A programmable oral bacterial hydrogel for controllable production and release of nanovaccine for tumor immunotherapy.

Oral protein vaccines are mainly used to prevent the infection of intestinal pathogens in clinic due to their high safety and strong compliance. However, it is necessary to design the efficient delivery systems to overcome the harsh gastrointestinal environment in the application process. Here we established a programmable oral bacterial hydrogel system for spatiotemporally controllable production and release of nanovaccines. The system was divided into three parts: (1) Engineered bacteria were encapsulated in chitosan-sodium alginate microcapsules, which offered protection against the extreme acid conditions in the stomach. (2) Microcapsules were dissolved, and then engineered bacteria were released and colonized in the intestine. (3) The release of nanovaccines was controlled periodically by a synchronous lysis genetic circuit for tumor immunotherapy. Compared to control groups, tumor volume of subcutaneous tumor-bearing mice treated with bacterial microgels releasing optimized nanovaccine was almost inhibited by 75% and T cell response was activated at least 2-fold. We believe that this programmable bacterial hydrogel will offer a promising way for the application of oral nanovaccines.

Hydrogel microcapsules containing engineered bacteria for sustained production and release of protein drugs.

Subcutaneous administration of sustained-release formulations is a common strategy for protein drugs, which avoids first pass effect and has high bioavailability. However, conventional sustained-release strategies can only load a limited amount of drug, leading to insufficient durability. Herein, we developed microcapsules based on engineered bacteria for sustained release of protein drugs. Engineered bacteria were carried in microcapsules for subcutaneous administration, with a production-lysis circuit for sustained protein production and release. Administrated in diabetic rats, engineered bacteria microcapsules was observed to smoothly release Exendin-4 for 2 weeks and reduce blood glucose. In another example, by releasing subunit vaccines with bacterial microcomponents as vehicles, engineered bacterial microcapsules activated specific immunity in mice and achieved tumor prevention. The engineered bacteria microcapsules have potential to durably release protein drugs and show versatility on the size of drugs. It might be a promising design strategy for long-acting in situ drug factory.

CRISPR-dcas9 Optogenetic Nanosystem for the Blue Light-Mediated Treatment of Neovascular Lesions.

Vascular endothelial growth factor (VEGF) is the key regulator in neovascular lesions. The anti-VEGF injection is a major way to relieve retinal neovascularization and treat these diseases. However, current anti-VEGF therapeutics show significant drawbacks. The reason is the inability to effectively control its therapeutic effect. Therefore, how to controllably inhibit the VEGF target is a key point for preventing angiogenesis. Here, a CRISPR-dCas9 optogenetic nanosystem was designed for the precise regulation of pathologic neovascularization. This system is composed of a light-controlled regulatory component and transcription inhibition component. They work together to controllably and effectively inhibit the target gene's VEGF. The opto-CRISPR nanosystem achieved precise regulation according to individual differences, whereby the expression and interaction of gene was activated by light. The following representative model laser-induced choroid neovascularization and oxygen-induced retinopathy were taken as examples to verify the effect of this nanosystem. The results showed that the opto-CRISPR nanosystem was more efficacious in the light control group (NV area effectively reduced by 41.54%) than in the dark control group without light treatment. This strategy for the CRISPR-optogenetic gene nanosystem led to the development of approaches for treating severe eye diseases. Besides, any target gene of interest can be designed by merely replacing the guide RNA sequences in this system, which provided a method for light-controlled gene transcriptional repression.

[Radiosensitization Induced by ANTP-SmacN7 Fusion Peptide in H460 Cell Line].

BACKGROUND: The curative effect of radiotherapy may be limited by the radioresistance of tumor. Mimetic compounds of Second mitochondria-derived activator of caspase (Smac) were hopeful to become new drugs of radiosensitization for tumor because they can increase radiation induced apoptosis in tumor cells. The aim of present study is to observe the radiosensitization effect of a new Smac mimetic ANTP-SmacN7 fusion peptide in H460 cell line. METHODS: In order to observe if the fusion peptide can enter into tumor cell, ANTP-SmacN7 fusion peptide was synthesized and linked by FITC. H460 cell was divided into control, radiation only, ANTP-SmacN7 only and ANTP-SmacN7 combined with radiation group. The cells were exposed by 0, 2, 4 and 6 Gy and the concentration of ANTP-SmacN7 was 20 µmol/L. Proliferation of H460 tumor cell was detected by WST-1 assay. There are four groups in the present study: control group, radiation group, ANTP-SmacN7 group and ANTP-SmacN7 combined with radiation group. Apoptosis was detected by flow cytometry at 24 and 48 hours after the treatment of all the groups. The level of caspase3 and cleaved caspase3 were detected by Western blot assay. RESULTS: ANTP-SmacN7 can enter into cells and promote the radiosensitization of H460 cell obviously (F=25.1, P<0.01, sensitivity enhancement ratio was 1.86). The treatment of ANTP-SmacN7 combined with radiation decreased the cloning forming efficiency (χ2=45.2, P<0.01; χ2=40.3, P<0.01), activated caspase3 by promoting the expression of cleaved caspase3 and increased the apoptosis of H460 cell line. CONCLUSIONS: ANTP-SmacN7 fusion peptide had remarkably radiosensitization effect on H460 cell line. ANTP-SmacN7 fusion peptide might be hopeful to be applied in radiosensitization therapy as a new Smac mimetic polypeptide in the future.