Hybrid Self-Assembling Nanoparticles Encapsulating Zoledronic Acid: A Strategy for Fostering Their Clinical Use

Abstract

Self-assembling nanoparticles (SANPs) promise an effective delivery of bisphosphonates or microRNAs in the treatment of glioblastoma (GBM) and are obtained through the sequential mixing of four components immediately before use. The self-assembling approach facilitates technology transfer, but the complexity of the SANP preparation protocol raises significant concerns in the clinical setting due to the high risk of human errors during the procedure. In this work, it was hypothesized that the SANP preparation protocol could be simplified by using freeze-dried formulations. An in-depth thermodynamic study was conducted on solutions of different cryoprotectants, namely sucrose, mannitol and trehalose, to test their ability to stabilize the produced SANPs. In addition, the ability of SANPs to deliver drugs after lyophilization was assessed on selected formulations encapsulating zoledronic acid in vitro in the T98G GBM cell line and in vivo in an orthotopic mouse model. Results showed that, after lyophilization optimization, freeze-dried SANPs encapsulating zoledronic acid could retain their delivery ability, showing a significant inhibition of T98G cell growth both in vitro and in vivo. Overall, these results suggest that freeze-drying may help boost the industrial development of SANPs for the delivery of drugs to the brain.

Keywords: bisphosphonates; differential scanning calorimetry; glioblastoma; lyophilization; scale-up; self-assembling nanoparticles; zoledronic acid.

Figure 1

SANP preparation procedure. Preparation protocol of hybrid self-assembling NPs encapsulating ZOL (PL-Tf-CaP-ZOL/SANPs-ZOL). PL = PEGylated liposomes; Tf = transferrin; CaP NPs = calcium phosphate nanoparticles; ZOL = zoledronic acid.

Figure 2

DSC studies of cryoprotectants. DSC thermograms under heating of: depurated water, used as a reference (A); mannitol solution (B); trehalose solution (C); sucrose solution (D). Exotherm is directed upward.

Figure 3

Cell viability assay on GBM cell line after treatment with the different formulations. T98G cells were seeded at a density of 2.0 × 103 cells/well in 96-well plates in serum-containing media. After 24 h incubation at 37 °C, cells were treated with increasing concentrations (1.6–200 μM) of the two different formulations SANP 1 (after preparation) and SANP 2 and the respective plain SANPs (used as reference compounds) for 72 h. MTT assay was performed as described in Section 3. The overlapping concentration points in the graph have the same percentage values of cell viability. Experiments were performed in triplicate. Data were expressed as mean value ± the standard deviation calculated for at least three repeats.

Figure 4

Antitumor efficacy of SANP formulation on orthotopic GBM xenograft model. U373-MG (Uppsala) LUC cells were injected into the brain of male nude mice. Real-time tumor growth was monitored using the IVIS imaging system 200 series (PerkinElmer). (A) Quantitative analysis of luciferase activity in vivo at various time points. Luminescent signals are expressed as mean ± SD of total flux of photons/s/cm²/steradian (p/s/cm²/sr). p values were calculated using an unpaired two-tailed t-test. * p < 0.05; n = 6. (B) Representative images of mice analyzed before administration of compounds (day 0) and during treatments on days 10, 23, 30 and 38. Mice with stable disease (SD) or complete response (CR) after SANPs 1 or SANPs 2-L treatment are shown. Data were acquired and analyzed using the Living Image software version 4.3 (PerkinElmer).