A multistage assembly/disassembly strategy for tumor-targeted CO delivery

Abstract

CO gas molecule not only could selectively kill cancer cells but also exhibits limited anticancer efficacy because of the lack of active tumor-targeted accumulation capability. In this work, a multistage assembly/disassembly strategy is developed to construct a new intelligent nanomedicine by encapsulating a mitochondria-targeted and intramitochondrial microenvironment-responsive prodrug (FeCO-TPP) within mesoporous silica nanoparticle that is further coated with hyaluronic acid by step-by-step electrostatic assembly, realizing tumor tissue-cell-mitochondria-targeted multistage delivery and controlled release of CO in a step-by-step disassembly way. Multistage targeted delivery and controlled release of CO involve (i) the passive tumor tissue-targeted nanomedicine delivery, (ii) the active tumor cell-targeted nanomedicine delivery, (iii) the acid-responsive prodrug release, (iv) the mitochondria-targeted prodrug delivery, and (v) the ROS-responsive CO release. The developed nanomedicine has effectively augmented the efficacy and safety of CO therapy of cancer both in vitro and in vivo. The proposed multistage assembly/disassembly strategy opens a new window for targeted CO therapy.

Fig. 1. Construction of the FeCO-TPP@MSN@HA nanomedicine by the multistage assembly/disassembly strategy for tumor-targeted CO delivery.

(A) Schematic illustration of multistage assembly method for construction of the FeCO-TPP@MSN@HA nanomedicine. (B) Schematic illustration of multistage disassembly strategy and mechanisms for tumor tissue–cell–mitochondria–targeted delivery and controlled release of CO by the FeCO-TPP@MSN@HA nanomedicine. The transmission electron microscopy (TEM) image of the MSN carrier (C); the TEM (D), high angle annular dark field (HADDF) (E), scanning electron microscopy (SEM) (F), and element mapping (G) images of the FeCO-TPP@MSN@HA nanomedicine; the zeta potentials (H), dynamic light scattering (DLS) particle size distributions (I), and FTIR spectra (J) of MSN, FeCO-TPP@MSN, and FeCO-TPP@MSN@HA are shown. a.u., arbitrary units.

Fig. 2. Controlled release profiles of FeCO-TPP@MSN.

Acid-responsive prodrug release profiles of the FeCO-TPP@MSN nanomedicine under in vitro simulated conditions (A) and intramitochondrial ROS-responsive CO release profiles of the FeCO-TPP prodrug at different ·OH concentrations (B) are shown.

Fig. 3. Targeted delivery and controlled release profiles in vitro.

(A) CD44-dependent targeted endocytosis behaviors of the FeCO-TPP@MSN@HA nanomedicine shown by representative confocal fluorescence microscopy images of HeLa, 4T1, B16, and MCF-10A cells incubated with the FeCO-TPP@MSN-RITC@HA for 4 hours and stained for cellular nuclei with 4,6-diamino-2-phenylindole (DAPI). Therein, the MSN carrier was labeled with red RITC and then used to construct the FeCO-TPP@MSN-RITC@HA nanomedicine. (B) Intralysosome acid-responsive prodrug release behaviors of the FeCO-TPP@MSN@HA nanomedicine shown by the fluorescence images of HeLa cells treated with QL-FeCO-TPP@MSN-RITC@HA for the different time periods and stained with LysoTracker (green). Therein, the FeCO-TPP prodrug was labeled with blue QL (8-mercaptoquinoline). The increases in yellow and blue represent the lysosomal uptake of the nanomedicine and the FeCO-TPP prodrug release from lysosome, respectively. (C) Mitochondria-targeted prodrug delivery behaviors of the FeCO-TPP@MSN@HA nanomedicine shown by the fluorescence images of HeLa cells treated with QL-FeCO-TPP@MSN@HA for the different time periods and stained with MitoTracker (red). The increase in purple represents the mitochondrial uptake of the released FeCO-TPP prodrug. (D) Intramitochondrial CO release behaviors of the FeCO-TPP drug released from the FeCO-TPP@MSN@HA nanomedicine shown by the fluorescence images of HeLa cells treated with QL-FeCO-TPP@MSN-RITC@HA for the different time periods and stained with the COP-1 probe for detection of CO (green). The increase in green represents the release of CO. Scale bars, 20 μm.

Fig. 4. In vitro selective anticancer effects and mechanisms of the FeCO-TPP@MSN@HA nanomedicine.

(A to D) Cytotoxicity of the FeCO-TPP@MSN@HA nanomedicine to cancer (HeLa and 4T1 cells) and normal (MCF-10A and HEK-293T cells) model cell lines. (E to H) Cell energy metabolism evaluation by ATP production (E), basal respiration (F), maximal respiration (G), and proton leak (H). (I and K) Confocal images of treated HeLa and MCF-10A cells where nuclei and mitochondria were stained into blue and red, respectively. (J) and (L) were the quantification of red fluorescence intensity in (I) and (K), respectively. Mean value and error bar were represented as means ± SD. P values were calculated by the two-tailed Student’s t test (**P < 0.01 and ***P < 0.001; NS, no significant difference). Scale bars (I and K), 20 μm. OCR, oxygen consumption rate.

Fig. 5. In vivo tumor-targeted therapy efficacy of the FeCO-TPP@MSN@HA nanomedicine.

The tumor-targeted delivery behaviors testified by fluorescence images of the tumor-bearing mice intravenously injected with the FeCO-TPP prodrug, the MSN carrier, and the FeCO-TPP@MSN@HA nanomedicine, respectively (A), and the CD31-stained slice of FeCO-TPP@MSN@HA–treated tumor (B and C) where green, red, and blue represent vessel, MSN, and FeCO-TPP, respectively. The outcomes of B16 tumor therapy with the nanomedicine: The tumor volume change with time (D), the tumor weight comparison after 20 days of treatment (E), the mouse weight change with time (F), and the histological analysis of treated tumors by the H&E staining (G) are shown. Mean value and error bar were represented as means ± SD. P values were calculated by one-way analysis of variance (ANOVA) with Tukey post hoc testing to correct for multiple comparisons (*P < 0.05, **P < 0.005, and ***P < 0.0005). Scale bars, 200 (B), 20 (C), and 500 μm (G).

Fig. 6. Inhibition of the growth and lung metastasis of primary breast tumor by the FeCO-TPP@MSN@HA nanomedicine.

The volume change of primary 4T1 tumor with time (A), the survival rate of 4T1 tumor–bearing mice with and without drug treatment (B), the fluorescence tracking of 4T1 tumor growth and metastasis (C), the histological analysis of lung metastasis tumors (D) and primary tumors (F) by the H&E staining (scale bar. 500 μm), and the statics of the lung metastasis tumor number from the histological analysis (E) are shown. Lung metastasis tumors in (D) were indicated by blue dashed lines. Mean value and error bar were represented as means ± SD. P values were calculated by one-way ANOVA with Tukey post hoc testing to correct for multiple comparisons (**P < 0.005 and ***P < 0.0005).