Halloysite Nanotube-Based Delivery of Pyrazolo[3,4- d]pyrimidine Derivatives for Prostate and Bladder Cancer Treatment

Abstract

Background/objectives: The development of therapies targeting unregulated Src signaling through selective kinase inhibition using small-molecule inhibitors presents a significant challenge for the scientific community. Among these inhibitors, pyrazolo[3,4-d]pyrimidine heterocycles have emerged as potent agents; however, their clinical application is hindered by low solubility in water. To overcome this limitation, some carrier systems, such as halloysite nanotubes (HNTs), can be used.

Methods: Herein, we report the development of HNT-based nanomaterials as carriers for pyrazolo[3,4-d]pyrimidine molecules. To achieve this objective, the clay was modified by two different approaches: supramolecular loading into the HNT lumen and covalent grafting onto the HNT external surface. The resulting nanomaterials were extensively characterized, and their morphology was imaged by high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). In addition, the kinetic release of the molecules supramolecularly loaded into the HNTs was also evaluated. QSAR studies were conducted to elucidate the physicochemical and pharmacokinetic properties of these inhibitors, and structure-based virtual screening (SBVS) was performed to analyze their binding poses in protein kinases implicated in cancer.

Results: The characterization methods demonstrate successful encapsulation of the drugs and the release properties under physiological conditions. Furthermore, QSAR studies and SBVS provide valuable insights into the physicochemical, pharmacokinetic, and binding properties of these inhibitors, reinforcing their potential efficacy.

Conclusions: The cytotoxicity of these halloysite-based nanomaterials, and of pure molecules for comparison, was tested on RT112, UMUC3, and PC3 cancer cell lines, demonstrating their potential as effective agents for prostate and bladder cancer treatment.

Keywords: bladder cancer; carrier; halloysite; prostate cancer; pyrazolo[3,4-d]pyrimidine derivatives; structure-based virtual screening.

Figure 1

(a) Pyrazolo[3,4-d]pyrimidine derivatives studied; and (b) cartoon representation of HNT-based pyrazolo[3,4-d]pyrimidine nanomaterials.

Scheme 1

Synthesis of key intermediate 3. Reagents and conditions: (i) allyl bromide, acetone, K₂CO₃, pyridine, r.t., 4 h, 67%; and (ii) DMF an., POCl₃, CHCl₃ an., reflux, 12 h, 60%.

Scheme 2

Synthesis of final compounds 5–7. Reagents and reaction conditions: (i) PEA, toluene an., r.t., 14 h, 58%; (ii) EtOH, NaOH, reflux, 5 h, 52%; (iii) 3-bromoaniline, EtOH abs., reflux, 5 h, 47%; and (iv) EtOH, NaOH, reflux, 5 h, 43%.

Figure 2

Effect of increasing concentrations of the compounds 5, 6, 7 and Si113, Si306, Si27 and Si306 on the RT112 (a), UMUC3 (b) and PC3 (c) cell viability after 72 h of treatment. Each result represents the average value of four different experiments performed in quadruplicate. Cell viability is expressed as a percentage compared to untreated cells.

Figure 3

Proposed 3D binding mode inside the active site of human tyrosine, determined by FLAP analysis; in particular, the 3D binding pose in pocket one for compound Si113 on the left and compound 6 on the right side.

Figure 4

Two-dimensional binding pose in pocket one for compound Si113 on the left and compound 6 on the right side. N1 (Amide NH) probe describes the hydrogen bond donor character of interaction (blu areas in figures). DRY (Hydrophobic) probe describes the hydrophobic character of interaction (green areas in figure). O (Carbonyl Oxygen) probe describes the hydrogen bond acceptor character of interaction (red areas in figure).

Scheme 3

Synthesis of pyrazolo[3,4-d] pyrimidine derivative-based halloysite nanomaterials.

Figure 5

(a,b) FT-IR spectra of the synthetized nanomaterials; and (c) thermogravimetric curves (straight line) and their derivatives (dots line) of the HNTs, HNTs/Si306 and HNTs-6 nanomaterials.

Figure 6

HAADF/STEM images of (A) HNTs/Si27, (B,C) HNTs-6 and (D–F) HNTs-7 nanomaterials.

Figure 7

Kinetic release of Si27 from HNTs/Si27 nanomaterial in phosphate buffer pH 7.4 at 37 °C.

Figure 8

Effect of increasing concentrations of loaded HNTs on (a) RT112, (b) UMUC3 and (c) PC3 cell viability after 72 h of treatment. Each result represents the average value of four different experiments performed in quadruplicate. Cell viability is expressed as percentage compared to untreated cells.