Inhalable oridonin-loaded poly(lactic- co-glycolic)acid large porous microparticles for in situ treatment of primary non-small cell lung cancer

Abstract

Non-small cell lung cancer (NSCLC) accounts for about 85% of all lung cancers. Traditional chemotherapy for this disease leads to serious side effects. Here we prepared an inhalable oridonin-loaded poly(lactic-co-glycolic)acid (PLGA) large porous microparticle (LPMP) for in situ treatment of NSCLC with the emulsion/solvent evaporation/freeze-drying method. The LPMPs were smooth spheres with many internal pores. Despite a geometric diameter of ~10 µm, the aerodynamic diameter of the spheres was only 2.72 µm, leading to highly efficient lung deposition. In vitro studies showed that most of oridonin was released after 1 h, whereas the alveolar macrophage uptake of LPMPs occurred after 8 h, so that most of oridonin would enter the surroundings without undergoing phagocytosis. Rat primary NSCLC models were built and administered with saline, oridonin powder, gemcitabine, and oridonin-loaded LPMPs via airway, respectively. The LPMPs showed strong anticancer effects. Oridonin showed strong angiogenesis inhibition and apoptosis. Relevant mechanisms are thought to include oridonin-induced mitochondrial dysfunction accompanied by low mitochondrial membrane potentials, downregulation of BCL-2 expressions, upregulation of expressions of BAX, caspase-3 and caspase-9. The oridonin-loaded PLGA LPMPs showed high anti-NSCLC effects after pulmonary delivery. In conclusion, LPMPs are promising dry powder inhalations for in situ treatment of lung cancer.

Keywords: BSA, bovine serum albumin; DAB, 3,3ʹ-diaminobenzidine; DAPI, 4ʹ,6-diamidino-2-phenylindole; DPI, dry powder inhalation; EGFR, epidermal growth factor receptor; FPF, fine particle fraction; HPLC, high performance liquid chromatography; HRP, horseradish peroxidase; LPMP, large porous microparticle; Large porous microparticle; NSCLC, non-small cell lung cancer; Non-small cell lung cancer; Oridonin; PLGA, poly(lactic-co-glycolic)acid; PVA, polyvinyl alcohol; Poly(lactic-co-glycolic)acid; Pulmonary delivery; SEM, scanning electron microscopy; SLF, simulated lung fluid; TCM, traditional Chinese medicine; XRD, X-ray diffraction; qPCR, quantitative polymerase chain reaction.

Graphical abstract

Figure 1

SEM images of oridonin-loaded LPMPs (A) and grinded LPMPs (B), and the oridonin release profile from the PLGA LPMPs and the corresponding SEM images of microparticles (C). The LPMPs have smooth sphere surfaces with many pores on the surfaces and cavities in the inner spaces. Oridonin is rapidly released from the LPMPs and the microparticles show time-dependent erosions.

Figure 2

Lung deposition of microparticles shown by the 2D images (a), 3D images (b), and lung tissue section images (c, 100×) of conventional intact Cy7-loaded PLGA microparticles (A) and Cy7-loaded PLGA LPMPs (B) after pulmonary administration to the rats for 2 h. The arrows and circle in the images (c) show the deposited microparticles in lung tissues.

Figure 3

Lung appearances and CT images of the lung cancer rats treated with saline (A), oridonin powders (B), gemcitabine (C), and oridonin-loaded LPMPs (D) after pulmonary delivery. The light points indicate the tumor nodes shown in the CT images.

Figure 4

CD31 expressions in the primary NSCLC tissues from rats treated with saline (A), oridonin powders (B), gemcitabine (C), and oridonin-loaded LPMPs (D) via pulmonary delivery. The arrows indicate the CD31 expressions shown with brown points.

Figure 5

Apoptosis of NSCLC cells and pathological sections of the lung tissues of rats treated with saline (A), oridonin powders (B), gemcitabine (C), and oridonin-loaded LPMPs (D) after pulmonary delivery. Apoptosis is indicated by tunel staining (400×). The nuclei are shown by DAPI staining (400×). The merged images of tunel and DAPI staining show the apoptosis in the NSCLC cells. Hematoxylin and eosin (H&E) staining (100×) shows the states of NSCLC cells.

Figure 6

Apoptosis related expressions in the NSCLC cells. Flow cytometric graphs of mitochondrial membrane potentials (MMPs) (A); regulation of pro-apoptotic protein BAX and anti-apoptotic protein BCL-2 levels (B); and stimulation of caspase-3 and caspase-9 mRNA (C) in the NSCLC cells. Columns (a), (b), (c) and (d) indicate the rats treated with inhaled saline, oridonin powders, gemcitabine and oridonin-loaded LPMPs, respectively. Q2 and Q3 area in (A) indicates high and low MMPs, respectively. Data are expressed as mean ± SD (n=3). ^**P<0.01; ^*P<0.05.