Preformulation Characterization of Griffithsin, a Biopharmaceutical Candidate for HIV Prevention.

Griffithsin (GRFT) has shown potent anti-HIV activity, and it is being developed as a drug candidate for HIV prevention. Successful implementation requires thorough understanding of its preformulation characterization. In this work, preformulation assessments were conducted to characterize GRFT and identify its degradation pathways under selected conditions of temperature, light, pH, shear, ionic strength, and oxidation. Compatibility with vaginal fluid simulant, vaginal enzymes, Lactobacillus spp., and human cervicovaginal secretions was assessed. The purity, melting temperature, and HIV gp120-binding affinity of GRFT stored at 4°C and 25°C in phosphate-buffered saline (PBS) were assessed for 2 years. Chemical modifications were evaluated by intact mass analysis and peptide sequencing. Excised human ectocervical tissue permeability and localization of GRFT were evaluated. Our results demonstrated GRFT to be safe and stable under all the preformulation assessment conditions studied except oxidative stress. When GRFT was exposed to hydrogen peroxide or human cervicovaginal secretion, methionine 78 in the protein sequence underwent oxidation. GRFT did not permeate through human cervical tissue but adhered to the superficial epithelial tissue. The 2-year stability study revealed no significant change in GRFT's aggregation, degradation, melting temperature, or gp120-binding affinity despite a slow increase in oxidation over time. These studies elucidated desirable safety and bioactivity profile for GRFT, showing promise as a potential drug candidate for HIV prevention. However, susceptibility to oxidative degradation was identified. Effective protection of GRFT from oxidation is required for further development.

Efficacy of silk fibroin biomaterial vehicle for in vivo mucosal delivery of Griffithsin and protection against HIV and SHIV infection ex vivo.

INTRODUCTION: The majority of new HIV infections occur through mucosal transmission. The availability of readily applicable and accessible platforms for anti-retroviral (ARV) delivery is critical for the prevention of HIV acquisition through sexual transmission in both women and men. There is a compelling need for developing new topical delivery systems that have advantages over the pills, gels and rings, which currently fail to guarantee protection against mucosal viral transmission in vulnerable populations due to lack of user compliance. The silk fibroin (SF) platform offers another option that may be better suited to individual circumstances and preferences to increase efficacy through user compliance. The objective of this study was to test safety and efficacy of SF for anti-HIV drug delivery to mucosal sites and for viral prevention. METHODS: We formulated a potent HIV inhibitor Griffithsin (Grft) in a mucoadhesive silk fibroin (SF) drug delivery platform and tested the application in a non-human primate model in vivo and a pre-clinical human cervical and colorectal tissue explant model. Both vaginal and rectal compartments were assessed in rhesus macaques (Mucaca mulatta) that received SF (n = 4), no SF (n = 7) and SF-Grft (n = 11). In this study, we evaluated the composition of local microbiota, inflammatory cytokine production, histopathological changes in the vaginal and rectal compartments and mucosal protection after ex vivo SHIV challenge. RESULTS: Effective Grft release and retention in mucosal tissues from the SF-Grft platform resulted in protection against HIV in human cervical and colorectal tissue as well as against SHIV challenge in both rhesus macaque vaginal and rectal tissues. Mucoadhesion of SF-Grft inserts did not cause any inflammatory responses or changes in local microbiota. CONCLUSIONS: We demonstrated that in vivo delivery of SF-Grft in rhesus macaques fully protects against SHIV challenge ex vivo after two hours of application and is safe to use in both the vaginal and rectal compartments. Our study provides support for the development of silk fibroin as a highly promising, user-friendly HIV prevention modality to address the global disparity in HIV infection.

Exploring the carbohydrate-binding ability of Canavalia bonariensis lectin in inflammation models.

Lectins are a group of proteins of non-immune origin recognized for their ability to bind reversibly to carbohydrates. Researchers have been intrigued by oligosaccharides and glycoconjugates for their involvement as mediators of complex cellular events and then many biotechnological applications of lectins are based on glycocode decoding and their activities. Here, we report a structural and biological study of a ConA-like mannose/glucose-specific lectin from Canavalia bonariensis seeds, CaBo. More specifically, we evaluate the binding of CaBo with α-methyl-D-mannoside (MMA) and mannose-1,3-α-D-mannose (M13) and the resultant in vivo effects on a rat model of acute inflammation. A virtual screening was also carried out to cover a larger number of possible bindings of CaBo. In silico analysis demonstrated the stability of CaBo interaction with mannose-type ligands, and the lectin was able to induce acute inflammation in rats with the participation of the carbohydrate recognition domain (CRD) and histamine release. These results confirm the ability of CaBo to interact with hybrid and high-mannose N-glycans, supporting the hypothesis that CaBo's biological activity occurs primarily through its interaction with cell surface glycosylated receptors.

Interspecies metabolic diversity of artocarpin in vitro mammalian liver microsomes.

Artocarpin has shown anti-inflammation and anticancer activities. However, the metabolism differences among different species have not been reported. In this work, we used liver microsomes to explore the metabolic characteristics and possible metabolites of artocarpin among different species. The structures of six metabolites were characterized by LC-MS/MS, and hydroxylated artocarpin was the main metabolite. Enzyme kinetics and depletion studies of artocarpin among different species proved that artocarpin metabolism exhibited significant species differences; rats and monkeys showed a great metabolic ability to artocarpin, and minipigs showed the highest similarity to humans. The in vivo hepatic clearances of artocarpin in rats and humans were predicted that artocarpin was classified as a high-clearance drug in humans and rats. The glucuronidation assay of artocarpin in different liver microsomes also proved that artocarpin metabolism showed significant species difference. These findings will support further pharmacological or toxicological research on artocarpin.Abbreviations: UGT: UDP-glucuronosyltransferase; CYP: cytochrome P450; LC-MS/MS: liquid chromatography-tandem mass spectrometry; HPLC: high-performance liquid chromatography; HLMs: human liver microsomes; MLMs: monkey liver microsomes; RAMs: rabbit liver microsomes; RLMs: rat liver microsomes; DLMs: dog liver microsomes; PLMs: minipig liver microsomes; Vmax: maximum velocity; Km: Michaelis constant; CLint: intrinsic clearance; CLH: hepatic clearance; QH: hepatic blood flow.

Wisteria floribunda agglutinin staining for the quantitative assessment of cardiac fibrogenic activity in a mouse model of dilated cardiomyopathy.

Cardiac fibrosis is a typical phenomenon in failing hearts for most cardiac diseases, including dilated cardiomyopathy (DCM), and its specific detection and quantification are crucial for the analysis of cardiac remodeling. Since cardiac fibrosis is characterized by extensive remodeling of the myocardial extracellular matrix (ECM), in which glycoproteins are the major components, we assumed that fibrosis-related alterations in the cardiac glycome and glycoproteome would be suitable targets for the detection of cardiac fibrosis. Here, we compared protein glycosylation between heart tissues of normal and DCM model mice by laser microdissection-assisted lectin microarray. Among 45 lectins, Wisteria floribunda agglutinin (WFA) was selected as the most suitable lectin for staining cardiac fibrotic tissues. Although the extent of WFA staining was highly correlated (r > 0.98) with that of picrosirius red staining, a common collagen staining method, WFA did not bind to collagen fibers. Further histochemical analysis with N-glycosidase revealed that WFA staining of fibrotic tissues was attributable to the binding of WFA to N-glycoproteins. Using a mass spectrometry-based approach, we identified WFA-binding N-glycoproteins expressed in DCM hearts, many of which were fibrogenesis-related ECM proteins, as expected. In addition, the identified glycoproteins carrying WFA-binding N-glycans were detected only in DCM hearts, suggesting their cooperative glycosylation alterations with disease progression. Among these WFA-binding ECM N-glycoproteins, co-localization of the collagen α6(VI) chain protein and WFA staining in cardiac tissue sections was confirmed with a double-staining analysis. Collectively, these results indicate that WFA staining is more suitable for the quantitative assessment of cardiac fibrogenic activity than current collagen staining methods. Furthermore, given that plasma WFA-binding glycoprotein levels were significantly correlated with the echocardiographic parameters for left ventricular remodeling, cardiac WFA-binding glycoproteins are candidate circulating glyco-biomarkers for the quantification and monitoring of cardiac fibrogenesis.

Mechanical load increase-induced changes in cytoskeletal structure and cellular barrier function in human cerebral endothelial cells.

Globally, approximately a billion patients are estimated to suffer from neurological disorders. Although there are many therapeutic candidates for the central nervous system, treatment of brain disorders is restricted by the blood-brain barrier (BBB), which is a highly selective membrane that protects the brain from exogenous substances. This study was undertaken to develop a novel strategy to overcome the BBB and improve the efficiency of drug delivery to the brain by mechanical load increase using hypergravity. Human cerebral microvascular endothelial cells were exposed three times to 20 min hypergravity (10g), with a 20-min rest period between each exposure. The applied hypergravity reversibly decreased the cellular metabolic activity and increased the permeation rate of fluorescein sodium salt, fluorescein isothiocyanate-labeled dextran (FD-4), and fluorescein-labeled jacalin. Following the exposure to hypergravity, we also observed structural changes of the cytoskeleton and tight junctions, and an alteration in the expression levels of related genes. These results indicate that increased mechanical load due to the applied hypergravity affects the cytoskeletal arrangement and tight junctions, thereby weakening the cell barrier function and enhancing the permeability of the paracellular pathway. Thus, the mechanical load increase by hypergravity has the potential of being used as a novel strategy to overcome the BBB for brain drug delivery.

Identification of cytochrome P450 isoforms involved in the metabolism of artocarpin and assessment of its drug-drug interaction.

Artocarpin isolated from an agricultural plant Artocarpus communis has shows anti-inflammation and anticancer activities. In this study, we utilized recombinant human UDP-glucuronosyltransferasesupersomes (UGTs) and human liver microsomes to explore its inhibitory effect on UGTs and cytochrome p450 enzymes (CYPs). Chemical inhibition studies and screening assays with recombinant human CYPs were used to identify if CYP isoform is involved in artocarpin metabolism. Artocarpin showed strong inhibition against UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, CYP2C8 and CYP3A4. In particular, artocarpin exhibited competitive inhibition against CYP3A4 and noncompetitive inhibition against UGT1A3 and UGT1A7. The half inhibition concentration values for CYP3A4, UGT1A3 and UGT1A7 were 4.67, 3.82 and 4.82 µm, and the inhibition kinetic parameters for them were 0.78, 2.67 and 3.14 µm, respectively. After artocarpin was incubated in human liver microsomes and determined by HPLC, we observed its main metabolites (M1 and M2). In addition, we proved that CYP2D6 played the key role in the biotransformation of artocarpin in human liver microsomes. The result of molecular docking further confirmed that artocarpin interacted with CYP2D6, CYP2C8 and CYP3A4 through hydrogen bonds. This study provided preliminary results for further research on artocarpin or artocarpin-containing herbs.

Biodistribution of Viscumin after Subcutaneous Injection to Mice and In Vitro Modeling of Endoplasmic Reticulum Stress.

Viscumin (mistletoe lectin I, MLI) in concentrations of 10-11-10-7 M causes endoplasmic reticulum stress and triggers unfolded protein response, a modulator of antitumor immunity, in target cells.

Pharmacokinetics of the Antiviral Lectin Griffithsin Administered by Different Routes Indicates Multiple Potential Uses.

Griffithsin (GRFT) is a red alga-derived lectin with demonstrated broad spectrum antiviral activity against enveloped viruses, including severe acute respiratory syndrome-Coronavirus (SARS-CoV), Japanese encephalitis virus (JEV), hepatitis C virus (HCV), and herpes simplex virus-2 (HSV-2). However, its pharmacokinetic profile remains largely undefined. Here, Sprague Dawley rats were administered a single dose of GRFT at 10 or 20 mg/kg by intravenous, oral, and subcutaneous routes, respectively, and serum GRFT levels were measured at select time points. In addition, the potential for systemic accumulation after oral dosing was assessed in rats after 10 daily treatments with GRFT (20 or 40 mg/kg). We found that parenterally-administered GRFT in rats displayed a complex elimination profile, which varied according to administration routes. However, GRFT was not orally bioavailable, even after chronic treatment. Nonetheless, active GRFT capable of neutralizing HIV-Env pseudoviruses was detected in rat fecal extracts after chronic oral dosing. These findings support further evaluation of GRFT for pre-exposure prophylaxis against emerging epidemics for which specific therapeutics are not available, including systemic and enteric infections caused by susceptible enveloped viruses. In addition, GRFT should be considered for antiviral therapy and the prevention of rectal transmission of HIV-1 and other susceptible viruses.

Bauhinia purprea agglutinin-modified liposomes for human prostate cancer treatment.

Bauhinia purprea agglutinin (BPA) is a well-known lectin that recognizes galactosyl glycoproteins and glycolipids. In the present study, we firstly found that BPA bound to human prostate cancer specimens but not to normal prostate ones. Therefore, we sought to develop BPA-PEG-modified liposomes (BPA-PEG-LP) encapsulating anticancer drugs for the treatment of prostate cancer. We examined the tumor targetability of BPA-PEG-LP with human prostate cancer DU145 cells, and observed that fluorescently labeled BPA-PEG-LP dominantly associated with the cells via the interaction between liposome-surface BPA and cell-surface galactosyl molecules. We also observed that BPA-PEG-LP accumulated in the prostate cancer tissue after the i.v. injection to DU145 solid cancer-bearing mice, and strongly bound to the cancer cells. In a therapeutic study, DU145 solid cancer-bearing mice were i.v. injected thrice with BPA-PEG-LP encapsulating doxorubicin (BPA-PEG-LPDOX, 2 mg/kg/day as the DOX dosage) or PEG-modified liposomes encapsulating DOX (PEG-LPDOX). As a result, BPA-PEG-LPDOX significantly suppressed the growth of the DU145 cancer cells, whereas PEG-LPDOX at the same dosage as DOX showed little anti-cancer effect. The present study suggested that BPA-PEG-LP could be a useful drug carrier for the treatment of human prostate cancers.

Determination of the glycosylation-pattern of the middle ear mucosa in guinea pigs.

In the present study the glycosylation pattern of the middle ear mucosa (MEM) of guinea pigs, an approved model for middle ear research, was characterized with the purpose to identify bioadhesive ligands which might prolong the contact time of drug delivery systems with the middle ear mucosa (MEM). To assess the utility of five fluorescein labeled plant lectins with different carbohydrate specificities as bioadhesive ligands, viable MEM specimens were incubated at 4°C and the lectin binding capacities were calculated from the MEM-associated relative fluorescence intensities. Among all lectins under investigation, fluorescein-labeled wheat germ agglutinin (F-WGA) emerged as the highest bioadhesive lectin. In general, the accessibility of carbohydrate moieties of the MEM followed the order: sialic acid and N-acetyl-d-glucosamine (WGA)>>mannose and galactosamine (Lensculinaris agglutinin)>N-acetyl-d-glucosamine (Solanumtuberosum agglutinin)>fucose (Ulexeuropaeus isoagglutinin I)>>terminal mannose α-(1,3)-mannose (Galanthusnivalis agglutinin). Competitive inhibition studies with the corresponding carbohydrate revealed that F-WGA-binding was inhibited up to 90% confirming specificity of the F-WGA-MEM interaction. The cilia of the MEM were identified as F-WGA binding sites by fluorescence imaging as well as a z-stack of overlays of transmission, F-WGA- and nuclei-stained images of the MEM. Additionally, co-localisation experiments revealed that F-WGA bound to acidic mucopolysaccharides of the MEM. All in all, lectin-mediated bioadhesion to the MEM is proposed as a new concept for drug delivery to prolong the residence time of the drug in the tympanic cavity especially for successful therapy for difficult-to-treat diseases such as otitis media.

Activity of and effect of subcutaneous treatment with the broad-spectrum antiviral lectin griffithsin in two laboratory rodent models.

Griffithsin (GRFT) is a red-alga-derived lectin that binds the terminal mannose residues of N-linked glycans found on the surface of human immunodeficiency virus type 1 (HIV-1), HIV-2, and other enveloped viruses, including hepatitis C virus (HCV), severe acute respiratory syndrome coronavirus (SARS-CoV), and Ebola virus. GRFT displays no human T-cell mitogenic activity and does not induce production of proinflammatory cytokines in treated human cell lines. However, despite the growing evidence showing the broad-spectrum nanomolar or better antiviral activity of GRFT, no study has reported a comprehensive assessment of GRFT safety as a potential systemic antiviral treatment. The results presented in this work show that minimal toxicity was induced by a range of single and repeated daily subcutaneous doses of GRFT in two rodent species, although we noted treatment-associated increases in spleen and liver mass suggestive of an antidrug immune response. The drug is systemically distributed, accumulating to high levels in the serum and plasma after subcutaneous delivery. Further, we showed that serum from GRFT-treated animals retained antiviral activity against HIV-1-enveloped pseudoviruses in a cell-based neutralization assay. Overall, our data presented here show that GRFT accumulates to relevant therapeutic concentrations which are tolerated with minimal toxicity. These studies support further development of GRFT as a systemic antiviral therapeutic agent against enveloped viruses, although deimmunizing the molecule may be necessary if it is to be used in long-term treatment of chronic viral infections.

Synthesis of stable carboxy-terminated NaYF4: Yb3+, Er3+@SiO2 nanoparticles with ultrathin shell for biolabeling applications.

Here, a two-step method has been developed for synthesizing carboxy-terminated NaYF(4): Yb(3+), Er(3+)@SiO(2) core@shell nanoparticles (UCNP@SiO(2)) with ultrathin shell (1.5 nm). First, the NaYF(4): Yb(3+), Er(3+) upconverting nanoparticles (UCNPs) were prepared using solvothermal technology; then, silica shells (SiO(2)) were deposited on the nanocrystals to form core-shell structures by the hydrolysis of tetraethylorthosilicate (TEOS). The ultrathin SiO(2) shell was obtained by increasing surfactant amount and decreasing TEOS amount in the reaction mixture. Carboxyethylsilanetriol (CTES) was used to generate the carboxy group on the particle surface. The carboxy-terminated UCNP@SiO(2) are ideally suited for biolabeling and bioimaging applications because the as-prepared nanoparticles have extreme colloidal and optical stabilities, and the carboxy groups on the particle surface easily react with amino residues of biomolecules. As an example, we reported on the interactions of Ricinus Communis Agglutinin (RCA 120) conjugated UCNP@SiO(2) with HeLa cells. The excellent performance of the RCA 120 conjugated UCNP@SiO(2) in cellular fluorescence imaging was demonstrated.

Solanum tuberosum lectin-conjugated PLGA nanoparticles for nose-to-brain delivery: in vivo and in vitro evaluations.

Solanum tuberosum lectin (STL) conjugated poly (DL-lactic-co- glycolic acid) (PLGA) nanoparticle (STL-NP) was constructed in this paper as a novel biodegradable nose-to-brain drug delivery system. The in vitro uptake study showed markedly enhanced endocytosis of STL-NP compared to unmodified PLGA nanoparticles (NP) in Calu-3 cells and significant inhibition of uptake in the presence of inhibitor sugar (chitin hydrolysate). Following intranasal administration, coumarin-6 carried by STL-NP was rapidly absorbed into blood and brain. The AUC((0→12 h)) of coumarin-6 in blood, olfactory bulb, cerebrum and cerebellum were about 0.77-, 1.48-, 1.89- and 1.45-fold of those of NP, respectively (p < 0.05). STL-NP demonstrated 1.89-2.45 times (p < 0.01) higher brain targeting efficiency in different brain tissues than unmodified NP. Enhanced accumulation of STL-NP in the brain was also observed by near infrared fluorescence probe image following intranasal administration. The fluorescence signal of STL-NP appeared in olfactory bulb, cerebrum and brainstem early at 0.25 h. The signal in olfactory bulb decreased gradually after 2 h, while the obvious signal in brainstem, cerebrum and cerebellum lasted for more than 8 h. The STL-NP safety experiments showed mild cytotoxicity and negligible cilia irritation. These intriguing in vitro and in vivo results suggest that STL-NP might serve as a promising brain drug delivery system.

Lineage tracing evidence for transdifferentiation of acinar to duct cells and plasticity of human pancreas.

BACKGROUND & AIMS: Animal studies have indicated that pancreatic exocrine acinar cells have phenotypic plasticity. In rodents, acinar cells can differentiate into ductal precursors that can be converted to pancreatic ductal adenocarcinoma or insulin-producing endocrine cells. However, little is known about human acinar cell plasticity. We developed nongenetic and genetic lineage tracing methods to study the fate of human acinar cells in culture. METHODS: Human exocrine tissue was obtained from organ donors, dissociated, and cultured. Cell proliferation and survival were measured, and cell phenotypes were analyzed by immunocytochemistry. Nongenetic tracing methods were developed based on selective binding and uptake by acinar cells of a labeled lectin (Ulex europaeus agglutinin 1). Genetic tracing methods were developed based on adenoviral introduction of a Cre-lox reporter system, controlled by the amylase promoter. RESULTS: Both tracing methods showed that human acinar cells can transdifferentiate into cells that express specific ductal markers, such as cytokeratin 19, hepatocyte nuclear factor 1ß, SOX9, CD133, carbonic anhydrase II, and cystic fibrosis transmembrane conductance regulator. Within 1 week of culture, all surviving acinar cells had acquired a ductal phenotype. This transdifferentiation was decreased by inhibiting mitogen-activated protein kinase signaling. CONCLUSIONS: Human acinar cells have plasticity similar to that described in rodent cells. These results might be used to develop therapeutic strategies for patients with diabetes or pancreatic cancer.

Mistletoe lectin transport by M-cells in follicle-associated epithelium (FAE) and IL-12 secretion in dendritic cells situated below FAE in vitro.

A galactose- and N-acetyl-D-galactosamine-specific lectin (Viscum album L. var. coloratum agglutinin, VCA), which is known for its anti-tumor activity, was isolated from Korean mistletoe. Mistletoe preparations have been given subcutaneously because of the unstability and poor absorption in the GI tract. In this study, we investigated the effect of incubation time and glucose on the VCA transport across the in vitro model of human FAE (follicle-associated epithelium) by two different cell models: Caco-2 cell monolayers mimicking human enterocytes and a model of the human FAE which is mainly composed of M-cells and enterocytes. As a result, the VCA transport across the FAE monolayer cells was higher and faster than the transport across the Caco-2 monolayer cells, and glucose increased VCA transport across both monolayer cells. In addition, IL-12 was secreted from myeloid DC1 and lymphoid DC2.4 cells which were co-treated simultaneously with LPS and VCA. Furthermore, the FAE model associated with underlying immune cells was established and VCA was added to the inserts apically. There was a greater IL-12 secretion in dendritic cells situated below FAE monolayer than Caco-2 monolayer. The results from this study provide important insight into the possible oral application of mistletoe on anti-tumor therapeutics.

Pharmacokinetics of natural mistletoe lectins after subcutaneous injection.

PURPOSE: Knowledge of natural mistletoe lectins (nML) pharmacokinetics can be regarded as essential for further rational studies with mistletoe preparations. Studies with intravenous application of a recombinant type II ribosome inactivating protein (rML) analogous to nML revealed a short half-life of about 13 min in cancer patients. This open-label, phase I, monocenter clinical trial was performed in order to describe the pharmacokinetics of nML. METHODS: In 15 healthy male volunteers aged 18-42 years, nML were detected with a modified sandwich immuno-polymerase chain reaction (PCR) technique (Imperacer, Chimera Biotec) after single subcutaneous injection of a mistletoe extract (abnobaVISCUM(R) Fraxini 20 mg) with marketing authorization containing about 20 microg nML/ml. Secondary objectives were safety and the number of activated natural killer cells (CD54(+)/CD94(+)). RESULTS: In none of the volunteers were nML detectable before the injection, and in all volunteers, nML were detected in serum samples after injection. Individual variability, however, was large. Mean and median peak concentrations were reached 1 and 2 h after injection, respectively. In some volunteers, nML were still detectable at the final investigation 2 weeks after injection. The injection resulted in fever and flu-like symptoms in all volunteers, but no serious adverse events occurred. All symptoms and local reactions at the injection site completely disappeared within a range of 4-95 days. The number of activated natural killer (NK) cells did not change. CONCLUSIONS: Natural ML from abnobaVISCUM Fraxini 20 mg are detectable in serum after a single subcutaneous injection. Detectability is considerably longer compared with intravenously administered rML. The subcutaneous injection of this preparation without usual pretreatment with lower doses results in short-lasting fever and other flu-like symptoms.

Characterization of two blood-brain barrier mimicking cell lines: distribution of lectin-binding sites and perspectives for drug delivery.

In the present study plant lectins with distinct sugar specificities were applied to two blood-brain barrier (BBB) mimicking cell lines, namely human ECV304 and porcine brain microvascular endothelial cells PBMEC/C1-2 in order to elucidate their glycosylation pattern and to evaluate the lectin-cell interaction for lectin-mediated targeting. The bioadhesive properties of fluorescein-labeled lectins were investigated with monolayers as well as single cells using fluorimetry and flow cytometry, followed by confirmation of the specificity of binding. For PBMEC/C1-2 layers highest binding capacity was found for wheat germ agglutinin (WGA), followed by Dolichus biflorus agglutinin (DBA) whereas single cell experiments revealed a predominance of DBA only. Analyzing ECV304 monolayers and single cells, WGA yielded the strongest interaction without any changes during cultivation. The binding capacities of the other lectins increased significantly during differentiation. As similar results to primary cells and brain sections were observed, both cell lines seem to be suitable as models for lectin-interaction studies. Thus, an additional focus was set on the mechanisms involved in uptake and intracellular fate of selected lectins. Cytoinvasion studies were performed with WGA for human ECV304 cells and WGA as well as DBA for PBMEC/C1-2 cells. For both lectins, the association rate to the cells was dependent on temperature which indicated cellular uptake.

Simian immunodeficiency virus is susceptible to inhibition by carbohydrate-binding agents in a manner similar to that of HIV: implications for further preclinical drug development.

Carbohydrate-binding agents (CBAs), such as the plant lectins Hippeastrum hybrid agglutinin (HHA) and Urtica dioica agglutinin (UDA), but also the nonpeptidic antibiotic pradimicin A (PRM-A), inhibit entry of HIV into its target cells by binding to the glycans of gp120. Given the high sequence identity and similarity between the envelope gp120 glycoproteins of HIV and simian immunodeficiency virus (SIV), the inhibitory activity of a variety of CBAs were evaluated against HIV-1, HIV-2, and SIV. There seemed to be a close correlation for the inhibitory potential of CBAs against HIV-1, HIV-2, and SIV replication in cell culture and syncytia formation in cocultures of persistently SIV-infected HUT-78 cell cultures and uninfected CEM cells. CBAs also inhibit transmission of the SIV to T lymphocytes after capture of the virus by dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN)-expressing cells. A total of 8 different SIV strains were isolated after prolonged HHA, UDA, and PRM-A exposure in virus-infected cell cultures. Each virus isolate consistently contained at least 2 or 3 glycan deletions in its gp120 envelope and showed decreased sensitivity to the CBAs and cross-resistance toward all CBAs. Our data revealed that CBAs afford SIV and HIV-1 inhibition in a similar manner regarding prevention of virus infection, DC-SIGN-directed virus capture-related transmission, and selection of drug-resistant mutant virus strains. Therefore, SIV(mac251)-infected monkeys might represent a relevant animal model to study the efficacy of CBAs in vivo.

Efficient intracellular delivery of functional proteins using cationic polymer core/shell nanoparticles.

Cationic core/shell nanoparticles self-assembled from biodegradable, cationic and amphiphilic copolymer poly{N-methyldietheneamine sebacate)-co-[(cholesteryl oxocarbonylamido ethyl) methyl bis(ethylene) ammonium bromide] sebacate}, P(MDS-co-CES), were fabricated and employed to deliver lectin A-chain, an anticancer glycoprotein. Lectin A-chain was efficiently bound onto the surfaces of the nanoparticles at high mass ratios of nanoparticles to lectin A-chain. The nanoparticle/lectin A-chain complexes had an average size of approximately 150 nm with zeta potential of about +30 mV at the mass ratio of 50 or above while the BioPorter/lectin A-chain complexes had a larger particle size and relatively lower zeta potential (150 nm vs. 455 nm; +30 mV vs. +20 mV). Therefore, the cellular uptake of nanoparticle/lectin A-chain complexes was much greater than that of BioPorter/lectin A-chain complexes. The results obtained from cytotoxicity tests show that lectin A-chain delivered by the nanoparticles was significantly more toxic against MDA-MB-231, HeLa, HepG2 and 4T1 cell lines when compared to BioPorter, and IC50 of lectin A-chain delivered by the nanoparticles was 0.2, 0.5, 10 and 50 mg/l, respectively, while that of lectin A-chain delivered by BioPorter was higher than 100 mg/l in all cell lines tested. These nano-sized particles may provide an efficient approach for intracellular delivery of biologically active proteins.