[Preliminary Investigation on Difference of Protein Compositions Between DC2.4 Cells and Their Derived Exosomes by nanoLC-MS/MS].

OBJECTIVE: To preliminarily investigate the differences of protein composition between immature dendritic cells (DC2.4) and their derived exosomes (DC-Exo) using a relatively rapid and sample-saving method based on nano-flow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). METHODS: The supernatant of DC2.4 cells culture medium was collected and gradient centrifugation was applied to primarily extract and isolate DC-Exo; then sucrose density gradient ultracentrifugation was adopted to purify the DC-Exo. Bradford protein assay was used to determine the total protein content of the purified DC-Exo, and dynamic light scattering and transmission electron microscope were conducted to characterize the morphology and size distribution of the DC-Exo. Afterwards, protein samples including DC2.4 cells and DC-Exo were prepared by FASP enzymolysis method. Samples were performed nanoLC-MS/MS assay. The µLPickUp sample loading mode was used and only 1 µg of protein sample was required for each assay. The phase of Transport liquid and Micro A were both 0.05% trifluoroacetic acid-2% acetonitrile (ACN) aq. ( V/ V). Acclaim ® PepMap RSLC column was used to separate sample compositions and the gradient elute was adopted where the mobile phase consisted of (A) 0.1% formic acid (FA) and (B) 0.08% FA-80% ACN aq. ( V/ V) with flow rate of 0.3 µL/min. Positive APCI nanospray interface was used and "one-drive-ten" schema was set to collect primary information. The collected data was then searched and matched based on Uniport Mouse Fasta file as protein database in this case, and the re-annotated data was further sorted out and analyzed. RESULTS: In the current study, relatively high yield of DC-Exo samples with sizes of 40-200 nm were obtained. The lyophilized protein samples prepared by FASP method could be loaded directly after redissolution, and only 1 µg of protein sample is required. The annotated results showed that DC2.4 cells contained 998 kinds of proteins, among which 227 were highly expressed and 535 were unique; while DC-Exo contained only 348 types of proteins, among which 18 were uniquely and highly expressed. There were 306 kinds of consensus proteins in both DC2.4 cells and DC-Exo, among them 7 kinds were highly expressed. CONCLUSION: The nanoLC-MS/MS method developed in this study only requires very small amount of protein samples, and it could primarily differentiate the protein compositions between DC2.4 cells and their derived exosomes rapidly.

Mechanistic and therapeutic study of novel anti-tumor function of natural compound imperialine for treating non-small cell lung cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Bulbus Fritillaria cirrhosa D. Don (BFC) is a Chinese traditional herbal medicine that has long been used as an indispensable component in herbal prescriptions for bronchopulmonary diseases due to its well-established strong anti-inflammation and pulmonary harmonizing effects. Interestingly, there are few case reports in traditional Chinese medicine available where they found it to contribute in anti-tumor therapies. Imperialine is one of the most favored active substances extracted from BFC and has been widely recognized as an anti-inflammatory agent. AIM OF THE STUDY: The aim of the current work is to provide first-hand evidences both in vitro and in vivo showing that imperialine exerts anti-cancer effects against non-small cell lung cancer (NSCLC), and to explore the molecular mechanism of this anti-tumor activity. It is also necessary to examine its systemic toxicity, and to investigate how to develop strategies for feasible clinical translation of imperialine. MATERIALS AND METHODS: To investigate anti-NSCLC efficacy of imperialine using both in vitro and in vivo methods where A549 cell line were chosen as in vitro model NSCLC cells and A549 tumor-bearing mouse model was constructed for in vivo study. The detailed underlying anti-cancer mechanism has been systematically explored for the first time through a comprehensive set of molecular biology methods mainly including immunohistochemistry, western blot and enzyme-linked immunosorbent assays. The toxicity profile of imperialine treatments were evaluated using healthy nude mice by examining hemogram and histopathology. An imperialine-loaded liposomal drug delivery system was developed using thin film hydration method to evaluate target specific delivery. RESULTS: The results showed that imperialine could suppress both NSCLC tumor and associated inflammation through an inflammation-cancer feedback loop in which NF-κB activity was dramatically inhibited by imperialine. The NSCLC-targeting liposomal system was successfully developed for targeted drug delivery. The developed platform could favorably enhance imperialine cellular uptake and in vivo accumulation at tumor sites, thus improving overall anti-tumor effect. The toxicity assays revealed imperialine treatments did not significantly disturb blood cell counts in mice or exert any significant damage to the main organs. CONCLUSIONS: Imperialine exerts anti-cancer effects against NSCLC both in vitro and in vivo, and this previously unknown function is related to NF-κB centered inflammation-cancer feedback loop. Imperialine mediated anti-cancer activity is not through cytotoxicity and exhibit robust systemic safety. Furthermore, the liposome-based system we commenced would dramatically enhance therapeutic effects of imperialine while exhibiting extremely low side effects both on cellular and in NSCLC model. This work has identified imperialine as a promising novel anti-cancer compound and offered an efficient target-delivery solution that greatly facilitate practical use of imperialine.

Exosome-like nanoplatform modified with targeting ligand improves anti-cancer and anti-inflammation effects of imperialine.

Currently, most anti-cancer therapies are still haunted by serious and deleterious adverse effects. Here, we report a highly biocompatible tumor cell-targeting delivery systems utilizing exosome-like vesicles (ELVs) that delivers a low-toxicity anti-cancer agent imperialine against non-small cell lung cancer (NSCLC). First, we introduced a novel micelle-aided method to efficiently load imperialine into intact ELVs. Then, integrin α3ß1-binding octapeptide cNGQGEQc was modified onto ELV platform for tumor targeting as integrin α3ß1 is overexpressed on NSCLC cells. This system not only significantly improved imperialine tumor accumulation and retention, but also had extremely low systemic toxicity both in vitro and in vivo. Our discoveries offer new ways to utilize ELV more efficiently for both drug loading and targeting. The solid pharmacokinetics improvement and extraordinary safety of this system also highlight possibilities of alternative long course cancer therapies using similar strategies.

Liver-Targeted Co-delivery of Entecavir and Glycyrrhetinic Acid Based on Albumin Nanoparticle To Enhance the Accumulation of Entecavir.

Hepatitis B, one of the most common contagious viral hepatitis with high infection rate, is challenging to treat. Although the treatment for hepatitis B has been improved over the years, many therapeutic drugs still have either severe adverse effects or insufficient effectiveness via systemic administration. In this study, we confirmed that glycyrrhetinic acid can enhance the accumulation of entecavir in HepaRG cell and liver. Then we constructed a novel albumin nanoparticle co-loading entecavir and glycyrrhetinic acid (ETV-GA-AN) to improve liver accumulation of entecavir and investigated its ability to deliver both drugs to liver. In vitro cellular uptake study and in vivo tissue distribution experiment showed that these negatively charged ETV-GA-AN (112 ± 2 nm in diameter) can increase the accumulation of entecavir in hepatic HepaRG cells and improve entecavir distribution in liver. We also revealed the mechanism that glycyrrhetinic acid enhances intracellular accumulation of entecavir by inhibiting the activity of specific efflux transporters. Our delivery system is the first liver-targeted albumin nanoparticle that utilizes the site-specific co-delivery strategy to delivery entecavir and glycyrrhetinic acid. As it combines high efficiency and low toxicity, it possess great potential for treating hepatitis B.

Dopamine-loaded blood exosomes targeted to brain for better treatment of Parkinson's disease.

Parkinson's disease (PD), one of the most common movement and neurodegenerative disorders, is challenging to treat, largely because the blood-brain barrier blocks passage of most drugs. Here we find exosomes from blood showing natural brain targeting ability which involved the transferrin-transferrin receptor interaction. Thus, we develop a biocompatible platform based on blood exosomes for delivering drugs across the blood-brain barrier. Blood exosomes show sizes between 40 and 200 nm and spherical morphology, and dopamine can be efficiently loaded into blood exosomes by a saturated solution incubation method. Further in vitro and in vivo studies demonstrates these exosomes successfully delivered dopamine to brain, including the striatum and substantia nigra. Brain distribution of dopamine increased >15-fold by using the blood exosomes as delivery system. Dopamine-loaded exosomes show much better therapeutic efficacy in a PD mouse model and lower systemic toxicity than free dopamine after intravenous administration. These results suggest that blood exosomes can be used as a promising drug delivery platform for targeted therapy against PD and other diseases of the central nervous system.

A novel α-enolase-targeted drug delivery system for high efficacy prostate cancer therapy.

Prostate cancer, one of the leading causes of disease and death in men all over the world, is challenging to treat. α-Enolase, a multifunctional protein, is overexpressed on human prostate carcinoma cells, and thereby it is a potential target for treatment of prostate cancer. In the current study, the pHCT74 peptide was used to construct a kind of highly targeted liposome (pHCT74-lipo) loaded with doxorubicin (pHCT74-lipo-Dox), which specifically targeted α-enolase on prostate tumour cells. Compared with liposomes without pHCT74 modification, pHCT74-lipo-Dox displayed a superior intracellular internalization with enhanced tumour cytotoxicity. In the in vivo study, pHCT74-lipo showed much higher tumour accumulation. In addition, loaded into pHCT74-lipo, doxorubicin demonstrated significantly improved anti-tumour activity on prostate tumour-bearing mice. These results suggest that the pHCT74 peptide has potential to be used in the development of a novel drug delivery system for targeted therapy against prostate cancer.

A brain targeting functionalized liposomes of the dopamine derivative N-3,4-bis(pivaloyloxy)-dopamine for treatment of Parkinson's disease.

Parkinson's disease (PD) remains one of the most common neurodegenerative movement disorders with limited treatment options available. A dopamine derivative N-3,4-bis(pivaloyloxy)-dopamine (BPD) previously developed in our group has demonstrated superior therapeutic outcome compared to levodopa in a PD mice model. To further improve the therapeutic performance of BPD, a brain targeted drug delivery system was designed using a 29 amino-acid peptide (RVG29) derived from rabies virus glycoprotein as the targeting ligand. RVG29 functionalized liposomes (RVG29-lip) showed significantly higher uptake efficiency in murine brain endothelial cells and dopaminergic cells, and high penetration efficiency across the blood brain barrier (BBB) in vitro. In vivo and ex vivo distribution studies demonstrated RVG29-lip selectively distributed to the brain, striatum and substantia nigra. Furthermore, BPD loaded RVG29-lip (BPD-RVG29-lip) exhibited improved therapeutic efficacy in a PD mouse model, while causing no obvious systemic toxicity after intravenous administration. Thus, BPD-RVG29-lip represents a highly promising approach for the brain targeted treatment of PD.

A (polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer)-dispersed sustained-release tablet for imperialine to simultaneously prolong the drug release and improve the oral bioavailability.

Imperialine, extracted from Bulbus Fritillariae Cirrhosae, is an efficient antitussive and expectorant medicine. However, its short half-life and stomach degradation limited imperialine from further clinical use. The current study was conducted to develop a sustained-release tablet for imperialine both to prolong absorption time and to improve the oral bioavailability of the drug. The tablets were prepared by a direct compression method formulated on optimized solid dispersion (SD) for imperialine based on polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus(®)) with imperialine/Soluplus(®) ratio of 1:8 (w/w). In order to obtain the optimized formulation, factors that affected the drug release were investigated by in vitro dissolution studies in the media of pH1.2, 5.8, 7.0 and 7.4. Powder X-ray diffraction and scanning electron microscope confirmed that the imperialine in SD was amorphous instead of crystalline, and still stayed amorphous even after the direct compression. And besides, pharmacokinetic study in Beagle dogs was performed to inspect the in vivo sustained release. Plasma concentration-time curves and pharmacokinetic parameters were gained. As a result, the Cmax of imperialine was one-fold reduced and Tmax was two-fold prolonged, and the mean AUC0-24 was expressed as 89.581±21.243µgh/L, which showed that the oral bioavailability of imperialine was 2.46-fold improved. Moreover, the in vitro-in vivo correlation was recommended to carry out, demonstrating the percentages of drug release in vitro were well-correlated with the absorptive fraction in vivo with the correlation coefficients above 0.9900. By mathematically modeling and moment imaging of the drug release, Peppas equation was selected as the most fitted model for the sustained-release tablets with the diffusional coefficient in the range of 0.59-0.62, indicating the release of imperialine from the sustained-release tablets was an anomalous process involving polymer swelling, drug diffusion and matrix erosion.