We thank the Imperial College London Department of Chemistry and the Medical Research Council Institute of Life Sciences for their support.

1. Synthesis of redox-responsive crosslinker
<ol>
	<li>Add 2-aminoethylmethacrylate hydrochloride (44.79 mg, 0.270 mmol) and 0.063 mL of triethylamine to 3 mL of anhydrous dichloromethane and allow to mix for 20 min under an inert N2 gas in a 25 mL round bottomed flask.</li>
	<li>Dissolve 4,7,10,13,16,19,22,25,32,35,38,41,44,47,50,53-Hexadecaoxa-28,29-dithiahexapentacontanedioic acid di-N-succinimidyl ester (100 mg, 0.045 mmol) in 1 mL of anhydrous dichloromethane and add to the flask.</li>
	<li>Add 4 mL of anhydrous dichloromethane to the flask. Wrap the reaction flask in aluminum foil and leave to stir under N2 at room temperature.</li>
	<li>Confirm product formation by thin-layer chromatography. Ensure correct references for the starting material while confirming product formation by this method.
	<ol>
		<li>Thin-layer chromatography is an affinity-based method used to separate desired compounds from the reaction mixture. Here, use the highly polar and adsorbent silica plate as a stationary phase. Spot the starting material sample and the reaction mixture sample onto one end of the silica plate and place vertically into a closed glass beaker with the eluent (mobile phase: 5% methanol: dichloromethane).</li>
		<li>By capillary action, the mobile phase moves up the silica plate and the sample compounds migrate varying distances depending on their affinities for the stationary and mobile phases. When the solvent front has crossed 3/4th of the plate, remove the plate and dry it. The separated compounds appear as spots on the plate. Quantify the retention factor (Rf) of the desired compound as the distance travelled by compound/distance travelled by solvent. Here, Rf of the product was 0.35. UV light or staining can be used to visualize the spots (potassium permanganate).</li>
	</ol>
	</li>
	<li>Concentrate the mixture in vacuo and purify via flash column chromatography (isocratic mixture of 5% methanol: dichloromethane)36. Introduce the reaction mixture onto the top of a glass column packed with silica (stationary phase) and then elute with the solvent system (mobile phase)36. Collect all fractions and assay using TLC to distinguish the product fractions using the previously determined Rf value.</li>
	<li>Concentrate the purified fractions by evaporating the solvent using a rotary evaporator (temperature: 40 &#176;C), set vacuum pressure to atmospheric pressure to remove lower boiling dichloromethane first. When the level of the solvent stops decreasing, set the vacuum pressure to 337 mbar to remove methanol and attain a pale-yellow oil, which will then be subjected to further characterization to confirm product formation37.</li>
	<li>Conduct Fourier-Transform Infrared (FT-IR) analysis38, 1H&#160;NMR (in deuterated dimethylsulfoxide (DMSO-d6))39, 13C NMR39 and matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF)40 analysis to confirm product formation before performing next steps.
	<ol>
		<li>For FT-IR analysis, load 1 mg of the oil onto the instrument to obtain the spectra.</li>
		<li>For NMR analyses, re-dissolve 10 mg of the product in 0.75 mL of the relevant NMR solvent, transfer to an NMR tube and seal tightly to prevent leakage. Load the sample onto the NMR instrument, after which the spectra are obtained39.</li>
		<li>For the mass spectrometry analysis, seal 1 mg of the product in a vial, dissolve in DMSO and load onto the instrument. 
		NOTE: Infrared analysis provides information regarding the key functional groups in the desired product, namely the carbonyl stretches (C=O) identified by the sharp peak at 1650 cm-1 indicates the formation of the amide bond, thereby highlighting product formation. The 1H NMR and the 13C provide information on the chemical environments and thereby can be used to determine the chemical composition of the desired product via the identification of chemical environments present in the compound. Finally, the use of mass spectrometry can verify the exact mass of the desired product.</li>
	</ol>
	</li>
</ol>
2. Test cleavage of disulphide crosslinker with glutathione
<ol>
	<li>Incubate the disulphide crosslinker (to a final concentration of 4.55 mg/mL) with the reducing agent glutathione (final [GSH]: 10 mM), to a final volume of 1 mL, for 24 h in a 1.5 mL glass vial.</li>
	<li>To confirm cleavage of the disulphide linker, submit the vial for MALDI-ToF analysis26,40.</li>
</ol>
3. Redox-responsive nanogel synthesis
<ol>
	<li>Ensure all glassware to be used for the reaction is free of dust by washing with filtered (0.20 &#181;m) deionized water and subsequently drying the glassware using compressed air that can be accessed via the compressed air utility on the side of the fumehood. Attach a secure nozzle to the compressed air outlet to ease accessibility. Hold the vial securely and position the nozzle into it. Activate the compressed air, releasing a controlled stream into the vial. Observe the process to ensure effective drying, checking for any remaining contaminants or moisture.</li>
	<li>Prepare a 10 mL glass vial by filling it with deionized water and seal the flask with a rubber septum. Deoxygenate the water by bubbling N2 through it for at least 30 min prior to starting the reaction. To do this, fill a balloon with N2 gas and attach a needle to the balloon; place it on the septum and use the needle to pierce the rubber septum and allow N2 flow. Attach an exit needle to the flask to ensure constant flow of N2.</li>
	<li>Dissolve bovine serum albumin (BSA; 2 mg, 30 nmol) or Cy7-tagged BSA (2 mg, 30 nmol) in 1 mL of deoxygenated deionized water and add to another clean 10 mL glass vial.</li>
	<li>Add [2-(acryloyloxy)ethyl]trimethylammonium chloride (AETC) solution (12.00 mg/10.86 &#181;L, 0.060 mmol) to the flask and seal it using another rubber septum. Ensure the reaction mixture is kept under a constant flow of N2 gas and stir for 30 min.</li>
	<li>Dissolve acrylamide (9.21 mg, 0.13 mmol) in 1 mL of deoxygenated deionized water and add to the mixture in the vial.</li>
	<li>Dissolve disulphide crosslinker (4.44 mg, 0.004 mmol) in 1 mL of deionized water and add to the mixture in the vial. Flush the mixture with N2 gas for 20 min.</li>
	<li>Dissolve sodium dodecyl sulfate (2 mg, 0.007 mmol) in 1 mL of deoxygenated deionized water and add to the mixture via a N2 gas flushed syringe and stir the mixture for 30 min. 
	NOTE: By flushing the syringe with N2 we minimize the exposure of the reaction mixture to oxygen in the atmosphere which can inhibit the radical polymerization reaction.</li>
	<li>Add N,N,N&#39;,N&#39;-tetramethylethylenediamine (1.5 &#181;L, 0.010 mmol) and let the reaction mixture deoxygenate for 3 min under N2 gas.</li>
	<li>Dissolve ammonium persulfate (1 mg, 0.004 mmol) in 1 mL of deoxygenated deionized water and add to the reaction mixture. Ensure that N2 gas is bubbling through the reaction mixture for 10 min. 
	NOTE: If the N2 addition step is omitted, it is likely that white particulates will form in the reaction flask. These are larger-sized particles and/or aggregates and can confound any further characterization.</li>
	<li>Seal the flask under a N2 atmosphere and leave to stir for 3 h and 30 min at room temperature.</li>
	<li>To end the reaction, remove the seal from the flask and let the reaction mixture stir for at least 10 min. This would expose the reaction mixture to atmospheric levels of O2 gas, which would then quench any remaining radicals in the reaction mixture. The reaction ends when the mixture assumes a clear blue opalescent appearance.</li>
	<li>In order to purify the nanogels, add the reaction mixture to a 15 mL centrifugal filter unit (100 kDa MWCO) and add deionized water to fill the centrifugal unit. Centrifuge the reaction mixture at 1000 x g for 10 min. This will remove any unreacted chemicals (or unencapsulated payload) that are smaller than 100 kDa and concentrate the nanogel sample.</li>
	<li>Once the level of water in the centrifugal unit decreases, add 2 mL deionized water to dilute the nanogel sample and&#160;centrifuge to concentrate the product. Repeat this step 3x.</li>
	<li>Store the sample at 2-8 &#176;C to prevent the degradation of the encapsulated protein.</li>
	<li>Conduct FT-IR and 1H&#160;NMR (in deuterated dimethyl sulfoxide (DMSO-d6)) analysis to confirm polymerization39,41. To prepare the sample, freeze-dry 1&#160;mL of the nanogel solution in a 1.5 mL vial. The freeze-drying process involves freezing the sample and then applying a vacuum to remove the frozen solvent through sublimation, leaving behind a powdered residue. After freeze-drying, re-dissolve the sample in the relevant NMR solvent, transfer to an NMR tube and tightly seal it. Load the sample onto the NMR machine, as described previously39. For FT-IR analysis, 1 mg of the dry powder should be loaded onto the instrument to obtain the spectra.</li>
</ol>
4. Nanogel morphological characterization
<ol>
	<li>Dynamic light scattering (DLS) 
	NOTE: Hydrodynamic diameter (nm), polydispersity index (PI), and zeta potential were measured at 25 &#176;C, 632.8 nm laser wavelength and 173&#176; signal detector using a DLS machine. Each run measured 25 sub-scanning cycles.
	<ol>
		<li>Hydrodynamic size (nm) measurement
		<ol>
			<li>Prepare a cuvette or a disposable folded capillary cell by cleaning it with filtered deionized water. Blow compressed air into the cuvette (suitable for both DLS and zeta potential measurements) to ensure it is free of large dust particles.</li>
			<li>Fill the cuvette with at least 50-100 &#181;L of the sample and dilute in 750-1000 &#181;L of filtered deionized water. While using a zeta potential cuvette, ensure that the cuvette has been filled with sample without bubble formation, which can perturb measurements.</li>
			<li>Put the cuvette inside the DLS instrument, ensuring that it has been correctly inserted. To do so, open the sample compartment cover of the DLS instrument. Hold the cuvette by its top edges to avoid fingerprints on the optical windows. Ensure the cuvette is properly seated in the cuvette holder making sure it is level and aligned with the instrument&#39;s optical path. Close the sample compartment to prevent ambient light interference. 
			NOTE: The dilution media is filtered using 0.45 &#181;m pore size syringe filter.</li>
			<li>Click to start the particle size measurement on the relevant DLS machine software.</li>
			<li>Select the results corresponding to the sample to obtain the z-average (average particle size), average PI, kilo-counts per second (kcps), zeta potential and relevant graphics. Ensure that the correlation function exhibits a smooth sigmoidal curve, as this suggests a uniform sample.</li>
			<li>Once analysis is complete, remove the cuvette from the DLS.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Transmission electron microscopy (TEM)
	<ol>
		<li>Prepare samples by placing 10 &#181;L of the nanoparticle solution (0.077 mg/mL) onto carbon-coated copper grids. Air dry at room temperature for 1 h.</li>
		<li>Wash the grids with filtered deionized water and stain with 5 &#181;L of filtered 1% uranyl acetate in water for 2 min.</li>
		<li>After soaking the excess staining agent with filter paper, dry the grids overnight at room temperature. Capture TEM images the next day at 45,000x magnification using a bright field camera.</li>
		<li>Use the provided software for image processing (resolution: 2048 x 2048 pixels).</li>
	</ol>
	</li>
</ol>
5. Quantification of protein (BSA) encapsulated within the nanogels using a micro-bicinchoninic acid (BCA) assay
<ol>
	<li>According to the microBCA assay protocol (given on the manufacturer&#39;s website), generate a standard curve based on a pre-defined protein concentration42.</li>
	<li>Following their synthesis, purify the protein-encapsulating nanogels using 15 mL centrifugal filter units with 100 kDa MWCO (1000 x g, 10 min) as described in steps 3.12-3.14.</li>
	<li>Collect the filtrate and analyze using the microBCA assay (protocol available on the manufacturer&#39;s website). Measure the absorbance at 562 nm using a plate reader. As this measurement correlates to the unencapsulated protein concentration, determine the mass of the encapsulated protein. To calculate the encapsulation efficiency (EE) %, use the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/63981/63981eq01.jpg" style="margin: auto;" /></li>
</ol>
6. Quantification of protein release from nanogels in the presence of glutathione
<ol>
	<li>Incubate 200 &#181;L of 5 mg/mL of Cy7BSA-loaded nanogels with 200 &#181;L of 10 mM glutathione (GSH) in the 0.5 mL centrifugal filter units (100 kDa MWCO). Place the filter units in a water bath to maintain the temperature at 37 &#176;C.</li>
	<li>Centrifuge at 1000 x g for 3 min every 10 min for at least 1 h - then every 2-8 h.</li>
	<li>Collect the filtrate and use for further analysis. Maintain the volume of the filtrate within the centrifugal filter units at 500 &#181;L by adding more deionized water or PBS.</li>
	<li>Measure the amount of protein in the filtrates by referring to a standard curve for Cy7BSA-loaded nanogels, where the absorbance of the fluorescent protein was measured using a nanodrop instrument at 750 nm. 
	NOTE: This standard curve can be calculated by preparing serial dilutions of Cy7BSA within the range of 0 mg/mL to 2 mg/mL. Values obtained from the nanodrop can be interpolated against this calibration curve to determine the encapsulation efficiency.</li>
</ol>

Authors have no conflicts of interest to disclose. There are no financial details to declare.

The growing demand for target-specific biologics in the biopharmaceutical industry has driven a need for technologies that can improve their in vivo pharmacological profiles, while preventing their rapid physiological degradation and offsetting any unwanted side effects. With this in mind, a straightforward procedure for the synthesis of protein-loaded nanogels is described. As indicated in the protocol, the redox-responsive crosslinker needs to be synthesized prior to nanogel synthesis. Then, the key remaining monomers are added and after adding sodium dodecyl sulfate (SDS), in situ radical polymerization is initiated by N,N,N&#39;,N&#39;-tetramethylethylenediamine (TEMED) and ammonium persulfate (APS). The highly soluble acrylamide (AM) maintains nanogel stability in aqueous settings, the 2-(acryloyloxy)ethyl trimethylammonium chloride (AETC) salt maintains electrostatic templating of desired protein payload and reduces premature release26. Further, the PEG disulphide diacrylate crosslinker enables controlled release of payload28,32. SDS is later removed by purification methods; centrifugation with three washes against deionized water or dialysis.
Many researchers worldwide have synthesized polymeric nanoparticles using a variety of different synthetic strategies1,48,49. Our synthetic protocol differs from the commonly used methods in a fundamental way, as it does not involve the use of organic solvents or high temperatures. Taking this into account, the novelty of our procedure does mean that the user&#39;s ability may play a key role in successful synthesis of the nanogels. An important step in nanogel synthesis is ensuring the maintenance of inert atmosphere, which must take place in a controlled manner in order to avoid loss of the desired product, as oxygen in the air can hinder the polymerization of acrylates and methacrylates. Therefore, the mixing and the addition of each reagent under inert conditions may require some practice to be achieved correctly. Nonetheless, the protocol outlined offers a versatile methodology, as numerous other monomers can be added to the synthesis to endow the nanogels with specific properties, for instance the use of acrylated&#160;heterobifunctional monomers to make the nanogels amenable towards further bio-conjugative procedures. Further, it is possible, with enough optimization, to explore the encapsulation of nucleic acid-based payloads, as there has been an immense resurgence in the use of gene therapies to treat not only rare monogenic diseases, but also chronic and progressive diseases50.
In summary, we have presented a mild aqueous-based strategy to reproducibly synthesize nanogels with sub-100 nm sizes and efficient payload-retaining abilities. In addition, by incorporating a novel disulphide crosslinker within the nanogels, we have demonstrated the controlled release of the entrapped payload in response to an intracellular reducing agent. We have also demonstrated that this synthetic strategy does not compromise the payload. This protocol will provide a foundation for further innovation in the field of stimuli-responsive nano-therapy. Due to their unique and flexible synthetic conditions, nanogels are expected to play a critical role in the drug delivery field going forward.

Nanogels are three-dimensional, sub-micron-sized hydrogels with crosslinked polymer network structures that can hold large quantities of fluids within their core shell without affecting their morphological integrity1. In general, nanogels are synthesized by the polymerization of functional monomers via physical or chemical crosslinking in heterogeneous colloidal systems, such as water-in-oil inverse microemulsions2,3. Amphiphilic copolymers can self-assemble into nanoscale structures in aqueous environments. However, they must be stabilized using chemical crosslinking strategies involving disulfides or amide-based coupling, click chemistry, or can be physically induced (hydrophobic, electrostatic, or hydrogen-bonding strategies) or photo-induced4. Among these strategies, the physical self-assembly of polymers followed by chemical crosslinking has been reported as a successful nanogel fabrication technique5. While historically, the first nanogel was introduced in the 1990s by Vingradov et al.6, Akiyoshi et al.7, and Lemieux et al.8, lately, a variety of smart nanogels composed of both natural and synthetic polymers have been developed and explored for diverse biomedical applications9.
Nanogels possess extensive cargo-retaining capacity, large surface area, in vivo stability, as well as customizable chemical and mechanical properties10. The synthesis of nanogels is also scalable and can be aqueous-based. In addition, the enhanced water content of the nanogels makes them effective carriers of sensitive biological payloads11. Furthermore, the high surface area can satisfy multiple bioconjugation needs, thereby permitting the attachment of targeting modalities to enable active targeting. Notably, the versatility of nanogel design allows the use of a wide range of stimuli-responsive monomers that permits precise control of their physicochemical properties9. This unique engineerability enables the rational improvement of nanogel design, which is difficult to achieve with conventionally used liposomes, micelles, or polymerosomes12,13. By incorporating stimuli-responsive moieties within specifically designed monomers, nanogels can be engineered to trigger the controlled release of their payload in response to various physiologically-relevant stimuli, such as pH, redox conditions, enzymes, etc.9,14. Such smart nanogels are more useful than conventional nanogels, as they possess superior stability for extended blood circulation, and they can endure physiological conditions to maintain the integrity of their cargo and mediate its controlled release at desired target sites15. Indeed, due to their versatile nature, nanogels have gained traction in the biomedical arena, with notable advances in the development of stimuli-responsive nanogels for numerous theranostic and diagnostic applications2,16,17.
Biologics can represent a category of pharmaceutical products consisting of proteins, peptides, and/or nucleic acids and have revolutionized the therapeutic landscape due to their remarkable selectivity, thereby becoming the fastest-growing class of therapeutics18. Indeed, the growing market for such therapeutics is evident in the steep increase in their approval by the US Federal Drug Association (FDA), where biologics represented ~40% of total drug approvals, in 202319. In addition to their specificity and potency, rapid discoveries of novel drug targets, more efficient bioengineering processes and greater knowledge of the in vivo fate of these therapeutics has led to their increased use20. Traditional biologics include interfering RNA, replacement proteins, cytokines and hormones that are usually generated using recombinant DNA technology21. Since the approval of human recombinant insulin in 1982, biologics have been developed for many conditions including cancer (e.g., trastuzumab, avelumab), inflammatory bowel disease (e.g., adalimumab, certolizumab) and rare genetic diseases (e.g., mipomersan, myozyme, aldurazyme, fabrazyme)21. While the high specificity of the interactions of biologics with their targets should theoretically offset any off-target effects, several clinical concerns have emerged with their use relating to unwanted side effects22. These side effects can be grouped into two categories, including exaggerated pharmacology (over-stimulation of targets) and immunogenicity. Further to this, their short half-lives, limited bioavailability, protease damage, short shelf-life and costly production processes limit their therapeutic benefits21. Conventional methods of mitigating these issues, involve covalent modification of these biologics that can compromise their function, and therefore, efficacy23. Alternatively, the nanomedicine approach to encapsulating therapeutic payloads can confer numerous advantages to pharmacological properties, most importantly, passive targeting to the inflamed site via the enhanced permeation and retention (EPR) effect24. Other nanoparticle-associated benefits can include enhanced circulation times, reduced clearance rate, greater formulation flexibility, improved vasculature permeation and cellular uptake25. While a huge variety of nanoparticle formulations are currently under investigation for the delivery of biological payloads, few can emulate the multifunctionality of nanogels. Indeed, nanogels exceed the loading capacities achieved by liposomal- and micelle-based nanoparticles, and they exhibit greater colloidal stability than most inorganic nanoparticles. As such, nanogels present a valuable platform for the delivery of various biological therapeutics.
We have previously successfully delivered an anti-oxidative enzyme within novel matrix metalloproteinase-responsive crosslinked polymeric nanogels, where the mild encapsulation strategy used maintained the protein&#39;s bioactivity upon release26. In this work, we demonstrate the optimized synthesis of redox-responsive nanogels for the delivery of protein-based payloads. Notably, the synthetic methodology enables nanogel synthesis using mild conditions to encapsulate the desired payload, without the use of harsh organic solvents or high temperatures. We exploited the redox homeostasis within the intracellular environment to regulate the release of the encapsulated payload27,28. Typically, the naturally abundant antioxidant glutathione (GSH) controls the extracellular and intracellular redox potentials, where its concentration ranges between 2-20 &#181;M and 1-10 mM, respectively29,30. To date, numerous redox-sensitive nanoparticles have been reported, making this a proven and reliable strategy to enable controlled released of drugs in vivo27,28. Indeed, disulphide bonds have been installed within polymeric nanomaterials by using disulphide-containing crosslinkers31,32, self-assembly of biodegradable polymers from disulphide-containing monomers33, and redox-responsive polymer prodrugs or drug/polymer conjugates34,35. Therefore, this study investigates the incorporation of a unique, highly GSH-sensitive disulphide crosslinker within the polymeric nanoparticles, thereby enabling the controlled release of an encapsulated protein payload.
In this study, nanogel design was centered on the following criteria to address specificity and payload delivery: small size (~100 nm) and a uniform size distribution (polydispersity index (PI)&#60;0.3) to ensure efficient penetration of the endothelium and in vivo stability27; efficient encapsulation of protein payload, and controlled release of payload in response to GSH. We report the synthesis of GSH-responsive crosslinked nanogels, that demonstrated homogenous sub-100 nm sized nanoparticles, with a 76% encapsulation efficiency of the desired protein payload.

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-(&lt;br/&gt; acryloyloxy)ethyl]trimethylammonium chloride solution&amp;nbsp;</td><td>Sigma Aldrich</td><td>496146</td><td></td></tr><tr><td>2-aminoethyl methacrylatehydrochloride&amp;nbsp;</td><td>Sigma Aldrich</td><td>516155</td><td></td></tr><tr><td>4,7,10,13,16,19,22,25,32,35,38,41,&lt;br/&gt; 44,47,50,53-Hexadecaoxa-28,29-dithiahexapentacontanedioic acid di-N-succinimidyl ester&amp;nbsp;</td><td>Sigma Aldrich</td><td>671630</td><td></td></tr><tr><td>Acrylamide</td><td>Sigma Aldrich</td><td>23701</td><td></td></tr><tr><td>Ammonium persulfate</td><td>Sigma Aldrich</td><td>248614</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>B6917</td><td></td></tr><tr><td>Cy7- labelled bovine serum albumin</td><td>Nanocs</td><td>BS1-S7-1</td><td></td></tr><tr><td>Deuterated dimethyl sulfoxide</td><td>Sigma Aldrich</td><td>547239</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma Aldrich</td><td>270997 (anhydrous) and D65100</td><td></td></tr><tr><td>Glutathione</td><td>Sigma Aldrich</td><td>G4251</td><td></td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>34860</td><td></td></tr><tr><td>N,N,N&amp;rsquo;,N&amp;rsquo;-tetramethylethylenediamine&amp;nbsp;</td><td>Sigma Aldrich</td><td>411019</td><td></td></tr><tr><td>Phosphate buffered saline&amp;nbsp;</td><td>ThermoFisher</td><td>10010023</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Sigma Aldrich</td><td>436143</td><td></td></tr><tr><td>Triethylamine</td><td>Sigma Aldrich</td><td>471283</td><td></td></tr><tr><td>Uranyl Acetate</td><td>Agar Scientific&amp;nbsp;</td><td>AGR1260A</td><td></td></tr><tr><td>&lt;strong&gt;Equipment necessary for nanogel synthesis and characterisation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal filter units (100kDa MWCO)&amp;nbsp;</td><td>Merck Millipore</td><td>C7715</td><td></td></tr><tr><td>Camera</td><td>Olympus&amp;nbsp;</td><td>Veleta</td><td></td></tr><tr><td>Carbon-coated copper grids</td><td>Agar Scientific&amp;nbsp;</td><td>AGS160</td><td></td></tr><tr><td>Dialysis tubing (100kDa MWCO)</td><td>Spectrum labs</td><td>11405949</td><td></td></tr><tr><td>Dynamic Light Scattering&amp;nbsp;</td><td>Malvern&amp;nbsp;</td><td>Zetasizer Nano Ultra</td><td></td></tr><tr><td>Freeze dryer</td><td>Labconco</td><td>WZ-03336-01</td><td></td></tr><tr><td>Infrared spectroscopy</td><td>Agilent&amp;nbsp;</td><td>Cary 630 FTIR</td><td></td></tr><tr><td>iTEM software</td><td>Olympus&amp;nbsp;</td><td></td><td></td></tr><tr><td>Mass spectrometry</td><td>Waters</td><td>Micromass MALDI microMX MALDI Q-ToF&amp;nbsp;</td><td></td></tr><tr><td>MF-MilliporeTM membrane filter (0.45/0.2&amp;mu;m pore size)</td><td>Merck Millipore, UK</td><td>HAWP04700, GSWP04700&amp;nbsp;</td><td></td></tr><tr><td>Micro BCA Protein Assay Kit</td><td>ThermoFisher</td><td>23235</td><td></td></tr><tr><td>Plate reader</td><td>Beckman</td><td>Coulter-PARADIGM</td><td></td></tr><tr><td>Proton and Carbon-13 nuclear magnetic resonance data</td><td>Bruker</td><td>400MHz AV-400 NMR spectrometer</td><td></td></tr><tr><td>Rotary evaporator&amp;nbsp;</td><td>Buchi&amp;nbsp;</td><td>R-114 Rotary Vap System</td><td></td></tr><tr><td>Single-use needles&amp;nbsp;</td><td>Sterican</td><td>4665643</td><td></td></tr><tr><td>Suba-Seal&amp;nbsp;septa</td><td>Sigma Aldrich</td><td>Z124575</td><td></td></tr><tr><td>Transmission electron microscopy</td><td>Phillips&amp;nbsp;</td><td>CM 100 TEM&amp;nbsp;</td><td></td></tr><tr><td>UV-vis spectrophotometer</td><td>Nanodrop</td><td>Nanodrop One/One C microvolume&amp;nbsp;</td><td></td></tr></tbody></table>

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.

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 ...

synthesis-stimuli-responsive-nanogels-using-aqueous-one-step

Nanyang Technological University

Research

JoVE Journal

Chemistry

945 Views.  Imperial College London. 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.

Video: Synthesis of Stimuli-responsive Nanogels using Aqueous One-step Crosslinking and Co-nanopolymerization

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

Bioengineering

Synthesis and Characterization of Functionalized Metal-organic Frameworks

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and technological applications. Their signature property is their ultrahigh porosity, which however imparts a series of challenges when it comes to both constructing them and working with them. Securing desired MOF chemical and physical functionality by linker/node assembly into a highly porous framework of choice can pose difficulties, as less porous and more thermodynamically stable congeners (e.g., other crystalline polymorphs, catenated analogues) are often preferentially obtained by conventional synthesis methods. Once the desired product is obtained, its characterization often requires specialized techniques that address complications potentially arising from, for example, guest-molecule loss or preferential orientation of microcrystallites. Finally, accessing the large voids inside the MOFs for use in applications that involve gases can be problematic, as frameworks may be subject to collapse during removal of solvent molecules (remnants of solvothermal synthesis). In this paper, we describe synthesis and characterization methods routinely utilized in our lab either to solve or circumvent these issues. The methods include solvent-assisted linker exchange, powder X-ray diffraction in capillaries, and materials activation (cavity evacuation) by supercritical CO2 drying. Finally, we provide a protocol for determining a suitable pressure region for applying the Brunauer-Emmett-Teller analysis to nitrogen isotherms, so as to estimate surface area of MOFs with good accuracy.

Synthesis, activation, and characterization of intentionally designed metal-organic framework materials is challenging, especially when building blocks are incompatible or unwanted polymorphs are thermodynamically favored over desired forms. We describe how applications of solvent-assisted linker exchange, powder X-ray diffraction in capillaries and activation via supercritical CO2 drying, can address some of these challenges.

Metal-organic frameworks have attracted extraordinary amounts of research attention, as they are attractive candidates for numerous industrial and ...

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 ...

Using Polystyrene-block-poly(acrylic acid)-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are encapsulated in polystyrene-block-poly(acrylic acid) (PSPAA) shells and then used as monomers for their self-assembly. Spherical PSPAA micelles upon acid treatment are known to assemble into cylindrical micelles. Exploiting this tendency, the core-shell nanoparticles are induced to aggregate, coalesce, and then transform into long chains. When more than one type of nanoparticles are used, random and block &#8220;copolymers&#8221; of nanoparticles can be obtained. Detailed procedures are reported for the PSPAA encapsulation of nanoparticles, homo- and co-polymerization of the core-shell nanoparticles, separation and purification of the resulting nanoparticle chains. Transformations of single-line chains into double- and triple-line chains are also presented. The synergy between the polymer shell and the embedded nanoparticles leads to an unusual chain-growth polymerization mode, giving long nanoparticle chains that are distinct from the products of the traditional step-growth aggregation process.

Using Polystyrene-block-polyacrylic acid-coated Metal Nanoparticles as Monomers for Their Homo- and Co-polymerization

We report protocols for &#8220;polymerizing&#8221; various types of polymer-encapsulated metal nanoparticles into long chains of &#8220;homo-&#8220; and &#8220;co-polymers&#8221;.

We present a template-free method for &#8220;polymerizing&#8221; nanoparticles into long chains without side branches. A variety of nanoparticles are ...

Synthesis and Reaction Chemistry of Nanosize Monosodium Titanate

This paper describes the synthesis and peroxide-modification of nanosize monosodium titanate (nMST), along with an ion-exchange reaction to load the material with Au(III) ions. The synthesis method was derived from a sol-gel process used to produce micron-sized monosodium titanate (MST), with several key modifications, including altering reagent concentrations, omitting a particle seed step, and introducing a non-ionic surfactant to facilitate control of particle formation and growth. The resultant nMST material exhibits spherical-shaped particle morphology with a monodisperse distribution of particle diameters in the range from 100 to 150&#160;nm. The nMST material was found to have a Brunauer-Emmett-Teller (BET) surface area of 285 m2g-1, which is more than an order of magnitude higher than the micron-sized MST. The isoelectric point of the nMST measured 3.34 pH units, which is a pH unit lower than that measured for the micron-size MST. The nMST material was found to serve as an effective ion exchanger under weakly acidic conditions for the preparation of an Au(III)-exchange nanotitanate. In addition, the formation of the corresponding peroxotitanate was demonstrated by reaction of the nMST with hydrogen peroxide.

The surfactant mediated sol-gel synthesis of nanosized monosodium titanate is described, along with preparation of the corresponding peroxide modified material. An ion-exchange reaction with Au(III) is also presented.

This paper describes the synthesis and peroxide-modification of nanosize monosodium titanate (nMST), along with an ion-exchange reaction to load the ...

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

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

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

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

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

Nanosponge Tunability in Size and Crosslinking Density

We describe a protocol for the synthesis of linear polyesters containing pendant epoxide functionality and their incorporation into a nanosponge with controlled dimensions. This approach begins with synthesis of a functionalized lactone which is key to the pendant functionalization of the resulting polymer. Valerolactone (VL) and allyl-valerolactone (AVL) are then copolymerized using ring-opening polymerization. Post-polymerization modification is then used to install an epoxide moiety on some or all of the pendant allyl groups. Epoxy-amine chemistry is employed to form nanoparticles in a dilute solution of both polymer and small molecule diamine crosslinker based on the desired nanosponge size and crosslinking density. Nanosponge sizes can be characterized by transmission electron microscopy (TEM) imaging to determine the dimension and distribution. This method provides a pathway by which highly tunable polyesters can create tunable nanoparticles, which can be used for small molecule drug encapsulation. Due to the nature of the backbone, these particles are hydrolytically and enzymatically degradable for a controlled release of a wide range of hydrophobic small molecules.

This article describes a process for tuning the size and crosslinking density of covalently crosslinked nanoparticles from linear polyesters containing pendant functionality. By tailoring synthesis parameters (polymer molecular weight, pendant functionality incorporation, and crosslinker equivalents), a desired nanoparticle size and crosslinking density can be achieved for drug delivery applications.

We describe a protocol for the synthesis of linear polyesters containing pendant epoxide functionality and their incorporation into a nanosponge with ...

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.), ...

Microfluidic Synthesis of Microgel Building Blocks for Microporous Annealed Particle Scaffold

The microporous annealed particle (MAP) scaffold platform is a subclass of granular hydrogels. It is composed of an injectable slurry of microgels that can form a structurally stable scaffold with cell-scale porosity in situ following a secondary light-based chemical crosslinking step (i.e.,&#160;annealing). MAP scaffold has shown success in a variety of regenerative medicine applications, including dermal wound healing, vocal fold augmentation, and stem cell delivery. This paper describes the methods for synthesis and characterization of poly(ethylene glycol) (PEG) microgels as the building blocks to form a MAP scaffold. These methods include the synthesis of a custom annealing macromer (MethMAL), determination of microgel precursor gelation kinetics, microfluidic device fabrication, microfluidic generation of microgels, microgel purification, and basic scaffold characterization, including microgel sizing and scaffold annealing. Specifically, the high-throughput microfluidic methods described herein can produce large volumes of microgels that can be used to generate MAP scaffolds for any desired application, especially in the field of regenerative medicine.

This protocol describes a set of methods for synthesizing the microgel building blocks for microporous annealed particle scaffold, which can be used for a variety of regenerative medicine applications.

The microporous annealed particle (MAP) scaffold platform is a subclass of granular hydrogels. It is composed of an injectable slurry of microgels that ...

Hydrogel Synthesis

Source: Amber N. Barron, Ashlea Patterson, and <a href="https://www-jove-com.remotexs.ntu.edu.sg/author/Taylor+D._Sparks">Taylor D. Sparks</a>, Department of Materials Science and Engineering, The University of Utah, Salt Lake City, UT
Hydrogels are a versatile class of cross-linked polymers produced through relatively simple procedures and with generally inexpensive materials. They can be formed from solution and involve a polymer backbone formed from monomer reagents, an initiator which makes the polymer reactive and a crosslinking species which binds the polymer chains together. An important aspect of these materials is that they swell in the presence of water, but this response can be tuned further to enhance swelling as a function of salinity, pH, or other signals. As a final product, hydrogels can be used in aqueous or dry environments, with a range of useful properties such as flexibility, high absorbance, transparency and thermal insulation. They are commonly used for liquid absorbance, sensors, consumer products, and drug delivery.

Synthesis, Crosslinking and Characterization of Hydrogels

Source: Amber N. Barron, Ashlea Patterson, and Taylor D. Sparks, Department of Materials Science and Engineering, The University of Utah, Salt Lake City, ...