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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

D-phenylglycine-L-dopa, a dipeptide synthesized in this laboratory for improving the oral absorption of L-dopa, showed better absorption and distribution in rats. We assumed that the extensive distribution might explain its sustained release of brain dopamine. Since protein binding is a main factor of drug distribution, we investigated the effect of D-phenylglycine on the binding of L-dopa to human serum albumin. The degree of the binding of D-phenylglycine, L-dopa and D-phenylglycine-L-dopa to serum albumin was determined. Free and bound portion of test compounds were separated with ultrafiltration method and the assay of free drug portion was conducted with reversed phase HPLC. The LOQ for D-phenylglycine, L-dopa and D-phenylglycine-L-dopa was 0.5 μg/mL, 0.1 μg/mL and 0.5 μg/mL, respectively. Assay methods were validated by determining the precision and accuracy of interday and intraday variations. The coefficient of variation (CV) was within 12% and the relative error (RE) was within 10% (n = 3). The recovery rate was 95.4% - 98.1% for D-phenylglycine, 91.9 % - 98.8% for L-dopa and 96.8% - 97.9% for D-phenylglycine-L-dopa, respectively (n = 3). D-Phenylglycine showed higher serum albumin binding than L-dopa did at various concentrations. At a concentration of 600 μM, the degree of albumin binding of D-phenylglycine, L-dopa, D-phenylglycine-L-dopa was 27.98%, 8.20% and 19.18%, respectively. The albumin binding of L-dopa at this concentration increased by 2.4 folds when chemically bound to D-phenylglycine. The number of the binding sites of L-dopa increased by 5.8 folds and the binding constant Ka1 increased by 3.8 folds when L-dopa was chemically bound to D-phenylglycine. With the affinity to serum albumin, D-phenylglycine showed its possibility as a delivery moiety for drugs with limited distribution to use the body protein as a reservoir.

The effect of Povacoat on the solid state characteristics and polymorphic transformation of γ-form of indomethacin (γ-IMC) after solvent co-evaporation via two drying methods was investigated by thermoanalytical and FTIR spectroscopic studies. The types of solvents (water, ethanol, and water:ethanol (1:9 ~ 2:8, v/v)), weight ratios of Povacoat:IMC (4:1 ~ 4:10, w/w), and drying methods (air-drying and microwave-drying) were selected as variables in this study. A physical mixture of Povacoat and γ-IMC was also carried out by neat grinding process. The results indicate that there was no interaction between Povacoat and γ-IMC or no polymorphic change of γ-IMC after the neat co-grinding process. Since Povacoat was only soluble in water or

The effect of Soluplus on the solid-state characteristics and physical transformation of indomethacin (IMC), saccharin (SAC), IMC-SAC physical mixture, and the previously prepared IMC-SAC co-crystal was investigated after solvent evaporation via air-drying process. Soluplus with γ-IMC or SAC (weight ratio = 1:1) was respectively prepared by completely dissolving both components in acetone by ultrasonication, and then evaporated via air-drying process. Soluplus was fully co-dissolved with the previously prepared IMC-SAC co-crystal (procedure I) or completely dissolved together with IMC-SAC physical mixture (procedure II) in acetone by ultrasonication, and then evaporated through air-drying process. The physical stability of both Soluplus/IMC-SAC systems was also carried out under accelerated storage conditions. All the samples were determined by thermoanalytical and FTIR spectroscopic studies with spectral curve-fitting technique. Soluplus caused the amorphous formation of IMC or SAC in the Soluplus solid dispersion, in which the intermolecular hydrogen bonding interaction between SAC and Soluplus occurred. Although Soluplus did not influence the co-crystal formation between IMC and SAC in the solid dispersion, the polymorphic transformation and physical stability of Soluplus/IMC-SAC solid dispersion were markedly influenced by procedures I and II after evaporation via air-drying process. Soluplus influences the amorphous or crystalline IMC-SAC co-crystal formulated in the solid dispersion prepared by procedure I or II via air-drying process. The amorphous IMC-SAC co-crystal in the Soluplus solid dispersion prepared by procedure I gradually transformed to a crystalline IMC-SAC co-crystal after storage at 40 ± 2 °C/75 ± 5 % RH conditions for 28 days, whereas the crystalline IMC-SAC co-crystal in the Soluplus solid dispersion prepared by procedure II via air-drying process maintained a long-term storage stability.

The amorphous form of a drug has higher water solubility and faster dissolution rate than its crystalline form. However, the amorphous form is less thermodynamically stable and may recrystallize during manufacturing and storage. Maintaining the amorphous state of drug in a solid dosage form is extremely important to ensure product quality. The purpose of this study was to quantitatively determine the amount of amorphous indomethacin (INDO) formed in the Soluplus® solid dispersions using thermoanalytical and Fourier transform infrared (FTIR) spectral curve-fitting techniques. The INDO/Soluplus® solid dispersions with various weight ratios of both components were prepared by air-drying and heat-drying processes. A predominate IR peak at 1683cm(-1) for amorphous INDO was selected as a marker for monitoring the solid state of INDO in the INDO/Soluplus® solid dispersions. The physical stability of amorphous INDO in the INDO/Soluplus® solid dispersions prepared by both drying processes was also studied under accelerated conditions. A typical endothermic peak at 161°C for γ-form of INDO (γ-INDO) disappeared from all the differential scanning calorimetry (DSC) curves of INDO/Soluplus® solid dispersions, suggesting the amorphization of INDO caused by Soluplus® after drying. In addition, two unique IR peaks at 1682 (1681) and 1593 (1591)cm(-1) corresponded to the amorphous form of INDO were observed in the FTIR spectra of all the INDO/Soluplus® solid dispersions. The quantitative amounts of amorphous INDO formed in all the INDO/Soluplus® solid dispersions were increased with the increase of γ-INDO loaded into the INDO/Soluplus® solid dispersions by applying curve-fitting technique. However, the intermolecular hydrogen bonding interaction between Soluplus® and INDO were only observed in the samples prepared by heat-drying process, due to a marked spectral shift from 1636 to 1628cm(-1) in the INDO/Soluplus® solid dispersions. The INDO/Soluplus® solid dispersions prepared by both drying processes could keep the amorphous state of INDO in the INDO/Soluplus® solid dispersions at the accelerated storage condition.

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.

Line width roughness (LWR) is one of the most critical performance indexes for low k1 ArF immersion Lithography. Several factors are impacting LWR performance during Lithography process, such as structures, anti-reflective coating (ARC), photoresist, baking condition, illumination condition and track process. However, the structures and ARCs are strongly related to integration and Etch processes. The illumination conditions, including mask bias, are decided by simulation software but the track condition usually follows the pervious node at initial step normally. Therefore, it sometimes shows poor results because of the difference of the under-layer condition. For example, it has been uncovered that LWR and local CD uniformity (LCDU) became worse while structure changed to Carbon-DARC-Resist (CDR) from Multi-Layer Resist (MLR). Generally, photoresist evaluation with baking condition optimization is a typical way to improve LWR performance. The photoresist formulation contains photosensitive polymers, photoacid generators (PAGs), quenchers, additives, and solvents. Based on photoresist's point of view, the first two are the most important factors of LWR performance control. Several designs of experiments (DOEs) were planned with polymers, PAGs, and PEB conditions. The target is to achieve LWR of CDR under the result of MLR. In this paper, Polymer DOE1 and its statistical analyses have finished. Compared to de-composition efficiency of each unit, small protection unit and PEB effect are the most important factors, and bulky protection unit shows less influence for LWR improvement. Small protection unit is more important than bulky protection unit, and high Ea monomers of both are good for LWR. Based on previous experiences, we have chosen the acid diffusion length / amount of PAG and PEB temperature for PAG DOE2 plan. Higher PEB is the most effective to LWR. Shorter diffusion length PAG with fewer loading amounts is better for MEEF and EL by DOE2 result. The final DOE3 is combined by optimized polymers by DOE1 and optimized PAG by DOE2. The polymer is used DOE1 Tr2, DOE1 Tr4, and estimated new polymer by DOE1 result, and the PAG is chosen shorter diffusion PAG-O with the split of loading amount and PEB temperature. Based on the statistical analyses of DOE3 result, higher PEB temperature is still the most effective to LWR, and new polymer and DOE1 Tr2 polymer are better for LWR reduction. However, higher PEB temperature would suffer a little bit of DOF, EL, and MEEF. In order to achieve the lower LWR, it needs to use DOE1 Tr2 polymer with higher PEB, but insufficient DOE and MEEF would get at the same time. New polymer by DOE1 combined fewer PAG-O loading amount with lower PEB could get slower sensitivity for all Litho index's requirements. Through DOE works, we found out the volume of protection unit and PEB were key factors for good LWR. However, these factors are trades off DOF, EL, and MEEF against LWR, and they need to optimize for the best balanced Litho performances.

Small contact hole patterning had become the most difficult task in optical lithography as design rule of semiconductor continuously shrinks below 65nm. Conventional contact hole scheme need to avoid side-lobe and conduct complicated dense-isolated bias for resolution enhancement and depth of focus (DOF) improvement. To overcome this issue, some RETs (Resolution Enhancement Techniques) by process had been investigated, like RELACS (resolution enhancement lithography assisted by chemical shrink). RELACS is one of feasible procedures which could provide enough improvement in resolution, photo-resist profile, DOF, and CD uniformity (CDU). Proximity effect is one of significant topics to evaluate chemical shrink bias of different type contacts. Research of shrink bias of different size and pitch contacts had been investigated broadly in the past. In general, the constant bias of shrinkage for difference pattern sizes was an assumption. However, according to our evaluative results, we had characterized the correlation about the shrink bias versus pattern size. In this paper, we not only show DOF, CDU, shrink bias of RELACS, but also present chemical shrink bias of different size and different pitch contact holes and then we could follow this correlation rule to define general rule for proximity effect correction.

In order to reduce the overall size of device features, continuing development in the low k1 lithography process is essential for achieving the feature reduction. Although ArF immersion lithography has extended the feature size scaling to 45nm node, investigation of low k1 lithography process is still important for either ArF dry or wet lithography. Double patterning is one procedure pushing down the k1 limit below 0.25. It combines the multilayer hard mask application and resist shrinkage process to get the feature size reduced to quarter pitch of the illumination limit. In recent spin-on hard mask studies, silicon containing bottom antireflective coatings (BARC) have been developed to combine the function of reflective control and great etching selectivity to the photoresist. Trilayer resist including the photoresist, silicon containing BARC and planarizing organic underlay can improve the reflectivity by optical index tuning of dual hard mask layer effectively and reduce photoresist thickness to avoid the pattern collapse with small features. In our study, we found some interesting characteristics of trilayer resist could be used for double patterning technology and made the low k1 process more feasible. This procedure we investigated can make the feature size of half pitch reduce to 37nm and beyond at 0.92NA under ArF dry lithography. Among the resolution enhancement for ArF dry illumination, double patterning scheme, overlay controllability and pattern transfer process by reactive ion etching (RIE) will be discussed in this paper.

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.