## 4 Drug Delivery
### Polyplex Delivery
Interestingly, lysozyme complexation in AB\({}_{3}\) miktoarm stars containing B blocks, each being the same MW as the B block in the linear copolymer, showed only marginal improvement. Based on tryptophan fluorescence, it was found that no detectable protein unfolding occurred when bound to the polymers, and so, lysozyme's structural conformation was conserved. Despite this, lower enzymatic activity was observed for bound lysozyme, with a more noticeable decrease in lytic activity at higher polymer/protein ratios. When assessed using _Microoccus lisodeikticus_, the lysozyme showed decreased cleavage of \(N\)-acetylmuramic acid and \(N\)-acetylglucosamine in cell walls, due to obstructed access of substrates to its active site. AB\({}_{3}\) (A = PEG, B = PGA) miktoarm polymers have also been prepared for the pulmonary delivery of complexed lysozyme as a dry powder [68, 76]. The miktoarm star/lysozyme nanocomplexes were synthesized at a molar charge ratio of 2.5, with mannitol, trehalose, and leucine as aerosol excipients. Fine particle fractions of up to 68% were seen for powders consisting of the polyplexes with trehalose and leucine. Trehalose was used as a bulk agent in formulating the dry powders, which aided in stabilizing the 3D protein structure of lysozyme through the formation of hydrogen bonds. Leucine functioned as a dispersion enhancer, which was essential in forming powders with spherical morphology and an average diameter of 2.5 um, which is ideal for inhalation. The complexes were found to be well incorporated into dry powders with encapsulation efficiencies between 80 and 100%, while retaining enzymatic activity. Additionally, conjugation of vitamin B\({}_{12}\), cobalamin, to PEG termini via CuAAC click coupling, allowed targeting of B\({}_{12}\)-receptors in epithelial cells containing the vitamin B\({}_{12}\)-internalization receptor (CD320), _in vitro_ and _in vivo_[76]. This significantly increased the cellular internalization of miktoarm star/lysozyme complexes compared to those without conjugated B\({}_{12}\). Using the Calu-3 epithelial cell model, it was further established that the topology of miktoarm star polyplexes resulted in greater lysozyme internalization compared to linear complexes, despite a slightly stronger immunogenic response. Dry powders that incorporated the polymer/protein polyplexes showed non-homogenous dispersion in mouse lungs, yet deposition data showed that delivered lysozyme was present in the lungs 14 h after administration, in contrast to the B\({}_{12}\)-lacking non-targeting polyplexes. Although not a strict example of polyplex-based delivery, a system composed of cationic bis-hydrophilic AB\({}_{4}\) miktoarm stars (A = PEG, B = quaternized poly(2-(dimethylamino)ethyl methacrylate) (qPDMAEMA)) was combined with anionic poly(styrenesulfonate) to form stable polymersomes with inter-polyelectrolyte complexes [64]. Their hydrophilic interiors allowed the encapsulation of rhodamine B, a cationic dye molecule, and as a result of the polymersomes' polyelectrolyte complexes, there was no obvious leakage of rhodamine B from their cores, but when dialyzed against a 2 M NaCl solution, the complete release was seen in 5 h. These polymersomes were used to construct multilayered microcapsules by the sequential adsorption of tannic acid and polymersome layers, on the surface of bare negatively charged silica nanoparticles, followed by silica core dissolution. PEG chains were able to strongly interact with tannic acid layers because of hydrogen bonding. A change of pH from 5 to 9 resulted in a microcapsule size change from 4.51 to 4.12 um, which resulted in the opening of microcapsule pores (greater permeability) that could accommodate FITC-dextran in its shell at pH 5 (or below 7). Loading FITC-dextran in 8-layer microcapsule shells and subsequently switching the pH back to 9 resulted in the desorption of the dextran from the shell and its loading into the microcapsule cores.