Biodegradable Core–Multishell Nanocarriers: Influence of Inner Shell Structure on the Encapsulation Behavior of Dexamethasone and Tacrolimus

We here present the synthesis and characterization of a set of biodegradable core–multishell (CMS) nanocarriers. The CMS nanocarrier structure consists of hyperbranched polyglycerol (hPG) as core material, a hydrophobic (12, 15, 18, 19, and 36 C-atoms) inner and a polyethylene glycol monomethyl ether (mPEG) outer shell that were conjugated by ester bonds only to reduce the toxicity of metabolites. The loading capacities (LC) of the drugs, dexamethasone and tacrolimus, and the aggregate formation, phase transitions, and degradation kinetics were determined. The intermediate inner shell length (C15) system had the best overall performance with good LCs for both drugs as well as a promising degradation and release kinetics, which are of interest for dermal delivery.

The compound C18b is a mixture of different isomers of the dimeric fraction of the product EMPOL 1026 from Cognis. The dimeric fraction was isolated by column chromatography with hexane/ethyl acetate on acidified (acetic acid) silica. The dimeric fraction comprises of the compounds that are given in Scheme S1 and its isomers. Table S1. Estimation of the degree of functionalization from GPC analytical data based on Mw.

Calculation of DF via NMR
All signals between 4.48 ppm and 3.32 ppm and an additional peak at 5.20 ppm can be attributed to all methylene and methine protons of hPG ( Figure S1, a) and is partially overlaid with peaks assigned to the PEG backbone. (Figure S1, e,f,g) The aliphatic signal at 1.34 ppm, which has 18 protons (d), is needed to estimate the fraction that is assigned to the hPG backbone (a).
Per glycerol unit, the signal (a) originates from 5 protons of the hPG backbone and, depending on the degree of functionalization (DF), another 28.8 protons are in position e + f at the PEG backbone and additional 3 at position g (Equation S1 and S2). The signal of the 18 protons in (d) also depends on the DF (Equation S3).
Having established these relations (S1-3), the focus is now on the single double-shell chain. Scaling the peak (d) ppm to 18, one can now calculate the relation between DF and , , , (Equation S4) using Equation S2 and S3. This equation is then simplified to Equation S5 and later solved for DF to yield Equation S6, which now can be used to calculate DF from , , , after having set to 18. This procedure works equally for CMS-A18, for CMS-E12 set to 12, for CMS-E18 to 24, and for CMS-E19 to 26. An adapted form of this equation (Equation S7) was used to calculate DF of CMS-E18b. In this case, the methoxy peak at 3.36 ppm was set at 3.

Accuracy of the NMR experiment
Assuming an average measurement error (ME) of 3 %, the deviation for the measured DF was calculated depending on the real DF (see Figure S2) Figure S2. Estimated range of deviation (red) based on a measuring error of the NMR-experiment of 3% for CMS with mPEG350 (left) and mPEG750 (right).   The hydrodynamic diameter was measured before and after filtration with a 450 nm RC filter and after the dissolution of PBS salt. The sizes and populations widely remained the same, only in CMS-E12 the aggregates' diameters decreased. Figure S4. Loading capacities of the investigated carrier architectures for dexamethasone (top) and tacrolimus (bottom) plotted against the melting temperature of their respective inner shell. No correlation could be found. a) Encapsulation performed at 60 °C.

CMS-A18
CMS-E19 a) 0.0% 0.5%   Figure S4. Relevant NMR signals for determining the rate of degradation in DMSO-d6: The signal at 4.07 ppm vanishes over time, indicating a cleavage of the inter-shell ester. The signal at 2.23 decreases upon any ester cleavage and reappears either as a triplet at 2.18 ppm (cleavage of the inter-shell ester) or as a broad signal at 2.14 ppm (cleavage of core ester). Figure S5. Degree of cleavage of total ester bonds (blue) and inter-shell ester bonds of CMS-E15 as determined by NMR (DMSO-d6). total esters Inter-shell esters the triplet at 2.18 ppm to the α-protons of the acid. The broad peak between 2.12 ppm and 1.97 ppm is assigned to the amide α-protons and is overlapped by an impurity at 2.08 ppm. There is no decrease of the amide or ester peaks over time in favor of the signal of the acid protons over time. Figure S7. Cumulative Release (CR) of the absolute amounts of dexamethasone from the solutions of loaded carriers (polymer concentration 10 g/L) in comparison to the untreated control (UC) without enzyme, n=3.