Preparation of Betulinic Acid Galactosylated Chitosan Nanoparticles and Their Effect on Liver Fibrosis.

Aim: Liver fibrosis is mainly characterized by the formation of fibrous scars. Galactosylated chitosan (GC) has gained increasing attention as a liver-targeted drug carrier in recent years. The present study aimed to investigate the availability of betulinic acid-loaded GC nanoparticles (BA-GC-NPs) for liver protection. Covalently-conjugated galactose, recognized by asialoglycoprotein receptors exclusively expressed in hepatocytes, was employed to target the liver. Materials and Methods: Galactose was coupled to chitosan by chemical covalent binding. BA-GC-NPs were synthesized by wrapping BA into NPs via ion-crosslinking method. The potential advantage of BA-GC-NP as a liver-targeting agent in the treatment of liver fibrosis has been demonstrated in vivo and in vitro. Results: BA-GC-NPs with diameters <200 nm were manufactured in a virtually spherical core-shell arrangement, and BA was released consistently and continuously for 96 h, as assessed by an in vitro release assay. According to the safety evaluation, BA-GC-NPs demonstrated good biocompatibility at the cellular level and did not generate any inflammatory reaction in mice. Importantly, BA-GC-NPs showed an inherent liver-targeting potential in the uptake behavioral studies in cells and bioimaging tests in vivo. Efficacy tests revealed that administering BA-GC-NPs in a mouse model of liver fibrosis reduced the degree of liver injury in mice. Conclusion: The findings showed that BA-GC-NPs form a safe and effective anti-hepatic fibrosis medication delivery strategy.

Vitamin A - modified Betulin polymer micelles with hepatic targeting capability for hepatic fibrosis protection.

Targeting hepatic stellate cells (HSCs) can improve the therapeutic efficacy of medicines used to treat hepatic fibrosis. The present work aimed to study the feasibility of homing devices with vitamin A(VA) chemically attached for delivering betulin(Bt)specifically to HSCs. The manufacture and characterisation of VA modified poly (ethylene glycol) -poly (lactide-co-glycolide) block copolymer micelles loaded with Bt (Bt/ VAPPMs) and their potential therapeutic benefits in vitro and in vivo are described in this paper. Bt/VAPPMs were made in a nearly spherical core-shell configuration with diameters under 200nm.In vitro release study showed that Bt/VAPPMs exhibited steady and continuous release for over 168 hours. Bt/VAPPMs had good biocompatibility at the cellular level, according to the safety evaluation, and elicited no inflammatory response in mice. More importantly, as uptake behavior studied in cells and bioimaging experiments in vivo, Bt/VAPPMs exhibited an instinctive liver- targeting capability to focus on activated HSCs. Efficacy tests revealed that administering Bt/VAPPMs effectively inhibits collagen I expression in LX-2 cells in vitro, and this effect was also seen in a mouse model of liver fibrosis. Overall, results demonstrated that Bt/VAPPMs is a promising drug delivery system that possesses specific HSCs targeting ability for treating hepatic fibrosis.

Arctigenin ameliorates depression-like behaviors in Toxoplasma gondii-infected intermediate hosts via the TLR4/NF-κB and TNFR1/NF-κB signaling pathways.

Toxoplasma gondii (T. gondii) is a known neurotropic protozoan that remains in the central nervous system and induces neuropsychiatric diseases in intermediate hosts. Arctigenin (AG) is one of the major bioactive lignans of the fruit Arctium lappa L. and has a broad spectrum of pharmacological activities such as neuroprotective, anti-inflammatory and anti-T. gondii effects. However, the effect of AG against depressive behaviors observed in T. gondii-infected hosts has not yet been clarified. In the present study, we analyzed the effects of AG against T. gondii-induced depressive behaviors in intermediate hosts using a microglia cell line (BV2 cells) and brain tissues of BALB/c mice during the acute phase of infection with the RH strain of T. gondii. AG attenuated microglial activation and neuroinflammation via the Toll-like receptor/nuclear factor-kappa B (NF-κB) and tumor necrosis factor receptor 1/NF-κB signaling pathways, followed by up-regulating the dopamine and 5-hydroxytryptamine levels and inhibiting the depression-like behaviors of hosts. AG also significantly decreased the T. gondii burden in mouse brain tissues. In conclusion, we elucidated the effects and underlying molecular mechanisms of AG against depressive behaviors induced by T. gondii infection.

Ultrasensitive MRI detection of spontaneous pancreatic tumors with nanocage-based targeted contrast agent.

Contrast agents with greater specificity and sensitivity are required for the diagnosis of pancreatic cancers by magnetic resonance imaging (MRI). In this study, small heat shock protein 16.5 (Hsp16.5)-based nanocages conjugated to gadolinium(III)-chelated contrast agents and iRGD peptides (which target neuropilin-1 expressed on pancreatic cancer cells) were developed. To investigate whether template size influences relaxivity, nanocages with one to four hydrophobic domains were designed. MRI data showed that larger nanocages had higher T1 relaxivity than smaller nanocages, which resulted from a reduction in molecular tumbling rates caused by an increase in nanocage size, and a robust cage structure resulting from the introduction of hydrophobic domains. For in vivo MRI studies, the engineered nanocages were evaluated using the KrasG12D; Trp53R172H; Pdx-1Cre (KPC) transgenic mouse models, which develop clinically relevant pancreatic tumor under normal processes of angiogenesis, immune function and inflammation. Molecular MRI with protein nanocages was enabled to detect neuropilin-1-positive cells and to produce strong signal enhancement of spontaneous pancreatic tumors in KPC genetically engineered mouse models. Novel iRGD-modified nanocages displayed potential as a specific and sensitive MRI contrast agent for the diagnosis of pancreatic tumors for clinical translation.

Noninvasive mapping of the redox status of dimethylnitrosamine-induced hepatic fibrosis using in vivo dynamic nuclear polarization-magnetic resonance imaging.

Hepatic fibrosis is a chronic disorder caused by viral infection and/or metabolic, genetic and cholestatic disorders. A noninvasive procedure that enables the detection of liver fibrosis based on redox status would be useful for disease identification and monitoring, and the development of treatments. However, an appropriate technique has not been reported. This study describes a novel method for assessing the redox status of the liver using in vivo dynamic nuclear polarization-magnetic resonance imaging (DNP-MRI) with the nitroxyl radical carbamoyl-PROXYL as a molecular imaging probe, which was tested in dimethylnitrosamine-treated mice as a model of liver fibrosis. Based on the pharmacokinetics of carbamoyl-PROXYL in control livers, reduction rate mapping was performed in fibrotic livers. Reduction rate maps demonstrated a clear difference between the redox status of control and fibrotic livers according to the expression of antioxidants. These findings indicate that in vivo DNP-MRI with a nitroxyl radical probe enables noninvasive detection of changes in liver redox status.

Förster Resonance Energy Transfer-Based Self-Assembled Nanoprobe for Rapid and Sensitive Detection of Postoperative Pancreatic Fistula.

Postoperative pancreatic fistula (POPF) is the most serious and challenging complication following gastroenterological surgery. Activated pancreatic juice leaking from the organ remnant contains proteases that attack the surrounding tissue, potentially leading to severe inflammation, tissue necrosis, and fistula formation. However, it is difficult to observe pancreatic leakage during surgery and to evaluate the protease activity of leaked fluid at the patient's bedside. This report describes a protein nanocage-based protease ratiometric sensor comprising a pancreatic protease-sensitive small heat-shock protein (HSP) 16.5, which is a naturally occurring protein in Methanococcus jannaschii that forms a spherical structure by self-assembly of 24 subunits, and a chemically conjugated donor-acceptor dye pair for Förster resonance energy transfer (FRET). The HSP-FRET probe was constructed by subunit exchange of each dye-labeled engineered HSP, resulting in a spherical nanocage of approximately 10 nm in diameter, which exhibited very high stability against degradation in blood plasma and no remarkable toxicity in mice. The efficiency of FRET was found to depend on both the dye orientation and the acceptor/donor ratio. Pancreatic proteases, including trypsin, α-chymotrypsin, and elastase, were quantitatively analyzed by fluorescence recovery with high specificity using the HSP-FRET nanoprobe. Furthermore, the HSP-FRET nanoprobe was sufficiently sensitive to detect POPF in the pancreatic juice of patients using only the naked eye within 10 min. Thus, this novel nanoprobe is proposed as an effective and convenient tool for the detection of POPF and the visualization of activated pancreatic juice during gastroenterological surgery.

Splenectomy enhances the therapeutic effect of adipose tissue-derived mesenchymal stem cell infusion on cirrhosis rats.

BACKGROUND & AIMS: Clinical studies suggest that splenectomy improves liver function in cirrhotic patients, but the influence of splenectomy on stem cell transplantation is poorly understood. This study investigated the effect of splenectomy on stem cell infusion and elucidated its mechanism. METHODS: Rat adipose tissue-derived mesenchymal stem cells were infused into cirrhosis rats with or without splenectomy, followed by the assessment of the in vivo distribution of stem cells and pathological changes. Stromal cell-derived factor-1 and hepatocyte growth factor expression were also investigated in splenectomized cirrhosis patients and rats. RESULTS: Splenectomy, prior to cell infusion, improved liver function and suppressed fibrosis progression more efficiently than cell infusion alone in the experimental cirrhosis model. Stromal cell-derived factor-1 and hepatocyte growth factor levels after splenectomy were increased in patients and rats. These upregulated cytokines significantly facilitated stem cell motility, migration and proliferation in vitro. C-X-C chemokine receptor type 4 neutralization weakened the promotion of cell migration by these cytokines. The infused cells integrated into liver fibrosis septa and participated in regeneration more efficiently in splenectomized rats. Direct coculture with stem cells led to inhibition of hepatic stellate cell proliferation. In addition, hepatocyte growth factor induced hepatic stellate cell apoptosis via the c-jun N-terminal kinase-p53 pathway. CONCLUSIONS: Splenectomy prior to cell infusion enhanced the therapeutic effect of stem cells on cirrhosis, which involved upregulation of stromal cell-derived factor-1 and hepatocyte growth factor after splenectomy.

Design and Function of Engineered Protein Nanocages as a Drug Delivery System for Targeting Pancreatic Cancer Cells via Neuropilin-1.

We describe the development of neuropilin 1-binding peptide (iRGD)-nanocages that specifically target human pancreatic cancer cells in which an iRGD is joined to the surface of naturally occurring heat shock protein (HSP) cages. Using a genetic engineering approach, the iRGD domain was joined to the C-terminal region of the HSP cage using flexible linker moieties. The characteristics of the interdomain linkages between the nanocage and the iRGD domain play an important role in the specificity and affinity of the iRGD-nanocages for their target cells. An engineered L30-iRGD-nanocage with 30 amino acid linkers, (GGS)10, showed greater binding affinity for pancreatic cancer cells relative to that of other linkers. Furthermore, a moderately hydrophobic anticancer drug, OSU03012, was successfully incorporated into the L30-iRGD-nanocage by heating the mixture. The OSU03012-loaded L30-iRGD-nanocage induced cell death of pancreatic cancer cells by activating the caspase cascade more effectively than the same concentrations of free OSU03012. The iRGD-nanocages show great potential as a novel nanocarrier for pancreatic cancer-targeted drug delivery.

Basic fibroblast growth factor-treated adipose tissue-derived mesenchymal stem cell infusion to ameliorate liver cirrhosis via paracrine hepatocyte growth factor.

BACKGROUND AND AIM: Recent studies show that adipose tissue-derived mesenchymal stem cells have potential clinical applications. However, the mechanism has not been fully elucidated yet. Here, we investigated the effect of basic fibroblast growth factor-treated adipose tissue-derived mesenchymal stem cells infusion on a liver fibrosis rat model and elucidated the underlying mechanism. METHODS: Adipose tissue-derived mesenchymal stem cells were infused into carbon tetrachloride-induced hepatic fibrosis rats through caudal vein. Liver functions and pathological changes were assessed. A co-culture model was used to clarify the potential mechanism. RESULTS: Basic fibroblast growth factor treatment markedly improved the proliferation, differentiation, and hepatocyte growth factor expression ability of adipose tissue-derived mesenchymal stem cells. Although adipose tissue-derived mesenchymal stem cells infusion alone slightly ameliorated liver functions and suppressed fibrosis progression, basic fibroblast growth factor-treatment significantly enhanced the therapeutic effect in association with elevated hepatocyte growth factor expression. Moreover, double immunofluorescence staining confirmed that the infused cells located in fibrosis area. Furthermore, co-culture with adipose tissue-derived mesenchymal stem cell led to induction of hepatic stellate cell apoptosis and enhanced hepatocyte proliferation. However, these effects were significantly weakened by knockdown of hepatocyte growth factor. Mechanism investigation revealed that co-culture with adipose tissue-derived mesenchymal stem cells activated c-jun N-terminal kinase-p53 signaling in hepatic stellate cell and promoted apoptosis. CONCLUSIONS: Basic fibroblast growth factor treatment enhanced the therapeutic effect of adipose tissue-derived mesenchymal stem cells, and secretion of hepatocyte growth factor from adipose tissue-derived mesenchymal stem cells plays a critical role in amelioration of liver injury and regression of fibrosis.

Expression and characterization of myristoylated preS1-conjugated nanocages for targeted cell delivery.

Lipid modification of proteins plays key roles in cellular signaling pathways. We describe the development of myristoylated preS1-nanocages (myr-preS1-nanocages) that specifically target human hepatocyte-like HepaRG cells in which a specific receptor-binding peptide (preS1) is joined to the surface of naturally occurring ferritin cages. Using a genetic engineering approach, the preS1 peptide was joined to the N-terminal regions of the ferritin cage via flexible linker moieties. Myristoylation of the preS1 peptide was achieved by co-expression with yeast N-myristoyltransferase-1 in the presence of myristic acid in Escherichia coli cells. The myristoylated preS1-nanocages exhibited significantly greater specificity for human hepatocyte-like HepaRG cells than the unmyristoylated preS1-nanocages. These results suggest that the lipid-modified nanocages have great potential for effective targeted delivery to specific cells.

Systemic delivery of protein nanocages bearing CTT peptides for enhanced imaging of MMP-2 expression in metastatic tumor models.

Matrix metalloproteinase 2 (MMP-2) in metastatic cancer tissue, which is associated with a poor prognosis, is a potential target for tumor imaging in vivo. Here, we describe a metastatic cancer cell-targeted protein nanocage. An MMP-2-binding peptide, termed CTT peptide (CTTHWGFTLC), was conjugated to the surface of a naturally occurring heat shock protein nanocage by genetic modification. The engineered protein nanocages showed a binding affinity for MMP-2 and selective uptake in cancer cells that highly expressed MMP-2 in vitro. In near-infrared fluorescence imaging, the nanocages showed specific and significant accumulation in tumor tissue after intravenous injection in vivo. These protein nanocages conjugated with CTT peptide could be potentially applied to a noninvasive near-infrared fluorescence detection method for imaging gelatinase activity in metastatic tumors in vivo.

A nanocarrier based on a genetically engineered protein cage to deliver doxorubicin to human hepatocellular carcinoma cells.

Herein, we report the preparation of genetically engineered protein cages (HspG41C-SP94), taken up selectively by human hepatocellular carcinoma (HCC) cells. An engineered protein cage-doxorubicin (DOX) conjugate was as cytotoxic as free DOX against HCC cells but much less cytotoxic against normal hepatocytes.

Biological evaluation of protein nanocapsules containing doxorubicin.

This study describes the applications of a naturally occurring small heat shock protein (Hsp) that forms a cage-like structure to act as a drug carrier. Mutant Hsp cages (HspG41C) were expressed in Escherichia coli by substituting glycine 41 located inside the cage with a cysteine residue to allow conjugation with a fluorophore or a drug. The HspG41C cages were taken up by various cancer cell lines, mainly through clathrin-mediated endocytosis. The cages were detected in acidic organelles (endosomes/lysosomes) for at least 48 hours, but none were detected in the mitochondria or nuclei. To generate HspG41C cages carrying doxorubicin (DOX), an anticancer agent, the HspG41C cages and DOX were conjugated using acid-labile hydrazone linkers. The release of DOX from HspG41C cages was accelerated at pH 5.0, but was negligible at pH 7.2. The cytotoxic effects of HspG41C-DOX against Suit-2 and HepG2 cells were slightly weaker than those of free DOX, but the effects were almost identical in Huh-7 cells. Considering the relatively low release of DOX from HspG41C-DOX, HspG41C-DOX exhibited comparable activity towards HepG2 and Suit-2 cells and slightly stronger cytotoxicity towards Huh-7 cells than free DOX. Hsp cages offer good biocompatibility, are easy to prepare, and are easy to modify; these properties facilitate their use as nanoplatforms in drug delivery systems and in other biomedical applications.

Liver cell specific targeting by the preS1 domain of hepatitis B virus surface antigen displayed on protein nanocages.

Protein nanocages are self-organized complexes of oligomers whose three-dimensional architecture can been determined in detail. These structures possess nanoscale inner cavities into which a variety of molecules, including therapeutic or diagnostic agents, can be encapsulated. These properties yield these particles suitable for a new class of drug delivery carrier, or as a bioimaging reagent that might respond to biochemical signals in many different cellular processes. We report here the design, synthesis, and biological characterization of a hepatocyte-specific nanocage carrying small heat-shock protein. These nanoscale protein cages, with a targeting peptide composed of a preS1 derivative from the hepatitis B virus on their surfaces, were prepared by genetic engineering techniques. PreS1-carrying nanocages showed lower cytotoxicity and significantly higher specificity for human hepatocyte cell lines than other cell lines in vitro. These results suggested that small heat-shock protein-based nanocages present great potential for the development of effective targeted delivery of various agents to specific cells.

Development of human hepatocellular carcinoma cell-targeted protein cages.

We described herein a human hepatocellular carcinoma (HCC) cell-targeted protein cage for which the HCC-binding peptide termed SP94 was modified at the surface of a naturally occurred heat shock protein (Hsp) cage. Six types of HCC-targeted Hsp cages were chemically synthesized using two types of heterobifunctional linker (SM(PEG)(n)) with different lengths and two types of SP94 peptide, which contained a unique Cys residue at the N- or C-terminus of the peptide. These Hsp cages were characterized using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF MS) analyses, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, and dynamic light scattering (DLS) measurement. Fluorescence microscopic observations revealed that all the engineered protein cages bind selectively to HCC cells but not to the other cell lines tested (including normal liver cell). Moreover, the number of SP94 peptides on Hsp cages, conjugation site of SP94 peptide, and linker length between a Hsp cage and a SP94 peptide had important effects upon the binding of engineered Hsp cages to HCC cells. An engineered Hsp cage conjugated to the N-terminus of SP94 peptide via a longer linker molecule and containing high SP94 peptide levels showed greater binding toward HCC cells. Surprisingly, through optimization of these three factors, up to 10-fold greater affinity toward HCC cells was achieved. These results are critically important not only for the development of HCC cell-targeting devices using SP94 peptide, but also to create other cell-targeting materials that utilize other peptide ligands.