Geigerflex

X-ray diffraction diagrams were detected using an X-ray diffractometer (Geigerflex, Rigaku Co., Akishima, Japan) with Cu Kα radiation. Samples were scanned from 5 to 50° (2θ) at a scanning speed of 2°/min and step size of 0.02°. The X-ray system was operated at a potential of 30 kV and current of 30 mA.

The drug-loaded Bi(mPEG-S-S)-TPE micelles were obtained as follows. At first, the Bi(mPEG-S-S)-TPE dispersion in distilled water was prepared. Then, the PTX in the acetone solution was added. After being sonicated for 3 min (on for 3 s and off for 2 s), the mixture was dialyzed (MWCO of 3500 Da) against distilled water for 2 h and further filtrated with a 0.45 μm filter to obtain the drug-loaded micelles. The micelles could be further freeze-dried and stored as a white powder.
XRD analysis was used to analyze the crystalline characteristic of PTX in the micelles (Geigerflex, Rigaku Co., Tokyo, Japan). Data were collected from 5° to 50° with a step-scan mode. The dynamic light-scattering method was employed to determine the particle size and zeta potential of PTX-loaded micelles (Zetasizer Nano-ZS90, Malvern Instruments, Malvern, UK). The drug loading (DL) and encapsulation efficiency (EE) were calculated using Equations (2) and (3), respectively.

The functional groups were analyzed by Fourier transform infrared spectroscopy (FTIR, JASCO 410, Tokyo, Japan) in the scanning range of 400–4000 cm⁻¹ at a scanning rate of 400 nm/min.
The crystal structure was identified using an X-ray diffractometer (XRD, Rigaku Geigerflex, Tokyo, Japan). The XRD patterns were obtained at 30 kV and 15 mA, within the range of 10–70° at a scanning rate of 1°/min [15 (link)].
The morphology of the synthesized particles was examined using scanning electron microscopy (SEM, Philips XL30, Amsterdam, The Netherlands), with a voltage of 15 kV. Particles were mounted on the sample stage of the SEM and coated with a platinum-sputtered coating [16 (link)].
The particle sizes and zeta potential were detected by dynamic light scattering (DLS, Malvern, Worcestershire, UK) at 25 °C. The zeta potential was determined with electrophoretic mobility at pH 7.4. The sample was dispersed in ddH₂O to measure the mean size.
The pore size distribution and specific surface area were analysis by Brunauer, Emmett, and Teller (BET; Micromeritics ASAP2010, Norcross, GA, USA) using nitrogen gas adsorption–desorption isotherms.

The phase composition of C₃S and Fe/C₃S powders before and after immersion in SBF solution was analyzed by diffraction of X-rays (XRD, Geigerflex, Rigaku, Tokyo, Japan). Fourier transform infrared spectroscopy (FT-IR, NEXUS 670, Nicolet, Beijing, China) further determined the phase composition and chemical bonds of these powders. Surface morphology of discs immersed in SBF solution for 7 days was observed using scanning electron microscopy (SEM, JSM-IT300, JEOL, Tokyo, Japan).

The incorporation of PTX into the mPEG-CS-Hz-CH micelles was achieved by a sonication method. As is typical, a certain amount of mPEG-CS-Hz-CH was dispersed in deionized water (0.25 mg/mL). Afterwards, 1 mL of PTX–acetone solution (0.25 mg/mL) was added, and the mixture was sonicated at 400 W for 6 min (pulse on 2 s and pulse off 1 s). During this process, the sample was kept in an ice cooling bath to prevent overheating. After ultrasonication, the mixture was dialyzed against distilled water (MWCO: 14 kDa) for 3 h to remove acetone and the unloaded PTX. Finally, the resultant product was lyophilized to obtain the PTX-loaded mPEG-CS-Hz-CH micelle powder. The blank micelles were prepared by the same process without addition of PTX.
For characterization, X-ray diffraction analysis was performed (Geigerflex, Rigaku Co., Tokyo, Japan). The DLS technique was used to measure the particle size and zeta potential of the PTX-loaded mPEG-CS-Hz-CH micelles (Malvern Instruments Zetasizer Nano-ZS90, Malvern, UK). The morphology of the PTX-loaded micelles was observed by transmission electron microscopy (TEM, HT7700, Hitachi Ltd., Tokyo, Japan). The drug encapsulation efficiency (EE%) and drug loading (DL%) were determined by an HPLC system at 227 nm, and the following equations were used for calculation [29 (link)].

Our method of obtaining X-ray fiber diffraction data was described in our previous study [16 ,20 (link)]. Briefly, tendon chitosan was prepared from a highly oriented chitin specimen of a crab tendon, Chionecetes opilio O. Fabricius, by N-deacetylation with 67% aqueous sodium hydroxide at 100 °C for 2 h under a nitrogen atmosphere. The degree of N-acetylation of the tendon chitosan was found to be 0% by measurement of a colloidal titration, and the viscosity average polymerization was 10,800 [16 ,20 (link)]. The tendon chitosan was soaked in aqueous ZnCl₂. The X-ray fiber diffraction patterns were recorded with a box camera equipped with an imaging plate (Fujifilm HR-III), at 76% relative humidity in a helium atmosphere, with a Rigaku Geigerflex diffractometer equipped with Ni-filtered Cu Kα radiation. The X-ray diffraction image was read with an imaging plate detector (Rigaku R-AXIS), and the three-dimensional intensity profile was analyzed with Surfer (Golden Software, Inc., Golden, CO, USA) for resolution of overlapped profiles, background removal, and calculations of peak intensities. A set of the measured intensities was corrected for the Lorentz and polarization factors to provide a set of observed structure factors, F_o.
The density of the tendon chitosan–ZnCl₂ complex was measured by flotation with a carbon tetrachloride–m-xylene solution.

XRD analysis was used to study the existence state of PTX after loaded into micelles. Samples were measured by an X-ray diffractometer (Geigerflex, Rigaku Co., Tokyo, Japan) using Cu Kα radiation source at 30 kV and 30 mA. The relative intensity was recorded in the range of 5–50° (2θ) at scanning speed of 4°/min and step size of 0.02°.