Human serum albumin as the carrier to fabricate STING-activating peptide nanovaccine for antitumor immunotherapy.

Tumor vaccines are emerging as one of the most promising therapeutic strategies for cancer treatment. With the advantages of low toxicity, convenient production and stable quality control, peptide vaccines have been widely used in preclinical and clinical trials involving various malignancies. However, when used alone, they still suffer from significant challenges including poor stability and immunogenicity as well as the low delivery efficiency, leading to limited therapeutic success. Herein, the STING-activating peptide nanovaccine based on human serum albumin (HSA) and biodegradable MnO2 was constructed, which can improve the stability and immunogenicity of antigenic peptides as well as facilitate their uptake by dendritic cells (DCs). Meanwhile, Mn2+ degraded from the nanovaccine can activate the STING pathway and further promote DCs maturation. In this way, the prepared nanovaccine can efficiently mediate T-cell immune responses, thereby exerting the effects of tumor prevention and therapy. Moreover, the prepared nanovaccine possesses the advantages of low cost, convenient preparation and good biocompatibility, showing great potential for practical applications.

Simultaneous Determination of Rifamycin Antibiotics and Their Active Metabolites in Human Plasma Using UHPLC-MS/MS to Evaluate Their Impact on Target Peak Concentrations: A Short Communication.

BACKGROUND: Standard and proper antituberculosis (anti-TB) treatment is essential for patients with TB, and rifamycin antibiotics are key components of anti-TB therapy. Therapeutic drug monitoring (TDM) of rifamycin antibiotics can shorten the time to response and complete treatment of TB. Notably, antimicrobial activities of the major active metabolites of rifamycin are similar to those of their parent compounds. Thus, a rapid and simple assay was developed for simultaneous determination of rifamycin antibiotics and their major active metabolites in plasma to evaluate their impact on target peak concentrations. Here, the authors have developed and validated a method for simultaneous determination of rifamycin antibiotics and their active metabolites in human plasma using ultrahigh-performance liquid chromatography tandem mass spectrometry. METHODS: Analytical validation of the assay was performed in accordance with the bioanalytical method validation guidance for industry described by the US Food and Drug Administration and the guidelines for bioanalytical method validation described by the European Medicines Agency. RESULTS: The drug concentration quantification method for rifamycin antibiotics, including rifampicin, rifabutin, and rifapentine, and their major active metabolites was validated. Significant differences in the proportions of active metabolites in rifamycin antibiotics may affect the redefinition of their effective concentration ranges in the plasma. The method developed herein is expected to redefine the ranges of "true" effective concentrations of rifamycin antibiotics (including parent compounds and their active metabolites). CONCLUSIONS: The validated method can be successfully applied for high-throughput analysis of rifamycin antibiotics and their active metabolites for TDM in patients receiving anti-TB treatment regimens containing these antibiotics. Proportions of active metabolites in rifamycin antibiotics markedly varied among individuals. Depending on the clinical indications of patients, the therapeutic ranges for rifamycin antibiotics may be redefined.

Biodegradable MnO2-based gene-engineered nanocomposites for chemodynamic therapy and enhanced antitumor immunity.

Immune checkpoint blockade (ICB) is emerging as a promising therapeutic approach for clinical treatment against various cancers. However, ICB based monotherapies still suffer from low immune response rate due to the limited and exhausted tumor-infiltrating lymphocytes as well as tumor immunosuppressive microenvironment. In this work, the cell membrane with surface displaying PD-1 proteins (PD1-CM) was prepared for immune checkpoint blockade, which was further combined with multifunctional and biodegradable MnO2 for systematic and robust antitumor therapy. The MnO2-based gene-engineered nanocomposites can catalyze the decomposition of abundant H2O2 in TME to generate O2, which can promote the intratumoral infiltration of T cells, and thus improve the effect of immune checkpoint blockade by PD-1 proteins on PD1-CM. Furthermore, MnO2 in the nanocomposites can be completely degraded into Mn2+, which can catalyze the generation of highly toxic hydroxyl radicals for chemodynamic therapy, thereby further enhancing the therapeutic effect. In addition, the prepared nanocomposites possess the advantages of low cost, easy preparation and good biocompatibility, which are expected to become promising agents for combination immunotherapy.

Self-assembled traditional Chinese nanomedicine modulating tumor immunosuppressive microenvironment for colorectal cancer immunotherapy.

Colorectal cancer (CRC), mostly categorized as a low immunogenic microsatellite-stable phenotype bearing complex immunosuppressive tumor microenvironment (TME), is highly resistant to immunotherapy. Seeking safe and efficient alternatives aimed at modulating tumor immunosuppressive TME to improve outcome of CRC is highly anticipated yet remains challenging. Methods: Enlightened from the drug complementary art in traditional Chinese medicine, we designed a self-assembled nanomedicine (termed LNT-UA) by the natural active ingredients of ursolic acid (UA) and lentinan (LNT) through a simple nano-precipitation method, without any extra carriers, for CRC immunotherapy. Results: UA induces immunogenic cell death (ICD), while LNT further promotes dendritic cell (DC) maturation and repolarizes tumor-associated macrophage (TAM) from a protumorigenic M2 to an antitumor M1 phenotype. Co-delivery of UA and LNT by LNT-UA effectively reshapes the immunosuppressive TME and mobilizes innate and adaptive immunity to inhibit tumor progression in the CT26 CRC tumor model. Following the principle of integrative theoretical system of traditional Chinese medicine (TCM) on overall regulation, the further combination of LNT-UA and anti-CD47 antibody (αCD47) would reinforce the antitumor immunity by promoting phagocytosis of dying tumor cells and tumor-associated antigens (TAAs), leading to effective suppression of both primary and distant tumor growth with 2.2-fold longer of median survival time in the bilateral tumor model. Most notably, this combination effect is also observed in the spontaneous CRC model induced by chemical carcinogens, with much less and smaller size of tumor nodules after sequential administration of LNT-UA and αCD47 through gavage and intraperitoneal injection, respectively. Conclusions: This study provides a promising self-assembled traditional Chinese nanomedicine to improve immunotherapy for CRC, which might be applicable for future clinical translation.

Development and evaluation of a new test kit for determination of immunosuppressants in blood by UHPLC-MS/MS.

In this study, we have developed and validated a new test kit that applied pure solvents instead of blood samples for determination of immunosuppressant drugs in blood by ultra high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). We have used commercially available quality control samples to verify this new test kit, and the results showed that the performance of our new kit was comparable with commercially available kits by immunoassays. The new test kit is pure solvent-based, which saves the cost and can be directly loaded without pre-treatment, thus shortening the detection time. The agreement between pure solvent-based kit and blood-based kit was also evaluated by Bland-Altman plot and two-tailed Student's T-test. For 97.08% of the 137 Tacrolimus clinical samples, the concentration difference between these two kits was laid within 20% of the mean concentration. The concentrations of 12 cyclosporine samples and 15 sirolimus samples obtained by both kits showed no significant difference evaluated by the two-tailed Student's T-test. These results further demonstrated the good agreement between these two types of test kits. Meanwhile, our pure solvent-based test kit could be stored stably for 14 weeks at -30 °C. Therefore, the newly developed pure solvent-based test kit can be used for detecting the whole blood immunosuppressant concentration and therapeutic drug monitoring of immunosuppressants.

CD16/PD-L1 bi-specific aptamer for cancer immunotherapy through recruiting NK cells and acting as immunocheckpoint blockade.

It is well established that natural killer (NK) cells can be used as an alternative candidate of T cells for adoptive cell therapy (ACT) due to its high killing capacity, off-the-shelf utility, and low toxicity. Though NK cells provide rapid and potent immune effects, they still suffer from insufficient infiltration and tumor immunosuppression environment, which result in unsatisfactory therapeutic efficiency. Herein, a highly stable CD16/PD-L1 bi-specific aptamer (defined as CP-bi-apt) with high affinity and selectivity was introduced to overcome these obstacles. This CP-bi-apt can mediate a significant antitumor immunity by recruiting CD16-positive NK cells to directly contact with PD-L1 high-expressed tumor cells. In addition, the induced up-regulation of PD-L1 on tumor cells can inevitably occur as an adaptive response to most of the immunotherapeutic strategies. The prepared CP-bi-apt can be further used as an immune checkpoint inhibitor to specifically bind to PD-L1, thus reducing the negative impact of PD-L1 over-expression on the therapeutic efficacy. Furthermore, this CP-bi-apt-based immunotherapy is simple, highly efficient, and has low side effects, showing a promising potential for clinical translation.

A highly stable multifunctional aptamer for enhancing antitumor immunity against hepatocellular carcinoma by blocking dual immune checkpoints.

T-lymphocytes play a potent role in cancer immunotherapy; while, limited tumor infiltrating lymphocytes (TILs) combined with severe immunosuppression always significantly hinder their antitumor immune responses, especially in solid tumors such as hepatocellular carcinoma (HCC). Here, we prepared a highly stable multifunctional aptamer for strengthening antitumor immunity against HCC solid tumors through a dual immune checkpoint blockade of CTLA-4 and PD-L1. The engineered multifunctional aptamer (termed P1/C4-bi-apt) can block both CTLA-4/B7 and PD-1/PD-L1 signaling pathways and thus enhance the antitumor immune responses. Furthermore, it can direct CTLA-4-positive T cells to infiltrate into tumors to further enhance the antitumor efficacy compared to a single blockage of CTLA-4 or PD-L1. As a result, the multifunctional aptamer can significantly inhibit tumor growth and thus improve the long-term survival of HCC-bearing mice. The designed multifunctional aptamer is simple, stable and easy to prepare, and it can significantly strengthen the functionality of T cells, holding great potential for HCC immunotherapy.

A novel long-wavelength off-on fluorescence probe for nitroreductase analysis and hypoxia imaging.

Hypoxia is one of the most important features of various diseases including solid tumors. It is of significant clinical importance for hypoxia analysis in tumors, and the analysis of hypoxia can be moved away to the indirect detection of nitroreductase. Various fluorescence methods have been developed for the analysis of nitroreductase and tumor hypoxia due to their simplicity, non-invasiveness and excellent spatial-temporal resolution. Here, we synthesized a multifunctional fluorophore, which possesses larger π-conjugation structure, leading to the red-shift of absorption and fluorescence emission to long-wavelength region. This fluorophore (RCOH) can be further used to prepare the off-on hypoxia probe (RCO-NTR) by introducing the electron withdrawing group of nitrobenzyl, leading to the quenching of fluorescence emission through an electron transfer process. After reaction with NTR accompanied by NADH as an electron donor, the nitro group in the RCO-NTR probe can be reduced and then the RCOH fluorophore can be released through rearrangement and elimination, leading to the enhancement of fluorescence signal. Thus, the prepared RCO-NTR probe can be used for nitroreductase analysis, which can be further used for hypoxia imaging at both cellular and animal levels.

Protein-assisted formation of gold clusters-MnO2 nanocomposite for fluorescence imaging of intracellular glutathione.

Herein, a bovine serum albumin-stabilized gold clusters-MnO2 nanocomposite (BSA@AuNCs-MnO2) was constructed. Manganese dioxide (MnO2) was generated in situ on the gold clusters (BSA@AuNCs) based on the redox reaction between bovine serum albumin (BSA) and potassium permanganate (KMnO4). The fluorescence of BSA@AuNCs can be quenched by the in-situ grown MnO2, which has strong light absorption ability. It is worth noting that the quenched fluorescence of the BSA@AuNCs can be restored in the presence of glutathione (GSH), and MnO2 was reduced to Mn2+ in return. Encouragingly, 1 µM GSH can cause a detectable fluorescence change. This sensitivity is comparable to other nanomaterials based fluorescent probes. Furthermore, this nanocomposite has obvious superiorities, such as good uniformity, simple preparation and mild reaction. The nanocomposite also has good stability and specificity, which can be further used for visualizing of intracellular GSH.

Water-soluble organic probe for pH sensing and imaging.

pH value is one of the most important parameters, which show significant application in environmental monitoring, chemistry and biology. Abnormal pH values always associate with some serious diseases, including cancer and Alzheimer's disease. Thus, development of highly sensitive and selective method for pH sensing and imaging is of great importance. In this paper, we synthesized a water-soluble organic probe for pH sensing either through its absorption or through its fluorescent signals. The probe was synthesized from the intermediate containing a phenol group, and the reaction was carried out in concentrated H2SO4 at 90 °C. In this way, the probe can introduce a sulfonic acid group into its structure, and thus improve its water solubility. The synthesized probe is pH-responsive, and the response process is reversible, because that the phenol group in the probe can transfer to deprotonation state with increasing the pH values to improve the intramolecular charge transfer. Meanwhile, the synthesized probe also showed high specificity and excellent biocompatibility, which is suitable for cell imaging applications.

Artificial Engineered Natural Killer Cells Combined with Antiheat Endurance as a Powerful Strategy for Enhancing Photothermal-Immunotherapy Efficiency of Solid Tumors.

Although photothermal therapy (PTT) is preclinically applied in solid tumor treatment, incomplete tumor removal of PTT and heat endurance of tumor cells induces significant tumor relapse after treatment, therefore lowering the therapeutic efficiency of PTT. Herein, a programmable therapeutic strategy that integrates photothermal therapeutic agents (PTAs), DNAzymes, and artificial engineered natural killer (A-NK) cells for immunotherapy of hepatocellular carcinoma (HCC) is designed. The novel PTAs, termed as Mn-CONASHs, with 2D structure are synthesized by the coordination of tetrahydroxyanthraquinone and Mn2+ ions. By further adsorbing polyetherimide/DNAzymes on the surface, the DNAzymes@Mn-CONASHs exhibit excellent light-to-heat conversion ability, tumor microenvironment enhanced T1 -MRI guiding ability, and antiheat endurance ability. Furthermore, the artificial engineered NK cells with HCC specific targeting TLS11a-aptamer decoration are constructed for specifically eliminating any possible residual tumor cells after PTT, to systematically enhance the therapeutic efficacy of PTT and avoid tumor relapse. Taken together, the potential of A-NK cells combined with antiheat endurance as a powerful strategy for immuno-enhancing photothermal therapy efficiency of solid tumors is highlighted, and the current strategy might provide promising prospects for cancer therapy.

Sensitive fluorometric determination of glutathione using fluorescent polymer dots and the dopamine-melanin nanosystem.

A bioinspired fluorometric method has been developed for the detection of glutathione (GSH) in biological fluids. It is based on the use of near-infrared fluorescent semiconducting polymer dots (P-dots) and of the dopamine (DA)-melanin nanosystem. The P-dots were prepared from poly(styrene-co-maleic anhydride), the semiconducting polymer poly[(9,9'-dioctyl-2,7-divinylenefluorenylene)-alt-2-methoxy-5-(2-ethyl-hexyloxy)-1,4-phenylene] and the fluorescent dye tetraphenylporphyrin. They have excitation/emission maxima at 458/656 nm, and this enables measurement to be performed with low autofluorescence and scattering background. DA can self-polymerize on the surface of the P-dots to yield a poly-DA coating. This coating, at weak alkaline pH values, causes the quenching of the fluorescence of the P-dots. However, the polymerization of DA is inhibited by GSH. Hence, quenching of fluorescence is prevented. This effect was used to design a fluorometric assay for GSH that has good selectivity and sensitivity. Under optimal conditions, the method has a linear response in the 0.2 to 20 µM GSH concentration range and a 60 nM detection limit. It was successfully applied to the determination of GSH in HepG2 cells and in spiked human serum. Graphical abstract Schematic representation of using a NIR fluorescent P-dots and dopamine (DA)-melanin nanohybrid as a probe for glutathione (GSH) detection. The P-dots were prepared from poly(styrene-co-maleic anhydride) (PSMA), the semiconducting polymer poly[(9,9'-dioctyl-2,7-divinylenefluorenylene)-alt-2-methoxy-5-(2-ethyl-hexyloxy)-1,4-phenylene] (PEPV) and the fluorescent dye tetraphenylporphyrin (TPP).The GSH can inhibit the dopamine self-polymerization and prevented the formation of PDA and fluorescence quenching of P-dots.

Dual sensing of glutathione and acidic pH values by using MnO2 nanosheets and 3-acetyl-7-hydroxy-2H-chromen-2-one as a fluorescent pH probe.

A multifunctional nanoprobe is described for dual sensing of acidic pH values and glutathione (GSH) by combining the pH-responsive fluorescent probe 3-acetyl-7-hydroxy-2H-chromen-2-one (AHC) and MnO2 nanosheets. The fluorescence of the MnO2/AHC composite is weak due to an inner filter effect. If, however, the MnO2 nanosheets are reductively decomposed by GSH, the blue fluorescence of the pH probe AHC (with excitation/emission maximum at 417/456 nm) will be restored. The MnO2 nanosheets also are decomposed by acidic pH values, and the fluorescence of AHC is decreased. According to absorbance and fluorescence signal changes, the pH and GSH induced responses can be easily distinguished. Thus, the nanoprobe can be used for logical analysis of acidic pH values and GSH. The nanoprobe works in the pH range from 4 to 7, and GSH can be determined in the concentration range from 0.5 to 200 µM. Graphical abstract Schematic presentation of a multifunctional nanoprobe for dual sensing of acidic pH values and glutathione by combining a pH-responsive fluorescent probe and MnO2 nanosheets. According to absorbance and fluorescence signal changes, the nanoprobe can be used for logical analysis.

Highly efficient redox reaction between potassium permanganate and 3,3',5,5'-tetramethylbenzidine for application in hydrogen peroxide based colorimetric assays.

Potassium permanganate (KMnO4) is one of the most important oxidants, which plays important roles in many fields. Here, we found that KMnO4 could directly induce the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate an oxidized product with a color change. This redox reaction is highly efficient, and 1 µM KMnO4 is enough to cause detectable changes in the absorbance signal. Meanwhile, this reaction is very fast and the generated blue product can stabilize for a relatively long period, which has great advantages in practical applications. Since hydrogen peroxide (H2O2) is able to react with KMnO4 under acidic conditions, the KMnO4-TMB system can be used for the detection of H2O2; the absorbance signal induced by 5 µM H2O2 can be easily detected in this method. Meanwhile, the KMnO4-TMB system can also be used for the detection of glucose by monitoring the generation of H2O2, which is the main product of glucose oxidation; this method permits detection of concentrations as low as 10 µM glucose, and the sensitivity is comparable to or higher than most peroxidase mimetic based methods, but avoiding the preparation and storage of the nanomaterials. Furthermore, the KMnO4-TMB system can even be used for analyzing glucose in serum samples, which can also be expected to be used in immunoassays.

Photoresponsive Nanovehicle for Two Independent Wavelength Light-Triggered Sequential Release of P-gp shRNA and Doxorubicin To Optimize and Enhance Synergistic Therapy of Multidrug-Resistant Cancer.

Prerelease of RNA molecules than chemotherapeutic drugs with a sufficient interval is a vital prerequisite for RNA/drug co-delivery strategy to overcome multidrug resistance (MDR) of cancer cells, but how to precisely control their release at different time points is still a grand challenge up to now. This study aims to on-demand remotely manipulate RNA and drug release in real time through single delivery system to sequentially play their respective roles for optimizing and enhancing their synergistic antitumor effects. To this end, a photoresponsive mesoporous silica nanoparticle (PMSN) is fabricated as a co-delivery vehicle of P-glycoprotein (P-gp) short-hairpin RNA (shRNA) and photocaged prodrug of doxorubicin (DOX), by which the orthogonal and sequential release of shRNA and DOX can be achieved using an external light. In our design, the cationic poly[2-( N, N-dimethylaminoethyl)methacrylate] is introduced onto the PMSN surface through a light-sensitive coumarin ester derivative linker to adsorb P-gp shRNA, whereas the photocleavable o-nitrobenzyl ester derivative-caged DOX is loaded into the inner pores of the PMSN. The PMSN is found to be effectively internalized by MDR cancer cells, and the release of the shRNA and DOX is demonstrated to be independently regulated by 405 and 365 nm light irradiations due to selectively cleaved coumarin and o-nitrobenzyl ester, resulting in enhanced drug retention, and finally bring out optimized and significantly improved chemotherapeutic effects both in vitro and in vivo for MDR cancer treatment, which might hold extensive application prospects in MDR cancer treatment in future.

Polydopamine-assisted versatile modification of a nucleic acid probe for intracellular microRNA imaging and enhanced photothermal therapy.

MicroRNAs play an important role in various biological processes, and their aberrant expression is closely associated with various human diseases, especially cancer. Real-time monitoring of microRNAs in living cells may help us to understand their role in cellular processes, which can further provide a basis for diagnosis and treatment. In this study, polydopamine was used to assist the versatile modification of a nucleic acid probe for intracellular microRNA imaging and enhanced photothermal therapy. Polydopamine can be covalently linked with a thiol-terminated nucleic acid probe through the Michael addition reaction under slightly alkaline conditions. This modification is mild and can be performed directly in an aqueous solution, which can better resist hydrolysis than the traditional modification processes, resulting in a nanoprobe with better stability and higher loading of nucleic acids. This prepared nanoprobe can easily enter cells without transfection agents and then realize the imaging of intracellular miRNA through fluorescence restoration. Moreover, the coating of PDA can enhance the photothermal conversion efficiency of the nanoprobe, making it suitable for photothermal therapy of cancer. It is expected that the PDA-based versatile modification can help to construct a promising platform for tumor imaging and treatment.

A cancer cell specific targeting nanocomplex for combination of mRNA-responsive photodynamic and chemo-therapy.

We have developed a cancer cell specific targeting nanocomplex which combines photodynamic therapy with chemotherapy through precisely responding to the intracellular tumor-related mRNA. The combined treatment of these two modalities showed significantly enhanced therapeutic effects on cancer cells.

A fluorescent turn on nanoprobe for simultaneous visualization of dual-targets involved in cell apoptosis and drug screening in living cells.

Herein, a novel dual-responsive two-color fluorescent nanoprobe has been designed for the fluorescence activation imaging of cell apoptosis in living cells. The nanoprobe consists of a gold nanoparticle core functionalized with a dense layer of DNA aptamers and peptides, which shows high affinity and specific response to cytochrome c (Cyt c) and caspase-3, respectively. The formation of the aptamer-Cyt c complex and the cleavage of the specific peptide by caspase-3 can liberate the dye labelled aptamers and peptides from the surface of gold nanoparticles, and then recover their fluorescence. The turn-on and specific recognition properties of our nanoprobe allow for the sensitive and selective detection of Cyt c concentration and caspase-3 activity both in solutions and in living cells. The here proposed nanoprobe with the abilities of real-time monitoring the cell apoptosis and evaluating the apoptosis-related drug efficacy might serve as a potential interesting tool for studying the molecular mechanisms of apoptosis regulation or screening the apoptosis-based drugs.

Tumor Microenvironment Activable Self-Assembled DNA Hybrids for pH and Redox Dual-Responsive Chemotherapy/PDT Treatment of Hepatocellular Carcinoma.

Smart self-assembled "Turn-ON" DNA hybrids are employed, which could respond to tumor microenvironment stimuli for cancer cell specific real-time fluorescence imaging, tumor-specific synergistic photodynamic therapy and chemotherapy in hepatocellular carcinoma.

Smart Cu(II)-aptamer complexes based gold nanoplatform for tumor micro-environment triggered programmable intracellular prodrug release, photodynamic treatment and aggregation induced photothermal therapy of hepatocellular carcinoma.

This study describes smart Cu(II)-aptamer complexes based gold nanoplatform for tumor micro-environment triggered programmable prodrug release, in demand photodynamic therapy and aggregation induced photothermal ablation of hepatocellular carcinoma. The nanoplatform is consist of monodispersed gold nanoparticle (GNP) that is binding to HCC cell specific targeting aptamers (TLS11a) through Au-S bond; the aptamer is labeled with Ce6 at the 5'end and coordinated with Cu(II) through (GA)10 repeating bases to load AQ4N at the 3' end. In normal physiological conditions, the fluorescence and ROS generation ability of Ce6 are quenched by GNPs via RET; but in cancerous cells, the fluorescence and the ROS generation of Ce6 could be recovered by cleavage of Au-S bond through high level of intracellular GSH for real-time imaging and in demand PDT. Meanwhile, the prodrug AQ4N release could be triggered by acid-cleavage of coordination bonds, then accompanied by a release of Cu(II) that would induce the electrostatic aggregation of GNPs for photo-thermal ablation; furthermore, the significantly enhanced chemotherapy efficiency could be achieved by PDT produced hypoxia to convert AQ4N into AQ4. In summary, here described nanoplatform with tumor cell specific responsive properties and programmable PDT/PTT/chemotherapy functions, might be an interesting synergistic strategy for HCC treatment.