Integration of gold nanodendrites and immune checkpoint blockers to achieve highly efficient photothermal immunotherapy for eradicating primary and distant metastatic osteosarcoma
Introduction
Tumor metastases account for more than 90% of cancer-related deaths [1]. Currently, the goal of fighting against cancer is to develop effective treatment strategies with high specificity and low toxicity to eradicate tumors and further prevent their recurrence and metastasis. However, the current clinical treatment approaches to treating tumors, such as surgery, chemotherapy, and radiotherapy, have all failed to achieve the desired outcome [[2], [3], [4]]. Therefore, it is necessary to develop a comprehensive strategy that can remove or destroy the primary tumor while having the ability to identify, track, and destroy any tumor cells remaining at the distant metastatic sites.
Recently, the applications of immune checkpoint inhibitors (ICIs) in cancer therapy have attracted extensive attention which can bind tumor cells or tumor tissue infiltrating T cells surface antigen to block the inhibition of tumor cells on T cell activity [[5], [6], [7]]. Particularly, ICIs treatments blocking programmed death receptor 1 (PD-1) and programmed death-ligand 1 (PD-L1) have shown effectiveness against varieties of cancer [[8], [9], [10], [11], [12]]. Unfortunately, only a small amount of patients with cancer (10–30%) benefit from the current ICIs treatment because a considerable number of patients suffer from ‘cold’ tumors, resulting in insufficient T-cell infiltration and low immunogenicity [13]. In addition, long-term treatment with ICIs can cause immune-related side-effects, leading to premature cessation of tumor therapy, which is mainly related to the effect of the ‘on target but off tumor’ effect produced by ICIs receptors in non-targeted normal tissues [[14], [15], [16]]. Therefore, exploring strategies to reprogram the ‘cold’ tumor into a ‘hot’ one and improving the tumor-targeting ability of ICIs are effective methods to solve the current dilemma of ICIs tumor therapy.
Remodeling the tumor microenvironment by inducing immunogenic cell death (ICD) is expected to solve the dilemma of ICIs [17,18]. During the ICD process, tumor-associated antigen (TAA) released from killed tumor cells can act as a ‘danger’ signal of immune stimulation, enhancing the phagocytosis of tumor antigen by dendritic cells (DCs) and inducing DCs maturation and T cells activation. Although traditional radiotherapy and chemotherapy have been fully proven to induce ICD events, these strategies not only lack tissue specificity and reversibility but also have serious side-effects, ultimately causing new disease problems or being intolerable to patients [19,20]. Recent studies have shown that nanomaterial-based photothermal therapy (PTT) (NPTT) not only has an excellent photothermal conversion performance and drug loading ability but also shows a strong tumor homing ability [[21], [22], [23], [24], [25]]. Therefore, loading ICIs on the surface of nanomaterials to enhance their transmission to the tumor microenvironment will improve the specificity of ICIs for tumor therapy.
More importantly, tumor therapy based on NPTT has been verified to effectively induce ICD and turn ‘cold’ tumors into ‘hot’ [[26], [27], [28]]. The ablated tumor tissue by NPTT can produce TAAs to induce antigenic specific immune responses like tumor vaccines which provide long-term immune memory effects, thereby continuously inhibiting tumor growth, recurrence, and metastasis inhibition [29,30]. Recent studies have also shown that PTT-induced temperature rise can also upregulate PD-L1 expression of tumor cells, so tumor immune effect activated by PTT alone is difficult to completely inhibit primary and metastatic tumor growth [[31], [32], [33]]. Therefore, NPTT combined with targeted PD-1/PD-L1 axis can play a synergistic anti-tumor effect, alleviate the tumor inhibitory microenvironment, and allow the complete elimination of primary and metastatic tumors [[34], [35], [36]]. However, most of the current studies simply use NPTT and ICIs alone, and do not make full use of the targeted delivery performance of nanomaterials and antibodies, thus failing to solve the problem of ‘on target but off tumor’ faced by ICIs.
Here, we proposed a combined all-in-one strategy to prepare photothermal immune-integrated nanocomposites (Scheme 1). The nanocomposites, termed AuNDs@aPD-1, were made by chemically conjugating the immune checkpoint blocker, the programmed receptor 1 antibody (aPD-1), on the surface of a new type of gold nanoparticle, gold nanodendrites (AuNDs) (Scheme 1A). We used AuNDs as a near-infrared (NIR) light-activated nanomaterial for photothermal stimulation, in conjunction with the aPD-1-induced immunotherapy (Scheme 1B), due to two major considerations. First, among nanomaterials used in photothermal tumor therapy, like Au nanomaterials [37], transition metal sulfides [38], and carbon-based nanomaterials [35,37,38], Au nanoparticles show unique advantages in photothermal tumor treatment due to their inherent characteristics of low toxicity and high photothermal conversion efficiency [39]. Second, AuNDs are novel gold nanoparticles with hyperbranched nanostructures that have a larger specific surface area and a more irregular shape. Thus, they can generate stronger localized surface plasmon resonance signals and present a longer circulation time in vivo, resulting in a stronger drug loading capability and a more efficient photothermal conversion ability than the other Au nanoparticles with smooth surfaces [40,41].
Osteosarcoma (OS) was used as a model for PTT combined with aPD-1 tumor therapy owing to the following three aspects: (1) OS is commonly considered a typical ‘cold’ tumor; (2) the incidence rate of OS is the highest in primary malignant bone tumors, which are often accompanied with local invasion, early distant metastasis, seriously threatening the life and health of patients; (3) OS often occurs in the proximal tibia and distal femur, with less soft tissue coverage and shallow location, which is conducive to NIR light penetration. Efficient co-delivery of AuNDs@aPD-1 into tumor tissue via a combination of nanomaterial-based passive targeting (i.e. through enhanced permeability and retention effect) and aPD-1 antibody-based active targeting is expected to achieve tumor targeting, NIR-induced release of TAA and aPD-1, and immune enhancement (Scheme 1B). Therefore, the NIR-stimulated AuNDs@aPD-1 provides a new strategy that is promising for treating patients with metastasis.
Section snippets
Characterization of AuNDs@aPD-1
First, we used transmission electron microscopy (TEM) and ultraviolet-visible (UV–vis) spectroscopy to evaluate gold seeds. We detected that the diameter of the synthesized gold nanoseeds was about 13 nm and spherical with an absorption peak about 524 nm (Fig. S1a). Next, we synthesized AuNDs by successfully using gold nanoseeds as cores (Fig. 1b). To verify whether proteins could be attached to AuNDs by chemical cross-linking methods, we chose bovine serum albumin (BSA) as a model protein.
Conclusion
Novel Au nanodendrites (AuNDs) served as the photothermal transduction and loading carriers for the loading of immune checkpoint blocker aPD-1 to prepare a multi-functional biomaterial. The resultant AuNDs@aPD-1 nanocomposites could treat ‘cold’ tumors with low immunogenicity. The strategy of long-term retention of drugs on nanomaterials to construct the complex not only fundamentally solved the problems of rapid diffusion and metabolism of local injection of free drug aPD-1 and immune-related
Materials and reagents
Gold chloride trihydrate (16961-25-4), oleylamine (909831), polyvinylpyrrolidone (9003-39-8), and ascorbic acid were obtained from Sigma-Aldrich. Thiol polyethylene glycol (PEG) (T164398), thiol PEG Acid (T164359), EDC (E106172), NHS (H109330) was purchased from Aladdin. Anti-PD-1 (CD279) was obtained from Bioxcell. Micro-BCA (23235) and FITC (46425) were purchased from Thermo Scientific. Mito-Tracker(C1048), Lyso-Tracker(C1046). Cy5 was purchased from Beyotime. The endocytosis inhibitors
Author contributions
GP. He and YJ. Shuai contributed equally to this work. CB. Mao, MY. Yang, and Y. Hai conceived and designed the project. GP. He, YJ. Shuai, T. Yang, XY. Pan, YZ. Liu, and XL. Meng performed the experiments and analyzed the data. GP. He, YJ. Shuai, HH. Yang and C. M. wrote the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by Beijing Chao-Yang Hospital Golden Seeds Foundation (CYJZ202148) at Chao-Yang Hospital, Beijing, China. This research was also sponsored by the National Natural Science Foundation of China (8187482, 81871499 and 31800807), Zhejiang Provincial Natural Science Foundation of China (LY22E030004), State of Sericulture Industry Technology System (CARS-18-ZJ0501). G.H is grateful for the support from the Medical Research Center of Beijing Chaoyang Hospital Affiliated to
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These authors contributed equally to this paper.