Enhanced Antitumor Efficacy and Reduced Toxicity in Colorectal Cancer Using a Novel Multifunctional Rg3- Targeting Nanosystem Encapsulated with Oxaliplatin and Calcium Peroxide

Abstract

Purpose: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. Oxaliplatin (OXA) is currently the primary chemotherapeutic agent for CRC, but its efficacy is limited by the tumor microenvironment (TME). Here, we present a combined approach of chemotherapy and TME modulation for CRC treatment. A multifunctional nanosystem (Rg3-Lip-OXA/CaO₂) was established using Ginsenoside Rg3 liposomes targeting glucose transporter 1 overexpressed on the surface of CRC cells to co-deliver OXA and calcium peroxide (CaO₂).

Methods: The CaO₂ nanoparticles were synthesized via the CaCl₂-H₂O₂ reaction under alkaline conditions and characterized using X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). Rg3-Lip-OXA/CaO₂ was prepared through a thin-film hydration approach and characterized; additionally, its stability and release behavior were studied. The O₂, H₂O₂, and Ca²⁺ generation ability of Rg3-Lip-OXA/CaO₂ in solution and HCT116 cells were measured. The in vitro cellular uptake was observed via fluorescence microscope and flow cytometry. In vitro cytotoxicity was evaluated using the CCK-8 assay, flow cytometry, and live/dead cell staining. The in vivo targeting effect as well as antitumor efficacy were determined in HCT116 tumor-bearing mice. Finally, the acute toxicity of Rg3-Lip-OXA/CaO₂ was investigated in ICR mice to explore its safety.

Results: The XRD and XPS analyses confirmed the successful synthesis of CaO₂ nanoparticles. The Rg3-Lip-OXA/CaO₂ exhibited an average particle size of approximately 92.98 nm with good stability and sustained release behavior. In vitro and in vivo studies confirmed optimal targeting by Rg3-Lip and demonstrated that the nanosystem effectively produced O₂, H₂O₂ and Ca²⁺, resulting in significant cytotoxicity. Additionally, in vivo studies revealed substantial tumor growth suppression and reduced tumor-associated fibroblasts (TAFs) and collagen. Acute toxicity studies indicated that Rg3-Lip-OXA/CaO₂ markedly reduced the toxicity of chemotherapeutic drugs.

Conclusion: This multifunctional nanosystem enhances chemotherapy efficacy and reduces toxicity, offering a promising approach for optimizing CRC treatment and potential clinical application.

Keywords: anticancer therapy; calcium peroxide; multifunctional nanosystem; oxaliplatin; targeted drug delivery; tumor microenvironment.

Graphical abstract

Figure 1

Characterization of CaO₂ and Rg3-Lip-OXA/CaO₂. (A) XRD spectra and (B) XPS spectrum of CaO₂. (C) Particle sizes and (D) TEM images of CaO₂ and Rg3-Lip-OXA/CaO₂. (E) EDS elemental mapping of Rg3-Lip-OXA/CaO₂. (F) O₂ release profiles of Lip-OXA, Lip-OXA/CaO₂, and Rg3-Lip-OXA/CaO₂ in PBS at pH 7.4. (G) Photographs and (H) UV–vis absorption spectra of KMnO₄ treated with Lip-OXA, Lip-OXA/CaO₂, or Rg3-Lip-OXA/CaO₂. (I) Cumulative Ca²⁺ release profiles of Lip-OXA, Lip-OXA/CaO₂, and Rg3-Lip-OXA/CaO₂ in PBS at pH 7.4. (J) Changes in EE of OXA in Lip-OXA/CaO₂ and Rg3-Lip-OXA/CaO₂ and (K) Rg3 in Rg3-Lip-OXA/CaO₂ within 48 h at different temperatures. (L) Particle sizes, PDI, and (M) zeta potential changes of Rg3-Lip-OXA/CaO₂ stored at 4°C for 14 days. (N) Particle sizes and (O) PDI of Rg3-Lip-OXA/CaO₂ mixed with different dispersion medium for 48 h. Drug release behavior of (P) OXA and (Q) Rg3 in diverse formulations. Data are presented as mean ± SD (n = 3).

Figure 2

Intracellular uptake and mechanism of Rg3-Lip-Rh B in HCT116 cells. (A) Fluorescence images of HCT116 cells incubated with different Rh B formulations for 6 h. (B) Flow cytometry analysis and (C) quantification of the cellular uptake of different Rh B formulations. (D) Examination of Rg3-Lip-Rh B cellular uptake in the presence of various Glut inhibitors, including glucose and quercetin. (E) Flow cytometry analysis and (F) quantification of the cellular uptake of Rg3-Lip-Rh B in the presence of different Glut inhibitors. Data are expressed as mean ± SD (n = 3). **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

Detection of intracellular O₂, ROS, Ca²⁺ levels, and mitochondrial membrane potential (MMP) in HCT116 cells treated with a CaO₂-loaded nanosystem. Fluorescence images of cellular (A) O₂, (C) ROS, and (E) Ca²⁺ levels in HCT116 cells treated with Lip-OXA, Lip-OXA/CaO₂, or Rg3-Lip-OXA/CaO₂ for 12 h. Scale bar = 50 μm. The fluorescence quantitative analysis of cellular (B) O₂, (D) ROS, and (F) Ca²⁺ levels. (G) MMP in HCT116 cells treated with Lip-OXA, Lip-OXA/CaO₂, and Rg3-Lip-OXA/CaO₂ for 12 h. (H) The fluorescence quantitative analysis of (G). Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4

In vitro cytotoxicity and apoptosis of different formulations in HCT116 cells. (A) Cell viability of HCT116 cells treated with different formulations for 48 h. (B) IC₅₀ of various formulations. (C) The apoptosis quantitative analysis and (D) apoptosis assay results of HCT116 cells treated with different formulations for 48 h by flow cytometry. (E) Live/dead cell staining images of HCT116 cells treated with different formulations for 48 h. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

In vivo fluorescence imaging of Rg3-Lip-DiR. (A) In vivo fluorescence imaging of different DiR formulations in HCT116 tumor-bearing athymic mice at predetermined time points. (B) Ex vivo fluorescence images of the main organs and tumors at 6 h. (C) Quantification of fluorescence intensity at tumor sites of (A). (D) Quantification of fluorescence intensity at tumor sites of (B). Data are presented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6

In vivo antitumor evaluation on the HCT116 tumor-bearing athymic mice. (A) Tumor inoculation and therapeutic schedule over 24 days. (B) The tumor growth curves, (C) tumor photographs, (D) tumor weight, and (E) tumor inhibition rate of tumor-bearing mice following the treatments. (F) The H&E staining and TUNEL assay of tumor tissues. (G) Masson’s trichrome assay and α-SMA immunohistochemistry staining of tumor tissues. Data are expressed as mean ± SD (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 7

The safety evaluation of different formulations on the HCT116 tumor-bearing athymic mice. (A) Body weight change of mice throughout the 24-day experiment. (B) The organ coefficients and (C) H&E staining of major organs. (D) Blood routine analysis and biochemical analysis results of WBC, RBC, HGB, PLT, BUN, Cre, ALT, and AST. Data are presented as mean ± SD (n = 5).

Figure 8

Acute toxicity assessment. Survival curves and body weight changes after intravenous administration with (A and D) OXA, (B and E) Rg3-OXA/CaO₂, and (C and F) Rg3-Lip-OXA/CaO₂ at various doses. Data are presented as mean ± SD (n = 10).

Figure 9

Acute toxicity assessment. (A) The blood routine analysis and (B) biochemical analysis results of low dose groups. (C) H&E staining of main organs of low dose groups. Data are expressed as mean ± SD (n = 10). *p < 0.05, **p < 0.01, ***p < 0.001.