Nonmetallic magnetic hyperthermia and chemo-immunotherapy of tumors

Abstract

Combined therapy based on magnetic hyperthermia holds significant promise for tumor treatment. Nevertheless, the existing magnetic hyperthermia platforms are often constrained by suboptimal heating efficiency and concerns over the potential metal ion toxicity. Herein, a minimalistic and nonmetallic graphite-agarose gel-drug (Gra-Aga-Drug) implant is prepared by mold-assisted casting method for magnetic hyperthermia-based combination therapy of tumors. This implant can achieve magnetic hyperthermia through the strong eddy current thermal effect of Gra, while the agarose gel provides the implant with universal and efficient drug-loading capacity and magnetic hyperthermia-responsive release ability. The chemotherapeutic drug doxorubicin (DOX) and the immune adjuvant imiquimod (R837) are chosen to prepare Gra-Aga-DOX-R837 implant for combination therapy of tumors in vivo. The magnetic hyperthermia and DOX induce immunogenic cell death (ICD) of tumors, combined with the immune adjuvant R837, eliciting a robust anti-tumor immune response to efficiently ablate primary tumors and suppress distant tumors growth. Importantly, the implant platform effectively addresses the challenges of limited heating efficiency and metal ion toxicity associated with traditional magnetic hyperthermia materials. This study marks the pioneering construction of nonmetallic combination therapy platform with excellent magnetothermal performance and favorable biocompatibility.

Keywords: Controlled drug release; Graphite; Nonmetallic magnetic hyperthermia; Tumor combination therapy; Universal and efficient drug loading.

Graphical abstract

Scheme 1

Schematic diagram of nonmetallic graphite-agarose gel-drug implants for combined magnetic hyperthermia therapy of tumors.

Fig. 1

(a) Photos of Gra in different shapes (rectangular prisms, cylinders, and cubes); photos of Gra cubes with different sizes (length of side: 0.75, 1, 1.25, 1.5, 1.75, 2 mm). (b) The XRD pattern of Gra. (c) Experimental pictures of the conductivity of Gra. (d) Photos of Aga-DOX at different temperature. (e) Photos of preparation of Gra-Aga-DOX-R837 implant. (f) Photos of Gra-Aga-DOX-R837 implant. (g) SEM images of Aga-DOX and Aga-R837. (h) Photos of Aga-DOX-R837 stored at 4 °C for different time. (i) Drug release of Gra-Aga-DOX in room temperature and heating conditions (37 °C, 45 °C, 65 °C, 3 min) for different time.

Fig. 2

(a) Thermal images of Gra cubes in AMF with various sizes. (b) Temperature–time curves of Gra cubes in AMF with various sizes. (c) Thermal images of Gra cubes heating in AMF with various magnetic field strengths. (d) Temperature–time curves of Gra cubes heating in AMF with various magnetic field strengths. (e) Heating and cooling curves of Gra cubes in AMF. (f) Temperature–time curves of different Gra cubes in AMF with 8 repeats. (g) Thermal images of Gra-Aga/Gra-Aga-DOX-R837 heating in AMF. (h) Temperature–time curves of Gra-Aga/Gra-Aga-DOX-R837 heating in AMF. (i) Photos of Gra cube coated in pork tissue before and after heating in AMF (The image below shows the opened pork tissue before and after heating. During the AMF heating process, the pork tissue encloses the Gra embedded in a centrifuge tube filled with thermochromic materials in the middle, resembling a hamburger. The image above provides a magnified view of the pork tissue surrounding the Gra after the tissue is opened).

Fig. 3

(a) Thermal images of cell heating in AMF. (b) Temperature–time curves of cell heating in AMF. (c) Cell viabilities of CT26 cells after different operations. (d) Fluorescent images of CT26 cells after different operations. (e) Concentration of ATP in cellular supernatant of CT26 cells after different operations. (f) Fluorescent images of CRT expression in CT26 cells after different operations. (g) Flow analysis results of BMDCs after different treatments. Statistical analysis was performed using one-way ANOVA; ∗∗∗∗p < 0.0001.

Fig. 4

(a) Liver and kidney function indicators of Kunming mice following subcutaneous implantation of Gra over different periods. (b) Images of vital organs stained with H&E of Kunming mice following subcutaneous implantation of Gra over different periods. (c) Body weight monitoring of Kunming mice following subcutaneous implantation of Gra over different periods. (d) XRD patterns and photos of Gra dissected from Kunming mice following subcutaneous implantation over different periods.

Fig. 5

(a) A schematic representation for in vivo combined magnetic hyperthermia and chemotherapy of tumors using Gra-Aga-DOX. (b) Thermal imaging of CT26 tumor-bearing mice under various treatments. (c) Heating curves of CT26 tumor-bearing mice with different operations. (d) Representative images of CT26 tumor-bearing mice subjected to various treatments over a 14-d period. (e) Images of tumors excised from mice under various operations on the 14th d. (f) Tumor growth curves of mice following different treatments for 14 d. (g) The weight of tumors excised from mice following different treatments on the 14th d. Statistical analysis was performed using one-way ANOVA; ∗∗∗∗p < 0.0001, ∗p < 0.05.

Fig. 6

(a) Timeline for combined magnetic hyperthermia, chemotherapy and immunotherapy using Gra-Aga-DOX-R837 of tumors in vivo. (b) Thermal imaging of bilateral CT26 tumor-bearing mice in AMF. (c) Temperature rise curves of bilateral CT26 tumor-bearing mice in AMF. (d) Photos of primary tumors excised from mice undergoing various procedures on the 10th d. (e) Primary tumor growth curves of mice undergoing various procedures for 10 d. (f) The weight of primary tumors excised from mice undergoing various procedures on the 10th d. (g) Photos of distant tumors excised from mice undergoing various procedures on the 10th d. (h) Distant tumor growth curves of mice undergoing various procedures for 10 d. (i) The weight of distant tumors excised from mice undergoing various procedures on the 10th d. Statistical analysis was carried out using one-way ANOVA; ∗∗∗∗p < 0.0001.

Fig. 7

(a) The level of TNF-α from serum in mice with different operations. (b) The level of IL-6 from serum in mice undergoing various procedures. (c) The level of IFN-γ from serum in mice with different operations. (d) Flow analysis results of CD3⁺ CD8⁺ T cells from distant tumors of mice undergoing various procedures. Statistical analysis was carried out using one-way ANOVA; ∗∗∗∗p < 0.0001.