Facile Preparation of Monodisperse Pharmaceutical Colloidal Spheres of Atorvastatin Calcium via Self‐Assembly

Colloidal spheres of atorvastatin calcium (see picture) with tunable size and enhanced dissolution rate are obtained by a solvation-driven molecular assembly method. This technique enables the large-scale synthesis of monodisperse pharmaceutical spheres, to facilitate their delivery and maximize their therapeutic effectiveness. Monodisperse colloidal spheres have continuously attracted research interest due to their interesting properties tunable by their size, composition, and structure.1-3 Such materials are of great interest for drug delivery, biodiagnostics, combinatorial synthesis, photonic crystals, rheological fluids, and building complex structures.2-6 To date, most of the monodisperse colloidal spheres are, however, limited to inorganic (e.g., Pb, CdS, ZnS, TiO2, C, Si, and Se) and polymeric spheres.3, 7-12 It is known that the size and dispersity of pharmaceutical particles can significantly affect their cellular interactions and biodistribution;13-15 making pharmaceutical molecules into small spheres not only facilitates their delivery but also maximizes their therapeutic effectiveness.14-21 Nevertheless, the fabrication of monodisperse pharmaceutical spheres with controllable diameter has not yet been fully reported. Herein, we report a facile molecular assembly method towards the large-scale synthesis of monodisperse pharmaceutical spheres. Atorvastatin calcium (AC), a synthetic lipid-lowering drug with enormous medical and commercial significance,22 was used as a model drug. Molecular assembly, a process based on noncovalent interactions between building-block molecules, has been commonly used to generate assemblies with various morphologies, such as micelles,23 vesicles,24, 25 ribbons,26 fibers,27 and tubes.28 Among the various assembling systems, amphiphilic molecules often tend to self-organize in either aqueous or organic media to form a large variety of complex structures.29-32 As shown in Scheme 1, each AC molecule contains a hydrophilic segment (the carboxylic acid-containing segment) and hydrophobic segments, which allow their assembly into monodisperse particles in mixed solvents of methanol and isopropanol (IPA). Experimentally, the addition of a solution of AC in methanol to IPA immediately led to the formation of a suspension of monodisperse AC spheres. Manipulation of the AC concentration and the solvent composition allows effective control of the particle size and dissolution properties, thus opening a new avenue towards the fabrication of better and more effective pharmaceutical formulations. Molecular structure of AC (C33H34FN2O5)2Ca · 3H2O: [R-(R*,R*)]-2-(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrole-1-heptanoic acid calcium salt (2:1) trihydrate. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed to study the morphology of the spheres. Figure 1a provides a representative SEM image of the particles sampled from the suspension, which was prepared by adding a solution of AC in methanol (40 mg mL−1) to IPA at an IPA/methanol (IPA/MeOH) volume ratio of 10. It is clear that these particles are nearly monodisperse with an average diameter of 560 nm and a homogeneous structure (see the TEM image in Figure 1b). The suspension can be readily dried to form a powder of monodisperse particles. Figures 1c and d exhibit SEM images of the powder, and indicate that it consists of smooth, monodisperse spheres of similar sizes. The inset of Figure 1c shows a Petri dish containing 8 g of powder obtained in a single batch, which suggests an excellent scale-up possibility. a) SEM and b) TEM images of the AC spheres sampled from a suspension prepared with a solution of AC in methanol (40 mg mL−1) and an IPA/MeOH volume ratio of 10. c,d) SEM images of the dry powder from the suspension; inset in (c): photograph of a Petri dish containing 8 g of dry powder, which indicates the feasibility of scale-up production. The size of the spheres can be readily tuned by controlling the AC concentration and solvent composition. It was found that the sizes of the obtained particles are inversely proportional to the AC concentration. For example, Figure 2 depicts SEM images of the particles prepared at different AC concentrations and a fixed IPA/MeOH ratio of 10. Clearly, increasing the AC concentration from 15 to 40 mg mL−1 systematically decreases the particle size from 1000 (Figure 2a) to 560 nm (Figure 2d). Further increasing the AC concentration (e.g., 60 and 80 mg mL−1) led to the formation of polydisperse particles (Figure S2, Supporting Information), possibly due to aggregation of small particles.3, 7 SEM images of AC spheres prepared at different AC concentrations at a fixed IPA/MeOH ratio of 10: a) 15 mg mL−1 (1000 nm); b) 20 mg mL−1 (900 nm); c) 30 mg mL−1 (700 nm); d) 40 mg mL−1 (560 nm). To investigate the effect of solvent composition, AC particles were synthesized at different IPA/MeOH ratios. Figure 3a shows the evolution of particle size versus the IPA/MeOH ratio, and indicates a continuously decreasing size with increasing volume ratio (see Figure S3, Supporting Information). In particular, a rapid size decrease from 680 to 310 nm was observed when the ratio was increased from 5 to 15. Further increasing the ratio had less effect on size (e.g., 240-nm particles were achieved at a high volume ratio of 40). It is worth mentioning that monodisperse particles were generally obtained at volume ratios from 8 to 30. This work provides an effective approach towards the synthesis of pharmaceutical spheres with tunable size by simply controlling the initial drug concentration and the solvent composition. a) Plot showing the evolution of particle size as a function of IPA/MeOH ratio at the fixed AC concentration of 40 mg mL−1. b) XRD patterns of commercial AC and the AC spheres. c) Dissolution profiles of commercial AC and AC spheres with different particle sizes. Figure 3b compares the powder X-ray diffraction (XRD) patterns of commercial AC and the AC spheres, and indicates that the spheres are amorphous whereas the commercial material is highly crystallized. Such an observation is further confirmed by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and Fourier transform infrared (FTIR) studies (Figures S4–S6, Supporting Information). It has been recognized that drugs with an amorphous state may show better dissolution and bioactivity than crystalline ones.33, 34 Figure 3c compares the dissolution profiles of commercial AC and AC spheres with an average size of 310 nm. As expected, compared with the commercial AC that shows 45% dissolution in 10 min, the spheres show a faster dissolution of 86%. The enhanced dissolution of the spheres can be attributed to their amorphous structure, small size, and high surface area (4.8 m2 g−1 for commercial AC versus 14.1 m2 g−1 for the spheres).33-35 According to the Kelvin equation, a solid with high curvature has a high solubility in liquid; thus, as shown in Figure 3c, the solubility of the AC spheres increases with decreasing size, further providing an effective route to manipulate the drug dissolution rate. The formation mechanism involves self-organization of the AC molecules driven by the intermolecular interactions of the amphiphilic AC molecules and by solvation forces. Methanol and IPA are, respectively, good and poor solvents for the AC molecule. Adding a solution of AC in methanol to IPA enhances the intermolecular interactions of the AC molecules, promotes their assembly into molecular superstructures to minimize the surface energy,29, 31 and further organizes them into the spherical colloidal structure.25, 30, 36 To confirm the proposed mechanism, AC particles were formed in a capillary tube and observed in situ by using an optical microscope (Figure 4). When the AC solution was injected into a micropipette tube containing IPA, particles formed immediately (Figure 4a). These particles then rapidly diffused into the IPA and scattered as individual spheres with a clear-walled structure (Figure 4b–d). After 20 s, the particles were freestanding in the solution and assumed a spherical shape, which is a thermodynamically favorable state in terms of surface energy.3, 32, 37 As displayed in Figure S7 (Supporting Information), after storage for 7 days the particles still showed a similar morphology and size to the freshly prepared AC spheres (Figure 2d). The formation of highly stable colloidal spheres is of importance for pharmaceutical and other related purposes. More importantly, this strategy can be generalized to fabricate a large variety of pharmaceutical molecular spheres, such as sartans, statins, cephalosporins, macrolides, and glucocorticoids, with controlled structure and morphology (Figure S8, Supporting Information). Optical micrographs depicting the formation of the AC spheres at a) 0, b) 2, c) 6, d) 20, e) 60, and f) 180 s. In summary, monodisperse AC colloidal spheres with tunable size and enhanced dissolution rate were prepared conveniently through solvation-force-driven assembly. This method enables the large-scale production of monodisperse pharmaceutical colloidal spheres for better therapeutic effects at low cost, which is essential for the preparation, formulation, and delivery of pharmaceutical molecules. The commercial AC (Form I, purity 99.4%) was purchased from Beijing Mediking Biopharm Co., Ltd. (Beijing, China) and used without further purification. Methanol and IPA were of analytical grade and purchased from Beijing Chemical Reagents Company (Beijing, China). In a typical synthesis procedure, the addition of a clear solution of AC in methanol to IPA under stirring (1000 rpm) immediately created an opalescent suspension. After stirring for 30 s, the particles were separated from the suspension by vacuum evaporation at room temperature. Different AC concentrations in methanol and volume ratios of IPA/MeOH were used to prepare particles with different sizes and dispersities. Particle sizes were measured with a laser diffractometer (Malvern, Zetasizer-3000HS). TEM images were generated with a Hitachi H-800 transmission electron microscope. SEM images were obtained with a model JEOL JSM-6360LV scanning electron microscope. Optical micrographs were taken with an IX-51 microscope (Olympus Optical Co., Ltd., Japan). The crystalline structure was examined by using an X-ray diffractometer (XRD-6000, Shimadzu, Japan). Specific surface areas of the particles were analyzed with an ASAP 2010 surface area analyzer (Micromeritics) using nitrogen sorption at 77 K. Dissolution testing was carried out by following the USP Apparatus 2 (paddle) method (D-800LS, Tianjin, China). The paddle speed and bath temperature were set at 75 rpm and 37.0 ± 0.5 °C, respectively. Phosphate buffer (pH 6.8) was employed as the dissolution medium. An approximately 30 mg sample of commercial product or AC spheres was added to a vessel containing 900 mL of the dissolution medium. Aliquots (5 mL) were withdrawn at specific intervals and filtered using a 0.45-µm filter. The concentration of the samples was assayed by a UV spectrophotometer (UV-2501, Shimadzu, Japan) at 246 nm. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.