Blood-declustering excretable metal clusters assembled in DNA matrix

Abstract

We report polymeric DNA-supported gold clusters that achieve interparticle plasmon-coupling, generate immunotherapeutic effects at the tumor tissue, but decluster in the bloodstream. As immunostimulating DNA, we used polyCpG DNA, which could act as a supporting matrix for metal clusters, enabling the clusters to decluster in the bloodstream. We constructed polyCpG-supported gold nanoclusters (AuPCN). For comparison with AuPCN, monomer CpG-bound gold nanoparticles (AuMC) were used. Unlike AuMC, AuPCN showed an interparticle plasmon-coupling effect and a higher light-to heat conversion efficiency. In the serum, AuPCN declustered to subunits. The CT26 tumor rechallenge of mice pretreated with AuPCN(+NIR) was followed by 0% tumor recurrence and 100% survival for up to 80 days. Compared with other groups, AuPCN(+NIR)-treated mice revealed greater cytotoxic T cell-infiltration in distant tumors and higher memory T cells in the lymph nodes. Until 7 days post-dose, the urinary excretion of Au was observed in the AuPCN-treated group, but not in the Au nanoparticle-treated mice. Although we used gold clusters and concatemeric immunostimulatory CpG as components of AuPCN, the concept of declustering in the bloodstream can be applied to design other functional DNA scaffold-based metal clusters with reduced concerns for long-term retention in the body.

Introduction

Metal nanomaterials have been widely studied for biomedical applications, and numerous studies have examined the applications of gold, silver, and iron oxide nanoparticles in areas such as imaging [1,2], drug delivery [3], and phototherapies [4,5]. In these studies, the reported sizes of the metal nanomaterials have been in the ranges of 10–200 nm [6,7].

Despite reports of therapeutic applications of metal nanomaterials, there has been little clinical translation of these systems. One of the barriers to clinical translation is the nondegradable feature of metallic nanomaterials, which may be retained in the body for a prolonged period leading to side effects. Indeed, gold, iron oxide, and silver nanomaterials have been reported to induce toxicity upon chronic exposure [8]. The side effects of prolonged retention after administration include inflammation [9], pulmonary oxidative damage [10], neural damage [11], and tissue damage [12].

Among the various biomedical applications of metal nanomaterials, photothermal immunotherapy has gained research attention. The sizes of metal nanomaterials have been reported to play crucial roles in both photothermal properties and functionality [13]. A recent study found that the absorption cross-section is directly proportional to the size of a gold nanoparticle. Since the heat conversion efficiency depends on the absorption coefficient of the material, a larger gold nanoparticle can have increased photothermal efficacy. That said, it is difficult for particles bigger than 10 nm to undergo renal filtration [14]. Thus, it could be beneficial for metal nanomaterials with higher functionality at larger sizes to exert their functions at the target sites at full size and then rapidly shrink in the bloodstream to facilitate their renal elimination.

To achieve this size change between the injection site and the bloodstream, one might apply the concept of interparticle plasmon-coupling (IPC) effects of clusters. IPC occurs when plasmon particles are located closely, substantially magnifying the plasmon effect of single particle. IPC of clustered metal nanomaterials is known to be more efficient than surface plasmon effect of singular metal nanomaterials [15]. In the tumor tissues, the condensed metal clusters can exert IPC-mediated photothermal effects. They can then degrade to single-unit nanoparticles smaller than 10 nm, which should facilitate renal clearance.

To stimulate the induction of systemic immune responses after photothermal therapy, immune adjuvants have been loaded in various nanoparticles. Chemical adjuvants, such as imiquimod [16] and lipopolysaccharide [17,18], have been loaded inside or on the surfaces of nanoparticles. CpG, an oligomeric DNA, has been studied as an immune adjuvant that binds to toll-like receptor 9 [19,20] and induces the maturation and antigen presentation of dendritic cells (DC) [[21], [22], [23]]. However, the rapid degradation of CpG DNA sequences in the bloodstream has limited the widespread use of CpG as an immune adjuvant. Moreover, the use of CpG alone cannot inhibit the growth of primary tumors, which would still require surgical resection [24] or other modalities of primary tumor treatment [25,26].

In this study, we set out to leverage the rapid degradation of CpG DNA in the blood to design a serum-responsive size-shrinking metal nanomaterial as a photothermal immunotherapeutic. We aimed to design metal clusters that function effectively in tumor tissues but shrink to smaller unit sizes in the bloodstream. To enable this shrinkage at the bloodstream, we used biodegradable polymeric CpG DNA as a matrix for the metal clusters. Concatemeric polyCpG DNA was obtained by rolling-circle amplification (RCA); we hypothesized that it would play multiple roles by acting as a supporting matrix for the metal clusters, functioning as a toll-like receptor 9 (TLR9) immune adjuvant, and enabling size-shrinking at the bloodstream.

Here, we report that the immunostimulatory polyCpG DNA-supported gold clusters provided stronger photothermal and immunotherapeutic efficacy than monomeric CpG DNA-bound gold nanoparticles. The CpG DNA matrix-supported condensed assembly of gold clusters provided IPC effects and enhanced photothermal efficacy upon near infrared (NIR) light irradiation. As a TLR9 adjuvant, the CpG activated DC to take up NIR-irradiated tumor cells and present tumor antigens. Moreover, the polymeric CpG DNA-based gold clusters exhibited declustering at the serum, potentially reducing its undesirable retention in the body. Proposed working hypothesis is depicted in Fig. 1.