Surface-engineered gold nanorods: promising DNA vaccine adjuvant for HIV-1 treatment.

With the intense international response to the AIDS pandemic, HIV vaccines have been extensively investigated but have failed due to issues of safety or efficacy in humans. Adjuvants for HIV/AIDS vaccines are under intense research but a rational design approach is still lacking. Nanomaterials represent an obvious opportunity in this field due to their unique physicochemical properties. Gold nanostructures are being actively studied as a promising and versatile platform for biomedical application. Herein, we report novel surface-engineered gold nanorods (NRs) used as promising DNA vaccine adjuvant for HIV treatment. We have exploited the effects of surface chemistry on the adjuvant activity of the gold nanorod by placing three kinds of molecules, that is, cetyltrimethylammonium bromide (CTAB), poly(diallydimethylammonium chloride) (PDDAC), and polyethyleneimine (PEI) on the surface of the nanorod. These PDDAC- or PEI-modified Au NRs can significantly promote cellular and humoral immunity as well as T cell proliferation through activating antigen-presenting cells if compared to naked HIV-1 Env plasmid DNA treatment in vivo. These findings have shed light on the rational design of low-toxic nanomaterials as a versatile platform for vaccine nanoadjuvants/delivery systems.

A general strategy towards personalized nanovaccines based on fluoropolymers for post-surgical cancer immunotherapy.

Cancer metastases and recurrence after surgical resection remain an important cause of treatment failure. Here we demonstrate a general strategy to fabricate personalized nanovaccines based on a cationic fluoropolymer for post-surgical cancer immunotherapy. Nanoparticles formed by mixing the fluoropolymer with a model antigen ovalbumin, induce dendritic cell maturation via the Toll-like receptor 4 (TLR4)-mediated signalling pathway, and promote antigen transportation into the cytosol of dendritic cells, which leads to an effective antigen cross-presentation. Such a nanovaccine inhibits established ovalbumin-expressing B16-OVA melanoma. More importantly, a mix of the fluoropolymer with cell membranes from resected autologous primary tumours synergizes with checkpoint blockade therapy to inhibit post-surgical tumour recurrence and metastases in two subcutaneous tumour models and an orthotopic breast cancer tumour. Furthermore, in the orthotopic tumour model, we observed a strong immune memory against tumour rechallenge. Our work offers a simple and general strategy for the preparation of personalized cancer vaccines to prevent post-operative cancer recurrence and metastasis.

Nanoparticle-Enhanced Radiotherapy to Trigger Robust Cancer Immunotherapy.

External radiotherapy is extensively used in clinic to destruct tumors by locally applied ionizing-radiation beams. However, the efficacy of radiotherapy is usually limited by tumor hypoxia-associated radiation resistance. Moreover, as a local treatment technique, radiotherapy can hardly control tumor metastases, the major cause of cancer death. Herein, core-shell nanoparticles based poly(lactic-co-glycolic) acid (PLGA) are fabricate, by encapsulating water-soluble catalase (Cat), an enzyme that can decompose H2 O2 to generate O2 , inside the inner core, and loading hydrophobic imiquimod (R837), a Toll-like-receptor-7 agonist, within the PLGA shell. The formed PLGA-R837@Cat nanoparticles can greatly enhance radiotherapy efficacy by relieving the tumor hypoxia and modulating the immune-suppressive tumor microenvironment. The tumor-associated antigens generated postradiotherapy-induced immunogenic cell death in the presence of such R837-loaded adjuvant nanoparticles will induce strong antitumor immune responses, which together with cytotoxic T-lymphocyte associated protein 4 (CTLA-4) checkpoint blockade will be able to effectively inhibit tumor metastases by a strong abscopal effect. Moreover, a long term immunological memory effect to protect mice from tumor rechallenging is observed post such treatment. This work thus presents a unique nanomedicine approach as a next-generation radiotherapy strategy to enable synergistic whole-body therapeutic responses after local treatment, greatly promising for clinical translation.

Light-Triggered In Situ Gelation to Enable Robust Photodynamic-Immunotherapy by Repeated Stimulations.

Photodynamic therapy (PDT) has shown the potential of triggering systemic antitumor immune responses. However, while the oxygen-deficient hypoxic tumor microenvironment is a factor that limits the PDT efficacy, the immune responses after conventional PDT usually are not strong enough to eliminate metastatic tumors. Herein, a light-triggered in situ gelation system containing photosensitizer-modified catalase together with poly(ethylene glycol) double acrylate (PEGDA) as the polymeric matrix is designed. Immune adjuvant nanoparticles are further introduced into this system to trigger robust antitumor immune responses after PDT. Following local injection of the mixed precursor solution into tumors and the subsequent light exposure, polymerization of PEGDA can be initiated to induce in situ gelation. Such hybrid hydrogel with long-term tumor retention of various agents and the ability to enable persistent tumor hypoxia relief can enable multiple rounds of PDT, which results in significantly enhanced immune responses by multiround stimulation. Further combination of such gel-based multiround PDT with anticytotoxic T-lymphocyte antigen-4 checkpoint blockade offers not only the abscopal effect to inhibit growth of distant tumors but also effective long-term immune memory protection from rechallenged tumors. Therefore, such a light-triggered in situ gelation system by a single-dose injection can enable greatly enhanced photoimmunotherapy by means of repeated stimulations.

Photosensitizer-crosslinked in-situ polymerization on catalase for tumor hypoxia modulation & enhanced photodynamic therapy.

Tumor hypoxia is known to be one of critical factors that aggravate the tumor resistance to photodynamic therapy (PDT) in which oxygen is essential for tumor destruction. Herein, catalase, an enzyme to trigger hydrogen peroxide (H2O2) decomposition, is modified by in-situ free radical polymerization, using meso-tetra(p-hydroxyphenyl) porphine (THPP) as the cross-linker to enable condensed grafting of short polyethylene glycol (PEG) chains on the protein surface as a permeable brush-like safeguard. The formulated catalase-entrapped nanocapsules (CAT-THPP-PEG) with enhanced enzyme stability can be labeled with 99mTc4+, a radioisotope ion that is chelated by the porphyrin structure of THPP, to allow in vivo single-photon emission computed tomography (SPECT) imaging. It is found that such CAT-THPP-PEG nanoparticles exhibit efficient tumor passive retention after intravenous injection, and are able to greatly relieve tumor hypoxia by triggering the decomposition of tumor endogenous H2O2 into oxygen. With THPP functioning as a photosensitizer, in vivo PDT is further conducted, achieving a remarkable antitumor therapeutic effect. This work presents an enzyme modification strategy by in-situ polymerization with photosensitizer as the cross-linker to develop multifunctional nano-theranostics with strengthened enzymatic stability, efficient tumor passive homing, SPECT imaging capability, enhanced PDT efficacy as well as decreased immunogenicity, promising for clinical translation.

Cancer Cell Membrane-Coated Adjuvant Nanoparticles with Mannose Modification for Effective Anticancer Vaccination.

Tumor vaccines for cancer prevention and treatment have attracted tremendous interests in the area of cancer immunotherapy in recent years. In this work, we present a strategy to construct cancer vaccines by encapsulating immune-adjuvant nanoparticles with cancer cell membranes modified by mannose. Poly(d,l-lactide- co-glycolide) nanoparticles are first loaded with toll-like receptor 7 agonist, imiquimod (R837). Those adjuvant nanoparticles (NP-R) are then coated with cancer cell membranes (NP-R@M), whose surface proteins could act as tumor-specific antigens. With further modification with mannose moiety (NP-R@M-M), the obtained nanovaccine shows enhanced uptake by antigen presenting cells such as dendritic cells, which would then be stimulated to the maturation status to trigger antitumor immune responses. With great efficacy to delay tumor development as a prevention vaccine, vaccination with such NP-R@M-M in combination with checkpoint-blockade therapy further demonstrates outstanding therapeutic efficacy to treat established tumors. Therefore, our work presents an innovative way to fabricate cancer nanovaccines, which in principle may be applied for a wide range of tumor types.

One-pot synthesis of pH-responsive charge-switchable PEGylated nanoscale coordination polymers for improved cancer therapy.

Nanoscale coordination polymers (NCPs) are promising nanomedicine platforms featured with biodegradability and versatile functionalities. However, multi-step post-synthesis surface modification is usually required to functionalize as-made NCPs before their biomedical applications. Moreover, efforts are still required to design therapeutic NCPs responsive to the unique tumor microenvironment to achieve more specific and effective therapy. Herein, we uncover a simple yet general strategy to synthesize a series of polyethylene glycol (PEG) modified NCPs via a one-step method by adding poly-histidine-PEG co-polymer into the mixture of metal ions and organic ligands during NCPs formation. With NCPs consisting Ca2+/dicarboxylic cisplatin (IV) prodrug as the example, we show that such Ca/Pt(IV)@pHis-PEG NCPs are highly sensitive to pH changes. With slightly negative charges and compact structure under pH 7.4 during blood circulation, those NCPs exhibit efficient passive accumulation in the tumor, in which the reduced pH (c.a. 6.5) would trigger charge conversion and size expansion to enhance their tumor retention and cell internationalization. After cellular uptake, NCPs within cell endo-/lysosomes with further reduced pH would then lead to decomposition of those NCPs and thus drug release. Chemotherapy with Ca/Pt(IV)@pHis-PEG NCPs in our animal tumor model demonstrates great efficacy under low drug doses, and is found to be particularly effective towards solid tumors with reduced pH.

Hollow MnO2 as a tumor-microenvironment-responsive biodegradable nano-platform for combination therapy favoring antitumor immune responses.

Herein, an intelligent biodegradable hollow manganese dioxide (H-MnO2) nano-platform is developed for not only tumor microenvironment (TME)-specific imaging and on-demand drug release, but also modulation of hypoxic TME to enhance cancer therapy, resulting in comprehensive effects favoring anti-tumor immune responses. With hollow structures, H-MnO2 nanoshells post modification with polyethylene glycol (PEG) could be co-loaded with a photodynamic agent chlorine e6 (Ce6), and a chemotherapy drug doxorubicin (DOX). The obtained H-MnO2-PEG/C&D would be dissociated under reduced pH within TME to release loaded therapeutic molecules, and in the meantime induce decomposition of tumor endogenous H2O2 to relieve tumor hypoxia. As a result, a remarkable in vivo synergistic therapeutic effect is achieved through the combined chemo-photodynamic therapy, which simultaneously triggers a series of anti-tumor immune responses. Its further combination with checkpoint-blockade therapy would lead to inhibition of tumors at distant sites, promising for tumor metastasis treatment.MnO2 nanostructures are promising TME-responsive theranostic agents in cancer. Here, the authors develop a nano-platform based on hollow H-MnO2 nanoshells able to modulate the tissue microenvironment, release a drug and inhibit tumor growth alone or in combination with check-point blockade therapy.

Photothermal therapy with immune-adjuvant nanoparticles together with checkpoint blockade for effective cancer immunotherapy.

A therapeutic strategy that can eliminate primary tumours, inhibit metastases, and prevent tumour relapses is developed herein by combining adjuvant nanoparticle-based photothermal therapy with checkpoint-blockade immunotherapy. Indocyanine green (ICG), a photothermal agent, and imiquimod (R837), a Toll-like-receptor-7 agonist, are co-encapsulated by poly(lactic-co-glycolic) acid (PLGA). The formed PLGA-ICG-R837 nanoparticles composed purely by three clinically approved components can be used for near-infrared laser-triggered photothermal ablation of primary tumours, generating tumour-associated antigens, which in the presence of R837-containing nanoparticles as the adjuvant can show vaccine-like functions. In combination with the checkpoint-blockade using anti-cytotoxic T-lymphocyte antigen-4 (CTLA4), the generated immunological responses will be able to attack remaining tumour cells in mice, useful in metastasis inhibition, and may potentially be applicable for various types of tumour models. Furthermore, such strategy offers a strong immunological memory effect, which can provide protection against tumour rechallenging post elimination of their initial tumours.

Stimulation of immune systems by conjugated polymers and their potential as an alternative vaccine adjuvant.

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.

Graphene Oxide Selectively Enhances Thermostability of Trypsin.

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.

Immunological responses triggered by photothermal therapy with carbon nanotubes in combination with anti-CTLA-4 therapy to inhibit cancer metastasis.

Photothermal ablation of primary tumors with single-walled carbon nanotubes is demonstrated to be able to trigger significant adaptive immune responses, which are not observed if tumors are removed by surgical resection. Such a treatment in combination with anti-CTLA-4 antibody therapy is able to prevent the development of tumor metastasis, which is a major cause of cancer death.

Surface chemistry and aspect ratio mediated cellular uptake of Au nanorods.

Gold nanorods (Au NRs) have been recognized as promising materials for biomedical applications, like sensing, imaging, gene and drug delivery and therapy, but their toxicological issues are still controversial, especially for the Au NRs synthesized with seed-mediated method. In this study, we investigated the influence of aspect ratio and surface coating on their toxicity and cellular uptake. The cellular uptake is highly dependent on the aspect ratio and surface coating. However, the surface chemistry has the dominant roles since PDDAC-coated Au NRs exhibit a much greater ability to be internalized by the cells. The present data demonstrated shape-independent but coating-dependent cytotoxicity. Both the CTAB molecules left in the suspended solution and on the surface of Au NRs were identified as the actual cause of cytotoxicity. CTAB can enter cells with or without Au NRs, damage mitochondria, and then induce apoptosis. The effects of surface coating upon toxicity and cellular uptake were also examined using Au NRs with different coatings. When Au NRs were added into the medium, the proteins were quickly adsorbed onto the Au NRs that made the surface negatively charged. The surface charge may not directly affect the cellular uptake. We further demonstrated that the amount of serum proteins, especially for BSA, adsorbed on the Au NRs had a positive correlation with the capacity of Au NRs to enter cells. In addition, we have successfully revealed that the cationic PDDAC-coated Au NRs with an aspect ratio of 4 possess an ideal combination of both negligible toxicity and high cellular uptake efficiency, showing a great promise as photothermal therapeutic agents.