Transition from Amorphous Cyclosporin A Nanoparticles to Size-Reduced Stable Nanocrystals in a Poloxamer 407 Solution

Abstract

Amorphous drug nanoparticles usually exhibit low storage stability. A comprehensive understanding of the molecular states and physicochemical properties of the product is indispensable for designing stable formulations. In the present study, an amorphous cyclosporin A (CyA) nanosuspension with a mean particle size of approximately 370 nm was prepared by wet bead milling with poloxamer 407 (P407). Interestingly, the prepared amorphous CyA nanoparticles were transformed into uniform CyA nanocrystals with a reduced mean particle size of approximately 200 nm during storage at 25 °C. The CyA nanocrystals were stably maintained for at least 1 month. The particle morphologies and molecular structures of the CyA nanosuspensions before and after storage were thoroughly characterized by cryogenic transmission electron microscopy and magic-angle spinning nuclear magnetic resonance spectroscopy, respectively. They revealed that the freshly prepared amorphous CyA nanoparticles (∼370 nm) were secondary particles composed of aggregated primary particles with an estimated size of 50 nm. A portion of P407 was found to be entrapped at the gaps between the primary particles due to aggregation, while most of P407 was dissolved in the solution either adsorbing at the solid/liquid interface or forming polymeric micelles. The entrapped P407 is considered to play an important role in the destabilization of the amorphous CyA nanoparticles. The resultant CyA nanocrystals (∼200 nm) were uniform single crystals of Form 2 hydrate and showed corner-truncated bipyramidal features. Owing to the narrow particle size distribution of the CyA nanocrystals, the rate of Ostwald ripening was slow, giving long-term stability to the CyA nanocrystals. This study provides new insights into the destabilization mechanism of amorphous drug nanoparticles.

Multistep Crystallization of Pharmaceutical Amorphous Nanoparticles via a Cognate Pathway of Oriented Attachment: Direct Evidence of Nonclassical Crystallization for Organic Molecules

“…The freshly prepared amorphous CyA nanoparticles were irregularly shaped (Figure 2a) because they are aggregates of primary particles with a size of approximately 50 nm. 30 The CyA nanoparticles after storage for 24 h (Figure S2) showed morphologies similar to those of the freshly prepared ones. The freeze-dried sample exhibited an amorphous halo for CyA in its PXRD pattern (Figure 2g), suggesting that the CyA nanoparticles maintained the features of amorphous aggregates of primary particles at this point.…”

“…The nanocrystal aggregates could also form single nanocrystals by further crystallographic reorientation and fusion. Because P407 was not present in the lattice of the CyA nanocrystals as demonstrated in our previous study, the entrapped P407 should have been released into the liquid phase during crystallographic fusion. Crystallographic fusion probably started from the core of the mesocrystal (Figure a), allowing the entrapped P407 to be released from the mesostructure.…”

“…Therefore, we additionally performed powder X-ray diffraction (PXRD) for the freeze-dried samples at each time point to evaluate the crystallinity of nanoparticles (Figure g). The freshly prepared amorphous CyA nanoparticles were irregularly shaped (Figure a) because they are aggregates of primary particles with a size of approximately 50 nm . The CyA nanoparticles after storage for 24 h (Figure S2) showed morphologies similar to those of the freshly prepared ones.…”

“…Recently, we reported the abnormal crystallization of amorphous nanoparticles of an immunosuppressive API, cyclosporin A (CyA), in a poloxamer 407 (P407) aqueous solution (The chemical structures of CyA and P407 are shown in Figure S1.). 30 As shown in Figure 1, the amorphous CyA nanoparticles were prepared via wet bead milling, and they were secondary particles (∼370 nm) composed of aggregated primary particles (∼50 nm). A portion of P407 was entrapped in the gaps between the primary particles owing to aggregation.…”

“…In our previous study, we demonstrated that a portion of P407, the dispersant for CyA nanoparticles, was entrapped and solidified in the gaps between the aggregated amorphous primary particles. 30 Polymers are known to induce heterogeneous nucleation of organic compounds ranging from small molecules to proteins. 33−35 The entrapped P407 in the gaps may facilitate the heterogeneous nucleation of CyA.…”