Cell Killing Ability Research Articles

Nanozyme for tumor therapy: Surface modification matters.

As the next generation of artificial enzymes, nanozymes have shown unique properties compared to its natural counterparts, such as stability in harsh environment, low cost, and ease of production and modification, paving the way for its biomedical applications. Among them, tumor catalytic therapy mediated by the generation of reactive oxygen species (ROS) has made great progress mainly from the peroxidase-like activity of nanozymes. Fe3O4 nanozymes, the earliest type of nanomaterial discovered to possess peroxidase-like activity, has consequently received wide attention for tumor therapy due to its ROS generation ability and tumor cell killing ability. However, inconsistent results of cytotoxicity were observed between different reports, and some even showed the scavenging of ROS in some cases. By collectively studying these inconsistent outcomes, we raise the question whether surface modification of Fe3O4 nanozymes, either through affecting peroxidase activity or by affecting the biodistribution and intracellular fate, play an important role in its therapeutic effects. This review will go over the fundamental catalytic mechanisms of Fe3O4 nanozymes and recent advances in tumor catalytic therapy, and discuss the importance of surface modification. Employing Fe3O4 nanozymes as an example, we hope to provide an outlook on the improvement of nanozyme-based antitumor activity.

A Single-Domain Antibody-Based Anti-PSMA Recombinant Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Therapy.

Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.

Construction of Fe3O4-Loaded Mesoporous Carbon Systems for Controlled Drug Delivery.

Magnetite (Fe3O4) nanoparticles as drug carriers can achieve precise drug target due to their magnetic property. However, they are easy to aggregate in the physiological environment, which obviously limits their application in drug delivery. The development of the Fe-MIL-88B-derived method to construct the Fe3O4-loaded mesoporous carbon (Fe3O4/carbon) system is a feasible strategy to solve the issue. First, iron atoms evenly distribute in the organic links through coordination bonds in Fe-MIL-88B. After the carbonization of Fe-MIL-88B, mesoporous carbon acts as a barrier to prevent the aggregation of Fe3O4 nanoparticles. Herein, Fe-MIL-88B particles were fabricated by the hydrothermal method and then pyrolyzed to construct Fe3O4/carbon systems. Results showed that Fe3O4 nanoparticles uniformly in situ grew on mesoporous carbon generated by the carbonization of organic components. More encouragingly, the Fe3O4/carbon system loaded with DOX demonstrated pH-responsive DOX release, efficient delivery of DOX into cancer cells, and significant cancer cell killing ability. Therefore, the Fe3O4/carbon systems prepared by the Fe-MIL-88B-derived method might open up a way for targeted and controlled drug delivery.

Multifunctional PLGA nanoparticles combining transferrin-targetability and pH-stimuli sensitivity enhanced doxorubicin intracellular delivery and in vitro antineoplastic activity in MDR tumor cells

Targeted delivery aims to enhance cellular uptake and improve therapeutic outcome with higher disease specificity. The expression of transferrin receptor (TfR) is upregulated on tumor cells, which make the protein Tf and its receptor vastly relevant when applied to targeting strategies. Here, we proposed Tf-decorated pH-sensitive PLGA nanoparticles containing the chemosensitizer poloxamer as a carrier for doxorubicin delivery to tumor cells (Tf-DOX-PLGA-NPs), aiming at alleviating multidrug resistance (MDR). We performed a range of in vitro studies to assess whether targeted NPs have the ability to improve DOX antitumor potential on resistant NCI/ADR-RES cells. All evaluations of the Tf-decorated NPs were performed comparatively to the nontargeted counterparts, aiming to evidence the real role of NP surface functionalization, along with the benefits of pH-sensitivity and poloxamer, in the improvement of antiproliferative activity and reversal of MDR. Tf-DOX-PLGA-NPs induced higher number of apoptotic events and ROS generation, along with cell cycle arrest. Moreover, they were efficiently internalized by NCI/ADR-RES cells, increasing DOX intracellular accumulation, which supports the greater cell killing ability of these targeted NPs with respect to MDR cells. Altogether, these findings supported the effectiveness of the Tf-surface modification of DOX-PLGA-NPs for an improved antiproliferative activity. Therefore, our pH-responsive Tf-inspired NPs are a promising smart drug delivery system to overcome MDR effect at some extent, enhancing the efficacy of DOX antitumor therapy.

Comparative Evaluation of Natural Killer Cell-mediated Cell Killing Assay Based on the Leakage of an Endogenous Enzyme or a Pre-loaded Fluorophore.

A cell-killing ability of natural killer (NK) cells has been evaluated by the leakage of marker molecules from target cells. Lactate dehydrogenase (LDH) and calcein are two major non-radioisotope markers used for the killing assay. The spontaneous death of NK cells during the killing-assay of cells is a major issue in the assay because it provides background signals to increase errors. In this study, the effect of the spontaneous death of NK cells on the killing assays based on LDH and the calcein method was comparatively evaluated. We found that the calcein method is much less sensitive to the spontaneous death of NK cells to enable an accurate evaluation of the cell killing.

Therapeutic efficacy and cardioprotection of nucleolin-targeted doxorubicin-loaded ultrasound nanobubbles in treating triple-negative breast cancer

Targeted lipid nanobubbles as theranostic ultrasound molecular probes with both targeted contrast-enhanced ultrasound molecular imaging and synergistic treatment capabilities are expected to overcome severe challenges in the diagnosis and treatment of refractory triple-negative breast cancer (TNBC). In this study, AS1411 aptamer-functionalised nucleolin-targeted doxorubicin-loaded lipid nanobubbles (AS1411-DOX-NBs) were constructed, and their physicochemical properties as well as anti-tumour and cardioprotective efficacies were systematically tested and evaluated. The results showed that AS1411-DOX-NBs can carry and maintain the physicochemical and pharmacodynamic properties of doxorubicin (DOX) and show stronger tumour cell-killing ability in vitro by increasing the active uptake of drugs. AS1411-DOX-NBs also significantly inhibited the growth of TNBC xenografts while maintaining the weight and health of the mice. Echocardiography and pathological examination further confirmed that AS1411-DOX-NBs effectively caused tumour tissue apoptosis and necrosis while reducing DOX-induced cardiotoxicity. The AS1411-DOX-NBs constructed in this study enable both targeted contrast-enhanced ultrasound molecular imaging and synergistic therapeutic efficacy and can be used as safe and efficient theranostic ultrasound molecular probes for the diagnosis and treatment of TNBC.

Crystal Phase-Related Toxicity of One-Dimensional Titanium Dioxide Nanomaterials on Kidney Cells.

One-dimensional (1D) nanomaterials are widely used in different fields, and the increased application of 1D nanomaterials has drawn concerns about their unknown toxicity. 1D titanium oxide (TiO2) nanomaterials in different crystal phases are commonly applied in environmental remediation and solar energy conversion fields, but these materials pose a threat to human health, especially to the kidneys, an organ with abundant blood flow. To systematically evaluate the cytotoxicity to the kidneys, TiO2 nanofibers with TiO2(B), anatase, and rutile phases, as well as nanorods with anatase and rutile phases were synthesized and added to the culture medium of HK2 cells. Cell counting kit-8 assay, 2',7'-dichlorofluorescin diacetate assay, Hoechst 33342 staining experiments, and quantitative real-time reverse transcription polymerase chain reaction tests were used to explore the renal effects of the as-prepared TiO2 nanomaterials in the short term or long term. In the short-term evaluation, all the added TiO2 nanomaterials were toxic to HK2 cells, and the cytotoxicity was dose-dependent. Rutile TiO2 can widely attach to the cell surface and displays the most serious cell-killing and proapoptotic ability, while anatase induces the most serious oxidative stress. In long-term evaluation, all the as-prepared TiO2 nanomaterials led to epithelial mesenchymal transition (EMT), a mechanism of renal fibrosis. Consistent with the short-term results, rutile induced the most serious EMT. This study indicated that the renal toxicity of 1D TiO2 nanomaterials is crystal phase-dependent and that rutile induced the most significant renal cell injury. Oxidative stress is a crucial but not the only contributor to the renal toxicity of TiO2 nanomaterials in the short term.

Antioxidant and Anti-proliferative Activities of Smilax Zeylanica Root and Rhizome Extract against Liver Carcinoma Cells

ABSTRACT The antioxidant, cytotoxic, and anti-cancer potential of aqueous root and rhizome extract (ARRE) of Smilax zeylanica was investigated against human liver carcinoma cells. The phytochemical composition was evaluated by standard methods, and confirmed by thin-layer chromatography. Free radical scavenging activity was determined by DPPH assay, cytotoxic activity by brine shrimp lethality assay, and anti-cancer potential was investigated using liver carcinoma HepG2 cells. Phytochemical analysis revealed the presence of saponins, flavonoids, proanthocyanins, tannins, and polyphenols. The total phenolic and flavonoid content varied from 0.28 to 5.26 mg gallic acid equivalent 100 mg−1 and 0.25–50.26 quercetin equivalent 100 mg−1, respectively. There was a linear relationship between the free radical scavenging activity and phytochemical concentrations with an IC50 value of 108 ± 1.2 µg mL−1 compared to the standard (IC50: 2.59 ± 0.02 µg mL−1). The brine shrimp lethality assay showed strong cell killing ability (IC50: 84 ± 0.7 µg mL−1) and the extract induced apoptosis of HepG2 cells (77 ± 1.52%) at 300 µg mL−1, comparable to the positive control (camptothecin).

68Ga-Labeled Magnetic-NIR Persistent Luminescent Hybrid Mesoporous Nanoparticles for Multimodal Imaging-Guided Chemotherapy and Photodynamic Therapy.

Featured with a zero-autofluorescence background, superior signal-to-noise ratio, high sensitivity, and deep penetration ability, near-infrared persistent luminescence nanoparticle (NIR-PLNP)-based multimodal nanoprobes show great potential for full-scale noninvasive cancer diagnosis. However, direct synthesis of NIR-PLNP-based multimodal nanoprobes with high drug loading capacity to meet growing cancer theranostic demands remains a challenge. In this work, multifunctional hybrid mesoporous nanoparticles (HMNPs) that integrate NIR-PLNPs (Ga2O3:Cr3+, Nd3+), magnetic nanoparticles (Gd2O3), and radionuclides (68Ga) are designed and constructed via a large-pore (mesoporous silica nanoparticle) MSN-templated strategy. The ingenious composition design endows HMNPs with rechargeable NIR-PL, superior longitudinal relaxivity, and excellent radioactivity, making these versatile nanoparticles available for long-term in vivo NIR-PL imaging, magnetic resonance imaging (MRI), and positron emission tomography (PET) imaging. More importantly, the application of large-pore MSN templates maintains the mesoporous structure of HMNPs, promising excellent drug loading capacity of these nanoparticles. As a proof-of-concept, HMNPs loaded with a high dose of DOX (chemotherapy agent) and Si-Pc (photosensitizer) are rationally designed for chemotherapy and NIR-PL-sensitized photodynamic therapy (PDT), respectively. Studies with mice tumor models demonstrate that the DOX/Si-Pc-loaded HMNPs possess excellent cancer cell killing ability and an outstanding tumor suppression effect without systemic toxicity. This work shows the great potential of HMNPs as an "all-in-one" nanotheranostic tool for multimodal NIR-PL/MR/PET imaging-guided chemotherapy and NIR-PL-sensitized photodynamic cancer therapy and provides an innovative paradigm for the development of NIR-PLNP-based nanoplatforms in cancer theranostic.

Novel EGFR‑bispecific recombinant immunotoxin based on cucurmosin shows potent anti‑tumor efficiency invitro.

Epidermal growth factor receptor (EGFR) is overexpressed in various tumors and is associated with cancer initiation, progression, and poor prognosis. Despite the achievements made by tyrosine kinase inhibitors and monoclonal antibodies in certain cases, many patients have not benefited from such treatment due to resistance. Immunotoxins (ITs) are antibody-cytotoxin chimeric molecules with specific cell killing ability, which have achieved different degrees of success in the treatment of a wide range of cancers in clinical trials. The aim of the current study was to examine a novel targeting EGFR recombinant immunotoxin Bs/cucurmosin (CUS) generated by fusing CUS to the EGFR-specific nanobody 7D12-9G8. Bs/CUS was successfully expressed in Escherichia coli strain BL21 (DE3) in a soluble form. Furthermore, it retained binding capacity and specificity with EGFR and was superior to rE/CUS, a monospecific IT we reported previously. In vitro results showed that Bs/CUS could be internalized into the cytoplasm and selectively kill cells in the picomolar range. Flow cytometry showed that Bs/CUS killed the cells mediated by the apoptosis pathway. Taken together, results of the current study indicated that Bs/CUS is a promising candidate that should be further evaluated as a cancer therapeutic for the treatment of EGFR-positive tumors.

CircRNA-SORE mediates sorafenib resistance in hepatocellular carcinoma by stabilizing YBX1

Sorafenib is the first-line chemotherapeutic therapy for advanced hepatocellular carcinoma (HCC). However, sorafenib resistance significantly limits its therapeutic efficacy, and the mechanisms underlying resistance have not been fully clarified. Here we report that a circular RNA, circRNA-SORE (a circular RNA upregulated in sorafenib-resistant HCC cells), plays a significant role in sorafenib resistance in HCC. We found that circRNA-SORE is upregulated in sorafenib-resistant HCC cells and depletion of circRNA-SORE substantially increases the cell-killing ability of sorafenib. Further studies revealed that circRNA-SORE binds the master oncogenic protein YBX1 in the cytoplasm, which prevents YBX1 nuclear interaction with the E3 ubiquitin ligase PRP19 and thus blocks PRP19-mediated YBX1 degradation. Moreover, our in vitro and in vivo results suggest that circRNA-SORE is transported by exosomes to spread sorafenib resistance among HCC cells. Using different HCC mouse models, we demonstrated that silencing circRNA-SORE by injection of siRNA could substantially overcome sorafenib resistance. Our study provides a proof-of-concept demonstration for a potential strategy to overcome sorafenib resistance in HCC patients by targeting circRNA-SORE or YBX1.

In situ pepsin-assisted needle assembly of magnetic-graphitic-nanocapsules for enhanced gastric retention and mucus penetration

Gastric environment is an extreme pH and enzyme-rich condition, which together with gastric mucus barrier and short retention time of oral medicine militate against effective oral drug delivery to lesion areas of gastric diseases. Assembly, especially shape-control assembly of magnetic nanoparticles potentially helps the site-selective drug molecule delivery. However, the harsh gastric condition, and cumbersome and strict requirements make the in situ reversible assembly remain challenging. Herein, synergistically pepsin-bridged and magnetic field-mediated morphological control of magnetic-graphitic-nanocapsules (MGNs) needle assembly (MNA) is developed in stomach for prolonged gastric retention and enhanced mucus penetration. The MNAs with good biocompatibility, large magnetic moments and unique needle-shape are formed with the help of pepsin, demonstrating superior magnetic-driven capability to overcome gastric mucus barrier. Molecular dynamics simulations reveal binding modes between MGN and amino acid residues on either side of the pepsin, indicating pepsin acts as “bridge” allowing sufficient contact among MGNs and further facilitates the needle assembly. Moreover, anticancer drug doxorubicin (DOX)-loaded MNAs demonstrate superior targeted cancer cell killing ability and boosted DOX penetration in a mouse model, which promises good bioavailability of drug molecules. The versatile MNA-based platform offers a robust strategy for effective oral drug delivery and site-selective therapy of gastric diseases.

Synergistic Combination Chemotherapy of Lung Cancer: Cisplatin and Doxorubicin Conjugated Prodrug Loaded, Glutathione and pH Sensitive Nanocarriers.

PurposeProdrug technology-based combination drug therapy has been exploited as a promising treatment strategy to achieve synergistic lung cancer therapy, reduce drug dose, and decrease side effects. In the present study, we synthesized a pH and glutathione (GSH) sensitive prodrug, cisplatin (CIS) and doxorubicin (DOX) conjugates (CIS-DOXp). CIS-DOXp was loaded by nanocarriers and delivered into the tumor site.MethodspH and GSH sensitive CIS-DOX prodrug (CIS-DOXp) was synthesized by conjugating GSH responsive CIS prodrug with pH sensitive DOX prodrug. CIS-DOXp-loaded nanocarriers (CIS-DOXp NC) were prepared using emulsification and solvent evaporation method. The morphology, particle size, polydispersity index (PDI) and zeta potential of nanocarriers were measured. In vitro cytotoxicity of nanocarriers and the corresponding free drugs was examined using the MTT assay. In vivo anti-tumor efficiency and biodistribution behaviors were evaluated on lung cancer mice models.ResultsThe size, PDI, zeta potential, CIS loading efficiency, and DOX loading efficiency of CIS-DOXp NC were 128.6 ± 3.2 nm, 0.196 ± 0.021, 15.7 ± 1.7 mV, 92.1 ± 2.1%, and 90.4 ± 1.8%, respectively. The best cell killing ability (the lowest combination index of 0.57) was found at the combination ratio of 1:3 (CIS:DOX, w/w) in the drugs co-loaded formulations, indicating the strongest synergism effect. CIS-DOXp NC showed the best tumor inhibition efficiency (79.9%) in mice with negligible body weight lost.ConclusionCIS-DOXp NC could be applied as a promising system for the synergistic chemotherapy of lung cancer.

Antitumor Efficacy of Oncolytic Herpes Virus Type 1 Armed with GM-CSF in Murine Uveal Melanoma Xenografts.

BackgroundUveal melanoma (UM) is the most common primary intraocular tumor in adults with a high incidence of metastasis. Standard care therapies for UM include enucleation and radiation, which are minimally effective in prolonging patient survival. Oncolytic virus treatment has become a new trend in cancer field. Of which, oncolytic herpes simplex virus type 1 (HSV-1) therapy is one of the most effective antitumor treatments. Here, we established an oncolytic HSV-1 encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), tested its efficacy in UM therapy, and investigated the innate immune response induced by this virus.MethodsOncolytic HSV-1 expressing GM-CSF (HSV-GM-CSF) was constructed, then verified using qPCR and Western blot assays. Cell viability assays and transmission electron microscopy were conducted on three UM cell lines, MUM2B, 92.1, and MP41, to assess the cell-killing ability and virus infection of this virus. For in vivo experiments, BALB/c-nude mice in situ UM xenografts were established to testify the efficacy of the oncolytic virus, oncolytic HSV-1, and HSV-GM-CSF groups, respectively. IVIS images, ocular volumes, mice weights, and survivals were tracked to see the efficacy of the virus. Hematoxylin and eosin staining, immunohistochemistry, and flow cytometry analyses were conducted to demonstrate the immune activity after virus treatment.ResultsAll three tested UM cell lines were sensitive to infection by HSV-GM-CSF. In vivo xenograft experiments revealed that oncolytic virus HSV-1 reduced UM tumor volume and that oncolytic virus HSV-1 armed with GM-CSF enhanced the antitumor effect compared with unmodified HSV-1. The bodyweights of untreated control group mice were significantly lower than those of mice in either virus-treated group (HSV-1 or HSV-GM-CSF). Follow-up survivals were prolonged in the virus-treated groups compared with the control group and were prolonged to a greater extent in the HSV-GM-CSF group than in the HSV-1 group. Macrophage stimulation was observed following HSV-GM-CSF treatment.ConclusionOur results indicate that the recombinant oncolytic virus HSV-GM-CSF is a potential therapeutic treatment for UM.

Liquid-phase exfoliation of black sesame to create a nanoplatform for in vitro photoluminescence and photothermal therapy.

Aim: The present study aims to apply the facile liquid-phase exfoliation (LPE) strategy to fabricate 2D organic materials and thus to broaden the family of biocompatible and multifunctional 2D materials. Materials & methods: 2D material-organic melanin and cellulose nanosheets were synthesized from black sesame hull using LPE. Photoluminescence and photothermal properties of the nanosheets were assessed, as well as stability and cell killing ability. Results: The prepared 2D nanoplatform exhibited broad and multiple photoluminescent emission bands. It also demonstrated efficient photothermal cancer therapy with excellent biocompatibility. Conclusion: The present study could open an avenue in exfoliating organic materials using the LPE strategy. This could make the fabrication of multifunctional 2D organic materials more efficient and broaden the family of biocompatible 2D nanomaterials.

Facilitating drug release in mesoporous silica coated upconversion nanoparticles by photoacid assistance upon near-infrared irradiation

Near-infrared (NIR) light-triggered pH-manipulation has been realized by utilizing upconversion nanoparticle assisted ring-closing reactions of the conventional photoacid merocyanine (MC). This pH manipulation behavior was then used to regulate the switch of an acid-labile cap for facilitating drug release on the basis of mesoporous silica coated upconversion nanoparticles, in which the drug release rate and amount and the cell killing ability have been greatly improved upon NIR light irradiation due to the locally high concentration of H+ within pore channels that is generated by upconversion assisted MC. This proof of concept may provide a way to utilize NIR light to regulate pH change for new drug delivery system designs and applications in biomedical field.

Gold nanoparticles gated mesoporous carbon with optimal particle size for photothermal-enhanced thermochemotherapy

Mesoporous carbon nanoparticles (MCNs) have been widely used in tumor photothermal therapy. Herein, MCNs with different particle sizes (100 nm, 400 nm and 500 nm) were fabricated in our work by controlling the ethanol amounts to evaluate their photothermal conversion performance. Rationally, small-sized MCNs with the highest photothermal conversion efficiency were selected as the drug-loading carriers. Subsequently, the spherical gold nanoparticles with the same size (4 nm) as the mesopores of MCNs were modified on the opening of MCNs as the gated switches, which could not only achieve the redox-responsive controlled release of anticancer drugs at tumor location, but also realize the photothermal-enhanced synergetic thermochemotherapy under a low power irradiation. Meanwhile, the photothermal conversion efficiency (η) of MCN-Au was 31.2 %, which was higher than that of MCNs (η = 28.3 %). DOX release increased significantly under the condition of high concentration of GSH, low pH and NIR, suggesting that the delivery platform was characterized by multi-responsive drug release. In addition, the combined therapeutic index of DOX/MCN-Au was 0.509, proving that the combination group of thermochemotherapy had the highest cell killing ability. Meanwhile, confocal laser scanning microscopy (CLSM) showed that DOX/MCN-Au had an increased cellular uptake efficiency under NIR irradiation. In summary, the constructed DOX/MCN-Au system was expected to show a clinical treatment potential for synergistic thermochemotherapy of tumors.

Oxygen-producing catalase-based prodrug nanoparticles overcoming resistance in hypoxia-mediated chemo-photodynamic therapy

Extreme hypoxia inside solid tumors is the primary barrier against the advance of chemotherapy and photodynamic therapy (PDT). To address this problem, a hybrid nano-enzyme prodrug system was developed to alleviate hypoxia as well as simultaneously sensitize chemo-photodynamic therapy. Lactobionic acid (LA) and doxorubicin (DOX) precursor (cis-aconitic anhydride-linked doxorubicin, CAD) were pre-conjugated onto the side chain of catalase (CAT), then co-assembled with chlorin e6 (Ce6) to form LA-CAT-CAD@Ce6 nanoparticles (LCC@Ce6-NPs). LA as the active-targeting ligand increased cellular internalization, CAD as the pH-sensitive component triggered rapid drug release, Ce6 as the photosensitizer induced reactive oxygen species (ROS) generation, and CAT decomposed intracellular H2O2 to produce oxygen in situ. Oxygen production efficiently decreased the expression of hypoxia-inducible factor-1α (HIF-1α) and P-glycoprotein (P-gp), which enhanced chemotherapy efficiency. In addition, sufficient oxygen further amplified PDT-mediated cell-killing and apoptosis in hypoxic tumor. In vivo studies showed that combined chemo-photodynamic therapy by LCC@Ce6-NPs led to the most effective inhibition of tumor growth (TGI>90%), and even partially ablated tumor. Thus, this nano-enzyme prodrug platform can be a potentially effective treatment in clinical cancer therapy, and married to other therapeutic agents. Statement of SignificanceHypoxia in solid tumors seriously impedes the efficacy of chemotherapy or photodynamic therapy. Herein, we designed hybrid nano-enzyme prodrug particles to improve hypoxia-mediated limitations on cancer therapy. Lactobionic acid (LA) as the hydrophilic outer layer of particles increased cellular uptake by receptor-mediated endocytosis, and cis-aconitic anhydride-linked doxorubicin (CAD) as the pH sensitive component inside particles efficiently triggered DOX and Ce6 release. More importantly, catalase (CAT) as the backbone of particles was capable of greatly relieving tumor hypoxia through catalyzing the decomposition of H2O2 in situ. Oxygen re-generation not only prevented hypoxia-mediated chemo-resistance, but also amplified PDT-induced ROS cell-killing ability. As a result, the multiple combination action of this nano-system could simultaneously sensitize chemo-photodynamic therapy, thus significantly enhancing tumor therapy.

Increased NKG2D expressing NK cells in patients with chronic HBV infection

Abstract Introductions Hepatitis B virus (HBV) infection remains a significant health issue worldwide. It is a major cause of cirrhosis, HCC and liver failure. A large number of studies showed that host immune responses are the key factor for HBV clearance and persistence. In here, we investigated the role of NK cells in chronic HBV infections. Methods We compared the expression frequency, and functionality of NKGs and KIRs repertoire on NK cells in 170 samples by multi-colour flow cytometry. To further investigate the functionality of the NK cells, we performed K562 target cell killing assay and cytokine driven perforin released assay. Results The percentage of circulation and liver resident NK cells did not significantly alter in HBV patients compared to the healthy controls (HCs). Enrichment of NK cells was observed in the healthy controls but not in the HBV infected patients. The intracellular expression of Granzyme B was significantly reduced in NK cells derived from HBV-infected patients as compared to HCs. No differences were observed in the expression of NKG2A and NKG2C. Interestingly, the levels of NKG2D was significantly increased in the circulating NK cells of HBV infection but not in the liver resident NK cells. The K562 target cell killing ability and IFN-gamma production correlated with the NKG2D expression, particularly in the HBV patients as compared to HCs. Conclusions Our study demonstrated that NK cells activity and status might play an important role in controlling HBV infection.

Cell-based cancer immunotherapy using chimeric antigen receptor (CAR)-engineered natural killer (NK) cells surface-modified with drug-loaded nanoparticles targeting adenosine receptors

Abstract The immunosuppressive tumor microenvironment (TME) plays a key role in failed eradication of solid tumors and tumor recurrence upon cancer treatment, as resident immune cells lose their cancer surveillance abilities. One of the underlying mechanisms responsible for the loss of adoptively transferred immune cell function in the TME is the A2a adenosine receptor (A2aR) inhibitory pathway. Encapsulating SCH-58261 (SCH), a potent antagonists of A2aR, in crosslinked multilamellar liposomal vesicles (cMLVs) was shown to efficiently deliver the drug to adoptively transferred cells in tumors. We genetically modified human NK92 cells to express anti-mesothelin CAR (ameso.CAR.NK) to be used in this study. We hypothesized that the efficacy of NK cell adoptive transfer therapies could be enhanced if CAR.NK cells were used as active carriers to direct SCH-loaded cMLVs to the immunosuppressive TME to block the A2aR pathway. We first covalently attached cMLVs to NK cell surface. We next verified that cMLV conjugation did not have adverse effects on NK cell viability and migration. In addition, surface conjugation of cMLVs did not reduce IFN-γ secretion and cancer cell killing ability of NK cells, suggesting similar levels of cognate antigen-specific activation of ameso.CAR.NK cells by mesothelin-overexpressing ovarian cancer cells. In the future, we will test the in vitro function of irradiated CAR.NK cells, given the requirement of irradiating NK92 cell lines prior to clinical use. Next, we will test our platform in an ovarian cancer xenograft mouse model and examine the effects of our therapy on tumor growth. Finally, we will investigate the potential of this therapy to reverse immune suppression on the TME-resident tumor-infiltrating NK cells.