## 4 Drug Delivery
### Redox-Responsive Drug Delivery  
Oxidative stress is characteristic of many pathologies, such as neurodegenerative disorders, cancer, and diabetes, which results in heightened concentrations of reactive oxygen species (ROS), including hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)), superoxide (O\({}_{2}\)\({}^{-}\)), hydroxy radicals (-OH), and singlet oxygen (\({}^{1}\)O\({}_{2}\)) [137]. While normally present as regulators in redox-dependent signal transduction [138], elevated ROS concentrations at diseased sites are a sign of insufficient activity from the endogenous antioxidant defense mechanisms. One aspect of this system is the antioxidant glutathione (GSH), which is oxidized to glutathione disulfide (GSSG) upon exposure to ROS [139]. In cancer, tumor cells are known to contain elevated GSH levels that are thought to aid in tumor cell proliferation [139]. Additionally, evidence suggests that GSH protects cancer cells against chemotherapeutic drugs [140]. While a variety of cleavable polymer linkers have been applied to ROS-responsive drug delivery, including those based on thioketals, diselenides, phenylboronic acids, and esters, and vinyldithioethers [137], interestingly, only the \(\beta\)-aminoacrylate was used as a specifically \({}^{1}\)O\({}_{2}\)-labile linker in miktoarm polymers [74]. Much effort has been devoted to developing miktoarm stars responsive to GSH that is present at elevated concentrations intracellularly at disease sites. In preparing GSH-responsive systems, polymers are typically conjugated using disulfide linkers that can be cleaved through thiol-disulfide exchange. GSH can also participate in thiol-thioester exchange, facilitating polymer cleavage, and it is reactive to acrylates via Michael additions, which can be used to prepare GSH conjugates [141; 142]. An example of a miktoarm star containing a GSH-responsive linker was prepared using an AB\({}_{2}\) (A = PEG, B = PMMA) build-up, based on a dihydroxy-benzoic acid core with cystamine, a disulfide linker, connecting the core and the hydrophilic PEG arm [58]. The dihydroxy functionalities on the core were coupled to bromoisobutyrate ATRP initiators and used to polymerize PMMA. Aqueous self-assembly of these polymers led to micelles with a diameter of 130 nm and CMC of 0.91 mg/L. As a substitute for GSH, which would be present biologically, 10 mM of dithiotheitol (DTT) was used (mimicking intracellular GSH concentrations) to reduce the disulfide linker present in the micelles. This treatment resulted in a micelle size shift to 300 nm after 2 h, and \(>\)1000 nm after 24h, as a result of the aggregation of unlinked polymer chains, while no significant size change was seen in untreated samples. MTX was encapsulated with 64% efficiency, and the nanocarriers showed a 22% release in 48 h with no clear burst release. Meanwhile, DTT treatment, causing complete micellar dissociation, resulted in a much more significant (95%) release in 48 h.  
A series of miktoarm polymers based on a core with four branching PEG arms have been prepared, by selectively carrying out the ROP of caprolactone on individual PEG ends to yield A(AB)\({}_{3}\), A\({}_{2}\)(AB)\({}_{2}\), and A\({}_{3}\)(AB) (A = PEG, B = PCL) stars (Figure 9) [57]. PEG blocks were end-conjugated to folic acid (FA) while PEG-PCL ones were conjugated to camptothecin (CPT) via dithiopropionic acid, a GSH-cleavable disulfide bearing linker. Incubation with GSH not only led to the cleavage of CPT conjugated directly to miktoarm stars, but also to a several-hundred nanometer increase in micellar diameter--which was associated with core destabilization from the linker cleavage. Faster CPT release was seen in micelles that had the highest CPT content (more points of conjugation), with 76%, 69%, and 54% release coming from A(AB)\({}_{3}\), A\({}_{2}\)(AB)\({}_{2}\), and A\({}_{3}\)(AB) micelles, respectively. Cellular uptake of CPT was investigated with SKOV-3 cells that overexpressed FA receptors.