While immunotherapy has become a highly promising paradigm for cancer treatment in recent years, it has long been recognized that photodynamic therapy (PDT) has the ability to trigger antitumor immune responses. However, conventional PDT triggered by visible light has limited penetration depth, and its generated immune responses may not be robust enough to eliminate tumors. Herein, upconversion nanoparticles (UCNPs) are simultaneously loaded with chlorin e6 (Ce6), a photosensitizer, and imiquimod (R837), a Toll-like-receptor-7 agonist. The obtained multitasking UCNP-Ce6-R837 nanoparticles under near-infrared (NIR) irradiation with enhanced tissue penetration depth would enable effective photodynamic destruction of tumors to generate a pool of tumor-associated antigens, which in the presence of those R837-containing nanoparticles as the adjuvant are able to promote strong antitumor immune responses. More significantly, PDT with UCNP-Ce6-R837 in combination with the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) checkpoint blockade not only shows excellent efficacy in eliminating tumors exposed to the NIR laser but also results in strong antitumor immunities to inhibit the growth of distant tumors left behind after PDT treatment. Furthermore, such a cancer immunotherapy strategy has a long-term immune memory function to protect treated mice from tumor cell rechallenge. This work presents an immune-stimulating UCNP-based PDT strategy in combination with CTLA-4 checkpoint blockade to effectively destroy primary tumors under light exposure, inhibit distant tumors that can hardly be reached by light, and prevent tumor reoccurrence via the immune memory effect.Keywords: checkpoint blockade; immune memory; immunotherapy; photodynamic therapy; upconversion nanoparticles;

Benefiting from their unique physicochemical properties, graphene derivatives have attracted great attention in biomedicine. In this study, we carefully engineered graphene oxide (GO) as a vaccine adjuvant for immunotherapy using urease B (Ure B) as the model antigen. Ure B is a specific antigen for Helicobacter pylori, which is a class I carcinogen for gastric cancer. Polyethylene glycol (PEG) and various types of polyethylenimine (PEI) were used as coating polymers. Compared with single-polymer modified GOs (GO–PEG and GO–PEI), certain dual-polymer modified GOs (GO–PEG–PEI) can act as a positive modulator to promote the maturation of dendritic cells (DCs) and enhance their cytokine secretion through the activation of multiple toll-like receptor (TLR) pathways while showing low toxicity. Moreover, this GO–PEG–PEI can serve as an antigen carrier to effectively shuttle antigens into DCs. These two advantages enable GO–PEG–PEI to serve as a novel vaccine adjuvant. In the subsequent in vivo experiments, compared with free Ure B and clinically used aluminum-adjuvant-based vaccine (Alum-Ure B), GO–PEG–PEI–Ure B induces stronger cellular immunity via intradermal administration, suggesting promising applications in cancer immunotherapy. Our work not only presents a novel, highly effective GO-based vaccine nano-adjuvant, but also highlights the critical roles of surface chemistry for the rational design of nano-adjuvants.

Whether graphene and graphene oxide (GO) would affect the activities of bacteria has been under debate. Nevertheless, how graphene derivatives with biocompatible coatings interact with microorganisms and the underlying mechanisms are important issues for nanobiotechnology, and remain to be further explored. Herein, three new types of nano-GOs functionalized with polyethylene glycol (nGO-PEGs) were synthesized by varying the PEGylation degree, and their effects on Escherichia coli (E. coli) were carefully investigated. Interestingly, nGO-PEG (1:1), the one with relatively lower PEGylation degree, could significantly stimulate bacterial growth, whereas as-made GO and the other two nGO-PEGs showed no effect. Further analysis revealed that nGO-PEG (1:1) treatment significantly accelerated FtsZ-ring assembly, shortening Phase 1 in the bacterial cell cycle. Both DNA synthesis and extracellular polymeric substance (EPS) secretion were also dramatically increased. This unique phenomenon suggests promising potentials in microbial engineering as well as in clinical detection of bacterial pathogens. As a proof-of-concept, nGO-PEG (1:1) treatment could remarkably enhance (up to 6-fold) recombinant protein production in engineered bacteria cells. To our best knowledge, this is the first demonstration of functionalized GO as a novel, positive regulator in microbial engineering. Moreover, our work highlights the critical role of surface chemistry in modulating the interactions between nanomaterials and microorganisms.

As professional antigen presenting cells, dendritic cells (DCs) greatly determine the quality of the innate and adaptive immunities. Therefore, DC-based immunotherapy has been one of the hotspots in cancer immunotherapy in recent years. Although this unique therapeutic strategy has been approved by U.S. Food and Drug Administration for prostate cancer treatment, the efficacy of DC-based immunotherapy remains to be further improved. Moreover, it is still not completely clear about the immunological basis of DCs, which is another hurdle for the progress of DC-based immunotherapy. Due to their unique physicochemical properties, nanomaterials have shown potentials in addressing these above mentioned problems and have provided important guidelines for optimizing DC-based immunotherapy. However, it is still at the starting stage for this emerging field and there are many critical questions in the rational design of this therapeutic strategy to be answered. Therefore, it is greatly necessary to review and analyze recent progresses in this field. In this review, we mainly focus on the development of various types nanoparticles for DC-based immunotherapy. The existed challenges in this field are also discussed.专职性抗原呈递细胞-树突状细胞在天然免疫和获得性免疫反应的发生和调控方面发挥着极其重要的关键作用。近年来, 基于树突状细胞的免疫治疗成为肿瘤免疫治疗领域的研究热点。虽然该治疗策略已被批准应用于临床前列腺癌的治疗, 但其治疗效果尚有待于进一步提高。同时, 人类尚未完全揭示树突状细胞的免疫基础, 这成为该免疫治疗策略的另一重要瓶颈。近年来, 凭借其独特的理化性质, 纳米材料在提高树突状细胞免疫治疗效果以及示踪树突状细胞方面显示出良好的应用前景, 为优化该治疗策略提供了重要信息。然而, 这一新兴领域尚处于研究初期, 对于如何科学合理地设计该治疗策略尚有许多关键科学问题亟待研究。本综述主要集中于对近年来纳米材料应用于树突状细胞免疫治疗研究的总结与分析, 并对该领域中存在的挑战与机遇进行了深入讨论。

Recently, conjugated polymers have been widely explored in the field of nanomedicine. Careful evaluations of their biological effects are thus urgently needed. Hereby, we systematically evaluated the biological effects of different types of conjugated polymers on macrophages and dendritic cells (DCs), which play critical roles in the innate and adaptive immune systems, respectively. While naked poly-(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) (PEDOT:PSS) exhibits a high level of cytotoxicity, polyethylene glycol (PEG) modified PEDOT:PSS (PEDOT:PSS-PEG) shows greatly reduced toxicity to various types of cells. To our surprise, PEGylation of PEDOT:PSS could obviously enhance the cellular uptake of these nanoparticles, leading to subsequent immune stimulations of both macrophages and DCs. In contrast, another type of conjugated polymer, polypyrrole (PPy), is found to be an inert material with neither significant cytotoxicity nor noticeable immune-stimulation activity. Interestingly, utilizing ovalbumin (OVA) as a model antigen, it is further uncovered in our ex vivo experiment that PEDOT:PSS-PEG may serve as an adjuvant to greatly enhance the immunogenicity of OVA upon simple mixing. Our study on the one hand suggests the promise of developing novel nano-adjuvants based on conjugated polymers, and on the other hand highlights the importance of careful evaluations of the impacts of any new nanomaterials developed for nanomedicine on the immune systems.

In the past few years, graphene and its derivative, graphene oxide (GO), have been extensively studied for their applications in biotechnology. In our previous work, we reported certain PEGylated GOs (GO-PEGs) can selectively promote trypsin activity and enhance its thermostability. To further explore this, here we synthesized a series of GO-PEGs with varying PEGylation degrees. Enzymatic activity assay shows that both GO and GO-PEGs can protect trypsin, but not chymotrypsin, from thermal denaturation at high temperature. Surprisingly, the lower the PEGylation degree, the better the protection, and GO as well as the GO-PEG with the lowest PEGylation degree show the highest protection efficiency (∼70% retained activity at 70 °C). Fluorescence spectroscopy analysis shows that GO/GO-PEGs have strong interactions with trypsin. Molecular Dynamics (MD) simulation results reveal that trypsin is adsorbed onto the surface of GO through its cationic residues and hydrophilic residues. Different from chymotrypsin adsorbed on GO, the active site of trypsin is covered by GO. MD simulation at high temperature shows that, through such interaction with GO, trypsin’s active site is therefore stabilized and protected by GO. Our work not only illustrates the promising potential of GO/GO-PEGs as efficient, selective modulators for trypsin, but also provides the interaction mechanism of GO with specific proteins at the nano–bio interface.Keywords: enzyme thermostability; graphene oxide; molecular dynamics simulation; nano−bio interface; trypsin;

A dendritic cell (DC) vaccine, which is based on efficient antigen delivery into DCs and migration of antigen-pulsed DCs to draining lymph nodes after vaccination, is an effective strategy in initiating CD8+ T cell immunity for immunotherapy. Herein, antigen-loaded upconversion nanoparticles (UCNPs) are used to label and stimulate DCs, which could be precisely tracked after being injected into animals and induce an antigen-specific immune response. It is discovered that a model antigen, ovalbumin (OVA), could be adsorbed on the surface of dual-polymer-coated UCNPs via electrostatic interaction, forming nanoparticle–antigen complexes, which are efficiently engulfed by DCs and induce DC maturation and cytokine release. Highly sensitive in vivo upconversion luminescence (UCL) imaging of nanoparticle-labeled DCs is successfully carried out, observing the homing of DCs to draining lymph nodes after injection. In addition, strong antigen-specific immune responses including enhanced T cell proliferation, interferon gamma (IFN-γ) production, and cytotoxic T lymphocyte (CTL)-mediated responses are induced by a nanoparticle-pulsed DC vaccine, which is promising for DC-based immunotherapy potentially against cancer.Keywords: DC vaccine; immunotherapy; sensitive tracking; UCNP;

In this article, we present a kind of silicon-based antibacterial material made of silver nanoparticle (AgNP)-decorated silicon wafers (AgNP@Si), which is facilely and rapidly (30 min) synthesized via a one-step reaction. Significantly, such a resultant silicon-based antibacterial material features stable and high antibacterial activity, preserving >99% antibacterial efficiency against E. coli during 30 day storage.

Because circulating tumor cells (CTCs) have been proven to be an important clue of the tumor metastasis, their detection thus plays a pivotal role in the diagnosis and prognosis of cancer. Herein, we fabricate an electrochemical sensor by directly conjugating two cell-specific aptamers, TLS1c and TLS11a, which specifically recognize MEAR cancer cells, to the surface of a glassy carbon electrode (GCE) via the formation of amide bonds. The two aptamers are simultaneously conjugated to the GCE surface via precisely controlled linkers: TLS1c through a flexible linker (a single-stranded DNA T15; ss-TLS1c) and TLS11a through a rigid linker (a double-stranded DNA T15/A15; ds-TLS11a). It is found that such ss-TLS1c/ds-TLS11a dual-modified GCEs show greatly improved sensitivity in comparison with those modified with a single type of aptamer alone or ds-TLS1c/ds-TLS11a with both rigid linkers, suggesting that our optimized, rationally designed electrode–aptamer biosensing interface may enable better recognition and thus more sensitive detection of tumor cells. Through the utilization of this dual-aptamer-modified GCE, as few as a single MEAR cell in 109 whole blood cells can be successfully detected with a linear range of 1–14 MEAR cells. Our work demonstrates a rather simple yet well-designed and ultrasensitive tumor cell detection method based on the cell-specific aptamer-modified GCE, showing a promising potential for further CTC-related clinical applications.Keywords: biosensing interface; cell-specific aptamer; chemically modified electrode; circulating tumor cells; electrochemical detection;

The development of new antibacterial agents that are highly effective are of great interest. Herein, we present a recyclable and synergistic nanocomposite by growing both iron oxide nanoparticles (IONPs) and silver nanoparticles (AgNPs) on the surface of graphene oxide (GO), obtaining GO-IONP-Ag nanocomposite as a novel multifunctional antibacterial material. Compared with AgNPs, which have been widely used as antibacterial agents, our GO-IONP-Ag shows much higher antibacterial efficiency toward both Gram-negative bacteria Escherichia coli (E. coli) and Gram-positive bacteria Staphylococcus aureus (S. aureus). Taking the advantage of its strong near-infrared (NIR) absorbance, photothermal treatment is also conducted with GO-IONP-Ag, achieving a remarkable synergistic antibacterial effect to inhibit S. aureus at a rather low concentration of this agent. Moreover, with magnetic IONPs existing in the composite, we can easily recycle GO-IONP-Ag by magnetic separation, allowing its repeated use. Given the above advantages as well as its easy preparation and cheap cost, GO-IONP-Ag developed in this work may find potential applications as a useful antibacterial agent in the areas of healthcare and environmental engineering.Keywords: antibacterial; graphene nanocomposite; photothermal; recycling; synergistic effect;

Due to the high specificity, low side effects, and great efficacy, photothermal therapy using light energy to burn cancer has been proposed as an attractive alternative to traditional cancer therapies. In the recent few years, researchers have found that various conjugated polymers with strong absorbance in the near-infrared (NIR) window, if appropriately functionalized, could serve as highly effective photothermal agents, showing encouraging cancer ablation results both in vitro and in vivo. Those polymers could also be utilized as drug delivery carriers to load many aromatic therapeutic agents with high loading efficiencies, promising for combination cancer therapy. Thus, this mini-review article would discuss the latest progress in the development of conjugated polymers for photothermal therapy of cancer.

Detection of circulating tumor cells (CTCs) plays an important role in cancer diagnosis and prognosis. In this study, aptamer-conjugated upconversion nanoparticles (UCNPs) are used for the first time as nanoprobes to recognize tumor cells, which are then enriched by attaching with magnetic nanoparticles (MNPs) and placing in the presence of a magnetic field. Owing to the autofluorescencefree nature of upconversion luminescence imaging, as well as the use of magnetic separation to further reduce background signals, our technique allows for highly sensitive detection and collection of small numbers of tumor cells spiked into healthy blood samples, and shows promise for CTC detection in medical diagnostics.

Potential toxicity and risk of inducing allergy and inflammation have always been a great concern of using nanomaterials in biomedicine. In this work, we investigate the serum behaviors of graphene oxide (GO) and how such behaviors are affected by its surface modification such as PEGylation. The results show that, when incubated with human sera, unfunctionalized GO adsorbs a significant amount of serum proteins and strongly induces complement C3 cleavage (part of the complement activation cascade), generating C3a/C3a(des-Arg), an anaphylatoxin involved in local inflammatory responses, whereas PEGylated nano-GO (nGO-PEG) exhibits dramatic reductions in both protein binding in general and complement C3 activation. Moreover, we uncover that PEGylation on GO nanosheets apparently generates an interesting nanointerface, evidenced by the acquired certain selectivity and increased binding capacities of nGO-PEG toward a few serum proteins. Further mass spectrometry analysis identifies six nGO-PEG binding proteins, four of which are immune-related factors, including C3a/C3a(des-Arg). A series of Western blot analysis demonstrate that nGO-PEG binds up to 2-fold amount of C3a/C3a(des-Arg) than unfunctionalized GO, and can efficiently decrease the level of C3a/C3a(des-Arg) in treated sera, preventing the normal interaction of C3a with its receptor. In a proof-of-concept experiment, we demonstrate that nGO-PEG may serve to help eliminate the C3a/C3a(des-Arg) induced by other nanomaterials such as as-made GO, indicating a new strategy to modulate the immune responses evoked by one nanomaterial through the addition of another type of nanomaterial. Our results highlight the great importance of nanobio interface in regulating the biological effects of nanomaterials.Keywords: anaphylatoxin; C3a/C3a(des-Arg); graphene oxide; immune response; nanobio interface; serum behavior;

Recently, graphene oxide (GO) based nanocomposites have raised significant interests in many different areas, one of which being antibacterial agents where sliver nanoparticle (AgNPs) anchored GO (GO–Ag) has shown promising potential. However, to our best knowledge, factors affecting its antibacterial activity as well as the underlying mechanism remain unclear. In this study, we fabricate GO–Ag nanocomposites with different AgNPs to GO ratios and carefully investigate their antibacterial activities against both the Gram-negative (G−) bacteria Escherichia coli (E. coli) and the Gram-positive (G+) bacteria Staphylococcus aureus (S. aureus). We discover that, compared to AgNPs, GO–Ag nanocomposite with an optimal ratio of AgNPs to GO is much more effective and shows synergistically enhanced, strong antibacterial activities at rather low dose (2.5 μg/mL). The GO–Ag nanocomposite is more toxic to E. coli than that to S. aureus. The antibacterial effects of GO–Ag nanocomposite are further investigated, revealing distinct, species-specific mechanisms. The results demonstrate that GO–Ag nanocomposite functions as a bactericide against the G– E. coli through disrupting bacterial cell wall integrity, whereas it exhibits bacteriostatic effect on the G+ S. aureus by dramatically inhibiting cell division. Our work not only highlights the great promise of using GO–Ag as a highly effective antibacterial agent but also provides more in-depth understandings of the interactions between microorganisms and GO-based nanocomposites.Keywords: antibacterial; bactericide; bacteriostatic agent; graphene oxide; sliver nanoparticle anchored graphene oxide (GO−Ag); species-specific mechanism;

The understanding of interactions between nanomaterials and biomolecules is of fundamental importance to the area of nanobiotechnology. Graphene and its derivative, graphene oxide (GO), are two-dimensional (2-D) nanomaterials with interesting physical and chemical properties and have been widely explored in various directions of biomedicine in recent years. However, how functionalized GO interacts with bioactive proteins such as enzymes and its potential in enzyme engineering have been rarely explored. In this study, we carefully investigated the interactions between serine proteases and GO functionalized with different amine-terminated polyethylene glycol (PEG). Three well-characterized serine proteases (trypsin, chymotrypsin, and proteinase K) with important biomedical and industrial applications were analyzed. It is found that these PEGylated GOs could selectively improve trypsin activity and thermostability (60–70% retained activity at 80 °C), while exhibiting barely any effect on chymotrypsin or proteinase K. Detailed investigation illustrates that the PEGylated GO-induced acceleration is substrate-dependent, affecting only phosphorylated protein substrates, and that at least up to 43-fold increase could be achieved depending on the substrate concentration. This unique phenomenon, interestingly, is found to be attributed to both the terminal amino groups on polymer coatings and the 2-D structure of GO. Moreover, an enzyme-based bioassay system is further demonstrated utilizing our GO-based enzyme modulator in a proof-of-concept experiment. To our best knowledge, this work is the first success of using functionalized GO as an efficient enzyme positive modulator with great selectivity, exhibiting a novel potential of GO, when appropriately functionalized, in enzyme engineering as well as enzyme-based biosensing and detection.Keywords: enzyme engineering; graphene oxide; nano-bio interfaces; serine protease; trypsin

Interactions between DNA binding proteins and specific DNA elements are the fundamental basis of many biological pathways during gene expression and regulation. The sequence diversity of DNA elements leads to affinity variation, which could play important roles in regulatory and/or pathogenic processes. Therefore detection and analysis of such interactions, particularly the interactions between essential protein factors and their various DNA targets, are crucial for unveiling the molecular mechanisms behind these processes. For this purpose, a simple electrochemical method based on protein-modified electrode was developed. Transcription factor Sp1, an important and well-studied DNA binding protein, was used as a proof example and immobilized onto a glass carbon electrode (GCE) surface, with the wild type Sp1 consensus binding sequence (wtDNA) and a mutant sequence carrying two point mutations (mutDNA) within the Sp1 binding site served as testing samples. Binding of DNA samples to GCE-immobilized Sp1 was analyzed by electrochemical impedance spectroscopy (EIS), and the wtDNA showed a 3-fold higher change of the charge-transfer resistance (Rct) value than that of the mutDNA, correlating well with the known high affinity of wtDNA to Sp1. The reversible disassociation and re-association of Sp1-DNA complexes were also achieved and monitored closely by EIS. Further detection of a series of concentrations of wtDNA using difference pulse voltammetry (DPV) showed that as low as 1 × 10−10 mol/L wtDNA can be selectively detected in a DNA pool containing 0.05 g/L DNA fragments derived from salmon sperm. Exhibiting high affinity resolution, excellent selectivity, and low detection limit, this functionalized and reusable electrode provides a promising and convenient electrochemical approach for label-free, rapid monitoring, and comprehensive study of the specific interactions and/or affinity changes of DNA binding proteins with their various target DNAs under different conditions.Highlights► The detection of interactions between protein and DNA. ► Simple electrochemical method based on protein-modified electrode was developed. ► Binding of DNA to GCE-immobilized Sp1 was analyzed by EIS.

This article reviews the latest progresses regarding the applications of carbon nanotubes (CNTs), including single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs), as multifunctional nano-probes for biomedical imaging. Utilizing the intrinsic band-gap fluorescence of semi-conducting single-walled carbon nanotubes (SWNTs), fluorescence imaging in the near infrared II (NIR-II) region with enhanced tissue penetration and spatial resolution has shown great promise in recent years. Raman imaging based on the resonance Raman scattering of SWNTs has also been explored by a number of groups for in vitro and in vivo imaging of biological samples. The strong absorbance of CNTs in the NIR region can be used for photoacoustic imaging, and their photoacoustic signals can be dramatically enhanced by adding organic dyes, or coating with gold shells. Taking advantages of metal nanoparticle impurities attached to nanotubes, CNTs can also serve as a T2-contrast agent in magnetic resonance (MR) imaging. In addition, when labeled with radioactive isotopes, many groups have developed nuclear imaging with functionalized CNTs. Therefore CNTs are unique imaging probes with great potential in biomedical multimodal imaging.Download high-res image (304KB)Download full-size image

As professional antigen presenting cells, dendritic cells (DCs) greatly determine the quality of the innate and adaptive immunities. Therefore, DC-based immunotherapy has been one of the hotspots in cancer immunotherapy in recent years. Although this unique therapeutic strategy has been approved by U.S. Food and Drug Administration for prostate cancer treatment, the efficacy of DC-based immunotherapy remains to be further improved. Moreover, it is still not completely clear about the immunological basis of DCs, which is another hurdle for the progress of DC-based immunotherapy. Due to their unique physicochemical properties, nanomaterials have shown potentials in addressing these above mentioned problems and have provided important guidelines for optimizing DC-based immunotherapy. However, it is still at the starting stage for this emerging field and there are many critical questions in the rational design of this therapeutic strategy to be answered. Therefore, it is greatly necessary to review and analyze recent progresses in this field. In this review, we mainly focus on the development of various types nanoparticles for DC-based immunotherapy. The existed challenges in this field are also discussed.

In this article, we present a kind of silicon-based antibacterial material made of silver nanoparticle (AgNP)-decorated silicon wafers (AgNP@Si), which is facilely and rapidly (30 min) synthesized via a one-step reaction. Significantly, such a resultant silicon-based antibacterial material features stable and high antibacterial activity, preserving >99% antibacterial efficiency against E. coli during 30 day storage.