EGF‐modified Au NP–Pc 4 conjugates showed 10‐fold improved selectivity to the brain tumor compared to untargeted conjugates. The hydrophobic photodynamic therapy drug Pc 4 can be delivered efficiently into glioma brain tumors by EGF peptide‐targeted Au NPs. Compared to the untargeted conjugates, EGF–Au NP–Pc 4 conjugates showed 10‐fold improved selectivity to the brain tumor. This delivery system holds promise for future delivery of a wider range of hydrophobic therapeutic drugs for the treatment of hard‐to‐reach cancers.

Targeted drug delivery using epidermal growth factor peptide-targeted gold nanoparticles (EGFpep-Au NPs) is investigated as a novel approach for delivery of photodynamic therapy (PDT) agents, specifically Pc 4, to cancer. In vitro studies of PDT show that EGFpep-Au NP-Pc 4 is twofold better at killing tumor cells than free Pc 4 after increasing localization in early endosomes. In vivo studies show that targeting with EGFpep-Au NP-Pc 4 improves accumulation of fluorescence of Pc 4 in subcutaneous tumors by greater than threefold compared with untargeted Au NPs. Targeted drug delivery and treatment success can be imaged via the intrinsic fluorescence of the PDT drug Pc 4. Using Pc 4 fluorescence, it is demonstrated in vivo that EGFpep-Au NP-Pc 4 impacts biodistribution of the NPs by decreasing the initial uptake by the reticuloendothelial system (RES) and by increasing the amount of Au NPs circulating in the blood 4 h after IV injection. Interestingly, in vivo PDT with EGFpep-Au NP-Pc 4 results in interrupted tumor growth when compared with EGFpep-Au NP control mice when selectively activated with light. These data demonstrate that EGFpep-Au NP-Pc 4 utilizes cancer-specific biomarkers to improve drug delivery and therapeutic efficacy over untargeted drug delivery.

Descending input from the rostral ventromedial medulla (RVM) provides positive and negative modulation of spinal nociceptive transmission and has been proposed to be critical for maintaining neuropathic pain. This study tests the hypothesis that neuropathic pain requires the activity of a subset of RVM neurons that are distinguished by co-expression of mu opioid receptor (MOR) and cholecystokinin type 2 receptor (CCK2). Using male Sprague-Dawley rats, we demonstrate that discrete RVM neurons express MOR and CCK2; over 80% of these cells co-express both receptors. Agonist-directed cell lesion in the RVM with the cytotoxin, saporin, using either CCK-saporin to target CCK receptor expressing cells, or dermorphin-saporin to target MOR expressing cells, resulted in concomitant loss of CCK2 and MOR expressing cells, did not alter the basal sensory thresholds but abolished the hyperalgesia induced by microinjection of CCK into the RVM. The findings suggest that these CCK2-MOR co-expressing RVM neurons facilitate pain and can be directly activated by CCK input to the RVM. Furthermore, lesion of these RVM neurons did not affect the initial development of neuropathic pain in the hind paw upon injury to the sciatic nerve, but the abnormal pain states were short lived such that by about day 9 the sensory thresholds had reverted to pre-injury baselines despite the existing neuropathy. These data support our hypothesis and identify CCK2-MOR co-expressing neurons in the RVM as potential therapeutic targets for neuropathic pain.

The Mouse Defense Test Battery (MDTB) has been designed to assess defensive reactions in Swiss-Webster mice to situations associated with a natural predator, the rat. Primary measures taken before, during and after predator confrontation comprise escape attempts, predator assessment, defensive attack and flight. Previous reports from this laboratory have shown that the panic-promoting drug yohimbine potentiated flight behavior, while long-term treatment with the panicolytic agent alprazolam reduced this response. In order to evaluate further the possibility that the MDTB may represent an effective animal model of panic attacks, the present study investigated the behavioral effect of imipramine and fluoxetine, two serotonin reuptake inhibitors (SRIs) known to alleviate panic symptoms when given on a repeated basis. Both drugs were administered acutely and chronically (one daily IP injection for 21 days) at 5, 10 and 15 mg/kg. Our results showed that a single dose of imipramine or fluoxetine strongly potentiated flight reactions in response to an approaching predator and increased defensive attack toward the rat. This was in contrast to chronic treatment with each drug which dramatically decreased flight responses and defensive attack behaviors. In addition, long-term administration with both SRIs produced a reliable attenuation of predator assessment activities. Taken together, these findings suggest an acute anxiogenic-like effect of imipramine and fluoxetine followed by a fear/anxiety reducing effect after repeated administrations. These results support clinical observations revealing an acute anxiogenic effect of SRIs followed by an anxiolytic and/or panicolytic effect after chronic use, and support previous results suggesting that the MDTB may be useful for the investigation of panic-modulating agents.

A fluorescent sensor of protein kinase activity has been developed and used to characterize the compartmentalized location of cAMP-dependent protein kinase activity in mitochondria. The sensor functions via a phosphorylation-induced release of a quencher from a peptide-based substrate, producing a 150-fold enhancement in fluorescence. The quenching phenomenon transpires via interaction of the quencher with Arg residues positioned on the peptide substrate. Although the cAMP-dependent protein kinase is known to be present in mitochondria, the relative amount of enzyme positioned in the major compartments (outer membrane, intermembrane space, and the matrix) of the organelle is unclear. The fluorescent sensor developed in this study was used to reveal the relative matrix:intermembrane space:outer membrane (85:6:9) distribution of PKA in bovine heart mitochondria.Protein kinases are a large enzyme family that have been implicated in nearly every cell-based behavior, from ATP generation to unrestrained growth and division.1 These enzymes are linked by their ability to catalyze phosphoryl transfer from ATP to the hydroxyl moieties of serine, threonine, and/or tyrosine residues in proteins. A variety of factors limit protein kinasecatalyzed phosphorylation to intended protein targets: (a) the ability to phosphorylate serine/ threonine or tyrosine, but only rarely both aliphatic and aromatic residues, (b) differential expression as a function of cell type, (c) recognition of specific amino acid sequences encompassing the hydroxyl phosphoryl acceptor moiety, and (d) localization to specific intracellular sites. The cAMP-dependent protein kinase (PKA) exhibits many of these attributes as a serine/threonine-specific protein kinase with a special preference for sequences of the general form Arg-Arg-Xaa-Ser/Thr-Xaa in protein substrates.2 In addition, PKA is anchored to a variety of intracellular sites via coordination to A-Kinase Anchoring Proteins (AKAPs), and thus the biological consequences of its action are location-dependent.3 For example, mitochondrial PKA is implicated in the regulation of apoptosis and ATP synthesis.4 However, as is true for protein kinases in general, presumed intracellular PKA activity is commonly assessed in an indirect fashion: either by the mere presence of the enzyme (immunofluorescence or western blots) or by the effect of small molecule modulators, such as inhibitors, on the phosphorylation of presumed PKA protein substrates. Unfortunately, these commonly employed methods don't furnish a direct measure of kinase activity. Fluorescent sensors have been used to directly and continuously assess kinase action. 5 However, these either display a limited dynamic range or employ fluorophores with photophysical properties (short λ ex /λ em 6, small ε,7 low Φ) that are incompatible (due to interference from autofluorescence) with cells, cell lysates, or organelles. With the latter limitation in mind, we report herein the application of a quenched fluorescence strategy6 to create a kinase senso...