Improvement of pyrazolo[3,4-d]pyrimidines pharmacokinetic properties: nanosystem approaches for drug delivery

Pyrazolo[3,4-d]pyrimidines are a class of compounds with a good activity against several cancer cell lines. Despite the promising anticancer activity, these molecules showed a poor aqueous solubility. This issue could threat the future development of pyrazolo[3,4-d]pyrimidines as clinical drug candidates. With the aim of improving their solubility profile and consequently their pharmacokinetic properties, we have chosen four compounds (1–4) on the base of their anti-neuroblastoma activity and we have developed albumin nanoparticles and liposomes for the selected candidates. Albumin nanoparticles and liposomes were prepared and characterized regarding size and ζ-potential distribution, polidispersity index, entrapment efficiency and activity against SH-SY5Y human neuroblastoma cell line. The most promising nanosystem, namely LP-2, was chosen to perform further studies: confocal microscopy, stability and drug release in physiological conditions, and biodistribution. Altogether, the obtained data strongly indicate that the encapsulation of pyrazolo[3,4-d]pyrimidines in liposomes represent an effective method to overcome the poor water solubility.


HPLC-UV-MS method
A HPLC-UV-MS system was used for quantitative analysis. LC analysis were performed by Agilent 1100 LC/MSD VL system (G1946C) (Agilent Technologies, Palo Alto, CA) constituted by a vacuum solvent degassing unit, a binary high-pressure gradient pump, a 1100 series UV detector and a 1100 MSD model VL benchtop mass spectrometer. The Agilent 1100 series mass spectra detection (MSD) single-quadrupole instrument was equipped with the orthogonal spray API-ES (Agilent Technologies, Palo Alto, CA). Nitrogen was used as nebulizing and drying gas. The pressure of the nebulizing gas, the flow of the drying gas, the capillary voltage, the fragmentor voltage and the vaporization temperature were set at 40 psi, 9 L/min, 3000 V, 70 V and 350 °C, respectively. UV detection was monitored at 280 nm. The HPLC-ESI-MS determination was performed by operating the MSD in the positive ion mode. Spectra were acquired over the scan range m/z 50-1500 using a step size of 0.1 u. Chromatographic analysis were performed using a Phenomenex Kinetex C18-100A column (150 x 4.6 mm, 5 µm particle size) at room temperature. Analysis were carried out using gradient elution of a binary solution; (eluent A: ACN, eluent B: Water). The analysis started with 0% of A (from t = 0 to t = 3 min), then A was increased to 98% (from t = 3 to t = 12 min), then kept at 98% (from t = 12 to t = 18 min). The analysis were performed at a flow rate of 0.8 mL/min with 20 µL as injection volume. The quantification of all compounds was calculated by referring to the appropriate calibration curves in methanol.

ADME assays
Aqueous Solubility Each solid compound (1 mg where VA is the volume in the acceptor well (cm 3 ), VD is the volume in the donor well (cm 3 ), A is the "effective area" of the membrane (cm 2 ), t is the incubation time (s) and r the ratio between drug concentration in the acceptor and equilibrium concentration of the drug in the total volume (VD+VA).
Drug concentration is estimated by using the peak area integration.
Membrane Retention (MR) was calculated according to the following equation:

FESEM analysis of albumin nanoparticles
Albumin nanoparticles were analyzed by field emission scanning electron microscope (FESEM SIGMA VP Zeiss, Germany) equipped by a energy dispersive X-ray spectroscopy system with an In-Lens detector. Samples were diluted in water and a few drops were placed on carbon-filmed grids and introduced in the sample holder stub. Samples were dried and analyzed using an accelerating voltage between 10 and 20 kV. Figura S1: Albumin nanoparticles morphology study by FESEM.

Albumin-drug nanoparticles preparation
In order to define the best experimental conditions to obtain albumin nanoparticles, different experiments were performed, varying several parameters (concentration of HSA, drug concentration, ratio HSA/Cys). Table S1 shows some representative experiments regarding our best albumin candidate AL-4. The hydration volume was kept fixed at a value of 5 mL. To the optimized preparation column (2), D,L-glyceraldehyde was added as cross-linking agent.

6) Entrapment Efficacy (EE%)
The percentage of entrapment efficacy was calculated with the following equation: where, Fi is the concentration of compound determined in the nano-preparation (evaluated by HPLC-UV-MS, after disruption of nanoparticles) and Ft is the theoretical final concentration (Calculated on the basis of the total amount of compound dissolved in the organic solvent).

Liposome-drug nanoparticles preparations
In order to define the best experimental conditions to obtain liposomes, different experiments were performed, varying several parameters (lipid ratio, use of sonication, drug concentration). Table S2 shows some representative experiments regarding our best liposomal candidate LP-Si306. The hydration volume was kept fixed at a value of 3 mL.

Z-stack projection of SH-SY5Y cells incubated with fluorescent liposomes
Upper panel, Z-stack projection of SH-SY5Y cells incubated with fluorescent liposomes. Liposomes are visualized in green, cellular membrane in red and nuclei in blue. In the lower panel, dashed lines highlightes a liposome (green dot) and orthogonal projection of "X" and "Y" axis are shown. White arrows indicate the highlighted green dot inside the cell.