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Cyclo-(D-Trp-Tyr) peptide nanotubes (PNTs) were reported to be potential carriers for oral gene delivery in our previous study; however, the effect of the aspect ratio (AR) of these PNTs on gene delivery in vivo could affect penetration or interception in biological environments. The aim of this study was to assess the feasibility of cyclo-(D-Trp-Tyr) PNTs with two ARs as carriers for oral pMBP-bcl-xL-hRluc delivery to the spinal cord to treat spinal cord injury (SCI). We evaluated the biodistribution of oligodendrocyte (OLG)-specific myelin basic protein gene promoter-driven antiapoptotic DNA (pMBP-bcl-xL) to the brain and spinal cord delivered with cyclo-(D-Trp-Tyr) PNTs with large (L) and small (S) PNTs with two ARs. After complex formation, the length, width, and AR of the L-PNTs/DNA were 77.86 ± 3.30, 6.51 ± 0.28, and 13.75 ± 7.29 μm, respectively, and the length and width of the S-PNTs/DNA were 1.17 ± 0.52 and 0.17 ± 0.05 μm, respectively, giving an AR of 7.12 ± 3.17 as detected by scanning electron microscopy. Each of these three parameters exhibited significant differences (p 0.05) between the L-PNTs and S-PNTs for either their DNA encapsulation efficiency (29.72 ± 14.19 and 34.31 ± 16.78%, respectively) or loading efficiency (5.15 ± 2.58 and 5.95 ± 2.91%). The results of the in vitro analysis showed that the S-PNT/DNA complexes had a significantly higher DNA release rate and DNA permeation in the duodenum than the L-PNT/DNA complexes. Using Cy5 and TM-rhodamine to individually and chemically conjugate the PNTs with plasmid DNA, we observed, using laser confocal microscopy, that the PNTs and DNA colocalized in complexes. We further confirmed the complexation between DNA and the PNTs using fluorescence resonance energy transfer (FRET). Data from an in vivo imaging system (IVIS) showed that there was no significant difference (p > 0.05) in PNT distribution between L-PNTs/DNA and S-PNTs/DNA within 4 h. However, the S-PNT/DNA group had a significantly higher DNA distribution (p < 0.05) in several organs, including the ilium, heart, lungs, spleen, kidneys, testes, brain, and spinal cord. Finally, we determined the bcl-xL protein expression levels in the brain and spinal cord regions for the L-PNT/DNA and S-PNT/DNA complex formulations. These results suggested that either L-PNTs or S-PNTs may be used as potential carriers for oral gene delivery to treat SCI.

Self-assembled cyclic peptide nanotubes (cPNTs) have recently drawn particular attention as one of the most intriguing nanostructures in the field of nanotechnology. Given their unique features including high surface area, increased drug loading, environmental stability, enhanced permeation, and modifiable drug release, these hollow tubular structures can be constructed with cyclic di-, tri-, tetra-, hexa-, octa-, and decapeptides with various amino acid sequences, enantiomers, and functionalized side chains and can be applied for antiviral and antibacterial drugs, drug delivery and gene delivery vectors, organic electronic devices, and ionic or molecular channels. Recent publications have presented promising results regarding the use of cPNTs as drugs or biomedical devices. However, there is an urgent need for the further in vivo nanotoxicity and safety testing of these nanotubes to evaluate their suitability in different fields. Keywords: Cyclic peptide, Nanotube, Application

Cyclo-(D-Trp-Tyr) peptide nanotubes (PNTs) were reported to be potential carriers for oral gene delivery in our previous study; however, the effect of the aspect ratio (AR) of these PNTs on gene delivery

The stability and bio-distribution of genes or drug complexes with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO–PPO–PEO, Pluronic F-68) polymeric micelles (PM) are essential for an effective nanosized PM delivery system. We used Förster resonance energy transfer (FRET) pairs with PM and measured the FRET ratio to assess the stability of PM in vitro and in vivo on the cornea. The FRET ratio reached a plateau at 0.8 with 3% PM. Differential scanning calorimetry measurement confirmed the complex formation of FRET pairs with PM. Confocal imaging with the fluorophores fluorescein isothiocyanate isomer I (FITC) and rhodamine B base (RhB) also showed the occurrence of FRET pairs in vitro. The fluorophores were mixed with 3% PM solution or the FITC-labeled PEO–PPO–PEO polymers (FITC-P) were mixed with RhB-labeled plasmids (RhB–DNA). In addition, the in vitro corneal permeation of FRET pair complexes with PM reached a 0.8 FRET ratio. One hour after eye drop administration, FRET pairs colocalized in the cytoplasm, and surrounded and entered the nuclei of cells in the cornea, and the polymers were located in the corneal epithelial layers, as detected through anti-PEG immunohistochemistry. Furthermore, fluorescence colocalization in the cytoplasm and cell nucleus of the corneal epithelium was confirmed in tissues where RhB or RhB–DNA complexed with FITC-P was found to accumulate. We demonstrate that at a concentration of 3%, PM can encapsulate FRET pairs or RhB–DNA and retain their integrity within the cornea 1 h after administration, suggesting the feasibility and stability of PEO–PPO–PEO polymers as a vehicle for drug delivery. Keywords: Cornea, Eye drops, Polymeric micelles, Stability

This study attempts to investigate the transdermal permeability, the bioavailability and gene expression of plasmid formulated with nonionic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles (PM). Dynamic light scattering (DLS) and atomic force microscopy (AFM) were used to analyze the PM formulated pCMV-Lac Z (P/PM) containing the gene for β-galactosidase (β-Gal) driven by cytomegalovirus early promoter. Franz diffusion cell was used for in vitro transdermal permeability analysis. Real-time PCR was used to quantify the permeated plasmid in vitro and in vivo. β-Gal activity assay was performed to evaluate transgene expression in vivo. The size of P/PM was ~50 nm with round shape. PM significantly enhanced the in vitro transdermal permeability of plasmid in a direction- and temperature-dependent manner. Following transdermal application of P/PM, higher area under the curve (AUC(P/PM): 98.34 h·ng/mL) and longer half-life of plasmid were detected compared with that of plasmid alone (AUC(P): 10.12 h·ng/mL). Additionally, the β-Gal activity was significantly increased in skin, stomach, brain and spinal cord at both 48 and 72 h after P/PM application and in testis and spleen at 72 h postapplication. In conclusion, PM formulation enhanced the permeation of plasmid through skin into blood circulation, increasing its absorption and the transgene expression in various tissues.

Stromal keratocyte apoptosis triggered by epithelial injury is one mechanism of corneal disorders. A model of epithelial injury by epithelial debridement is established, and keratocyte apoptosis is evidenced by DNA fragmentation and cellular morphological changes in the anterior stroma underlying the injured epithelium. Delivery of plasmid (pCMV-bcl-x(L)-eGFP) encoding an anti-apoptotic gene, the bcl-x(L) with a nano-carrier, polymeric micelles (PM) via eye drop to cornea after epithelial debridement, the mRNA level of bcl-x(L) was significantly increased (2.2-fold, P0.05) at 48 h and the eGFP mRNA was detected (4571.7 ± 1194.5 copies/μg total RNA). The bcl-x(L)-eGFP fusion protein was also detected in wounded cornea at 48 h after delivery, accompanying with decreased DNA fragmentation and lower caspase-3 activity (P0.05). In conclusion, eye drop of pCMV-bcl-x(L)-eGFP/PM reduced corneal apoptosis following epithelial debridement.

To investigate the effect of PEO-PPO-PEO polymeric micelles (PM) formulation on the bioavailability of methylprednisolone (MP), a treatment of spinal cord injury (SCI), to the blood and spinal cord (SC) of rabbits.The characteristic of MP formulated with PM (MP/PM) was evaluated by critical micelles concentration (CMC), dynamic light scattering (DLS), atomic force microscopy (AFM) and in vitro kinetic release measurements. HPLC was used to analyze the MP disposition in plasma and SC of rabbits receiving single dose intravenous administration. After MP/PM delivery, the mRNA and protein levels of anti-apoptotic marker, Bcl-x(L), were monitored by Reverse Transcription -Real-Time -Polymerase Chain Reaction (RT-qPCR) and Western blotting analysis, respectively.At a concentration of 0.1% and at 25 degrees C, PEO-PPO-PEO copolymers formed micelles shown by fluorescence probe, DLS and solubility test. The size of the MP/PM was in an average of 60 nm with a single, rounded shape detected under AFM. Being formulated with 6% PM, MP had higher solubility (219.6 +/- 3.6 microg/ml) and release rate (11.1 +/- 0.4 ng min(1/2)) at 37 degrees C. After intravenously administrated with single dose of 1 mg/kg of MP/PM to rabbits, higher levels of MP in plasma and SC were detected compared to animals receiving an equal dose of MP, analyzed by HPLC. PM formulation markedly increased (7-fold) the plasma half-lives (t (1/2)) of MP (from 76.1 +/- 8.0 to 514.3 +/- 70.0 min). In addition, the SC t (1/2) of MP/PM also increased from 278 to 528 min. In SC, the mRNA level of Bcl-x(L) increased 4-fold in animals receiving MP/PM compared to that with MP alone at 7 h post-administration. Similar elevated Bcl-x(L) protein was also detected upon MP/PM administration compared to MP.PM vehicle was able to deliver MP to improve its pharmacokinetic profile in plasma and SC with higher expression of anti-apoptotic Bcl-x(L) at both mRNA and protein levels.

This study evaluates the eye drop delivery of genes with cornea-specific promoters, i.e., keratin 12 (K12) and keratocan (Kera3.2) promoters, by non-ionic poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) polymeric micelles (PM) to mouse and rabbit eyes, and investigates the underlying mechanisms.Three PM-formulated plasmids (pCMV-Lac Z, pK12-Lac Z and pKera3.2-Lac Z) containing the Lac Z gene for beta-galactosidase (beta-Gal) whose expression was driven by the promoter of either the cytomegalovirus early gene, the keratin 12 gene or the keratocan gene, were characterized by critical micelle concentration (CMC), dynamic light scattering (DLS), and atomic force microscopy (AFM). Transgene expression in ocular tissue after gene delivery was analyzed by 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-Gal) color staining, 1,2-dioxetane beta-Gal enzymatic activity measurement, and real-time polymerase chain reaction (PCR) analysis. The delivery mechanisms of plasmid-PM on mouse and rabbit corneas were evaluated by EDTA and RGD (arginine-glycine-aspartic acid) peptide.The sizes of the three plasmid-PM complexes were around 150-200 nm with unimodal distribution. Enhanced stability was found for three plasmid-PM formulations after DNase I treatment. After six doses of eye drop delivery of pK12-Lac Z-PM three times a day, beta-Gal activity was significantly increased in both mouse and rabbit corneas. Stroma-specific Lac Z expression was only found in pKera3.2-Lac Z-PM-treated animals with pretreatment by 5 mM EDTA, an opener of junctions. Lac Z gene expression in both pK12-Lac Z-PM and pKera3.2-Lac Z-PM delivery groups was decreased by RGD peptide pretreatment.Cornea epithelium- and stroma-specific gene expression could be achieved using cornea-specific promoters of keratin 12 and keratocan genes, and the gene was delivered with PM formulation through non-invasive, eye drop in mice and rabbits. The transfection mechanism of plasmid-PM may involve endocytosis and particle size dependent paracellular transport.

Corneal keratocyte apoptosis triggered by cornel debridement is one mechanism of corneal disorders. In this study, the feasibility of cyclo-(d-Trp-Tyr) peptide nanotubes (PNTs) as carriers of caspase 3 silence shRNA delivery was assessed. A model of epithelial injury by epithelial debridement was applied to investigate the feasibility of PNTs as gene delivery carriers on corneal injury. First, the PNTs were found within 2 μm in length and 300 nm in width by an atomic force microscope and confocal laser microscope system. Plasmid DNAs were observed to be associated with PNTs by atomic force microscope and confocal laser scanning microscope. The plasmids were associated with tyrosine of PNTs with a binding constant of 2.7 × 108 M−1. The stability of plasmid DNA with PNTs against the DNase was found at 60 min. Using thioflavin T pre-stained PNTs on the corneal eye drop delivery, the distribution of PNTs was in the epithelial and stroma regions. After corneal debridement, the rhodamine-labeled plasmid DNA and thioflavin T pre-stained PNTs were also delivered and could be observed in the stroma of cornea. PNTs complexed with anti-apoptotic plasmid caspase 3 silencing shRNA eye drop delivery decreased 41% of caspase 3 activity after the first dose by caspase 3 activity and Western blot analysis.

The main aim of this study was to investigate the feasibility of using nonionic polymeric micelles of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) as a carrier for oral DNA delivery in vivo. The size and appearance of DNA/PEO-PPO-PEO polymeric micelles were examined, respectively, by dynamic light scattering and atomic force microscopy, and their zeta potential was measured. Expression of the delivered lacZ gene in various tissues of nude mice was assessed qualitatively by 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining of sections and quantitatively by measuring enzyme activity in tissue extracts, using the substrate of beta-galactosidase, chlorophenol red-beta-D-galactopyranoside. In addition, the types of cells expressing the lacZ gene in the duodenum were identified by histological analysis. DNA/PEO-PPO-PEO polymeric micelles are a single population of rounded micelles with a mean diameter of 170 nm and a zeta potential of -4.3 mV. Duodenal penetration of DNA/PEO-PPO-PEO polymeric micelles was evaluated in vitro by calculating the apparent permeability coefficient. The results showed a dose-independent penetration rate of (5.75 +/- 0.37) x 10(-5) cm/sec at low DNA concentrations (0.026-0.26 microg/microl), but a decrease to (2.89 +/- 0.37) x 10(-5) cm/sec at a concentration of 1.3 microg/microl. Furthermore, when 10 mM RGD peptide or 10 mM EDTA was administered before and concurrent with the administration of DNA/PEO-PPO-PEO polymeric micelles, transport was inhibited ([0.95 +/- 0.57] x 10(-5) cm/sec) by blocking endocytosis or enhanced ([29.8 +/- 5.7] x 10(-5) cm/sec) by opening tight junctions, respectively. After oral administration of six doses at 8-hr intervals, the highest expression of transferred gene lacZ was seen 48 hr after administration of the first dose, with gene expression detected in the villi, crypts, and goblet cells of the duodenum and in the crypt cells of the stomach. Reporter gene activity was seen in the duodenum, stomach, and liver. Activity was also seen in the brain and testis when mice were administered 10 mM EDTA before and concurrent with DNA/PEO-PPO-PEO polymeric micelle administration. lacZ mRNA was detected in these five organs and in the blood by reverse transcription-polymerase chain reaction. Taken together, these results show efficient, stable gene transfer can be achieved in mice by oral delivery of PEO-PPO-PEO polymeric micelles.

A simple and sensitive high-performance liquid chromatographic method was developed for the determination of sildenafil transdermal permeation of nude mouse skin. A reversed-phase column with UV detection at 224 nm was used for chromatographic separation. The mobile phase consisted of 32% acetonitrile with 0.2% phosphoric acid in water at pH 5.3 adjusted with 10 M NaOH with the flow-rate set at 1.0 ml/min. The limit of quantitation achieved was 5 ng/ml, and the calibration curve showed good linearity over the concentration range of 5–500 ng/ml. The relative standard deviations of within- and between-day analyses were all within 15%. Sildenafil was found to be stable between pH 3 and 12 during 24-h incubation with skin. After transdermal administration of 15.8 μg/ml of sildenafil to nude mouse skin, it was detected as early as 15 min. The transport amount of sildenafil could be quantitated and, at pH 8–11, had the highest permeation rate in nude mouse skin.

The primary objective of this study was to investigate the effect of geniposide, a potent anti-inflammatory, on ovalbumin-antigen-induced tracheal permeability and transepithelial electrical resistance in guinea pigs. Two weeks after sensitization with ovalbumin (100 mg/ml), the permeability of guinea-pig tracheas was evaluated by flux measurements using the transcellular tracer, [ 14 C]estradiol, and the paracellular tracer, [ 14 C]mannitol. The effect of extracellular Ca 2+ with geniposide was also studied, using deletion of Ca 2+ in the donor chamber. The in vivo treatment effect of aerosolized geniposide on tracheal permeability in the ovalbumin-sensitized guinea pigs was also evaluated. The results indicate that tight junction permeability of ovalbumin-sensitized trachea was significantly dose dependent and decreased by geniposide (1–10 mM), as evidenced by substantial recovery of transepithelial electrical resistance and decreased transepithelial permeability of [ 14 C]mannitol at (1.32±0.12)×10 −5 cm/s. The effect of combination of the removal of extracellular Ca 2+ with geniposide had no effect on tight junction permeability of ovalbumin-sensitized trachea and revealed that transepithelial electrical resistance and junction permeability did not recover. In addition, the cAMP levels and phosphodiesterase activity were not significantly influenced in ovalbumin-sensitized tracheal tissues after geniposide treatment. Inhaled geniposide (50 mM, 30 min after ovalbumin sensitization) significantly restored junction permeability induced by ovalbumin (100 mg/ml, 2 min). Junction permeability did not recover on pretreatment with geniposide (50 mM for 30 min over 16 days consecutive before ovalbumin sensitization) after exposure of conscious guinea pigs to aerosol ovalbumin. In conclusion, geniposide has inhibitory effects on ovalbumin-induced junction permeability and recovery of transepithelial electrical resistance in guinea pig trachea, showing its potential as anti-asthma therapy.

Purpose. To determine aortic endothelial cells permeation ability and mechanisms of the aqueous block copolymeric micelles, poly(ethylene oxide)-poly ((β-benzyl L-aspartate) (PEO-PBLA) chemically conjugated with fluroescein isothiocyanate (FITC) by transport study and confocal laser scanning microscopy.

Purpose. To directly visualize and evaluate the aqueous block copolymeric micelles, poly(ethylene oxide)-poly(β-benzyl L-aspartate) (PEO-PBLA) chemically conjugated with pyrene fluorescence molecule, by nanotechnology of atomic force microscopy (AFM).

The feasibility of cyclo-(D-Trp-Tyr) peptide nanotubes (PNTs) as oral gene delivery carriers was investigated in nude mice with eight 40 μg doses of pCMV-lacZ in 2 days at 3 h intervals. The association between DNA and PNTs, the DNase I stability of PNTs-associated DNA, and in vitro permeability of DNA were estimated. The results showed that the cyclo-(D-Trp-Tyr) PNTs self-associated at concentrations above 0.01 mg/mL. Plasmid DNA associated with PNTs with a binding constant of 3.2 × 10(8) M(-1) calculated by a fluorescence quenching assay. PNTs were able to protect DNA from DNase I, acid, and bile digestion for 50 min, 60 min, and 180 min, respectively. The in vitro duodenal apparent permeability coefficient of pCMV-lacZ calculated from a steady state flux was increased from 49.2 ± 21.6 × 10(-10) cm/s of naked DNA to 395.6 ± 142.2 × 10(-10) cm/s of pCMV-lacZ/PNT formulation. The permeation of pCMV-lacZ formulated with PNTs was found in an energy-dependent process. Furthermore, β-galatosidase (β-Gal) activity in tissues was quantitatively assessed using chlorophenol red-β-D-galactopyranoside (CPRG) and was significantly increased by 41% in the kidneys at 48 h and by 49, 63, and 46% in the stomach, duodenum, and liver, respectively, at 72 h after the first dose of oral delivery of pCMV-lacZ/PNT formulation. The organs with β-Gal activity were confirmed for the presence of pCMV-lacZ DNA with Southern blotting analysis and intracellular tracing the TM-rhodamine-labeled DNA and the presence of mRNA by reverse transcription-real time quantitative PCR (RT-qPCR). Another plasmid (pCMV-hRluc) encoding Renilla reniformis luciferase was used to confirm the results. An increased hRluc mRNA and luciferase in stomach, duodenum, liver, and kidney were detected by RT-qPCR, ex vivo bioluminescence imaging, luciferase activity quantification, and immunostaining, respectively.

Alveolar macrophage (AM) plays important roles in lung homeostasis and pathogenesis of diseases. The study of macrophage gene function and regulation as well as its potential therapeutic intervention will require the development of vectors capable of safe and efficient transfer of DNA to the AM. In the present study, we report a new transfection system that utilizes Fc receptor-mediated endocytosis as a means to target DNA to the AM. This system employs molecular conjugates consisting of a cognate moiety, in this case IgG which recognizes the AM Fc receptor, covalently-linked to a DNA-binding moiety, such as a cationic polyamine. A Complex was formed between immunoglobulin G-polylysine conjugate (IgG-pL) and plasmid DNA carrying the LacZ reporter gene (pSV beta). The conjugate-DNA complex was added directly to the AMs in culture and incubated for 24 h, after which LacZ gene expression was analyzed for beta-galactosidase activity by microfluorometry using a fluorogenic beta-galactosidase substrate, 5-dodecanoylaminofluorescein di-beta-D-galactopyranoside (C12FDG). The AMs treated with the IgG-pL/DNA complex exhibited galactosidase activity significantly augmented over background levels. Effective gene transfer was shown to require both the DNA-binding moiety and cognate moiety for the cell surface receptor. Specific internalization of the complex by the Fc receptor pathway was verified by competitive inhibition using excess IgG. Under this condition, LacZ gene expression was inhibited, suggesting complex internalization through the Fc mediated endocytosis pathway. The requirement of Fc receptors for complex internalization was further demonstrated using cells that lack Fc receptors, e.g., alveolar epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to understand the effect of charge type on corneal transport of peptides, it is useful to first investigate the mechanism of transport of amino acids across corneal tissue. The transport characteristics of l-[14C]lysine and l-[14C]glutamic acid were based on in vitro flux measurements. Net flux of l-lysine occurs via a sodium-dependent cotransport mechanism (with a coupling ratio of 1:1). The transport of l-lysine involves the Na+-K+-ATPase pump and requires a stereospecific carrier-mediated transport system. The permeability coefficient (P) of l-lysine was decreased from 10.1 ± 0.80 to 2.34 ± 0.75 × 10−6 (cm/s by blocking the active pump and carrier-mediated system. However, l-glutamic acid was apparently absorbed by a passive aqueous transport mechanism which was oubain-insensitive, [Na+]-independent, temperature-insensitive and non-stereospecific. Overall, the cornea was 2–3-fold more permeable to a cation (l-lysine) than an anion (l-glutamic acid). In addition, the negatively charged drug, salicylic acid, was 2.3 times less permeable than the cationic drug, benzylamine.

Researchers have identified a number of peptide and protein drugs which may be of benefit in treating conditions of the eye. However, the clinical use of these agents may be limited by their low bioavailabilities of ocular tissues following topical application. Few studies to date have addressed the issue of corneal penetration enhancement of peptides, not least perhaps because of concerns over the local toxicity of such agents. This paper reviews the ocular delivery of peptides and the ocular use of penetration enhancement. Further, it reports a general approach to corneal penetration enhancement based on the principle that epithelial tight junctions, modulated by the cell cytoskeleton, are a major component of the permeability barrier. The cytoskeletal modulator, cytochalasin B, has been shown to give a similar increase in permeability to other conventional penetration enhancers, but with substantially less disruption of epithelial cell membranes. This agent may act via a paracellular cytoskeletally modulated mechanism, which may form a basis for developing relatively specific, low-toxicity corneal penetration enhancers.

The use of nonionic polymeric micelles orally to protect and deliver plasmid DNA in vivo was investigated. Parathyroid hormone (PTH)(1-34) gene (179 bp) was inserted into a human cytomegalovirus promoter (PCMV) and E. coli competent cells were used to amplify the cDNA. Polymeric micelle formations (100 microl) formed from PCMV-PTH(1-34) cDNA (7.2 microg/microl) and 6% (w/v) polyethylene oxide-polypropylene oxide-polyethylene oxide (PEO-PPO-PEO) was administered at 8-hr intervals for 48 hr and then at 8-hr intervals for 24 hr weekly for 3 weeks. Parathyroidectomized rats receiving 150 microl of EDTA (10 mM) before each dose of formation served as the study group; rats receiving drinking water, EDTA (10 mM), PCMV-PTH(1-34) cDNA and PCMV-PTH(1-34) cDNA plus EDTA at the same amount and time intervals served as the control groups. Serum levels of calcium and PTH(1-34) were measured weekly for 4 weeks. Immunohistochemical stain for PTH(1-34), reverse transcriptase polymerase chain reaction for PTH(1-34) mRNA and the relative density of PTH(1-34) mRNA were performed at 2 and 4 weeks after oral gene therapy in different organs. One third to three of five rats in the control groups died after parathyroidectomy. Serum levels of calcium and PTH(1-34) were higher in the study than in the control groups. In the study group, positive stain of PTH(1-34) and PTH(1-34) mRNA could be found in those organs. Relative densities of PTH(1-34) mRNA were higher in the study than in the drinking water group in different organs. Oral gene therapy can maintain calcium and PTH(1-34) levels in parathyroidectomized rats.

The present study investigates the effects of various penetration enhancers including EDTA, digitonin, deoxycholic acid, and a cytoskeleton-active agent, cytochalasin B, on the structure and electrophysiological properties of the rabbit cornea. Using confocal fluorescence microscopy, with the aid of a specific membrane probe (3,3′-dioctadecyloxacarbocyanine perchlorate), and a viability polar tracer (propidium iodide), structural changes and cell death as a result of the penetration enhancers can be detected. In addition to its great sensitivity, the technique provides direct observation, through optical sectioning, of the interior structures of living tissues without the usual artifacts associated with tissue preparation which is required in conventional microscopy. In conjunction with the microscopic method, an electrical method based on membrane resistance and capacitance measurements was used to provide quantitative assessment of the promoting effect of the enhancers. Since electrical resistance is a measure of charge flow and ionic membrane permeability, it can be used to indicate changes in dimension of the aqueous transport pathway, i.e., the tight junction and the intercellular space, as well as the degree of penetrability of the charged species. On the other hand, membrane capacitance reflects the ability of the membrane dielectric to store charges, and thus, it can be used to detect structural damage to the membrane surface. Results obtained from this study indicate that all enhancers significantly increase membrane permeability depending on the concentration and exposure time. However, with the exception of the cytoskeleton-active agent, all enhancers also cause severe membrane damage. Mechanistically, this agent acts specifically on the tight junction proteins and consequently increases intercellular permeability. The chelator, EDTA, acts not only on cell junctions by interfering with calcium ions and altering intercellular integrity, but also disrupts the plasma membrane. Digitonin and bile salt interact with membrane lipid structures and disrupt their organization.

The present study uses the cannulated dog to verify the major elements of gastrointestinal (GI) motility affecting the performance of oral sustained release dosage forms. It appears that the residence time of a Theo-dur tablet in the fasted canine stomach and small intestine is dictated by the phasic activity of the motility pattern at the time of ingestion. The tablet does not disintegrate in vivo but undergoes surface erosion. Onset of tablet discharge corresponded with late phase II and phase III activity. The discharged eroded tablet was entrapped within mucus plugs. Mucus may play a significant role in the dissolution of administered tablets. There is little variability in the duodenal and ileal effluent pH, observed after onset of Theo-dur tablet discharge.

Elevation of corticosteroids and excessive glutamate release are the two major stress responses that occur sequentially during traumatic CNS injury. We have previously reported that sequential application of corticosterone and kainic acid (CORT + KA) mimicking the nerve injury condition results in synergistic enhancement of neurite outgrowth and expression of growth-associated protein 43 (GAP-43) in cultured dorsal root ganglion (DRG). GAP-43 is known to promote neurite extension when phosphorylated by protein kinase C (PKC). In addition, PKC can phosphorylate the signal transducer and activator of transcription 3 (STAT3) at Ser727, which is phosphorylated primarily by Janus kinase (JAK) at Tyr705. In this study, we further examine the role of PKC in this stress-induced growth-promoting effect. In the cultured DRG neurons, the JAK inhibitor AG-490 and the PKC inhibitor Ro-318220 reduced the CORT + KA-enhanced neurite growth effect when applied prior to CORT and KA treatment, respectively. Both AG-490 and Ro-318220 diminished the CORT + KA-enhanced GAP-43 expression, phosphorylation, and axonal localization. Furthermore, CORT + KA treatment synergistically phosphorylated STAT3 at Ser727 but not at Tyr705. Similar phenomena were observed in an animal model of acute spinal cord injury (SCI), in which phosphorylation of GAP-43 and phospho-Ser727-STAT3 was elevated in the injured DRG 4 hr after the impact injury. Further treatment with the therapeutic glucocorticoid methylprednisolone enhanced the phosphorylation of GAP-43 in both the DRG and the spinal cord of SCI rats. These results suggest that elevated glucocorticoids and overexcitation following CNS injury contribute to nerve regeneration via induction of JAK/PKC-mediated GAP-43 and STAT3 activities.

Modifications of both carriers and host barriers have been investigated for efficient inhalation gene delivery to lung. Here we used a biocompatible, non-ionic poly(ethyleneoxide)-poly(propyleneoxide)-poly(ethyleneoxide) (PEO-PPO-PEO) polymeric micelles (PM) as a carrier and combined it with ethanol to enhance membrane penetration of delivered DNA. The inhalation delivery with six 100 microg doses of pCMV-Lac Z with PM co-formulated with 10%-40% ethanol to nude mice in 2 days at 8 h interval was performed. The beta-galatosidase (beta-Gal) activity was assessed using chlorophenol red-beta-d galactopyranoside (CPRG) and X-gal staining for quantitative and qualitative analysis in tissues. The results showed that beta-Gal activity was significantly increased by 38% in lung around bronchioles when inhalation with PM and 10% ethanol was given. The 10% ethanol also increased the intracellular apparent permeability by 42% in stomach and by 141% in intestine at 48 h after the first dosage of delivery. Also delivery of DNA encoding a functional human cystic fibrosis transmembrane protein (CFTR) using the same inhalation delivery method co-formulated with 10% ethanol, an increased expression of CFTR in lung was detected by immunostaining. We concluded that 10% ethanol co-formulated with the PM system could enhance inhaled gene delivery to airway and gastrointestinal (GI) tract.

The primary objective of this study was to investigate the feasibility of using PEO-PPO-PEO non-ionic copolymeric micelles as a carrier for eye-drop gene delivery of plasmid DNA with lacZ gene in vivo. Using pyrene fluorescence probe methods, zeta potential, and dynamic light scattering test (DLS), the ability of micelle formation of these block copolymers with plasmid was studied. Gene expressions were visualized by both the quality of enzymatic color reaction using X-gal staining and by the quantification of the substrate chlorophenol red galactopyranoside (CPRG) in enucleated eyes on day 2 after gene transfer. In addition, microscopy to identify the types of cell showing uptake and expression of the transferred gene was used. We found that the block polymeric micelles were formed above 0.1% (w/v) of block copolymer with a size of 160 nm and a zeta potential of -4.4 mV. After 2 days of topically delivery three times a day, the most intense gene expression was observed on days 2 and 3. Reporter expression was detected around the iris, sclera, conjunctiva, and lateral rectus muscle of rabbit eyes and also in the intraocular tissues of nude mice upon in vivo topical application for 48 h with a DNA/polymeric micelle formulation. Furthermore, after two enhancement treatments, the transport mechanisms of the block copolymeric micelles were found through endocytosis in tissues by enhancement through the tight junction pathway. Thus, efficient and stable transfer of the functional gene could be achieved with PEO-PPO-PEO polymeric micelles through topical delivery in mice and rabbits. These in vivo experiments indicate the possible potential use of block copolymers for DNA transfer.

The primary objective of this study was to investigate the feasibility of PEO-PPO-PEO copolymer gel as a release vehicle for percutaneous administration of fentanyl in vitro and in vivo. A cellulose membrane and nude mouse skin with series concentrations of PEO-PPO-PEO block copolymers were used to examine the sustained-release pattern and permeation of fentanyl. The in vivo percutaneous absorption was examined using rabbits to evaluate the preliminary pharmacokinetics of fentanyl with 46% PEO-PPO-PEO copolymer formulation patches. The micelle formation ability of this block copolymer and the penetration ability of PEO-PPO-PEO copolymer over time were also studied by pyrene fluorescence probe methods and the dynamic light scattering test. At a concentration of 46% at 37 degrees C, PEO-PPO-PEO copolymers formed a gel and showed a pseudo-zero-order sustained-release profile. With increasing concentration of copolymer in the cellulose membrane transport, the apparent release flux of fentanyl (200 microgram/ml) decreased to 1. 09+/-0.19 microgram cm(-2) h(-1). Assessment of the effect of the copolymer on nude mouse skin also showed a decrease in the apparent permeability coefficient [(P(H(2)O))=2.24+/-0.47x10(-6) cm s(-1) vs. (P(46% block copolymer))=0.93+/-0.23x10(-7) cm s(-1)]. The preliminary pharmacokinetics of the fentanyl patch was shown to be in steady state within 24 h, and this was maintained for at least 72 h with an elimination half-life (t(1/2)) of 10.5+/-3.4 h. A fluorescence experiment showed polymeric micelle formation of PEO-PPO-PEO copolymers at 0.1% (w/w) within 50 nm micelle size and the PEO-PPO-PEO copolymers were able to penetrate nude mouse skin within 24 h. Thus, it appears that fentanyl preparations based on PEO-PPO-PEO copolymer gel might be practical for percutaneous delivery.