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 fla...

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 ...

Our protocol offers a significant advancement in synthesizing sub 100-nanometer protein-loaded nanogels in a single step cross-linking and co-polymerization technique under mild aqueous-based reaction conditions. The main advantage of this technique is in its ability to synthesize protein-loaded nanogels under mild conditions, avoiding harsh organic solvents and high temperatures, thereby preserving the integrity of the biological payload. Begin by washing the glassware with filtered deionized water.To dry the glassware, attach a secure nozzle to the compressed air outlet located on the side of the fume hood. Hold the vial firmly, position the nozzle inside and activate the compressed air to emit a controlled stream. Next, fill a 10-milliliter glass vial with deionized water and seal it with a rubber septum.To deoxygenate the water, fill a balloon with nitrogen gas and attach a needle to the balloon. Place it on the septum and pierce the rubber septum to allow the nitrogen flow. Also, attach an exit needle to the flask to maintain a consistent nitrogen flow.Next, dissolve BSA or Cy7-tagged BSA in deoxygenated deionized water. Then add this solution to a clean 10-milliliter glass vial. Add AETC solution to the flask and seal it with another rubber septum.Dissolve acrylamide and deoxygenated deionized water and add this solution to the vial's mixture. Then dissolve the disulfide cross linker in deionized water and add it to the mixture in the vial. Flush the mixture with nitrogen gas for 20 minutes.Afterward, dissolve SDS in deoxygenated deionized water. Using a nitrogen gas-flushed syringe, introduce it into the mixture. Next, add TEMED to the reaction mixture.Let the mixture deoxygenate for three minutes under a continuous flow of nitrogen gas. Dissolve ammonium persulfate in one milliliter of deoxygenated deionized water and introduce it to the reaction mixture. Then seal the flask under a nitrogen atmosphere and stir the mixture for three hours and 30 minutes at room temperature.To terminate the reaction, remove the seal from the flask. The reaction concludes once the mixture becomes clear and colorless. To purify nanogels, add the reaction mixture to a 15-milliliter centrifugal filter unit.Fill the unit with deionized water and centrifuge to remove unreactive chemicals or unencapsulated payload. Add two milliliters of deionized water in the centrifugal unit to dilute the nanogel sample. Store the final sample at two to eight degrees Celsius to prevent degradation of the encapsulated protein.Take a cuvette or a disposable folded capillary cell for analysis. Clean it with filter deionized water, followed by blowing compressed air to remove any large dust particles. Then fill the cuvette with 50 to 100 microliters of the sample, diluting it with 750 to 1, 000 microliters of filtered deionized water.To insert the cuvette into the DLS instrument, open the sample compartment cover and hold the cuvette by its top edges to avoid leaving fingerprints to the optical windows. Place the cuvette properly aligned with the instrument's optical path. Then close the sample compartment to block ambient light.Initiate the particle size measurement using the relevant software on the DLS machine. After the measurement, select the results relevant to the sample, including the Z average, average PI, count rate per minute, zeta potential and related graphics. Confirm that the correlation function displays a smooth sigmoidal curve indicative of a uniform sample.Once the analysis is complete, carefully removed the cuvette from the DLS instrument. A gradual decrease in the intensity of the peak associated with the BSA protein was observed, suggesting encapsulation of the protein within the nanogel structure. The physical chemical characterization of the Cy7-tagged BSA-loaded nanogels showed a single defined peak at 50 to 100 nanometers.However, empty nanogels demonstrated inconsistent and larger sizes. Up to 86%of encapsulated Cy7-tagged BSA was released within 48 hours. On the other hand, control experiments with no added glutathione only led to 15%protein released.Fourier transform infrared analysis showed that BSA isolated during nanogel synthesis and post glutathione incubation retained its structure, indicated by the peak at 1, 662 centimeters inverse representing intact beta turns. However, BSA from nanogels stored for 30 days showed a peak at 1, 613 centimeters inverse, indicative of intermolecular beta sheet structures and protein aggregation. Circular dichroism showed unaltered spectra like native BSA, indicating preserved alpha helical structure.However, minor aberrations in the characteristic peaks for BSA isolated after 30 days suggested some aggregation. Maintaining a continuous nitrogen flow is critical to prevent oxygen from inhibiting the free radical polymerization. Additionally, the initiating system components require swift addition for a successful synthesis.After this procedure, nanogel-sized distribution, morphology and payload release kinetics can be further characterized through DLS, TEM and redox responsiveness assessment via incubation with the physiological reducing agent glutathione.

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

Research

JoVE Journal

Chemistry

390 Views.  Imperial College London. Our protocol offers a significant advancement in synthesizing sub 100-nanometer protein-loaded nanogels in a single step cross-linking and co-polymerization technique under mild aqueous-based reaction conditions. The main advantage of this technique is in its ability to synthesize protein-loaded nanogels under mild conditions, avoiding harsh organic solvents and high temperatures, thereby preserving the integrity of the biological payload. Begin by washing the glassware with filtered deionize...