Red blood cell membrane-coated functionalized Au nanocage as a biomimetic platform for improved MicroRNA delivery in hepatocellular carcinoma.

Dysregulation of microRNAs (miRNAs) expression is closely related to cancers and managing miRNA expression holds great promise for cancer therapy. However, their wide clinical application has been hampered by their poor stability, short half-life and non-specific biodistribution in vivo. Herein, a novel biomimetic platform designated as RHAuNCs-miRNA for improved miRNA delivery was prepared through wrapping miRNA-loaded functionalized Au nanocages (AuNCs) with red blood cell (RBC) membrane. RHAuNCs-miRNA not only successfully loaded miRNAs but also effectively protected them from enzymatic degradation. With good stability, RHAuNCs-miRNA had the characteristics of photothermal conversion and sustained release. Cellular uptake of RHAuNCs-miRNA by SMMC-7721 cells was in a time-dependent manner via clathrin- and caveolin-mediated endocytosis. The uptake of RHAuNCs-miRNAs was affected by cell types and improved by mild near infrared (NIR) laser irradiation. More importantly, RHAuNCs-miRNA exhibited a prolonged circulation time without the occurrence of accelerated blood clearance (ABC) in vivo, resulting in efficient delivery to tumor tissues. This study may demonstrate the great potential of RHAuNCs-miRNA for improved miRNAs delivery.

Gene Therapy and Photothermal Therapy of Layer-by-Layer Assembled AuNCs /PEI/miRNA/ HA Nanocomplexes.

BACKGROUND: MicroRNA (miRNA) therapy, which was widely considered to treat a series of cancer, has been confronted with numerous obstacles to being delivered into target cells because of its easy biodegradation and instability. METHODS: In this research, we successfully constructed 11-mercaptoundecanoic acid modified gold nanocages (AuNCs)/polyethyleneimine (PEI)/miRNA/hyaluronic acid (HA) complexes (abbreviated as AuNCs/PEI/miRNA/HA) using a layer-by-layer method for target-specific intracellular delivery of miRNA by HA receptor mediated endocytosis. RESULTS: The results of UV spectra, hydrodynamic diameter and zeta potential analyses confirmed the formation of AuNCs/PEI/ miRNA/HA complex with its average particle size of ca. 153 nm and surface charge of ca. -9.43 mV. Next, we evaluated the antitumor effect of the nanocomplex mediated by the combination of gene therapy and photothermal therapy (PTT) against hepatocellular carcinoma (HCC) in vitro. CONCLUSION: Our experimental results indicated that the AuNCs/PEI/miRNA/HA complex effectively delivered miRNA to the target cells and its antitumor effect was significantly enhanced by the combination of gene therapy and photothermal therapy. In addition, anti-miR-181b could promote Bel-7402 cell arrest in S phase and improve TIMP-3 mRNA expression. All these results suggested that AuNCs/PEI/miRNA/HA gene delivery system with combination of gene therapy and photothermal therapy might be exploited for HCC treatment.

Simultaneous determination of seven flavonoids, two phenolic acids and two cholesterines in Tanreqing injection by UHPLC-MS/MS.

A new ultra-high performance liquid chromatography combined with triple quadrupole mass spectrometry was developed to evaluate the quality of Tanreqing injection. Seven flavonoids (Rutin, Baicalin, Scutellarin, Chrysin-7-O-Beta-d-glucoronide, Oroxylin A-7-O-ß-d-glucoronide, Wogonin, Luteolin-7-O-glucoside), two phenolic acids (Chlorogenic acid, Caffeic acid) and two cholesterines (Ursodeoxycholic acid, Chenodeoxycholic acid) in Tanreqing injection could be measured simultaneously. For the determination of the eleven compounds, the conditions were set as follows: The mobile phase was a gradient of 0.1% aqueous formic acid solution (A) and acetonitrile (B); the flow rate was 0.2 mL min-1, the column was Acquity UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 µm); and the multiple-reaction monitoring (MRM) with a negative electro spray ionization interface (ESI-) was selected. Within the test ranges, all the standard regression curves showed excellent linear regression (r > 0.99). In terms of (relative standard deviation) RSDs, the precision, repeatability and stability of the eleven compounds were all lower than 3%. The recovery rates of Tanreqing injection and the RSD were 97.8-103.7% and 0.4%-2.0%, respectively. The RSD value was in accordance with the requirements of less than 3.0%. This method has been successfully used in the analysis of Tanreqing injection. In summary, a fast, accurate and reliable UPLC-ESI--MS/MS method was successfully developed for the simultaneous detection of the eleven major active ingredients with different chemical structures in Tanreqing injection, and can be used for the quality control of Tanreqing injection as well.

A54 peptide-mediated functionalized gold nanocages for targeted delivery of DOX as a combinational photothermal-chemotherapy for liver cancer.

The combination of photothermal therapy and chemotherapy (photothermal-chemotherapy) is a promising strategy for cancer therapy. Gold nanocages (AuNCs), with hollow and porous structures and unique optical properties, have become a rising star in the field of drug delivery. Here, we designed a novel targeted drug delivery system based on functionalized AuNCs and evaluated their therapeutic effects in vitro and in vivo. We then loaded doxorubicin into this promising system, designated as DHTPAuNCs consisting of hyaluronic acid-grafted and A54 peptide-targeted PEGylated AuNCs. Its formation was corroborated by ultraviolet-visible spectroscopy, transmission electron microscopy and dynamic light scattering. This delivery platform needed hyaluronidase to release encapsulated drugs, meanwhile the acidic pH and near-infrared irradiation could accelerate the release. In addition, the results of cellular uptake demonstrate that this system could bind specifically with BEL-7402 cells. In vitro, we evaluated therapeutic effects of the DHTPAuNCs in BEL-7402 cells by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide assay. Moreover, in BEL-7402 tumor-bearing nude mice, its therapy effect in vivo was also evaluated. As expected, DHTPAuNCs exhibited excellent therapeutic effect by photothermal-chemotherapy, both in vitro and in vivo. In short, DHTPAuNCs with low toxicity showed great potential as a drug delivery system for cancer therapy.

Production, Characterization, and Biological Evaluation of Well-Defined IgG1 Fc Glycoforms as a Model System for Biosimilarity Analysis.

Four different well-defined IgG1 Fc glycoforms are proposed as a model system to examine important biological and physicochemical features for protein drug biosimilar analyses. The IgG1 Fc glycoforms were produced by yeast expression combined with in vitro enzymatic synthesis as a series of sequentially truncated high-mannose IgG1 Fc glycoforms with an anticipated range of biological activity and structural stability. Initial characterization with mass spectrometry, SDS-PAGE, size exclusion HPLC, and capillary isoelectric focusing confirmed that the glycoproteins are overall highly similar with the only major difference being glycosylation state. Binding to the activating Fc receptor, FcγRIIIa was used to evaluate the potential biological activity of the IgG1 Fc glycoproteins. Two complementary methods using biolayer interferometry, 1 with protein G-immobilized IgG1 Fc and the other with streptavidin-immobilized FcγRIIIa, were developed to assess FcγRIIIa affinity in kinetic binding studies. The high-mannose IgG1 Fc and Man5-IgG1 Fc glycoforms were highly similar to one another with high affinity for FcγRIIIa, whereas GlcNAc-Fc had weak affinity, and the nonglycosylated N297Q-Fc had no measurable affinity for FcγRIIIa. These 4 IgG1 Fc glycoforms were also evaluated in terms of physical and chemical stability profiles and then used as a model system to mathematically assess overall biosimilarity, as described in a series of companion articles.