Elsevier

Biomaterials

Volume 35, Issue 37, December 2014, Pages 9963-9971
Biomaterials

Immunostimulatory oligonucleotides-loaded cationic graphene oxide with photothermally enhanced immunogenicity for photothermal/immune cancer therapy

https://doi.org/10.1016/j.biomaterials.2014.08.036Get rights and content

Abstract

Graphene oxide (GO) has attracted tremendous research interest due to its excellent electrical, thermal, and mechanical properties. Here, we apply the polyethylene glycol (PEG) and polyethylenimine (PEI) dual-polymer-functionalized GO (GO-PEG-PEI) as the carrier for efficient CpG delivery. GO-PEG-PEI can significantly promote the production of proinflammatory cytokines and enhance the immunostimulatory effect of CpG. In addition, the NIR optical absorbance of GO-PEG-PEI has been further applied to control the immunostimulatory activity of CpG ODNs, showing remarkably enhanced immunostimulation responses under NIR laser irradiation, owing to the photothermally induced local heating that accelerated intracellular trafficking of nanovectors. This is the first demonstration of using the photothermally enhanced intracellular transportation of nanocarriers for light-controllable CpG delivery. In vivo assay demonstrates that the GO-PEG-PEI-CpG complex provides synergistic photothermal and immunological effects under laser irradiation for cancer treatment, which shows the highest efficiency in tumor reduction, implying the excellent therapeutic efficacy of the GO-PEG-PEI-CpG complex in cancer therapy.

Introduction

Graphene, a one-atom-thick two-dimensional (2D) layer of sp2-bonded carbon, has attracted tremendous research interest since its discovery in 2004 [1], [2], [3], [4]. Due to their impressive electrical, thermal, and mechanical properties [5], [6], [7], [8], [9], [10], [11], graphene and its derivatives, such as graphene oxide (GO) and reduced graphene oxide (rGO), have been extensively studied for applications in a large variety of fields including nanoelectronics, molecule sensing, composite materials, energy research, catalysis, and more recently biomedicine [12], [13], [14], [15], [16], [17], [18], [19], [20]. Among these applications, the promise of using graphene and its functionalized derivatives as robust nanocarriers for molecular payloads delivery has become particularly attractive. The payloads can be small drug molecules or large biomolecules, like anticancer drugs [21], [22], [23], [24], [25], photosensitizers [26], proteins [14], [27], or nucleic acids [28], [29], [30], [31], [32], [33]. Recently, graphene-based materials have been reported that they can significantly activate macrophages and trigger the production of proinflammatory cytokines, and thus may be used as the available candidates of immunoadjuvants to promote vaccine efficacy [34], [35], [36], [37], [38]. Yet, despite these burgeoning developments, studies focusing on graphene-based platform for vaccines or adjuvants delivery have scarcely been reported [39].

In the past few decades, oligonucleotide therapy has been widely investigated for the treatment of various diseases, such as viral infections, cancer, and neurological disorders [40], [41], [42], [43]. In addition to conventional plasmid DNA-based gene therapy, artificial nucleic acids, including aptamers, antisense DNA, and small interfering RNAs (siRNAs) et al., have also been recognized as promising oligonucleotide drugs as a consequence of their easy synthesis with high reproducibility and purity, perfect sequence control, simple operation and conformational polymorphism nature [44], [45], [46], [47]. Especially, recent research activities are directed to unmethylated cytosine-phosphate-guanine (CpG) motifs, which act as a type of therapeutic nucleic acids with strong immunostimulatory activities [48]. The mammalian immune system can recognize the CpG oligodeoxynucleotides (ODNs) through Toll-like receptor 9 (TLR9) and secrete a number of proinflammatory cytokines, including tumor necrosis factors TNF-α and interleukin IL-6 [49], [50], which are able to stimulate a cascade of innate and adaptive immune responses. Although synthetic CpG ODNs have therefore become a favorable tool for immunotherapeutic applications in both basic research and clinical trials [51], [52], the delivery of these synthetic nucleic acids to disease sites still remains a great challenge [41]. For example, since nucleic acids are negatively charged, they cannot easily cross the electronegative cell membrane. Moreover, they can be rapidly degraded by nucleases [41]. Therefore, the development of new materials that can serve as efficacious CpG delivery vehicles capable of efficiently transferring CpG into target cells and preventing CpG degradation is of key importance [53].

The emergence of nanobiotechnology has provided unprecedented opportunities to develop effective vectors to transport CpG ODNs into target cells. For instance, various DNA assemblies have emerged as effective carriers for CpG ODN delivery [54], [55], [56], [57], [58], [59], [60], [61]. Alternatively, many novel nanomaterials such as nanoliposomes [62], gold nanoparticles [44], [63], [64], [65], carbon nanotubes [66], [67], boron nitride nanospheres [68], [69], and lanthanides-based core–shell nanoparticles [70] et al. have also been utilized as efficient carriers for CpG delivery. Although great progress has been achieved in this field, effective methods for facile synthesis of the simple yet efficient CpG delivery vehicles are still highly desirable. In this study, we synthesize the nanosized GO-PEG-PEI nanocomposite (GGI) consisting of GO sheets with covalently conjugated polyethyleneimine (PEI) and polyethylene glycol (PEG), which can be used as a promising carrier of CpG adjuvant. GO-PEG-PEI-CpG nanocomplex (GGIC) can significantly promote the production of proinflammatory cytokines and enhance the immunostimulatory effect. In addition, utilizing the NIR absorbance of GO, we show for the first time that the photothermal heating of GO can efficiently enhance the intracellular delivery of CpG ODNs, which can thus efficiently improve the immune responses (Scheme 1). Furthermore, we also investigate the synergistic effect of GGIC for combining photothermal therapy and immunotherapy in vivo, which shows the highest efficiency in tumor reduction, implying the excellent therapeutic efficacy of GGIC in cancer therapy.

Section snippets

Materials and measurements

Purified anti-mouse TNF-α, biotin conjugated anti-mouse TNF-α cocktail, TNF-α standard, anti-mouse IL-6, biotin anti-mouse IL-6 and IL-6 standard were purchased from eBioscience. OPD (o-phenylenediamine) substrate was obtained from DingGuo. PEI 25k and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma–Aldrich (USA). Graphite was obtained from Sinopharm Chemical Reagent Co., (Shanghai, China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was purchased from

Synthesis of the GO-PEG-PEI conjugates

To realize the GGI-based CpG delivery, the GGI was first prepared according to previous reports with a little modification [29], [30], as illustrated in Scheme 1. In brief, PEG with molecular weight (MW) of 10 kDa was firstly conjugated to GO to enhance the colloidal stability. Subsequently, PEI 25k was covalently coupled to GO using EDC chemistry. Excess unbound PEG and PEI was removed by centrifugation and repeated water washing. For the synthesis of GO-PEI (GI) and GO-PEG (GG), the whole

Conclusion

In summary, we have successfully used the biocompatible GGI as a multifunctional nanocarrier to load immune adjuvant CpG ODNs for photothermally enhanced immune response. The GGIC offers dramatically improved immunostimulatory activity due to the increased cellular uptake of CpG delivered by GGI and the immunogenicity of GGI. Moreover, the NIR optical absorbance of GGI has been further applied to control the immunostimulatory activity of CpG ODNs, showing remarkably enhanced immunostimulation

Acknowledgments

The authors are grateful for the referee's helpful comments on the manuscript. Financial support was provided by the National Basic Research Program of China (2011CB936004 and 2012CB720602) and the National Natural Science Foundation of China (Grants 21210002, 91213302, 21431007).

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