This work was financially supported by Ministry of Science and Technology of Taiwan (MOST 104-2221-E-131-010, MOST 105-2622-E-131-001-CC2), and partially supported by Institute of Atomic and Molecular Sciences, Academia Sinica.

1. Synthesis of Surface-modified, Water-dispersible, Magnetic Nanoparticles, Fe3O4 and Fe3O4-NH2
<ol>
	<li>Add 14.02 g of FeCl3, 8.6 g of FeCl2&#183;4H2O and 250 mL water to a 500-mL beaker.</li>
	<li>Connect the rotor and controller to set up mechanic stirring. Mix the solution at 300 rpm for 30 min at room temperature (RT).</li>
	<li>Add 25 mL of ammonium hydroxide (33%) into the solution at RT and keep stirring (300 rpm) for 30 min. Keep the beaker open. 
	CAUTION: Ammonium hydroxide may cause nose irritation if inhaled. This step must be performed inside an appropriate fume hood.</li>
	<li>To collect the magnetic iron oxides (Fe3O4), remove the mechanic stirring. Put a magnet under the beaker to collect the black particles.
	<ol>
		<li>After the Fe3O4 nanoparticles are entirely precipitated, carefully remove the supernatant. Do not shake the beaker while pouring the supernatant to avoid loss of Fe3O4.</li>
		<li>Remove the magnet and add 50 mL of fresh water to the beaker.</li>
		<li>Shake the beaker to re-disperse the Fe3O4. Repeat steps 1.4 to 1.4.2 three times to purify the Fe3O4.</li>
	</ol>
	</li>
	<li>After the last wash, transfer all the Fe3O4 (10 g) into a 100-mL glass bottle. Add water until the total solution volume is 100 mL. Shake the glass bottle vigorously until no lumps are visible. 
	NOTE: The protocol can be paused here. The Fe3O4 nanoparticles are prepared.</li>
	<li>Modify the Fe3O4 with aminosilane (Fe3O4-NH2).
	<ol>
		<li>Take the 100-mL solution from step 1.5 and transfer into a 1,000-mL beaker. Add 10 mL of ammonia solution, 90 mL of water, and 900 mL of ethanol to the beaker.</li>
		<li>Use a magnetic stir bar to mix the solution at 300 rpm. Add 500 &#181;L of (3-aminopropyl)triethoxysilane (APTES) dropwise to the beaker at RT and stir for another 12 h.</li>
	</ol>
	</li>
	<li>Purify and collect the Fe3O4-NH2 as described in section 1.4.</li>
	<li>Re-disperse 1 g of Fe3O4-NH2 (from step 1.7) in a 20-mL glass bottle with 20 mL water. 
	NOTE: The protocol can be paused here. The Fe3O4-NH2 nanoparticles are prepared.</li>
</ol>
2. Synthesis of Organic-inorganic Hybrid Microgels by Thermo-induced Emulsion
<ol>
	<li>Preparation of Solution 1-1 and 1-2.
	<ol>
		<li>For solution 1-1, add 0.25 g of PNIPAAm, 5 mL of Fe3O4 solution (from step 1.5), and 0.2 g of PEI to a 50-mL glass bottle. Add 20 mL of water and use a magnetic stir bar to stir at 300 rpm for 30 min.</li>
		<li>For solution 1-2, repeat step 2.1.1, but replace Fe3O4 as Fe3O4-NH2 solution (from step 1.8).</li>
	</ol>
	</li>
	<li>To prepare Solution 2, add 0.8 g of PEI and 18.2 mL of water to a 50-mL glass bottle. Use a water bath to heat up the solution to 70 &#176;C for 30 min. Prepare a second bottle of Solution 2.</li>
	<li>Preparation of PNIPAAm/Fe3O4.
	<ol>
		<li>Use an ultrasonic cell disruptor to sonicate (50 w), a magnetic stir bar to stir (300 rpm), and a water bath to heat Solution 2 (70 &#176;C).</li>
		<li>Add Solution 1-1 to the heated Solution 2 dropwise using a 3-mL syringe at a rate of 1 mL/min.</li>
		<li>Continue sonication, stirring and heating at 70 &#176;C for 30 min.</li>
		<li>Cool the solution to RT. Remove the solution from the cell disruptor and water bath.</li>
		<li>Collect the microgels by placing the magnet close to the glass bottle.</li>
		<li>Remove the supernatant after the microgels have precipitated to the bottom of the glass bottle.</li>
		<li>Add another 25 mL of water to the glass bottle and re-disperse the microgels by vortexing. This solution is PNIPAAm/Fe3O4. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
	<li>Preparation of PNIPAAm/Fe3O4-NH2.
	<ol>
		<li>Use an ultrasonic cell disruptor to sonicate (50 w), a magnetic stir bar to stir (300 rpm), and a water bath to heat Solution 2 (70 &#176;C).</li>
		<li>Add Solution 1-2 to the heated Solution 2 dropwise using a 3-mL syringe at a rate of 1 mL/min.</li>
		<li>Continue sonication, stirring and heating at 70 &#176;C for 30 min.</li>
		<li>Cool the solution to RT. Remove the solution from the cell disruptor and water bath.</li>
		<li>Collect the microgels by placing the magnet close to the glass bottle.</li>
		<li>Once the microgels precipitate, remove the supernatant.</li>
		<li>Add another 25 mL of water to the glass bottle and re-disperse the microgels by vortexing. This solution is PNIPAAm/Fe3O4-NH2. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
</ol>
3. Preparation of Curcumin-loaded Microgels (Cur-PNIPAAm/Fe3O4-NH 2 )
NOTE: These steps must be performed in the dark.
<ol>
	<li>Add 100 mg of Cur and 20 mL of ethanol to a 20-mL of glass bottle.</li>
	<li>Take 2 mL of the Cur solution and transfer to the PNIPAAm/Fe3O4-NH2 solution (step 2.4.7). Stir at 400 rpm and RT overnight.</li>
	<li>After stirring at 400 rpm and RT overnight, use the magnet to collect PNIPAAm/Fe3O4-NH2 as described in steps 2.4.5 and 2.4.6.</li>
	<li>Add another 25 mL of water to the glass bottle and re-disperse the microgels by vortexing. This solution is Cur-PNIPAAm/Fe3O4-NH2.</li>
</ol>
4. Magnetically Triggered Drug Release
<ol>
	<li>Transfer 10 mL of the Cur-PNIPAAm/Fe3O4-NH2 solution and add 2 mL of water to a 15-mL centrifugation tube.</li>
	<li>Place the centrifugation tube in the center of the coil for applying the HFMF20. Apply HFMF at 15 KHz for 20 min.</li>
	<li>Withdraw 0.5 mL of the HFMF solution and replace with fresh 0.5 mL of water at every 2 min interval while applying the HFMF.</li>
	<li>Transfer the withdrawn solution to the 1-mL cuvette.</li>
	<li>Measure the absorption of the withdrawn solution by UV/Vis at 482 nm21.</li>
	<li>Determine the concentration of the released drugs by using the relationship of absorption and concentration from a standard calibration curve22. 
	NOTE: The standard calibration relation is: 
	<img alt="Equation" src="/files/ftp_upload/55648/55648eq1.jpg" /> 
	where the correlation coefficient is 0.9993.</li>
</ol>
5. Characterization of the Magnetic Microgels
<ol>
	<li>Thermogravimetric analyzer (TGA)23.
	<ol>
		<li>Measure the weight loss of PNIPAAm/Fe3O4 and PNIPAAm/Fe3O4-NH2 vs. temperature under air atmosphere by TGA.
		<ol>
			<li>Heat the sample from RT to 100 &#176;C and keep at this temperature for 10 min to eliminate humidity. Heat the sample from 100 &#176;C to 800 &#176;C at a rate of 10 &#176;C/min. Weigh the samples.</li>
			<li>Plot the weight loss vs. temperature of both PNIPAAm/Fe3O4 and PNIPAAm/Fe3O4-NH2. 
			NOTE: The residue weight is either Fe3O4 or Fe3O4-NH2, while the lost weight is PNIPAAm.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>FT-IR24.
	<ol>
		<li>Dry 10 mg of sample with 1 g of KBr at 100 &#176;C overnight.</li>
		<li>Press the mixture from step 5.2.1 into pellets as described in the following steps (5.2.2.1 - 5.2.2.5):
		<ol>
			<li>Grind the materials from step 5.2.1 into a fine powder by using a mortar and pestle.</li>
			<li>Place the assembled apparatus (mortar and pestle) into the pellet press. Align the apparatus in the exact middle of the press.</li>
			<li>Pump the press until a pressure of 20,000 psi is reached. Let the pellet sit at that pressure for 5 min. 
			CAUTION: Align the apparatus in the exact middle of the press otherwise sample will disperse out of the mortar and cause injury from exposure.</li>
			<li>Remove the die containing the pellet and the piston from the press.</li>
			<li>Turn it upside down and pump the piston to force the pellet out.</li>
		</ol>
		</li>
		<li>Record the FT-IR absorption spectra of samples by FT-IR at frequencies ranging from 400 to 4,000 cm-1 with 4 cm-1 resolution24.</li>
	</ol>
	</li>
	<li>Morphology observations by TEM25.
	<ol>
		<li>Drop the sample solution onto a copper-grid coated with a collodion and then dry at RT or in a 70 &#176;C oven overnight.</li>
		<li>Take TEM images. 
		NOTE: Strong electron beams can damage the samples. Therefore, TEM images should be taken as quickly as possible.</li>
	</ol>
	</li>
	<li>Aqueous-dispersion abilities of polymers and microgels.
	<ol>
		<li>To prepare PNIPAAm solution, add 7 mg of PNIPAAm and 7 mL of water to a 7-mL glass bottle. Use a vortex to mix the solution until there are no aggregates.</li>
		<li>To prepare PNIPAAm/Fe3O4-NH2 solution, transfer 0.7 mL of PNIPAAm/Fe3O4-NH2 solution (step 2.4.7) to a 7-mL glass bottle and add 6.3 mL of water. Use a vortex to mix the solution until there is no precipitation.</li>
		<li>To prepare Cur-PNIPAAm/Fe3O4-NH2 solution, transfer 0.7 mL of Cur-PNIPAAm/Fe3O4-NH2 solution (step 3.4) to a 7-mL glass bottle and add 6.3 mL of water. Use a vortex to mix the solution until there is no precipitation.</li>
		<li>Take a picture of the solutions (steps 5.4.1 - 5.4.3) using a digital camera.</li>
		<li>Place the solutions into an oven and set the temperature to 70 &#176;C. Wait 2 h until equilibrium.</li>
		<li>Take another picture of the solutions. To maintain the temperature, take the picture within 1 min. Avoid shaking the glass bottle as this can re-disperse the precipitations.</li>
	</ol>
	</li>
	<li>For magnetic collection of microgels, place the strong magnet close to the Cur-PNIPAAm/Fe3O4-NH2 solution (step 5.4.3). Wait until the microgels are fully collected, then take a picture.
	<ol>
		<li>Remove the magnet and vortex the microgel solution until fully dispersed. Take another picture.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclosure.

The most important steps of the preparation are in protocol section 2, for the synthesis of the magnetic microgels by thermo-induced emulsion. As shown in Figure 2 (TEM images), the spherical structure of microgels could be maintained at RT (lower than the LCST) due to the physical crosslinking resulting from the strong H-bonding between PNIPAAm (amide groups), PEI (amine groups) and Fe3O4-NH2 (amine groups). Based on the comparison in Fi...

Hydrogels are three-dimensionally (3D) polymeric networks which cannot dissolve but can swell in aqueous solutions1. The polymeric networks have hydrophilic domains (which can be hydrated to provide the hydrogel structure), and a cross-linked conformation (which can prevent the collapse of the network). Various methods have been investigated for preparation of hydrogels, such as emulsion polymerization, anionic copolymerization, crosslinking of neighboring polymer chains, and inverse micro-emulsion polymerization2. Physical and chemical cross-linking are introduced through these methods to obtain structurally stable hydrogels1,3. Chemical crosslinking normally requires the participation of the crosslinking agent, which connects the backbone or the side-chain of the polymers. Compared to chemical crosslinking, physical crosslinking is a better choice to fabricate hydrogels due to the avoidance of a crosslinking agent, since these agents are often toxic for practical applications4. Several approaches have been investigated for synthesizing physically cross-linked hydrogels, like crosslinking with ionic interaction, crystallization, bonding between amphiphilic blocks or grafting on the polymer chains, and hydrogen bonding4,5,6,7.
Stimuli-sensitive polymers, which can undergo conformational, chemical or physical property changes in response to different environmental conditions (i.e., temperature, pH, light, ionic strength, and magnetic field), have recently attracted attention as a potential platform for controlled release systems, drug delivery, and anti-cancer therapy8,9,10,11,12. Researchers are focusing on thermo-sensitive polymers where intrinsic temperature can be easily controlled. PNIPAAm is a thermally sensitive polymer, which contains both hydrophilic amide groups and hydrophobic isopropyl groups, and has a lower critical solution temperature (LCST)13. Hydrogen bonding between amide groups and water molecules provides the dispersity of PNIPAAm in aqueous solution at low temperatures (below the LCST), while the hydrogen-bonding between polymer chains occurs at high temperatures (above the LCST) and excludes water molecules so that the polymer network collapses. Regarding this unique property, many reports have been published for preparing temperature-triggered, self-assembled hydrogels by adjusting the hydrophobic and hydrophilic ratio of the polymer chain length, such as copolymerization, grafting, or side-chain modification for pharmaceutical platforms14,15,16,17.
Magnetic materials such as iron, cobalt, and nickel have also received increased attention during the past decades for biochemical applications18. Among those candidates, iron oxide is the most widely used because of its stability and low toxicity. Nano-sized iron oxides respond instantly to the magnetic field and behave as superparamagnetic atoms. However, such small particles easily aggregate; this reduces the surface energy, and therefore they lose their dispersity. In order to improve the water-dispersity, grafting or coating to protect the layer are commonly applied not only to separate each individual particle for stability but also to further functionalize the reaction site19.
Here, we fabricated magnetic PNIPAAm-based microgels to serve as drug carriers for controlled release systems. The synthesis process is described and shown in Figure 1. Instead of complicated copolymerization and chemical crosslinking, the novel temperature-induced emulsion of PNIPAAm followed by physical crosslinking was employed for obtaining the microgels without additional surfactant or crosslinking agents. This simplified the synthesis and prevented undesired toxicity. Within such a simple preparation protocol, the as-synthesized microgels offered water-dispersity for both the magnetic iron oxide nanoparticles and the hydrophobic, anti-cancer drug, curcumin. FT-IR, TEM, and imaging provided evidence of dispersion and encapsulation. Due to the embedded Fe3O4-NH2, the magnetic microgels showed potential for serving as micro-devices for controlled release under HFMF.

<table><tbody><tr><td>Poly(N-isopropylacrylamide)</td><td>Polyscience, Inc</td><td>21458-10</td><td>Mw ~40,000</td></tr><tr><td>(3-aminopropyl)trimethoxysilane</td><td>Sigma-Aldrich</td><td>440140</td><td>&gt; 99 %</td></tr><tr><td>Iron(II) chloride tetrahydrate</td><td>Sigma-Aldrich</td><td>44939</td><td>99%</td></tr><tr><td>Iron(III) chloride</td><td>Sigma-Aldrich</td><td>157740</td><td>97%</td></tr><tr><td>Curcumin</td><td>Sigma-Aldrich</td><td>00280590</td><td></td></tr><tr><td>Ammonia hydroxide</td><td>Fisher Chemical</td><td>A/3240/PB15</td><td>35%</td></tr><tr><td>Phosphate Buffered Saline</td><td>Sigma-Aldrich</td><td>806552</td><td>pH 7.4, liquid, sterile-filtered</td></tr><tr><td>Polyethylenimine (PEI)</td><td>Sigma-Aldrich</td><td>P3143</td><td>50 % (w/v) in water</td></tr><tr><td>High-frequency magnetic field (HFMF)</td><td>Lantech Industrial Co., Ltd.,Taiwan</td><td>LT-15-80</td><td>15 kV, 50&amp;ndash;100 kHz</td></tr><tr><td>Ultraviolet-Visible Spectrophotometry</td><td>Thermo Scientific Co.</td><td>Genesys</td><td></td></tr><tr><td>Transmission electron microscopy (TEM)</td><td>JEM-2100</td><td>JEOL</td><td></td></tr><tr><td>Fourier transform infrared spectroscopy (FTIR)</td><td>PerkinElmer</td><td>Spectrum 100</td><td></td></tr><tr><td>Thermogravimetric analyzer</td><td>PerkinElmer</td><td>Pyris 1</td><td></td></tr><tr><td>Ultrasonic cell disruptor</td><td>Hielscher Ultrasonics</td><td>UP50H</td><td></td></tr></tbody></table>

magnetic and thermal-sensitive poly(n-isopropylacrylamide)-based microgels for magnetically triggered controlled release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were designed and fabricated for magnetically triggered release. PNIPAAm-based magnetic microgels with a spherical structure were produced via a temperature-induced emulsion followed with physical-crosslinking by mixing PNIPAAm, polyethylenimine (PEI), and Fe3O4-NH2 magnetic nanoparticles. Because of their dispersity, the Fe3O4-NH2 nanoparticles were embedded inside the polymer matrix. The amine groups exposed on the Fe3O4-NH2 and PEI surface supported the spherical structure by physically crosslinking with the amide groups of the PNIPAAm. The hydrophobic anti-cancer drug curcumin can be dispersed in water after encapsulation into the microgels. The microgels were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), and UV-Vis spectral analysis. Furthermore, magnetically triggered release was studied under an external high frequency magnetic field (HFMF). A significant &#34;burst release&#34; of curcumin was observed after applying the HFMF to the microgels due to the magnetic inductive heating (hyperthermia) effect. This manuscript describes the magnetically triggered controlled release of Cur-PNIPAAm/Fe3O4-NH2 encapsulated curcumin, which can be potentially applied for tumor therapy.

The schematic for synthesis of PNIPAAm/PEI/Fe3O4-NH2 microgels is shown in Figure 1. TGA was applied to estimate the relative composition of the organic compound against the whole microgel. Since only the organic compound PNIPAAm could be burned, the relative composition of PNIPAAm and Fe3O4 (or Fe3O4-NH2) was determined and is shown in Table 1. Why do PNI...

Watch this Scientific Journal Video about Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release at JoVE.com

Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

This manuscript describes the preparation of magnetic and thermal-sensitive microgels via a temperature-induced emulsion without chemical reaction. These sensitive microgels were synthesized by mixing poly(N-isopropylacrylamide) (PNIPAAm), polyethylenimine (PEI) and Fe3O4-NH2 nanoparticles for the potential use in magnetically and thermally triggered drug-release.

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

Magnetically and thermally sensitive poly(N-isopropylacrylamide) (PNIPAAm)/Fe3O4-NH2 microgels with the encapsulated anti-cancer drug curcumin (Cur) were ...

magnetic-thermal-sensitive-poly-n-isopropylacrylamide-based-microgels

Research

JoVE Journal

Bioengineering

8.9K Views.  National Taiwan University.  Dit manuscript beschrijft de bereiding van magnetische en thermische gevoelige microgels via een temperatuur geïnduceerde emulsie zonder chemische reactie. Deze gevoelige microgels werden gesynthetiseerd door poly ( N- isopropylacrylamide) (PNIPAAm), polyethyleenimine (PEI) en Fe304 -NH2 nanodeeltjes te mengen voor het potentiële gebruik in magnetisch en thermisch geactiveerde geneesmiddelafgifte. 

Video: Magnetic and Thermal-sensitive PolyN-isopropylacrylamide-based Microgels for Magnetically Triggered Controlled Release

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Chemistry

Synthesis of Poly(N-isopropylacrylamide) Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this unique property, Janus microparticles have been recognized as a new class of materials, thereby attracting a great deal of attention from various research fields. The versatility of these microparticles has been exemplified through their uses as building blocks for self-assembly, electrically responsive actuators, emulsifiers for painting and cosmetics, and carriers for drug delivery. This study introduces a detailed protocol that explicitly describes a synthetic method for designing novel Janus microhydrogels composed of a single base material, poly(N-isopropylacrylamide) (PNIPAAm). Janus microdroplets are firstly generated via a hydrodynamic focusing microfluidic device (HFMD) based on the separation of a supersaturated aqueous NIPAAm monomer solution and subsequently polymerized through exposure to UV irradiation. The resulting Janus microhydrogels were found to be entirely composed of the same base material, featured an easily identifiable compartmentalized morphology, and exhibited anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability. We believe that the proposed method introduces a novel hydrogel platform with the potential for advanced synthesis of multi-functional Janus microhydrogels.

Synthesis of PolyN-isopropylacrylamide Janus Microhydrogels for Anisotropic Thermo-responsiveness and Organophilic/Hydrophilic Loading Capability

We present a protocol to synthesize Janus microhydrogels composed entirely of the same base material, poly(N-isopropylacrylamide) (PNIPAAm), with a clearly compartmentalized structure base on the phase separation of a supersaturated NIPAAm monomer solution. The synthesized Janus microhydrogels show unique properties such as anisotropic thermo-responsiveness and organophilic/hydrophilic loading capability.

Janus microparticles are compartmentalized particles with differing molecular structures and/or functionality on each of their two sides. Because of this ...

Magnetically-Assisted Remote Controlled Microcatheter Tip Deflection under Magnetic Resonance Imaging

X-ray fluoroscopy-guided endovascular procedures have several significant limitations, including difficult catheter navigation and use of ionizing radiation, which can potentially be overcome using a magnetically steerable catheter under MR guidance.
	The main goal of this work is to develop a microcatheter whose tip can be remotely controlled using the magnetic field of the MR scanner. This protocol aims to describe the procedures for applying current to the microcoil-tipped microcatheter to produce consistent and controllable deflections.
	A microcoil was fabricated using laser lathe lithography onto a polyimide-tipped endovascular catheter. In vitro testing was performed in a waterbath and vessel phantom under the guidance of a 1.5-T MR system using steady-state free precession (SSFP) sequencing. Various amounts of current were applied to the coils of the microcatheter to produce measureable tip deflections and navigate in vascular phantoms.
	The development of this device provides a platform for future testing and opportunity to revolutionize the endovascular interventional MRI environment.

Current applied to an endovascular microcatheter with microcoil tip made by laser lathe lithography can achieve controllable deflections under magnetic resonance (MR) guidance, which may improve speed and efficacy of navigation of vasculature during various endovascular procedures.

X-ray fluoroscopy-guided  endovascular procedures have several significant limitations, including  difficult catheter navigation and use of ionizing ...

Controlled Synthesis and Fluorescence Tracking of Highly Uniform Poly(N-isopropylacrylamide) Microgels

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this work, we use single particle tracking of fluorescently labeled PNIPAM microgels as a showcase for tuning microgel size by a rapid non-stirred precipitation polymerization procedure. This approach is well suited for prototyping new reaction compositions and conditions or for applications that do not require large amounts of product. Microgel synthesis, particle size and structure determination by dynamic and static light scattering are detailed in the protocol. It is shown that the addition of functional comonomers can have a large influence on the particle nucleation and structure. Single particle tracking by wide-field fluorescence microscopy allows for an investigation of the diffusion of labeled tracer microgels in a concentrated matrix of non-labeled microgels, a system not easily investigated by other methods such as dynamic light scattering.

Controlled Synthesis and Fluorescence Tracking of Highly Uniform PolyN-isopropylacrylamide Microgels

Non-stirred precipitation polymerization provides a rapid, reproducible prototyping approach to the synthesis of stimuli-sensitive poly(N-isopropylacrylamide) microgels of narrow size distribution. In this protocol synthesis, light scattering characterization and single particle fluorescence tracking of these microgels in a wide-field microscopy setup are demonstrated.

Stimuli-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels have various prospective practical applications and uses in fundamental research. In this ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an integral part of normal tissue renewal, excessive amount of remodeling activity is involved in tumors, arthritis, and many other pathological conditions. During collagen remodeling, the triple helical structure of collagen molecules is disrupted by proteases in the extracellular environment. In addition, collagens present in many histological tissue samples are partially denatured by the fixation and preservation processes. Therefore, these denatured collagen strands can serve as effective targets for biological imaging. We previously developed a caged collagen mimetic peptide (CMP) that can be photo-triggered to hybridize with denatured collagen strands by forming triple helical structure, which is unique to collagens. The overall goals of this procedure are i)&#160;to image denatured collagen strands resulting from normal remodeling activities in vivo, and ii) to visualize collagens in ex vivo tissue sections using the photo-triggered caged CMPs. To achieve effective hybridization and successful in vivo and ex vivo imaging, fluorescently labeled caged CMPs are either photo-activated immediately before intravenous injection, or are directly activated on tissue sections. Normal skeletal collagen remolding in nude mice and collagens in prefixed mouse cornea tissue sections are imaged in this procedure. The imaging method based on the CMP-collagen hybridization technology presented here could lead to deeper understanding of the tissue remodeling process, as well as allow development of new diagnostics for diseases associated with high collagen remodeling activity.

Imaging Denatured Collagen Strands In vivo and Ex vivo via Photo-triggered Hybridization of Caged Collagen Mimetic Peptides

This procedure demonstrates in vivo near IR fluorescence imaging of collagen remodeling activities in mice as well as ex vivo staining of collagens in tissue sections using caged collagen mimetic peptides that can be photo-triggered to hybridize with denatured collagen strands.

Collagen is a major structural component of the extracellular matrix that supports tissue formation and maintenance. Although collagen remodeling is an ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

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.), ...

An Additive Manufacturing Technique for the Facile and Rapid Fabrication of Hydrogel-based Micromachines with Magnetically Responsive Components

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels have simple monolithic architectures and often function as scaffolding materials for tissue engineering applications. More sophisticated structures typically take a long time to fabricate and do not contain moving components. This protocol describes a photolithography method that allows for facile and rapid microfabrication of PEG structures and devices. This strategy involves an in-house developed fabrication stage that allows for the rapid fabrication of 3D structures by building upwards in a layer-by-layer fashion. Independent moving components can also be aligned and assembled onto support structures to form integrated devices. These independent components are doped with superparamagnetic iron oxide nanoparticles that are sensitive to magnetic actuation. In this manner, the fabricated devices can be actuated using external magnets to yield movement of the components within. Hence, this technique allows for the fabrication of sophisticated MEMS-like devices (micromachines) that are composed entirely out of a biocompatible hydrogel, able to function without an onboard power source, and respond to a contact-less method of actuation. This manuscript describes the fabrication of both the fabrication set-up as well as the step-by-step method for the microfabrication of these hydrogels-based MEMS-like devices.

An additive manufacturing strategy for processing UV-crosslinkable hydrogels has been developed. This strategy allows for the layer-by-layer assembly of microfabricated hydrogel structures as well as the assembly of independent components, yielding integrated devices containing moving components that are responsive to magnetic actuation.

Polyethylene glycol (PEG)-based hydrogels are biocompatible hydrogels that have been approved for use in humans by the FDA. Typical PEG-based hydrogels ...

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 ...

Magnetic and Thermal-sensitive Poly(N-isopropylacrylamide)-based Microgels for Magnetically Triggered Controlled Release

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