br Particle structure and crystallinity
3.3. Particle structure and crystallinity
Generally, fibroin has two polymorphs, hydrophilic random coil and alpha helix silk I and hydrophobic β-sheet silk II. During desolvation process, the fibroin conformation was changed from silk I to silk II . As the portion of silk II increases, fibroin crystallinity increases pro-portionally. Thus, FT-IR, XRD and DSC were performed to confirm this transformation and to elucidate the drug polymorphism. In-depth analysis on the eﬀects of crosslinker EDC and PEI on the molecular structure of blank FNPs were thoroughly discussed in our previous study .
All blank FNPs possess a rigid silk II conformation with AMG 925 β-sheet structure, and the degree of crystallinity follows EDChigh-FNP > EDClow-FNP > PEI-FNP . The similar order was observed in the α-mangostin loaded FNPs, Fig. 2. The silk II conformation in all formulations was confirmed by FT-IR specific peaks at 1622 cm−1 (amide I), 1517 cm−1 (amide II), and 1265 cm−1 (amide III) (Fig. 2A); XRD characterized peaks at approx. 20° and 24° (Fig. 2B); and the ab-sence of the transformation peak of silk I to silk II at 230 °C in DSC thermogram (Fig. 2C) .
Interestingly, α-mangostin loaded FNPs illustrated significant less crystallinity than the blank FNPs. To determine the degree of crystal-linity of FNPs, we utilized calculation methods derived from FT-IR, XRD, and DSC. Comparing between α-mangostin loaded FNPs and its blank counterparts, significant decrease in intensity was observed in (1) the FT-IR amide I and II peaks (1622 and 1517 cm−1, respectively, Fig. 2A); and (2) the XRD silk II peak of approx. 20° (Fig. 2B). Ad-ditionally, the DSC curves (Fig. 2C and the table below) of α-mangostin loaded FNPs show lower glass transition temperatures (Tg) and higher specific heat capacity ( Cp) at Tg than blank FNPs. Tg and Cp corre-spond proportionally to the degree of crystalline portions in fibroin . Thus, when Tg decreases and/or Cp increases, the crystalline portion of fibroin decreases . Thus, it is confirmed by FT-IR, XRD, and DSC that the loading drug α-mangostin decreased the crystallinity of the fibroin. This might be due to α-mangostin hydrophobic property and the hydrogen bonding between nitrogen/oxygen of the fibroin backbone and α-mangostin.
The polymorph of the α-mangostin in the FNPs was also examined. Clearly, the free α-mangostin is in crystalline form, as sharp signals appeared in both XRD and DSC graphs (Fig. 2D and E). However, in the α-mangostin loaded FNPs, no significant signals of the free drug were observed (Fig. 2A, B, and E). Two reasons were proposed, (1) the drug
Physicochemical properties of α-mangostin loaded FNPs. The values are expressed in mean ± SD (n = 3).
Formulation (FNPs) Size (nm) PI Charge (mV) EE% DL%
Fig. 1. TEM micrographs of α-mangostin loaded FNPs. Large scale bar, 1 μm; small scale bar, 200 nm.
was entrapped in the particles as an amorphous molecular dispersion; and (2) the amount of the entrapped drug was insuﬃcient to be de-tected. The latter can be ruled out by the analysis of physical mixtures of the free α-mangostin and the blank FNPs at the same amount of DL% as the drug loaded particles. As expected, these mixtures illustrated strong drug signals in both DSC (Fig. 2D) and XRD (Fig. 2E). Thus, the polymorph of α-mangostin in the FNPs is amorphous molecular dis-persion. This was the main reason for the solubility increase and con-trollable drug dissolution profiles of the systems (discussed in solubi-lity and dissolution sections).
Therefore, in this section, we firstly conclude that all formulas changed from water soluble silk I to insoluble rigid silk II, with the degree of crystallinity follows EDChigh-FNP > EDClow-FNP > PEI-FNP. Secondly, the entrapment of α-mangostin reduces the FNP crys-tallinity. Thirdly, the polymorph of α-mangostin in FNPs is a molecular dispersion.
3.4. Alpha mangostin aqueous solubility test
The aqueous solubility of α-mangostin loaded FNPs and the free drug itself in HEPES buﬀer, pH 7.4, are demonstrated in Fig. 3A. The
aqueous solubility of the free α-mangostin is 0.386 ± 0.047 μg/mL, in agreement with previous study (0.2 ± 0.2 μg/mL) . Interestingly, all formulations enhanced the drug solubility significantly from 2 to 3 times, as following order; EDClow-FNP > EDChigh-FNP, PEI-FNP > Free α-mangostin (p < 0.01). This was due to the enhanced surface-area-to-volume ratio of nanosized particles and the amorphous mole-cular dispersion morphology of the entrapped α-mangostin, as com-pared to the less soluble crystalline form of the free drug. The highest crystallinity formula, EDChigh-FNP, showed the lowest α-mangostin solubility due to the rigid and ordered structure, which could entrap the drug molecules in a denser manner. Consequently, it took longer and was more diﬃcult for the solvent molecules (i.e., water) to penetrate the particles and solvate/solubilize the drug molecules. Surprisingly, PEI-FNP, although possessing the lowest crystallinity, also had low drug solubility. This is because of the additional interactions such as hy-drophobic interactions and hydrogen bonding between α-mangostin and the branched PEI coating the particles. These interactions retained the drug molecules in the particles, consequently led to a low water solubility.