Lactate-Loaded Nanoparticles Induce Glioma Cytotoxicity and Increase the Survival of Rats Bearing Malignant Glioma Brain Tumor

Abstract

A glioblastoma is an aggressive form of a malignant glial-derived tumor with a poor prognosis despite multimodal therapy approaches. Lactate has a preponderant role in the tumor microenvironment, playing an immunoregulatory role as well as being a carbon source for tumor growth. Lactate homeostasis depends on the proper functioning of intracellular lactate regulation systems, such as transporters and enzymes involved in its synthesis and degradation, with evidence that an intracellular lactate overload generates metabolic stress on tumor cells and tumor cell death. We propose that the delivery of a lactate overload carried in nanoparticles, allowing the intracellular release of lactate, would compromise the survival of tumor cells. We synthesized and characterized silica and titania nanoparticles loaded with lactate to evaluate the cellular uptake, metabolic activity, pH modification, and cytotoxicity on C6 cells under normoxia and chemical hypoxia, and, finally, determined the survival of an orthotopic malignant glioma model after in situ administration. A dose-dependent reduction in metabolic activity of treated cells under normoxia was found, but not under hypoxia, independent of glucose concentration. Lactated-loaded silica nanoparticles were highly cytotoxic (58.1% of dead cells) and generated significant supernatant acidification. In vivo, lactate-loaded silica nanoparticles significantly increased the median survival time of malignant glioma-bearing rats (p = 0.005) when administered in situ. These findings indicate that lactate-loaded silica nanoparticles are cytotoxic on glioma cells in vitro and in vivo.

Keywords: cytotoxicity; glioma; in situ therapy; lactate-loaded nanoparticle; metabolism.

Figure 1

Silica and titania nanoparticles were efficiently loaded with lactate. (a) Five mg of each (dry weight) of the nanoparticles were analyzed on a spectrophotometer with an ATR detector, obtaining the infrared spectra of the silica nanoparticles (SNP-black line) and the lactate-loaded silica nanoparticles (LSNP-blue line) compared with the lactate spectrum (red line) (upper left panel), and the infrared spectra of the titania nanoparticles (TNP-black line) and the lactate-loaded titania nanoparticles (LTNP-blue line) compared with the lactate spectrum (red line) (lower left panel). (b) Nanoparticles were suspended in PBS at 250 µg/mL and analyzed with the red laser module (532 nm) of the Malvern Nanosight NS300 instrument. Histograms show mean particle size distribution, the black line represents mean size, and red bars show ±1 SEM.

Figure 2

Cumulative lactate release from LSNP. Nanoparticles were resuspended in DMEM and artificial CSF, obtaining samples over 72 h. The percentage of lactate released was calculated by spectrophotometry with FeCl₃ reaction assay. The graph shows the mean ± SEM of 3 independent experiments in triplicate in artificial CSF (blue) and DMEM (red).

Figure 3

Nanoparticle treatment induces the intracellular accumulation of nanometer-sized electron-dense granules. C6 cells were treated with 100 μg/mL of nanoparticles for 24 h, fixed in glutaraldehyde, and processed for visualization by transmission electron microscopy. Representative images of cells are stocked in columns from lower to higher magnification. Key: endoplasmic reticulum (ER), nucleus (N), mitochondria (m), nanoparticles (arrows).

Figure 4

Nanoparticle treatment increases the granularity of C6 cells. Cells were treated with 100 μg/mL of nanoparticles for 24 h and analyzed by flow cytometry, obtaining the mean granularity. (a) Percentage increase in the granularity of treated cells, bars show mean ± SEM of three independent experiments in triplicate, n = 9 (statistical analysis with one-way ANOVA after logarithmic transformation, ** p < 0.001 and *** p < 0.0001 vs. control). Representative dot plots (left) and bright-field images (right) of treated C6 cells: (b) untreated control, (c) SNP, (d) LSNP, (e) TNP, (f) LTNP.

Figure 5

Nanoparticle treatment induces a decrease in metabolic activity in a dose-dependent manner, and LSNP are highly cytotoxic and induce the acidification of the cellular supernatant. C6 cells were treated with 100 μg/mL of nanoparticles for 72 h. (a) Dehydrogenase-dependent metabolic activity was measured by MTT reduction assay. Lines show mean percentage vs. control ± SEM (n = 6). (b) Cytotoxicity was measured by propidium iodide staining. Bars show the mean percentage of dead cells ± SEM (n = 9). (c) The supernatant of treated cells was collected for pH measurement. Lines show mean pH value ± SEM over 96 h (n = 6). (d) Nanoparticles were resuspended in a culture medium for pH measurement. Lines show mean pH value ± SEM over 72 h (n = 6); (statistical analysis with one-way ANOVA after logarithmic transformation for cytotoxicity evaluation, and two-way ANOVA for pH measurement: * p < 0.05 and *** p < 0.0001 vs. control).

Figure 6

The decrease in cell metabolic activity induced by nanoparticles is inhibited during hypoxia. C6 cells were cultured under chemical hypoxia with 320 µM of CoCl₂ and a glucose concentration of 5.5 mM, 17 mM, and 25 mM and treated with 100 μg/mL of nanoparticles for 72 h. Dehydrogenase-dependent metabolic activity was measured by MTT reduction assay. Bars show mean percentage vs. control without CoCl₂ ± SEM of three independent experiments in triplicate, n = 9 (statistical analysis with one-way ANOVA after logarithmic transformation).

Figure 7

LSNP-induced cytotoxicity is inhibited by the interaction between metabolically conditioned tumor cells. C6 cells were cultured under normoxia and hypoxia for 3 weeks, normoxic cells (glucose 5.5 mM + lactate 32mM, blue bars) and hypoxic cells (glucose 25mM + CoCl₂ 320 µM, red bars), and then co-cultured and treated with 100 μg/mL of nanoparticles for 72 h for the evaluation of cytotoxicity with propidium iodide staining. Bars show mean percentage of dead cells ± SEM of two independent experiments in quadruplicate, n = 8 (statistical analysis with one-way ANOVA after logarithmic transformation, * p < 0.001 and ** p < 0.0001 vs. their respective untreated controls).

Figure 8

In situ administration of lactate-silica nanoparticles increases the median survival time in rats bearing orthotopic malignant glioma. Stereotactic implantation of 1 × 10⁶ C6 cells in the right cerebral hemisphere of male Wistar rats; after 7 days they were treated with the intratumoral administration of 1 mg of nanoparticles. Survival follow-up of rats surviving the surgical procedure. Data are shown in a Kaplan–Meier survival curve (statistical analysis by log-rank test, * p = 0.005).