(M1330-09-56) Neratinib Nanocrystals, Preparation, Characterization and In Vitro Evaluation Against Triple-Negative Breast Cancer

Purpose: Neratinib (NTB), a pan-HER tyrosine kinase inhibitor, exhibits limited aqueous solubility, bioavailability and efficacy against triple-negative breast cancer (TNBC). This study investigates how to overcome such challenges by developing nanocrystal formulation from highly crystalline NTB standard. This study uses a top-down nano crystallization (NTB NCs) approach with Pluronic® F-127 as a stabilizer. This formulation strategy was expected to enhance drug dissolution, cellular uptake, and anticancer efficacy, with reduced systemic toxicity. The study intended to present an in-depth comparison between the conventional and NCs formulations, underpinned by both physicochemical analyses and in vitro cytotoxicity assays against TNBC models (MDA-MB-231, 4T1) versus non-tumorigenic breast cells (MCF-10A).

Methods: NTB nanocrystals (NTB NCs) were formulated using a top-down approach: NTB (0.1%w/v) from Adooq BioScience, USA (Lot L10638B005) and Pluronic F-127 stabilizer (1%w/v) were dispersed in deionized water, followed by vortexing (5 min) and sequential probe sonication (30%, 50%, 80% amplitude; 10 min each with pulse mode). High-pressure homogenization (20 cycles, 700 psi) was used to make colloidal dispersion. Particle size, PDI, and zeta potential analysis was done using dynamic light scattering (DLS; Brookhaven 90 PALS). Aliquot of NCs was lyophilized and redispersed in 0.1% PF-127 for stability studies. Solid-state properties were determined by differential scanning calorimetry (DSC; TA Instruments, 5°C/min under N₂ purge) and X-ray diffraction (XRD) (Rigaku: SmartLab, 1°–60° 2θ). Morphology was assessed by scanning electron microscopy (SEM). In vitro efficacy was evaluated using MTT assays in MDA-MB-231, 4T1, (5k cells/well) and MCF-10A (10k cells/well) cells across an 8-dose range,2-fold serial dilution (50–0.39 μM, 24–72 hr exposure), with NTB standard (DMSO vehicle 0.5%) and PF-127 controls.

Results: Initial NCs exhibited a monodisperse particle size distribution (221±12 nm, PDI of 0.21) and zeta potential (23 ± 2 mV) (Figure: 1). However, after lyophilization and subsequent redispersion in a 0.1% w/v PF-127 aqueous solution, the particle size increased to approximately (342±28 nm, PDI 0.297, zeta potential: 17± 3 mV). This result suggests that the lyophilization process induced partial agglomeration of the NTB NCs. DSC analyses of the NTB standard exhibited a sharp endothermic peak @ 168°C as compared to the nanocrystal formulation. Enthalpy of fusion (ΔH) for both crystals and nanocrystals were 112 J/g and 85 J/g, respectively. Similarly, XRD data (Figure: 2) demonstrated that the characteristic diffraction peaks of NTB were maintained in the NTB NCs, with significantly reduced intensities and small peak shifts, reflective of a decrease in overall crystallinity and potential lattice strain effects. In vitro, MTT assay results indicated NTB NCs exhibited significant cytotoxicity in TNBC cells (Figure: 3). Both cancer cell lines, MDA-MB-231 and 4T1, show a dose-dependent decrease in cell viability upon treatment with the NTB NCs.  IC₅₀ values for NCs were 2.3-fold lower than NTB in 4T1 and 5-fold lower in MDA-MB-231. The NTB NCs formulation gives more cytotoxic activity as compared to NTB standard at all concentrations investigated, which is possibly due to higher cellular uptake of NTB NCs as compared to NTB standard.  However, NTB NCs showed negligible cytotoxic effect in MCF-10A cells.

Conclusion: NTB NCs can be successfully formulated from highly crystalline NTB standard. NTB NCs with PF-127 stabilization significantly enhances cytotoxic potency against TNBC cells (2.3 to 5 fold improvement in IC₅₀) while they were nontoxic to normal cells. This study validates nano crystallization as a transformative strategy to overcome solubility limitations in kinase inhibitors, with immediate translational potential for TNBC therapy. The absence of cytotoxicity in MCF-10A (non-tumorigenic breast cells) shows that the drug is selectively delivered to tumor cells. Future work will try to optimize the lyophilization process to minimize agglomeration and evaluate the pharmacokinetics and biodistribution of these formulations.

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Acknowledgements: We gratefully thank the Jack and Lois Wareham Research Award and Creighton University School of Pharmacy and Health Professions for funding this project. Thankful to the Department of Pharmacy Sciences for providing their support to use the Drug Development and Characterization Core facility for this research. Conflict of interest statements: The authors confirm that there are no conflicts of interest associated with this work and there has been no significant financial support for this work that could have influenced its outcome.

Fig 1: Physical Stability study of Neratinib Nanocrystal (NTB NCs) Suspension by DLS: NTB NCs stored at room temperature (RT) vs. refrigerated (-4°C) over 6 days. A. Particles size in nm. B. PDI of NTB NCs, C. zeta potential of NTB NCs. Mean ±SD; (n=3).

Fig 2: Overlay of powder X-ray diffraction patterns: Neratinib base (NTB L5, red), Neratinib nanocrystals (NTB NCS L5, Blue). Intensity (counts per second, cps) is plotted against diffraction angle (2θ, degrees).

Fig 3: MTT cytotoxicity of Neratinib Nanocrystals (NTB NCs) vs. Neratinib Standard (NTB St.) in triple-negative breast cancer (TNBC) and normal breast cell lines. *Data represents mean viability (%) ± SD (n=3)
A. Viability % of 4T1 after 48-hr exposure, B. Viability % of MCF-10A after 48-hr exposure, C. Viability % of MDA-MB-231after 48-hr exposure, D. Viability % of 4T1 after 72-hr exposure, E. % of MDA-MB-231after 72-hr exposure.