Strategy for Conjugating Oligopeptides to Mesoporous Silica Nanoparticles Using Diazirine-Based Heterobifunctional Linkers.

Successful strategies for the attachment of oligopeptides to mesoporous silica with pores large enough to load biomolecules should utilize the high surface area of pores to provide an accessible, protective environment. A two-step oligopeptide functionalization strategy is examined here using diazirine-based heterobifunctional linkers. Mesoporous silica nanoparticles (MSNPs) with average pore diameter of ~8 nm and surface area of ~730 m2/g were synthesized and amine-functionalized. Tetrapeptides Gly-Gly-Gly-Gly (GGGG) and Arg-Ser-Ser-Val (RSSV), and a peptide comprised of four copies of RSSV (4RSSV), were covalently attached via their N-terminus to the amine groups on the particle surface by a heterobifunctional linker, sulfo-succinimidyl 6-(4,4'-azipentanamido)hexanoate (sulfo-NHS-LC-diazirine, or SNLD). SNLD consists of an amine-reactive NHS ester group and UV-activable diazirine group, providing precise control over the sequence of attachment steps. Attachment efficiency of RSSV was measured using fluorescein isothiocyanate (FITC)-tagged RSSV (RSSV-FITC). TGA analysis shows similar efficiency (0.29, 0.31 and 0.26 mol peptide/mol amine, respectively) for 4G, RSSV and 4RSSV, suggesting a generalizable method of peptide conjugation. The technique developed here for the conjugation of peptides to MSNPs provides for their attachment in pores and can be translated to selective peptide-based separation and concentration of therapeutics from aqueous process and waste streams.

Target-directed evolution of novel modulators of the dopamine transporter in Lobelia cardinalis hairy root cultures.

The dopamine transporter (DAT) is targeted in substance use disorders (SUDs), and "non-classical"" DAT inhibitors with low abuse potential are therapeutic candidates. Lobinaline, from Lobelia cardinalis, is an atypical DAT inhibitor lead. Chemical synthesis of lobinaline is challenging; thus, "target-directed evolution" was used for lead optimization. A target protein is expressed in plant cells, and a mutant cell population is selected under conditions where target protein functional inhibition confers a survival advantage. Surviving mutants are "mined" for the targeted activity. Applied to a mutant L. cardinalis cell population expressing the human DAT, we identified 20 mutants overproducing DAT inhibitors. Microanalysis prioritized novel lobinaline derivatives, and we first investigated the more water-soluble lobinaline N-oxide. It inhibited rat synaptosomal [3H]DA uptake with an IC50 similar to lobinaline. Against repeated DA microinjections into the rat striatum, lobinaline produced transient DA clearance reductions. In contrast, lobinaline N-oxide prolongingly increased DA peak amplitudes, particularly in the ventral striatum. Lobinaline N-oxide also produced complex changes in post-peak DA clearance inconsistent with simple DAT inhibition. This unusual DAT interaction may prove therapeutically useful for treating SUDs. This study demonstrates the value of target-directed evolution of plant cells for optimizing lead compounds difficult to synthesize chemically.

Mechanism of Mesoporous Silica Nanoparticle Interaction with Hairy Root Cultures during Nanoharvesting of Biomolecules.

Cellular uptake and expulsion mechanisms of engineered mesoporous silica nanoparticles (MSNPs) are important in their design for novel biomolecule isolation and delivery applications such as nanoharvesting, defined as using nanocarriers to transport and isolate valuable therapeutics (secondary metabolites) out of living plant organ cultures (e.g., hairy roots). Here, temperature-dependent MSNP uptake and recovery processes in hairy roots are examined as a function of surface chemistry. MSNP uptake into hairy roots and time-dependent expulsion are quantified using Ti content (present for biomolecule binding) and fluorescence spectroscopy of fluorescently tagged MSNPs, respectively. The results suggest that functionalization and surface charge (regulated by amine group attachment) play the biggest role in the effectiveness of uptake and recovery. Comparison of MSNP interactions with hairy roots at 4 and 23 °C shows that weakly charged MSNPs functionalized only with Ti are taken up and expelled by thermally activated mechanisms, while amine-modified positively charged particles are taken up and expelled mainly by direct penetration of cell walls. Amine-functionalized MSNPs move spontaneously in and out of plant cells by dynamic exchange with a residence time of 20 ± 5 min, suggesting promise as a biomolecule nanoharvesting platform for plant organ cultures.

Novel multifunctional pharmacology of lobinaline, the major alkaloid from Lobelia cardinalis.

In screening a library of plant extracts from ~1000 species native to the Southeastern United States, Lobelia cardinalis was identified as containing nicotinic acetylcholine receptor (nicAchR) binding activity which was relatively non-selective for the α4ß2- and α7-nicAchR subtypes. This nicAchR binding profile is atypical for plant-derived nicAchR ligands, the majority of which are highly selective for α4ß2-nicAchRs. Its potential therapeutic relevance is noteworthy since agonism of α4ß2- and α7-nicAchRs is associated with anti-inflammatory and neuroprotective properties. Bioassay-guided fractionation of L. cardinalis extracts led to the identification of lobinaline, a complex binitrogenous alkaloid, as the main source of the unique nicAchR binding profile. Purified lobinaline was a potent free radical scavenger, displayed similar binding affinity at α4ß2- and α7-nicAchRs, exhibited agonist activity at nicAchRs in SH-SY5Y cells, and inhibited [(3)H]-dopamine (DA) uptake in rat striatal synaptosomes. Lobinaline significantly increased fractional [(3)H] release from superfused rat striatal slices preloaded with [(3)H]-DA, an effect that was inhibited by the non-selective nicAchR antagonist mecamylamine. In vivo electrochemical studies in urethane-anesthetized rats demonstrated that lobinaline locally applied in the striatum significantly prolonged clearance of exogenous DA by the dopamine transporter (DAT). In contrast, lobeline, the most thoroughly investigated Lobelia alkaloid, is an α4ß2-nicAchR antagonist, a poor free radical scavenger, and is a less potent DAT inhibitor. These previously unreported multifunctional effects of lobinaline make it of interest as a lead to develop therapeutics for neuropathological disorders that involve free radical generation, cholinergic, and dopaminergic neurotransmission. These include neurodegenerative conditions, such as Parkinson's disease, and drug abuse.

A nicotinic receptor-mediated anti-inflammatory effect of the flavonoid rhamnetin in BV2 microglia.

The alpha7 nicotinic acetylcholine receptor (nAChR) is a potential target in neuroinflammation. Screening a plant extract library identified Solidago nemoralis as containing methyl-quercetin derivatives that are relatively selective ligands for the alpha7 nAChR. Flavonoids are not known for this activity, so we screened a small library of pure flavonoids to confirm our findings. Some flavonoids, e.g. rhamnetin, displaced a selective alpha7 nAChR radioligand from rat brain membranes whereas similar structures e.g. sakuranetin, did not. To evaluate the contribution of this putative nAChR activity to the known anti-inflammatory properties of these flavonoids, we compared their effects on lipopolysaccharide induced release of inflammatory mediators from BV2 microglia. Both rhamnetin and sakuranetin reduced mediator release, but differed in potency (rhamnetin>sakuranetin) and the Hill slope of their concentration-response curves. For rhamnetin the Hill coefficient was >3.0 whereas for sakuranetin the coefficient was 1.0, suggesting that effects of rhamnetin are mediated through more than one mechanism, whereas sakuranetin has a single mechanism. nAChR antagonists decreased the Hill coefficient for rhamnetin toward unity, which suggests that a nAChR-mediated mechanism contributes cooperatively to its overall anti-inflammatory effect. In contrast nAChR antagonists had no effect on the potency or Hill coefficient for sakuranetin, but a concentration of nicotine (1µM) that had no effect alone, significantly increased the Hill coefficient of this flavonoid. In conclusion, the anti-inflammatory effects of rhamnetin benefit cooperatively from a nAChR-mediated mechanism. This action, together with potent free radical scavenging activity, suggests that flavonoids with alpha7 nAChR activity have therapeutic potential in neuroinflammatory conditions.

Reactive oxygen species regulate alkaloid metabolism in undifferentiated N. tabacum cells.

Plants produce an immense number of natural products and undifferentiated cells from various plant tissues have long been considered an ideal source for their synthesis. However, undifferentiated plant cells often either lose their biosynthetic capacity over time or exhibit immediate repression of the required pathways once dedifferentiated. In this study, freshly prepared callus tissue was employed to further investigate the regulation of a natural product pathway in undifferentiated tobacco cells. Putrescine N-methyltransferase (PMT) is a pathway-specific enzyme required in nicotinic alkaloid production in Nicotiana species. Callus derived from transgenic Nicotiana tabacum plants harboring PMT promoter-GUS fusions were used to study factors that influence PMT expression. Under normal callus growth conditions in the presence of light and auxin, PMT promoter activity was strongly repressed. Conversely, dark conditions and the absence of auxin were found to upregulate PMT promoter activity, with light being dominant to the repressive effects of auxin. Since reactive oxygen species (ROS) are known by-products of photosynthesis and have been implicated in signaling, their involvement was investigated in transgenic callus by treatment with the ROS scavenger, dimethylthiourea, or catalase. Under highly repressive conditions for alkaloid synthesis, including normal culture conditions in the light, both ROS scavengers resulted in significant induction of PMT promoter activity. Moreover, treatment of callus with catalase resulted in the upregulation of PMT promoter activity and alkaloid accumulation in this tissue. These results suggest that ROS impact the regulation of the alkaloid pathway in undifferentiated cells and have implications for regulation of the pathway in other plant tissues.

Neonatal ethanol exposure produces a hyperalgesia that extends into adolescence, and is associated with increased analgesic and rewarding properties of nicotine in rats.

RATIONALE: Drug exposure during CNS development may alter subsequent dependence liability. We postulated that early alcohol exposure might produce persistent alterations in responses to noxious stimuli. Because relief of physical discomfort may be negatively reinforcing, changes in responses to noxious stimuli produced by early alcohol exposure may increase the rewarding properties of nicotine, a potent analgesic. Such factors may contribute to the high level of alcohol and nicotine co-abuse in humans. OBJECTIVES: The purpose of this study was to determine whether neonatal ethanol exposure in rats altered responses to noxious stimuli, and whether nicotine would then be more rewarding to the alcohol-exposed offspring, perhaps via its analgesic actions. METHODS: Neonatal rats received ethanol by gavage (5.0 or 6.5 g/kg) on postnatal days (PND) 9-13. An iso-caloric control group was also included. Rats were then tested to assess responsiveness to a mild noxious heat stimulus, as measured in the tail-flick assay (PND 14 and PND 28), for their response to acute analgesic injections of either nicotine or ethanol (PND 28), and for nicotine induced conditioned place preference (CPP) (PND 36). RESULTS: Neonatal ethanol exposure produced hyperalgesia during the first 24 h after alcohol withdrawal (PND 14) that continued through PND 28. The analgesic effects of 12.5 microg/kg nicotine were enhanced approximately 2-fold in adolescent rats with previous ethanol histories, relative to controls. These ethanol-exposed rats also showed a significant CPP to nicotine, whereas controls showed no CPP. CONCLUSIONS: Persistent decreases in tail-flick response latencies suggestive of hyperalgesia were observed following neonatal ethanol exposure in the rat. These changes were accompanied by increases in the analgesic and place-conditioning effects of nicotine in adolescence. If similar effects occur in humans, prenatal alcohol exposure may play a role in an increased risk for the rewarding effects and dependence liability of nicotine later in life.