LK is thankful for funding from the National Science Foundation Graduate Research Fellowship Program (DGE-1445197) and Vanderbilt University Chemistry Department. LK and EH would like to thank the funding for the Osiris TEM instrument (NSF EPS 1004083).

1. Synthesis and Characterization of AVL
<ol>
	<li>Place a magnetic stir bar inside a 2 neck 500 mL round bottom flask (Flask 1) and seal with an appropriate sized rubber septum and steel wire. Flame dry the flask to remove moisture by purging with nitrogen gas connected through an inlet needle and open outlet needle in the septum, while using a butane flame torch to gently heat the outside of the flask by moving the flame along the surface.
	<ol>
		<li>Continue heating the entire flask by running the flame across the surface until moisture clouding the inside of the flask is not seen. Remove the flame and let the flask cool to room temperature while continuing the nitrogen flow throughout the reaction.</li>
	</ol>
	</li>
	<li>Add 156.25 mL anhydrous tetrahydrofuran (THF) via a 30 mL syringe to Flask 1. Cool Flask 1 to -78 &#176;C with a dry ice/acetone bath. Monitor the temperature with a thermometer throughout the reaction and add dry ice as needed to maintain -78 &#176;C. Begin stirring the magnetic stir bar using a magnetic stir plate to create a gentle vortex.</li>
	<li>Add N,N-diisopropylethylamine (DIPA) (3.30 mL, 2.3 x 10-2 mol) via a 5 mL syringe, followed by the dropwise addition of 2.5 M n-Butyllithium (9.34 mL, 2.3 x 10-2 mol) via a 10 mL glass syringe equipped with a needle locking mechanism and metal needle to Flask 1. Continue to stir using a magnetic stir plate for 15 min at -78 &#176;C.</li>
	<li>Flame dry (as described in step 1.1) and purge with argon gas (as described in step 1.1 with nitrogen gas) a 100 mL round bottom flask (Flask 2). Remove the argon gas inlet and outlet at the same time once the flask is cool enough to touch. Add 56 mL anhydrous THF via a 30 mL syringe and &#948;-valerolactone (VL) (1.97 mL, 2.1 x 10-2 mol) via a 5 mL syringe.</li>
	<li>Add the solution in Flask 2 to Flask 1 via a small cannula dropwise over 45 min while continuously stirring via a magnetic stir bar. Once Flask 2 is empty, remove the cannula and allow Flask 1 to continue stirring for 15 min at -78 &#176;C.</li>
	<li>Flame dry (as described in step 1.1) and purge with argon gas (as described in step 1.1 with nitrogen gas) a 25 mL round bottom flask (Flask 3). Remove the argon gas inlet and outlet at the same time once the flask is cool enough to touch, to maintain positive pressure inside the flask. Add ally bromide (2.02 mL, 2.3 x 10-2 mol) and hexamethyphosphoramide (HMPA) (4.43 mL, 2.5 x 10-2 mol) each via a 5 mL syringe.</li>
	<li>Add Flask 3 to Flask 1 via a small cannula dropwise. Once Flask 3 is empty, remove the cannula.
	<ol>
		<li>Remove as much dry ice and cold acetone as possible from the dry ice/acetone bath in which Flask 1 is submerged using a scoop and plastic cup. Replace with room temperature acetone to raise the bath to -40 &#176;C and allow Flask 1 to continue stirring for 2 h while monitoring the temperature. If the temperature increases above -40 &#176;C, add a few pieces of dry ice to cool the acetone.</li>
		<li>Warm to -10 &#176;C by removing the cold acetone and dry ice and replace with room temperature acetone.</li>
	</ol>
	</li>
	<li>Remove the septum from Flask 1 and quench the reaction with a 150 mL saturated ammonium chloride solution while stirring via a magnetic stir bar. Place Flask 1 in a freezer at -20 &#176;C overnight for storage.</li>
	<li>Remove Flask 1 from the freezer and allow to warm to room temperature. Pour the solution from Flask 1 into a 500 mL separatory funnel.</li>
	<li>Add 50 mL dichloromethane (DCM), cap the separatory funnel, and rock gently to mix the DCM and aqueous/THF mixture to extract the product into the organic layer. Collect the organic layer into a 500 mL beaker containing an appropriately sized magnetic stir bar by opening the separatory funnel stopcock and set aside. Repeat two times.</li>
	<li>Collect all the organics into the same beaker and stir over magnesium sulfate (to remove any residual water) using a magnetic stir plate until the magnesium sulfate no longer clumps when added to solution. Discard the aqueous waste.</li>
	<li>Remove the magnesium sulfate solid from the organic product solution by using a filter paper inside a glass funnel and collect the organics in a 250 mL round bottom flask. Rinse the filter paper and solid magnesium sulfate with excess DCM to ensure that all the product is collected.</li>
	<li>Remove the solvent from the product by rotary evaporation while heating at 30 &#176;C and using a water aspirator as the vacuum source.</li>
	<li>Purify the crude product by column chromatography10 using a silica gel as the stationary phase and eluting beginning with 3% ethyl acetate/hexanes for 1 column volume (CV), increasing the gradient to 29% ethyl acetate/hexanes over 5 CVs. Collect the fractions in 16 x 150 mm2 borosilicate glass tubes.</li>
	<li>Perform thin layer chromatography using 20% ethyl acetate/hexanes as eluent to determine the product fractions.</li>
	<li>Combine all product fractions into an appropriately sized round bottom flask and remove the solvent using rotary evaporation at 40 &#176;C. Transfer to a pre-weighed product vial and place under a high vacuum at 0.05 torr to obtain pure AVL. Characterize the product by 1H NMR11 (CDCl3, 400 MHz): &#948; 5.75 (m, 1H), 5.06 (m, 2H), 4.26 (m, 2H), 2.54 (m, 3H), 2.28 (m, 1H), 2.03 (m, 1H), 1.87 (m, 1H), 1.54 (m, 1H). Yield: 2.07 g (70.56%).</li>
</ol>
2. Synthesis and Characterization of VL- co- AVL
<ol>
	<li>Flame dry and purge with nitrogen gas a 25 mL round bottom flask equipped with a magnetic stir bar and septum.
	<ol>
		<li>Flame dry the flask to remove moisture by purging with nitrogen gas connected through an inlet needle and open outlet needle in the septum, while using a butane flame torch to gently heat the outside of the flask by moving the flame along the surface.</li>
		<li>Continue heating the entire flask by running the flame across the surface until moisture clouding the inside of the flask is not seen. Remove the flame and let the flask cool to room temperature while continuing the nitrogen flow throughout the reaction.</li>
	</ol>
	</li>
	<li>Quickly remove the septum from the round bottom and add tin(II) triflate (2.5 mg, 5.9&#160; x 10-6 mol) to the very bottom of the flask using a spatula. Replace the septum.</li>
	<li>Add 3-methyl-1-butanol (72.6 &#181;L, 6.6 x 10-3 mol) via a 100 &#181;L microsyringe and 1.33 mL anhydrous DCM via a 2 mL syringe sequentially. Stir via a magnetic stir plate for 10 min.</li>
	<li>Add AVL (0.48 mL, 3.7 x 10-3 mol) and VL (1.37 mL, 1.4 x 10-2 mol) sequentially via a syringe. Allow to continue stirring for 18 - 20 h. 
	NOTE: The AVL and VL percentage in the final copolymer can be altered by stoichiometry in this step.</li>
	<li>Quench the reaction by adding ~5 mL methanol (MeOH). Add ~100 mg solid-support tin scavenger and stir for 2 h. Filter by gravity filtration to remove the solid.</li>
	<li>Remove the solvent under rotary evaporation with a water aspirator as the vacuum source while heating at 30 &#176;C until the solution is viscous. Precipitate the solution dropwise into 500 mL cold MeOH (chilled with dry ice for at least 1 h) to produce flakes of white solid. Filter the solution by vacuum filtration into a funnel containing a fritted glass disc with filter paper to collect the solid. 
	NOTE: If precipitation produces a cloudy supernatant or fine powder which goes through the filter paper, then the product solution is too dilute. All supernatant solvent should be removed by rotary evaporation with a water aspirator as the vacuum source while heating at 30 &#176;C until the solution is viscous and then precipitation should be performed again.</li>
	<li>Transfer the solid product to a pre-weighed vial via a spatula and dry overnight via high vacuum pressure of 0.05 torr to collect the white, flaky solid.</li>
	<li>Characterize the polymer by 1H NMR11 (CDCl3, 400 MHz): &#948; 5.71 (m, 1H), 5.03 (t, 2H), 4.08 (m, 4H), 3.65 (t, 2H), 2.15 - 2.50 (m, 5H), 1.47-1.78 (m, 9H), 0.91 (d, 6H). 81.42% VL, 18.58% AVL, 2,940 g/mol, 578 g/mol repeat unit of AVL. Yield: 1.43 g, (73.43%).</li>
</ol>
3. Post-polymerization Epoxidation to Produce Epoxy-valerolactone (EVL) Copolymer Units
<ol>
	<li>Add VL-co-AVL copolymer (500 mg, 1.7&#160; x 10-4 mol, 2,940 g/mol) to a 6-dram vial with a magnetic stir bar. Add 6.15 mL anhydrous DCM to the vial and vortex to solubilize the polymer.</li>
	<li>Add meta-chloroperoxybenzoic acid (mCPBA) (74.53 mg, 4.3 x 10-4 mol) to a second 6-dram vial. Add 6.15 mL anhydrous DCM and vortex until the mCPBA is solubilized completely. 
	NOTE: The conversion ratio of allyls to epoxides can be varied by altering the stoichiometry in this step. The final allyl concentration in the solvent should be 0.065 M.</li>
	<li>Transfer the mCPBA solution to the VL-AVL solution. Cap the reaction and cover with a plastic paraffin film. Allow to stir for 48 h.</li>
	<li>Transfer the reaction mixture to a 50 mL separatory funnel. Add 15 mL saturated sodium bicarbonate, cap the separatory funnel, and rock gently to mix. Collect the organic layer containing the product into a 50 mL Erlenmeyer flask with an appropriate sized magnetic stir bar (product flask).
	<ol>
		<li>Add 5 mL DCM to the aqueous layer that is still in the separatory funnel, cap, and then rock gently. Collect the organics into the product flask. Discard the aqueous waste. Transfer the organic layer back into a separatory funnel. Repeat this aqueous wash two times.</li>
	</ol>
	</li>
	<li>Add magnesium sulfate (to remove any residual water) to the product flask while stirring over a magnetic stir plate. Continue adding small scoops of magnesium sulfate until it no longer clumps when added. Use a glass funnel fitted with a filter paper to remove the solid magnesium sulfate while transferring the mixture to a 50 mL round bottom flask.</li>
	<li>Remove the solvent by rotary evaporation with a water aspirator as the vacuum source and heating at 25 &#176;C. Transfer the contents of the round bottom flask to a pre-weighed product vial and remove the solvent by rotary evaporation. Place on high vacuum at 0.05 torr overnight to produce a white, waxy solid.</li>
	<li>Characterize by 1H NMR11 (CDCl3, 400 MHz): &#948; 5.71 (m, 1H), 5.03 (t, 2H), 4.08 (m, 4H), 3.65 (t, 2H), 2.94 (d, 1H), 2.90 (s, 1H), 2.74 (s, 1H), 2.15-2.50 (m, 5H), 1.47-1.78 (m, 9H), 0.92 (d, 6H). 84.53% VL, 9.65% AVL, 5.82% EVL, 2,855 g/mol, 1,841 g/mol repeat unit of EVL. Yield: 417.3 mg, (83.46%).</li>
</ol>
4. Nanosponge Synthesis and Characterization
<ol>
	<li>Dissolve the VL-co-AVL-co-EVL polymer (200 mg, 7.0 x 10-5 mol, 2,855 g/mol) in 20.01 mL anhydrous DCM for an epoxide concentration of 0.0054 M. Transfer to a 100 mL round bottom flask with a 14/20 neck. 
	NOTE: To catalyze the reaction, 100 &#181;L aqueous saturated sodium bicarbonate can be added in this step.</li>
	<li>Place the reaction flask in an oil bath at 50 &#176;C. Stir the solution with a fast vortex and add 2,2&#39;-(ethylenedioxy)bis(ethylamine) (21.45 &#181;L, 2.9 10-4 mol) via a microsyringe dropwise. Fit the neck of the flask with a water-jacketed condenser with cool water flowing through it, fitted with a 14/20 neck adapter, and reflux the solution for 12 h. Ensure a very tight seal between the neck of the flask and the condenser to avoid solvent evaporation.</li>
	<li>Remove excess solvent from the reaction flask by rotary evaporation at 25 &#176;C until a viscous solution is obtained.</li>
	<li>Place a large magnetic stir bar into a 2 L beaker. Cut an approximately six-inch section of 10 K molecular weight cut-off (MWCO) dialysis tubing and fold one end then close with a dialysis clip.
	<ol>
		<li>Transfer the product to 10 K MWCO dialysis tubing. Rinse the flask with excess DCM and transfer it to the tubing.Fold the top of the tubing and close with a dialysis clip that has a wire for hanging. Hang the dialysis tubing on the side of the beaker using the wire and fill the beaker with DCM until the dialysis tubing is completely submerged.</li>
		<li>Place on a magnetic stir plate and stir gently. Cover the beaker with aluminum foil to prevent solvent evaporation. Remove the solvent by pouring into a waste container and replace with fresh DCM three times daily for 48 h to remove the unreacted polymer and crosslinker.</li>
	</ol>
	</li>
	<li>Remove all solvent from the beaker and transfer the contents of the dialysis tubing to a 10 mL syringe fitted with a 0.45 &#181;m polytetrafluoroethylene (PTFE) syringe filter. Push the solution through the filter directly into a pre-weighed product vial to remove any remaining impurities. Remove the solvent by rotary evaporation at 25 &#176;C, then place on high vacuum at 0.05 torr overnight to collect the light yellow, waxy solid.</li>
	<li>Characterize by 1H NMR11 (CDCl3, 400 MHz): &#948; 5.71 (m, 1H), 5.03 (t, 2H), 4.08 (m, 4H), 3.60-3.67 (t, 14H), 2.94 (d, 1H), 2.90 (s, 1H), 2.74 (s, 1H), 2.15-2.50 (m, 5H), 1.47-1.78 (m, 9H), 0.92 (d, 6H). Yield: 176.9 mg (88.45%).</li>
</ol>
5. TEM Imaging of Nanosponge Morphology and Size
<ol>
	<li>Filter 5 mL cell culture water through a 0.2 &#181;m PTFE syringe filter.</li>
	<li>Place 0.5 mg nanosponges into a 1.5 mL centrifuge tube. Add 1 mL filtered cell culture water. Use a probe sonicator (20 kHz, 40 a) to sonicate the solution with 2 s bursts 4 - 5 times at room temperature until the particles have developed a fine suspension. Do not use prolonged sonication or let the solution heat up as this will cause aggregation.</li>
	<li>Add 30 mg phosphotungstic acid hydrate (PTA) to 1 mL filtered cell culture water in a 1.5 mL centrifuge tube. Vortex on the highest setting for 10 s or until the PTA is completely solubilized to produce 3% PTA solution.</li>
	<li>Use a 1 mL syringe with 22 G needle to draw up 0.5 mL 3% PTA solution. Add 4 drops of the 3% PTA solution to the particles and vortex on the highest setting for 10 s. Use a pair of self-closing tweezers to pick up a TEM grid then dip it into the particles solution quickly, three times. Let the grid dry for 5 h under a cover to reduce dust collection on the grid.</li>
	<li>Perform TEM imaging12 of the sample using high contrast and 40 &#181;m objective.</li>
</ol>

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The authors have nothing to disclose.

Obtaining reproducible nanosponge sizes is vital in drug delivery applications. Multiple parameters in polymerization and nanosponge synthesis affect the size and crosslink density of the resulting particle. Three important parameters were identified in our analysis: polymer molecular weight, epoxide pendant functionality, and crosslinker equivalents. In order to produce a range of molecular weights and epoxide functionalities for nanosponge synthesis, the stoichiometry of the VL-co-AVL copolymer must be altered. The concentration of the allyl functional group during epoxidation of the copolymer can be used to epoxidize either a desired percentage of allyls or all of them. If an excess of oxidizing agent is used, degradation of the polymer chain can occur; however, this can be remedied by reducing the amount of oxidizing agent. When all allyls are epoxidized, there are no pendant allyls on the surface of the nanosponge for further functionalization. It is also important for nanosponge synthesis that the concentration of epoxide in solution nanosponge synthesis is 0.0054 M.
The nanosponge reaction has been previously evaluated to determine an optimal concentration for desired nanosponge size ranges13. This concentration is calculated based on the repeat unit value for the epoxide functionality in the polymer. The repeat unit is the weight of polymer per one reactive unit, which is used to calculate the moles of reactive units in one polymer. For example, as shown below, if a polymer with a molecular weight of 2,000 g/mol contains 10 reactive monomer units (RMU) bearing pendant functionality, determined by quantitative NMR, the reactive unit of the polymer is 200 g/mol RMU. Using this value, the moles of reactive units can be calculated from the polymer weight in order to determine crosslinker equivalencies for nanosponge synthesis.
<img alt="Equation 1" src="/files/ftp_upload/56073/56073eq1.jpg" />
<img alt="Equation 2" src="/files/ftp_upload/56073/56073eq2.jpg" />
As a general trend, increasing both the polymer molecular weight and epoxide functionality contributed to an increased nanosponge size independently. A narrow polydispersity achieves a narrow nanosponge size distribution (~ 10% standard deviation) and improves reproducibility of nanosponge synthesis.
The presented approach achieves a narrow polymer dispersity by use of a tin triflate catalyst14. The crosslinking equivalencies are calculated based on the amine per epoxide equivalents, and an increase in crosslinker equivalents is shown to increase the nanosponge size. However, using an excess of crosslinker is important due to the goal of consuming all available epoxides. Remaining amine functionality on the nanosponge surface can be used for further functionalization of the particle surface.
Compared to conventional methods for nanoparticle preparation, the advantages in this approach are the multiple parameters by which precise size and density control can be achieved, the ability to further functionalize the surface of the nanosponge, and the solubility in the organics for hydrophobic drug encapsulation.

Precisely tuning the size and crosslinking density of nanoparticles based on intermolecular crosslinking is of great importance to influence and guide the drug release profile of these nanosystems1. Designing nanosponge tunability, i.e., preparing particles of different network densities, is reliant upon the pendant functionality of the precursor polymer and the equivalents of the hydrophilic crosslinker incorporated. In this approach, the concentration of the precursor and crosslinker in the solvent is important to form nanoparticles of a discrete size rather than a bulk gel. Utilizing quantitative nuclear magnetic resonance spectroscopy (NMR) as a characterization technique allows for the precise determination of incorporated pendant functionality and polymer molecular weight. Once nanoparticles are formed, they can be concentrated and solubilized in organics without having the character of a nanogel.
Recent work in nanoparticle drug delivery has focused on the use of poly(lactic-co-glycolic acid) (PLGA) self-assembled nanoparticles2,3,4,5,6. PLGA has degradable ester linkages which make it suitable for drug delivery applications and is often combined with poly(ethylene glycol) (PEG) due to its stealth properties7. However, due to the self-assembled nature of PLGA particle formation, the particles cannot be solubilized in organics for further functionalization. In contrast to PLGA nanoparticles, the proposed method provides covalent crosslinking forming a nanoparticle with defined sizes and morphology, which are stable in organics and degrade in aqueous solutions1. Advantages of this approach are the ability to further chemically functionalize the surface of the nanosponge8, and its stability in organic solvents can be used for the post-loading of the particles with pharmaceutical compounds1,9. With this method, encapsulation of hydrophobic small molecules can be achieved by precipitation into aqueous media. The hydrophobicity of the polyester backbone together with the hydrophilic short crosslinker gives these particles an amorphous character at body temperature. Furthermore, after drug loading, the particle can form fine suspensions in aqueous media to be readily injected in vivo. It is our goal in this work to evaluate the parameters for the synthesis of these polyester nanosponges and determine those which are vitally important to the design and control of size and morphology.

<table width="649">
	<tbody>
		<tr height="42">
			<td align="left" height="42" style="width:216px;height:42px;">2,2&#39;-(Ethylenedioxy)bis(ethylamine)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:119px;">385506-100ML</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">3-methyl-1-butanol</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>309435-100ML</td>
			<td align="left" style="width:187px;">anhydrous, &#8805;99%</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">Acetone</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>179124-4L</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">Allyl bromide</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>A29585-5G</td>
			<td align="left" style="width:187px;">&#8805;99%</td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="width:216px;height:42px;">Ammonium chloride</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td>A661-500</td>
			<td align="left" style="width:187px;">saturated solution in DI water</td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="width:216px;height:42px;">Cell culture water</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>W3500-500ML</td>
			<td align="left" style="width:187px;">Filtered through 0.45 &#956;m syringe filter</td>
		</tr>
		<tr height="63">
			<td align="left" height="63" style="width:216px;height:63px;">Dichloromethane (DCM)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>270997-100ML</td>
			<td align="left" style="width:187px;">anhydrous, &#8805;99%, contains 40-150 ppm amylene as stabilizer</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">Ethyl Acetate</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td>E145SK-4</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">EZFlow 0.2 &#956;m Syringe Filter</td>
			<td style="width:127px;">Foxx Life Sciences</td>
			<td>386-2116-OEM</td>
			<td align="left" style="width:187px;">Hydrophillic PTFE, 13 mm</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">EZFlow 0.45 &#956;m Syringe Filter</td>
			<td style="width:127px;">Foxx Life Sciences</td>
			<td>386-3126-OEM</td>
			<td align="left" style="width:187px;">Hydrophillic PTFE, 25 mm</td>
		</tr>
		<tr height="63">
			<td align="left" height="63" style="width:216px;height:63px;">Fisherbrand Disposable Borosilicate Glass Test Tubes with Plain End</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td>14-961-31</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="width:216px;height:42px;">Fisherbrand Microcentrifuge Tubes</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td>14-666-318</td>
			<td align="left" style="width:187px;">1.5 mL</td>
		</tr>
		<tr height="63">
			<td align="left" height="63" style="width:216px;height:63px;">Hamilton Microliter Syringe, 100 &#956;L</td>
			<td style="width:127px;">Hamilton Company</td>
			<td>80600</td>
			<td align="left" style="width:187px;">Model 710 N SYR, Cemented NDL, 22s ga, 2 in, point style 2</td>
		</tr>
		<tr height="63">
			<td align="left" height="63" style="width:216px;height:63px;">Hexamethylphosphoramide</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>H11602-100G</td>
			<td align="left" style="width:187px;">&#8805;99%, contains &#8804;1000 ppm propylene oxide as stabilizer</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">Hexanes</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td>H292-4</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">Magnesium sulfate anhydrous</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td>M65-500</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="width:216px;height:42px;">Meta-chloroperoxybenzoic acid</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>273031-100G</td>
			<td align="left" style="width:187px;">Purified to &#8805;99% by buffer wash</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">Methanol (MeOH)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>322415-100ML</td>
			<td align="left" style="width:187px;">anhydrous, &#8805;99%</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">N-butyllithium solution</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>230707-100ML</td>
			<td align="left" style="width:187px;">2.5 M in hexanes</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">N,N-diisopropylethylamine</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>550043-500ML</td>
			<td align="left" style="width:187px;">&#8805;99%</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">Parafilm M</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>P7793-1EA</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="width:216px;height:42px;">PELCO Pro Reverse (Self-Closing) Tweezers</td>
			<td style="width:127px;">Ted Pella, Inc.</td>
			<td>5375-NM</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">Phosphotungstic acid hydrate</td>
			<td style="width:127px;">Alfa Aesar</td>
			<td>40116</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">Q55 Sonicator</td>
			<td style="width:127px;">Qsonica</td>
			<td>Q55-110</td>
			<td align="left" style="width:187px;">55 Watts, 20 kHz</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">SiliaMetS Cysteine</td>
			<td style="width:127px;">Silicycle</td>
			<td>R80530B-10g</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">SnakeSkin Dialysis Clips</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td>68011</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="width:216px;height:42px;">SnakeSkin Dialysis Tubing, 10K MWCO</td>
			<td style="width:127px;">Thermo Scientific</td>
			<td>68100</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="width:216px;height:42px;">Sodium bicarbonate</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td>5233-500</td>
			<td align="left" style="width:187px;">saturated solution in DI water</td>
		</tr>
		<tr height="84">
			<td align="left" height="84" style="width:216px;height:84px;">TEM grid</td>
			<td style="width:127px;">Ted Pella, Inc.</td>
			<td>01822-F</td>
			<td align="left" style="width:187px;">Ultrathin Carbon Type-A, 400 mesh, Copper, approx. grid hole size: 42&#181;m</td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="width:216px;height:42px;">Tetrahydrofuran (THF)</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>401757-1L</td>
			<td align="left" style="width:187px;">Anhydrous, &#8805;99.9%, inhibitor-free</td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="width:216px;height:42px;">Tin(II) trifluoromethanesulfonate</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>388122-1G</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="width:216px;height:42px;">Vortex-Genie 2</td>
			<td style="width:127px;">Scientific Industries</td>
			<td>SI-0236</td>
			<td align="left" style="width:187px;"></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="width:216px;height:21px;">Whatman Filter Paper, Grade 1</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td>09-805H</td>
			<td align="left" style="width:187px;">Circles, 185 mm</td>
		</tr>
		<tr height="42">
			<td align="left" height="42" style="width:216px;height:42px;">&#948;-valerolactone</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td>389579-100ML</td>
			<td align="left" style="width:187px;">Purified by vacuum distillation</td>
		</tr>
	</tbody>
</table>

nanosponge tunability in size and crosslinking density

We describe a protocol for the synthesis of linear polyesters containing pendant epoxide functionality and their incorporation into a nanosponge with controlled dimensions. This approach begins with synthesis of a functionalized lactone which is key to the pendant functionalization of the resulting polymer. Valerolactone (VL) and allyl-valerolactone (AVL) are then copolymerized using ring-opening polymerization. Post-polymerization modification is then used to install an epoxide moiety on some or all of the pendant allyl groups. Epoxy-amine chemistry is employed to form nanoparticles in a dilute solution of both polymer and small molecule diamine crosslinker based on the desired nanosponge size and crosslinking density. Nanosponge sizes can be characterized by transmission electron microscopy (TEM) imaging to determine the dimension and distribution. This method provides a pathway by which highly tunable polyesters can create tunable nanoparticles, which can be used for small molecule drug encapsulation. Due to the nature of the backbone, these particles are hydrolytically and enzymatically degradable for a controlled release of a wide range of hydrophobic small molecules.

To evaluate the relationship between the synthesis parameters of the nanosponge and its resultant size, the concentration and pendant functionality of each polymer precursor is important. In Figure 1, a successfulsynthetic scheme of nanosponges is carried out under reflux conditions after incorporating both precursor polymer and diamine crosslinker in DCM for 12 h. The concentration of epoxides in the solution is also critical to forming discrete particles. Once nanosponges were synthesized, TEM imaging was used to determine the precise dimensions of a set of particles. In Figure 2, a collection of various nanosponge experiments was analyzed based on their polymer precursor molecular weight and pendant functionality incorporation to determine if a relationship between the two could have an effect on the nanosponge size. In Figure 2, a trend of increasing particle size is seen as molecular weight is increased for both a 6% and 8% EVL incorporation with one diamine crosslinker per epoxide (2 amines per epoxide).
Figure 3 shows that increasing both the epoxide percentage and crosslinker equivalents would have a similar effect while maintaining a similar molecular weight between nanosponge sets. Again, a trend in increasing nanosponge size while changing these parameters is seen. It is important to understand how the synthesis of polymer precursors can play a vital role in the resultant nanoparticle size to precisely tune nanosponges for various applications. It is also important to maintain a reproducible and reliable method for nanosponge synthesis which has small deviations among individual particle size, as shown by Figure 4. By utilizing these parameters, a range of sizes and a formula for reliably reproducing a nanosponge of a particular size can be developed for a given application or desired goal, proving this to be a versatile and practical nanosponge chemistry.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56073/56073fig1.jpg" /> 
Figure 1: Reaction Scheme for Nanosponge Synthesis. A linear polyester copolymer containing pendant allyl and epoxide functional groups is reacted with a diamine crosslinker to form discrete nanoparticles with size dimensions of approximately 100 nm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56073/56073fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56073/56073fig2.jpg" /> 
Figure 2: Analysis of Nanosponge Tunability Based on Molecular Weight and Pendant Functionality. By evaluating the change in the nanosponge size based on the molecular weight of the precursor polymer while keeping the relative pendant functionality the same, an increase in the particle size as the molecular weight increases can be shown for both 6% and 8% EVL polymers. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56073/56073fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56073/56073fig3.jpg" /> 
Figure 3: Analysis of Nanosponge Tunability by Crosslinker and Pendant Functionality Equivalencies. By holding the crosslinker equivalent steady, a higher pendant functionality will result in higher crosslinker incorporation. In this figure, four amines per epoxide (two diamine crosslinker equivalents per epoxide) were added to both a 6% and 10 % EVL polymer. As more crosslinker is incorporated into the nanosponge due to more epoxides per polymer and higher crosslinker equivalents, the size increases. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56073/56073fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56073/56073fig4.jpg" /> 
Figure 4: TEM Image of Nanosponges. A TEM image of covalently linked nanoparticles formed during synthesis. Indicated size of 79 &#177; 12 nm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56073/56073fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Nanosponge Tunability in Size and Crosslinking Density at JoVE.com

Nanosponge Tunability in Size and Crosslinking Density

This article describes a process for tuning the size and crosslinking density of covalently crosslinked nanoparticles from linear polyesters containing pendant functionality. By tailoring synthesis parameters (polymer molecular weight, pendant functionality incorporation, and crosslinker equivalents), a desired nanoparticle size and crosslinking density can be achieved for drug delivery applications.

We describe a protocol for the synthesis of linear polyesters containing pendant epoxide functionality and their incorporation into a nanosponge with ...

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7.5K Views.  Vanderbilt University. This article describes a process for tuning the size and crosslinking density of covalently crosslinked nanoparticles from linear polyesters containing pendant functionality. By tailoring synthesis parameters (polymer molecular weight, pendant functionality incorporation, and crosslinker equivalents), a desired nanoparticle size and crosslinking density can be achieved for drug delivery applications.

Video: Nanosponge Tunability in Size and Crosslinking Density

A Simple Method for the Size Controlled Synthesis of Stable Oligomeric Clusters of Gold Nanoparticles under Ambient Conditions

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like oligomeric clusters of these yellow nanoparticles of narrow size distribution are synthesized under ambient conditions via two methods. The delay-time method controls the number of subunits in the oligoclusters by varying the time between the addition of HAuCl4 to alkaline solution and the subsequent addition of reducing agent, NaSCN. The yellow oligoclusters produced range in size from ~3 to ~25 nm. This size range can be further extended by an add-on method utilizing hydroxylated gold chloride (Na+[Au(OH4-x)Clx]-) to auto-catalytically increase the number of subunits in the as-synthesized oligocluster nanoparticles, providing a total range of 3 nm to 70 nm. The crude oligocluster preparations display narrow size distributions and do not require further fractionation for most purposes. The oligoclusters formed can be concentrated &#62;300 fold without aggregation and the crude reaction mixtures remain stable for weeks without further processing. Because these oligomeric clusters can be concentrated before derivatization they allow expensive derivatizing agents to be used economically. In addition, we present two models by which predictions of particle size can be made with great accuracy.

We describe a simple method for producing highly stable oligomeric clusters of gold nanoparticles via the reduction of chloroauric acid (HAuCl4) with sodium thiocyanate (NaSCN). The oligoclusters have a narrow size distribution and can be produced with a wide range of sizes and surface coats.

Reducing dilute aqueous HAuCl4 with sodium thiocyanate (NaSCN) under alkaline conditions produces 2 to 3 nm diameter nanoparticles. Stable grape-like ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications.

This protocol outlines the fabrication of a large-scale, multiplexed two-dimensional DNA or antibody array, with potential applications in cell signaling studies and biomarker detection.

Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this ...

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)1 and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH4 (KOH-sol Fr) as described by Ratnayake et al. (2014)2. Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSn. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

Multiscale Sampling of a Heterogeneous Water/Metal Catalyst Interface using Density Functional Theory and Force-Field Molecular Dynamics

A significant number of heterogeneously-catalyzed chemical processes occur under liquid conditions, but simulating catalyst function under such conditions is challenging when it is necessary to include the solvent molecules. The bond breaking and forming processes modeled in these systems necessitate the use of quantum chemical methods. Since molecules in the liquid phase are under constant thermal motion, simulations must also include configurational sampling. This means that multiple configurations of liquid molecules must be simulated for each catalytic species of interest. The goal of the protocol presented here is to generate and sample trajectories of configurations of liquid water molecules around catalytic species on flat transition metal surfaces in a way that balances chemical accuracy with computational expense. Specifically, force field molecular dynamics (FFMD) simulations are used to generate configurations of liquid molecules that can subsequently be used in quantum mechanics-based methods such as density functional theory or ab initio molecular dynamics. To illustrate this, in this manuscript, the protocol is used for catalytic intermediates that could be involved in the pathway for the decomposition of glycerol (C3H8O3). The structures that are generated using FFMD are modeled in DFT in order to estimate the enthalpies of solvation of the catalytic species and to identify how H2O molecules participate in catalytic decompositions.

The goal of the protocol presented here is to generate and sample trajectories of configurations of liquid water molecules around catalytic species on a flat transition metal surface. The sampled configurations can be used as starting structures in quantum mechanics-based methods.

A significant number of heterogeneously-catalyzed chemical processes occur under liquid conditions, but simulating catalyst function under such conditions ...

Synthesis of Graphene Nanofluids with Controllable Flake Size Distributions

A method for synthesizing graphene nanofluids with controllable flake size distributions is presented. Graphene nanoflakes can be obtained by the exfoliation of graphite in the liquid phase, and the exfoliation time is used to control the lower limits of the graphene nanoflake size distributions. Centrifugation is successfully used to control the upper limits of the nanoparticle size distributions. The objective of this work is to combine exfoliation and centrifugation to control the graphene nanoflake size distributions in the resulting suspensions.

A method for synthesizing graphene nanofluids with controllable flake size distributions is presented.