## 2 Synthetic Approaches to Miktoarm Star Polymers
### Miktoarm Polymer Characterization  
Owing to their branched architecture, the characterization of miktoarm polymers is important to establish their compositions, and it requires careful consideration and interpretation of data. Almost universally, the characterization of miktoarm star build-up is obtained through a combination of NMR spectroscopy and GPC chromatography. Despite its prowess in characterizing polymer homoarms, MALDI-TOF has not been successfully employed for the characterization of miktoarm polymers [53; 67; 115]. Many of the earlier miktoarm polymers, generally synthesized using chlorosilane-based methodologies, had ABC type structures, which were simple to elucidate. For example, as discussed in Section 2.1, the construction of the first miktoarm polymer required separate anionic polymerization of PI, PS, and PB arms--each of which were characterized using GPC. The stepwise conjugation of the polymers with living anionic ends, onto a chlorosilane core, was then followed by GPC, and a downwards shift in retention time, upon addition of each arm, was observed. The inclusion of each arm was further determined using \({}^{1}\)H NMR, and through this combination, miktoarm polymer's overall composition could be accurately calculated [88]. Due to its reliability and simplicity, this characterization methodology has been applied in recent ABC type miktoarm polymers, which have been used for applications in drug delivery [42; 45]. For example, the synthesis of a PEG-PNBM-PNIPAM miktoarm polymer, which involved sequential ATRP of NBM, and CuAAC of alkynyl-PNIPAM onto a PEG(-Br)-N\({}_{3}\) macroinitiator, was followed stepwise, using GPC, \({}^{1}\)H NMR, as well as FT-IR [66]. Characterization of miktoarm polymers with AB\({}_{n}\) composition typically involves characterization using a careful comparison of \({}^{1}\)H NMR peak integrations, together with the analysis of their GPC chromatograms. For example, in an AB\({}_{2}\) polymer where B segments are added by click chemistry,  
Figure 3: Schematic representation of PEG-PHLG CCS polymer synthesis using an in-out methodology. Reprinted with permission from reference [61]. Copyright 2016 Royal Society of Chemistry. the relative integrations of B-derived protons become double, compared to those from the A segment. Analysis of its GPC chromatogram showed a unimodal peak, implying that there are no unconjugated segments [24; 38; 46; 53; 55]. Often, miktoarm polymer development is carried out using polymerization from heteromulfunctional cores. When there are equivalent initiating functional groups present in multifunctional or polymer segment-conjugated cores, it is generally assumed that the polymerization is initiated from every such functional group present. For example, in the development of a PEG-PLA\({}_{2}\) miktoarm polymer from a PEG macroinitiator conjugated to a serinol core, L-Lactide ROP was initiated from both hydroxyls present in the core [34]. In such cases, GPC is used to verify A and B segment addition. The \({}^{1}\)H NMR is then used to verify the overall polymer structure and calculate the degree of polymerization (DP) of A and B arms [32; 34; 40; 43; 44; 54; 62; 65; 67; 72; 74; 116]. Such strategies have been applied to other miktoarm star architectures, including A\({}_{2}\)B\({}_{2}\) and A\({}_{3}\)B\({}_{3}\)[50; 51]. Depending on the core structure, \({}^{1}\)H NMR spectra can also provide more accurate confirmation of initiator usage, as with the disappearance of the corresponding protons [59; 63]. For example, ethyl-\(\beta\)-d-glucopyranoside, a sugar (containing one secondary and three primary hydroxyls) was used to initiate the ROP ofcaprolactone, specifically at its secondary hydroxyl group, with the aid of the catalyst Novozyme 435. After the hydroxyl group was terminated with vinyl acetate,caprolactone ROP was initiated from the remaining hydroxyl groups, using a Sn(Oct)\({}_{2}\) catalyst. PEG was then conjugated to free PCL-OH ends.