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

<ol>
	<li>Yu, X., 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+nanoparticles+with+high+phenolic+content+as+efficient+antioxidant+and+sun-blocker+for+food+and+cosmetics.">Lignin nanoparticles with high phenolic content as efficient antioxidant and sun-blocker for food and cosmetics.</a> ACS Sustainable Chem. Eng. 11 (10), 4082-4092 (2023).</li><li>Boarino, A., Klok, H. -. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opportunities+and+challenges+for+lignin+valorization+in+food+packaging,+antimicrobial,+and+agricultural+applications.">Opportunities and challenges for lignin valorization in food packaging, antimicrobial, and agricultural applications.</a> Biomacromolecules. 24 (3), 1065-1077 (2023).</li><li>Aadil, K., Barapatre, A., Jha, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+characterization+of+Acacia+lignin-gelatin+film+for+its+possible+application+in+food+packaging.">Synthesis and characterization of Acacia lignin-gelatin film for its possible application in food packaging.</a> Bioresour. Bioprocess. 3 (27), 1-11 (2016).</li><li>Sharma, S., 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=Valorization+of+lignin+into+nanoparticles+and+nanogel:+characterization+and+application.">Valorization of lignin into nanoparticles and nanogel: characterization and application.</a> Bioresour. Technol. Reports. 18, 101041 (2022).</li><li>Zadeh, E. M., O'Keefe, S. F., Kim, Y. -. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+lignin+in+biopolymeric+packaging+films.">Utilization of lignin in biopolymeric packaging films.</a> ACS Omega. 3 (7), 7388-7398 (2018).</li><li>Beaucamp, A., 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+for+energy+applications+-+state+of+the+art,+life+cycle,+technoeconomic+analysis+and+future+trends+(Critical+Review).">Lignin for energy applications - state of the art, life cycle, technoeconomic analysis and future trends (Critical Review).</a> Green Chem. 24, 8193-8226 (2022).</li><li>Antunes, F., 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=From+sugarcane+to+skin:+Lignin+as+a+multifunctional+ingredient+for+cosmetic+application.">From sugarcane to skin: Lignin as a multifunctional ingredient for cosmetic application.</a> Int J Biol Macromol. 234, 123592 (2023).</li><li>Garg, J., 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=Applications+of+lignin+nanoparticles+for+cancer+drug+delivery:+An+update.">Applications of lignin nanoparticles for cancer drug delivery: An update.</a> Materials Letters. 311, 131573 (2022).</li><li>Anushikha, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignin+as+a+UV+blocking,+antioxidant,+and+antimicrobial+agent+for+food+packaging+applications.">Lignin as a UV blocking, antioxidant, and antimicrobial agent for food packaging applications.</a> Biomass Conv. Bioref. , 1-14 (2023).</li><li>Freitas, F. M. C., 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=synthesis+of+lignin+nano-+and+micro-particles:+Physicochemical+characterization,+bioactive+properties+and+cytotoxicity+assessment.">synthesis of lignin nano- and micro-particles: Physicochemical characterization, bioactive properties and cytotoxicity assessment.</a> Int J Biol Macromol. 163, 1798-1809 (2020).</li><li>Rismawati, R., Nurdin, I. A., Pradiptha, M. N., Maulidiyah, A., Mubarakati, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+lignin+nanoparticles+from+rice+straw+after+biosynthesis+using+Lactobacillus+bulgaricus.">Preparation and characterization of lignin nanoparticles from rice straw after biosynthesis using Lactobacillus bulgaricus.</a> Journal of Physics: Conference Series. 9th International Seminar on New Paradigm and Innovation of Natural Sciences and its Application. 1524, 012070 (2020).</li><li>Worku, L. A., 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=Synthesis+of+lignin+nanoparticles+from+Oxytenanthera+abyssinica+by+nanoprecipitation+method+followed+by+ultrasonication+for+the+nanocomposite+application.">Synthesis of lignin nanoparticles from Oxytenanthera abyssinica by nanoprecipitation method followed by ultrasonication for the nanocomposite application.</a> Journal of King Saud University - Science. 35 (7), 102793 (2023).</li><li>Gala Morena, A., Tzanov, T. z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibacterial+lignin-based+nanoparticles+and+their+use+in+composite+materials.">Antibacterial lignin-based nanoparticles and their use in composite materials.</a> Nanoscale Adv. 4, 4447-4469 (2022).</li><li>Ivanova, D., Nikolova, G., Karamalakova, Y., Marutsova, V., 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=Water-soluble+alkali+lignin+as+a+natural+radical+scavenger+and+anticancer+alternative.">Water-soluble alkali lignin as a natural radical scavenger and anticancer alternative.</a> Int J Mol Sci. 24 (16), 12705 (2023).</li><li>Ivanova, D., Toneva, M., Simeonov, E., Antov, G., 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=Newly+synthesized+lignin+microparticles+as+bioinspired+oral+drug-delivery+vehicles:+Flavonoid-carrier+potential+and+in+vitro+radical-scavenging+activity.">Newly synthesized lignin microparticles as bioinspired oral drug-delivery vehicles: Flavonoid-carrier potential and in vitro radical-scavenging activity.</a> Pharmaceutics. 15 (4), 1067 (2023).</li><li>Yaneva, Z., 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=Antimicrobial+potential+of+conjugated+lignin/morin/chitosan+combinations+as+a+function+of+system+complexity.">Antimicrobial potential of conjugated lignin/morin/chitosan combinations as a function of system complexity.</a> Antibiotics. 11, 650 (2022).</li><li>Handral, H. K., Wyrobnik, T. A., Lam, A. T. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+trends+in+biodegradable+microcarriers+for+therapeutic+applications.">Emerging trends in biodegradable microcarriers for therapeutic applications.</a> Polymers. 15 (6), 1487 (2023).</li><li>Figueiredo, P., Lintinen, K., Hirvonen, J. T., Kostiainen, M. A., Santos, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+and+chemical+modifications+of+lignin:+Towards+lignin-based+nanomaterials+for+biomedical+applications.">Properties and chemical modifications of lignin: Towards lignin-based nanomaterials for biomedical applications.</a> Prog. Mater. Sci. 93, 233-269 (2018).</li><li>Tang, Q., 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-based+nanoparticles:+a+review+on+their+preparations+and+applications.">Lignin-based nanoparticles: a review on their preparations and applications.</a> Polymers. 12 (11), 2471 (2020).</li><li>Zhao, W., Simmons, B., Singh, S., Ragauskas, A., Cheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+lignin+association+to+nano-/micro-particle+preparation:+extracting+higher+value+of+lignin.">From lignin association to nano-/micro-particle preparation: extracting higher value of lignin.</a> Green Chemistry. 18 (21), 5693-5700 (2016).</li><li>Stewart, H., Golding, M., Matia-Merino, L., Archer, R., Davies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manufacture+of+lignin+microparticles+by+anti-solvent+precipitation:+Effect+of+preparation+temperature+and+presence+of+sodium+dodecyl+sulfate.">Manufacture of lignin microparticles by anti-solvent precipitation: Effect of preparation temperature and presence of sodium dodecyl sulfate.</a> Food Res Int. 66, 93-99 (2014).</li><li>Beisl, S., Friedl, A., Miltner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lignin+from+micro-+to+nanosize:+Applications.">Lignin from micro- to nanosize: Applications.</a> Int. J. Mol. Sci. 18, 2367 (2017).</li><li>Mishra, P. K., Ekielski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+to+synthesize+lignin+nanoparticles.">A simple method to synthesize lignin nanoparticles.</a> Colloids Interfaces. 3, 52 (2019).</li><li>Qian, Y., Deng, Y., Qiu, X., Li, H., Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+uniform+colloidal+spheres+from+lignin,+a+renewable+resource+recovered+from+pulping+spent+liquor.">Formation of uniform colloidal spheres from lignin, a renewable resource recovered from pulping spent liquor.</a> Green Chem. 16, 2156-2163 (2014).</li><li>Tardy, B. 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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;ACS reagent, &#8805;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, &#8805;99.8%</td>
		</tr>
		<tr>
			<td>Folin&#8211;Ciocalteu&#8217;s phenol reagent</td>
			<td>Sigma</td>
			<td>F9252</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;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>HNO3</td>
			<td>Sigma</td>
			<td>438073</td>
			<td>&#160;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, &#8805;99.0%</td>
		</tr>
		<tr>
			<td>Na2CO3</td>
			<td>Sigma</td>
			<td>223530</td>
			<td>powder, &#8805;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&#160; 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

588 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

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.
In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges21-23 and (b) grippers that self-fold due to residual stress powered hinges24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales22, 26 and with a range of materials including metals21, semiconductors9 and polymers27. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 &mu;m to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films5,7 to possibly create even smaller nanoscale grasping devices.

We describe experimental details of the synthesis of patterned and reconfigurable particles from two dimensional (2D) precursors. This methodology can be used to create particles in a variety of shapes including polyhedra and grasping devices at length scales ranging from the micro to centimeter scale.

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

Exfoliation of Egyptian Blue and Han Blue, Two Alkali Earth Copper Silicate-based Pigments

In a visualized example of the ancient past connecting with modern times, we describe the preparation and exfoliation of CaCuSi4O10 and BaCuSi4O10, the colored components of the historic Egyptian blue and Han blue pigments. The bulk forms of these materials are synthesized by both melt flux and solid-state routes, which provide some control over the crystallite size of the product. The melt flux process is time intensive, but it produces relatively large crystals at lower reaction temperatures. In comparison, the solid-state method is quicker yet requires higher reaction temperatures and yields smaller crystallites. Upon stirring in hot water, CaCuSi4O10 spontaneously exfoliates into monolayer nanosheets, which are characterized by TEM and PXRD. BaCuSi4O10 on the other hand requires ultrasonication in organic solvents to achieve exfoliation. Near infrared imaging illustrates that both the bulk and nanosheet forms of CaCuSi4O10 and BaCuSi4O10 are strong near infrared emitters. Aqueous CaCuSi4O10 and BaCuSi4O10 nanosheet dispersions are useful because they provide a new way to handle, characterize, and process these materials in colloidal form.

The preparation and exfoliation of CaCuSi4O10&#160;and BaCuSi4O10 are described. Upon stirring in hot water, CaCuSi4O10 spontaneously exfoliates into monolayers, whereas BaCuSi4O10&#160;requires ultrasonication in organic solvents. Near infrared (NIR) imaging illustrates the NIR emission properties of these materials, and aqueous dispersions of these nanomaterials are useful for solution processing.

In a visualized example of the ancient past connecting with modern times, we describe the preparation and exfoliation of CaCuSi4O10 and BaCuSi4O10, the ...

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the hydroxylmethylene phosphine precursor with ammonia in methanol under a nitrogen atmosphere. The N-triphosPh ligand precipitates from the solution after approximately 1 hr of reflux and can be isolated analytically pure via simple cannula filtration procedure under nitrogen. Reaction of the N-triphosPh ligand with [Ru3(CO)12] under reflux affords a deep red solution that show evolution of CO gas on ligand complexation. Orange crystals of the complex [Ru(CO)2{N(CH2PPh2)3}-&#954;3P] (2) were isolated on cooling to RT. The 31P{1H} NMR spectrum showed a characteristic single peak at lower frequency compared to the free ligand. Reaction of a toluene solution of complex 2 with oxygen resulted in the instantaneous precipitation of the carbonate complex [Ru(CO3)(CO){N(CH2PPh2)3}-&#954;3P] (3) as an air stable orange solid. Subsequent hydrogenation of 3 under 15 bar of hydrogen in a high-pressure reactor gave the dihydride complex [RuH2(CO){N(CH2PPh2)3}-&#954;3P] (4), which was fully characterized by X-ray crystallography and NMR spectroscopy. Complexes 3 and 4 are potentially useful catalyst precursors for a range of hydrogenation reactions, including biomass-derived products such as levulinic acid (LA). Complex 4 was found to cleanly react with LA in the presence of the proton source additive NH4PF6 to give [Ru(CO){N(CH2PPh2)3}-&#954;3P{CH3CO(CH2)2CO2H}-&#954;2O](PF6) (6).

The Synthesis, Characterization and Reactivity of a Series of Ruthenium N-triphosPh Complexes

Ruthenium phosphine complexes are widely used for homogeneous catalytic reactions such as hydrogenations. The synthesis of a series of novel tridentate ruthenium complexes bearing the N-triphos ligand N(CH2PPh2)3 is reported. Additionally, the stoichiometric reaction of a dihydride Ru&#8211;N-triphos complex with levulinic acid is described.

Herein we report the synthesis of a tridentate phosphine ligand N(CH2PPh2)3 (N-triphosPh) (1) via a phosphorus based Mannich reaction of the ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Extraction of Ramie Fiber in Alkali Hydrogen Peroxide System Supported by Controlled-release Alkali Source

This protocol demonstrates a method for ramie fiber extraction by scouring raw ramie in an alkali hydrogen peroxide system supported by a controlled-release alkali source. The fiber extracted from ramie is a type of textile material of great importance. In previous studies, ramie fiber was extracted in an alkali hydrogen peroxide system supported only by sodium hydroxide.However, due to the strong alkalinity of sodium hydroxide, the oxidation reaction speed of hydrogen peroxide was difficult to control and thus resulted in great damage to the treated fiber. In this protocol, a controlled-release alkali source, which is composed of sodium hydroxide and magnesium hydroxide, is used to provide an alkali condition and buffer the pH value of the alkali hydrogen peroxidesystem. The substitution rate of magnesium hydroxide can adjust the pH value of the hydrogen peroxide system and has great influence on the fiber properties. The pH value and oxidation-reduction potential (ORP) value, which represents the oxidation ability of alkali hydrogen peroxide system, were monitored using a pH meter and ORP meter, respectively. The residual hydrogen peroxide content in the alkali hydrogen peroxide system during the extraction process and the chemical oxygen demand (COD) value of wastewater after fiber extraction are tested by KMnO4 titration method. The yield of fiber is measured using a precision electronic balance, and residual gums of fiber are tested by a chemical analysis method. The polymerization degree (PD value) of fiber is tested by an intrinsic viscosity method using the Ubbelohde viscometer. The tensile property of fiber, including tenacity, elongation, and rupture, is measured using a fiber strength instrument. Fourier transform infrared spectroscopy and X-ray diffraction are used to characterize the functional groups and crystal property of fiber. This protocol proves that the controlled-release alkali source can improve the properties of the fiber extracted in an alkali hydrogen peroxide system.

Presented here is a protocol for extraction of ramie fiber in alkali hydrogen peroxide system supported by controlled-release alkali source.

This protocol demonstrates a method for ramie fiber extraction by scouring raw ramie in an alkali hydrogen peroxide system supported by a ...

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

High Resolution Physical Characterization of Single Metallic Nanoparticles

Individual molecules can be detected and characterized by measuring the degree by which they reduce the ionic current flowing through a single nanometer-scale pore. The signal is characteristic of the molecule&#39;s physicochemical properties and its interactions with the pore. We demonstrate that the nanopore formed by the bacterial protein exotoxin Staphylococcus aureus alpha hemolysin (&#945;HL) can detect polyoxometalates (POMs, anionic metal oxygen clusters), at the single molecule limit. Moreover, multiple degradation products of 12-phosphotungstic acid POM (PTA, H3PW12O40) in solution are simultaneously measured. The single molecule sensitivity of the nanopore method allows for POMs to be characterized at significantly lower concentrations than required for nuclear magnetic resonance (NMR) spectroscopy. This technique could serve as a new tool for chemists to study the molecular properties of polyoxometalates or other metallic clusters, to better understand POM synthetic processes, and possibly improve their yield. Hypothetically, the location of a given atom, or the rotation of a fragment in the molecule, and the metal oxidation state could be investigated with this method. In addition, this new technique has the advantage of allowing the real-time monitoring of molecules in solution.&#160;

Here, we present a protocol to detect discrete metal oxygen clusters, polyoxometalates (POMs), at the single molecule limit using a biological nanopore-based electronic platform. The method provides a complementary approach to traditional analytical chemistry tools used in the study of these molecules.