibrium, and kinetic mechanism of Azure A biosorption by Zea mays biomass.</a> Journal of Dispersion Science and Technology. 35 (2), 193-204 (2014).</li></ol>

The authors have no conflicts of interest to disclose.

Among the main critical issues of modern synthesis methodologies for the design of drug-carrier formulations based on biopolymers is the application of hazardous organic reagents - volatile and flammable solvents, such as tetrahydrofuran, acetone, methanol, and even DMSO in high concentrations - which limits their applicability in biomedicine, pharmaceutical industry, and food technology due to the manifestation of possible toxic effects20,21,<sup clas...

Nowadays inclination towards biopolymers such as cellulose, chitosan, collagen, dextran, gelatin, and lignin as precursors for the design of micro-/submicron carriers with customizable size, physicochemical properties, and biofunctionalities has increased in the biomedical, pharmaceutical, and food technology industries due to their applicability in tissue engineering, 3D bioprinting, in vitro disease modeling platforms, packaging industry, emulsion preparation, and nutrient delivery among others1,2,3.
Novel studies highlight the aspects of lignin-based hydrogels as well as micro- and nano- formulations4 as advantageous vehicles used for food packaging materials5, energy storage6, cosmetics7, thermal/light stabilizers, reinforced materials, and drug-carrier matrices8 for the delivery of hydrophobic molecules, improvement of UV barriers9, as reinforcing agents in nanocomposites, and as an alternative to inorganic nanoparticles due to some recent safety issues10,11,12. The reason behind this tendency is the biocompatibility, biodegradability, and non-toxicity of the natural hetero biopolymer, as well as its proven bioactivities of lignin-antioxidant potential and radical scavenging, anti-proliferative, and antimicrobial activities13,14,15,16,17.
Scientific literature reports various methods for synthesis (self-assembly, anti-solvent precipitation, acid precipitation, and solvent shifting)18 and characterization of lignin-based micro-/nano- scaled formulations, including the application of expensive or harmful solvents such as tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and acetone, and complicated, indirect, and tedious processes that use a lot of equipment and toxic substances12,19,20.
To overcome the latter disadvantages, the following protocols present novel methodologies for the synthesis of lignin-based micro-/submicron particles using cheap and green solvents; easy, straightforward, quick, and sensitive processes requiring little equipment, non-toxic substances, and simple methods for their characterization and the determination of encapsulation capacity towards poorly water-soluble bioactive compounds and in vitro release potential of the lignin matrices. The presented lab-scale production methods are advantageous for the manufacture of functional lignin carriers with tunable sizes, high encapsulation capacity, and sustainable in vitro release behavior utilizing simple characterization procedures and eco-friendly chemicals that can find application in various areas of biomedical sciences and food technology. Two flavonoids were applied as target molecules encapsulated into the lignin particles: morin-into the microparticles, and quercetin-into the submicron particles. The difference in the structures of both flavonoids Is only the position of the second -OH group in the B-aromatic ring: the -OH group is on the 2&#39; position in morin and on the 3&#39; position in quercetin, thus both organic compounds are positional isomers. The latter fact presumes similar behavior of both bioactive natural compounds in the processes of encapsulation and/or release.

<table><tbody><tr><td>automatic-cell counter</td><td>EVE, NanoEnTek</td><td></td><td></td></tr><tr><td>Citric acid</td><td>Sigma</td><td>251275</td><td>&amp;nbsp;ACS reagent, &amp;ge;99.5%</td></tr><tr><td>digital water bath</td><td>Memmert</td><td></td><td></td></tr><tr><td>Eppendorf tubes, 1.5-2 mL</td><td></td><td></td><td></td></tr><tr><td>Ethanol</td><td>Sigma</td><td>34852-M</td><td>absolute, suitable for HPLC, &amp;ge;99.8%</td></tr><tr><td>Folin&amp;ndash;Ciocalteu&amp;rsquo;s phenol reagent</td><td>Sigma</td><td>F9252</td><td></td></tr><tr><td>&amp;nbsp;freeze dryer</td><td>Biobase</td><td></td><td></td></tr><tr><td>gallic acid</td><td>Sigma-</td><td>BCBW7577</td><td>monohydrate</td></tr><tr><td>HCl</td><td>Sigma</td><td>258148</td><td>ACS reagent, 37%</td></tr><tr><td>HNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>438073</td><td>&amp;nbsp;ACS reagent, 70%</td></tr><tr><td>lignin, alkali</td><td>Sigma</td><td>370959</td><td></td></tr><tr><td>morin</td><td>Sigma</td><td>PHL82601</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S9888</td><td>ACS reagent, &amp;ge;99.0%</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;CO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>223530</td><td>powder, &amp;ge;99.5%, ACS reagent</td></tr><tr><td>NaOH</td><td>Sigma</td><td>655104</td><td>reagent grade, 97%, powder</td></tr><tr><td>orbital shaker</td><td>IKA</td><td>KS 130 basic</td><td></td></tr><tr><td>pH-meter</td><td>Consort</td><td></td><td></td></tr><tr><td>phosphate-buffered saline (PBS)</td><td>Sigma</td><td>RNBH7571</td><td></td></tr><tr><td>Quercetin hydrate</td><td>Sigma</td><td>STBG3815V</td><td></td></tr><tr><td>statistical software for Excel</td><td>Microsoft Corporation</td><td></td><td>XLSTAT&amp;nbsp; Version 2022.4.5.</td></tr><tr><td>Tween 80</td><td>Sigma</td><td>P8074</td><td>BioXtra, viscous liquid</td></tr><tr><td>ultracentrifuge</td><td>Hermle</td><td>Z 326 K</td><td></td></tr><tr><td>Ultrapure water system</td><td>Adrona</td><td>INTEGRITY+</td><td></td></tr><tr><td>ultrasound homogenizer</td><td>Bandelin Sonopuls</td><td>HD 2070</td><td></td></tr><tr><td>UV/Vis spectrophotometer</td><td>Hach-Lange</td><td>DR 5000</td><td></td></tr></tbody></table>

green synthesis, characterization, encapsulation, and measurement of the release potential of novel alkali lignin micro-/submicron particles

The applicability of biopolymer micro-/nano- technology in human, veterinary medicine, pharmaceutical, and food technology is rapidly growing due to the great potential of biopolymer-based particles as effective carrier systems. The use of lignin as a basic heteropolymer biomatrix for the design of innovative micro-/submicron formulations allows the achievement of increased biocompatibility and offers various active functional groups presenting opportunities for customization of the physicochemical properties and bioactivities of the formulations for diverse applications. The aim of the present study was to develop a simple and ecofriendly methodology for the synthesis of lignin particles with micro- and submicron size; to evaluate their physicochemical, spectral, and structural characteristics; and to examine their capacity for encapsulation of biologically active molecules and potential for in vitro release of bioflavonoids in simulated gastrointestinal media. The presented methodologies apply cheap and green solvents; easy, straightforward, quick, and sensitive processes requiring little equipment, non-toxic substances, and simple methods for their characterization, the determination of encapsulation capacity towards the poorly water-soluble bioactive compounds morin and quercetin, and the in vitro release potential of the lignin matrices.

An anti-solvent precipitation technique was executed to produce alkali lignin micro-/submicron particles. An aqueous solution of diluted inorganic acid-nitric acid/organic acid-citric acid was dispersed into an alkali lignin aqueous solution, enriched with an eco-friendly surfactant/ethanol, which resulted in the gradual precipitation of the biopolymer solute and, after sonication, a suspension of compact micro-/submicron particles was finally produced (Figure 1).
<p class="jove_content ...

Watch this Scientific Journal Video about Author Spotlight: Development and Characterization of Eco-Friendly Lignin-Based Microparticles for Enhanced Delivery of Bioflavonoids at JoVE.com

Author Spotlight: Development and Characterization of Eco-Friendly Lignin-Based Microparticles for Enhanced Delivery of Bioflavonoids

We describe novel, simple methodologies of synthesis and characterization of biocompatible lignin micro- and submicron particles. These formulations provide a facile approach for the utilization of the heteropolymer, as well as an alternative for the rational design of multifunctional carrier matrices with potential applicability in biomedicine, pharmaceutical technology, and the food industry.

Green Synthesis, Characterization, Encapsulation, and Measurement of the Release Potential of Novel Alkali Lignin Micro-/Submicron Particles

The applicability of biopolymer micro-/nano- technology in human, veterinary medicine, pharmaceutical, and food technology is rapidly growing due to the ...

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Research

JoVE Journal

Chemistry

597 Views.  Trakia University. We describe novel, simple methodologies of synthesis and characterization of biocompatible lignin micro- and submicron particles. These formulations provide a facile approach for the utilization of the heteropolymer, as well as an alternative for the rational design of multifunctional carrier matrices with potential applicability in biomedicine, pharmaceutical technology, and the food industry.

Video: Author Spotlight: Development and Characterization of Eco-Friendly Lignin-Based Microparticles for Enhanced Delivery of Bioflavonoids

Methods for Facilitating Microbial Growth on Pulp Mill Waste Streams and Characterization of the Biodegradation Potential of Cultured Microbes

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high quality paper. Black liquor is a pulping waste generated by the kraft process that has potential for downstream bioconversion. However, the recalcitrant nature of the lignocellulose resources, its chemical derivatives that constitute the majority of available organic carbon within black liquor, and its basic pH present challenges to microbial biodegradation of this waste material. Methods for the collection and modification of black liquor for microbial growth are aimed at utilization of this pulp waste to convert the lignin, organic acids, and polysaccharide degradation byproducts into valuable chemicals. The lignocellulose extraction techniques presented provide a reproducible method for preparation of lignocellulose growth substrates for understanding metabolic capacities of cultured microorganisms. Use of gas chromatography-mass spectrometry enables the identification and quantification of the fermentation products resulting from the growth of microorganisms on pulping waste. These methods when used together can facilitate the determination of the metabolic activity of microorganisms with potential to produce fermentation products that would provide greater value to the pulping system and reduce effluent waste, thereby increasing potential paper milling profits and offering additional uses for black liquor.

Industrial wastes can be collected and modified to analyze microbial growth. Lignocellulose extraction techniques provide components to analyze specific biodegradation ability. Gas chromatography-mass spectrometry identifies fermentation products of microorganisms grown on pulping waste. These methods determine the metabolic capacity of microorganisms to degrade pulping waste.

The kraft process is applied to wood chips for separation of lignin from the polysaccharides within lignocellulose for pulp that will produce a high ...

Environment

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.&#160; Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

Experimental Protocol to Investigate Particle Aerosolization of a Product Under Abrasion and Under Environmental Weathering

The present article presents an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental weathering, which is a fundamental element to the approach of nanosafety-by-design of nanostructured products for their durable development. This approach is basically a preemptive one in which the focus is put on minimizing the emission of engineered nanomaterials' aerosols during the usage phase of the product's life cycle. This can be attained by altering its material properties during its design phase without compromising with any of its added benefits. In this article, an experimental protocol is presented to investigate the nanosafety-by-design of three commercial nanostructured products with respect to their mechanical solicitation and environmental weathering. The means chosen for applying the mechanical solicitation is an abrasion process and for the environmental weathering, it is an accelerated UV exposure in the presence of humidity and heat. The eventual emission of engineered nanomaterials is studied in terms of their number concentration, size distribution, morphology and chemical composition. The purpose of the protocol is to study the emission for test samples and experimental conditions which are corresponding to real life situations. It was found that the application of the mechanical stresses alone emits the engineered nanomaterials' aerosols in which the engineered nanomaterial is always embedded inside the product matrix, thus, a representative product element. In such a case, the emitted aerosols comprise of both nanoparticles as well as microparticles. But if the mechanical stresses are coupled with the environmental weathering, the experimental protocol reveals then the eventual deterioration of the product, after a certain weathering duration, may lead to the emission of the free engineered nanomaterial aerosols too.

In this article, an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental weathering is presented. Results on the emission of engineered nanomaterials, in the form of aerosols are presented. The specific experimental set-up is described in detail.

The present article presents an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental ...

Engineering

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

Testing of Nanoparticle Release from a Composite Containing Nanomaterial Using a Chamber System

With the rapid development of nanotechnology as one of the most important technologies in the 21st century, interest in the safety of consumer products containing nanomaterials is also increasing. Evaluating the nanomaterial release from products containing nanomaterials is a crucial step in assessing the safety of these products, and has resulted in several international efforts to develop consistent and reliable technologies for standardizing the evaluation of nanomaterial release. In this study, the release of nanomaterials from products containing nanomaterials is evaluated using a chamber system that includes a condensation particle counter, optical particle counter, and sampling ports to collect filter samples for electron microscopy analysis. The proposed chamber system is tested using an abrasor and disc-type nanocomposite material specimens to determine whether the nanomaterial release is repeatable and consistent within an acceptable range. The test results indicate that the total number of particles in each test is within 20% from the average after several trials. The release trends are similar and they show very good repeatability. Therefore, the proposed chamber system can be effectively used for nanomaterial release testing of products containing nanomaterials.

Nanoparticle release is tested using a chamber system that includes a condensation particle counter, an optical particle counter and sampling ports to collect filter samples for microscopy analysis. The proposed chamber system can be effectively used for nanomaterial release testing with a repeatable and consistent data range.

With the rapid development of nanotechnology as one of the most important technologies in the 21st century, interest in the safety of consumer products ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Bioengineering

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the emerging elegant procedures to obtain specific aromatic products rely on the lignin substrate having a high content of the readily cleavable &#946;-O-4 linkage as present in the native lignin structure. This provides a miss-match with typical technical lignins that are highly degraded and therefore are low in &#946;-O-4 linkages. Therefore, the extraction yields, and the quality of the obtained lignin are of utmost importance to access new lignin valorization pathways. In this manuscript, a simple protocol is presented to obtain lignins with high &#946;-O-4 content by relatively mild ethanol extraction that can be applied to different lignocellulose sources. Furthermore, analysis procedures to determine the quality of the lignins are presented together with a depolymerization protocol that yields specific phenolic 2-arylmethyl-1,3-dioxolanes, which can be used to evaluate the obtained lignins. The presented results demonstrate the link between lignin quality and potential for the lignins to be depolymerized into specific monomeric aromatic chemicals. Overall, the extraction and depolymerization demonstrates a trade-off between the lignin extraction yield and the retention of the native aryl-ether structure and thus the potential of the lignin to be used as the substrate for the production of chemicals for higher-value applications.

Here, we present a protocol to perform ethanol extraction of lignin from several biomass sources. The effect of the extraction conditions on the lignin yield and &#946;-O-4 content are presented. Selective depolymerization is performed on the obtained lignins to obtain high aromatic monomer products.

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls and is an aromatic heteropolymer primarily composed of three monolignols with significant industrial importance. Lignin plays an important role in plant growth and development, protects&#160;from biotic and abiotic stresses, and in the quality of animal fodder, the wood, and industrial lignin products. Accurate estimation of lignin content is essential for both fundamental understanding of the lignin biosynthesis and industrial applications of biomass. The thioglycolic acid (TGA) method is a highly reliable method of estimating the total lignin content in the plant biomass. This method estimates the lignin content by forming thioethers with the benzyl alcohol groups of lignin, which are soluble in alkaline conditions and insoluble in acidic conditions. The total lignin content is estimated using a standard curve generated from commercial bamboo lignin.

Estimation of Plant Biomass Lignin Content using Thioglycolic Acid TGA

Here, we present a modified TGA method for estimation of lignin content in herbaceous plant biomass. This method estimates the lignin content by forming specific thioether bonds with lignin and presents an advantage over the Klason method, as it requires a relatively small sample for lignin content estimation.

Lignin is a natural polymer that is the second most abundant polymer on Earth after cellulose. Lignin is mainly deposited in plant secondary cell walls ...

Biochemistry

Designed for Molecular Recycling: A Lignin-Derived Semi-aromatic Biobased Polymer

The development of chemically recyclable biopolymers offers opportunities within the pursuit of a circular economy. Chemically recyclable biopolymers make a positive effort to solve the issue of polymer materials in the disposal phase after the use phase. In this paper, the production of biobased semi-aromatic polyesters, which can be extracted entirely from biomass such as lignin, is described and visualized. The polymer poly-S described in this paper has thermal properties similar to certain commonly used plastics, such as PET. We developed a Green Knoevenagel reaction, which can efficiently produce monomers from aromatic aldehydes and malonic acid. This reaction has been proven to be scalable and has a remarkably low calculated E-factor. These polyesters with ligno-phytochemicals as a starting point show an efficient molecular recycling with minimal losses. The polyester poly(dihydrosinapinic acid) (poly-S) is presented as an example of these semi-aromatic polyesters, and the polymerization, depolymerization, and re-polymerization are described.

An example of a closed-loop approach towards a circular materials economy is described here. A whole sustainable cycle is presented where biobased semi-aromatic polyesters are designed by polymerization, depolymerization, and then re-polymerized with only slight changes in their yields or final properties.

The development of chemically recyclable biopolymers offers opportunities within the pursuit of a circular economy. Chemically recyclable biopolymers make ...

Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements as well as power and heat consumption and by using environment-friendly solvents. Deep eutectic solvents (DESs) are key, green, and low-cost solvents in sustainable biorefineries. They are transparent mixtures characterized by low freezing points resulting from at least one hydrogen bond donor and one hydrogen bond acceptor. Although DESs are promising solvents, it is necessary to combine them with an economic heating technology, such as microwave irradiation, for competitive profitability. Microwave irradiation is a promising strategy to shorten the heating time and boost fractionation because it can rapidly attain the appropriate temperature. The aim of this study was to develop a one-step, rapid method for biomass fractionation and lignin extraction using a low-cost and biodegradable solvent. 
In this study, a microwave-assisted DES pretreatment was conducted for 60 s at 800 W, using three kinds of DESs. The DES mixtures were facilely prepared from choline chloride (ChCl) and three hydrogen-bond donors (HBDs): a monocarboxylic acid (lactic acid), a dicarboxylic acid (oxalic acid), and urea. This pretreatment was used for biomass fractionation and lignin recovery from marine residues (Posidonia leaves and aegagropile), agri-food byproducts (almond shells and olive pomace), forest residues (pinecones), and perennial lignocellulosic grasses (Stipa tenacissima). Further analyses were conducted to determine the yield, purity, and molecular weight distribution of the recovered lignin. In addition, the effect of DESs on the chemical functional groups in the extracted lignin was determined by Fourier-transform infrared (FTIR) spectroscopy. The results indicate that the ChCl-oxalic acid mixture affords the highest lignin purity and the lowest yield. The present study demonstrates that the DES-microwave process is an ultrafast, efficient, and cost-competitive technology for lignocellulosic biomass fractionation.

Deep eutectic solvent-based, microwave-assisted pretreatment is a green, fast, and efficient process for lignocellulosic fractionation and high-purity lignin recovery.

Pretreatment is still the most expensive step in lignocellulosic biorefinery processes. It must be made cost-effective by minimizing chemical requirements ...

Green Synthesis, Characterization, Encapsulation, and Measurement of the Release Potential of Novel Alkali Lignin Micro-/Submicron Particles

Summary

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Estimation of Plant Biomass Lignin Content using Thioglycolic Acid (TGA)

Designed for Molecular Recycling: A Lignin-Derived Semi-aromatic Biobased Polymer

Ultrafast Lignin Extraction from Unusual Mediterranean Lignocellulosic Residues