Targeted destruction of cancer stem cells using multifunctional magnetic nanoparticles that enable combined hyperthermia and chemotherapy

Cancer stem cells (CSCs) have been implicated in cancer recurrence and therapy resistance. Therefore, a CSC-targeted therapy that disrupts the maintenance and survival of CSCs may offer an effective approach in killing tumor cells in primary tumors and preventing the metastasis caused by CSCs. Nanoparticles (NPs)-based thermotherapy and/or chemotherapy are promising therapeutic methods for cancer treatment. Methods: A silica-based multifunctional NP system was present, which encapsulated a chemotherapeutic agent and magnetic cores and coated with a specific antibody against the lung CSCs. The efficacy of this novel therapeutic strategy was systematically studied both in vitro and in vivo by simultaneous activating the combined thermotherapy and chemotherapy via CSC-targeted NPs. Results: These NPs were systematically administered and activated for targeted chemotherapy and thermotherapy by using an externally applied alternating magnetic field (AMF). The antibody-modified NPs targeted to lung CSCs with enhanced cellular uptake in vitro and extended accumulation in tumor in vivo. Up to 98% of lung CSCs was killed in vitro with 30-min application of AMF, due to the combined effects of hyperthermia and chemotherapeutic drug treatment. In in vivo models, this combined therapy significantly suppressed tumor growth and metastasis in lung CSC xenograft-bearing mice, with minimal side effects and adverse effects. Conclusion: With good biocompatibility and targeting capability, the nanodrug delivery system may offer a promising clinical platform for the combined thermotherapy and chemotherapy. This work demonstrated the feasibility of developing multifunctional nanomedicine targeting CSCs for effective cancer treatment.

Briefly, an aqueous solution of FeCl 3 (2 mL, 1 mol/L) and FeCl 2 (0.5 mL, 2 mol/L, Sigma-Aldrich) in 2 mol/L HCl was mixed and added into diluted NH 3 solution (25 mL, 0.7 mol/L) and stirred for 30 min. Then, the precipitate was isolated by magnetic decantation and centrifugation. Solutions of Heat Shock Protein Inhibitor (HSPI, Selleckchem) were prepared by dissolving in PSB. Silica coating on the Fe 3 O 4 NPs was accomplished by a water-in-oil reverse micelle method [2] . Typically, 350 μL Fe 3 O 4 nanoparticles (2.0 mg/mL) and 50 μg/mL HSPI were dispersed into 7.7 mL cyclohexane, 2 mL Triton X-100, and 1.6 mL hexanol and stirred for 30 min to generate the microemulsion system. Then, 40 µL tetraethoxy orthosilicate (TEOS) was added to the mixture, followed by the addition of 100 µL aqueous ammonia for the TEOS hydrolysis under stirring for 24 h. After that, 50 µL tetraethoxy orthosilicate (TEOS) and 50 µL 3-aminopropyl triethoxysilane (APTS)/50 µL carboxyethylsilane triol sodium salt (CETS) were added to the mixture for another 24 h stirring. Finally, acetone was added to destabilize the microemulsion system. The HSPI-loaded Fe 3 O 4 @SiNPs were isolated via centrifugation and washed in sequence with ethanol and D.I. water for purification. All the reagents were obtained from Sigma-Aldrich.

Conjugation of Specific Antibody with HSPI-loaded Fe 3 O 4 @SiNPs
For specific antibody conjugation, the carboxyl functional groups were activated by The loading efficiency (LE) was evaluated by using the following formula: For drug release, 1 mL medium dispersed-HSPI-loaded Fe 3 O 4 @SiNPs was placed in a dialysis bag (weight cut-off of 10 kDa), and then immersed in 9 mL PBS and kept in an AMF at a constant temperature. Samples (300 mL) were periodically collected and the same volume of fresh medium was added. The amount of released HSPI was analyzed via UV-Visible spectrophotometry (U-3900, Hitachi) and the concentration-absorbance standard equation.

Lung Cancer Stem Cell Culture
Human lung cancer stem cell line (LCSC) originated from human small cell lung cancer tissue was purchased from Celprogen (Cat# 36107-34, Celprogen, USA). Cell culture completed medium, 0.05% Trypsin-EDTA, and culture plates were purchased from Celprogen. Antibiotic solution (penicillin and streptomycin) were purchased from Invitrogen. LCSCs were maintained in a serum-free medium (DMEM/F12) supplemented with 2% B27, 10 ng/ml bFGF and EGF, 1% N2, and 1% antibiotic solution and cultured in a humidified atmosphere containing 5% CO 2 at 37 o C. The differentiated LCSCs (dLCSCs) were obtained by culturing LCSCs in the completed medium with 10% fetal bovine serum (FBS). The fresh medium was replaced ever three days of culture.

Tumor Sphere Formation Assay
LCSCs (3 rd and 10 th generation) were plated at a density of 10,000 cells/well in 6-well nonadherent plates (Corning Inc.) in DMEM/F12 cell medium, supplemented with human EGF (10 ng/mL, Invitrogen), N2 (1% v/v, Invitrogen), and human bFGF (10 ng/mL, Invitrogen). Fresh aliquots of EGF and bFGF were added every other day, and the spheres were collected at day 12 following gravity-based sedimentation and/or low-speed centrifugation to remove any remaining single cells. Spheres were then washed once with PBS, followed by gentle resuspension in cell detachment solution and constant trituration for at least 20 min to obtain single cells. The dissociated single cells of the primary spheres were washed at least three times with the medium and then plated for sequential sphere-formation for an additional 12 days.

In vitro Cytotoxicity of Multifunctional Nanoparticles
To study the cytotoxicity of designed nanoparticles, LCSCs were seeded at 5×10 3 cells/well in a 96-well plate, pre-incubated for 24 h, then incubated with

Hemolysis Assay and Biochemical Analysis
The whole blood was centrifuged at 3000 rpm for 5 min to harvest the red blood cells       Immunohistochemically stained with PE-conjugated CD20 antibodies.