Preparation and evaluation of a novel complex of cefixime by mechanochemical activation

2.1. Materials

Pharmaceutical-grade CEF (CAS no. 79350-37-1) was obtained from Bide Pharmatech Ltd., Shanghai, China. AG (CAS no. 9036-66-2), with a purity greater than 99.0%, was supplied by Jiangsu Yuanshengtong Bioengineering Co., Ltd., Nanjing, China. Na₂GA (CAS no. 71277-79-7) with 98% purity was sourced from ShaanXi Sciphar Biotechnology Co. Ltd., Xi’an, China. Hexane (CAS no. 110-54-3) and hexadecane (CAS no. 544-76-3) were provided by Aladdin Industrial Co., Ltd., Shanghai, China. Cephradine (CAS no. 38821-53-3), also sourced from Aladdin Industrial Co. Ltd., Shanghai, China, had a purity exceeding 98%. All reagent-grade chemicals were utilized as received, without additional purification.

2.2. Preparation of solid-state CEF complexes with excipients

The supramolecular complexes were synthesized using a JC-GMJ roll mill (Qingdao Juchuang Environmental Co.,Ltd., China). The milling conditions involved a 300 mL drum rotating at 157 rpm, grinding media consisting of steel balls (22 mm diameter, total mass 700 g), and a 22 g sample load, with grinding acceleration set at 1 g (free fall). The mechanical processing durations ranged from 2 to 24 h (specifically at 2, 4, 8, 16, and 24 h), and samples were periodically collected for subsequent analysis. The complexes of CEF with AG and Na₂GA were synthesized at a mass ratio of 1:10. Samples demonstrating the greatest increase in solubility were selected for further comprehensive physicochemical and pharmacological evaluations. The procedure used to prepare these CEF complexes with excipients is analogous to that previously described by our group [36].

Physical mixtures (PMs) were prepared through simple blending. Collectively, 0.2 g of CEF and 2 g of either Na₂GA or AG were combined in a 10 mL reagent bottle and manually shaken for several minutes until homogeneous.

2.3. HPLC analyses

An Agilent 1200 HPLC system (Agilent Technologies, USA) equipped with dual pumps and a UV detector was utilized to quantify CEF concentrations. Chromatographic separation was conducted at room temperature using a Hedera ODS-2-C₁₈ reversed-phase column (5 μm, 4.6 × 150 mm). The mobile phase consisted of acetonitrile and tetrabutylammonium hydroxide solution (40:60%, v/v), adjusted to pH 7.0 with phosphoric acid. Detection occurred at a wavelength of 254 nm. Each sample (20 μL) was injected, with a flow rate maintained at 1 mL/min. Prior to analysis, all samples were filtered through a 0.45 μm membrane filter. The retention time of CEF was 3.94 min. AG did not produce any chromatographic peak under these conditions, whereas Na₂GA exhibited a retention time of 7.01 min. Calibration curves were prepared under the aforementioned chromatographic parameters. The calibration curve for pure CEF concentration was linear in the range of 0.01-0.1 mg/mL (y=34316x-16.669, r²=0.9999, where x=drug concentration, y=peak area). The calibration curve for CEF plasma concentration was linear in the range of 0.1-2.0 μg/mL (Y=607.233x-1.594, r²=0.9995, where X=CEF concentration, Y=ratio of cephradine peak area to CEF peak area).

2.4. Quantitative analysis of CEF and its complexes

For CEF quantification, approximately 10 mg of each sample was precisely weighed and dissolved in 25 mL of methanol. The resulting solutions were diluted appropriately and analyzed for CEF concentration via high-performance liquid chromatography (HPLC), as described previously [51].

2.5. Solubility analysis of CEF and its complexes

Mechanochemically prepared samples obtained after various milling intervals were transferred into 25 mL flat-bottomed flasks and dissolved in 10 mL of distilled water. To ensure equilibrium, these flasks were agitated for 3 h at 200 rpm in an orbital shaker maintained at 37°C. After shaking, the mixtures were centrifuged for 5 min at 12,000 rpm. The clear supernatants were filtered through a 0.45 μm membrane filter, and the filtrates were analyzed by HPLC according to the method described above. Additionally, the remaining clear supernatants were transferred into 10 mL reagent bottles, and their pH was measured at room temperature using a PHS-3G pH meter (Shanghai Leici, China).

2.6. Dissolution studies

Dissolution characteristics of pure CEF, as well as its PMs and mechanochemically prepared complexes with Na₂GA and AG, were investigated. Dissolution tests were performed using a ZRS-8G rotating paddle system (Tianjin Tianda Tianfa, China). The dissolution medium consisted of 900 mL of 0.05 M phosphate buffer (pH 7.2), maintained at 37 ± 0.5°C with paddle rotation at 50 rpm. Samples equivalent to 100 mg of pure CEF were dispersed in the dissolution medium and allowed to dissolve over a period of 1 h. Aliquots of 5 mL were withdrawn at predetermined intervals (5, 10, 15, 30, 45, and 60 min) and immediately replaced with an equal volume of fresh, preheated dissolution medium [36]. Following filtration through a 0.45 μm membrane, CEF content was determined by HPLC as previously described. All tests were conducted in triplicate, and results were expressed as mean ± standard deviation (SD).

2.7. Phase solubility assay

A modified version of the Higuchi and Connors [52] method was applied to perform the phase solubility study. Mechanochemically obtained samples with enhanced solubility were dissolved in 10 mL of distilled water in flat-bottom flasks. These flasks were then sealed and shaken for 3 h at three temperatures (30, 37, and 42°C) using a thermostatic orbital shaker. Upon reaching equilibrium, mixtures were centrifuged at 12,000 rpm for 5 min, and the clear supernatants were subsequently filtered through a 0.45 μm membrane. The concentration of CEF in the filtrates was quantified using HPLC. Phase solubility diagrams were created by plotting the molar concentration of CEF versus the total molar concentration of AG or Na₂GA. The stability constants (K_c) (Eq. 1) and complexation efficiency (CE) (Eq. 2) were calculated according to previously established equations [53]. The CE parameter is considered more reliable than K_c, as it is less sensitive to measurement errors in solubility determinations [54]. Additionally, thermodynamic parameters $\Delta G$ (Eq. 3) at each temperature were computed using the equations below:

where S₀ represents the intrinsic solubility of the drug.

2.8. Powder X-ray diffraction (XRD)

XRD analyses of pure CEF, excipients, and complexation products were conducted using an ARL EQUINOX 3000 X-ray diffractometer (Thermo Fisher Scientific, USA) [36]. XRD scans were performed from 2θ = 5° to 60° using 40 kV and 40 mA. Measurements were collected at an intensity scale up to 1000 counts and at a scan speed of 2° per minute. The resulting peak positions and intensities were analyzed using Origin 9 software.

2.9. Differential scanning calorimetry (DSC)

Thermal analyses of CEF and its complexes were conducted using a TA Instruments Discovery DSC 25 under a nitrogen atmosphere. Samples (5 mg each) were sealed in aluminum pans and heated at a rate of 10°C min^-1 from 20°C to 300°C [12].

2.10. Scanning electron microscopy (SEM)

Surface and cross-sectional morphologies of the samples were examined using a Carl Zeiss Gemini SEM500 (Carl Zeiss, Germany). Specimens were fixed on stubs, coated with gold, and subsequently placed onto the SEM stage for morphological analysis at various magnifications [36].

2.11. Transmission electron microscopy (TEM)

The nanostructure of the CEF/Na₂GA micelle complex was analyzed via TEM (JEOL JEM-2100F, JEOL, Japan). Samples were prepared using a negative staining method with aqueous uranyl acetate. Specifically, a drop of the sample solution was placed on a formvar-coated copper grid, excess solution was removed to create a thin film, and the grids were air-dried at room temperature before being analyzed under TEM to characterize their structural features [48].

2.12. Fourier transform infrared spectroscopy (FT-IR)

Spectroscopic analysis of CEF, its excipients, and their dispersions was conducted using a Nicolet iS50 Fourier spectrophotometer (Thermo Fisher Scientific, USA). Spectral data were collected over a range from 500 to 4000 cm⁻¹ employing the KBr pellet technique [16]. This spectral range was crucial to elucidate potential molecular interactions between CEF and AG or Na₂GA within the resulting supramolecular assemblies. Infrared spectra from each finely milled complex formulation were carefully compared with their respective PMs.

2.13. In vitro parallel artificial membrane permeability assay (PAMPA)

Compound permeability across membranes was assessed using 12-well plates equipped with polycarbonate membranes (12 mm diameter inserts, 0.4 μm pores, 1.12 cm² area; Corning Incorporated) [35]. Initially, 60 µL of a 5% hexadecane solution in hexane was introduced into each donor well to saturate the synthetic membrane. The assembly was left overnight in a ventilated fume hood, allowing complete evaporation of hexane. Subsequently, each acceptor well was filled with 1.5 mL of distilled water, and the treated donor plate was placed above it. Then, 0.5 mL of CEF or its complex solutions was dispensed into the donor wells. The assembled PAMPA plates were shaken for 6 h at 200 rpm at 30°C using an orbital shaker. At predetermined intervals (every 0.5 h up to 6 h), 1 mL of solution was withdrawn from the acceptor plate for HPLC analysis and replaced with an equivalent volume of distilled water.

2.14. Particle characterization and zeta potential

Particle size characterization, including polydispersity index (PDI) and zeta potential measurements of the ultrasonically treated CEF/Na₂GA, was performed using a dynamic light scattering (DLS) device (Zetasizer NanoZS, Malvern Instruments, Malvern, UK). Prior to analysis, the CEF/Na₂GA sample was solubilized in deionized water and subjected to a 5-min ultrasonication treatment [55].

2.15. Stability test for CEF and mechanically processed complexes

The original CEF and mechanochemically treated samples were stored in airtight containers at 40°C and 75% humidity in a stability chamber (model LHH-80SD, Shanghai Yiheng Scientific Instruments Co., Ltd., Shanghai, China) to evaluate the stability of mechanochemically prepared CEF solid complexes. Storage conditions were maintained for 3 months. Following storage, samples underwent comprehensive evaluation of their physicochemical properties, particularly content uniformity and dissolution capability. Additionally, their crystalline structures were meticulously analyzed during the final month of the study [49].

2.16. Molecular dynamics (MD) studies

Initial structural models exhibiting the lowest affinity were identified as stable candidates for subsequent MD simulations. Structures of CEF, Na₂GA, and the AG fragment were generated using ChemDraw software and optimized with Open Babel. MD simulations of CEF/AG and CEF/Na₂GA complexes were performed using the GROMACS software suite (2019 version) and the GAFF force field [56]. A TIP3P explicit solvation model was applied, embedding the structures within an orthorhombic box and ensuring a solvent layer thickness of at least 20 Å. The simulation protocol began with energy minimization of the entire system over 10,000 steps. This was followed by a gradual temperature increase to 300 K over 50 picoseconds (ps). Equilibration under constant volume and temperature (NVT ensemble) with periodic boundary conditions was conducted for an additional 50 ps, followed by a 100 ns production MD simulation under constant pressure and temperature (NPT ensemble). Simulation data were recorded at intervals of 20 ps for subsequent detailed analysis.

2.17. In vivo pharmacokinetic study

3.1. Properties of solid compositions CEF/excipients

3.1.1. FT-IR analysis

FT-IR spectroscopy was employed to elucidate the molecular interactions between CEF and its excipients. The IR spectra of both the ball-milled preparations and their corresponding PMs have been presented in Figure 2. The CEF spectrum displayed distinct absorption peaks, including N-H stretching at 3294.21 cm⁻¹, O-H stretching at 3562.94 cm⁻¹, C-H stretching at 2947.43 cm⁻¹, C=O stretching (COOH) at 1773.20 cm⁻¹, C=O stretching (CONH) at 1669.75 cm⁻¹, ring stretching vibrations at 1590.83 cm⁻¹, aromatic C-N stretching at 1336.27 cm⁻¹, C-H bending at 747.43 cm⁻¹, and C=C stretching at 1542.62 cm⁻¹, consistent with findings from a previous study [4]. In the PMs, these characteristic peaks of CEF remained unchanged, indicating that no chemical interactions occurred between the drug and excipients. Notably, the sharp C=O stretching peak at 1669.75 cm⁻¹ in CEF was diminished in the milled CEF/AG complex, suggesting a potential interaction between the acrylamide groups of CEF and the side chains of AG. Moreover, the near-complete absence of the C=O stretching peak at 1773.20 cm⁻¹ associated with carboxyl groups in the milled CEF/Na₂GA complex implied the formation of hydrogen bonds.

Close

Figure 2.

FT-IR spectra of free CEF, AG, Na₂GA, CEF/AG PM (1/10), the CEF/AG (1/10) mixture treated in the mill for 24 h, CEF/Na₂GA PM (1/10), and the CEF/Na₂GA (1/10) mixture treated in the mill for 2 h.

Export to PPT

3.1.2. XRD analysis

XRD serves as a powerful analytical method for rapidly identifying novel crystalline phases in solid samples. The XRD patterns for all studied samples are shown in Figure 3. CEF exhibited diffraction peaks at 2θ values of 5.82°, 8.92°, 15.09°, 19.48°, 22.18°, 26.42°, and 27.22°, confirming its crystalline nature. The PM samples displayed weak CEF-related peaks without any new diffraction signals, indicating that simply mixing CEF with excipients did not trigger interactions, and thus, the PMs preserved their crystalline structure. In contrast, the XRD profiles of the milled CEF/AG and CEF/Na₂GA complexes exhibited a complete absence of the characteristic crystalline peaks of CEF, indicating their transformation into an amorphous state due to mechanical activation.

Close

Figure 3.

XRD patterns of unprocessed CEF, AG, Na₂GA, CEF/AG PM (1/10), the CEF/AG (1/10) mixture treated in the mill for 24 h, CEF/Na₂GA PM (1/10), and the CEF/Na₂GA (1/10) mixture treated in the mill for 2 h.

Export to PPT

3.1.3. DSC analysis

DSC analyses were conducted to further elucidate the physicochemical properties of the CEF samples, as illustrated in Figure 4. The DSC thermogram of free CEF showed an endothermic peak at 118.5°C, indicating water loss from its trihydrate crystal lattice. Additionally, exothermic peaks at 187.7°C and 251.2°C were observed, likely attributable to crystal phase transition and compound decomposition, respectively, aligning with findings from previous studies [12]. Thermograms of the excipients exhibited an endothermic peak at approximately 80°C, corresponding to dehydration. Specifically, the AG thermogram revealed another endothermic peak at 240.5°C, indicative of decomposition. In the PM systems, the phase transition and decomposition peaks of CEF were detected but with diminished intensity, suggesting the absence of significant interactions between CEF and the excipients, thus precluding complex formation. In contrast, the milled products displayed a broadened and less distinct thermal profile, indicating the transformation of CEF into an amorphous state following mechanochemical treatment.

Close

Figure 4.

DSC of unprocessed CEF, AG, Na₂GA, CEF/AG PM (1/10), the CEF/AG (1/10) mixture treated in the mill for 24 h, CEF/Na₂GA PM (1/10), and the CEF/Na₂GA (1/10) mixture treated in the mill for 2 h.

Export to PPT

3.1.4. Morphological analysis

SEM was utilized to examine the surface morphology of pristine CEF crystals, individual excipients, and their mechanocomplexes (Figure 5). SEM images showed pristine CEF crystals with an irregular, block-like structure. AG particles appeared spherical with a distinctly wrinkled texture, whereas Na₂GA particles exhibited a smooth outer surface and hollow spherical morphology (Figure 5d). In contrast, samples subjected to mechanochemical synthesis displayed significantly altered morphologies characterized by smaller, irregularly shaped particles, and the original block-like CEF crystals were no longer evident (Figures 5c, e).

Close

Figure 5.

SEM of (a) unprocessed CEF, (b) AG, (c) the CEF/AG (1/10) mixture treated in the mill for 24 h, (d) Na₂GA, and (e) the CEF/Na₂GA (1/10) mixture treated in the mill for 2 h.

Export to PPT

In this study, the physicochemical characteristics of micelles formed from mechanically milled CEF/Na₂GA were evaluated to determine their effects on the drug’s biopharmaceutical properties. Utilizing DLS and TEM (Figures 6a,b), the mean micelle diameter after dispersion in water was measured as 168 nm, with a PDI of 0.468 and a zeta potential of approximately –14.9 mV. TEM imaging further revealed micelles as spherical entities with smooth contours and an estimated diameter of 50 nm. Differences in particle sizes observed between DLS and TEM may be attributed to variations in measurement conditions inherent to each analytical method. Nevertheless, the formation of nanomicelles upon the dispersion of milled CEF/Na₂GA complexes in water was confirmed by the above-mentioned methods. These results align with previous findings indicating that GA inherently forms micelles with a core-shell structure in aqueous media due to its amphiphilic nature. This characteristic facilitates the encapsulation of hydrophobic molecules within the micelle core, thus enhancing drug solubility and potentially reducing the required therapeutic dosage [36-38,51]. The nanoscale dimensions of these micelles are expected to facilitate rapid drug absorption following ingestion due to enhanced solubility, diffusivity, and dispersibility within the gastrointestinal mucosal layer [57].

Close

Figure 6.

(a) DLS size measurement of CEF/Na₂GA micelles, (b) TEM of CEF/Na₂GA micelles, (c) Dissolution profiles of CEF/AG formulation, and (d) Dissolution profiles of CEF/Na₂GA formulation.

Export to PPT

3.2. Characteristics of CEF aqueous solution and prepared CEF/excipient complexes

3.2.3. Phase-solubility study

The phase solubility diagrams for the mechanically treated formulations are presented in Figures 7(a, b), and the calculated K_c, molar concentrations of CEF in CE, and ΔG values have been summarized in Table 2. The phase solubility results demonstrated a proportional increase in CEF solubility with rising concentrations of AG and Na₂GA. The graphical profiles exhibited an A_L-type, suggesting a probable 1:1 stoichiometry for complex or micelle formation. The slope values were below unity, further indicating a 1:1 association between CEF and either AG or Na₂GA [52]. These findings suggest that the GA micelle likely forms a stable, “single macromolecule” structure encapsulating CEF within its core. Specifically, in the CEF/AG system, each CEF molecule presumably interacts individually with an AG macromolecule. Moreover, the K_c values for the CEF/Na₂GA system were significantly higher than those for CEF/AG, indicating greater stability of the CEF/Na₂GA complex. Additionally, ΔG values were consistently negative across all conditions, demonstrating the thermodynamically spontaneous formation of these complexes.

Close

Figure 7.

Phase solubility diagrams of complexes (a) CEF/AG (1/10, 24 h), and (b) CEF/Na₂GA (1/10, 2 h) at different temperature in aqueous solution, (c) PAMPA assay of CEF and its milling complexes, (d) Time-concentration curve after oral introduction of CEF and its compositions CEF/AG (1/10, 24 h) and CEF/Na₂GA (1/10, 2 h).

Export to PPT

Table 2. The apparent stability constant, the complexation efficiency, and the thermodynamic parameters of CEF in its mechanical processed complexes.

Composition	Kc, M-1			CE			ΔG, KJ/mol
	30°C	37°C	42°C	30°C	37°C	42°C	30°C	37°C	42°C
CEF/AG (1/10, 24 h)	54.83	71.91	51.19	0.032	0.042	0.030	–10.09	–11.03	–10.32
CEF/Na2GA (1/10, 2 h)	122.5	130.5	127.0	0.072	0.076	0.074	–12.11	–12.55	–12.68

3.3. Stability of CEF and corresponding complexes

Although amorphous forms of poorly soluble drugs can significantly enhance dissolution rates, they may recrystallize during storage, adversely affecting dissolution performance and resulting in inconsistent oral absorption. Amorphous systems, characterized by disordered structures and higher free energy, are known to degrade more readily than their crystalline counterparts [57,64]. To evaluate the physicochemical stability of mechanically processed formulations, accelerated storage conditions (40°C and 75% relative humidity) were employed for pure CEF and its mechanically prepared derivatives. Drug content and solubility data have been summarized in Table 3. Over the storage period, pure CEF exhibited decreased drug content, whereas mechanically processed formulations retained their initial drug content after 90 days, indicating improved stability due to their incorporation into inclusion complexes or micelles. Moreover, the solubility of the mechanically processed products remained stable throughout the three-month study period. XRD analysis (Figure 8) demonstrated that the amorphous state was effectively maintained post-storage without signs of recrystallization, suggesting that AG and GA serve as efficient crystallization inhibitors in amorphous solid-state formulations.

Close

Figure 8.

(a) Stability test for the XRD patterns of the mechanical processed products during 3 months of storage, and (b) The interaction energy between each component

Export to PPT

3.4. MD analysis

MD simulations were performed to elucidate interactions between CEF and the excipients GA or AG. During the 100 ns simulations, conformational spaces of the complexes were explored, and non-covalent interactions were analyzed. The simulations revealed that formation of stable micellar or inclusion structures, with CEF encapsulated within micellar systems or stably interacting with AG side chains, after approximately 50 ns (Figure 9).

Close

Figure 9.

Snapshots of complexes (a-c) CEF/Na₂GA system, and (d-f) CEF/AG system at the various time frames 0, 50, and 100 ns during MD simulation.

Export to PPT

Root mean square displacement (RMSD) values (Figure 10) indicated system stabilization after 10 ns, with equilibrium maintained throughout the simulation, reflecting the stability and equilibration of the complexes. Similarly, the radius of gyration (Rg) values remained consistent, confirming the formation of stable CEF-excipient complexes. A reduction in solvent-accessible surface area was noted in both CEF/Na₂GA and CEF/AG systems, indicating tighter molecular packing and potential water exclusion from molecular clusters, particularly pronounced in the CEF/Na₂GA system.

Close

Figure 10.

Quantitative analysis of complexes (a-d) CEF/Na₂GA system, and (e-h) CEF/AG system during 100 ns MD simulation.

Export to PPT

The role of hydrogen bonding in enhancing solubility has been extensively documented [32,53]. As illustrated in Figure 10, the number of hydrogen bonds formed between CEF and the excipients remained constant during MD simulations, suggesting the establishment of multiple stable hydrogen bonds. Additionally, energy distribution analysis revealed that both Van der Waals (Vdw) and electrostatic interactions (EIE) significantly contributed to the stabilization of complexes (Figure 8b).

3.5. Pharmacokinetic study

Plasma concentrations of CEF over time following oral administration of pure CEF and its complexes with AG and Na₂GA have been depicted in Figure 7(d), with the corresponding pharmacokinetic parameters summarized in Table 4. The pharmacokinetic profiles clearly indicated enhanced bioavailability of CEF when administered as mechanical complexes relative to the free drug. The C_max values of CEF/Na₂GA and CEF/AG complexes were nearly double those of pure CEF.

Furthermore, the T_max was reduced from 1 h (free drug) to 0.5 h (complexes), and the AUC_0-inf of the complexes was significantly greater compared to pure CEF.

The improved oral bioavailability of CEF may result from several factors. Firstly, the amorphous state of CEF in mechanical complexes, as opposed to its crystalline form, likely enhances dispersibility and wettability, contributing to increased bioavailability. Secondly, the strong mucoadhesive properties of polysaccharides [65] or the inhibition of P-glycoprotein (P-gp) efflux pumps [66] could increase drug absorption.

It is hypothesized that the CEF/Na₂GA system forms micelles in aqueous media, encapsulating CEF within a hydrophobic core and hydrophilic shell, thereby protecting the drug from degradation and enhancing oral absorption. Although the CEF/Na₂GA complex exhibited superior aqueous solubility, the CEF/AG complex notably increased plasma CEF concentrations more significantly. This discrepancy may be due to the metabolism of GA into 18β-glycyrrhetinic acid by intestinal bacterial β-glucuronidase [67], affecting the stability of the GA complex in vivo. Under in vitro PAMPA conditions lacking intestinal flora, the notable permeability enhancement observed for the CEF/Na₂GA complex can thus be rationalized.