Development of a Mitochondrial Targeting Lipid Nanoparticle Encapsulating Berberine.

Delivering drugs to mitochondria, the main source of energy in neurons, can be a useful therapeutic strategy for the treatment of neurodegenerative diseases. Berberine (BBR), an isoquinoline alkaloid, acts on mitochondria and is involved in mechanisms associated with the normalization and regulation of intracellular metabolism. Therefore, BBR has attracted considerable interest as a possible therapeutic drug for neurodegenerative diseases. While BBR has been reported to act on mitochondria, there are few reports on the efficient delivery of BBR into mitochondria. This paper reports on the mitochondrial delivery of BBR using a lipid nanoparticle (LNP), a "MITO-Porter" that targets mitochondria, and its pharmacological action in Neuro2a cells, a model neuroblastoma. A MITO-Porter containing encapsulated BBR (MITO-Porter (BBR)) was prepared. Treatment with MITO-Porter (BBR) increased the amount of BBR that accumulated in mitochondria compared with a treatment with naked BBR. Treatment with MITO-Porter (BBR) resulted in increased ATP production in Neuro2a cells, which are important for maintaining life phenomena, compared with treatment with naked BBR. Treatment with MITO-Porter (BBR) also increased the level of expression of mitochondrial ubiquitin ligase (MITOL), which is involved in mitochondrial quality control. Our findings indicate that increasing the accumulation of BBR into mitochondria is important for inducing enhanced pharmacological actions. The use of this system has the potential for being important in terms of the regulation of the metabolic mechanism of mitochondria in nerve cells.

Effect and Mechanism of Pharmaceutical Excipients on Berberine to Alleviate Ulcerative Colitis via Regulating Gut Microbiota.

BACKGROUND: Various potential effect of drugs on alleviating diseases by regulating intestinal microbiome as well as the pharmaceutical excipients on gut microbiota has been revealed. However, the interaction between them is rarely investigated. METHODS: Histological analysis, immunohistochemistry analysis, enzyme-linked immunosorbent assay (ELISA) analysis, RT-qPCR, and 16S rRNA analysis were utilized to explore the effect mechanism of the five excipients including hydroxypropyl methylcellulose (HPMC) F4M, Eudragit (EU) S100, chitosan (CT), pectin (PT), and rheum officinale polysaccharide (DHP) on berberine (BBR) to cure UC. RESULTS: The combined BBR with PT and DHP group exhibited better therapeutic efficacy of UC with significantly increased colon length, and decreased hematoxylin-eosin (H&E) scores than other groups. Furthermore, the expression of tight junction ZO-1 and occludin in colon tissue were upregulated, and claudin-2 was downregulated. Ultimately, the serum content of tumor necrosis (TNF)-α, interleukin (IL)-1ß, and IL-6 was decreased. Moreover, the combined BBR with PT significantly promoted the restoration of gut microbiota. The relative abundance of Firmicutes and Lactobacillus was significantly increased by the supplement of PT and DHP, and the relative abundance of Proteobacteria was downregulated. CONCLUSIONS: Our study may provide a new perspective that the selection of pharmaceutical excipients could be a crucial factor affecting the drugs' therapeutic efficiency outcome.

Berberine-Loaded Thiolated Pluronic F127 Polymeric Micelles for Improving Skin Permeation and Retention.

BACKGROUND: Challenges associated with local antibacterial and anti-inflammatory drugs include low penetration and retention of drugs at the expected action site. Additionally, improving these challenges allows for the prevention of side effects that are caused by drug absorption into the systemic circulation and helps to safely treat local skin diseases. METHODS: In the current study, we successfully prepared a thiolated pluronic F127 polymer micelles (BTFM), which binds to keratin through a disulphide bond, to produce skin retention. In addition, the small particle size of polymer micelles promotes the penetration of carriers into the skin. The current study was divided into two experiments: an in vitro experiment; an in vivo experiment that involved the penetration of the micelle-loaded drugs into the skin of rats, the skin irritation test and the anti-inflammatory activity of the drug-loaded micelles on dimethyl benzene-induced ear edema in mice. RESULTS: Results from our in vitro transdermal experiment revealed that the amount of drug absorbed through the skin was decreased after the drug was loaded in the BTFM. Further, results from the vivo study, which used fluorescence microscopy to identify the location of the BTFM after penetration, revealed that there was strong fluorescence in the epidermis layer, but there was no strong fluorescence in the deep skin layer. In addition, the BTFM had a very good safety profile with no potentially hazardous skin irritation and transdermal administration of BTFM could significantly suppress ear edema induced by dimethyl benzene. Therefore, these findings indicated that BTFM reduced the amount of drug that entered the systemic circulation. Our results also demonstrated that the BTFM had a certain affinity for keratin. CONCLUSION: Our experimental results suggest that the BTFM may be an effective drug carrier for local skin therapy with good safety profile.

Development and in-vitro evaluation of co-loaded berberine chloride and evodiamine ethosomes for treatment of melanoma.

Berberine chloride (BBR) and evodiamine (EVO) are two main active ingredients of "ZuoJinWan", a classical Chinese herbal medicine, and these compounds are known to have a synergistic inhibitory effect on various cancer cell lines. Several recent studies have reported anti-melanoma effects for both BBR and EVO. However, topical delivery of the two compounds has been challenging, due to their poor aqueous solubility and their low skin penetration. In the current study, we have combined BBR and EVO into an ethosomes delivery system with the future aim to design a novel topical anti-melanoma formulation. The ethosomes formulations were characterized using particle size, entrapment efficiency and an in vitro skin drug deposition study. The ethosome formulation displaying maximum drug deposition in the epidermis was selected for further study. This formulation contained ethosomes with mean size of 171 nm and 90% or above entrapment efficiency for both BBR and EVO. Cell viability tests proved the optimized ethosomes increased the inhibitory effect on B16 melanoma cells. These results corroborate that ethosomes containing a combination of BBR and EVO are a promising delivery system for potential use in melanoma therapy.

Baicalin and berberine ultradeformable vesicles as potential adjuvant in vitiligo therapy.

0.5-1% of the world's population is affected by vitiligo, a disease characterized by a gradual depigmentation of the skin. Baicalin and berberine are natural compounds with beneficial activities, such as antioxidant, anti-inflammatory and proliferative effects. These polyphenols could be useful for the treatment of vitiligo symptoms, and their efficacy can be improved by loading in suitable carriers. The aim of this work was to formulate and characterize baicalin or berberine loaded ultradeformable vesicles, and demonstrate their potential as adjuvants in the treatment of vitiligo. The vesicles were produced using a previously reported simple, scalable method. Their morphology, size distribution, surface charge and entrapment efficiency were assessed. The ability of the vesicles to promote the permeation of the polyphenols was evaluated. The antioxidant and photoprotective effects were investigated in vitro using keratinocytes and fibroblasts. Further, the stimulation of melanin production and tyrosinase activity in melanocytes after treatment with the vesicles were assessed. Ultradeformable vesicles were small in size, homogeneously dispersed, and negatively charged. They were able to incorporate high amounts of baicalin and berberine, and promote their skin permeation. In fact, the polyphenols concentration in the epidermis was higher than 10%, which could be indicative of the formation of a depot in the epidermis. The vesicles showed remarkable antioxidant and photoprotective capabilities, presumably correlated with the stimulation of melanin production and tyrosinase activity. In conclusion, baicalin or berberine ultradeformable vesicles, and particularly their combination, may represent promising nanosystem-based adjuvants for the treatment of vitiligo symptoms.

Berberine Encapsulated PLGA-PEG Nanoparticles Modulate PCSK-9 in HepG2 Cells.

BACKGROUND: The developments of new parenteral approaches to target PCSK-9 for the treatment of LDL-Cholesterol has yielded impressive results; and have shown significant decreases in the risk of mortality associated with hypercholesterolemia. However improved and convenient alternate approaches that exploit the beneficial drug target properties of PCSK-9 also need to be explored and developed. One such approach is the oral administration of Berberine using nanotechnology. METHODS: Nanoprecipitation encapsulation and physiochemical characterization of Berberine Chloride in PLGA-PEG-PLGA block copolymer has been developed and characterized in Hep-G2 cells using Berberine Chloride encapsulated nanoparticle (Bc-NP). Evaluation of PCSK-9, SREBP- 1, LDL-r, HNF-1alpha mRNAs and PCSK-9 protein expression was performed using quantitative real-time PCR (qPCR) and median fluorescence intensity (MFI) of flow cytometric studies respectively. Pearson's correlation analysis of PCSK-9 mRNA and protein levels in Berberine chloride delivery was performed. RESULTS: The PCSK-9 mRNA and protein expression shows a relationship to the release of Berberine from the encapsulating PLGA-PEG-PLGA polymer in a time dependent manner. CONCLUSION: PCSK-9 modulation by oral administration of Berberine using nanotechnology approach can improve its pharmacokinetic profile. Further studies are needed to maximize its delivery efficiency.

The single berberine bridge enzyme homolog of Physcomitrella patens is a cellobiose oxidase.

The berberine bridge enzyme from the California poppy Eschscholzia californica (EcBBE) catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine, that is, the formation of the berberine bridge in the biosynthesis of benzylisoquinoline alkaloids. Interestingly, a large number of BBE-like genes have been identified in plants that lack alkaloid biosynthesis. This finding raised the question of the primordial role of BBE in the plant kingdom, which prompted us to investigate the closest relative of EcBBE in Physcomitrella patens (PpBBE1), the most basal plant harboring a BBE-like gene. Here, we report the biochemical, structural, and in vivo characterization of PpBBE1. Our studies revealed that PpBBE1 is structurally and biochemically very similar to EcBBE. In contrast to EcBBE, we found that PpBBE1 catalyzes the oxidation of the disaccharide cellobiose to the corresponding lactone, that is, PpBBE1 is a cellobiose oxidase. The enzymatic reaction mechanism was characterized by a structure-guided mutagenesis approach that enabled us to assign a catalytic role to amino acid residues in the active site of PpBBE1. In vivo experiments revealed the highest level of PpBBE1 expression in chloronema, the earliest stage of the plant's life cycle, where carbon metabolism is strongly upregulated. It was also shown that the enzyme is secreted to the extracellular space, where it may be involved in later steps of cellulose degradation, thereby allowing the moss to make use of cellulose for energy production. Overall, our results suggest that the primordial role of BBE-like enzymes in plants revolved around primary metabolic reactions in carbohydrate utilization. DATABASE: Structural data are available in the PDB under the accession numbers 6EO4 and 6EO5.

Enhanced production of the polysaccharide arabinogalactan using immobilized cultures of Tinospora cordifolia by elicitation and in situ adsorption.

Immobilized callus cultures of Tinospora cordifolia (Willd) Miers ex Hooks and Thoms were investigated to find out the combined effect of elicitation, cell permeabilization with chitosan and in situ product recovery by polymeric neutral resin-like Diaion HP 20. In this study, callus cultures of T. cordifolia were immobilized using sodium alginate and calcium chloride and the beads were cultured in Murashige and Skoog's basal medium along with benzyl adenine (BA), 2,4-dichlorophenoxy acetic acid (2,4-D) and 3% sucrose. The immobilized cultures, when subjected to elicitation and cell permeabilization with chitosan and in situ removal of the secondary metabolites by addition of resin, showed a 10-fold increase in production of arabinogalactan (0.490% dry weight) as compared to respective controls devoid of resin and chitosan. This indicates that in situ adsorption may have reduced the feedback inhibition caused by accumulation of secondary metabolites in the media, while the dual effect of elicitation and cell permeabilization by chitosan may have released the intracellular (secreted) berberine and the polysaccharide arabinogalactan, respectively.

Characterization of bacterial communities in hybrid upflow anaerobic sludge blanket (UASB)-membrane bioreactor (MBR) process for berberine antibiotic wastewater treatment.

Biodegradation of berberine antibiotic was investigated in upflow anaerobic sludge blanket (UASB)-membrane bioreactor (MBR) process. After 118days of operation, 99.0%, 98.0% and 98.0% overall removals of berberine, COD and NH4(+)-N were achieved, respectively. The detailed composition of the established bacterial communities was studied by using 16S rDNA clone library. Totally, 400 clones were retrieved and grouped into 186 operational taxonomic units (OTUs). UASB was dominated by Firmicutes and Bacteroidetes, while Proteobacteria, especially Alpha- and Beta-proteobacteria were prevalent in the MBRs. Clostridium, Eubacterium and Synergistes in the UASB, as well as Hydrogenophaga, Azoarcus, Sphingomonas, Stenotrophomonas, Shinella and Alcaligenes in the MBRs were identified as potential functional species in biodegradation of berberine and/or its metabolites. The bacterial community compositions in two MBRs were significantly discrepant. However, the identical functions of the functional species ensured the comparable pollutant removal performances in two bioreactors.

A novel microsphere with a three-layer structure for duodenum-specific drug delivery.

Owing to the quick elimination of drug from duodenum and the depth of Helicobacter pylori (H. pylori) colonized in mucus, antibiotic therapy often fails in the eradication of H. pylori infection for duodenal ulcer. A novel duodenum-specific microsphere (DSM) consisting of three-layer structure was developed to enhance the drug concentration and retention time in duodenal mucus layer. Firstly a core-shell mucoadhesive microsphere was prepared with a novel emulsification/coagulation coating method by introducing drug loaded Eudragit cores into a thiolated chitosan mucoadhesive layer. Then the obtained core-shell mucoadhesive microspheres were further coated with hydroxypropyl methylcellulose acetate maleate as the pH-sensitive layer for the trigger of mucoadhesion and drug release in duodenum. From the fluorescence microscopic and scanning electron microscopic images, the three-layer structure was successfully established. The microspheres exhibited a duodenum-specific trigger performance, good mucoadhesive property and pH-dependent drug release. In vivo study performed in rats demonstrated that DSM exhibited about 3-fold augmentation of AUC and about 5-fold augmentation of C(max) for duodenal mucus drug concentration compared with free drug suspension. These results suggest that the three-layer structure microspheres may provide a promising approach for duodenum-targeting drug delivery system.

Integrated bioprocessing for plant cell cultures.

Plant cell suspension culture has become the focus of much attention as a tool for the production of secondary metabolites including paclitaxel, a well-known anticancer agent. Recently, it has also been regarded as one of the host systems for the production of recombinant proteins. In order to produce phytochemicals using plant cell cultures, efficient processes must be developed with adequate bioreactor design. Most of the plant secondary metabolites are toxic to cells at the high concentrations required during culture. Therefore, if the product could be removed in situ during culture, productivity might be enhanced due to the alleviation of this toxicity. In situ removal or extractive bioconversion of such products can be performed by in situ extraction with various kinds of organic solvents. In situ adsorption using polymeric resins is another possibility. Using the fact that secondary metabolites are generally hydrophobic, various integrated bioprocessing techniques can be designed not only to lower toxicity, but also to enhance productivity. In this article, in situ extraction, in situ adsorption, utilization of cyclodextrins, and the application of aqueous two-phase systems in plant cell cultures are reviewed.