Nanogel

The synthesis of nanogels can be achieved using a vast array of different methods. However, two critical steps typically included in each method are polymerization and crosslinking, with physical and chemical crosslinking the most common.^[4] These steps can be completed concomitantly or in sequential order depending on the synthesis method and eventual nanogel application.^[4] Here, several different synthesis mechanisms are described briefly.

In desolvation or coacervation, a non-solvent is added to a homogeneous polymer solution to produce individual, nanosized polymer complexes dispersed in the same solution.^[3] These complexes then undergo crosslinking to form nanogels with surface functionalization an optional next step.^[3] In precipitation, initiators and crosslinking agents are added to a homogenous monomer solution to induce a polymerization reaction.^[4] When the polymer chain reaches the desired length, the reaction is halted and a polymer colloidal suspension is formed.^[2][3] Surfactants are the final addition to produce nanosized polymers.^[2][3]

Electrostatic interactions can form nanogels through the combination of anionic and cationic polymers in an aqueous solution.^[3] The size and surface charge of the resulting nanogels can be modulated by changing the molecular weight or the charge ratio of the two different polymers.^[3] Ionotropic gelation can also leverage electrostatic interactions between multivalent anions and cations to form nanogels.^[3] Hydrophobic interactions rely heavily on physical crosslinking to form nanogels.^[3] In this method, hydrophobic groups are added to hydrophilic polymers in an aqueous solution to induce their self-assembly into nanogels.^[3] When thiolated polymers (thiomers) are used for this preparation process, nanogels can be further stabilized by the formation of inter- and intrachain disulfide bonds due to oxidation. In the following the oppositely charged oligo- or polymers can even be removed.^[5][6]

Inverse-emulsion, or reverse miniemulsion, requires an organic solvent and a surfactant or emulsifying agent.^[3][4] Nanosized droplets are produced when an aqueous monomer solution is dispersed in the organic solvent in the presence of the surfactant or emulsifying agent.^[3][4] Upon removal of the organic solvent and further chemical and physical crosslinking of the droplets, nanogels are formed.^[3][4] The size of nanogels synthesized using this method can vary greatly depending on the type of surfactant and reaction medium used.^[3] Purifying nanogels produced using an emulsifying agent may also pose a challenge.^[3]

The addition of a monomer precursor solution and crosslinking agent to a microtemplate, or mold-type device, can initiate polymerization and the formation of nanogels.^[4] This method can be used to create nanogels in specific shapes and load them with various small molecules.^[4] Lithographic microtemplate polymerization is a similar process that uses a photoinitiator and light to trigger the formation of nanogels.^[4] Lithographic microtemplate polymerization can produce smaller nanogels on a length scale of <200 nm, which has a higher resolution compared to microtemplate polymerization that does not require a photoinitiator.^[4]

Polymer-based micelles that undergo crosslinking reactions can induce the formation of nanogels.^[3][7] Crosslinking either the core or the shell of a preexisting micelles can synthesize nanogels with a "high degree of spatial organization".^[3][7]

Since biodegradability is an important characteristic of nanogels, these hydrogels are typically composed of natural or degradable synthetic polymers.^[3] Polysaccharides and proteins largely dominate the natural forms of polymers used to synthesize nanogels.^[2][3] Due to the use of thiolated polysaccharides (thiomers) such as thiolated chitosan or thiolated hyaluronic acid^[8][9] nanogels can be stabilized via intra- and interchain disulfide bonding. Advantages of natural polymer-based nanogels include biocompatibility and degradability by cellular mechanisms in vivo.^[3] Natural polymers also tend to be nontoxic and bioactive in which they are more likely to induce biological cues that govern various aspects of cellular behavior.^[3] However, natural-based polymers can still cause an immune response and possess other disadvantages such as variable degradation rates and heterogeneous structures.^[3] Conversely, synthetic-based polymers have more defined structures, increased stability, and controlled degradation rates.^[3] In comparison to natural-based polymers, synthetic polymers lack biological cues that may be necessary for specific therapeutic applications.^[3] Given that natural and synthetic polymers are defined by their own set of advantages and disadvantages, an ongoing area of research aims to create composite hydrogels for nanogel synthesis that combines synthetic and natural polymers to leverage the benefits of both in one nanogel formulation.^[3]

The structure of a nanogel is dependent upon the synthesis mechanism and its application. Simple or traditional nanogels are nanoparticle-sized crosslinked polymer networks that swell in water.^[3][10] Hollow nanogels consisting only of an outer shell can increase the amount of cargo loaded into the platform.^[3][10] In other nanogel structures, the inner core and outer shell can be made of two different materials, such as a hydrophobic inner core to surround drugs or other small molecules and a hydrophilic outer shell that interacts with the external environment.^[2][3] The addition of a second linear monomer crosslinked to a nanogel is deemed a "hairy nanogel".^[2][7] Different nanogel synthesis methods can be completed in sequential order to create multilayered nanogels, such as starting with ionotropic gelation and then combining anionic and cationic polymers in an aqueous solution.^[7][10] Functionalized nanogels, in which targeting ligands or stimuli-sensitive functional groups are conjugated to the outer shell of a nanogel, are also important for certain nanogel applications.^{[2][3][7][10]}

Nanogels can be designed to respond to various stimuli including changes in pH and temperature or the presence of redox and light cues. Thoughtfully designed stimuli-responsive nanogels can be leveraged to transport and release different types of cargo to specific tissues within the body with increasing spatiotemporal resolution.

pH responsive nanogels are an attractive form of nanogel technology due to the different pH levels found within the body. Healthy tissues exhibit a pH of 7.4 whereas tumors can be as low as 6.5 and the stomach as low as 1.0.^[10] The protonation or deprotonation of certain functional groups can change the swelling rate and stability of a nanogel, thus resulting in the release of encapsulated cargo when exposed to different pH ranges.^[2][10] For example, anionic nanogels with carboxylic acid groups will collapse upon exposure to a pH that is smaller than the pKa of the nanogel polymer.^[2][10] Similarly, cationic nanogels with terminal amino groups will become protonated if the pH of the environment is less than the pKa of the hydrogel.^[2][10] In this case, the swelling rate of the nanogel will change and it will become more hydrophilic.^[2][10] Other groups have also previously cross-linked pH-responsive hydrazone linkages to polysaccharide-based nanogels that released a payload in an acidic environment.^[11]

The usage of thermoresponsive polymers in nanogel synthesis allows these systems to respond to changes in temperature.^[1] Depending on the chemical groups present, thermoresponsive polymers can either respond to a decrease in temperature or an increase in temperature.^[10] Both hydrophobic and hydrophilic groups are typically present in thermoresponsive polymer nanogels that react to temperature decreases, whereas nanogels that respond to temperature increases often have to be prepared by a hydrogen-bonded layering technique.^[10] Temperature-responsive nanogels are a potential strategy when a therapeutic is targeting the skin, which has a natural temperature gradient, or a region experiencing inflammation.^[1][2][10]

Redox-responsive nanogels generally contain crosslinks formed by disulfide bonds^[12] or specific crosslinking agents.^[3][10] Nanogels made of bioreducible and bifunctional monomers can also be used. In the presence of reducing agents such as glutathione, thioredoxin and peroxiredoxin, these nanogels respond by releasing their cargo.^[10][13] Given that these reducing agents and several others are found in larger concentrations inside cells compared to their external environment, redox-responsive nanogels are a promising strategy for targeted intracellular delivery.^[10]

Light-responsive nanogels can be triggered to release their cargo with exposure to light at a certain wavelength.^[10] These nanogels are synthesized to contain specific acrylic or coumarin-based bonds that cleave during a photoreaction.^[10] With the tunability of the wavelength of light, energy, and time of irradiation, light-responsive nanogels can be triggered to degrade with an increased control over crosslinking density.^[10] For example, both the swelling and size of light-responsive nanogels with vinyl groups were found to decrease and produce a sustained release of drugs after irradiation with UV light.^[10]

One major concern with any form of drug delivery system, including nanogels, is potential side effects and damage to healthy tissue in addition to causing a negative immune response with the introduction of a foreign substance.^[3][7] This has to be balanced with the need for nanogels to remain within circulation for an adequate period to deliver cargo and produce a therapeutic effect.^[3][7] To combat a significant immune response, degradable nanogels are the typical default since they are considered less toxic compared to non degradable nanogels.^[3][7] The compliance and small size of degradable nanogels also allows them to travel through blood vessels and reach their target area before consumption by immune cells or filtration by the liver and spleen.^[3][7]

After nanogels exit the vasculature, they diffuse through the interstitial space into their target tissue.^[7] At the cellular level, nanogels can be internalized by a large number of different types of endocytosis that depend on the particle's size, shape, and surface properties.^[3][7] Endocytosis is the most common mechanism that starts with the nanogels engulfed by the cellular membrane.^[3][7] The nanogels are transported in intracellular vesicles for delivery to endosomes that eventually combine with lysosomes.^[3][7] Once lysosomes are released into the cytosol of a cell, they deliver their cargo immediately or move to the appropriate cellular compartment.^[3][7]