Endogenous lung surfactant inspired pH responsive nanovesicle aerosols: pulmonary compatible and site-specific drug delivery in lung metastases

Abstract

Concerns related to pulmonary toxicity and non-specificity of nanoparticles have limited their clinical applications for aerosol delivery of chemotherapeutics in lung cancer. We hypothesized that pulmonary surfactant mimetic nanoparticles that offer pH responsive release specifically in tumor may be a possible solution to overcome these issues. We therefore developed lung surfactant mimetic and pH responsive lipid nanovesicles for aerosol delivery of paclitaxel in metastatic lung cancer. 100-200 nm sized nanovesicles showed improved fusogenicity and cytosolic drug release, specifically with cancer cells, thereby resulting in improved cytotoxicity of paclitaxel in B16F10 murine melanoma cells and cytocompatibility with normal lung fibroblasts (MRC 5). The nanovesicles showed airway patency similar to that of endogenous pulmonary surfactant and did not elicit inflammatory response in alveolar macrophages. Their aerosol administration while significantly improving the biodistribution of paclitaxel in comparison to Taxol (i.v.), also showed significantly higher metastastes inhibition (~75%) in comparison to that of i.v. Taxol and i.v. Abraxane. No signs of interstitial pulmonary fiborisis, chronic inflammation and any other pulmonary toxicity were observed with nanovesicle formulation. Overall, these nanovesicles may be a potential platform to efficiently deliver hydrophobic drugs as aerosol in metastatic lung cancer and other lung diseases, without causing pulmonary toxicity.

Figure 1. Illustrative schematic representing aerosol delivery of paclitaxel loaded lipid nanovesicles and their pH responsive release in cancer cell cytosol.

Figure 2. Physiochemical characterization of paclitaxel loaded nanovesicles.

(a) Size distribution of LN-PTX obtained using dynamic light scattering (DLS) (b) Transmission electron microscopy (TEM) image of LN-PTX. Scale bar: 200 nm. (c) Encapsulation and loading efficiency of paclitaxel in LN-PTX, and their zeta potential. (d) In vitro release of paclitaxel from LN-PTX at 37°C temperature and different pH conditions. *p < 0.05 in comparison to pH 7.4.

Figure 3. Airway patency and aerodynamic behavior of aerosolized nanovesicles (LN-PTX).

(a) Airway patency of LN-PTX, Taxol and Abraxane measured in terms of percentage opening time of the capillary using a Capillary Surfactometer. *p < 0.05 in comparison to other groups. (b) Percentage deposition of paclitaxel in different stages of twin impinger as a result of 1 min nebulization of LN-PTX. *p < 0.05 in comparison to deposition in throat and stage I.

Figure 4. pH dependent membrane fusogenicity of nanovesicles.

(a) Change in surface pressure (Δπ) of DPPC monolayer as a result of exogenous addition of LN-PTX measured at 37°C and different pH conditions. *p < 0.05 in comparison to pH 7.4; **p < 0.05 in comparison to pH 6.0. (b) Change in surface pressure (Δπ) of DPPC monolayer as a result of exogenous addition of DPPC-PTX measured at 37°C and different pH conditions. (c) Change in surface pressure (Δπ) of DPPC monolayer as a result of exogenous addition of Taxol measured at 37°C and different pH conditions.

Figure 5. In vitro cytotoxicity, cellular uptake and immunogenic potential of nanovesicles.

(a) Percentage cell viability of B16F10 cells following incubation with different concentrations of LN-PTX, LN-B, Taxol and Abraxane. *p < 0.05 in comparison to other groups. (b) 48 h IC₅₀ values for LN-PTX, Taxol and Abraxane in B16F10 cells. *p < 0.05 in comparison to other groups. (c) CLSM images of B16F10 cells after incubation with Rh-6G loaded nanovesicles for different time points. (100 ×) (d) CLSM images of cells incubated with Rh-6G loaded nanovesicles for 3 h in Z scan mode with the scanning done from −10 μm to +10 μm. (e) Cellular levels of Rh-6G after incubation of B16F10 cells with Rh-6G loaded nanovesicles under normal and ATP depleted conditions. *p < 0.05 in comparison to 37°C + 0.1% sodium azide and 4°C. (f) Comparison of intracellular uptake of paclitaxel loaded nanovesicles (LN-PTX) by B16F10 and MRC 5 cells. *p < 0.05. (g) Levels of TNF-α in cell supernatant in response to LN-B, LPS and TCP. *p < 0.05 in comparison to LPS. (h) Levels of IL-1β in cell supernatant in response to LN-B, LPS and TCP. *p < 0.05 in comparison to LPS. (i) Levels of IL-6 in cell supernatant in response to LN-B, LPS and TCP. *p < 0.05 in comparison to LPS.

Figure 6. Biodistribution of aerosolized nanovesicles (LN-PTX).

(a) Paclitaxel accumulation in lungs.*p < 0.05 in comparison to corresponding time points for Taxol. (b) Paclitaxel accumulation in liver. *p < 0.05 in comparison to corresponding time points for Taxol. (c) Paclitaxel accumulation in spleen. *p < 0.05 in comparison to corresponding time points for Taxol. (d) Paclitaxel concentration in plasma. *p < 0.05 in comparison to corresponding time points for Taxol.

Figure 7. In vivo metastases inhibition potential and pulmonary toxicity.

(a) Average lung weights of different groups as observed after sacrifice. n = 6; *p < 0.05 in comparison to control group; **p < 0.05 in comparison to Taxol and Abraxane. (b) Percentage inhibition of pulmonary metastasis for different groups. n = 6; *p < 0.05 in comparison to Taxol and Abraxane. (c) Number of metastatic tumor nodules as observed in different groups upon sacrifice. n = 6; *p < 0.05 in comparison to control group; **p < 0.05 in comparison to Taxol and Abraxane. (d) Percentage area of lung occupied by tumor nodules as observed in different groups upon sacrifice. n = 6; *p < 0.05 in comparison to control group; **p < 0.05 in comparison to Taxol and Abraxane. (e) Kaplan-Meir survival curves of animals from different treatment groups. (f) Histopathology images (20×) of hemtoxylin and eosin (H&E) stained lung tissue sections from different groups. Scale bar: 200 μm.