In vitro and in vivo anti-tumor activities of a gemcitabine derivative carried by nanoparticles - PubMed (original) (raw)

Comparative Study

In vitro and in vivo anti-tumor activities of a gemcitabine derivative carried by nanoparticles

Brian R Sloat et al. Int J Pharm. 2011.

Abstract

Gemcitabine (Gemzar(®)) is the first line treatment for pancreatic cancer and often used in combination therapy for non-small cell lung, ovarian, and metastatic breast cancers. Although extremely toxic to a variety of tumor cells in culture, the clinical outcome of gemcitabine treatment still needs improvement. In the present study, a new gemcitabine nanoparticle formulation was developed by incorporating a previously reported stearic acid amide derivative of gemcitabine into nanoparticles prepared from lecithin/glyceryl monostearate-in-water emulsions. The stearoyl gemcitabine nanoparticles were cytotoxic to tumor cells in culture, although it took a longer time for the gemcitabine in the nanoparticles to kill tumor cells than for free gemcitabine. In mice with pre-established model mouse or human tumors, the stearoyl gemcitabine nanoparticles were significantly more effective than free gemcitabine in controlling the tumor growth. PEGylation of the gemcitabine nanoparticles with polyethylene glycol (2000) prolonged the circulation of the nanoparticles in blood and increased the accumulation of the nanoparticles in tumor tissues (>6-fold), but the PEGylated and un-PEGylated gemcitabine nanoparticles showed similar anti-tumor activity in mice. Nevertheless, the nanoparticle formulation was critical for the stearoyl gemcitabine to show a strong anti-tumor activity. It is concluded that for the gemcitabine derivate-containing nanoparticles, cytotoxicity data in culture may not be used to predict their in vivo anti-tumor activity, and this novel gemcitabine nanoparticle formulation has the potential to improve the clinical outcome of gemcitabine treatment.

Copyright © 2011 Elsevier B.V. All rights reserved.

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Figures

Fig. 1

Fig. 1. Preparation and characterization of GemC18-NPs

(A). In GPC, GemC18-free NPs (○) and GemC18-NPs (□) eluted about two fractions earlier than GemC18 in Tween 20 micelles (□). The concentration of the GemC18 in the micelles and GemC18-NPs was 100 μg/mL. (B). Gel permeation chromatographs of GemC18-NPs prepared with 0, 0.1, 0.5, 1, 2.5, and 5 mg/mL of GemC18. In A & B, gemcitabine was measured at 248 nm. (C). TEM micrograph of the GemC18-NPs (with 5 mg/mL of GemC18). (D). Chromatographs of GemC18-NPs (●) and PEGylated GemC18-NPs (△) prepared with 5 mg/mL of GemC18. (E). The size and zeta potential of the GemC18-NPs and the PEG-GemC18-NPs (F). The dynamic light scattering spectra of the GemC18-in-Tween 20 micelles (left), GemC18-NPs, and PEG-GemC18-NPs (far right) overlaid. (G). The release of the GemC18 from the GemC18-NPs (●) or PEG-GemC18-NPs (△). (H). The release or hydrolysis of the gemcitabine from the GemC18-NPs when incubated in PBS, mouse serum, or human serum (values in the Y-axis are natural log product). (I). The size of the GemC18-NPs and PEG-GemC18-NPs after 30 min of incubation at 37°C in FBS in normal saline. Except in C and F, all data presented were the mean from at least 3 independent determinations. Standard deviations were not included in some figures for clarity.

Fig. 2

Fig. 2. The uptake of GemC18-NPs by TC-1 tumor cells in culture

(A). Fluorescence micrographs. Cells were incubated with fluorescein-labeled GemC18-NPs for 6 h at 37°C or 4°C and observed under a bright-field microscope (left panel) or a fluorescence microscope (right panel). Photos were taken at 20 × magnification. (B). Comparison of the uptakes of PEGylated and un-PEGylated GemC18-NPs. * p < 0.001, PEG-GemC18-NPs vs. GemC18-NPs at 37°C.

Fig. 3

Fig. 3. GemC18-NPs were cytotoxic to tumor cells in culture

(A). The IC50 values of gemcitabine, GemC18-NPs, and PEG-GemC18-NPs in TC-1 and BxPC-3 cells. Cells were incubated with gemcitabine HCl or nanoparticles for 48 h. * For both cell lines, p < 0.05, Gemcitabine vs. GemC18-NPs. (B). It took the GemC18-NPs a longer time than the gemcitabine HCl to kill tumor cells. TC-1 cells were incubated with gemcitabine HCl or GemC18-NPs at 28.7 nM for 24 or 48 h, and the % of surviving cells was determined. Data are mean ± S.D. (n = 3-4).

Fig. 4

Fig. 4. In vivo and ex vivo imaging of GemC18-NPs and PEG-GemC18-NPs

(A). IVIS images of athymic mice 24 h after injection of fluorescein-labeled GemC18-NPs or PEG-GemC18-NPs. (B). Relative fluorescence intensity values in BxPC-3 tumors (circular ROI in A). a p = 0.0006, GemC18-NPs vs. PEG-GemC18-NPs. (C). Tissue distribution of fluorescein-labeled GemC18-NPs and PEG-GemC18-NPs 24 h after injection. b GemC18-NPs vs. PEG-GemC18-NPs, p = 0.003, 0.021, and 0.002 for blood, liver, and spleen, respectively.

Fig. 5

Fig. 5. In vivo anti-tumor activity of the GemC18-NPs against TC-1 tumors in C57BL/6 mice

(A). TC-1 tumor growth curves in C57BL/6 mice. Tumor cells were implanted on day 0. On days 4 and 13, mice (n = 4) were i.v. injected with GemC18-NPs, gemcitabine HCl, or sterile mannitol. Data reported are mean ± S.D. * The values of Gemcitabine and GemC18-NPs were different starting from day 8 (p < 0.05). This experiment was repeated 3 times to confirm the anti-tumor activity of the GemC18-NPs, and similar result was obtained. (B). Photographs of TC-1 tumors 21 days after tumor cell injection. (C). (Immuno)histograms of TC-1 tumors after treatment with gemcitabine HCl or GemC18-NPs. CAS3, caspase 3 staining.

Fig. 6

Fig. 6. In vivo anti-tumor activity of GemC18-NPs against BxPC-3 tumors in athymic mice

(A). BxPC-3 tumor growth curves. Tumor cells were seeded on day 0, and mice were i.v. injected on days 6 and 19. (B). Average weight of BxPC-3 tumor-bearing mice after different treatments. * p = 0.0007 (ANOVA on week 3). (C). GemC18-free nanoparticles lack anti-tumor activity. BxPC-3 cells were seeded on day 0, and mice were i.v. injected once on day 4. NPs, GemC18-free nanoparticles.

Fig. 7

Fig. 7. Comparison of the in vivo anti-tumor activities of GemC18-NPs and PEGylated GemC18-NPs

(A). TC-1 tumors in C57BL/6 mice. Mice (n = 5-7) were injected (i.v.) with GemC18-NPs or PEG-GemC18-NPs once (1 mg GemC18 per mouse). (B). BxPC-3 tumors in athymic mice. Mice (n = 5) were injected (i.v.) with GemC18-NPs or PEG-GemC18-NPs 3 times (days 0, 12, and 21). In A&B, tumor sizes were reported starting from the day of the injection of the nanoparticles. (C). TC-1 tumors in C57BL/6 mice. The nanoparticles were injected peritumorally (0.25 mg of GemC18 per mouse at each injection). Data shown are mean ± S.E.M. Statistical analysis did not reveal any differences between the GemC18-NPs and PEG-GemC18-NPs in A, B, and C.

Fig. 8

Fig. 8. The GemC18-NPs were more effective than the GemC18-in-Tween 20 micelles

PEG-GemC18-NPs or GemC18-in-Tween 20 micelles were injected twice a week for 5 times (150 μg of GemC18 per mouse). *, p < 0.05, PEG-GemC18-NPs vs. GemC18-in-Tween 20 micelles starting on day 12. Data shown are mean ± S.E.M.

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