Homotypic Cell Membrane-Cloaked Biomimetic Nanocarrier for the Targeted Chemotherapy of Hepatocellular Carcinoma

Goals: Hepatocellular carcinoma (HCC) has been reported to be the third most common malignant tumor and has the highest rate of mortality. To increase the chemotherapy efficacy of HCC, a drug delivery system featured with desirable active targeting ability, delivery efficiency and immune evasion is in high demand. Methods: We have developed a drug nanocarrier by utilizing a homotypic cancer cell membrane for targeted chemotherapy of HCC. Structurally, the homotypic HepG2 cell membrane was used as the cloak, and a poly (lactic-co-glycolic acid) (PLGA) nanoparticle as the core, resulting in the nanocarrier HepM-PLGA. Results: The HepM-PLGA nanoparticles exhibit excellent targeting ability toward HepG2 cells. Doxorubicin (Dox) carried by HepM-PLGA possesses high delivery efficiency and a remarkable in vitro therapeutic effect. In in vivo experiments, HepM-PLGA delivers Dox directly to the tumor lesion of nude mice, and tumor volume decreases by approximately 90% after treatment. Conclusion: We have developed a drug nanocarrier by utilizing a homotypic cancer cell membrane for targeted chemotherapy of HCC with excellent active targeting ability. This biomimetic platform not only effectively treats HCC but also provides a sound strategy for the treatment of other cancers via changes in the corresponding homotypic cancer cell membrane.

Isopropyl alcohol and DMSO were purchased from Sinopharm Chemical Reagent Co., Ltd.
Solvents and regents were all of analytical grade and purchased from China National

TEM characterization.
Negative staining (1% phosphotungstic acid) for TEM was used to characterize the bare PLGA nanoparticles and the cell membrane-cloaked PLGA nanoparticles and HepG2 cell membrane. The nanoparticles were dropped on a copper grid for 5 min, and then another sample was negatively stained with 2 drops of 1 wt% phosphotungstic acid. After the solution was completely dry, TEM characterization was carried out on a JEM-2100.

Gel electrophoresis analysis and western blot analysis.
HepG2 cell lysates and HepG2 cell membrane protein extraction solution was added to SDS loading buffer in a 4:1 ratio and then heated at 95-100 °C for 5 min for protein denaturation.
Twenty microliters of protein sample and protein marker were sequentially and slowly added to the loading wells of an SDS-PAGE gel containing 12% separation gel and 5% spacer gel, the voltage was then set to 80 V for electrophoresis in the electrophoresis apparatus, and finally the voltage was changed to 120 V until the sample reached the spacer gel. The proteins in the resulting polyacrylamide gel were stained with Coomassie brilliant blue stain. The gel was imaged by the ChemiDoc TM Touch Imaging System (Bio-Rad, Hercules, CA, USA).
The proteins in the resulting polyacrylamide gel were then transferred to a PVDF membrane (PerkinElmer) at 100 V for 60 min, while the Trans-Blot system was placed in ice water. The obtained PVDF membrane was blocked with 5% nonfat milk powder at room temperature for 1 h.
Then, the PVDF membranes were incubated with primary antibodies overnight at 4°C. The primary antibodies included antibodies to galectin-1, galectin-3, CD47, cytochrome c oxidase (cytoplasmic markers), and histone H3 (nuclear marker). The next day, the rewarmed PVDF membrane was washed with TBST three times, incubated with goat anti-mouse lgG HRP conjugate (1:5000 dilution in 5% nonfat milk powder) for 1 h, and then washed thrice with TBST.
Finally, ECL chemiluminescent coloring solution was added dropwise, and the PVDF membranes were imaged by ChemiDoc TM Touch Imaging system (Bio-Rad, Hercules, CA, USA). incubation buffer was discarded, and the cells were washed three times with PBS (pH 7.4). Then, the cells were imaged immediately using a confocal microscope with an objective lens (× 63).

Validating the homologous targeting property of
Excitation of the probe-treated cells at 488 nm was performed using an argon laser, and the emitted light was collected with a META detector between 520 and 550 nm.

Generation of stably transfected cell lines
The HepG2 cells were seeded onto a 24-well plate with 8×104 cells peer well. After 12 hours, the cells are completely attached to the wall. Replace the original culture medium with fresh culture medium and add 10 μL mCherry-loaded-lentiviral vector suspension. After mixing, the culture was continued for 24 hours. The culture medium containing virus was replaced with fresh medium to culture the cells. Stable transfected mCherry-HepG2 cell lines were obtained by purinomycin (1.0 μg /mL) screening 72 h after transfection.

The time-dependent internalization of HepM-PLGA by HepG2 cells
HepG2 cells or L02 cells were seeded in a confocal cell culture dish and cultured for 24 h in 2 mL of DMEM with 10% FBS. After the supernatant was discarded, HepG2 cells or L02 cells were incubated with FITC-HepM-PLGA for 1 h, 2 h, 3 h or 4 h. To prepare the incubation buffer, 100 μL of FITC-HepM-PLGA was mixed with 900 μL of DMEM containing 10% FBS. HepG2 cells or L02 cells were incubated with 200 μL of the incubation buffer for in every well. The incubation buffer was discarded, and the cells were washed three times with PBS (pH 7.4). Then, the cells were imaged immediately using a confocal microscope with an objective lens (× 63). Excited at 488 nm using an argon laser, and the emitted light was collected with a META detector between 520 and 550 nm. The relative fluorescence intensity was measured by Zen software.