Ciprofloxacin Derivative-Loaded Nanoparticles Synergize with Paclitaxel Against Type II Human Endometrial Cancer

Abstract

Combinations of chemotherapeutic agents comprise a clinically feasible approach to combat cancers that possess resistance to treatment. Type II endometrial cancer is typically associated with poor outcomes and the emergence of chemoresistance. To overcome this challenge, a combination therapy is developed comprising a novel ciprofloxacin derivative-loaded PEGylated polymeric nanoparticles (CIP2b-NPs) and paclitaxel (PTX) against human type-II endometrial cancer (Hec50co with loss of function p53). Cytotoxicity studies reveal strong synergy between CIP2b and PTX against Hec50co, and this is associated with a significant reduction in the IC₅₀ of PTX and increased G2/M arrest. Upon formulation of CIP2b into PEGylated polymeric nanoparticles, tumor accumulation of CIP2b is significantly improved compared to its soluble counterpart; thus, enhancing the overall antitumor activity of CIP2b when co-administered with PTX. In addition, the co-delivery of CIP2b-NPs with paclitaxel results in a significant reduction in tumor progression. Histological examination of vital organs and blood chemistry was normal, confirming the absence of any apparent off-target toxicity. Thus, in a mouse model of human endometrial cancer, the combination of CIP2b-NPs and PTX exhibits superior therapeutic activity in targeting human type-II endometrial cancer.

Keywords: PEGylated polymeric nanoparticles; ciprofloxacin derivative; endometrial cancer; nanomedicine; paclitaxel.

Figure 1.. Physicochemical properties of ciprofloxacin derivative (CIP2b).

(a) Chemical structures of ciprofloxacin (CIP) and its derivative (CIP2b). (b) UV-VIS spectrum shows that CIP has one UV maximum at 281 nm while CIP2b has two UV maxima at 247 and 281 nm. (c) and (d) FT-IR spectra of CIP and CIP2b, respectively. Compared to CIP, CIP2b has one extra functional group of C-Cl in the fingerprint region (piperazine group). (e) and (f) FT-IR frequency range and functional groups of CIP and CIP2b, respectively. (g) Solubility of CIP2b in organic solvents and solubilizers (data are plotted as means ± SD, n=3). (h) Solubility of CIP2b in aqueous buffered solutions at different pH where CIP2b solubility gradually decreased from pH 2 to pH 6, then started to gradually increase from pH 7 and showed a peak at pH 10 (data are plotted as means ± SD, n=3). (i) Solubility (Log S)–pH profile of CIP2b. The experimental intrinsic solubility (log So) was found to be –0.26 (0.5 μg/mL) and this value is lower than the solubility predicted by ChemAxon. Two pKa values were found to be 5.4 and 6.0. There was another inflection point near pH 12 which could be another potential pKa.

Figure 2.. In vitro antitumor activity of combined CIP2b and PTX treatment against Hec50co endometrial cancer cells.

a-c) Cytotoxicity assays of Hec50co cells following treatment for 72 hours with: a) different concentrations of PTX and fixed concentrations of CIP2b (10, 25, and 50 μM) (n=4); b) different concentrations of CIP2b, and fixed concentrations of PTX (1, 5, and 10 nM) (n=4); and c) different concentrations of PTX and a fixed concentration (10 μM) of either CIP2b or CIP (n=4). d) The synergy between CIP2b and PTX is calculated using the Combination Index (CI) method, with a fixed concentration of PTX (5 nM) being added to CIP2b (CI values less than 1 indicate synergy). e) The effect of the addition of 10 μM CIP2b to 1, 5, and 10 nM of PTX for 72 h (n=4). f) The effect of the addition of 5 nM PTX to 1, 10, and 25 μM of CIP2b for 72 h (n=4). g) IC₅₀ values for PTX following the addition of 10, 25, and 50 μM of CIP2b (n=4). h) IC₅₀ values for CIP2b following the addition of 1, 5, and 10 nM of PTX (n=4). i) Distribution of cells in each stage of the cell cycle after receiving the indicated treatment for 24 hours. j) Histograms of Hec50co cells with indicated treatments representing cell cycle phases following the cell cycle analysis using ModFit LT (ver. 5.0.9, Verity Software House) (n=1). Statistical analysis was carried out by one-way ANOVA followed by Tukey’s post-hoc test; *, ***, and **** indicate p<0.05, p<0.001, and p<0.0001, respectively; ns, not significant.

Figure 3.. Development and characterization of CIP2b-loaded NPs.

a) Proposed structure of CIP2b-loaded NPs (CIP2b NPs) with an outer shell composed of PLGA and coated with TPGS as a surfactant. b) Scanning electron photomicrograph showing the shape and morphology of CIP2b-loaded NPs. Scale bar = 100 nm. c) Table showing properties of CIP2b-loaded NPs (n=3). d) Particle size distribution of CIP2b NPs. e) Zeta potential distribution of CIP2b NPs. f) Cumulative in vitro release of CIP2b from NPs in release medium containing PBS and 0.1% w/v Tween-80 for 10 days (n=3). g) Hec50co intracellular accumulation of CIP2b when administered in either soluble form or as CIP2b-loaded NPs (n=3). Statistical analysis was carried out using a two-tailed Student’s t-test (*** indicates p<0.001). h) Quantitation of flow cytometric analysis of the intracellular accumulation of PTX-OG (400 nM) in Hec50co cells in the presence or absence of either CIP2b-loaded NPs (equivalent to 4 μM CIP2b) or blank NPs (equivalent to the amount of CIP2b-loaded NPs used) (n=3). i) Representative flow cytometric histograms of intracellular accumulation of PTX-OG by Hec50co cells. j) Cytotoxicity assay following the treatment of Hec50co cells with different concentrations of PTX, and fixed concentrations of either CIP2b-loaded NPs (equivalent to 0.1 or 0.5 μM of CIP2b) or blank NPs (equivalent to the amount of CIP2b-loaded NPs used) for 72 hours (n=4). k) The effect of adding CIP2b-loaded NPs (equivalent to 0.1 or 0.5 μM CIP2b) or blank NPs (equivalent to the amount of CIP2b-loaded NPs used) to 0.1 nM PTX for 72 hours (n=4). l) Results from a cytotoxicity assay following the treatment of Hec50co cells with indicated concentrations of CIP2b NPs or blank NPs (equivalent to the amount of CIP2b NPs used) for 72 h (n=4). m) Distribution of cells in each stage of the cell cycle after receiving the indicated treatment for 24 hours. n) Cell cycle analysis of Hec50co cells treated with either 10 nM PTX + CIP2b NPs (0.5 μM CIP2b), or 10 nM PTX + blank NPs (equivalent to the amount used for the CIP2b NP treatment). Statistical analyses were performed using either one-way or two-way ANOVA followed by Tukey’s post-hoc test unless noted otherwise. **, ***, and **** indicate p<0.01, p<0.001, and p<0.0001, respectively.

Figure 4:. Pharmacokinetic and biodistribution profiles of CIP2b and CIP2b NPs in mice.

a) Pharmacokinetics following IV injection of CIP2b solution or CIP2b NPs (75 μg/mouse) in female BALB/c mice for up to 24 hours (n=3 mice/time point, data represent mean ± SD). b) close-up view of the pharmacokinetic profile represented in the dashed rectangle in a). c) Pharmacokinetic parameters following non-compartmental analysis (PK Solver) following IV injection of CIP2b solution or CIP2b NPs (75 μg/mouse) in female BALB/c mice for up to 24 hours (n=3 mice/time point, data represent mean ± SD). Biodistribution profiles fitted to 1-compartment model following the IV injection of CIP2b solution or CIP2b NPs (75 μg/mouse) in female BALB/c mice for up to 24 hours (n=3 mice/time point) in d) lungs, e) livers, f) kidneys, g) hearts, h) spleens, and i) pancreas of mice (PK Solver). j) Areas under the curve at 0-t (AUC 0-t) and 0-inf (AUC 0-inf) in lungs, livers, kidneys, hearts, spleens, and pancreas obtained following 1-compartmental fitting of organ concentrations-time profiles represented in d) to i).

Figure 5.. Antitumor efficacy of PTX + CIP2b.

a) Hec50co tumor progression in athymic Nu/Nu mice injected IV with indicated treatment (n=6–7). b) Body weight monitoring of the mice following their IV treatment with PBS, PTX + blank NPs, PTX + CIP2b solution, or PTX + CIP2b NPs. c) Photographic image of representative tumors collected from mice treated with IV treatments. d) Average tumor weights following mice euthanasia and tumor collection from mice treated with the previously mentioned IV treatments (n=6–7). e) Intra-tumoral accumulation of CIP2b following the IV injection of either CIP2b solution or CIP2b NPs in Hec50co tumor-bearing athymic Nu/Nu mice (n=3). f) Immunohistochemistry analysis of specimens stained with H&E, or antibodies against CD31 (indicating vascular endothelial cells), or ki-67 (indicating proliferating cells). Yellow arrows point to either the necrotic areas (H&E) or where antibodies showed abundant binding to their specific target. Scale bar = 50 μm. Statistical analyses were carried out by one-way ANOVA followed by Tukey’s post-hoc test or a two-tailed Student’s t-test. * and ** indicate p<0.05 and p<0.01, respectively; ns, not significant.

Figure 6.. In vivo toxicity profile of CIP2b + PTX combination.

a) Histological evaluation of major vital organs following the IV administration of PBS, PTX + blank NPs, PTX + CIP2b solution, or PTX + CIP2b NPs; H&E staining was performed on sections from the organs collected from representative mice from each group. Scale bar = 50 μm. b) Serum parameters measured in athymic Nu/Nu mice following IV injection of PBS, PTX + blank NPs, PTX + CIP2b solution, or PTX + CIP2b NPs. Statistical analysis was carried out by one-way ANOVA followed by Tuckey’s post-hoc test (n = 3 mice selected randomly from each group, ‘ns’ indicates not significant).

Figure 7.

Graphical representation of the proposed combination therapy to treat endometrial cancer. CIP2b which was found to strongly synergize with PTX against resistant endometrial cancer cells was encapsulated into PEGylated NPs to provide longer circulation and enhanced tumor accumulation. When given with PTX, these NPs provided higher tumor accumulation of CIP2b, which in turn augmented the intracellular cytotoxic activity of PTX. This proposed treatment may lead to the successful reduction of the commonly used PTX doses without compromising its activity.