Discovery and characterization of hydroxylysine O-glycosylation in an engineered IL-2 fusion protein.

In the present study, an engineered interleukin-2 (IL-2) fusion protein consisting of an anti-human serum albumin nanobody linked by ASTKG and a (G4S)2 linker to IL-2 was constructed. Liquid chromatography-mass spectrometry (LC-MS) characterization was performed on the intact molecule and at the peptide level. The LC-MS molecular mass analysis for the engineered fusion protein showed the appearance of unreported +340 Da peaks, apart from the expected O-glycosylation-related peaks in the IL-2 domain. Through a combination analysis of a K120R mutated molecule (The lysine at the position of 120 was mutated to arginine while the rest amino acid sequence remain unchanged), the possibility of a non-cleaved valine-histidine-serine signal peptide was ruled out and the presence of hydroxylysine (HyK) O-glycosylation in the ASTKG linker was confirmed. HyK O-glycosylation have been reported in other proteins such as collagen, which occurs in the conserved Gly-Xaa-HyK motif and is catalyzed by lysyl hydroxylase-3 complex. The present study showed high similar conserved motif of HyK-O-glycosylation in collagen, implying the HyK O-glycosylation in the engineered IL-2 possibly was catalyzed by the Chinese hamster ovary homolog of enzymes promoting HyK O-glycosylation in collagen. Bioactivity testing results revealed that HyK-O-glycosylation had no obvious effect on the in vitro activity of engineered IL-2. Our study is the first to report HyK-O-glycosylation modifications in therapeutic proteins through LC-MS characterization and in vitro activity analysis, which expands the scope of post-translational modification knowledge of therapeutic proteins.

Aptamer-functionalized targeted siRNA delivery system for tumor immunotherapy.

Programmed death ligand 1 (PD-L1) overexpressed on the surface of tumor cells is one of the reasons for tumor immune escape. Reducing PD-L1 expression has been proved to be an effective strategy to facilitate immune system activation and inhibit tumor progression. RNA interference (RNAi) is a promising technology for gene regulation in tumor therapy. In this study, we constructed a targeted siRNA delivery system NPs@apt to transfect PD-L1 siRNA into human non-small-cell lung carcinoma cell line (A549) for inhibiting tumor immune evasion. NPs@apt was prepared by compressing PD-L1 siRNA with cationic Lipofectamine 2000, fusing with erythrocyte membrane-derived nanovesicles, and further modifying with targeting AS1411 aptamer. The introduction of erythrocyte membrane endowed the siRNA delivery system with lower cytotoxicity and the ability to escape from the phagocytosis of macrophages. The stability of NPs@apt and the protection to loaded siRNA were confirmed.In vitrostudies after NPs@apt treatment demonstrated that PD-L1 siRNA was selectively delivered into A549 cells, and further resulted in PD-L1 gene knockdown, T cell activation and tumor cell growth inhibition. This study offered an alternative strategy for specific siRNA transfection for improving anti-tumor immunity.

Metal-Organic Frameworks with Enhanced Photodynamic Therapy: Synthesis, Erythrocyte Membrane Camouflage, and Aptamer-Targeted Aggregation.

Here, ferric oxide-loaded metal-organic framework (FeTCPP/Fe2O3 MOF) nanorice was designed and constructed by the liquid diffusion method. The introduction of iron metal nodes and the loading of Fe2O3 can effectively catalyze the Fenton reaction to produce hydroxyl radicals (•OH) and overcome the hypoxic environment of tumor tissue by generating oxygen. The monodispersity and porosity of the porphyrin photosensitizers in the MOF structure exposed more active sites, which promoted energy exchange between porphyrin molecules and oxygen molecules for photodynamic therapy (PDT) treatment. Therefore, the generated hydroxyl radicals and singlet oxygen (1O2) can synergistically act on tumor cells to achieve the purpose of improving tumor therapy. Then the erythrocyte membrane was camouflaged to enhance blood circulation and tissue residence time in the body, and finally, the targeted molecule AS1411 aptamer was modified to achieve the high enrichment of MOF photosensitizers on a tumor domain. As a result, the MOF nanorice camouflaged by the erythrocyte membrane can effectively reduce side effects and improve the therapeutic effect of PDT and chemo-dynamic therapy (CDT). The study not only improved the efficacy of PDT and CDT in essence from the MOF nanorice but also used the camouflage method to further concentrate FeTCPP/Fe2O3 on the tumor sites, achieving the goal of multiple gains. These results will provide theoretical and practical directions for the development of tumor-targeted MOF nanomaterials.

Tumor Acid Microenvironment-Triggered Self-Assembly of ESIONPs for T1/T2 Switchable Magnetic Resonance Imaging.

Smart magnetic resonance imaging (MRI) contrast agents (CAs), whose MRI contrasting enhancement is variable in response to the specific stimulus from tumor tissues, possess great potential in precise tumor diagnosis. Herein, we design a type of extremely small iron oxide nanoparticle (ESIONP)-based pH-responsive system for activatable T2 MRI in the tumor acid microenvironment. The ESIONP system is composed of ESIONP-PEG-PGA and ESIONP-PEG-PDC, which were respectively constructed through the surface modification with poly (l-glutamic acid) (PGA) and poly(N-{N'-[N″-(2-carbox aminoethyl)]-2-aminoethyl}glutamide) (PDC) on the surface of ESIONP. The pH-responsive system exhibits the dispersed state under the neutral condition, and when it is exposed to the weakly acid environment, ESIONP-PEG-PDC switches from the neutral to positive charge, finally leading to the aggregation by the electrostatic interaction between the positively charged ESIONP-PEG-PDC and negatively charged ESIONP-PEG-PGA. On the basis of the aggregation, the T1 contrasting effect of the pH-responsive system switches to a T2 contrasting effect, which can be employed to realize the selective enhancement of imaging contrast at the tumor location owing to the weakly acid microenvironment. Moreover, on the basis of size increase originated from the aggregation effect, the residence time of extremely small iron oxide nanoparticles (ESIONPs) in the tumor site is effectively prolonged, which is beneficial for the MRI of tumors. Therefore, the pH-responsive system based on the ESIONPs is a potential smart MRI contrast agent for accurate tumor diagnosis.

Aptamer-Based Erythrocyte-Derived Mimic Vesicles Loaded with siRNA and Doxorubicin for the Targeted Treatment of Multidrug-Resistant Tumors.

Multidrug resistance (MDR) remains one of the most important challenges to clinical chemotherapeutics. In this study, versatile mimic vesicles (MVs) derived from erythrocytes were investigated as delivery systems for siRNA and doxorubicin (DOX) to treat MDR tumors. The carriers could be readily obtained through extruding erythrocyte membranes and had the advantages of biological homogeneity, high output, controllable size, low cost, and excellent biocompatibility. Moreover, aptamers modified on the MVs endowed the carriers with tumor-targeting capacity. DOX and P-glycoprotein (P-gp) siRNA were loaded onto the MVs through incubation and cholesterol-mediated methods, achieving high loading rates and targeted tumor delivery. The drug-loaded carriers could successfully overcome drug resistance and synergistically kill MDR tumors through P-gp silencing and DOX-induced growth inhibition. This MV-based drug delivery system therefore provides new insights into the synergistic targeting of MDR tumors and offers an alternative delivery strategy to overcome MDR.

Exploration of Catalytic Nucleic Acids on Porphyrin Metalation and Peroxidase Activity by in Vitro Selection of Aptamers for N-Methyl Mesoporphyrin IX.

To develop a novel light-up probe and DNAzyme, we selected aptamers for N-methyl mesoporphyrin IX (NMM), a common fluorogenic analogue of coenzyme hemin, by a modified affinity chromatography-based systematic evolution of ligands by exponential enrichment (SELEX). Two truncated aptamers Nm1 and Nm2 with low micromolar dissociation constants (0.75 and 13.27 µM) were obtained after 11 rounds of selection and the final minimized 39-mer aptamer Nm2.1 showed 24-fold fluorescence enhancement for NMM at saturated concentration. Study of the interactions between aptamers and other porphyrin compounds by circular dichroism (CD) and absorption spectroscopy showed that Nm1 mainly assembled as a stem-loop structure, which exhibited a catalytic activity for the metal insertion reaction of mesoporphyrin IX with 3.3-fold rate enhancement. In contrast, the G-rich Nm2 and Nm2.1 were likely to form G-quadruplexes in the presence of alkali metal cations (K+ and Na+), which displayed excellent peroxidase activity exhibiting 19-fold higher catalytic efficiency than hemin alone. The selected aptamers could therefore be used as novel light-up fluorescent probes and DNAzymes by pairing with porphyrin compounds that have potential to construct sensors for various applications.

Self-Assembled saRNA Delivery System Based on Rolling Circle Transcription for Aptamer-Targeting Cancer Therapy.

With the capacity of gene promotion, RNA activation (RNAa) has been supposed to be a powerful technique in the field of biomedicine, especially in an antitumor aspect. However, one of the pressing challenges for clinical application is how to efficiently deliver therapeutic probes to cancer. Herein, we synthesized a carrier through rolling circle transcription (RCT) to deliver p21 saRNA with high loading rate and targeting capacity. This carrier could be condensed from a micron dimension to about 200 nm by polyethylenimine (PEI). In addition, the trait of robust tumor targeting and improved cytotoxicity was demonstrated when the aptamer was modified through layer-by-layer self-assembly. Moreover, the 4-fold activation of the p21 gene could be obviously detected in a targeting group. Meanwhile, the cell apoptosis induced by p21 promotion was also exhibited, which indicated that this highly efficient saRNA delivery carrier had a specific antitumor effect and could reduce side effects to normal cells. Therefore, this delivery system had the potential to be used in RNAa applications and cancer-targeted treatment.