This study was supported by Farris Family Innovation Award and NIH 1R01NR015674-01 to MK. The authors thank Josep Nayfach (Qteris, Inc) for providing an electro-magnetic generator system as well as his technical consultation. The authors also thank Huan Yan (LCI &#38; Chemical Physics Interdisciplinary Program, Kent State University) for her technical assistants.

Note: The overall process of fabricating magnetic field-responsive gelatin microgels is illustrated in Figure 1A.
1. Preparing Solutions and Suspensions
<ol>
	<li>Prepare a crosslinker genipin (1% w/v) solution by dissolving 20 mg of genipin in 2 ml of phosphate buffered saline (1x PBS; pH 7.4). Vortex the solution and place in a 50 oC water bath for 2 hr to completely dissolve the solution.</li>
	<li>Prepare a surfactant solution by dissolving 20 mg of poly(ethylene glycol)-poly(propylene glycol)-poly(ethylene glycol) (Mw = 2,900 Da; referred to as L64) in 200 ml of PBS to be at the concentration of 100 ppm.</li>
	<li>Prepare a 15% (w/v) gelatin solution by dissolving 64.5 mg of gelatin in 0.43 ml of PBS. Vortex the solution and place it on water bath at 37 oC until it reaches a sol phase, where the solution becomes fluidic. Then, vortex the gelatin solution 2 - 3 times to ensure the homogeneity of the sample.</li>
	<li>Preparation of p(NIPAM-co-AAm)/MNPs solution with a model drug (BSA):
	<ol>
		<li>Disperse 10.75 mg of hydrophilic MNPs in 0.43 ml of PBS and then dissolve 12.9 mg of p(NIPAM-co-AAm) in the MNP suspension to make the concentration at 3% (w/v). The increased concentration of p(NIPAM-co-AAm) can be used to achieve an increased deswelling behavior of microgels.</li>
		<li>Use Texas-Red conjugated bovine serum albumin (TR-BSA; Mw ~66 kDa) as a model drug. Dissolve 0.5 mg of TR-BSA in the mixture of p(NIPAM-co-AAm)/MNPs.</li>
	</ol>
	</li>
	<li>Prepare mixtures of gelatin/p(NIPAM-co-AAm)/MNPs/BSA solution (0.86 ml) by adding mixture of p(NIPAM-co-AAm)/MNPs (0.43 ml) into the gelatin solution (0.43 ml) and then thoroughly vortex them to make a homogeneous mixture. Thus, the concentrations of polymers and MNP become half of the initial concentrations in the final mixture.</li>
</ol>
2. Emulsification
<ol>
	<li>Pour 15 ml of silicone oil [polydimethylsiloxane (viscosity 350 cSt)] into a clean and sterile beaker.</li>
	<li>Immediately add the pre-prepared aqueous mixtures of gelatin/p(NIPAM-co-AAm)/MNPs/BSA solution (0.86 ml) into the silicone oil and emulsify the aqueous mixture in the oil phase by stirring with a magnetic stirring bar at 900 rpm at 30 oC for 30 min.</li>
</ol>
3. Gelation and Transfer of Micro-droplets to an Aqueous Solution
<ol>
	<li>Transfer the emulsion (~16 ml) from beaker into a 50 ml tube.</li>
	<li>Cool down the tube for 10 min at 4 &#176;C for gelation of the micro-droplets in the oil.</li>
	<li>Fill the tube with the prepared L64 solution (at 4 oC) up to 50 ml and vigorously shake the tube. It may be possible that a portion of L64 surfactants would be within the microgels.</li>
	<li>Centrifuge the tube for 20 min at 2,300 x g at 4 oC.</li>
	<li>Regularly check for the presence of the pellet of gel particles on the side of the tube. If the particles are not seen, centrifuge for another 20 min at the&#160;same speed and temperature. Proceed to carefully remove supernatant without disturbing the pellet formed on the inner wall of the tube.</li>
	<li>Repeat steps (3.3) through (3.5) once more. Each time, transfer the sample to a new tube to avoid the inclusion of any oil droplets in the microgel suspension. After this step, ensure that surfactants or oil droplets are not present in the sample suspension. However, the repeated separation steps may lead to loss of initial materials.</li>
</ol>
4. Covalent Crosslinking of the Microgels
<ol>
	<li>Add 2 ml of genipin solution (prepared in section 1) to the pellet of gel particles and mix them well by vortexing the solution.</li>
	<li>Quickly transfer the tube of the suspension in water bath at 23 oC to initiate a covalent crosslinking reaction during a desired crosslinking time (e.g., 5 - 120 min).</li>
	<li>After crosslinking, immediately remove any excessive crosslinkers by discarding the genipin solution, resuspending the microgels in PBS, and centrifuging the tube for 20 min at 2,300 x g (4 oC). If needed, cautiously break apart formed pellet with a pipette tip. This washing step can be repeated up to 3 times if the genipin is still remaining in the solution.</li>
	<li>Discard the supernatant and resuspend the microgels in PBS at a desired density (e.g., 5 &#215; 106 microgels/ml) by counting the number with a hemocytometer.</li>
	<li>For microscopic observations, load the microgel suspension in the space between a slide glass and a cover slip and seal the boundary of the cover slip with epoxy resin.</li>
</ol>
5. Application of Alternating Magnetic Field for Triggering Drug Release
<ol>
	<li>Place the tube with desired concentration of microgels in aqueous media into the chamber of magnetic coils. If necessary, insert a fiber optic temperature probe into the tube to monitor temperature change of the media during the application of AMF.</li>
	<li>Apply high frequency (&#62;100 kHz) AMF at a defined field strength (&#62;5 kA/m) and for a specified duration. Following the application of AMF, centrifuge the sample tube for 20 min at 2,273 x g (4 oC) and collect the supernatant to quantify the amount of TR-BSA released from microgel to the surrounding media using spectrophotometry. The excitation and emission wavelengths for Texas Red are 584 nm and 612 nm, respectively.</li>
</ol>

<ol>
	<li>Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+in+drug+delivery+and+tissue+engineering:+one+laboratory's+experience.">Biomaterials in drug delivery and tissue engineering: one laboratory's experience.</a> Acc. Chem. Res. 33, 94-101 (2000).</li><li>Rivest, C. M., Morrison, D., Ni, B., Rubib, J., Yadav, V., Mahdavi, A., Karp, J., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+hydrogels+for+medicine+and+biology:+synthesis,+characteristics+and+applications.">Microscale hydrogels for medicine and biology: synthesis, characteristics and applications.</a> J Mech Mater Struct. 2, 1103-1119 (2007).</li><li>Kawaguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoresponsive+microhydrogels:+preparation,+properties+and+applications.">Thermoresponsive microhydrogels: preparation, properties and applications.</a> Polym. Int. 63, 925-932 (2014).</li><li>Vinogradov, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colloidal+microgels+in+drug+delivery+applications.">Colloidal microgels in drug delivery applications.</a> Curr. Pharm. Des. 12, 4703-4712 (2006).</li><li>Liechty, W. B., Kryscio, D. R., Slaughter, B. V., Peppas, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymers+for+drug+delivery+systems.">Polymers for drug delivery systems.</a> Annu Rev Chem Biomol Eng. 1, 149-173 (2010).</li><li>Kumari, A., Yadav, S. K., Yadav, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+polymeric+nanoparticles+based+drug+delivery+systems.">Biodegradable polymeric nanoparticles based drug delivery systems.</a> Colloids Surf. B Biointerfaces. 75, 1-18 (2010).</li><li>Shibayama, M., Tanaka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+Phase-Transition+and+Related+Phenomena+of+Polymer+Gels.">Volume Phase-Transition and Related Phenomena of Polymer Gels.</a> Adv Polym Sci. 109, 1-62 (1993).</li><li>Sung, B., Kim, C., Kim, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+colloidal+microgels+with+tunable+thermosensitive+volume+phase+transitions+for+controllable+drug+delivery.">Biodegradable colloidal microgels with tunable thermosensitive volume phase transitions for controllable drug delivery.</a> J Colloid Interface Sci. 450, 26-33 (2015).</li><li>Kumar, C. S., Mohammad, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+nanomaterials+for+hyperthermia-based+therapy+and+controlled+drug+delivery.">Magnetic nanomaterials for hyperthermia-based therapy and controlled drug delivery.</a> Adv. Drug Deliv. Rev. 63, 789-808 (2011).</li><li>Mura, S., Nicolas, J., Couvreur, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli-responsive+nanocarriers+for+drug+delivery.">Stimuli-responsive nanocarriers for drug delivery.</a> Nat. Mater. 12, 991-1003 (2013).</li><li>Kong, S. D., 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=Magnetic+field+activated+lipid-polymer+hybrid+nanoparticles+for+stimuli-responsive+drug+release.">Magnetic field activated lipid-polymer hybrid nanoparticles for stimuli-responsive drug release.</a> Acta biomaterialia. 9, 5447-5452 (2013).</li><li>Hayashi, K., 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=Magnetically+responsive+smart+nanoparticles+for+cancer+treatment+with+a+combination+of+magnetic+hyperthermia+and+remote-control+drug+release.">Magnetically responsive smart nanoparticles for cancer treatment with a combination of magnetic hyperthermia and remote-control drug release.</a> Theranostics. 8, 834-844 (2014).</li><li>Suzuki, D., Kawaguchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli-sensitive+core/shell+template+particles+for+immobilizing+inorganic+nanoparticles+in+the+core.">Stimuli-sensitive core/shell template particles for immobilizing inorganic nanoparticles in the core.</a> Colloid Polym Sci. 284, 1443-1451 (2006).</li><li>Bhattacharya, S., Eckert, F., Boyko, V., Pich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-,+pH-,+and+magnetic-field-sensitive+hybrid+microgels.">Temperature-, pH-, and magnetic-field-sensitive hybrid microgels.</a> Small. 3, 650-657 (2007).</li><li>Wong, J. E., Gaharwar, A. K., Muller-Schulte, D., Bahadur, D., Richtering, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-stimuli+responsive+PNiPAM+microgel+achieved+via+layer-by-layer+assembly:+Magnetic+and+thermoresponsive.">Dual-stimuli responsive PNiPAM microgel achieved via layer-by-layer assembly: Magnetic and thermoresponsive.</a> J Colloid Interf Sci. 324, 47-54 (2008).</li><li>Zhao, X., 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=Active+scaffolds+for+on-demand+drug+and+cell+delivery.">Active scaffolds for on-demand drug and cell delivery.</a> Proc. Natl. Acad. Sci. U.S.A. 108, 67-72 (2011).</li><li>Xu, F., 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=Release+of+magnetic+nanoparticles+from+cell-encapsulating+biodegradable+nanobiomaterials.">Release of magnetic nanoparticles from cell-encapsulating biodegradable nanobiomaterials.</a> ACS nano. 6, 6640-6649 (2012).</li><li>Li, Y. H., 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=Magnetic+Hydrogels+and+Their+Potential+Biomedical+Applications.">Magnetic Hydrogels and Their Potential Biomedical Applications.</a> Adv Funct Mater. 23, 660-672 (2013).</li><li>Cooperstein, M. A., Canavan, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+cytotoxicity+of+(N-isopropyl+acrylamide)+and+poly(N-isopropyl+acrylamide)-coated+surfaces.">Assessment of cytotoxicity of (N-isopropyl acrylamide) and poly(N-isopropyl acrylamide)-coated surfaces.</a> Biointerphases. 8, 19 (2013).</li><li>Jorgensen, L., Moeller, E. H., van de Weert, M., Nielsen, H. M., Frokjaer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+and+evaluating+delivery+systems+for+proteins.">Preparing and evaluating delivery systems for proteins.</a> Eur J Pharm Sci. 29, 174-182 (2006).</li><li>Holland, T. A., Tabata, Y., Mikos, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+release+of+transforming+growth+factor-beta+1+from+gelatin+microparticles+encapsulated+in+biodegradable,+injectable+oligo(poly(ethylene+glycol)+fumarate)+hydrogels.">In vitro release of transforming growth factor-beta 1 from gelatin microparticles encapsulated in biodegradable, injectable oligo(poly(ethylene glycol) fumarate) hydrogels.</a> J Control Release. 91, 299-313 (2003).</li><li>Liang, H. C., Chang, W. H., Lin, K. J., Sung, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genipin-crosslinked+gelatin+microspheres+as+a+drug+carrier+for+intramuscular+administration:+in+vitro+and+in+vivo+studies.">Genipin-crosslinked gelatin microspheres as a drug carrier for intramuscular administration: in vitro and in vivo studies.</a> J Biomed Mater Res. Part A. 65, 271-282 (2003).</li><li>Solorio, L., Zwolinski, C., Lund, A. W., Farrell, M. J., Stegemann, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gelatin+microspheres+crosslinked+with+genipin+for+local+delivery+of+growth+factors.">Gelatin microspheres crosslinked with genipin for local delivery of growth factors.</a> J Tissue Eng Regen Med. 4, 514-523 (2010).</li><li>Regmi, R., 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=Hyperthermia+controlled+rapid+drug+release+from+thermosensitive+magnetic+microgels.">Hyperthermia controlled rapid drug release from thermosensitive magnetic microgels.</a> J Mater Chem. 20, 6158-6163 (2010).</li><li>Kim, M. H., 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=Magnetic+nanoparticle+targeted+hyperthermia+of+cutaneous+Staphylococcus+aureus+infection.">Magnetic nanoparticle targeted hyperthermia of cutaneous Staphylococcus aureus infection.</a> Ann Biomed Eng. 41, 598-609 (2013).</li><li>Ivkov, R., 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=Application+of+high+amplitude+alternating+magnetic+fields+for+heat+induction+of+nanoparticles+localized+in+cancer.">Application of high amplitude alternating magnetic fields for heat induction of nanoparticles localized in cancer.</a> Clin Cancer Res. 11, 7093s-7103s (2005).</li><li>Huang, S., Fu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+derived+materials-based+cell+and+drug+delivery+systems+in+skin+regeneration.">Naturally derived materials-based cell and drug delivery systems in skin regeneration.</a> J Control Release. 142, 149-159 (2010).</li><li>Malafaya, P. B., Silva, G. A., Reis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural-origin+polymers+as+carriers+and+scaffolds+for+biomolecules+and+cell+delivery+in+tissue+engineering+applications.">Natural-origin polymers as carriers and scaffolds for biomolecules and cell delivery in tissue engineering applications.</a> Adv. Drug Deliv. Rev. 59, 207-233 (2007).</li><li>Shah, R., Kim, J., Agresti, J., Weitz, D., Chu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+monodisperse+thermosensitive+microgels+and+gel+capsules+in+microfluidic+devices.">Fabrication of monodisperse thermosensitive microgels and gel capsules in microfluidic devices.</a> Soft Matter. 4, 2303-2309 (2008).</li><li>Hoare, 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=Magnetically+triggered+nanocomposite+membranes:+a+versatile+platform+for+triggered+drug+release.">Magnetically triggered nanocomposite membranes: a versatile platform for triggered drug release.</a> Nano letters. 11, 1395-1400 (2011).</li></ol>

The authors have nothing to disclose.

The technology described here demonstrates a proof of concept on the use of nanoparticle-microgel hybrids for magneto-thermally triggered drug release. This was achieved by physically entrapping MNPs and p(NIPAM-co-AAm) chains within a microscale three-dimensional gelatin network crosslinked by genipin. The magnetic field-responsive platform was sufficient to generate heat within the microgel in response to a remotely applied AMF, which in turn triggered the release of a model drug, BSA.
A strategy to embed both MNPs and p(NIPAM-co-AAm) appears to be critical for achieving a desired drug release from the gelatin microgel, since the extent of BSA release from gelatin/MNPs microgel in the absence of p(NIPAM-co-AAm) was significantly lower than the one from microgel incorporating p(NIPAM-co-AAm), even though the extent of temperature increase to AMF application were similar in both types of microgels (Figure 2C and 2D). We demonstrated that the deswelling behavior of gelatin/p(NIPAM-co-AAm) microgel is induced by the shrinkage of p(NIPAM-co-AAm) polymer chains in response to an increase in temperature; moreover, the deswelling behavior positively correlates with the extent of drug release from the microgel8. Taken together, this supports that the shrinkage of p(NIPAM-co-AAm) due to MNPs heating may be the main driving force for the BSA release from gelatin/p(NIPAM-co-AAm)/MNPs microgels.
We previously reported that the application of a high frequency AMF to MNPs could trigger increase in temperature on the surface of MNPs in a manner that is proportional to the magnitude of AMF strength and concentrations of MNPs25. It has been shown that AMF strength of 5 - 30 kA/m is sufficient to induce an appropriate heating on the surface of MNPs9,25,26. Thus, the extent of drug release from gelatin/p(NIPAM-co-AAm)/MNPs microgel can be tuned by appropriately changing parameters, which include the amount of p(NIPAM-co-AAm), crosslinking time, the amount of MNPs, and AMF parameters (field intensity, frequency, and duration of exposure).
In our protocol for fabricating magneto-thermally responsive microgels, the most critical step is the covalent crosslinking of gelatin gel particles in genipin solution. Careful control of the crosslinking time and temperature, as well as maintaining homogeneity of the microgel suspension in the genipin solution, is required to achieve desired gel elasticity, which may influence the microgel responsivity. After crosslinking, the removal of unreacted genipin molecules is also an important step.
The gelatin has been shown to be biocompatible with low immunogenicity and enzymatically degradable8,27. The chemical crosslinker, genipin, has been considered to be non-toxic28. Thus, our gelatin-based drug delivery platform exhibiting characteristics of magnetic responsivity and suitable biodegradability may offer a useful tool for tissue engineering application as an on-demand drug carrier.
However it should be also noted that the current protocol has limitations. First, the microgels made by water-in-oil emulsification method generally exhibit a polydispersity, which may result in heterogeneity in encapsulation of drugs and MNPs among particles. Microfluidics can be a good alternative to overcome this limitation29. Second, the current drug delivery system still has a limitation that it must be used at a body temperature of 37 &#730;C due to a small degree of deswelling characteristic at the temperature, associated with a relatively low value of LCST (~ 34 &#730;C) for poly(NIPAM-co-AAm) polymer used in this study. The use of thermoresponsive polymer that can exhibit a higher LCST may overcome this issue30.

Stimuli-responsive drug delivery systems that enable a tightly controlled drug delivery in response to either endogenous or exogenous stimuli (e.g., temperature or pH) have been extensively investigated as new types of smart soft devices for drug delivery. Microscale hydrogels have been widely employed as a drug delivery platform in that they confer controllable and sustainable drug release profiles as well as tunable chemical and mechanical properties1-3. In particular, the colloidal microgels exhibit many advantages as a vehicle for drug delivery due to their rapid responsiveness to external stimuli and suitable injectability to local tissue in a minimally invasive manner4. The poly(N-isopropylacrylamide) (pNIPAM) or its copolymers have been widely adopted in synthesizing thermo-responsive microgels by grafting pNIPAM with biodegradable/ biocompatible polymers including gelatin, chitosan, alginate acid, or hyaluronic acid5,6, in which a phase transition characteristic of pNIPAM at its lower critical solution temperature (LCST) can be used as a trigger of drug release7. We recently demonstrated a fabrication of biodegradable, gelatin-based thermo-responsive microgel by incorporating poly(N-isopropylacrylamide-co-acrylamide) [p(NIPAM-co-AAm)] chains as a minor component within three-dimensional gelatin networks8. The gelatin/p(NIPAM-co-AAm) microgel exhibited a tunable deswelling to temperature increase, which positively correlated to the release of bovine serum albumin (BSA).
During the last several years, there have been increasing efforts to develop a magnetically responsive drug delivery platform that can trigger the release of drug in an on-demand fashion9,10. The basic principle for the synthesis of magnetically responsive drug delivery platform utilizes the characteristic of superparamagnetic nanoparticles (MNPs) to generate heat when they receive a high frequency alternating magnetic field (AMF), which triggers a temperature-sensitive drug release. This holds promise for future clinical applications in that this system can target deep into the tissue, enables a non-invasive and remotely controlled drug release and can be combined with hyperthermia treatment and magnetic resonance imaging system10-12. Such platforms include: (1) MNPs/pNIPAM hybrid microgel particles 13-15 and (2) macroscopic hydrogel scaffolds incorporating immobilized MNPs16-18. The pNIPAM-based microgel platforms demonstrated a finely-tunable volume phase transition responsiveness to magneto-thermal stimuli. However, they still rely on complex and sophisticated techniques in the fabrication and the use of pNIPAM polymers with a high content can be potentially cytotoxic to cells19, which may limit their in vivo applications. The macroscopic scaffolds exhibit a relatively slow response to external stimuli and require an invasive surgical transplantation compared to colloidal microgels.
The water-in-oil emulsification has been the standard method to produce submillimeter or micrometer-sized gel particles20. At the water-oil interface of the emulsion, microgel particle forms a spherical shape due to the minimization of surface energy of the water droplet under mechanical shear force. This method allows the production of a large amount of aqueous spherical gel droplets in a simple fabrication procedure and has been successfully adopted for fabricating gelatin-based microgels for drug delivery applications21-23.
Here, we present a facile method to synthesize a magnetothermally responsive gelatin-based microgels for drug delivery application by employing the water-in-oil emulsification method. This was achieved by physically incorporating iron oxide MNPs and p(NIPAM-co-AAm) chains as a minor component within a spherical microscale gelatin network that is covalently crosslinked by a naturally-derived crosslinker genipin, in conjunction with a high frequency alternating magnetic field (AMF) application system.

<table><tbody><tr><td>Gelatin</td><td>Sigma-Aldrich, MO, USA</td><td>G2500</td><td>Gelatin type A, porcine skin</td></tr><tr><td>poly(N-isopropylacrylamide-co-acrylamide)&amp;nbsp;</td><td>Sigma-Aldrich, MO, USA</td><td>738727</td><td>MW = 20,000, LCST = 34 - 38 &amp;#176;C</td></tr><tr><td>Silicone oil</td><td>Sigma-Aldrich, MO, USA</td><td>378372</td><td>Viscosity 350 cSt</td></tr><tr><td>Pluoronic L64</td><td>Sigma-Aldrich, MO, USA</td><td>435449</td><td>poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)</td></tr><tr><td>genipin</td><td>TimTec LLC, DE, USA</td><td>ST080860</td><td>MW = 226.23</td></tr><tr><td>Magnetic nanoparticles (MNPs)</td><td>Micromod Inc, Germany</td><td>79-00-102</td><td>nanomag-D-spio, 100 nm</td></tr><tr><td>TR-BSA</td><td>Life Technologies, NY USA</td><td>A23017</td><td>Albumin from Bovine Serum (BSA), Texas Red conjugate</td></tr></tbody></table>

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.

When the protocol is performed correctly, the fabricated microgels should exhibit a well-characterized spherical morphology and colloidal dispersibility with diameters in the range between 5 &#956;m to 20 &#956;m (Figure 1B and C). Either fluorescent MNPs or fluorescent BSA can be used to confirm whether MNPs or drugs (BSA in this study) are properly encapsulated within the microgel (Figure 1D). The fabricated microgels can be stable and stored at 4 oC for up to 4 weeks in the absence of any enzymatically degrading agents including collagenase. The incorporation of p(NIPAM-co-AAm) in the gelatin microgel enables it to exhibit a temperature dependent volume change (Figure 2A), in which the increase of temperature of the media from 22 oC to 42 oC resulted in the deswelling of gelatin microgels incorporating p(NIPAM-co-AAm) by ~40% in volume, in contrast to only ~10% volume change for gelatin microgel without p(NIPAM-co-AAm) (Figure 2B). The extent of deswelling of the gelatin/p(NIPAM-co-AAm) microgels can be tuned as functions of the crosslinking degree of the gelatin matrix and the concentration of p(NIPAM-co-AAm)8.
If MNPs are properly incorporated in the gelatin/p(NIPAM-co-AAm) microgel, the microgel should experience a temperature increase within the gel upon the application of an appropriate AMF, which may induce an increase of the solution temperature as well. In this study, the application of a short exposure of AMF (10 min) at a magnetic field strength of 20 kA/m resulted in gradual increases in temperature by 10 oC (from 20 oC to 30 oC) in the media (Figure 2C). It is expected that the actual temperature increase within the microgel would be much higher than the one observed in the media, since the matrix of the microgel may hinder heat dissipation to the surrounding area. The extent of TR-BSA release from gelatin/p(NIPAM-co-AAm)/MNPs was measured to be ~35%, while the TR-BSA release from gelatin/MNPs microgel without incorporating p(NIPAM-co-AAm) was significantly low at ~10% (Figure 2D). Thus, our results indicate that the release of BSA in response to AMF application was induced by the deswelling of gelatin/p(NIPAM-co-AAm)/MNPs microgel, associated with the shrinkage of p(NIPAM-co-AAm) polymer chains within the microgel (Figure 3). Since the extent of microgel deswelling is proportional to the both extent of temperature increase and concentration of p(NIPAM-co-AAm)8, a strategy to increase either amount of MNPs24 or p(NIPAM-co-AAm)8 in step 1 in the protocol section may result in increased release of BSA at a given field strength and frequency of AMF application.
<img alt="Figure 1" src="/files/ftp_upload/53680/53680fig1.jpg" /> 
Figure 1. Preparation of Gelatin Hybrid Microgels. (A) Schematic overview of the fabrication of magnetically responsive microgels. (B) Phase contrast microscopic image of a colloidal suspension of magneto-responsive microgels incorporating superparamagnetic nanoparticles (MNPs) and thermo-responsive p(NIPAM-co-AAm) chains. Scale bar = 50 &#956;m. (C) Differential interference contrast (DIC) image of a single microgel. Scale bar = 5 &#956;m. (D) Fluorescence microscopic image of the single microgel encapsulating TR-BSA. The fluorescence signals in the image are from TR-BSA. Scale bar = 5 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53680/53680fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53680/53680fig2.jpg" /> 
Figure 2. Thermo-responsive Deswelling Behavior and BSA Release from Gelatin Hybrid Microgels. (A) Representative DIC images showing the deswelling of gelatin/p(NIPAM-co-AAm) microgel induced by temperature increase from 22 oC to 42 oC. This figure has been modified from reference [8]. (B) Comparison of the deswelling ratio (final volume / initial volume) of the pure gelatin microgels and the gelatin microgels embedding p(NIPAM-co-AAm) chains in response to temperature increase from 22 oC to 42 oC. This figure has been modified from reference [8]. (C) Changes in ambient temperature in the media during the AMF application (at field strength of 20kA/m and at frequency of 2.1 MHz). (D) The release of TR-BSA (%) in response to AMF (20 kA/m at 2.1 MHz) application for 10 min. The extent of TR-BSA release from microgels was quantified by measuring the ratio of fluorescence intensity of TR-BSA, in the media of microgels solution after AMF stimulus, to total fluorescence intensity of TR-BSA within microgles before AMF stimulus at 22 oC, using a spectrophotometer *: p &#60;0.05 between groups. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53680/53680fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53680/53680fig3.jpg" /> 
Figure 3. A Schematic Illustration of the Potential Mechanism by Which Gelatin/p (NIPAM-co-AAm)/MNPs Microgel Releases Drugs in Response to AMF Application. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53680/53680fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release at JoVE.com

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

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 ...

alternating-magnetic-field-responsive-hybrid-gelatin-microgels-for

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.7K Views.  Kent State University.  We presenteren een gemakkelijke methode om een ​​biologisch afbreekbare op gelatinebasis geneesmiddelafgifte platform dat magneto-thermisch gevoelige fabriceren. Dit werd bereikt door superparamagnetische ijzeroxide nanodeeltjes en poly (N-isopropylacrylamide- co-acrylamide) in een bolvormig gelatine micro-netwerk verknoopt door genipin, samen met een wisselend magnetisch veld aanbrengsysteem. 

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

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 ...

Chemistry

Generation of Alginate Microspheres for Biomedical Applications

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and physical architecture. Applications include cell encapsulation, drug delivery, stem cell culture, and tissue engineering scaffolds. In fact, clinical trials are currently being performed in which islets are encapsulated in PLO coated alginate microbeads as a treatment of type I diabetes. However, large numbers of islets are required for efficacy due to poor survival following transplantation. The ability to locally stimulate microvascular network formation around the encapsulated cells may increase their viability through improved transport of oxygen, glucose and other vital nutrients. Fibroblast growth factor-1 (FGF-1) is a naturally occurring growth factor that is able to stimulate blood vessel formation and improve oxygen levels in ischemic tissues. The efficacy of FGF-1 is enhanced when it is delivered in a sustained fashion rather than a single large-bolus administration. The local long-term release of growth factors from islet encapsulation systems could stimulate the growth of blood vessels directly towards the transplanted cells, potentially improving functional graft outcomes. In this article, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. In addition, we describe a method we developed for generating multilayered alginate microbeads. Cells can be encapsulated in the inner alginate core, and angiogenic proteins in the outer alginate layer. The release of proteins from this outer layer would stimulate the formation of local microvascular networks directly towards the transplanted islets.

In the following sections, we outline procedures for the preparation of alginate microspheres for use in biomedical applications. We specifically illustrate a technique for creating multilayered alginate microspheres for the dual purpose of cell and protein encapsulation as a potential treatment for type 1 diabetes.

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

Medicine

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

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.), ...

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.

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.

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

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 ...

Injectable Supramolecular Polymer-Nanoparticle Hydrogels for Cell and Drug Delivery Applications

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are composed of two components: hydrophobically modified cellulose as the network polymer and self-assembled core-shell nanoparticles that act as non-covalent cross linkers through dynamic, multivalent interactions. These methods describe both the formation of these self-assembled nanoparticles through nanoprecipitation as well as the formulation and mixing of the two components to form hydrogels with tunable mechanical properties. The use of dynamic light scattering (DLS) and rheology to characterize the quality of the synthesized materials is also detailed. Finally, the utility of these hydrogels for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery is demonstrated through in vitro experiments to characterize&#160;drug release, thermal stability, and cell settling and viability. Due to its biocompatibility, injectability, and mild gel formation conditions, this hydrogel system is a readily tunable platform suitable for a range of biomedical applications.

This protocol describes the synthesis and formulation of injectable, supramolecular polymer-nanoparticle (PNP) hydrogel biomaterials. Applications of these materials for drug delivery, biopharmaceutical stabilization, and cell encapsulation and delivery are demonstrated.

These methods describe how to formulate injectable, supramolecular polymer-nanoparticle (PNP) hydrogels for&#160;use as biomaterials. PNP hydrogels are ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

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

Samenvatting

Meer video's verkennen