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 op...

<ol>
	<li>Hennink, W. E., van Nostrum, C. 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L., Chiu, W. Y., Lee, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+thermoresponsive+core-shell+copolymer+latex+with+potential+use+in+drug+targeting.">Preparation of thermoresponsive core-shell copolymer latex with potential use in drug targeting.</a> J Colloid Interface Sci. 290 (2), 397-405 (2005).</li><li>Ma, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+to+in+situ+synthesis+of+magnetic+poly(N-isopropylacrylamide-co-acrylic+acid)+nanogels.">A novel method to in situ synthesis of magnetic poly(N-isopropylacrylamide-co-acrylic acid) nanogels.</a> Colloid Polym Sci. 294 (8), 1251-1257 (2016).</li><li>Du, G. H., Liu, Z. L., Xia, X., Chu, Q., Zhang, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+application+of+Fe3O4/SiO2+nanocomposites.">Characterization and application of Fe3O4/SiO2 nanocomposites.</a> Journal of Sol-Gel Science and Technology. 39 (3), 285-291 (2006).</li><li>Moroz, P., Jones, S. K., Gray, B. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetically+mediated+hyperthermia:+current+status+and+future+directions.">Magnetically mediated hyperthermia: current status and future directions.</a> International Journal of Hyperthermia. 18 (4), 267-284 (2002).</li><li>Silva-Buzanello, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+an+Ultraviolet-visible+(UV-Vis)+technique+for+the+quantitative+determination+of+curcumin+in+poly(l-lactic+acid)+nanoparticles.">Validation of an Ultraviolet-visible (UV-Vis) technique for the quantitative determination of curcumin in poly(l-lactic acid) nanoparticles.</a> Food Chemistry. 172, 99-104 (2015).</li><li>Kim, H. J., Jang, Y. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+analysis+of+curcumin+in+turmeric+by+DART-MS.">Direct analysis of curcumin in turmeric by DART-MS.</a> Phytochemical Analysis. 20 (5), 372-377 (2009).</li><li>Horowitz, H. 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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 border="0" cellpadding="0" cellspacing="0" width="773">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">Poly(N-isopropylacrylamide)</td>
			<td style="width:139px;">Polyscience, Inc</td>
			<td style="width:119px;">21458-10</td>
			<td style="width:205px;">Mw~40000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(3-aminopropyl)trimethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>440140</td>
			<td>&#62; 99 %</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iron(II) chloride tetrahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>44939</td>
			<td>99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iron(III) chloride</td>
			<td>Sigma-Aldrich</td>
			<td>157740</td>
			<td>97%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Curcumin</td>
			<td>Sigma-Aldrich</td>
			<td>00280590</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonia hydroxide</td>
			<td>Fisher Chemical</td>
			<td>A/3240/PB15</td>
			<td>35%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>806552</td>
			<td>pH 7.4, liquid, sterile-filtered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyethylenimine (PEI)</td>
			<td>Sigma-Aldrich</td>
			<td>P3143</td>
			<td>50 % (w/v) in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High-frequency magnetic field (HFMF)</td>
			<td>Lantech Industrial Co., Ltd.,Taiwan</td>
			<td>LT-15-80</td>
			<td>15 kV, 50&#8211;100 kHz</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultraviolet-Visible Spectrophotometry</td>
			<td>Thermo Scientific Co.</td>
			<td>Genesys</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transmission electron microscopy (TEM)</td>
			<td>JEM-2100</td>
			<td>JEOL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fourier transform infrared spectroscopy (FTIR)</td>
			<td>PerkinElmer</td>
			<td>Spectrum 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermogravimetric analyzer</td>
			<td>PerkinElmer</td>
			<td>Pyris 1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ...

The overall goal of this procedure is to investigate the behaviors of curcumin-loaded magnetically and thermally sensitive hydrogels before and during magnetically triggered curcumin release. This method can help answer key questions in the anti-cancer drug field about the encapsulation of hydrophobic anti-cancer drugs such as curcumin for localized delivery to tumor cells. The main advantage of this technique is hydrophobic anti-cancer drugs are encapsulated by a spherical thermal-induced physical-crosslinking between polyethylenime-PNIPAAm and magnetic nanoparticles enabling targeting delivery with high-frequency magnetic fields.Though this method can provide insight into thermal-induced self-assemblies of PNIPAAm, it can also be applied to other substances such as emulsions, or polymerizations, or nanoparticle preparations. To begin the procedure, place 0.2 grams of PEI, 0.25 grams of polyNIPAM, five milliliters of 9.1 weight percent iron two three oxide nanoparticles dispersed in water, and 20 milliliters of deionized water in a glass vial. Use the same stirring conditions to combine in another vial 0.2 grams of PEI, 0.25 grams of polyNIPAM, five milliliters of 4.76 weight percent aminosilane functionalized iron two three oxide nanoparticles dispersed in water, and 20 milliliters of deionized water.Next, prepare two batches of 0.8 grams of PEI and 18.2 milliliters of deionized water. Heat both solutions at 70 degrees Celsius, in a water bath, for 30 minutes, while stirring. Then, while still stirring in the water bath, begin sonicating one batch, with a 50-watt probe sonicator.Using a three milliliter syringe, add this batch to the polyNIPAM and iron oxide suspension drop-wise, at a rate of one milliliter per minute. The addition should be as slow as possible to keep the physical cross-linking minimal. Otherwise, it will need a large magnetic microgel, that is difficult to disperse in water.Continue sonicating and stirring the suspension at 70 degrees Celsius, for 30 minutes. Then, allow the suspension to cool to room temperature. Turn off the sonicator and remove the suspension from the water bath.Remove the magnetic stir bar from the suspension. Place a magnet next to the vial and wait for the magnetic polyNIPAM iron oxide microgels to collect at the bottom of the vial. Then, remove the supernatant, and add 25 milliliters of deionized water to the microgels.Remove the magnet and vortex the mixture to obtain a dispersion of polyNIPAM iron oxide microgels in water. Repeat this process with the polyNIPAM aminosilane functionalized iron oxide suspension and the second batch of the PEI solution, to produce the corresponding functionalized microgel dispersion. In an aluminum foil-wrapped vial, dissolve 100 milligrams of curcumin, and 20 milliliters of HPLC-grade ethanol.Add two milliliters of the solution to the polyNIPAM aminosilane functionalized iron oxide dispersion. Stir the mixture at 400 RPM, overnight at room temperature. Then, remove the stir bar.Place a magnet next to the vial, and wait for the curcumin-loaded microgels to collect at the bottom of the vial. Replace the supernatant with 25 milliliters of deionized water, and disperse the microgels by vortexing. Next, to test the magnetically triggered curcumin release, first transfer 10 milliliters of the curcumin-loaded microgel dispersion to a 15 milliliter conical centrifuge tube.Add two milliliters of deionized water to the tube and vortex to re-disperse the microgels. Place the tube at the center of the coil of a high frequency magnetic field. Apply the magnetic field at 15 kilohertz for 20 minutes.Every two minutes, transfer 0.5 milliliters of the upper layer of the dispersion to a one milliliter cuvette. Replace each aliquot with 0.5 milliliters of deionized water. Use a thermometer to mix the water into the dispersion and to measure the dispersion temperature.Measure the absorption of each aliquot at 482 nanometers. Calculate the concentration of released curcumin in each aliquot from a standard calibration curve. Organic inorganic microgels were prepared from polyNIPAM and iron two three oxide nanoparticles.Microgels prepared with the 3-aminopropyltriethyloxysilane iron oxide particles were found to have a much lower relative iron oxide composition than non-functionalized particles, suggesting that the functionalized particles had cross-linked a larger amount of polyNIPAM than the non-functionalized particles. Aminosilane functionalized microgels, loaded with curcumin, showed increased isolation compared to their precursor microgels, suggesting that the hydrophobic curcumin was at least partially present in the microgel surfaces. Encapsulation of curcumin was confirmed by the appearance of characteristic absorption peaks in the FTIR spectrum of the loaded microgels.The rate of curcumin release was evaluated with and without exposure to a high frequency magnetic field. The curcumin release rate over a period of 20 minutes was 2.5 times greater in the presence of a high frequency magnetic field. Exposure to the magnetic field increased the bulk solution temperature to approximately 50 degrees Celsius, thus altering the microgel properties to exclude curcumin.Thermostability tests showed that the microgels remain dispersed in water in temperatures up to 70 degrees Celsius, despite aggregation. After watching this video, you should have a good understanding of how to encapsulation, hydrophobic, and high cancer drugs in polyNIPAM-based magnetic microgels by temperature-induced immersion, and how to perform a rapid targeted delivery by the manipulation of high frequency magnetic fields.

magnetic-thermal-sensitive-polyn-isopropylacrylamide-based-microgels

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Bioengineering

8.8K visualizações.  National Taiwan University. The overall goal of this procedure is to investigate the behaviors of curcumin-loaded magnetically and thermally sensitive hydrogels before and during magnetically triggered curcumin release. This method can help answer key questions in the anti-cancer drug field about the encapsulation of hydrophobic anti-cancer drugs such as curcumin for localized delivery to tumor cells. The main advantage of this technique is hydrophobic anti-cancer drugs are encapsulated by a spherical thermal-induced ph...