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 ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-( 
			acryloyloxy)ethyl]trimethylammonium chloride solution&#160;</td>
			<td>Sigma Aldrich</td>
			<td>496146</td>
			<td></td>
		</tr>
		<tr>
			<td>2-aminoethyl methacrylatehydrochloride&#160;</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, 
			44,47,50,53-Hexadecaoxa-28,29-dithiahexapentacontanedioic acid di-N-succinimidyl ester&#160;</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&#8217;,N&#8217;-tetramethylethylenediamine&#160;</td>
			<td>Sigma Aldrich</td>
			<td>411019</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline&#160;</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&#160;</td>
			<td>AGR1260A</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment necessary for nanogel synthesis and characterisation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 Centrifugal filter units (100kDa MWCO)&#160;</td>
			<td>Merck Millipore</td>
			<td>C7715</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Olympus&#160;</td>
			<td>Veleta</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon-coated copper grids</td>
			<td>Agar Scientific&#160;</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&#160;</td>
			<td>Malvern&#160;</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&#160;</td>
			<td>Cary 630 FTIR</td>
			<td></td>
		</tr>
		<tr>
			<td>iTEM software</td>
			<td>Olympus&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mass spectrometry</td>
			<td>Waters</td>
			<td>Micromass MALDI microMX MALDI Q-ToF&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>MF-MilliporeTM membrane filter (0.45/0.2&#956;m pore size)</td>
			<td>Merck Millipore, UK</td>
			<td>HAWP04700, GSWP04700&#160;</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&#160;</td>
			<td>Buchi&#160;</td>
			<td>R-114 Rotary Vap System</td>
			<td></td>
		</tr>
		<tr>
			<td>Single-use needles&#160;</td>
			<td>Sterican</td>
			<td>4665643</td>
			<td></td>
		</tr>
		<tr>
			<td>Suba-Seal&#160;septa</td>
			<td>Sigma Aldrich</td>
			<td>Z124575</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscopy</td>
			<td>Phillips&#160;</td>
			<td>CM 100 TEM&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-vis spectrophotometer</td>
			<td>Nanodrop</td>
			<td>Nanodrop One/One C microvolume&#160;</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.

Synthesis and characterization of poly(ethylene glycol) (PEG) disulphide diacrylate crosslinker The redox-responsive crosslinker was synthesized by the nucleophilic substitution of an N-hydroxysuccinimide (NHS) ester by the 2-aminoethyl methacrylate via the formation of an amide linkage (Figure 1). The synthesis of the required product was validated primarily by 1H NMR (Supplementary Figure 1), that was carried out by dissolving the produ...

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

Research

JoVE Journal

Chemistry

710 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

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

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 ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

Synthesis and Characterization of Amphiphilic Gold Nanoparticles

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of their interactions with cell membranes, lipid bilayers, and viruses. The hydrophilic ligands make these particles colloidally stable in aqueous solutions and the combination with hydrophobic ligands creates an amphiphilic particle that can be loaded with hydrophobic drugs, fuse with the lipid membranes, and resist nonspecific protein adsorption. Many of these properties depend on nanoparticle size and the composition of the ligand shell. It is, therefore, crucial to have a reproducible synthetic method and reliable characterization techniques that allow the determination of nanoparticle properties and the ligand shell composition. Here, a one-phase chemical reduction, followed by a thorough purification to synthesize these nanoparticles with diameters below 5 nm, is presented. The ratio between the two ligands on the surface of the nanoparticle can be tuned through their stoichiometric ratio used during synthesis. We demonstrate how various routine techniques, such as transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and ultraviolet-visible (UV-Vis) spectrometry, are combined to comprehensively characterize the physicochemical parameters of the nanoparticles.

Amphiphilic gold nanoparticles can be used in many biological applications. A protocol to synthesize gold nanoparticles coated by a binary mixture of ligands and a detailed characterization of these particles is presented.

Gold nanoparticles covered with a mixture of 1-octanethiol (OT) and 11-mercapto-1-undecane sulfonic acid (MUS) have been extensively studied because of ...

Hierarchical and Programmable One-Pot Oligosaccharide Synthesis

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for generating potential synthetic solutions. The programmable one-pot oligosaccharide synthesis approach is designed to empower fast oligosaccharide synthesis of large amounts using thioglycoside building blocks (BBLs) with the appropriate sequential order of relative reactivity values (RRVs). Auto-CHO is a cross-platform software with a graphical user interface that provides possible synthetic solutions for programmable one-pot oligosaccharide synthesis by searching a BBL library (containing about 150 validated and &#62;50,000 virtual BBLs) with accurately predicted RRVs by support vector regression. The algorithm for hierarchical one-pot synthesis has been implemented in Auto-CHO and uses fragments generated by one-pot reactions as new BBLs. In addition, Auto-CHO allows users to give feedback for virtual BBLs to keep valuable ones for further use. One-pot synthesis of stage-specific embryonic antigen 4 (SSEA-4), which is a pluripotent human embryonic stem cell marker, is demonstrated in this work.

This protocol demonstrates how to use the Auto-CHO software for hierarchical and programmable one-pot synthesis of oligosaccharides. It also describes the general procedure for RRV determination&#160;experiments and one-pot glycosylation of SSEA-4.

This article presents a general experimental protocol for programmable one-pot oligosaccharide synthesis and demonstrates how to use Auto-CHO software for ...

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of ...

Preparation of Expanded Chitin Foams and their Use in the Removal of Aqueous Copper

Chitin is an underexploited, naturally abundant, mechanically robust, and chemically resistant biopolymer. These qualities are desirable in an adsorbent, but chitin lacks the necessary specific surface area, and its modification involves specialized techniques and equipment. Herein is described a novel chemical procedure for expanding chitin flakes, derived from shrimp shell waste, into foams with higher surface area. The process relies on the evolution of H2 gas from the reaction of water with NaH trapped in a chitin gel. The preparation method requires no specialized equipment. Powder X-ray diffraction and N2-physisorption indicate that the crystallite size decreases from 6.6 nm to 4.4 nm and the specific surface area increases from 12.6 &#177; 2.1 m2/g to 73.9 &#177; 0.2 m2/g. However, infrared spectroscopy and thermogravimetric analysis indicate that the process does not change the chemical identity of the chitin. The specific Cu adsorption capacity of the expanded chitin increases in proportion to specific surface area from 13.8 &#177; 2.9 mg/g to 73.1 &#177; 2.0 mg/g. However, the Cu adsorption capacity as a surface density remains relatively constant at an average of 10.1 &#177; 0.8 atom/nm2, which again suggests no change in the chemical identity of the chitin. This method offers the means to transform chitin into a higher surface area material without sacrificing its desirable properties. Although the chitin foam is described here as an adsorbent, it can be envisioned as a catalyst support, thermal insulator, and structural material.

This study describes a method to expand chitin into a foam by chemical techniques that require no specialized equipment.

Chitin is an underexploited, naturally abundant, mechanically robust, and chemically resistant biopolymer. These qualities are desirable in an adsorbent, ...

Stable Aqueous Suspensions of Manganese Ferrite Clusters with Tunable Nanoscale Dimension and Composition

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse applications. Here we describe how to form these materials in a hydrothermal process that permits the independent control of product cluster size (from 30 to 120 nm) and manganese content of the resulting material. Parameters such as the total amount of water added to the alcoholic reaction media and the ratio of manganese to iron precursor are important factors in achieving multiple types of MFC nanoscale products. A fast purification method uses magnetic separation to recover the materials making production of grams of magnetic nanomaterials quite efficient. We overcome the challenge of magnetic nanomaterial aggregation by applying highly charged sulfonate polymers to the surface of these nanomaterials yielding colloidally stable MFCs that remain non-aggregating even in highly saline environments. These non-aggregating, uniform, and tunable materials are excellent prospective materials for biomedical and environmental applications.

We report a one-pot hydrothermal synthesis of manganese ferrite clusters (MFCs) that offers independent control over material dimension and composition. Magnetic separation allows rapid purification while surface functionalization using sulfonated polymers ensures the materials are non-aggregating in biologically relevant medium. The resulting products are well positioned for biomedical applications.

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse ...

Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper tumor cells, leading to tumor recurrence. To conquer the limited penetration of biological barriers, cell-penetrating peptides (CPPs) have been discovered with excellent membrane translocation ability and have emerged as useful molecular transporters for delivering various cargoes into cells. However, conventional linear CPPs generally show compromised proteolytic stability, which limits their permeability across biological barriers. Thus, the development of novel molecular transporters that can penetrate biological barriers and exhibit enhanced proteolytic stability is highly desired to promote drug delivery efficiency in biomedical applications. We have previously synthesized a panel of short cyclic CPPs with aromatic crosslinks, which exhibited superior permeability in cancer cells and tissues compared to their linear counterparts. Here, a concise protocol is described for the synthesis of the fluorescently labeled cyclic polyarginine R8 peptide and its linear counterpart, as well as key steps for investigating their cell permeability.

This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper ...