Matrix metalloproteinase 2/9-triggered-release micelles for inhaled drug delivery to treat lung cancer: preparation and in vitro/in vivo studies

Abstract

Background: Improvement in drug accumulation in the lungs through inhalation administration and high expression of MMP2 and MMP9 in lung tumors have both been widely reported.

Methods: MMP2/9-triggered-release micelles were constructed and in vitro and in vivo studies of inhalation administration against lung tumor carried out. Pluronic P123 (P123) was modified with GPLGIAGQ-NH2 (GQ8) peptide to obtain P123-GQ8 (PG). MMP2/9-triggered-release micelles were constructed using PG and succinylated gelatin (SG) and loading paclitaxel (Ptx). To study biodistribution of micelles, DiR encapsulated in micelles was dosed to rats via intravenous injection or inhalation before ex vivo imaging for detecting DiR quantity in lungs. And B16F10 lung cancer-bearing nude mice were chosen as animal models to evaluate in vivo efficacy of MMP2/9-triggered-release micelles.

Results: Ptx-release efficiency from PG-SG-Ptx micelles was MMP2/9-concentration-dependent. For A549 cells, PG-SG-Ptx cytotoxicity was significantly greater (P<0.001) compared to P123-Ptx. Aerosol inhalation was chosen as the method of administration. In biodistribution experiment, DiR quantity in lungs was 5.8%±0.4% of that in major organs, while the ratio was 38.8%±0.5% for inhalation. For B16F10 lung cancer-bearing nude mice, the efficacy of inhalation of PG-SG-Ptx was significantly higher (P<0.001) than Taxol inhalation and injected PG-SG-Ptx. Inhaled PG-SG-Ptx also significantly inhibited the expression of Pgp in lung cancer.

Conclusion: Inhalation of MMP2/9-triggered-release micelles increased tumor sensitivity to chemotherapeutics and reduced the toxicity of chemotherapy to healthy lung cells, which has great potential in lung cancer therapy.

Keywords: P-glycoprotein; Pluronic; inhalation; matrix metalloproteinases; paclitaxel.

Figure 1

Device delivering drug to animal and device testing droplet-size distribution of nebulized micelles.

Figure 2

Characteristics of PG. Notes: (A) ¹H NMR spectra in D₂O: GQ8 (a), P123 (b), PG (c). (B) MALDI-TOF MS: P123 (a), PG (b). (C) Plot of UV absorption of I₂ versus concentration of P123 and PG. When absorbance increased dramatically, the corresponding concentration was the CMC of PG. Values expressed as means (n=3). (D) Western blotting analysis of Pgp expression in B16F10 cells after incubation with P123 and PG at various concentrations. Abbreviations: P123, Pluronic P123; GQ8, GPLGIAGQ-NH₂ peptide; PG, P123 modified with GQ8; NMR, nuclear magnetic resonance; MALDI-TOF MS, matrix-assisted laser desorption–ionization time-of-flight mass spectrometry; CMC, critical micelle concentration.

Figure 3

PG-SG-Ptx micelle formation and drug-release mechanism. Abbreviations: Ptx, paclitaxel; GQ8, GPLGIAGQ-NH₂ peptide; PG, P123 modified with GQ8; SG, succinylated gelatin; PPO, polyoxypropylene; PEO, polyoxyethylene; PG-Ptx, PG micelles loaded with Ptx.

Figure 4

Stability and in vitro release profile of micelles. Notes: (A) Amount of Ptx retained in P123-Ptx micelles, PG-Ptx micelles, and PG-SG-Ptx micelles in 48 hours. The percentage of Ptx remaining in the micelles indicated stability. (B) Ptx-release profile of PG-SG-Ptx micelles incubated with different mediums at 37°C. Values expressed as means ± SD (n=3). Abbreviations: P123, Pluronic P123; Ptx, paclitaxel; PG, P123 modified with GPLGIAGQ-NH₂; SG, succinylated gelatin; P123-Ptx, P123 micelles loaded with Ptx.

Figure 5

Cytotoxicity of micelles on cells and cellular uptake of micelles. Notes: (A) Cytotoxicity of placebo P123, PG, PG-SG and SG on HUVECs; (B) cytotoxicity of Ptx-loaded P123 micelles and PG-SG micelles on A549 cells; (C) cellular uptake of P123-Cou6 and PG-SG-Cou6 micelles in A549 cells; (D) cellular uptake of P123-Cou6 and PG-SG-Cou6 micelles in MCF7 and MCF7/ADR cells in the presence of GM6001. ^**P<0.01, ^***P<0.001 compared with P123-Ptx. Values expressed as means ± SD (n=3). Abbreviations: P123, Pluronic P123; PG, P123 modified with GPLGIAGQ-NH₂; SG, succinylated gelatin; Ptx, paclitaxel; PG-SG, micelles composed of PG and SG; P123-Ptx, P123 micelles loaded with Ptx; Cou6, coumarin 6; PG-SG-Cou6, PG/SG micelles loaded with coumarin 6; HUVECs, human umbilical vein endothelial cells.

Figure 6

Histopathological analysis of inhalation toxicity. Notes: H&E staining of lung section isolated at 1 hour after injection with 1% Pluronic P123 solution (B), inhalation of saline (C) and inhalation of 1% P123 solution (D), normal lung section (A) as control.

Figure 7

Biodistribution of intravenous injection and inhalation of micelles in rats. Notes: (A) Dissected organs of rats killed at 1, 6, and 12 hours after inhalation and intravenous injection of DiR-labeled P123 micelles; (B) distribution of DiR in the lung after inhalation of DiR-labeled P123 micelles; (C) ratio of fluorescence intensity in the lung to all organs (heart, liver, spleen, lung, and kidney) after inhalation and intravenous injection of DiR-labeled P123 micelles; (D) fluorescence intensity in the lung after inhalation and intravenous injection of DiR-labeled P123 micelles. ^***P<0.001 compared with inhalation. Values expressed as means ± SD (n=3). Abbreviation: P123, Pluronic P123.

Figure 8

Antitumor efficacy against melanoma pulmonary metastatic tumor-bearing nude mice. Notes: (A) Lung-mass weight of each treatment group. ^**P<0.01, ^***P<0.001 compared with saline; ^∆P<0.001 compared with Ptx injection; ^#P<0.001 compared with intravenous (IV) PG-SG-Ptx. (B) Body-weight changes as a function of time. Values expressed as means ± SD (n=6). (C) Lung sections isolated on Day 14 after treatment with different Ptx formulations and stained with H&E. Original magnification 4×. Locations of tumors marked with arrowheads. (D) Western blotting analysis: MMP2/9 protein expression in the lungs. (E) Pgp expression in the lungs after inhalation with different Ptx formulations: saline, Ptx injection, P123-Ptx, PG-SG-Ptx. Abbreviations: P123, Pluronic P123; Ptx, paclitaxel; SG, succinylated gelatin; P123-Ptx, P123 micelles loaded with Ptx; GAPDH, phosphoglyceraldehyde dehydrogenase.