6.09.2.1.3(i) Formation of disulfide bonds

CS-based thiolated microgels with an average size of 18 µm have been prepared either by water-in-oil (w/o) emulsification solvent evaporation⁴¹ or by precipitation-micronization technique.⁴² For thiolation, 2-iminothiolane (Trout’s reagent) was covalently linked to CS via an amidine bond leading to the formation of CS-4-thiobutylamidine. Such polymers show higher mucoadhesive and permeation-enhancing properties than unmodified CS,^43–45 which makes them ideal precursors for nasal or oral drug delivery systems.

The formation of nanogels with a mean diameter of 125 nm by the interaction of thiolated CS with DNA and the formation of disulfide bonds have been achieved in aqueous solution.⁴⁶ Nanogels cross-linked by disulfide bonds showed an increased stability to artificial intestinal fluid, a triggered release in a reducing environment, as well as an improved transfection efficiency compared to physically cross-linked CS/DNA particles. Chitosan-N-acetylcysteine (NAC) nanoparticles (NPs) prepared by the coupling of N-acetylcysteine to CS mediated through carbodiimide reaction were investigated as a nonviral gene carrier.⁴⁷ A similar thiolation procedure of CS was applied by Atyabi et al.⁴⁸ Subsequently, amikacin-loaded NPs with an average diameter of 280 nm were prepared by TPP gelation, where disulfide bond formation was achieved by time-dependent oxidation.

CS-based pH-sensitive nanogels with an average size of 170 nm were prepared by utilizing the formation of polyelectrolyte complexes.⁴⁹ In this procedure, CS formed polyelectrolyte complexes with cysteamine-functionalized polyaspartic acid and resulting polymer aggregates were cross-linked by chloramine-T-mediated disulfide formation.

Adipic acid dihydrazide-grafted hyaluronic acid (HAADH) was used for the preparation of HA microgels. Under addition of sodium tetrathionate as a specific cross-linking reaction accelerator, microgels with a diameter of 2.3 µm were prepared by spray drying.⁵⁰

In another study, thiol-functionalized HA was used for the preparation of disulfide cross-linked nanogels with physically entrapped green fluorescence protein siRNA by an inverse w/o emulsion approach.⁵¹ The same group prepared disulfide-cross-linked HA microgels and prepared shell cross-linked HA/polylysine layer-by-layer polyelectrolyte microcapsules through layer-by-layer approach followed by the removal of the reducible HA microgel cores with dithiothreitol (DTT).⁵²

Nanogels based on disulfide-cross-linked heparin were prepared to promote efficient cellular uptake of heparin in order to induce apoptotic cell death.⁵³ Thiol-functionalized heparin was synthesized by selective oxidation of d-glucuronic acid residues in heparin structure with sodium periodate followed by the addition of cysteamine hydrochloride.

The preparation of nanogels from synthetic polymers based on the formation of disulfide bonds was demonstrated by Aliyar et al.⁵⁴ Thiol-functionalized polyacrylamide (PAAm) was synthesized by the copolymerization of acrylamide (AAm) and N,N′-bisacryloylcystamine and subsequent reduction of the obtained gel.

Nanogels based on star-shaped poly(ethylene oxide-stat-propylene oxide) or linear PG were reported recently.⁵⁵ Both polymer precursors were functionalized with thiol groups through carbodiimide-mediated Steglich esterification between the free hydroxyl groups of the polymers and the disulfide-containing dicarboxyacid. The obtained bulk hydrogels were subsequently reduced by cleaving the disulfide cross-links to thiol groups. Nanogels were prepared in inverse w/o miniemulsion and the disulfide bond formation was accelerated by the addition of catalytic amounts of hydrogen peroxide.

6.09.2.1.3(ii) Michael addition

Michael addition reaction was frequently used for the cross-linking of polymer chains and formation of microgels. Hydroxypropylcellulose (HPC) nanogels were synthesized in aqueous solution by the association of HPC above its lower critical solution temperature (LCST) (∼41 °C) to metastable aggregates followed by cross-linking after addition of divinylsulfone (DVS).^56–58

HA microgel particles with a diameter of 900 nm were synthesized by DVS cross-linking in inverse miniemulsion.⁵⁹ The particles were further modified with aldehyde functionalities by oxidizing the hydroxyl groups with sodium periodate. Aldehyde-functionalized HA nanogels were used for the fabrication of bulk hydrogels by cross-linking with adipic dihydrazide for soft tissue regeneration.

Microgels with an average size of 100 nm were formed upon Michael-type conjugation between amine functionalities of bovine serum albumin (BSA) and vinylsulfone end-groups of eight-arm star-shaped PEG.⁶⁰

Cross-linking of acrylate group-modified cholesterol-bearing PuL nanogel with thiol group-modified star-PEG by Michael addition yielded raspberry-like colloidal assemblies with narrow size distribution (Figure 5).⁶¹ The size of microgels was controlled in the range of 40–120 nm by changing the concentration of PuL nanogel and star-PEG. Microgels gradually degraded by hydrolysis under physiological condition and dissociated back to the original nanogel. The microgels encapsulated interleukin-12 (IL-12) efficiently (96%) and stably kept it in the presence of BSA (50 mg ml⁻¹).

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Figure 5. Schematic illustration of the synthesis of raspberry microgels.

Reprinted with permission from Hasegawa, U.; Sawada, S.; Shimizu, T.; et al. J. Controlled Release 2009, 140, 312–317.⁶¹ Copyright 2009 Elsevier.

Nanogels with an average diameter of ∼100 nm synthesized by cross-linking vinylsulfone-modified PEG with eight-arm amine-terminated PEG-octamine have been prepared by Elbert et al.^60,62

The preparation of disulfide-stabilized nanogel PEG/DNA complexes with a diameter of around 100 nm was reported by Mok et al.⁶³ Spontaneous PEG/DNA nanocomplex formation took place during solubilization of DNA by a thiol-functionalized six-arm branched PEG in DMSO. Subsequent addition of the thiol-reactive dithio-bis-maleinidoethane (DTME) disulfide cross-linker led to stabilized nanogels (Figure 6) that showed high gene transfection efficiency.

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Figure 6. Schematic of PEG-assisted DNA solubilization in DMSO and preparation of PEG/DNA nanogels. (a) Plasmid DNA in water; (b) PEG/DNA nanocomplex in DMSO; and (c) stable DTME-cross-linked PEG/DNA nanogel in water.

Reprinted with permission from Mok, H.; Park, T. G. Bioconjugate Chem. 2006, 17, 1369–1372.⁶³ Copyright 2006 American Chemical Society.

6.09.2.1.3(iii) Condensation reactions

Glutaraldehyde has been mainly used as an effective cross-linker for the preparation of CS particles. It reacts with the primary amine groups of CS to form Schiff bases.⁶⁴ CS-based microgels were prepared by cross-linking of CS chains with glutaraldehyde in aqueous droplets.^65–70 A membrane emulsification technique allows preparation of highly monodisperse microgel particles. The Shirasu porous glass (SPG) membrane with a pore size from 0.1 to 18 µm was used to prepare uniform-sized water droplets containing CS in organic solvent and polymer chains were cross-linked by glutaraldehyde.⁷¹ Various drugs including 5-fluorouracil (5-FU), phenobarbitone, theophylline, aspirin, griseofulvin, and progesterone were loaded into such particles of sizes in the range of 30–450 µm.^66–68,70 Kumbar et al. prepared CS particles loaded with diclofenac sodium using cross-linking with glutaraldehyde, sulfuric acid, and heat treatment.⁶⁵ Mitra et al. prepared glutaraldehyde cross-linked CS NPs encapsulating doxorubicin–Dex conjugate.⁷² Nanogels were prepared by a reverse micellar method that resulted in spherical, highly monodispersed hydrogel particles with a diameter of 100 ± 10 nm.

In order to prepare uniformly glutardialdehyde-cross-linked gelatin NPs also with high cross-linking degrees, a convenient synthetic route based on the concept of nanoreactors (individual nanosized homogeneous entities) using the miniemulsion technique has been used.^73,74

Homogeneously cross-linked gelatin NPs were prepared by mixing of two inverse miniemulsions, one containing gelatin aqueous droplets and other containing aqueous droplets with glutaraldehyde.⁷⁵ The cross-linking reaction was performed by a fission and fusion process between the different droplet species. In this system, the amount of gelatin in the droplet and the degree of cross-linking can be varied over a wide range.

A membrane emulsification method has been applied for the preparation of glutaraldehyde-cross-linked CS particles loaded with BSA as a model drug. The diameter of the particles depended on the membrane pore size and was in the range of 0.65–26.06 µm.⁷⁶

CS nanogels were synthesized by carbodiimide coupling of CS with an oligo(ethylene glycol) dicarboxylic acid cross-linker in an aqueous homogeneous chemical gelation process.⁷⁷ The hydrodynamic diameter of the resulting particles was in the range between 60 and 98 nm depending on the ratio of cross-linking agent to CS.

Chemically cross-linked nanogels based on synthetic polymers PEG-cl-PEI were introduced by Kabanov et al.⁷⁸ They exhibit combined properties of swollen polyelectrolyte and hydrophilic nonionic networks where the PEI chains bind oppositely charged macromolecules, leading to the collapse of the network, while nonionic PEG fragments prevent precipitation and stabilize the nanogels in aqueous dispersions.⁷⁹ The particles were prepared by cross-linking branched PEI with bis-carbonyldiimidazole-activated PEG using emulsion techniques followed by evaporation of the solvent, maturation of the nanogels in aqueous solution, and final fractionation of the obtained particles by gel-permeation chromatography.

Lee et al.⁸⁰ reported the synthesis of nanogels in oil-in-water (o/w) emulsion by chemical cross-linking between 4-nitrophenyl chloroformate (p-NPC) terminally activated oligo(l-lactic acid)-block-poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide)-block-oligo(l-lactic acid) penta-block-copolymer and amine groups of poly(ethylene oxide) grafted poly(l-lysine).

The main advantage of the chemical cross-linking methods is the use of reactive polymers with predefined architecture, chemical composition, and functionalities. The selection of reactive building blocks allows very exact adjustment of nano- and microgel properties. During cross-linking step, one can vary flexibly swelling and mechanical properties of nano- and microgels. Since the cross-linking reactions can occur at very mild conditions, this is probably the best strategy to design biohybrid nanogels (networks consisting of biological and synthetic polymer chains) or to direct loading of polymer colloids with proteins or enzymes.