Precision USPIO-PEG-SLe^x Nanotheranostic Agent Targeted Photothermal Therapy for Enhanced Anti-PD-L1 Immunotherapy to Treat Immunotherapy Resistance

Abstract

Background

The anti-Programmed Death-Ligand 1 (termed aPD-L1) immune checkpoint blockade therapy has emerged as a promising treatment approach for various advanced solid tumors. However, the effect of aPD-L1 inhibitors limited by the tumor microenvironment makes most patients exhibit immunotherapy resistance.

Methods

We conjugated the Sialyl Lewis X with a polyethylene glycol-coated ultrasmall superparamagnetic iron oxide (USPIO-PEG) to form UPS nanoparticles (USPIO-PEG-SLe^x, termed UPS). The physicochemical properties of UPS were tested and characterized. Transmission electron microscopy and ICP-OES were used to observe the cellular uptake and targeting ability of UPS. Flow cytometry, mitochondrial membrane potential staining, live-dead staining and scratch assay were used to verify the in vitro photothermal effect of UPS, and the stimulation of UPS on immune-related pathways at the gene level was analyzed by sequencing. Biological safety analysis and pharmacokinetic analysis of UPS were performed. Finally, the amplification effect of UPS-mediated photothermal therapy on aPD-L1-mediated immunotherapy and the corresponding mechanism were studied.

Results

In vitro experiments showed that UPS had strong photothermal therapy ability and was able to stimulate 5 immune-related pathways. In vivo, when the PTT assisted aPD-L1 treatment, it exhibited a significant increase in CD4⁺ T cell infiltration by 14.46-fold and CD8⁺ T cell infiltration by 14.79-fold, along with elevated secretion of tumor necrosis factor-alpha and interferon-gamma, comparing with alone aPD-L1. This PTT assisted aPD-L1 therapy achieved a significant inhibition of both primary tumors and distant tumors compared to the alone aPD-L1, demonstrating a significant difference.

Conclusion

The nanotheranostic agent UPS has been introduced into immunotherapy, which has effectively broadened its application in biomedicine. This photothermal therapeutic approach of the UPS nanotheranostic agent enhancing the efficacy of aPD-L1 immune checkpoint blockade therapy, can be instructive to address the challenges associated with immunotherapy resistance, thereby offering potential for clinical translation.

Keywords:

• immunotherapy resistance
• immune checkpoint blockade
• nanotheranostic agent
• photothermal therapy
• enhancing efficacy

Abbreviations

aPD-L1, anti-Programmed Death-Ligand 1; USPIO-PEG, polyethylene glycol-coated ultrasmall superparamagnetic iron oxide; UPS, polyethylene glycol-coated ultrasmall superparamagnetic iron oxide nanoparticles coupled with sialyl Lewis X; ICP-OES, Inductively Coupled Plasma Optical Emission Spectroscopy; PTT, photothermal therapy; ROS, reactive oxygen species; ICB, immune checkpoint blockade; PD-L1, programmed death-ligand 1; PD-1, programmed cell death 1 receptor; mAb, monoclonal antibodies; FDA, Food and Drug Administration; ICD, immunogenic cell death; CRT, calreticulin; HMGB1, high mobility group box 1; PEG, Polyethylene glycol; HNSCC, head and neck squamous cell carcinoma; CTLs, cytotoxic T lymphocytes; SLe^x, Sialyl Lewis X; MES buffer, 2- (N-morpholino) ethanesulfonic acid buffer; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; VSM, Vibration Sample Magnetometer; NIR, near-infrared; SCC7, squamous cell carcinoma 7; KEGG, kyoto encyclopedia of genes and genomes; ICP-OES, inductively coupled plasma optical emission spectroscopy; ELISA, Enzyme-linked immunosorbent assay; TNF-α, tumor necrosis factor-alpha; IFN-γ, interferon-gamma; TEM, transmission electron microscopy; Ms, saturation magnetization; NPs, nanoparticles; τ, time constant; ɳ, photothermal conversion efficiency; MRI, magnetic resonance imaging; JC-1, mitochondrial membrane potential assay kit with JC-1; 0.1UPS, UPS nanoparticles aqueous solution with a concentration of 100 μg mL⁻¹; 0.2UPS, UPS nanoparticles aqueous solution with a concentration of 200 μg mL⁻¹; Cccp, carbonyl cyanide m-chlorophenyl hydrazone; TNF, tumor necrosis factor; HSPA6, heat shock 70 kDa protein 6; HSPA1A, heat shock 70 kDa protein 1A; HSPA1B, heat shock 70 kDa protein 1B; HSP70, heat shock protein 70; DCs, dendritic cells; USPIO, ultrasmall superparamagnetic iron oxide; SEM, standard error of the mean; ALT, alanine aminotransferase; AST, aspartate aminotransferase; TBIL, total bilirubin; ALB, albumin; TP, total protein; BUN, blood urea nitrogen; CREA, creatinine; H&E, hematoxylin and eosin; RBCs, red blood cells; PBS, phosphate-buffered saline; IHC, immunohistochemical; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; PCNA, proliferating cell nuclear antigen.

Ethics Approval and Consent to Participate

The ethical review of animal experiments adhered to the Guiding Opinions on the Humane Treatment of Laboratory Animals issued by the Ministry of Science and Technology of the People’s Republic of China, as well as the National Standard GB/T35892-2018, titled Ethical Standards for Animal Welfare in Experimental Research. It was approved by the Animal Experiment Ethics Committee of the Affiliated Tumor Hospital of Guangxi Medical University.

Consent for Publication

All authors give consent for the publication of the manuscript in International Journal of Nanomedicine.

Disclosure

The authors declare no competing interests for this work.

Additional information

Funding

Financial support for this study was provided by the Natural Science Foundation of Guangxi Province (2018GXNSFAA281095), the Guangxi Clinical Research Center for Medical Imaging Construction (Grant No. Guike AD20238096), and Guangxi Health Commission Key Laboratory of Tumor Molecular Imaging (Guangxi Medical University Cancer Hospital, Grant No. ZPZH2020004).