Dramatically Increased Binding Constant of Water-Soluble Cyclodextrin Hyperbranched Polymers: Explored with Diffusion Ordered NMR Spectroscopy (DOSY)

We report that the polymerization of cyclodextrin (CD) with epichlorohydrin (ECH) dramatically increases the binding constant of CD to vanillin, from 55 to 8.4 × 103 M–1, by approximately 100 times, as determined by diffusion ordered spectroscopy (DOSY)–1H NMR. The binding constant increased with an increase of the ECH content of the polymer, although ECH polymers without CDs showed no affinity at all, suggesting that the hydrophobicity of the ECH network outside of CDs helps to enhance the binding. This increased binding constant allows CD–ECH polymers to increase the drug loading ratio, which may be one of the most critical issues for drug delivery systems.


Determination of the weight average molecular weight (M w ) and hydrodynamic radius (R h )
Size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) and others SEC-MALS measurements were carried out for the CDNPs using a Shodex column (SB802.5 and SB806) at 40 °C with 10 mM aqueous NaCl as the eluent to measure M w . We prepared a sample solution of 1 mg/mL in 10 mM aqueous solution. The solution was optically purified with a PTFE (Polytetrafluoroethylene) membrane with 0.2 μm pores and injected into the column. The output from the column was passed sequentially through an MALS detector (Wyatt Technology, Dawn Heleos II, wavelength: λ = 658 nm) and an RI detector (Wyatt Technology, 1.2 -1.8 RIU, wavelength: λ = 488 -690 nm). The specific refractive index increments (∂n/∂c) of the CDNPs in 10mM aqueous NaCl was determined by a differential refractometer (Otsuka Electronics DRM-1020, wavelength: λ = 633 nm). Data acquisition and molecular weight calculation were performed using the ASTRA software, version 7.3.2 (Wyatt Technology).
The DLS measurement was operated using the facility of DelsaMax Pro (Beckman Coulter, Inc.) to determine the hydrodynamic radius R h . We prepared a sample solution of 10 mg/mL in aqueous solution.
The typical example of GPC chromatogram and the angular dependence of static light scattering to determine M w is shown in the Figure S1A. The distribution of R h was unimodal as shown in the Figure S1B.

S3
The CD weight percent in CDNPs was determined by the phenol sulfuric acid method. Dried CDNP (0.025 g) was refluxed at 100 °C for 8 h in 0.5M H 2 SO 4 (15 mL) to break down the reacted CD to a pentose. Then, the solution was reacted with 5% (w/w) phenol and concentrated H 2 SO 4 .
The final product was brown-orange in color, and the absorbance was detected by a UV-Vis spectrophotometer (Jasco, V-630) at 488 nm. The absorbance was converted into the CD weight percent (wt% CD) by use of a calibration curve established with glucose D(+). Here, we confirmed that polymerized ECH with poly vinyl alcohol samples did not show any absorbance after we treated them in the same manner. From the CD weight percent and M w of CDNP, we calculated the number of CDs in one CDNP particle (N CD ).

NMR of CDNP in DMSO
We carried out 1 H-NMR spectra of βCDNP 0.9 in DMSO and its results showed disappearance of all -OH peaks in as shown in Figure S2.

Complex formation ability of CDNP and DXNP
An amount of vanillin (3.35 mg) was added into the βCDNP 0.9 or DXNP solution (1ml, 50 mg/mL).
The mixture was stirred in 1 day at room temperature. After centrifugation (12000 rpm for 5 min), the upper solution was collected and purified by spin dialysis (cut-off M w = 3500 g/mol). The solution containing only complex finally was filtered before measuring UV-vis. The absorbance of vanillin was detected by UV-vis spectrophotometer. Figure S5. The UV-vis spectra of the βCDNP 0.9 -Val and DXNP-Val complex S6 Figure S6. The NOESY spectra of βCDNP 0.9 in DMSO-d 6 , 25°C Figure S7. The 1 H-NMR spectra of mixture βCDNP 0.9 /βCD at 10mM, 20°C in D 2 O 2 3 4 5 6 ppm