Characterization and Immunological Activities of Polysaccharides from Polygonatum sibiricum.

In this study, we investigated the physicochemical properties and composition of monosaccharidex from Polygonatum sibiricum. Simultaneously, we explored the in vivo and in vitro immunomodulatory activity and mechanism of Polygonatum sibiricum polysaccharide (PSP) activity by monitoring changes in immune organs, immune cells, and cytokines. The average molecular weight (Mw) of PSP was 9.514 × 104 Da. The monosaccharide components of PSP were galactose, rhamnose, arabinose, mannose, and glucose at a molar ratio of 11.72 : 1.78 : 4.15 : 1.00 : 2.48. PSP increased thymus and spleen indices, enhance the proliferative responses of splenocytes, and increased the phagocytosis of mononuclear macrophages. Simultaneously, PSP could recover the body mass of immunosuppressed mice, and increased blood erythrocyte counts in the sera of cyclophosphamide (Cy)-treated and normal mice, whilst blood leukocytes and platelet counts of Cy-treated mice recovered. PSP elevated the CD4+/CD8+ ratio is a dose-dependent manner and increased the levels of interleukin-2 (IL-2) and tumor necrosis factor-α (TNF-α) in the sera of Cy-treated mice. PSP further enhanced the expression of IL-2 and TNF-α in spleen lymphocytes. Additionally, PSP treatment accelerated the recovery of natural killer cell activity in a dose-dependent manner. Taken together, PSP not only regulated the immune function of normal mice, but participated in the protection against immunosuppression in Cy-treated mice, highlighting its potential as an immunostimulant.

Nanoparticles Targeted against Cryptococcal Pneumonia by Interactions between Chitosan and Its Peptide Ligand.

Inspired by the fact that chitosan is a representative constituent of the ectocellular structure of Cryptococcus neoformans and a typical biomaterial for improving drug oral absorption, we designed an elegant and efficient C. neoformans-targeted drug delivery system via oral administration. A chitosan-binding peptide screened by phage display was used as the targeting moiety, followed by conjugation to the surface of poly(lactic- co-glycolic acid) nanoparticles as the drug carrier, which was then incubated with free chitosan. The noncovalently bound chitosan adheres to mucus layers and significantly enhances penetration of nanoparticles through the oral absorption barrier into circulation and then re-exposed the targeting ligand for later recognition of the fungal pathogen at the site of infection. After loading itraconazole as a model drug, our drug delivery system remarkably cleared lung infections of C. neoformans and increased survival of model mice. Currently, targeted drug delivery is mainly performed intravenously; however, the system described in our study may provide a universal means to facilitate drug targeting to specific tissues and disease sites by oral administration and may be especially powerful in the fight against increasingly severe fungal infections.

[Effect of polysaccharides from Polygonatum sibiricum on lipid-metabolism related mRNA and protein expression in hyperlipidemic mice].

To study the effect of polysaccharides from Polygonatum sibiricum on mRNA and protein expressions of blood lipid metabolism in hyperlipidemic mice. The mice were randomly divided into 6 groups, namely the blank control group, the hyperlipidemia model group, the simvastatin group, and low, middle and high-dose PSP groups (200, 400, 800 mg·kg⁻¹·d⁻¹). Each group of the mice was administrated intragastrically for 14 days, respectively. Subsequently, every group of mice, except for the blank control group, was intraperitoneally injected with 75% fresh egg yolk emulsion for establishing the hyperlipidemic mice model. Upon completion of the administration, the contents of TC, TG, LDL-C and HDL-C in serum of each group were investigated in details. In particular, the mRNA expression levels of PPAR-α, PPAR-ß, PPAR-γ, SREBP-1c, IL-6 and TNF-α of the liver tissues were detected by Real-time PCR, and the protein expression levels (including PPAR-α, PPAR-ß, PPAR-γ, SREBP-1c, IL-6, TNF-α) were examined by Western blot. Consequently, the obtained results showed that the contents of the serum TC, TG, LDL-C of low, middle and high-dose PSP groups significantly decreased compared with those of the hyperlipidemia model group. Simultaneously, there were significant differences between middle-dose and high-dose PSP groups (P<0.01). In striking contrast, the contents of serum HDL-C of low, middle and high-dose PSP groups significantly increased, while obvious differences were also observed between middle-dose and high-dose PSP groups (P<0.01). Moreover, middle-dose and high-dose PSR groups could up-regulate the protein and mRNA expressions of PPAR-α, PPAR-ß (P<0.05) compared with those of the hyperlipidemia model group, and down-regulate the expressions of PPAR-γ,SREBP-1c, IL-6 and TNF-α(P<0.05) compared with those of liver tissues of the hyperlipidemia model group. In conclusion, all of the above results suggested that PSP could inhibit the oxidation of the liver lipid, and regulate the expression levels of the corresponding genes and proteins relating to the lipid metabolism, so as to play a critical role for preventing hyperlipidemia.

[Novel liposomal drug delivery system actively targeting Cryptococcus neoformans and elimination of infection].

The purpose of this study is to develop a liposomal drug delivery system actively targeting Cryptococcus neoformans and explore its feasibility in therapy of cryptococcal infection. The specific fungi-binding peptide was screened from 12-mer random phage display library, and linked to PEG-DSPE as the functional material of liposomes. The targeting capability of peptide-modified liposomes were investigated by fungi binding assay in vitro and fluorescence imaging in vivo. Itraconazole as a model drug were then encapsulated in the liposomes and were evaluated in pharmacodynamic test in vitro and for therapeutic effects against cryptococcal meningitis complicated with pulmonary cryptococcosis in vivo. The results showed that the peptide (sequence: NNHREPPDHRTS) could selectively recognize Cryptococcus and effectively mediate the corresponding liposomal formulation to accumulate in the infection site in vivo. This peptide-modified liposome has a small particle size (mean diameter of 88.25 ± 2.43 nm) with a homogeneous distribution and high encapsulation efficiency (88.05 ± 0.25 %) of itraconazole. After intravenous administration, the pathogens were obviously eliminated in lung and brain, and the life-span of model mice were significantly prolonged, suggesting a promising potential of this cryptococcosis targeting strategy.

[Construction of biotin-modified polymeric micelles for pancreatic cancer targeted photodynamic therapy].

In this study, we explored the feasibility of biotin-mediated modified polymeric micelles for pancreatic cancer targeted photodynamic therapy. Poly (ethylene glycol)-distearoyl phosphatidyl ethanolamine (mPEG2000-DSPE) served as the drug-loaded material, biotin-poly(ethylene glycol)-distearoyl phosphatidyl ethanolamine (Biotin-PEG3400-DSPE) as the functional material and the polymeric micelles were prepared by a thin-film hydration method. The targeting capability of micelles was investigated by cell uptake assay in vitro and fluorescence imaging in vivo and the amounts of Biotin-PEG-DSPE were optimized accordingly. Hypocrellin B (HB), a novel photosensitizer was then encapsulated in biotinylated polymeric micelles and the anti-tumor efficacy was evaluated systemically in vitro and in vivo. The results showed that micelles with 5 mol % Biotin-PEG-DSPE demonstrated the best targeting capability than those with 20 mol % or 0.5 mol % of corresponding materials. This formulation has a small particle size [mean diameter of (36.74 ± 2.16) nm] with a homogeneous distribution and high encapsulation efficiency (80.06 ± 0.19) %. The following pharmacodynamics assays showed that the biotinylated micelles significantly enhanced the cytotoxicity of HB against tumor cells in vitro and inhibited tumor growth in vivo, suggesting a promising potential of this formulation for treatment of pancreatic cancer, especially those poorly permeable, or insensitive to radiotherapy and chemotherapy.

Active targeting of tumors through conformational epitope imprinting.

Inspired by the knowledge that most antibodies recognize a conformational epitope because of the epitope's specific three-dimensional shape rather than its linear structure, we combined scaffold-based peptide design and surface molecular imprinting to fabricate a novel nanocarrier harboring stable binding sites that captures a membrane protein. In this study, a disulfide-linked α-helix-containing peptide, apamin, was used to mimic the extracellular, structured N-terminal part of the protein p32 and then serve as an imprinting template for generating a sub-40 nm-sized polymeric nanoparticle that potently binds to the target protein, recognizes p32-positive tumor cells, and successfully mediates targeted photodynamic therapy in vivo. This could provide a promising alternative for currently used peptide-modified nanocarriers and may have a broad impact on the development of polymeric nanoparticle-based therapies for a wide range of human diseases.

Apamin-mediated actively targeted drug delivery for treatment of spinal cord injury: more than just a concept.

Faced with the complex medical challenge presented by spinal cord injuries (SCI) and considering the lack of any available curative therapy, the development of a novel method of delivering existing drugs or candidate agents can be perceived to be as important as the development of new therapeutic molecules. By combining three ingredients currently in clinical use or undergoing testing, we have designed a central nervous system targeted delivery system based on apamin-modified polymeric micelles (APM). Apamin, one of the major components of honey bee venom, serves as the targeting moiety, poly(ethylene glycol) (PEG) distearoylphosphatidylethanolamine (DSPE) serves as the drug-loaded material, and curcumin is used as the therapeutic agent. Apamin was conjugated with NHS (N-hydroxysuccinimide)-PEG-DSPE in a site-specific manner, and APM were prepared by a thin-film hydration method. A formulation comprising 0.5 mol % targeting ligand with 50 nm particle size showed strong targeting efficiency in vivo and was evaluated in pharmacodynamic assays. A 7-day treatment by daily intravenous administration of low doses of APM (corresponding to 5 mg/kg of curcumin) was performed. Significantly enhanced recovery and prolonged survival was found in the SCI mouse model, as compared to sham-treated groups, with no apparent toxicity. A single dose of apamin-conjugated polymers was about 700-fold lower than the LD50 amount, suggesting that APM and apamin have potential for clinical applications as spinal cord targeting ligand for delivery of agents in treatment of diseases of the central nervous system.

Long-circulating liposomal daptomycin enhances protection against systemic methicillin-resistant Staphylococcus aureus infection with improved therapeutic potential.

In the face of escalating problems with pathogen control, the development of proper formulations of existing antibiotics is as important as the development of novel antibiotics. Daptomycin is a lipopeptide antibiotic with potent activity against Gram-positive bacteria. Currently, only injectable solution of daptomycin has been approved for clinical use. In the present study, the formulation of PEGylated liposomal daptomycin (PLD) was prepared and optimized, and its efficacy against methicillin-resistant Staphylococcus aureus (MRSA252) strains was investigated. The obtained PLD had a mean vesicle diameter of (111.5 +/- 15.4) nm and a mean percent drug loading of (5.81 +/- 0.19) % with high storage stability. Potent activity of PLD against MRSA was demonstrated in vitro with a more sustained effect than that of conventional liposomal daptomycin and daptomycin solution. In addition, intravenous administration of a single dose (equal to human use) of PLD significantly increased the survival of mice in a MRSA252 systemic infection model compared with other formulations. Drug distribution in the lung was significantly enhanced following administration of PLD, and no measurable tissue lesions or pathological changes were detected during PLD treatment. Taken together, PEGylated liposomes loaded with daptomycin may represent a promising approach to reduce MRSA252 infections, especially those involving bloodstream dissemination, such as hematogenous pulmonary infection.

Effects of wet and dry seasons on the aquatic bacterial community structure of the Three Gorges Reservoir.

This study investigated effects of wet and dry seasons on the bacterial community structure of the Three Gorges Reservoir by using denaturing gradient gel electrophoresis analysis of the PCR-amplified bacterial 16S rRNA gene. Bacterial diversity, as determined by the Shannon index, the Simpson's index, and the Richness, dramatically changed in between the dry and wet seasons. The changes in the diversity and relative abundance of microbial populations among the five sites during the wet season have become more marked than those observed during the dry season. Furthermore, cluster analysis also showed these changes. The phylogenetic analysis indicated that Betaproteobacteria is the dominant population, followed by Actinobacterium, in both the wet season and dry season. The water quality parameters were quite stable at all five sites during the same season but noticeably varied from season to season. Canonical correspondence analysis also indicated that the changes in the bacterial community composition were primarily correlated with the variations in temperature, transparency, and the concentrations of NH4 (+)-N. Slight changes in bacterial community composition among the five sites during the dry season were correlated with different environments. However, during the wet season, major changes were correlated not only with environments, but also it may be associated with the bacterial populations from the surrounding areas and tributaries of the Three Gorges Reservoir.

[Silica-coated ethosome as a novel oral delivery system for enhanced oral bioavailability of curcumin].

The aim of this study is to investigate the feasibility of silica-coated ethosome as a novel oral delivery system for the poorly water-soluble curcumin (as a model drug). The silica-coated ethosomes loading curcumin (CU-SE) were prepared by alcohol injection method with homogenization, followed by the precipitation of silica by sol-gel process. The physical and chemical features of CU-SEs, and curcumin release were determined in vitro. The pharmacodynamics and bioavailability measurements were sequentially performed. The mean diameter of CU-SE was (478.5 +/- 80.3) nm and the polydispersity index was 0.285 +/- 0.042, while the mean value of apparent drug entrapment efficiency was 80.77%. In vitro assays demonstrated that CU-SEs were significantly stable with improved release properties when compared with curcumin-loaded ethosomes (CU-ETs) without silica-coatings. The bioavailability of CU-SEs and CU-ETs was 11.86- and 5.25-fold higher, respectively, than that of curcumin suspensions (CU-SUs) in in vivo assays. The silica coatings significantly promoted the stability of ethosomes and CU-SEs exhibited 2.26-fold increase in bioavailablity relative to CU-ETs, indicating that the silica-coated ethosomes might be a potential approach for oral delivery of poorly water-soluble drugs especially the active ingredients of traditional Chinese medicine with improved bioavailability.

Noncovalent Complexation of Amphotericin B with Poly(ß-Amino Ester) Derivates for Treatment of C. Neoformans Infection.

Our goal was to improve treatment outcomes for C. neoformans infection by designing nanocarriers that enhance drug-encapsulating capacity and stability. Thus, a noncovalent complex of methoxy poly(ethylene glycol)-poly(lactide)-poly(ß-amino ester) (MPEG-PLA-PAE) and amphotericin B (AMB) was developed and characterized. The MPEG-PLA-PAE copolymer was synthesized by a Michael-type addition reaction; the copolymer was then used to prepare the AMB-loaded nanocomplex. AMB was in a highly aggregated state within complex cores. A high encapsulation efficiency (>90%) and stability of the AMB-loaded nanocomplex were obtained via electrostatic interaction between AMB and PAE blocks. This nanocomplex retained drug activity against C. neoformans in vitro. Compared with micellar AMB, the AMB nanocomplex was more efficient in terms of reducing C. neoformans burden in lungs, liver, and spleen, based on its improved biodistribution. The AMB/MPEG-PLA-PAE complex with enhanced drug-loading capacity and stability can serve as a platform for effective treatment of C. neoformans infection.

N-Benzyl-2-(2-chloro-4-methyl-phen-oxy)acetamide.

The structure determination of the title compound, C(16)H(16)ClNO(2), was performed as part of a project on the inter-actions between small organic mol-ecules and proteins. In the crystal structure, the dihedral angle between the two aromatic rings is 16.14 (12)°. The molecules are connected via N-H⋯O hydrogen bonding into chains, which extend in the direction of the b axis.

Synthesis of Carbon Dots Originated from Hydroxypropylmethyl Cellulose for Sensing Ciprofloxacin.

Hereby, one kind of facile carbon dots (CDs) from hydroxypropylmethyl cellulose (HPMC) has been successfully provided, which obviously emitted blue fluorescence. With the related characterizations in detail, the CDs prepared here mainly consisted of C and O, owing to the functional groups of -OH and C=O on their surfaces. Likewise, the CDs also showed multiple advantages, including excellent photostability, superior dispersity and desirable stability. Moreover, the CDs were applied for sensing ciprofloxacin due to forming complexes with ciprofloxacin, thus leading to the fluorescence quenching of CDs. This proposed method was permitted to sense ciprofloxacin in a linear range of 10 nM L-1 - 90 µM L-1, suggesting that it may broaden the sensing ways for assaying ciprofloxacin.

Construction of a two-in-one liposomal system (TWOLips) for tumor-targeted combination therapy.

In oncology, there is a growing need for simpler, more selective methods to deliver drug therapies directly to the tumor site. For combination therapies, simultaneous targeted delivery of multiple drugs would represent a significant improvement. In contrast to previous work that took a de novo approach, we constructed a novel two-in-one liposomal system (TWOLips) from two single drug-loaded liposomes. Our results demonstrated that TWOLips could be prepared by a simple process, through silica coating of one liposome and incubation with the second liposome. TWOLips had a mean diameter of 100 nm, relatively high drug loading rates (96.8%±0.9% for doxorubicin and 78.4%±1.2% for combretastatin), and high storage stability. TWOLips modification by adding a targeting moiety, an all D-amino acid peptide derived from a natural vascular endothelial growth factor, resulted in strong, selective binding to vascular endothelial growth factor receptor 2, a tumorigenesis marker, in vitro and in vivo. TWOLips significantly inhibited tumor growth and angiogenesis and enhanced survival in mice with A375 melanoma xenografts. The TWOLips system had a low potential risk of toxicity. Since the stepwise assembly could be carried further (additional drug-loaded liposomes), TWOLips shows potential as a treatment for many cancers, especially those that require multiple drugs.

A novel peptide delivers plasmids across blood-brain barrier into neuronal cells as a single-component transfer vector.

There is no data up to now to show that peptide can deliver plasmid into brain as a single-component transfer vector. Here we show that a novel peptide, RDP (consisted of 39 amino acids), can be exploited as an efficient plasmid vector for brain-targeting delivery. The plasmids containing Lac Z reporter gene (pVAX-Lac Z) and BDNF gene (pVAX-BDNF) are complexed with RDP and intravenously injected into mice. The results of gel retardation assay show that RDP enables to bind DNA in a dose-dependent manner, and the X-Gal staining identity that Lac Z is specifically expressed in the brain. Also, the results of Western blot and immunofluorescence staining of BDNF indicate that pVAX-BDNF complexed with RDP can be delivered into brain, and show neuroprotective properties in experimental Parkinson's disease (PD) model. The results demonstrate that RDP enables to bind and deliver DNA into the brain, resulting in specific gene expression in the neuronal cells. This strategy provides a novel, simple and effective approach for non-viral gene therapy of brain diseases.

Tumor-targeted liposomal drug delivery mediated by a diseleno bond-stabilized cyclic peptide.

Peptide ligands have played an important role in tumor-targeted drug delivery as targeting moieties. The in vivo fate of peptide-mediated drug delivery systems and the following antitumor effects may greatly depend on the stability of the peptide ligand. In the current study, a tumor-targeting cyclic peptide screened by phage display, Lyp-1 (a peptide that specifically binds to tumor and endothelial cells of tumor lymphatics in certain tumors), was structurally modified by replacement of the original intramolecular disulfide bond with a diseleno bond. The produced analog Syp-1 (seleno derivative of Lyp-1) maintained specific binding ability to the target protein p32 (Kd = 18.54 nM), which is similar to that of Lyp-1 (Kd = 10.59 nM), indicated by surface plasmon resonance assay. Compared with Lyp-1, Syp-1 showed significantly improved stability against serum. After the peptide attached onto the surface of fluorophore-encapsulating liposomes, the more efficient tumor uptake of liposomal fluorophore mediated by Syp-1 was observed. Furthermore, Syp-1 modified liposomal doxorubicin presented the most potent tumor growth inhibitory ability among all the therapeutic groups, with a low half maximal inhibitory concentration of 588 nM against MDA-MB-435 cells in vitro and a high tumor inhibition rate of 73.5% in vivo. These findings clearly indicated that Syp-1 was a stable and effective tumor targeting ligand and suggest that the sulfur-to-selenium replacement strategy may help stabilize the phage-displayed cyclic peptide containing disulfide-bond under physiological conditions and strongly support the validity of peptide-mediated drug targeting.

Preparation and characterization of flexible nanoliposomes loaded with daptomycin, a novel antibiotic, for topical skin therapy.

The purpose of this study was to investigate flexible nanoliposomes for mediating topical delivery of daptomycin, and to document permeation rates and bacteriostatic activity towards skin infections. Response surface methodology was used to optimize the daptomycin-loaded flexible nanoliposomes (DAP-FL), and the amount of drug loaded into the particles was evaluated as the investigation index. The optimal lipid ratio was lecithin to sodium cholate 17:1 (w/w) and the lipid to drug ratio was 14:1 (w/w). The hydration temperature was set at 37°C and the duration of treatment with ultrasound was 20 minutes. The DAP-FL obtained had a small mean particle size (55.4 nm) with a narrow size distribution (polydispersity index 0.15). The mean entrapment efficiency was 87.85% ± 2.15% and the mean percent drug loading was 5.61% ± 0.14%. Using skin mounted between the donor and receptor compartments of a modified Franz diffusion cell, the percentage and quantity of cumulative daptomycin permeation from DAP-FL within 12 hours were measured at 96.28% ± 0.70% and (132.23 ± 17.73) µg/cm(2) *5 = 661.15 ± 88.65 µg/cm(2), directly, showing rapid and efficient antibacterial activity against Staphylococcus aureus. Following local administration of DAP-FL, daptomycin was detected in multilayer tissues within the skin and underlying structures in the dorsal skin of the mouse. Effective therapeutic concentrations were maintained for several hours, and significantly inhibited bacterial growth and injury-induced biofilms. These results demonstrate that the DAP-FL can enhance the ability of daptomycin to permeate the skin efficiently, where it has a powerful antibacterial action and activity against biofilms. This novel formulation of daptomycin has potential as a new approach in the clinical application of daptomycin.

Silica-coated flexible liposomes as a nanohybrid delivery system for enhanced oral bioavailability of curcumin.

We investigated flexible liposomes as a potential oral drug delivery system. However, enhanced membrane fluidity and structural deformability may necessitate liposomal surface modification when facing the harsh environment of the gastrointestinal tract. In the present study, silica-coated flexible liposomes loaded with curcumin (CUR-SLs) having poor water solubility as a model drug were prepared by a thin-film method with homogenization, followed by the formation of a silica shell by the sol-gel process. We systematically investigated the physical properties, drug release behavior, pharmacodynamics, and bioavailability of CUR-SLs. CUR-SLs had a mean diameter of 157 nm and a polydispersity index of 0.14, while the apparent entrapment efficiency was 90.62%. Compared with curcumin-loaded flexible liposomes (CUR-FLs) without silica-coatings, CUR-SLs had significantly higher stability against artificial gastric fluid and showed more sustained drug release in artificial intestinal fluid as determined by in vitro release assays. The bioavailability of CUR-SLs and CUR-FLs was 7.76- and 2.35-fold higher, respectively, than that of curcumin suspensions. Silica coating markedly improved the stability of flexible liposomes, and CUR-SLs exhibited a 3.31-fold increase in bioavailability compared with CUR-FLs, indicating that silica-coated flexible liposomes may be employed as a potential carrier to deliver drugs with poor water solubility via the oral route with improved bioavailability.