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...

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><tbody><tr><td>2,2&#039;-(Ethylenedioxy)bis(ethylamine)</td><td>Sigma-Aldrich</td><td>385506-100ML</td><td></td></tr><tr><td>3-methyl-1-butanol</td><td>Sigma-Aldrich</td><td>309435-100ML</td><td>anhydrous, &amp;ge;99%</td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>179124-4L</td><td></td></tr><tr><td>Allyl bromide</td><td>Sigma-Aldrich</td><td>A29585-5G</td><td>&amp;ge;99%</td></tr><tr><td>Ammonium chloride</td><td>Fisher Scientific</td><td>A661-500</td><td>saturated solution in DI water</td></tr><tr><td>Cell culture water</td><td>Sigma-Aldrich</td><td>W3500-500ML</td><td>Filtered through 0.45 &amp;mu;m syringe filter</td></tr><tr><td>Dichloromethane (DCM)</td><td>Sigma-Aldrich</td><td>270997-100ML</td><td>anhydrous, &amp;ge;99%, contains 40 - 150 ppm amylene as stabilizer</td></tr><tr><td>Ethyl Acetate</td><td>Fisher Scientific</td><td>E145SK-4</td><td></td></tr><tr><td>EZFlow 0.2 &amp;mu;m Syringe Filter</td><td>Foxx Life Sciences</td><td>386-2116-OEM</td><td>Hydrophillic PTFE, 13 mm</td></tr><tr><td>EZFlow 0.45 &amp;mu;m Syringe Filter</td><td>Foxx Life Sciences</td><td>386-3126-OEM</td><td>Hydrophillic PTFE, 25 mm</td></tr><tr><td>Fisherbrand Disposable Borosilicate Glass Test Tubes with Plain End</td><td>Fisher Scientific</td><td>14-961-31</td><td></td></tr><tr><td>Fisherbrand Microcentrifuge Tubes</td><td>Fisher Scientific</td><td>14-666-318</td><td>1.5 mL</td></tr><tr><td>Hamilton Microliter Syringe, 100 &amp;mu;L</td><td>Hamilton Company</td><td>80600</td><td>Model 710 N SYR, Cemented NDL, 22s ga, 2 in, point style 2</td></tr><tr><td>Hexamethylphosphoramide</td><td>Sigma-Aldrich</td><td>H11602-100G</td><td>&amp;ge;99%, contains &amp;le;1,000 ppm propylene oxide as stabilizer</td></tr><tr><td>Hexanes</td><td>Fisher Scientific</td><td>H292-4</td><td></td></tr><tr><td>Magnesium sulfate anhydrous</td><td>Fisher Scientific</td><td>M65-500</td><td></td></tr><tr><td>Meta-chloroperoxybenzoic acid</td><td>Sigma-Aldrich</td><td>273031-100G</td><td>Purified to &amp;ge;99% by buffer wash</td></tr><tr><td>Methanol (MeOH)</td><td>Sigma-Aldrich</td><td>322415-100ML</td><td>anhydrous, &amp;ge;99%</td></tr><tr><td>N-butyllithium solution</td><td>Sigma-Aldrich</td><td>230707-100ML</td><td>2.5 M in hexanes</td></tr><tr><td>N,N-diisopropylethylamine</td><td>Sigma-Aldrich</td><td>550043-500ML</td><td>&amp;ge;99%</td></tr><tr><td>Parafilm M</td><td>Sigma-Aldrich</td><td>P7793-1EA</td><td></td></tr><tr><td>PELCO Pro Reverse (Self-Closing) Tweezers</td><td>Ted Pella, Inc.</td><td>5375-NM</td><td></td></tr><tr><td>Phosphotungstic acid hydrate</td><td>Alfa Aesar</td><td>40116</td><td></td></tr><tr><td>Q55 Sonicator</td><td>Qsonica</td><td>Q55-110</td><td>55 Watts, 20 kHz</td></tr><tr><td>SiliaMetS Cysteine</td><td>Silicycle</td><td>R80530B-10g</td><td></td></tr><tr><td>SnakeSkin Dialysis Clips</td><td>Thermo Scientific</td><td>68011</td><td></td></tr><tr><td>SnakeSkin Dialysis Tubing, 10K MWCO</td><td>Thermo Scientific</td><td>68100</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Fisher Scientific</td><td>5233-500</td><td>saturated solution in DI water</td></tr><tr><td>TEM grid</td><td>Ted Pella, Inc.</td><td>01822-F</td><td>Ultrathin Carbon Type-A, 400 mesh, Copper, approx. grid hole size: 42 &amp;micro;m</td></tr><tr><td>Tetrahydrofuran (THF)</td><td>Sigma-Aldrich</td><td>401757-1L</td><td>Anhydrous, &amp;ge;99.9%, inhibitor-free</td></tr><tr><td>Tin(II) trifluoromethanesulfonate</td><td>Sigma-Aldrich</td><td>388122-1G</td><td></td></tr><tr><td>Vortex-Genie 2</td><td>Scientific Industries</td><td>SI-0236</td><td></td></tr><tr><td>Whatman Filter Paper, Grade 1</td><td>Fisher Scientific</td><td>09-805H</td><td>Circles, 185 mm</td></tr><tr><td>&amp;delta;-valerolactone</td><td>Sigma-Aldrich</td><td>389579-100ML</td><td>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. O...

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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

Fabrication, Densification, and Replica Molding of 3D Carbon Nanotube Microstructures

The introduction of new materials and processes to microfabrication has, in large part, enabled many important advances in microsystems, lab-on-a-chip devices, and their applications. In particular, capabilities for cost-effective fabrication of polymer microstructures were transformed by the advent of soft lithography and other micromolding techniques 1, 2, and this led a revolution in applications of microfabrication to biomedical engineering and biology. Nevertheless, it remains challenging to fabricate microstructures with well-defined nanoscale surface textures, and to fabricate arbitrary 3D shapes at the micro-scale. Robustness of master molds and maintenance of shape integrity is especially important to achieve high fidelity replication of complex structures and preserving their nanoscale surface texture. The combination of hierarchical textures, and heterogeneous shapes, is a profound challenge to existing microfabrication methods that largely rely upon top-down etching using fixed mask templates. On the other hand, the bottom-up synthesis of nanostructures such as nanotubes and nanowires can offer new capabilities to microfabrication, in particular by taking advantage of the collective self-organization of nanostructures, and local control of their growth behavior with respect to microfabricated patterns. 
Our goal is to introduce vertically aligned carbon nanotubes (CNTs), which we refer to as CNT "forests", as a new microfabrication material. We present details of a suite of related methods recently developed by our group: fabrication of CNT forest microstructures by thermal CVD from lithographically patterned catalyst thin films; self-directed elastocapillary densification of CNT microstructures; and replica molding of polymer microstructures using CNT composite master molds. In particular, our work shows that self-directed capillary densification ("capillary forming"), which is performed by condensation of a solvent onto the substrate with CNT microstructures, significantly increases the packing density of CNTs. This process enables directed transformation of vertical CNT microstructures into straight, inclined, and twisted shapes, which have robust mechanical properties exceeding those of typical microfabrication polymers. This in turn enables formation of nanocomposite CNT master molds by capillary-driven infiltration of polymers. The replica structures exhibit the anisotropic nanoscale texture of the aligned CNTs, and can have walls with sub-micron thickness and aspect ratios exceeding 50:1. Integration of CNT microstructures in fabrication offers further opportunity to exploit the electrical and thermal properties of CNTs, and diverse capabilities for chemical and biochemical functionalization 3.

We present methods for fabrication of patterned microstructures of vertically aligned carbon nanotubes (CNTs), and their use as master molds for production of polymer microstructures with organized nanoscale surface texture. The CNT forests are densified by condensation of solvent onto the substrate, which significantly increases their packing density and enables self-directed formation of 3D shapes.

The introduction of new materials and processes to microfabrication has, in large part, enabled many important advances in microsystems, lab-on-a-chip ...

Engineering

Fine-tuning the Size and Minimizing the Noise of Solid-state Nanopores

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the creation of a nanopore in a thin insulating membrane remains challenging. Fabrication methods involving specialized focused electron beam systems can produce well-defined nanopores, but yield of reliable and low-noise nanopores in commercially available membranes remains low2,3 and size control is nontrivial4,5. Here, the application of high electric fields to fine-tune the size of the nanopore while ensuring optimal low-noise performance is demonstrated. These short pulses of high electric field are used to produce a pristine electrical signal and allow for enlarging of nanopores with subnanometer precision upon prolonged exposure. This method is performed in situ in an aqueous environment using standard laboratory equipment, improving the yield and reproducibility of solid-state nanopore fabrication.

A methodology for preparing solid-state nanopores in solution for biomolecular translocation experiments is presented. By applying short pulses of high electric fields, the nanopore diameter can be controllably enlarged with subnanometer precision and its electrical noise characteristics significantly improved. This procedure is performed in situ using standard laboratory equipment under experimental conditions.

Solid-state nanopores have emerged as a versatile tool for the characterization of single biomolecules such as nucleic acids and proteins1. However, the ...

Functionalization of Single-walled Carbon Nanotubes with Thermo-reversible Block Copolymers and Characterization by Small-angle Neutron Scattering

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous solution. In a carbon nanotube/PEO105-PPO70-PEO105 (poloxamer 407) aqueous solution, the amphiphilic poloxamer 407 adsorbs onto the carbon nanotube surfaces and self-assembles into continuous layers, driven by intermolecular interactions between constituent molecules. The addition of 5-methylsalicylic acid changes the self-assembled structure from spherical-micellar to a cylindrical morphology. The fabricated poloxamer 407/carbon nanotube hybrid particles exhibit thermo-responsive structural features so that the density and thickness of poloxamer 407 layers are also reversibly controllable by varying temperature. The detailed structural properties of the poloxamer 407/carbon nanotube particles in suspension can be characterized by small-angle neutron scattering experiments and model fit analyses. The distinct curve shapes of the scattering intensities depending on temperature control or addition of aromatic additives are well described by a modified core-shell cylinder model consisting of a carbon nanotube core cylinder, a hydrophobic shell, and a hydrated polymer layer. This method can provide a simple but efficient way for the fabrication and in-situ characterization of carbon nanotube-based nano particles with a structure-tunable encapsulation.

A method for the functionalization of carbon nanotubes with structure-tunable polymeric encapsulation layers and structural characterization using small-angle neutron scattering is presented.

We demonstrate a protocol for single-walled carbon nanotube functionalization using thermo-sensitive PEO-PPO-PEO triblock copolymers in an aqueous ...

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

Grafting Multiwalled Carbon Nanotubes with Polystyrene to Enable Self-Assembly and Anisotropic Patchiness

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a free-radical polymerization strategy to enable the modulation of the nanotube surface properties and produce supramolecular self-assembly of the nanostructures. First, a selective hydroxylation of the pristine nanotubes through a biphasic catalytically mediated oxidation reaction creates superficially distributed reactive sites at the sidewalls. The latter reactive sites are subsequently modified with methacrylic moieties using a silylated methacrylic precursor to create polymerizable sites. Those polymerizable groups can address further polymerization of styrene to produce a hybrid nanomaterial containing PS chains grafted to the nanotube sidewalls. The polymer-graft content, amount of silylated methacrylic moieties introduced and hydroxylation modification of the nanotubes are identified and quantified by Thermogravimetric Analysis (TGA). The presence of reactive functional groups hydroxyl and silylated methacrylate are confirmed by Fourier Transform Infrared Spectroscopy (FT-IR). Polystyrene-grafted carbon nanotube solutions in tetrahydrofuran (THF) provide wall-to-wall collinearly self-assembled nanotubes when cast samples are analyzed by transmission electron microscopy (TEM). Those self-assemblies are not obtained when suitable blanks are similarly cast from analogous solutions containing non-grafted counterparts. Therefore, this method enables the modification of the nanotube anisotropic patchiness at the sidewalls which results into spontaneous auto-organization at the nanoscale.

A procedure for the synthesis of polystyrene-grafted multiwalled carbon nanotubes using successive chemical modification steps to selectively introduce the polymer chains to the sidewalls and their self-assembly via anisotropic patchiness is presented.

We demonstrate a straightforward protocol to graft pristine multiwalled carbon nanotubes (MWCNTs) with polystyrene (PS) chains at the sidewalls through a ...

Multiscale Structures Aggregated by Imprinted Nanofibers for Functional Surfaces

Multiscale surface structures have attracted increasing interest owing to several potential applications in surface devices. However, an existing challenge in the field is the fabrication of hybrid micro-nano structures using a facile, cost-effective, and high-throughput method. To overcome these challenges, this paper proposes a protocol to fabricate multiscale structures using only an imprint process with an anodic aluminum oxide (AAO) filter and an evaporative self-aggregation process of nanofibers. Unlike previous attempts that have aimed to straighten nanofibers, we demonstrate a unique fabrication method for multiscale aggregated nanofibers with high aspect ratios. Furthermore, the surface morphology and wettability of these structures on various liquids were investigated to facilitate their use in multifunctional surfaces.

Presented is an easy method to fabricate nano-micro multiscale structures, for functional surfaces, by aggregating nanofibers fabricated using an anodic aluminum oxide filter.

Multiscale surface structures have attracted increasing interest owing to several potential applications in surface devices. However, an existing ...

Fabricating Degradable Thermoresponsive Hydrogels on Multiple Length Scales via Reactive Extrusion, Microfluidics, Self-assembly, and Electrospinning

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), their ultimate clinical use has been hampered by the lack of biologically-relevant degradation observed for most smart materials. This is particularly true for temperature-responsive hydrogels, which are almost uniformly based on polymers that are functionally non-degradable (e.g., poly(N-isopropylacrylamide) (PNIPAM) or poly(oligoethylene glycol methacrylate) (POEGMA)). As such, to effectively translate the potential of thermoresponsive hydrogels to the challenges of remote-controlled or metabolism-regulated drug delivery, cell scaffolds with tunable cell-material interactions, theranostic materials with the potential for both imaging and drug delivery, and other such applications, a method is required to render the hydrogels (if not fully degradable) at least capable of renal clearance following the required lifetime of the material. To that end, this protocol describes the preparation of hydrolytically-degradable hydrazone-crosslinked hydrogels on multiple length scales based on the reaction between hydrazide and aldehyde-functionalized PNIPAM or POEGMA oligomers with molecular weights below the renal filtration limit. Specifically, methods to fabricate degradable thermoresponsive bulk hydrogels (using a double barrel syringe technique), hydrogel particles (on both the microscale through the use of a microfluidics platform facilitating simultaneous mixing and emulsification of the precursor polymers and the nanoscale through the use of a thermally-driven self-assembly and cross-linking method), and hydrogel nanofibers (using a reactive electrospinning strategy) are described. In each case, hydrogels with temperature-responsive properties similar to those achieved via conventional free radical cross-linking processes can be achieved, but the hydrazone cross-linked network can be degraded over time to re-form the oligomeric precursor polymers and enable clearance. As such, we anticipate these methods (which may be generically applied to any synthetic water-soluble polymer, not just smart materials) will enable easier translation of synthetic smart materials to clinical applications.

Protocols are described for the fabrication of degradable thermoresponsive hydrogels based on hydrazone cross-linking of polymeric oligomers on the bulk scale, microscale, and nanoscale, the latter for preparation of both gel nanoparticles and nanofibers.

While various smart materials have been explored for a variety of biomedical applications (e.g., drug delivery, tissue engineering, bioimaging, etc.), ...

Bioengineering

Expanding Nanopatterned Substrates Using Stitch Technique for Nanotopographical Modulation of Cell Behavior

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a large area of nanopatterned substrate is desirable so that enough cells can be cultured on the nanotopography for subsequent biochemical and molecular biology analyses. However, current nanofabrication techniques have limitations to generate highly defined nanopatterns over a large area. Herein, we present a method to expand nanopatterned substrates from a small, highly defined nanopattern to a large area using stitch technique. The method combines multiple techniques, involving soft lithography to replicate poly(dimethylsiloxane) (PDMS) molds from a well-defined mold, stitch technique to assemble multiple PDMS molds to a single large mold, and nanoimprinting to generate a master mold on polystyrene (PS) substrates. With the PS master mold, we produce PDMS working substrates and demonstrate nanotopographical modulation of cell spreading. This method provides a simple, affordable yet versatile avenue to generate well-defined nanopatterns over large areas, and is potentially extended to create micro-/nanoscale devices with hybrid components.

A protocol for producing a large area of nanopatterned substrate from small nanopatterned molds for study of nanotopographical modulation of cell behavior is presented.

Substrate nanotopography has been shown to be a potent modulator of cell phenotype and function. To dissect nanotopography modulation of cell behavior, a ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to their versatile bottom-up synthesis and biocompatibility. While various methods have been employed for nanogel synthesis to date, very few have achieved it without the use of harsh organic solvents or high temperatures that can damage the integrity of the biological payload. In contrast, the methodology presented here accomplishes the synthesis of sub-100 nm sized, protein-loaded nanogels using mild reaction conditions. Here, we present a method for the non-covalent encapsulation of protein-based payloads within nano-gels that were synthesized using an aqueous-based, single-step, crosslinking copolymerization technique. In this technique, we initially electrostatically bind a protein-based payload to a cationic quaternary ammonium monomer and simultaneously cross-link and co-polymerize it using ammonium persulfate and N,N,N&#39;,N&#39;-tetramethylethylenediamine to form nanogels that entrap the protein payload. The size and polydispersity index of the nanogels is determined using dynamic light scattering (DLS), while the surface morphology is assessed by transmission electron microscopy (TEM). The mass of protein entrapped within nanogels is determined by calculating the encapsulation efficiency. Furthermore, the controlled-release ability of the nanogels via the gradual degradation of redox-responsive structural elements is also assessed in bioreduction assays. We provide examples of nanoparticle optimization data to demonstrate all caveats of nanogel synthesis and characterization using this technique. In general, uniformly sized nanogels were obtained with an average size of 57 nm and a polydispersity index value of 0.093. A high encapsulation efficiency of 76% was achieved. Furthermore, the nanogels exhibited controlled release of up to 86% of the encapsulated protein by gradual degradation of novel redox-responsive components in the presence of glutathione over 48 h.

Nanogels are an excellent and versatile nanoparticle platform for the delivery of biologics. Stimuli-responsive poly(ethylene) glycol-based polymeric nano-gels, capable of encapsulating protein-based payloads, were synthesized using a one-step cross-linking co-nanopolymerization strategy in aqueous conditions. The optimal fabrication and characterization of these novel nanoparticles are presented here.

Nanogels consisting of crosslinked-polymeric nanoparticles have been developed for the delivery of numerous chemical and biological therapeutics, owing to ...