Stepwise targeting and responsive lipid-coated nanoparticles for enhanced tumor cell sensitivity and hepatocellular carcinoma therapy

Abstract

Rationale: Antitumor drug delivery faces multiple barriers that require consecutively achieving tumor targeting, selective cellular uptake and sufficient intracellular drug dosage. Methods: Herein, we designed smart nanoparticles (GPDC-MSNs) that can accumulate stepwise in tumor tissues, selectively enter cancer cells by responding to the acidic tumor extracellular environment, and achieve considerable drug release in the intracellular microenvironment. The GPDC-MSNs comprise the synthesized material galactosyl-conjugated PEO-PPO-PEO (Gal-P123) for hepatocellular carcinoma (HCC) targeting, the tumor extracellular pH-responsive lipid (2E)-4-(dioleostearin)-amino-4-carbonyl-2-butenonic (DC) for selective cellular internalization, and antitumor drug irinotecan (CPT-11)-loaded mesoporous silica nanoparticles (MSNs) for on-demand intracellular drug release. Results: GPDC-MSNs are negatively charged at pH 7.4 and promote active HCC targeting mediated by the asialoglycoprotein receptor. Upon reaching the weakly acidic tumor microenvironment, the nanoparticles undergo charge conversion to neutral, enhancing cellular internalization. Moreover, the encapsulated CPT-11 can be retained within GPDC-MSNs in the blood circulation but undergo intracellular burst release, which facilitates the apoptosis of tumor cells. GPDC-MSNs significantly increased HCC selectivity in vivo and exhibited up to 25 times higher accumulation in tumor tissue than in normal hepatic tissue, thus achieving superior antitumor efficacy and low systemic toxicity. Conclusion: This stepwise-responsive nanoparticle should serve as a valuable platform and promising strategy for HCC treatment.

Keywords: charge conversion; drug delivery; hepatocellular carcinoma; pH sensitive; tumor targeting.

Scheme 1

Illustration of the stepwise targeting and responsiveness of the lipid-coated nanoparticles GPDC-MSNs.

Figure 1

(A) Schematic diagram of the structure and pH sensitivity of CPT-11@GPDC-MSNs. (B) TEM images of MSNs and GPDC-MSNs. Scale bar: 50 nm. (C) SAXRD pattern and nitrogen adsorption-desorption isotherms of the synthetic MSNs. (D) Drug loading efficiency of MSNs and GPDC-MSNs. (E) Particle size and zeta potential of GPDC-MSNs with various DC content in lipid film. (F) Particle size, zeta potential and (G) stability of MSNs and GPDC-MSNs. (H) Representative flow cytometry histograms and (I) quantitative fluorescence intensities of Huh-7 cells after incubation with GPDC-MSNs with various Gal-P123 content. (J) Zeta potential changes and (K) exposed primary amines of GPDC-MSNs and GP-MSNs at different pH values. All data are presented as the mean ± SD (n=3).

Figure 2

(A) CLSM images of Huh-7 and LO2 cells incubated with GPDC-MSNs, PDC-MSNs and GP-MSNs with or without galactose ligand inhibition. Nanoparticles were labeled with PI (red), and fluorescamine (blue) was added to the culture medium to visualize the amine exposure. Scale bar: 40 μm. (B) Quantitative measurement of the fluorescence intensity in Huh-7 cells by flow cytometry at pH 6.5 and (C) pH 7.4 with or without galactose ligand inhibition. ***p<0.001. (D) Representative flow cytometry histogram of Huh-7 and LO2 cells treated with GPDC-MSNs. (E) Quantitative measurement of the fluorescence intensity in LO2 cells after incubation with DC-MSNs, PDC-MSNs and GP-MSNs. (F) Nanoparticle penetration into the Huh-7 MCSs. Z-stack images were obtained starting from the top and proceeding into the core of the spheroid at intervals of 20 μm. Scale bar: 100 μm. (G) Quantification of the fluorescence intensity in the inside area of MCSs. **p<0.01. All data are presented as the mean ± SD (n=3).

Figure 3

(A) CPT-11 release from CPT-11-loaded MSNs, PDC-MSNs, GP-MSNs and GPDC-MSNs when exposed to simulated body fluid (pH 7.4) and acidic endosome/lysosome buffer (pH 5.0) at 37 °C. (B) Drug release from CPT-11@ GPDC-MSNs upon lowering the pH to 5.0 after exposure to pH 7.4 and pH 6.8 buffers for 12 h. (C) Real-time confocal microscopy images monitoring intracellular cargo release in Huh-7 cells after incubation with PI-labeled GPDC-MSNs at a pH gradient from 7.4 to 6.5. After recording at pH 7.4 for 20 min, the pH of the incubation medium was adjusted to 7.2, 7.0, 6.8 and 6.5 at 20 min intervals. Fluorescamine was added to the culture medium to visualize the amine exposure. Scale bar: 40 μm. (D) Real-time images of the intracellular cargo release of PI-labeled GPDC-MSNs in Huh-7 cells after bafilomycin A1 (an endosome/lysosome acidification inhibitor) treatment. Scale bar: 40 μm. (E) Quantification of CPT-11 accumulation in Huh-7 cells after various treatments at pH 6.5 and pH 7.4. All data are presented as the mean ± SD (n=3).

Figure 4

(A) Cell viability of Huh-7 cells after incubation with CPT-11 formulations at pH 7.4 and pH 6.5. (B) IC₅₀ values of CPT-11 formulations in Huh-7 cells after exposure to pH 7.4 and pH 6.5 culture media. ***p<0.001, **** p<0.0001. (C) Flow cytometric examination and (D) quantitative analysis of Huh-7 cell apoptosis after different treatments for 24 h. Early apoptotic cells appeared in the lower right quadrant, and late apoptotic cells appeared in the upper right quadrant. *p<0.05, **** p<0.0001. All data are presented as the mean ± SD (n=3).

Figure 5

(A) In vivo pharmacokinetics of free CPT-11, CPT-11 loaded GPDC-MSNs, PDC-MSNs and GP-MSNs. (B) Fluorescence images of mice treated with DiR solution, DiR-labeled PDC-MSNs, GP-MSNs and GPDC-MSNs. Images were taken 1, 2, 4, and 8 h after intravenous administration. (C) Ex vivo fluorescence images and (D) quantitative biodistribution of important organs excised 8 h post injection. ****p<0.0001. (E) Images of the excised HCC-bearing liver showing the colocalization of tumor tissues and nanoparticles. The black patch in the liver is HCC tissue (left in every image), and the fluorescence images show the distribution of DiR-labeled formulations (right in every image). (F) Liver sections and (G) quantitative analysis of fluorescence intensity showing PDC-MSNs, GP-MSNs and GPDC-MSNs accumulation in the interior of HCC and normal hepatic cells. N: normal hepatic tissue; T: tumor tissue. Original magnification: 200×, scale bar: 50 μm. ***p<0.001. All data are presented as the mean ± SD (n=3).

Figure 6

In vivo antitumor efficacy of CPT-11 formulations in Huh-7 ectopic and orthotopic xenograft tumor models. (A) Tumor growth curve and (B) average body weight of mice bearing ectopic Huh-7 tumors treated with free CPT-11, CPT-11-loaded PDC-MSNs, GP-MSNs or GPDC-MSNs. ****p<0.0001. (C) Representative liver photographs, (D) overall survival, (E) TUNEL assay of HCC tissue and (F) H&E staining of major organs in mice bearing Huh-7 orthotopic tumors after treatment with CPT-11 formulations. scale bar: 50 μm. All data are presented as the mean ± SD (n=6).