Funding: This research is funded through the support of the Rowland Fellowship at the Rowland Institute at Harvard University, the Harvard PSE Accelerator Fund, and the Gordon and Betty Moore Foundation. A portion of this work was performed at the Harvard Center for Nanoscale Systems (CNS), a member of the National Nanotechnology Coordinated Infrastructure Network (NNCI), which is supported by the National Science Foundation under NSF, Award No. 1541959. A portion of this work was performed at the Stanford Nano Shared Facilities (SNSF), supported by the National Science Foundation under award ECCS-2026822. A portion of this work was performed at the Stanford ChEM-H Macromolecular Structure Knowledge Center.
Acknowledgements: THS and SNS acknowledge the support of Arnold O. Beckman Postdoctoral Fellowships. MS acknowledges financial support through a Doc. Mobility Fellowship from the Swiss National Science Foundation (Project No. P1SKP2 187676). PN acknowledges the support of a Stanford Graduate Fellowship in Science &#38; Engineering (SGF) as a Gabilan Fellow. MH was partially supported by the Defense Advanced Research Projects Agency under Grant No. HR00112220010. AOG acknowledges the support of a National Science Foundation Graduate Research Fellowship under Grant DGE-1656518 and a Stanford Graduate Fellowship in Science &#38; Engineering (SGF) as a Scott A. and Geraldine D. Macomber Fellow.

1. Large-scale upconversion nanocapsule synthesis
<ol>
	<li>In a glovebox (see Table of Materials) with an inert atmosphere under red lighting, prepare saturated solutions of the sensitizer (PdTPTBP) and annihilator (TIPS-anthracene) (see Table of Materials) in 99% oleic acid at room temperature (~22 &#176;C).
	<ol>
		<li>Add 2 mL of oleic acid to 20 mg of PdTPTBP in a vial with a stir bar. Then, cover the vial with foil to protect from ambient light. Add 2 mL of oleic acid to 25 mg of TIPS-anthracene in a vial with a stir bar.</li>
		<li>Stir the mixtures at 600 rpm for at least 4 h before filtering with a 0.45 &#181;m PTFE syringe filter. Each solution should visibly have undissolved solid to be removed by filtration, signifying that each solution is saturated.</li>
		<li>Using a syringe, prepare 1.75 mL of the upconversion material stock solution by mixing 0.7 mL of the filtered TIPS-anthracene solution, 0.35 mL of the filtered PdTPTBP solution, and 0.7 mL of oleic acid. 
		NOTE: The upconversion solution used for the nanocapsules has a ratio of 2:1:2 of TIPS-anthracene to PdTPTBP to oleic acid by volume.</li>
	</ol>
	</li>
	<li>Measure 4 g of 10K MPEG-silane in a clean 20 mL vial so that it is ready for use during synthesis. This can be conducted inside or outside of the glovebox. If this material is measured outside of the glovebox, secure the vial lid with sealing film or electrical tape before bringing it into the glovebox.</li>
	<li>In a 250 mL Erlenmeyer flask sealed with a septum, chill 200 mL of ultrapure deionized water in an ice bath for at least 1 h to reach ~5 &#176;C . Typically, this takes a few hours.</li>
	<li>Secure the septum to the flask using at least six pieces of sealing film. This is to ensure that the septum remains affixed when the flask is under vacuum in the glovebox antechamber.</li>
	<li>Bring the chilled water into the glovebox immediately before preparing the nanocapsules. Only pull a light vacuum on the antechamber when bringing in the water by pulling 20% vacuum based on the measurement on the antechamber pressure gauge.</li>
	<li>After bringing the water into the glovebox, immediately turn on the glovebox purge feature to bypass the column. This removes the oxygen introduced when bringing in the water under light vacuum and prolongs the life of the column. Keep the purge on until the synthesis is complete, and all waste has been removed from the glovebox.</li>
	<li>Ensure all chemicals and consumables are ready to use, including syringes and needles for dispensing (3-aminopropyl)triethoxysilane (APTES) and tetraethyl orthosilicate. Make sure the 10K MPEG-silane is within reach. For cleaning, nylon cloths are also useful to have available.</li>
	<li>Plug in the blender (see Table of Materials). Cover the electrical sockets with a plastic bin or nylon cloth. This barrier allows for protection in the case of an unexpected blender leak. Make sure the blender is powered off.</li>
	<li>Carefully pour the water into the blender. Add 1.45 mL of the upconversion material stock solution (prepared in step 1.1.3) in one portion with a syringe into the center of the water in the blender.</li>
	<li>Affix the lid and cover it with a nylon wipe in the case of an unexpected leak. Blend at the maximum speed (22,600 rpm) for exactly 60 s while holding the blender lid to prevent small leaks.</li>
	<li>Turn off the blender and move it out of the way to ensure adequate working space.</li>
	<li>Transfer the emulsion to a 500 mL round bottom flask. Secure the flask to a stir plate with a clamp. Mix the emulsion vigorously at 1200 rpm with an egg-shaped stir bar (see Table of Materials).</li>
	<li>Using a syringe, add 0.75 mL of APTES to the emulsion to generate a clear solution of micelles.</li>
	<li>Add 4 g of 10K MPEG-silane to prevent capsule aggregation. Shake the flask if needed to ensure it is dispersed. Stir at 1200 rpm for approximately 10 min.</li>
	<li>During this time, dry the blender and lid with a nylon cloth. Use tongs to keep hands far away from the sharp blender blades.</li>
	<li>After 10 min have passed, add 15 mL of tetraethyl orthosilicate in one portion using a 20 mL syringe. Add another 15 mL of tetraethyl orthosilicate in one portion using a 20 mL syringe for a total of 30 mL. Affix a septum to the flask and stir at 1200 rpm for 30 min.</li>
	<li>Remove the flask and waste from the glovebox and turn off the glovebox purge.</li>
	<li>Affix the flask to a stir plate with a heating element, such as an oil bath or aluminum heating block. Connect the flask to a Schlenk line so the reaction is held at a constant pressure under an inert gas such as nitrogen or argon.</li>
	<li>Stir and heat the reaction at 65 &#176;C at a speed of 1200 rpm for 40 h.</li>
	<li>After 40 h, disconnect the reaction from the Schlenk line to add 4 g of 10K MPEG-silane. Reconnect the reaction to the Schlenk line. Stir and heat the reaction at 65 &#176;C at 1200 rpm for 8 h.</li>
	<li>After 8 h, turn off the heat and allow the reaction to cool to room temperature while stirring at 1200 rpm.</li>
	<li>When the reaction is cool, transfer the reaction into centrifuge tubes.
	<ol>
		<li>For a centrifuge (see Table of Materials) that holds 50 mL centrifuge tubes, split the reaction equally among 10 centrifuge tubes.</li>
		<li>For a centrifuge that holds 0.5 L centrifuge tubes, split the reaction equally among two centrifuge tubes.</li>
	</ol>
	</li>
	<li>Centrifuge the suspension at 8670 x g for 1 h at a temperature of 20-22 &#176;C. Discard the pellet and retain the supernatant containing the nanocapsules.</li>
	<li>Centrifuge the supernatant at 8670 x g for 14-16 h at 20-22 &#176;C.</li>
	<li>Discard the supernatant and collect the pellet containing upconversion nanocapsules.
	<ol>
		<li>Using a pipette, carefully rinse the top surface of the nanocapsule pellet with ultrapure deionized water (2 x 10 mL). This should be conducted at a low flow so the pellet is not dislodged from the centrifuge tube.</li>
		<li>Transfer the nanocapsule paste into two or three separate 20 mL scintillation vials with a spatula and immediately bring the vials into the glovebox. Approximately 7-10 g of nanocapsule paste should be recovered. 
		NOTE: For further use, it is recommended that the nanocapsules are dispersed into a solvent such as a monomer for 3D printing or deoxygenated ultrapure deionized water within 48 h of synthesis. Water will evaporate from the nanocapsule paste and leave the nanocapsules unusable after 48 h.</li>
	</ol>
	</li>
	<li>Perform scanning electron microscopy (SEM), dynamic light scattering (DLS), and upconversion photoluminescence to characterize the nanocapsule preparation.</li>
</ol>
2. Small-scale upconversion nanocapsule synthesis
<ol>
	<li>Prepare the stock solutions of the sensitizer and the annihilator as described in step 1.1. Scale down the volume of the solution used to make upconversion nanocapsules to 250 &#956;L instead of the 1.75 mL described in step 1.1. Mix 100 &#956;L of the filtered TIPS-an solution with 50 &#956;L of the filtered PdTPTBP solution and 100 &#956;L of oleic acid.</li>
	<li>Vigorously sparge 20 mL of ultrapure deionized water in a 40 mL scintillation vial (see Table of Materials) with an inert gas, such as nitrogen or argon, using a Schlenk line for at least 10 min. Affix the lid with electrical tape or sealing film prior to bringing the vial into the glovebox. 
	NOTE: If making multiple small-scale samples at once, larger volumes of water can be sufficiently degassed by blending 200 mL of chilled water as outlined in section 1 using a clean, unused blender pitcher. Sparging water with an inert gas on a Schlenk line is not effective at volumes greater than 20 mL.</li>
	<li>Measure 400 mg of 10K MPEG-silane so that it is ready for use during synthesis into a clean 10 mL vial. This can be conducted inside or outside of the glovebox. If this is measured outside of the glovebox, secure the vial&#39;s lid with sealing film or electrical tape before bringing it into the glovebox.</li>
	<li>Bring the sparged water into the glovebox, and immediately turn on the glovebox purge feature to bypass the column. This whisks away oxygen introduced when bringing in the water under light vacuum and prolongs the life of the column. The purge should remain on until the synthesis is complete, and all waste has been removed from the glovebox.</li>
	<li>Ensure all chemicals and consumables (5 mL syringes and a micropipette with tips) are ready to use.
	<ol>
		<li>Using a syringe, remove 1 mL of (3-aminopropyl)triethoxysilane from the bottle and dispense it into a clean, labeled 20 mL vial for later use.</li>
		<li>Using a syringe, remove 5 mL of tetraethyl orthosilicate and dispense it into a clean, labeled 20 mL vial for later use.</li>
		<li>Make sure the 10K MPEG-silane is within reach in the glovebox.</li>
		<li>For cleaning, extra nylon cloths are also useful to have available.</li>
	</ol>
	</li>
	<li>Plug in the vortex mixer (see Table of Materials) and set the speed to the highest setting (3200 rpm).</li>
	<li>Using a micropipette, add 145 &#181;L of sensitizer/annihilator stock solution to a vial of water (20 mL). Affix the lid with electrical tape or sealing film.</li>
	<li>Vortex the solution at the highest speed of the vortex mixer (3200 rpm) for 7 min to ensure nanodroplet formation similar to the large-scale synthesis. Hold the vial close to the base and never hold on to the vial lid during vortexing, as the lid can become loose and detach from the vial.</li>
	<li>Affix the vial to a stir plate. Stir the emulsion at 1200 rpm with an octagon-shaped stir bar (see Table of Materials).</li>
	<li>Using a micropipette, add 75 &#181;L of APTES to generate a clear solution of micelles.</li>
	<li>After generating the clear solution, immediately add 400 mg of 10K MPEG-silane. Affix the lid and shake the vial to efficiently mix the reaction. Return the vial to the stir plate.</li>
	<li>Using a syringe, add 3 mL of tetraethyl orthosilicate in sequence while the reaction is being stirred at 1200 rpm. Affix the lid and shake the vial to efficiently mix the reaction. Stir the reaction at 1200 rpm until it is removed from the glovebox.</li>
	<li>Seal the vial with electrical tape or sealing film and remove the vial from the glovebox.</li>
	<li>Heat the solution at 65 &#176;C using an oil bath or aluminum heating block. Stir the reaction at 1200 rpm for 40 h.</li>
	<li>After 40 h, add 400 mg of 10K MPEG-silane. Reseal the vial with electrical tape or sealing film. Stir the reaction at 1200 rpm for 8 h.</li>
	<li>Allow the reaction to cool to room temperature while stirring at 1200 rpm. When the reaction is cool, combine the reaction mixtures into one 50 mL centrifuge tube.</li>
	<li>Centrifuge the suspension at 8670 x g for 1 h at a temperature of 20-22 &#176;C. Discard the pellet and retain the supernatant containing the nanocapsules.</li>
	<li>Centrifuge the supernatant at 8670 x g for 14-16 h at 20-22 &#176;C.</li>
	<li>Discard the supernatant and retain the pellet containing upconversion nanocapsules. Using a pipette, carefully rinse the top surface of the nanocapsule pellet with 2 x 1 mL of ultrapure deionized water. This should be conducted at a low flow so the pellet is not dislodged from the centrifuge tube.</li>
	<li>Transfer the nanocapsule paste into a 20 mL scintillation vial with a spatula and bring the vials into the glovebox immediately. Approximately 700-1000 mg of nanocapsule paste should be recovered. 
	NOTE: For further use, it is recommended that the nanocapsules are dispersed into a solvent, such as a monomer for 3D printing or deoxygenated ultrapure deionized water, within 48 h. Water will evaporate from the nanocapsule paste and will leave the nanocapsules unusable after 48 h.</li>
</ol>

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Harvard University has filed several patents based on this work. SNS, RCS, and DNC are co-founders of Quadratic3D, Inc.

There are several considerations when preparing bright upconverting nanocapsules. First, the synthesis is completed in a glovebox because the upconversion materials must be protected from oxygen-it is well established that upconverted light output is reduced in the presence of oxygen13,14,15,16. Additionally, the sensitizer and annihilator stock solutions should be prepared fresh for every batch. PdTPTBP and other metalated porphyrins have been shown to demetalate in ambient lighting in the presence of acid18, and anthracenes are known to aggregate over time19. These effects can be minimized by preparing fresh solutions under red lighting for each synthesis. The authors note that rigorous red lighting is no longer required once the metalated porphyrin and anthracene are mixed, and ambient lighting is acceptable to use after this step. Finally, for the large-scale synthesis, it is recommended that at least 1.75 mL of the upconverting stock solution is prepared, since adding less than 1.45 mL of this solution to make UCNCs will alter the proportions of all other required reagents as well as the concentration-dependent nanodroplet formation. Similarly, for the small-scale synthesis, it is recommended that 250 &#181;L of the upconverting stock solution is prepared in the same proportions. Finally, when using a micropipette for dispensing the oleic acid stock solutions, slowly release the plunger and wait for it to fully rise to dispense the desired volume. The oleic acid will slowly fill the pipette tip due to its high viscosity and it is easy to inadvertently dispense less solution than expected.
It is important to understand that the oleic acid nanodroplet generation is sensitive to blending time, speed, and significant temperature changes. For instance, the blender selection is significant and can impact the formation of oleic acid nanodroplets. Multiple blender brands were tested in the initial development stages. The blender recommended in the Table of Materials led to the generation of relatively superior and reproducible nanocapsules described in this protocol. Notably, powerful blending increases the temperature of the emulsion and reduces the oleic acid nanodroplet formation efficiency. The blender blades must be completely submerged in water to best control the temperature, which was one consideration for determining the required water volume presented here10. Additionally, chilling the water in advance reduces droplet aggregation in the emulsion, which ultimately improves the nanocapsule yield for the large-scale synthesis. On the other hand, for the small-scale synthesis, chilling the water does not significantly alter the oleic nanodroplet formation, probably because holding the 40 mL vial does not increase the temperature of the water as much as the blender blades.
The APTES addition is a significant synthetic step, as APTES stabilizes the oleic acid nanodroplets generated by blending or vortexing. The initial nanodroplet emulsion is a cloudy, turbid dispersion. Upon addition of APTES, the solution becomes clear and transparent as the nanodroplets are stabilized. On average, the APTES volumes required are very close to what is presented in the protocol, but sometimes slightly less or slightly more APTES is required for the solution to become clear. Thus, the APTES addition should be treated in an analogous manner to conducting other titrations20. Adding too much APTES (i.e., beyond a &#34;just clear&#34; solution) will disrupt nanocapsule shell formation and decrease yield. To that end, if significantly different volumes of APTES are required to produce a clear suspension, or a clear suspension is never reached, this indicates troubleshooting is required to optimize the oleic acid nanodroplet formation. For instance, if the nanodroplet generation is inefficient, the droplet volume and thus surface area of the nanodroplet will be larger than expected and may require more APTES. This has been observed in the small-scale synthesis, and can be remedied in a variety of ways, such as the force used to hold a vial against the vortex mixer or by increasing the vortexing time.
Additionally, the 10K MPEG-silane must be added immediately after APTES to prevent aggregation and cannot be omitted10. Without the addition of 10K MPEG-silane, irreversible aggregation is observed within ~30 min in the form of precipitate generation. Although 5K MPEG-silane can be substituted for 10K MPEG-silane, lower molecular weight MPEG-silanes do not sufficiently prevent aggregation at a constant concentration.
The silica shell formation is key to impart UCNC durability when dispersed in various solutions. While silica shell growth is generally well studied21,22,23, the often-used21 acid or base catalysis to promote silica growth is not used here, as the heating is sufficient for generating a durable, cross-linked silica shell. To monitor the silica shell formation over time, bright upconversion should be observed after 100x dilution of a nanocapsule reaction aliquot in an organic solvent, such as acetone, with minimal sensitizer phosphorescence for the PdTPTBP/TIPS-an system (Figure 2D and reference10). Typically, bright upconversion is observable after about 24 h, but 48 h will increase the relative emission, signifying that a larger population of the UCNCs possess a durable shell. Note that the UC emission is dependent upon the irradiation power and sufficient power densities should be employed. For instance, in the system described here, power densities on the order of ~65 W cm-2 are required to see bright upconverted PL.
The second addition of 10K MPEG-silane after 40 h of silica growth improves nanocapsule dispersibility in organic solvents. While the UCNCs will still be dispersible in multiple solvents without this second 10K MPEG-silane addition, the second addition is highly recommended to increase the UCNC loadings by mass in solution. For instance, for use in a 3D printing resin, 0.67 g mL-1 of nanocapsule paste was dispersed in acrylic acid10.
Exposing the UCNCs to oxygen during the entire multi-day fabrication process results in the ingress of oxygen in concentrations that significantly reduce upconversion photoluminescence. To ensure an inert atmosphere is maintained during the 48 h of stirring in an ambient atmosphere, different protocols are invoked depending upon the reaction scale. At large scales, the ethanol generated during silica growth can produce significant pressures which can lead to the removal of an affixed septum or the loss in structural integrity of the reaction vessel24. Thus, the 500 mL flask should be connected to a Schlenk line to allow for a pressure release in an inert atmosphere. At small scales, sealing a 40 mL glass vial with sealing film or electrical tape maintains the seal&#39;s structural integrity. Without sealing the vial&#39;s lid, the increase in pressure will slowly unthread the lid and allow for the ingress of oxygen.
The reaction purification by centrifugation separates the UCNCs from other undesired side products. Multiple centrifuge brands and rotors are compatible with this purification if the g force provided in the protocol is accessible. The g force can be converted to rotations per minute based upon the centrifuge rotor dimensions25. Exposing the UCNCs to an ambient atmosphere briefly during centrifugation is acceptable as long as they are stored in an inert atmosphere after purification. One limitation of this synthesis is that atom yield is difficult to quantify in relation to the input chemicals. After centrifugation, this large-scale nanocapsule synthesis should yield roughly 10 g of paste and the small-scale synthesis should yield roughly 1.0 g of capsule paste. It is unclear how much of the TEOS is incorporated into making the UCNC shell. The pellet discarded after the first centrifugation is comprised of large molecular weight silica that is not incorporated into the UCNCs. After the second centrifugation, the supernatant can be centrifuged again to increase the mass collected. It is not recommended to increase the centrifugation time beyond 16 h, as the soft capsule paste will solidify into a compact film that cannot be dispersed in other solvents. Even so, the capsule paste masses collected from batch to batch are consistent and are sufficient for subsequent use and characterization.
The UCNC durability can vary from solvent to solvent as well as with storage conditions. While the UCNC paste collected by centrifugation is unusable after 48 h as water evaporates, the nanocapsules are durable in a variety of solvents. In water, the UCNC durability is in the order of several months. In acrylic acid, the durability is reduced to days mostly because the acrylic acid solvent is unstable and can undergo polymerization when stored in oxygen-free conditions10,26. Further solvent-dependent investigations of UCNC durability are ongoing.
The small-scale synthesis is especially useful for relative comparisons of upconversion photoluminescence among different formulations. The NC paste collected after the second centrifugation should be dispersed in water at concentration of 100-200 mg mL-1 and diluted in acetone (or another solvent as desired). A minimum of 25% of the solution volume must contain water (e.g., 25/75 water/acetone v/v) to keep the NCs suspended and prevent precipitates from forming. Comparing the relative upconversion emission among batches was required to determine the concentrations of sensitizer and annihilator in this protocol. Perhaps counterintuitively, the ratio of sensitizer to annihilator required to maximize the light output in UC nanocapsules for 3D printing may not be equivalent to the ratio that maximizes the UC quantum yield27 in oleic acid stock solutions.
In conclusion, a detailed protocol and best practices for synthesizing upconversion nanocapsules is expanded upon in a step-by-step fashion10. Since other methods to encapsulate upconversion materials for use in real-life applications are only compatible with aqueous environments16, this synthesis is significant because it allows for upconversion materials to be deployed into diverse chemical environments, such as organic solvents. These methods will serve to increase approaches to access volumetric 3D printing for precision additive manufacturing and in any application requiring high-energy light beyond the surface.

Moving away from subtractive manufacturing processes (i.e., complex shapes made by carving blocks of raw material) can reduce waste and increase production rates. Accordingly, many industries are moving toward additive manufacturing processes, where objects are built layer-by-layer1 by means of three-dimensional (3D) printing. Many are working to develop additive manufacturing processes for numerous classes of materials (e.g., glass2, ceramics3,4, metals5, and plastics6,7).
This layer-by-layer curing limits resin selection and impacts the print&#39;s mechanical properties6,7. Considering light-based 3D printing for making plastics, two-photon absorption (2PA)-based printing moves away from the layer-by-layer processes by printing volumetrically8. The 2PA process requires simultaneous absorption of two photons to initiate polymerization. This not only increases the requisite power inputs, but also increases the complexity and cost of the printing system, limiting the print sizes to the mm3 scale or smaller9.
Recently, a new 3D printing methodology using triplet fusion upconversion (UC) has made volumetric 3D printing with UC possible on the cm3 scale10. Excitingly, this process requires relatively low power density irradiation10 as compared to 2PA-based printing9,11,12. The upconversion process converts two low energy photons into one high energy photon13, and the upconverted light is absorbed by the photoinitiator to initiate polymerization. Deploying triplet fusion UC materials has traditionally been challenging due to high material concentration requirements, poor solubility, and oxygen sensitivity13,14,15. Encapsulating UC materials using a variety of nanoparticle schemes has been well studied16 but falls short of the durability required in organic solvents. The silica-coated oleic acid upconversion nanocapsule (UCNC) synthetic protocol described here overcomes this durability challenge for dispersion of UC materials in a wide variety of organic solvents, including 3D printing resins10. The upconverted light generated from materials inside of the nanocapsules is patterned in multiple dimensions to generate support structure-free solid objects, which allows for printing high resolution structures with a resolution as small as 50 &#181;m10. By removing support structures and printing in an oxygen-free environment, new resin chemistries are accessible to achieve both improved and novel material properties inaccessible with traditional stereolithography.
Here, the UCNC synthetic protocol is outlined for encapsulating the sensitizer (palladium (II) meso-tetraphenyl tetrabenzoporphine, PdTPTBP) and the annihilator (9,10-bis((triisopropylsilyl)ethynyl)anthracene, TIPS-an) at two different scales. Synthesis on a large scale provides material to provide ~10 g of upconversion nanocapsule paste for use in 3D printing resins. Synthesis on a small scale for ~1 g of upconversion nanocapsule paste allows for the optimization of new nanocapsule contents. This protocol will support the successful integration of triplet fusion UCNCs into a variety of 3D printing workflows and other applications.

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>(3-aminopropyl)triethoxysilane, anhydrous</td><td>Acros Organic/Fisher Scientific&amp;nbsp;</td><td>AC430941000</td><td></td></tr><tr><td>10K MPEG-Silane</td><td>Nanosoft Polymers</td><td>2526</td><td></td></tr><tr><td>Oleic acid (99%)</td><td>Beantown Chemical</td><td>126125</td><td></td></tr><tr><td>Pd (II) meso-tetraphenyl tetrabenzoporphine (PdTBTP)</td><td>Frontier Scientific&amp;nbsp;</td><td>41217</td><td></td></tr><tr><td>tetraethyl orthosilicate, anhydrous</td><td>Millipore Sigma</td><td>86578</td><td></td></tr><tr><td>TIPS-Anthracene</td><td>Millipore Sigma</td><td>731439</td><td></td></tr><tr><td>&lt;strong&gt;Representative Ultracentrifuge for Nanocapsule Purification&lt;/strong&gt;</td><td></td><td></td><td>While a smaller centrifuge can be used, the ultracentrifuge is convenient for the 12-14 h centrifugation to isolate upconversion nanocapsule paste.</td></tr><tr><td>500 mL, Polycarbonate Bottle with Cap Assembly, 69 x 160 mm - 6Pk</td><td>Beckman-Coulter</td><td>355605</td><td></td></tr><tr><td>Avanti J-26S XP High-Performance Centrifuge</td><td>Beckman-Coulter</td><td>Avanti J-26S XP</td><td></td></tr><tr><td>JA-10 Fixed-Angle Aluminum Rotor- 6 x 500 mL; 10,000 rpm; 17,700 x g</td><td>Beckman-Coulter</td><td>369687</td><td></td></tr><tr><td>&lt;strong&gt;Specialized Fabrication Equipment and Consumable Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>3M 03429NA 051131034297 Scotch Electrical Tape, 3/4-in by 66-ft, Black, 1-Roll, 3/4 Foot</td><td>Amazon</td><td></td><td></td></tr><tr><td>40 mL scintillation vials (28 mm OD x 95 mm Height, 24-400 thread size)</td><td>Fisher Scientific</td><td>CG490006</td><td>Small-scale synthesis</td></tr><tr><td>500 mL Single Neck RBF, 24/40 Outer Joint</td><td>Chemglass</td><td>CG-1506-20</td><td>Large-scale synthesis</td></tr><tr><td>Egg-shaped stir bar for use in a 500 mL round bottom flask (6.35 mm diameter, 16 mm length)</td><td>Fisher Scientific</td><td>14-512-122</td><td>Large-scale synthesis</td></tr><tr><td>Glovebox</td><td>Mbraun</td><td>LabStar Pro</td><td>This is the glovebox used by the authors. However, as long as the oxygen can be maintained at levels below ~10 ppm, any model is acceptable.</td></tr><tr><td>Magnetic stir plate - inside of glovebox</td><td>Any brand</td><td></td><td></td></tr><tr><td>Magnetic stir plate with temperature control (oil bath or heating blocks) - outside of glovebox</td><td>Any brand</td><td></td><td></td></tr><tr><td>Octagon-shaped stir bar for use in a 40 mL scintillation vial (3 mm diameter, 12 mm length)</td><td>VWR</td><td>58947-140</td><td>Small-scale synthesis</td></tr><tr><td>Parafilm M Wrapping Film</td><td>Fisher Scientific</td><td>&amp;nbsp;S37440</td><td></td></tr><tr><td>Precision Seal rubber septa</td><td>Millipore Sigma</td><td>Z554103-10EA</td><td>Large-scale synthesis</td></tr><tr><td>Vitamix Blender</td><td>Vitamix.com</td><td>E310</td><td>Large-scale synthesis</td></tr><tr><td>Vortex Genie 2</td><td>Millipore Sigma</td><td>Z258415</td><td>Small-scale synthesis</td></tr><tr><td>&lt;strong&gt;Representative Characterization Instrumentation and Accessories&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Brookhaven Instruments 90Plus Nanoparticle Size Analyzer</td><td>Brookhaven Instruments</td><td></td><td></td></tr><tr><td>M Series 635nm Laser 300-500mW</td><td>Dragon Lasers</td><td></td><td>Incident wavelength for upconversion photoluminescence characterization. The laser should only be used by trained researchers in a dedicated optics space with appropriate safety protocols. The laser should be focused using a lens to increase the incident power density.</td></tr><tr><td>P50-1-UV-VIS</td><td>Ocean Insight</td><td>P50-1-UV-VIS</td><td>Patch cord for QE Pro</td></tr><tr><td>QE Pro Spectrometer</td><td>Ocean Insight</td><td>QEPRO-VIS-NIR</td><td>Spectrometer for collecting upconversion photoluminescence.</td></tr><tr><td>Supra55VP Field Emission Scanning Electron Microscope (FESEM)</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

triplet fusion upconversion nanocapsule synthesis

Triplet fusion upconversion (UC) allows for the generation of one high energy photon from two low energy input photons. This well-studied process has significant implications for producing high energy light beyond a material's surface. However, the deployment of UC materials has been stymied due to poor material solubility, high concentration requirements, and oxygen sensitivity, ultimately resulting in reduced light output. Toward this end, nanoencapsulation has been a popular motif to circumvent these challenges, but durability has remained elusive in organic solvents. Recently, a nanoencapsulation technique was engineered to tackle each of these challenges, whereupon an oleic acid nanodroplet containing upconversion materials was encapsulated with a silica shell. Ultimately, these nanocapsules (NCs) were durable enough to enable triplet fusion upconversion-facilitated volumetric three-dimensional (3D) printing. By encapsulating upconversion materials with silica and dispersing them in a 3D printing resin, photopatterning beyond the surface of the printing vat was made possible. Here, video protocols for the synthesis of upconversion NCs are presented for both small-scale and large-scale batches. The outlined protocols serve as a starting point for adapting this encapsulation scheme to multiple upconversion schemes for use in volumetric 3D printing applications.

Figure 1 shows a cartoon depiction of the upconversion nanocapsule synthesis protocol. The parallels among the small scale and large-scale UCNC preparation are emphasized, such as the oil in water emulsion generation and the addition of chemicals to synthesize the silica shell. From the small-scale synthesis, 700-1000 mg of UCNC paste is typically collected, while 7-10 g of the UCNC is typically collected from the large-scale synthesis.
The nanocapsules were characterized using a combination of spectroscopic and microscopy techniques10. To prepare samples for SEM, a film was drop-cast from a solution of 100 mg mL-1 nanocapsule paste dispersed in water onto an appropriate conductive SEM substrate and allowed to dry. The conductivity of the nanocapsules is inherently low, but still sufficient for characterization without the addition of another conductive material. A representative SEM image (Figure 2A) shows the relatively monodisperse nanocapsules with diameters of ~50 nm obtained with this protocol. One limitation of using SEM to characterize the morphology of the UCNCs is that they are unstable under ultrahigh vacuum for long periods of time. Under ultrahigh vacuum necessary for SEM measurements, the UCNCs can be successfully imaged if working efficiently, typically within 30 min. UCNCs fuse under high vacuum after approximately 30 min under ultrahigh vacuum (Figure 2B). This fusion is not observed under ambient conditions following the procedure outlined in this protocol (vide infra). Even in light of the stability considerations under vacuum, electron microscopy is still a beneficial method to assess the typical morphology of the UCNCs.
Dynamic light scattering (DLS) is another useful technique to characterize the average nanocapsule hydrodynamic diameter in solution. The samples for DLS can be easily prepared with a sample of diluted UCNCs. Here, a sample of the supernatant recovered after the first centrifuge (step 1.23 or 2.17) was characterized by DLS. The supernatant was diluted by a factor of 10x with ultrapure deionized water and filtered with a 0.2 &#956;m PVDF filter to remove large particulates and dust. Alternatively, one can characterize the UCNC paste at a concentration of 100 mg mL-1 in ultrapure deionized water diluted 10x and filtered with a 0.2 &#956;m PVDF filter. The hydrodynamic diameter was measured using DLS to be &#60;100 nm from batch to batch, typically in the range of 65-90 nm10. Nanoparticle aggregation is not observed under these characterization conditions, removing the need for an additional electrolyte10. Similar UCNC diameters can be generated from large scale or small-scale protocols; representative traces from one scan are presented in Figure 2C.&#160;Due to Brownian motion and the mathematical fitting process to the Stokes-Einstein equation, many scans are averaged together to determine the average hydrodynamic diameters17. The average hydrodynamic diameters for the samples shown in Figure 2C are ~75 nm for the large batch (polydispersity, PDI: 0.21) and ~66 nm (PDI: 0.15) for the small batch presented. This variation in hydrodynamic diameter is typical from batch to batch, irrespective of the reaction scale.
Finally, optical characterization is vital to assess the integrity of the silica shell encapsulation (Figure 2D). Here, a sample of the supernatant recovered after the first centrifuge was diluted by 10x in deoxygenated acetone in the glovebox. The sample was diluted in acetone to test the structural integrity of the UCNCs. In Figure 2D, the anthracene upconversion emission is clearly present upon irradiation with a 635 nm laser, signifying the average silica shell remains intact. If the silica shells are too thin, bright upconversion is extremely low upon irradiation with a 635 nm laser. This is due to the upconversion contents being dissolved and diluted in acetone to a concentration too low to generate bright upconverted emission10.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64374/64374fig01.jpg" /> 
Figure 1: A cartoon depiction of the upconversion nanocapsule synthetic process on the small and large scale. This figure was created with Biorender.com. <a href="https://www.jove.com/files/ftp_upload/64374/64374fig01large.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/64374/64374fig02v2.jpg" /> 
Figure 2: Representative nanocapsule characterization using microscopy and spectroscopy. (A) SEM of the UCNCs shows the scale and uniformity of the upconversion nanocapsule synthesis. Scale bar = 200 nm. (B) SEM of the UCNCs that have fused under ultra-high vacuum over the course of ~30 min. SEM samples were prepared by drop-casting solutions of UCNCs in deionized ultrapure water. Scale bar = 20 &#956;m. (C) Representative DLS traces of upconversion nanocapsules prepared on a small scale and large scale. UCNCs were diluted in deionized ultrapure water. (D) The upconversion emission of TIPS-an in UCNCs diluted in acetone was generated upon irradiation with a 635 nm laser at ~65 W cm-2. This bright upconversion signifies the silica shells are thick enough to prevent the nanocapsule contents from spilling out. <a href="https://www.jove.com/files/ftp_upload/64374/64374fig02largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Triplet Fusion Upconversion Nanocapsule Synthesis at JoVE.com

Triplet Fusion Upconversion Nanocapsule Synthesis

This protocol details the synthesis of upconversion nanocapsules for subsequent use in photopolymerizable resins for triplet fusion upconversion-facilitated volumetric 3D printing.

Triplet fusion upconversion (UC) allows for the generation of one high energy photon from two low energy input photons. This well-studied process has ...

triplet-fusion-upconversion-nanocapsule-synthesis

Research

JoVE Journal

Engineering

2.3K Views.  Harvard University. This protocol details the synthesis of upconversion nanocapsules for subsequent use in photopolymerizable resins for triplet fusion upconversion-facilitated volumetric 3D printing.

Video: Triplet Fusion Upconversion Nanocapsule Synthesis

Integrating a Triplet-triplet Annihilation Up-conversion System to Enhance Dye-sensitized Solar Cell Response to Sub-bandgap Light

The poor response of dye-sensitized solar cells (DSCs) to red and infrared light is a significant impediment to the realization of higher photocurrents and hence higher efficiencies. Photon up-conversion by way of triplet-triplet annihilation (TTA-UC) is an attractive technique for using these otherwise wasted low energy photons to produce photocurrent, while not interfering with the photoanodic performance in a deleterious manner. Further to this, TTA-UC has a number of features, distinct from other reported photon up-conversion technologies, which renders it particularly suitable for coupling with DSC technology. In this work, a proven high performance TTA-UC system, comprising a palladium porphyrin sensitizer and rubrene emitter, is combined with a high performance DSC (utilizing the organic dye D149) in an integrated device. The device shows an enhanced response to sub-bandgap light over the absorption range of the TTA-UC sub-unit resulting in the highest figure of merit for up-conversion assisted DSC performance to date.

An integrated device, incorporating a dye-sensitized solar cell and triplet-triplet annihilation up-conversion unit was produced, affording enhanced light harvesting, from a wider section of the solar spectrum. Under modest irradiation levels a significantly enhanced response to low energy photons was demonstrated, yielding a record figure of merit for dye-sensitized solar cells.

The poor response of dye-sensitized solar cells (DSCs) to red and infrared light is a significant impediment to the realization of higher photocurrents ...

A 'Plug and Play' Method to Create Water-dispersible Nanoassemblies Containing an Amphiphilic Polymer, Organic Dyes and Upconverting Nanoparticles

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate amphiphilic polymers in situ, and describe a protocol to encapsulate the prepared UCNPs and different organic dye molecules (porphyrins and diarylethenes) using polymer shells to form stable water-dispersible nanoassemblies. The nanoassembly samples containing both the UCNPs and the diarylethene organic dyes have interesting photochemical and photophysical properties. Upon 365 nm UV irradiation, the diarylethene group undergoes a visual color change. When the samples are irradiated with visible light of another specific wavelength, the color fades and the samples return to the initial colorless state. The samples also emit visible light from the UCNPs upon irradiation with 980 nm near-infrared light. The emission intensity of the samples can be tuned through alternate irradiation with UV and visible light. Modulation of fluorescence can be performed for many cycles without observable degradation of the samples. This versatile encapsulation procedure allows for the transfer of hydrophobic molecules and nanoparticles from an organic solvent to an aqueous medium. The polymer helps to maintain a lipid-like microenvironment for the organic molecules to aid in preservation of their photochemical behavior in water. Thus this method is ideal to prepare water-dispersible photoresponsive systems. The use of near-infrared light to activate upconverting nanoparticles allows for lower energy light to be used to activate photoreactions instead of more harmful ultraviolet light.

Organic dye molecules and oleic acid coated upconverting nanoparticles are not water-soluble. This protocol describes a &#8216;plug and play&#8217; method that enables the transfer of organic dye molecules and upconverting particles from their initial hydrophobic solvent to water.

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate ...

Chemistry

Combinatorial Synthesis of and High-throughput Protein Release from Polymer Film and Nanoparticle Libraries

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with conventional one-sample-at-a-time synthesis techniques, a more recent high-throughput approach has been developed enabling the synthesis and testing of large libraries of polyanhydrides1. This will facilitate more efficient optimization and design process of these biomaterials for drug and vaccine delivery applications. The method in this work describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries. In this robotically operated method (Figure 1), linear actuators and syringe pumps are controlled by LabVIEW, which enables a hands-free automated protocol, eliminating user error. Furthermore, this method enables the rapid fabrication of micro-scale polymer libraries, reducing the batch size while resulting in the creation of multivariant polymer systems. This combinatorial approach to polymer synthesis facilitates the synthesis of up to 15 different polymers in an equivalent amount of time it would take to synthesize one polymer conventionally. In addition, the combinatorial polymer library can be fabricated into blank or protein-loaded geometries including films or nanoparticles upon dissolution of the polymer library in a solvent and precipitation into a non-solvent (for nanoparticles) or by vacuum drying (for films). Upon loading a fluorochrome-conjugated protein into the polymer libraries, protein release kinetics can be assessed at high-throughput using a fluorescence-based detection method (Figures 2 and 3) as described previously1. This combinatorial platform has been validated with conventional methods2 and the polyanhydride film and nanoparticle libraries have been characterized with 1H NMR and FTIR. The libraries have been screened for protein release kinetics, stability and antigenicity; in vitro cellular toxicity, cytokine production, surface marker expression, adhesion, proliferation and differentiation; and in vivo biodistribution and mucoadhesion1-11. The combinatorial method developed herein enables high-throughput polymer synthesis and fabrication of protein-loaded nanoparticle and film libraries, which can, in turn, be screened in vitro and in vivo for optimization of biomaterial performance.

This method describes the combinatorial synthesis of biodegradable polyanhydride film and nanoparticle libraries and the high-throughput detection of protein release from these libraries.

Polyanhydrides are a class of biomaterials with excellent biocompatibility and drug delivery capabilities. While they have been studied extensively with ...

Bioengineering

Synthesis of Core-shell Lanthanide-doped Upconversion Nanocrystals for Cellular Applications

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical properties, which allow for the absorption of near-infrared (NIR) light and can subsequently convert it into multiplexed emissions that span over a broad range of regions from the UV to the visible to the NIR. This article presents detailed experimental procedures for high-temperature co-precipitation synthesis of core-shell UCNs that incorporate different lanthanide ions into nanocrystals for efficiently converting deep-tissue penetrable NIR excitation (808 nm) into a strong blue emission at 480 nm. By controlling the surface modification with biocompatible polymer (polyacrylic acid, PAA), the as-prepared UCNs acquires great solubility in buffer solutions. The hydrophilic nanocrystals are further functionalized with specific ligands (dibenzyl cyclooctyne, DBCO) for localization on the cell membrane. Upon NIR light (808 nm) irradiation, the upconverted blue emission can effectively activate the light-gated channel protein on the cell membrane and specifically regulate the cation (e.g., Ca2+) influx in the cytoplasm. This protocol provides a feasible methodology for the synthesis of core-shell lanthanide-doped UCNs and subsequent biocompatible surface modification for further cellular applications.

A protocol is presented for the synthesis of core-shell lanthanide-doped upconversion nanocrystals (UCNs) and their cellular applications for channel protein regulation upon near-infrared (NIR) light illumination.

Lanthanide-doped upconversion nanocrystals (UCNs) have attracted much attention in recent years based on their promising and controllable optical ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Biology

Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic porous silicon nanoparticles (F-pSiNPs) have recently demonstrated high in vivo gene silencing efficacy due to its high oligonucleotide loading capacity and unique cellular uptake pathway that avoids endocytosis. The synthesis of F-pSiNPs is a multi-step process that includes: (1) loading and sealing of oligonucleotide payloads in the silicon pores; (2) simultaneous coating and sizing of fusogenic lipids around the porous silicon cores; and (3) conjugation of targeting peptides and washing to remove excess oligonucleotide, silicon debris, and peptide. The particle&#8217;s size uniformity is characterized by dynamic light scattering, and its core-shell structure may be verified by transmission electron microscopy. The fusogenic uptake is validated by loading a lipophilic dye, 1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI), into the fusogenic lipid bilayer and treating it to cells in vitro to observe for plasma membrane staining versus endocytic localizations. The targeting and in vivo gene silencing efficacies were previously quantified in a mouse model of Staphylococcus aureus pneumonia, in which the targeting peptide is expected to help the F-pSiNPs to home to the site of infection. Beyond its application in S. aureus infection, the F-pSiNP system may be used to deliver any oligonucleotide for gene therapy of a wide range of diseases, including viral infections, cancer, and autoimmune diseases.

We demonstrate the synthesis of fusogenic porous silicon nanoparticles for effective in vitro and in vivo oligonucleotide delivery. Porous silicon nanoparticles are loaded with siRNA to form the core, which is coated by fusogenic lipids through extrusion to form the shell. Targeting moiety functionalization and particle characterization are included.

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Rapid, Scalable Assembly and Loading of Bioactive Proteins and Immunostimulants into Diverse Synthetic Nanocarriers Via Flash Nanoprecipitation

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging applications. This increased specificity can have significant clinical implications, including decreased side effects and lower dosages with higher potency. Furthermore, the in situ targeting and controlled modulation of specific cell subsets can enhance in vitro and in vivo investigations of basic biological phenomena and probe cell function. Unfortunately, the required expertise in nanoscale science, chemistry and engineering often prohibit laboratories without experience in these fields from fabricating and customizing nanomaterials as tools for their investigations or vehicles for their therapeutic strategies. Here, we provide protocols for the synthesis and scalable assembly of a versatile non-toxic block copolymer system amenable to the facile formation and loading of nanoscale vehicles for biomedical applications. Flash nanoprecipitation is presented as a methodology for rapid fabrication of diverse nanocarriers from poly(ethylene glycol)-bl-poly(propylene sulfide) copolymers. These protocols will allow laboratories with a wide range of expertise and resources to easily and reproducibly fabricate advanced nanocarrier delivery systems for their applications. The design and construction of an automated instrument that employs a high-speed syringe pump to facilitate the flash nanoprecipitation process and to allow enhanced control over the homogeneity, size, morphology and loading of polymersome nanocarriers is described.

Nanomaterials provide versatile mechanisms of controlled therapeutic delivery for both basic science and translational applications, but their fabrication often requires expertise that is unavailable in most biomedical laboratories. Here, we present protocols for the scalable fabrication and therapeutic loading of diverse self-assembled nanocarriers using flash nanoprecipitation.

Nanomaterials present a wide range of options to customize the controlled delivery of single and combined molecular payloads for therapeutic and imaging ...

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical cores. Following adsorption of a prime layer and DNA plasmids, the formation of robust microcapsules was facilitated by inducing &#946;-sheets in silk secondary structure during acute dehydration of a single silk layer. Hence, the layering occurred via multiple hydrogen bonding and hydrophobic interactions. Upon adsorption of multilayered shells, the core-shell structures can be further functionalized with gold nanoparticles (AuNPs) and/or antibodies (IgG) to be used for remote sensing and/or targeted delivery. Adjusting several key parameters during sequential deposition of key macromolecules on silica cores such as the presence of a polymer primer, the concentration of DNA and silk protein, as well as a number of adsorbed layers resulted in biocompatible, DNA-laden microcapsules with variable permeability and DNA loadings. Upon dissolution of silica cores, the protocol demonstrated the formation of hollow and robust microcapsules with DNA plasmids immobilized to the inner surface of the capsule membrane. Creating a selectively permeable biocompatible membrane between the DNA plasmids and the external environment preserved the DNA during long-term storage and played an important role in the improved output response from spatially confined plasmids. The activity of DNA templates and their accessibility were tested during in vitro transcription and translation reactions (cell-free systems). DNA plasmids encoding RNA light-up aptamers and riboswitches were successfully activated with corresponding analytes, as was visualized during localization of fluorescently labeled RNA transcripts or GFPa1 protein in the shell membranes.

The protocol describes the formation of robust and biocompatible DNA-laden microcapsules as multiplexed in vitro biosensors capable of tracking several ligands.

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical ...