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

Summary

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.

Abstract

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 "burst release" 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.

Introduction

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 hydrogels^1,3. 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 bonding^4,5,6,7.

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 therapy^8,9,10,11,12. 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 platforms^14,15,16,17.

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.

Protocol

1. Synthesis of Surface-modified, Water-dispersible, Magnetic Nanoparticles, Fe₃O₄ and Fe3O4-NH₂

1. Add 14.02 g of FeCl₃, 8.6 g of FeCl₂·4H₂O and 250 mL water to a 500-mL beaker.
2. Connect the rotor and controller to set up mechanic stirring. Mix the solution at 300 rpm for 30 min at room temperature (RT).
3. 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.
4. To collect the magnetic iron oxides (Fe₃O₄), remove the mechanic stirring. Put a magnet under the beaker to collect the black particles.
   1. 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.
   2. Remove the magnet and add 50 mL of fresh water to the beaker.
   3. Shake the beaker to re-disperse the Fe₃O₄. Repeat steps 1.4 to 1.4.2 three times to purify the Fe₃O₄.
5. 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.
6. Modify the Fe₃O₄ with aminosilane (Fe₃O₄-NH₂).
   1. 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.
   2. Use a magnetic stir bar to mix the solution at 300 rpm. Add 500 µL of (3-aminopropyl)triethoxysilane (APTES) dropwise to the beaker at RT and stir for another 12 h.
7. Purify and collect the Fe₃O₄-NH₂ as described in section 1.4.
8. 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.

2. Synthesis of Organic-inorganic Hybrid Microgels by Thermo-induced Emulsion

1. Preparation of Solution 1-1 and 1-2.
   1. 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.
   2. For solution 1-2, repeat step 2.1.1, but replace Fe₃O₄ as Fe₃O₄-NH₂ solution (from step 1.8).
2. 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 °C for 30 min. Prepare a second bottle of Solution 2.
3. Preparation of PNIPAAm/Fe₃O₄.
   1. 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 °C).
   2. Add Solution 1-1 to the heated Solution 2 dropwise using a 3-mL syringe at a rate of 1 mL/min.
   3. Continue sonication, stirring and heating at 70 °C for 30 min.
   4. Cool the solution to RT. Remove the solution from the cell disruptor and water bath.
   5. Collect the microgels by placing the magnet close to the glass bottle.
   6. Remove the supernatant after the microgels have precipitated to the bottom of the glass bottle.
   7. 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.
4. Preparation of PNIPAAm/Fe₃O₄-NH₂.
   1. 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 °C).
   2. Add Solution 1-2 to the heated Solution 2 dropwise using a 3-mL syringe at a rate of 1 mL/min.
   3. Continue sonication, stirring and heating at 70 °C for 30 min.
   4. Cool the solution to RT. Remove the solution from the cell disruptor and water bath.
   5. Collect the microgels by placing the magnet close to the glass bottle.
   6. Once the microgels precipitate, remove the supernatant.
   7. 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.

3. Preparation of Curcumin-loaded Microgels (Cur-PNIPAAm/Fe₃O₄-NH ₂ )

NOTE: These steps must be performed in the dark.

1. Add 100 mg of Cur and 20 mL of ethanol to a 20-mL of glass bottle.
2. 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.
3. 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.
4. Add another 25 mL of water to the glass bottle and re-disperse the microgels by vortexing. This solution is Cur-PNIPAAm/Fe₃O₄-NH₂.

4. Magnetically Triggered Drug Release

1. Transfer 10 mL of the Cur-PNIPAAm/Fe₃O₄-NH₂ solution and add 2 mL of water to a 15-mL centrifugation tube.
2. Place the centrifugation tube in the center of the coil for applying the HFMF²⁰. Apply HFMF at 15 KHz for 20 min.
3. 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.
4. Transfer the withdrawn solution to the 1-mL cuvette.
5. Measure the absorption of the withdrawn solution by UV/Vis at 482 nm²¹.
6. 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:
   
   where the correlation coefficient is 0.9993.

5. Characterization of the Magnetic Microgels

1. Thermogravimetric analyzer (TGA)²³.
   1. Measure the weight loss of PNIPAAm/Fe₃O₄ and PNIPAAm/Fe₃O₄-NH₂ vs. temperature under air atmosphere by TGA.
      1. Heat the sample from RT to 100 °C and keep at this temperature for 10 min to eliminate humidity. Heat the sample from 100 °C to 800 °C at a rate of 10 °C/min. Weigh the samples.
      2. 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.
2. FT-IR²⁴.
   1. Dry 10 mg of sample with 1 g of KBr at 100 °C overnight.
   2. Press the mixture from step 5.2.1 into pellets as described in the following steps (5.2.2.1 - 5.2.2.5):
      1. Grind the materials from step 5.2.1 into a fine powder by using a mortar and pestle.
      2. Place the assembled apparatus (mortar and pestle) into the pellet press. Align the apparatus in the exact middle of the press.
      3. 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.
      4. Remove the die containing the pellet and the piston from the press.
      5. Turn it upside down and pump the piston to force the pellet out.
   3. 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.
3. Morphology observations by TEM²⁵.
   1. Drop the sample solution onto a copper-grid coated with a collodion and then dry at RT or in a 70 °C oven overnight.
   2. Take TEM images.
      NOTE: Strong electron beams can damage the samples. Therefore, TEM images should be taken as quickly as possible.
4. Aqueous-dispersion abilities of polymers and microgels.
   1. 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.
   2. 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.
   3. 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.
   4. Take a picture of the solutions (steps 5.4.1 - 5.4.3) using a digital camera.
   5. Place the solutions into an oven and set the temperature to 70 °C. Wait 2 h until equilibrium.
   6. 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.
5. 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.
   1. Remove the magnet and vortex the microgel solution until fully dispersed. Take another picture.

Results

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...

Discussion

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...

Materials

List of materials used in this article

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