The Pyrazolo[3,4- d]Pyrimidine Derivative Si306 Encapsulated into Anti-GD2-Immunoliposomes as Therapeutic Treatment of Neuroblastoma

Abstract

Si306, a pyrazolo[3,4-d]pyrimidine derivative recently identified as promising anticancer agent, has shown favorable in vitro and in vivo activity profile against neuroblastoma (NB) models by acting as a competitive inhibitor of c-Src tyrosine kinase. Nevertheless, Si306 antitumor activity is associated with sub-optimal aqueous solubility, which might hinder its further development. Drug delivery systems were here developed with the aim to overcome this limitation, obtaining suitable formulations for more efficacious in vivo use. Si306 was encapsulated in pegylated stealth liposomes, undecorated or decorated with a monoclonal antibody able to specifically recognize and bind to the disialoganglioside GD2 expressed by NB cells (LP[Si306] and GD2-LP[Si306], respectively). Both liposomes possessed excellent morphological and physio-chemical properties, maintained over a period of two weeks. Compared to LP[Si306], GD2-LP[Si306] showed in vitro specific cellular targeting and increased cytotoxic activity against NB cell lines. After intravenous injection in healthy mice, pharmacokinetic profiles showed increased plasma exposure of Si306 when delivered by both liposomal formulations, compared to that obtained when Si306 was administered as free form. In vivo tumor homing and cytotoxic effectiveness of both liposomal formulations were finally tested in an orthotopic animal model of NB. Si306 tumor uptake resulted significantly higher when encapsulated in GD2-LP, compared to Si306, either free or encapsulated into untargeted LP. This, in turn, led to a significant increase in survival of mice treated with GD2-LP[Si306]. These results demonstrate a promising antitumor efficacy of Si306 encapsulated into GD2-targeted liposomes, supporting further therapeutic developments in pre-clinical trials and in the clinic for NB.

Keywords: anti-GD2 monoclonal antibody; c-Src inhibitor; immunoliposomes; liposomes; neuroblastoma.

Figure 1

Development of Si306-loaded, untargeted and GD2-targeted pegylated stealth liposomes. (a) Chemical structure of Si306; schematic representation of (b) LP[Si306] and (c) GD2-LP[Si306].

Figure 2

Representative Cryo-TEM micrographs of Si306-loaded liposomes. Images of (a) LP[Si306] and (b) GD2-LP[Si306], obtained by Cryo-TEM analysis. Bar: 80 nm.

Figure 3

In vitro release of Si306-loaded untargeted (blue curve) and GD2-targeted (green curve) liposomes.

Figure 4

Cellular association of untargeted and GD2-targeted liposomes on neuroblastoma cell lines. Neuroblastoma cells (HTLA-230, IMR-32, SK-N-AS, and SH-SY5Y) and healthy fibroblasts (Fibro 2–93) were incubated at 4 °C, for 1 h, with Carboxy fluorescein-labelled liposomes at 400 nmol phospholipid/mL concentration. Fluorescence associated with cells was evaluated by flow cytometry. Binding is expressed as mean relative fluorescence intensity (MRFI) normalized over control cells (no liposomes incubation). Columns: MRFI ± SD. ** p ˂ 0.01: GD2-LP[Si306] vs. LP[Si306]; *** p ˂ 0.001: GD2-LP[Si306] vs. LP[Si306].

Figure 5

Pharmacokinetic profiles of Si306 in vivo. Plasma concentration-time curves (mean ± S.E.M., n = 5) in healthy mice, after i.v. administration of a single dose of 5 mg/kg Si306, either free (Si306-Tween80) or encapsulated into untargeted and GD2-targeted liposomes (LP[Si306] and GD2-LP[Si306], respectively). The plasma concentration in the y-axis is expressed as log₁₀ scale.

Figure 6

In vivo Si306 tumor uptake. IMR-32-bearing mice (n = 3/group) were treated with 5 mg/kg Si306, either free (Si306-Tween80) or encapsulated into untargeted and GD2-targeted liposomes (LP[Si306] and GD2-LP[Si306], respectively). 24 h after treatments, mice were euthanized, tumors removed and processed as described in the Materials and Methods section. Columns: mean ± SD. * p ˂ 0.05: GD2-LP[Si306] vs. LP[Si306]; *** p ˂ 0.001: GD2-LP[Si306] vs. Si306-Tween80.

Figure 7

Si306 antitumor effect against an orthotopic animal model of neuroblastoma. Mice were injected in the adrenal gland with IMR-32-luc cells and treated with 5 mg/kg Si306, either free (Si306-Tween80) or encapsulated into untargeted and GD2-targeted liposomes (LP[Si306] and GD2-LP[Si306], respectively), as described in the Materials and Methods section. (a) Representative images of histology (H/E) analysis and IHC staining (brown) for Src and p-Src proteins from tumor tissues of untreated (CTR) or treated mice (more brown staining, more Src and p-Src proteins expression). Bar: 200 μm. (b,c) Digital analyses of IHC grading of tumor tissues from the different experimental conditions. Histogram results from analysis of acquired images after Src (b) and p-Src (c) staining. Columns: mean ± SD. ** p ˂ 0.01 and *** p ˂ 0.001: vs. CTR. (d) Tumor growth delay followed by BLI imaging. Photon counts in the tumor Region of Interest (ROI) are reported. ** p ˂ 0.01: GD2-LP[Si306] vs. CTR, Si306-Tween80 and LP[Si306].

Figure 7

Si306 antitumor effect against an orthotopic animal model of neuroblastoma. Mice were injected in the adrenal gland with IMR-32-luc cells and treated with 5 mg/kg Si306, either free (Si306-Tween80) or encapsulated into untargeted and GD2-targeted liposomes (LP[Si306] and GD2-LP[Si306], respectively), as described in the Materials and Methods section. (a) Representative images of histology (H/E) analysis and IHC staining (brown) for Src and p-Src proteins from tumor tissues of untreated (CTR) or treated mice (more brown staining, more Src and p-Src proteins expression). Bar: 200 μm. (b,c) Digital analyses of IHC grading of tumor tissues from the different experimental conditions. Histogram results from analysis of acquired images after Src (b) and p-Src (c) staining. Columns: mean ± SD. ** p ˂ 0.01 and *** p ˂ 0.001: vs. CTR. (d) Tumor growth delay followed by BLI imaging. Photon counts in the tumor Region of Interest (ROI) are reported. ** p ˂ 0.01: GD2-LP[Si306] vs. CTR, Si306-Tween80 and LP[Si306].

Figure 8

Survival curves of neuroblastoma-bearing mice treated with Si306. Mice were injected in the adrenal gland with IMR-32-luc cells and treated with 25 mg/kg Si306, either free (Si306-Tween80) or encapsulated into untargeted and GD2-targeted liposomes (LP[Si306] and GD2-LP[Si306], respectively), as described in the Materials and Methods section. Survival times of untreated (CTR) and treated mice.