Multi-Smart and Scalable Bioligands-Free Nanomedical Platform for Intratumorally Targeted Tambjamine Delivery, a Difficult to Administrate Highly Cytotoxic Drug

Abstract

Cancer is one of the leading causes of mortality worldwide due, in part, to limited success of some current therapeutic approaches. The clinical potential of many promising drugs is restricted by their systemic toxicity and lack of selectivity towards cancer cells, leading to insufficient drug concentration at the tumor site. To overcome these hurdles, we developed a novel drug delivery system based on polyurea/polyurethane nanocapsules (NCs) showing pH-synchronized amphoteric properties that facilitate their accumulation and selectivity into acidic tissues, such as tumor microenvironment. We have demonstrated that the anticancer drug used in this study, a hydrophobic anionophore named T21, increases its cytotoxic activity in acidic conditions when nanoencapsulated, which correlates with a more efficient cellular internalization. A biodistribution assay performed in mice has shown that the NCs are able to reach the tumor and the observed systemic toxicity of the free drug is significantly reduced in vivo when nanoencapsulated. Additionally, T21 antitumor activity is preserved, accompanied by tumor mass reduction compared to control mice. Altogether, this work shows these NCs as a potential drug delivery system able to reach the tumor microenvironment, reducing the undesired systemic toxic effects. Moreover, these nanosystems are prepared under scalable methodologies and straightforward process, and provide tumor selectivity through a smart mechanism independent of targeting ligands.

Keywords: amphoteric nanocapsules; lung cancer treatment; pH-tunable; polymer nanocapsules; targeted drug delivery systems; tumor microenvironment.

Scheme 1

Steps involved in the preparation of the polymer. THF: tetrahydrofuran. rt: room temperature.

Scheme 2

Graphic representation of the nanoencapsulation process.

Figure 1

Characterization of the nanocapsules. (A,B) representative TEM micrographs (scale bar: 100 nm); (C) particle size distributions by DLS; (D) statistical analysis of different NCs.

Figure 2

Zeta potential measurements. (A) Zeta potential values of 4 samples of NCs and (B) comparative analysis to a control NCs (NC-DiO-AN).

Figure 3

Internalization of NCs by confocal microscopy. A549 cells were incubated with NC-DiO (green) for 1, 8, 16, 24, and 48 h, fixed and incubated with LysoTracker (red) to dye the late endosomes and lysosomes. Merge images show colocalization between NCs and acidic organelles (yellow). Scale bar 30 µm.

Figure 4

Study of cell viability after NC-T21 treatment dependent on pH. A549 and LLC1 cells were incubated with increasing concentrations of encapsulated T21 (NC-T21) or empty nanocapsules (NC) at pH 6.8 (blue) or 7.8 (black and grey) for 24, 48, and 72 h. (A). Cell viability was measured by triplicated MTT assay obtaining cell viability curves where IC₅₀ value was extrapolated. (B) Comparison of the IC₅₀ values. * p < 0.05. (C) IC₅₀ values (µg/mL ± SD), obtained from dose-response curves.

Figure 5

Cell viability in non-tumorigenic cells after treatment with free or encapsulated T21. Non-tumorigenic MCF10A cells were incubated with increasing concentrations of free (T21) and encapsulated T21 (NC-T21) or empty nanocapsules (NC) at pH 7.8 for 24, 48, and 72 h. (A) Cell viability was measured by MTT assay obtaining cell viability curves where IC₅₀ was extrapolated. (B) Comparison of the IC₅₀ values. * p < 0.05. (C) IC₅₀ values (µg/mL ± SD), obtained from dose-response curves.

Figure 6

Effect of pH on cell death. A549 cells were incubated with increasing concentrations of (A) free (T21) or encapsulated T21 (NC-T21), (B) cisplatin, and (C) staurosporine at pH 6.8 and 7.8 for 24 h. Dead cells were counted by trypan blue staining, average ± SD of 3 independent experiments is represented. ** p > 0.01, **** p > 0.0001.

Figure 7

Nanocapsules uptake by flow cytometry. A549 cells were incubated with DiO loaded amphoteric (NC-DiO) or anionic (NC-DiO-AN) NCs at pH 6.8 or pH 7.8 for 1, 15, 24, 48, and 72 h. Fluorescence intensity was measured by flow cytometry. (A) Mean intensity fluorescence is represented ± SD of three independent experiments. (B) Mean intensity fluorescence ratio at pH 6.8 respect to pH 7.8 is represented. **** p > 0.0001; ** p > 0.01; * p > 0.05 ns: not significant.

Figure 8

Nanocapsules tissue biodistribution. Mice were administered i.v. with NC-DiR or NC-DiR-T21.Nanoparticle tissue accumulations were determined by ex vivo DiR fluorescence imaging monitoring 72 h post-injection. (A) Representative images of the different organs and tumors analyzed. (B) Percentage of biodistribution among the indicated tissues after injecting NC-DiR or NC-DiR-T21 nanoparticles. Graph represents the average of total REU/area/tissue ± SD.

Figure 9

Effect of treatment in mouse growth and organ weight. Mice were administered i.v. with 6 mg/kg or 3 mg/kg of nanoencapsulated T21 (NC-T21), PBS (V), empty nanocapsules without T21 (NC), 6 mg/kg of free T21 (T21) administered i.p. or PBS 7.5% DMSO and 0.8% Tween (Vip). The mice received 10 i.v. doses in a twice a week regimen or every other day for i.p administration. Mouse weight was monitored during treatment; (A) Percentage of weight variation during treatment and (B) relative organs weight are represented.

Figure 10

Analysis of the tolerability and therapeutic effect of NC-T21 treatment. Lung tumors were induced in mutant mice by virus inhalation, as described in Materials and Methods. 12 weeks after, mice were treated twice a week, with 6 mg/kg of nanoencapsulated T21 (NC-T21); with PBS (V), with empty nanocapsules without T21 (NC) or with free T21 (T21). (A) Mice weight was monitored during treatment. (B) Macroscopic differences in epiploon, seminal vesicle, and liver were observed in mice treated with T21. Scale bar 1 cm (C) Organs and (E) Lungs were weighed at final point and relativized to mice total weight. (D) Pictures of hematoxylin/eosin staining of lung tumors were taken. Scale bar 0.5 mm. ** p > 0.01, *** p > 0.001.