RESUMEN
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 L-PNTs/DNA and S-PNTs/DNA. However, there were no 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.
Asunto(s)
Encéfalo/metabolismo , ADN/farmacocinética , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Nanotubos de Péptidos/química , Péptidos Cíclicos/química , Médula Espinal/metabolismo , Proteína bcl-X/metabolismo , Administración Oral , Animales , ADN/administración & dosificación , ADN/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Regiones Promotoras Genéticas , Distribución Tisular , Proteína bcl-X/administración & dosificación , Proteína bcl-X/genéticaRESUMEN
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.
Asunto(s)
Nanotecnología/métodos , Nanotubos de Péptidos/química , Animales , Sistemas de Liberación de Medicamentos/instrumentación , Sistemas de Liberación de Medicamentos/métodos , Humanos , Nanotecnología/instrumentación , EstereoisomerismoRESUMEN
Three thermal analytical techniques such as differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA) using five heating rates, and DSC-Fourier Transform Infrared (DSC-FTIR) microspectroscopy using one heating rate, were used to determine the thermal characteristics and the dehydration process of aspartame (APM) hemihydrate in the solid state. The intramolecular cyclization process of APM anhydrate was also examined. One exothermic and four endothermic peaks were observed in the DSC thermogram of APM hemihydrate, in which the exothermic peak was due to the crystallization of some amorphous APM caused by dehydration process from hemihydrate to anhydride. While four endothermic peaks were corresponded to the evaporation of absorbed water, the dehydration of hemihydrate, the diketopiperazines (DKP) formation via intramolecular cyclization, and the melting of DKP, respectively. The weight loss measured in TGA curve of APM hemihydrate was associated with these endothermic peaks in the DSC thermogram. According to the Flynn-Wall-Ozawa (FWO) model, the activation energy of dehydration process within 100-150 °C was about 218 ± 11 kJ/mol determined by TGA technique. Both the dehydration and DKP formation processes for solid-state APM hemihydrate were markedly evidenced from the thermal-responsive changes in several specific FTIR bands by a single-step DSC-FTIR microspectroscopy.
RESUMEN
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.
Asunto(s)
Indometacina/química , Rastreo Diferencial de Calorimetría , Estabilidad de Medicamentos , Polietilenglicoles/química , Polivinilos/química , Solubilidad , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
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, ß-galatosidase (ß-Gal) activity in tissues was quantitatively assessed using chlorophenol red-ß-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 ß-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.
Asunto(s)
Nanotubos de Péptidos/química , Animales , Southern Blotting , Técnicas de Transferencia de Gen , Masculino , Ratones , Ratones Desnudos , Microscopía de Fuerza Atómica , Microscopía Electrónica de Rastreo , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Nanotubos de Péptidos/ultraestructura , Plásmidos/químicaRESUMEN
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.