Establishment of a TaqMan-based real-time quantitative PCR method for detection of exogenous fowl adenovirus type â , type â ¢ and avian leukosis virus in human cold adapted live attenuated influenza vaccines.

Cold adapted live attenuated influenza vaccines can effectively prevent human disease and death caused by influenza virus. Since chicken embryos are used as the culture substrate for the large-scale production of influenza vaccines, cold adapted live attenuated influenza vaccines may be contaminated by exogenous avian viruses. Rapid and sensitive methods such as TaqMan-based quantitative PCR are needed for the detection of exogenous avian viruses during cold adapted live attenuated influenza vaccines production. In this study, a TaqMan-based quantitative PCR method was established for the detection of three common exogenous avian viruses, including fowl adenovirus type I, type Ⅲ and avian leukosis virus. Avian virus-encoding plasmids purified in high-performance liquid chromatography were essential for sensitivity analysis. The sensitivity reached 1 copy per reaction for each of the avian virus plasmids. Standard curves showed a strong linear relationship. The TaqMan-based quantitative PCR method had high specificity and no cross-reactivity with other irrelevant viruses. Furthermore, the established TaqMan-based quantitative PCR can effectively detect 0.1 TCID50 of each avian virus without or with interference from the influenza virus nucleic acid. Ultimately, this method was used to test three master seed lots of monovalent cold adapted live attenuated influenza vaccine, and the results showed that no fowl adenovirus type I, type Ⅲ or avian leukosis virus contamination, which were consistent with serological methods. The TaqMan-based quantitative PCR method for the determination of extraneous avian viruses in cold adapted live attenuated influenza vaccines met the requirement for both conventional and emergency inspection on cold adapted live attenuated influenza vaccines.

Gene-guided OX40L anchoring to tumor cells for synergetic tumor "self-killing" immunotherapy.

The low objective response rates and severe side effects largely limit the clinical outcomes of immune checkpoint blockade (ICB) therapy. Here, a tumor "self-killing" therapy based on gene-guided OX40L anchoring to tumor cell membrane was reported to boost ICB therapy. We developed a highly efficient delivery system HA/PEI-KT (HKT) to co-deliver the OX40L plasmids and unmethylated CG-enriched oligodeoxynucleotide (CpG). On the one hand, CpG induced the expression of OX40 on T cells within tumors. On the other hand, OX40L plasmids achieved the OX40L anchoring on the tumor cell membrane to next promote T cells responses via OX40/OX40L axis. Such synergistic tumor "self-killing" strategy finally turned "cold" tumors to "hot", to sensitize tumors to programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) blockade therapy, and promoted an immune-mediated tumor regression in both B16F10 and 4T1 tumor models, with prevention of tumor recurrence and metastasis. To avoid the side effects, the gene-guided OX40L anchoring and PD-L1 silencing was proposed to replace the existing antibody therapy, which showed negligible toxicity in vivo. Our work provided a new possibility for tumor "self-killing" immunotherapy to treated various solid tumors.

Cationic Flexible Organic Framework for Combination of Photodynamic Therapy and Genetic Immunotherapy Against Tumors.

Photodynamic therapy is a new type of anti-tumor therapy with excellent therapeutic effects and minor side effects. The key factor for photodynamic therapy is highly efficient loading and protection of photosensitizers. Covalent organic framework is a new type of organic porous material with rich sources and has huge development potential in the loading of photosensitizers. However, the π-π interaction between the rigid monomers inevitably causes aggregation and quenching between photosensitizers, which in turn affects the rate of reactive oxygen production. Here, newly designed cationic flexible organic framework nanoparticles (PEI-Por NPs) are synthesized via one-step method with PEI25K and meso-tetra(p-formylphenyl)porphyrin under microwave irradiation. The structure of the flexible organic framework can effectively inhibit the aggregation and quenching of porphyrin. In addition, PEI-Por NPs had excellent gene transfection ability both in vitro and in vivo. Excellent antitumor effect can be achieved by combining PEI-Por NPs' photodynamic therapy capacity and PEI-Por NPs-mediated PD-L1 gene silencing with the guidance of fluorescence imaging and photoacoustic imaging. This cationic flexible organic framework material combines the advantages of flexible building units and rigid monomers, which provides a basis for the development of nano-photosensitizers and excellent gene carriers, and has great potential for clinical application.

An immune cocktail therapy to realize multiple boosting of the cancer-immunity cycle by combination of drug/gene delivery nanoparticles.

Immune checkpoint blockade therapy (ICT) has shown potential in the treatment of multiple tumors, but suffers poor response rate in clinic. We found that even combining ICT with chemotherapy, which was wildly used in clinical trials, failed to achieve satisfactory tumor inhibition in the B16F10 model. Thus, we further constructed a previously unexplored immune cocktail therapy and realized multiple boosting of the cancer-immunity cycle. Cocktail therapy consisted of two kinds of tumor microenvironment-responsive drug and gene delivery nanoparticles to achieve specific delivery of doxorubicin and codelivery of plasmids expressed small hairpin RNA of PD-L1 (pshPD-L1) and hyaluronidase (pSpam1) in the tumor area. Experimental evidences proved that any component in the cocktail therapy was indispensable, and the cocktail therapy exhibited excellent antitumor effects against different types of tumors. The cocktail therapy presented here offers a searching strategy for more synergistic units with ICT and is meaningful for developing more efficient antitumor immunotherapy.

Helix Self-Assembly Behavior of Amino Acid-Modified Camptothecin Prodrugs and Its Antitumor Effect.

For effective antitumor treatment, it is important to increase the water solubility of hydrophobic antitumor drugs and improve their cell absorption efficiency and nuclear transmission capacity. Here, we use endogenous hydrophilic arginine to modify camptothecin (CPT) to increase its water solubility. Surprisingly, the modified CPT can self-assemble into helical nanofibers through intermolecular π-π stacking and hydrophilic-hydrophobic interactions. Prodrug-based nanofibers were better endocytosed into the nucleus than their nonassembled CPT. Moreover, in vivo, such nanofibers had a longer blood circulation time and a better ability to accumulate in the tumor site. Further, we found that the cationic nanofibers can be combined with the anionic cisplatin-polyglutamic acid through electrostatic interaction to achieve a combined antitumor effect. This provides a new idea for achieving more effective cancer chemotherapy effects.

Efficient PD-L1 gene silence promoted by hyaluronidase for cancer immunotherapy.

The immune checkpoint blockade of programmed death ligand-1 (PD-L1) or programmed death-1 (PD-1) has been a promising strategy to restore T cell mediated tumor suppression. In this study, a plasmid DNA which expressed small hairpin RNA of PD-L1 (shPD-L1) was loaded in the ultrasensitive pH triggered charge/size dual-rebound P[(GP)D] nanoparticles (NPs) to silence the PD-L1 gene for reducing the PD-L1/PD-1 interactions between tumors and T cells. To increase the penetration of the shPD-L1 loaded P[(GP)D] NPs (shPD-L1@NPs) in tumors, hyaluronidase (HAase) was utilized to degrade the overexpressed hyaluronicacid (HA) in the extracellular matrix (ECM) of the tumor tissues. The HAase-enhanced tumor accumulation and penetration of the P[(GP)D] NPs were carefully explored. Further in vivo antitumor therapy was carried out in the malignant melanoma mouse tumor model, and a significant tumor inhibition effect was achieved by the combination treatment of HAase and shPD-L1@NPs. Our results verified that the HAase could effectively degrade the HA in tumors and increase the penetration of the shPD-L1@NPs for achieving more efficient PD-L1 gene silence and finally realizing potent tumor suppression. This combination treatment strategy has great potentials to be adopted for other nanomedicines, and it will have broad applications for cancer therapy in the future.

Molecular Strings Significantly Improved the Gene Transfection Efficiency of Polycations.

High transfection efficiency and low cytotoxicity are the two key factors to be considered in the design of gene carriers. Herein, a novel and versatile gene carrier (PLL-RT) was prepared by introducing "molecular string" RT (i.e., p-toluylsulfonyl arginine) onto the polylysine backbone. The introduction of RT string contributed to the formation of multiple interactions between the polycationic gene carriers and cell membrane or DNA, as well as adopting α-helix conformation, all of which would be beneficial to enhance the gene transfection. In addition, RT string grafted onto other polycations such as hyperbranced PEI25k and dendrimer PAMAM could also acquire improved transfection efficiency and low cytotoxicity. Moreover, PLL-RT presented significant tumor inhibition effect in vivo. This work provided an effective strategy for constructing novel gene carriers with high transfection and low cytotoxicity.

Highly enhanced cancer immunotherapy by combining nanovaccine with hyaluronidase.

Tumor vaccine has been one of the research hotspots for cancer immunotherapy in recent years. By introducing tumor antigens into the body, the patient's own immune system will be specifically activated to induce effective immune responses for controlling or eliminating the malignant tumor cells. In this study, a simple nanovaccine was developed to induce antigen-specific anti-tumor immune responses. Polycationic polyethylenimine (PEI) was utilized to co-deliver the antigen ovalbumin (OVA) and the adjuvant unmethylated cytosine-phosphate-guanine (CpG) by electrostatic binding. The positively charged PEI could be beneficial to augment the PEI/CpG/OVA nanovaccine uptake in dendritic cells (DCs) and facilitate the endosomal escape of the nanovaccine for antigen delivering into the cytoplasm. The nanovaccine showed significant stimulation on DCs' maturation in vitro, and it was further applied for in vivo anti-tumor immunotherapy. To enhance the tumor infiltration of the nanovaccine-generated tumor-specific T cells, hyaluronidase (HAase) was employed to increase the permeability of the tumor tissues by breaking down the hyaluronan (HA) in the extracellular matrix (ECM) of tumors. Highly enhanced in vivo anti-tumor therapeutic efficiency was achieved by combining the PEI/CpG/OVA nanovaccine with HAase, which was attributed to the increased quantity of OVA-specific T cells in tumor tissues. The combination of nanovaccine with HAase has offered a simple and efficient strategy for inducing powerful anti-tumor effect in cancer immunotherapy.

pH Triggered Size Increasing Gene Carrier for Efficient Tumor Accumulation and Excellent Antitumor Effect.

High efficiency and serum resistant capacity are important for gene carrier in vivo usage. In this study, transfection efficiency and cell toxicity of polyethylenimine (PEI) (branched, Mw = 25K) was remarkably improved, when mixed with polyanion (polyethylene glycol-polyglutamic acid (PEG-PLG) or polyglutamic acid (PLG)). Different composite orders of PEI, polyanion, and gene, for example, PEI is first complexed with DNA, and then with polyanion, or PEI is first complexed with polyanion, and then with DNA, were studied. Results showed that only the polyanion/PEI complexes exhibited additional properties, such as decreased pH, resulting in increased particle size, as well as enhanced serum resistance capability and improved tumor accumulation. The prepared gene carrier showed excellent antitumor effect, with no damage on major organs, which is suitable for in vivo gene antitumor therapy.

Gold-Nanorods-Based Gene Carriers with the Capability of Photoacoustic Imaging and Photothermal Therapy.

Multifunctional nanoparticles with high gene transfection activity, low cytotoxicity, photoacoustic imaging ability, and photothermal therapeutic properties were prepared by conjugating low-molecular-weight polyethylenimine onto the surfaces of gold nanorods through the formation of stable S-Au bonded conjugates. Results revealed that the gene transfection efficiency of the prepared polyethylenimine-modified gold nanorods (GNRs-PEI1.8k) was higher and their cytotoxicity was less than those of the commercial reagent PEI25k. GNRs-PEI1.8k could also be potentially used as a photoacoustic and photothermal reagent to evaluate the pharmacokinetics, biodistribution, and antitumor effects of gene/drug nanoparticles. Therefore, GNRs-PEI1.8k can be considered a promising candidate for the clinical diagnosis and treatment of tumors.