Dissolving Microneedle Arrays as a Hepatitis B Vaccine Delivery System Adjuvanted by APC-Targeted Poly (Lactic-co-Glycolic Acid) (PLGA) Nanoparticles.

The objective of this study is to develop a new hepatitis B surface antigen (HBsAg) delivery system by coating soluble microneedle arrays with mannose-modified PLGA nanoparticles (MNPs). MNPs of different sizes were synthesized. The effects these nanoparticles on the maturation of dendritic cells were studied by flow cytometry. HBsAg-containing MNPs (HBsAg/MNPs) of the appropriate sizes were coated into water-soluble microneedle arrays. The in vitro characteristics of microneedles arrays and the immune responses after subcutaneous administration in mice were studied. The results showed that PLGA nanoparticles with an average size of about 800 nm showed the most significant effects in stimulating the maturation of dendritic cells. In the water-soluble microneedle array, the targeted PLGA nanoparticles containing HBsAg were distributed discretely with a maximum distribution height of about 280 µm with a drug load of 0.98 ± 0.05 µg/mg. The drug-containing microneedle arrays exhibited excellent mechanical properties and improved biosafety. The results of immune responses in vivo showed that the subcutaneous administration of the microneedle arrays induced the proliferation of splenocyte, secreted specific IL-12 and IFN-γ, and promote the production of IgG in mice. This study verifies the feasibility of soluble composited microneedles administration in hepatitis B immunization, and provides new ideas for the development and application of non-injectable vaccine delivery systems.

Preparation and Biological Property Evaluation of Novel Cationic Lipid-Based Liposomes for Efficient Gene Delivery.

Novel cationic lipid-based liposomes prepared using an amphiphilic cationic lipid material, N,N-dimethyl-(N',N'-di-stearoyl-1-ethyl)1,3-diaminopropane (DMSP), have been proposed to enhance the transfection of nucleic acids. Herein, we designed and investigated liposomes prepared using DMSP, soybean phosphatidylcholine, and cholesterol. This novel gene vector has high gene loading capabilities and excellent protection against nuclease degradation. An in vitro study showed that the liposomes had lower toxicity and superior cellular uptake and transfection efficiency compared with Lipofectamine 2000. An endosomal escape study revealed that the liposomes demonstrated high endosomal escape and released their genetic payload in the cytoplasm efficiently. Mechanistic studies indicated that the liposome/nucleic acid complexes entered cells through energy-dependent endocytosis that was mediated by fossa proteins. These results suggest that such cationic lipid-based liposome vectors have potential for clinical gene delivery.

Mannose-Modified PLGA Nanoparticles for Sustained and Targeted Delivery in Hepatitis B Virus Immunoprophylaxis.

The launched hepatitis B vaccine could induce powerful antibodies, whereas it failed to improve potent cellular immune responses due to that the Th2-type response-induced aluminum adjuvant was adopted. Here, to target antigen-presenting cells under the epidermis and induce potent cellular and humoral immune responses, mannose-modified poly D,L-lactide-co-glycolic acid (PLGA) was synthesized and nanoparticle (MNP)-loaded hepatitis B surface antigen (HBsAg) protein was prepared. HBsAg could be slowly released and highly presented to lymphocytes which facilitated to produce long-lasting immunity based on characters of PLGA. In vitro uptake test results showed that MNPs could enhance internalization in bone marrow-derived dendritic cells (BMDCs) and RAW 264.7 cells. Subcutaneous delivery of MNPs into mice kept humoral immune and strengthened cellular immune responses. Experimental results indicated that MNPs showed significantly modified properties compared with parental PLGA nanoparticles. Thus, the obtained MNPs could be a promising vehicle for hepatitis B vaccine delivery.

Five Active Components Compatibility of Astragali Radix and Angelicae Sinensis Radix Protect Hematopoietic Function Against Cyclophosphamide-Induced Injury in Mice and t-BHP-Induced Injury in HSCs.

Although the compatibility of Astragali Radix (AR) and Angelicae Sinensis Radix (ASR) has favorable effect on promoting hematopoiesis in traditional Chinese medicine (TCM), the main active components and pharmacological mechanism are unknown. We investigated the five active components and its mechanisms in vitro and in vivo. Five active components of Astragalus glycosides (AST), Formononetin (FRM), Ferulic acid (FRA), Calycosin (CAL), and Calycosin-7-glucoside (CLG), which could be absorbed in intestinal tract, were detected in this study. The peripheral blood, hematopoietic growth factors (HGFs), and hematopoietic progenitor cells (HPCs) colony were observed to evaluate the effect of these five active components promoting hematopoiesis. Furthermore, hematopoietic stem cell (HSC) proliferation, aging, cycle, and related proteins were detected to explore the mechanism of these five components promoting HSC proliferation. i) The in vivo experiments showed that the combination of the five active components could remarkably increase the number of RBCs, WBCs, PLTs, and content of Hb in peripheral blood and the area of bone marrow hematopoietic tissue, as well as thrombopoietin (TPO), erythropoietin (EPO), granulocyte-macrophage colony stimulating factor (GM-CSF), and colony of CFU-GM, CFU-MK, CFU-E, and BFU-E in serum. Each of these five components promoted the recovery of RBCs and Hb, and increased TPO, CFU-MK, and CFU-E. All components except for AST increased the CFU-GM. FRA increased the number of WBCs, the area of bone marrow hematopoietic tissue, and BFU-E. FRA and AST promoted PLT recovery. FRA and CAL improved the content of GM-CSF. FRA, CAL, and CLG improved the content of EPO. ii) The in vitro experiments showed that FRA, FRM, and AST significantly promoted cell proliferation, reduced the positive rate and G0/G1 cells, and increased G2/M + S cells and the expression of cyclin D1 and CDK4 proteins in aging HSCs. Furthermore, the combination of five components had the best effect. Taken together, the five active components of AST, FRM, FRA, CAL, and CLG were the main pharmacodynamic substances of the AR-ASR compatibility, which promoted hematopoiesis. The combination of them had a synergistic effect. The mechanism of promoting hematopoiesis may be relevant to regulating cyclin-related proteins, promoting cell cycle transformation, and promoting HSC proliferation.

Inhibition of Aortic Intimal Hyperplasia and Vascular Smooth Muscle Proliferation and Extracellular Matrix Protein Expressions by Astragalus-Angelica Combination.

VSMC proliferation and ECM deposition always resulted in intimal hyperplasia. Astragalus-Angelica combination has a protective effect on the cardiovascular system. The inhibition effect of different Astragalus-Angelica combination on the hyperplastic intima after vascular balloon injury in rats was investigated in this study. Astragalus-Angelica combination can inhibit the intima hyperplasia after balloon injury, in which a 1:1 ratio shows excellent results. Astragalus-Angelica combination can enhance the expression of smooth muscle α-actin (SMа-actin) and inhibit the expression of proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin E, collagen I (Col-I), fibronectin (FN), and matrix metallopeptidase-9 (MMP-9) in hyperplastic intima, suggesting that Astragalus-Angelica combination can inhibit the intimal hyperplasia of blood vessels in rats. The mechanism is related to the inhibition of PI3K/Akt signaling pathway activation and thereby inhibits the phenotypic transformation and cell proliferation of VSMCs and thus inhibits the extracellular matrix (ECM) deposition of vascular wall during intimal hyperplasia.

Multicoloured fluorescent indicators for live-cell and in vivo imaging of inorganic mercury dynamics.

Engineered fluorescent indicators for visualizing mercury ion (Hg2+) are powerful tools to illustrate the intracellular distribution and serious toxicity of the ion. However, the sensitive and specific detection of Hg2+ in living cells and in vivo is challenging. This paper reported the development of fluorescent indicators for Hg2+ in green or red color by inserting a circularly permuted fluorescent protein into a highly mercury-specific repressor. These sensors provided a rapid, sensitive, specific, and real-time read-out of Hg2+ dynamics in solutions, bacteria, subcellular organelles of mammalian cells, and zebrafish, thereby providing a useful new method for Hg2+ detection and bioimaging. In conjunction with the hydrogen peroxide sensor HyPer, we found mercury uptake would trigger subcellular oxidative events at the single-cell level, and provided visual evidence of the causality of mercury and oxidative damage. These sensors would paint the landscape of mercury toxicity to cell functions.

Co-delivery of dihydroartemisinin and docetaxel in pH-sensitive nanoparticles for treating metastatic breast cancer via the NF-κB/MMP-2 signal pathway.

Metastasis is a major barrier in cancer chemotherapy. Prolonged circulation and rapid, specific intracellular drug release are two main goals in the development of nanoscale drug delivery systems to treat metastatic breast cancer. In this study, we investigated the anti-metastasis effect of docetaxel (DTX) in combination with dihydroartemisinin (DHA) in metastatic breast cancer 4T1 cells. We synthesized a pH-sensitive material 4-arm-PEG-DTX with a hydrazone bond and used it to construct nanoparticles that co-deliver DTX and DHA (D/D NPs). The D/D NPs had a mean size of 142.5 nm and approximately neutral zeta potential. The pH-sensitive nanoparticles allowed acid-triggered drug release at the tumor site, showing excellent cytotoxicity (IC50 = 7.0 µg mL-1), cell cycle arrest and suppression of cell migration and invasion. The mechanisms underlying the anti-metastasis effect of the D/D NPs involved downregulation of the expression of p-AKT, NF-κB and MMP-2. Therefore, D/D NPs represent a new nanoscale drug delivery system for treating metastatic breast cancer, responding to the acidic tumor microenvironment to release the chemotherapeutic drugs.

Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is essential for biosynthetic reactions and antioxidant functions; however, detection of NADPH metabolism in living cells remains technically challenging. We develop and characterize ratiometric, pH-resistant, genetically encoded fluorescent indicators for NADPH (iNap sensors) with various affinities and wide dynamic range. iNap sensors enabled quantification of cytosolic and mitochondrial NADPH pools that are controlled by cytosolic NAD+ kinase levels and revealed cellular NADPH dynamics under oxidative stress depending on glucose availability. We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis, which is regulated by glucose-6-phosphate dehydrogenase and AMP kinase. Moreover, using the iNap sensors we monitor NADPH fluctuations during the activation of macrophage cells or wound response in vivo. These data demonstrate that the iNap sensors will be valuable tools for monitoring NADPH dynamics in live cells and gaining new insights into cell metabolism.