Matrix metalloproteinase-inspired suicidal treatments of diabetic ulcers with siRNA-decorated nanofibrous meshes.

Matrix metalloproteinase (MMP)-assisted siRNA treatment was accomplished with a nanofibrous matrix for suicidal gene therapy of diabetic ulcers. We fabricated a MMP-responsive nanofibrous matrix to control release of small interfering RNA (siRNA) in response to a high concentration of MMPs in diabetic ulcers. For MMP-responsive release of siRNA, linear polyethyleneimine (LPEI) was chemically conjugated on the surface of the nanofibrous matrix via a MMP-cleavable linker. To control the abnormally elevated MMP-2 expression in diabetic ulcers, MMP-2 siRNA was electrostatically incorporated into LPEI-immobilized nanofibrous meshes with various nitrogen/phosphate (N/P) ratios. The release profiles of siRNA and LPEI were monitored to confirm that MMP responsiveness of the matrix and MMP-2 significantly increased the release of both siRNA and LPEI for 72 h. The released fractions were transfected to dermal fibroblasts. Quantitative reverse transcription (qRT)-PCR for endogenous MMP-2 expression confirmed that the gene-silencing effects of siRNA were dependent on the charge ratio of LPEI to siRNA on the mesh. Diabetic animals with dorsal burns were treated with siRNA-incorporated nanofibrous mesh for 7days. siRNA-incorporated nanofibrous meshes dramatically increased the MMP-2 gene-silencing effects of the siRNA and neo-collagen accumulation at the wound sites. RT-PCR also confirmed the highest expression levels of the keratinocyte-specific markers and the lowest expression levels of MMP-2 in the nanofibrous mesh-treated groups, suggesting that wound recovery is restored to normal levels. The wound recovery rates of diabetic ulcers were significantly increased when siRNA-incorporated nanofibrous meshes were administered. Thus, the suicidal treatment with the MMP-2 siRNA-decorated nanofibrous mesh is expected to improve prognosis of diabetic ulcers with reduced side effects.

The role of psychological factors in the development of burning mouth syndrome.

The psychiatric profiles of 50 patients diagnosed with burning mouth syndrome (BMS) were compared to those of 50 age- and sex-matched individuals as the control group. The Symptom Checklist-90-Revised (SCL-90-R) questionnaire was used to evaluate the role of psychological factors in the development of BMS. Somatization, obsessive-compulsive, depression, anxiety, hostility, phobic anxiety, psychoticism, global severity index (GSI), positive symptom total (PST), and positive symptom distress index (PSDI) scores were significantly higher in the patients with BMS than in the control group. In a subgroup analysis according to sex, women with BMS had higher T-scores for somatization, obsessive-compulsive, paranoid ideation, GSI, PST, and PSDI than women in the control group. In contrast, only the PSDI score was significantly higher in men with BMS compared to men in the control group. There was a significant difference in the T-scores for somatization, psychoticism, and GSI between the three age subgroups (≤50, 51-65, and ≥66 years). The obsessive-compulsive and PSDI scores were significantly higher in patients with BMS who also had at least one chronic disease than in patients with BMS who had no chronic disease. In conclusion, psychological factors are correlated with BMS.

Hydro-nanofibrous mesh deep cell penetration: a strategy based on peeling of electrospun coaxial nanofibers.

A two-step strategy for coaxial electrospinning and postelectrospinning is an effective method for fabricating superfine nanofibers composed of highly swellable hydrogels. Alginate and poly(ε-caprolactone) [PCL] were coelectrospun via fibrous meshes with a coaxial nozzle; alginate at the core was subsequently cross-linked in calcium chloride solution. The PCL sheath was removed from the meshes by repeated organic-phase washing. The peeling process was monitored by scanning electron microscopy, transmission electron microscopy, and differential scanning calorimetry, and the complete removal of the PCL outer layers was confirmed by the thinning of the fiber volume. The obtained alginate hydronanofiber showed extreme water-swellability and mass erosion depending on the degree of cross-linking. We also measured the nanoscale and macroscale mechanical properties of a single nanofiber and of the whole mesh by atomic force microscopy and rheometry. Quantitative analysis of nanomechanical properties indicated that the hydronanofiber with higher cross-linking density had higher stiffness and Derjaguin-Müller-Toporov modulus. Cells laid on the mesh and the vertical infiltration distance were visualized and quantified by confocal laser scanning microscopy. Cells on the mesh with higher cross-linking density infiltrated deeply to the bottom of the mesh. Thus, hydrogel-like nanofibrous meshes are versatile matrices allowing for deep infiltration of cells throughout the mesh via manipulation of the mechanical properties of the nanofiber.

Biodegradable polymeric micelles composed of doxorubicin conjugated PLGA-PEG block copolymer.

Biodegradable polymeric micelles containing doxorubicin in the core region were prepared from a di-block copolymer composed of doxorubicin-conjugated poly(DL-lactic-co-glycolic acid) (PLGA) and polyethyleneglycol (PEG). The di-block copolymer of PLGA-PEG was first synthesized and the primary amino group of doxorubicin was then conjugated to the terminal hydroxyl group of PLGA, which had been pre-activated using p-nitrophenyl chloroformate. The resulting polymeric micelles in aqueous solution were characterized by measurement of size, drug loading, and critical micelle concentration. The micelles containing chemically-conjugated doxorubicin exhibited a more sustained release profile than PEG-PLGA micelles containing physically-entrapped doxorubicin. The cytotoxic activity of the micelles against HepG2 cells was greater than free doxorubicin, suggesting that the micelles containing conjugated doxorubicin were more effectively taken up cellularly, by an endocytosis mechanism rather than by passive diffusion. Confocal microscopic observation and flow cytometry analysis supported the enhanced cellular uptake of the micelles.

In vitro and in vivo anti-tumor activities of nanoparticles based on doxorubicin-PLGA conjugates.

Doxorubicin was chemically conjugated to a terminal end group of poly(D,L-lactic-co-glycolic acid) [PLGA] by an ester linkage and the doxorubicin-PLGA conjugate was formulated into nanoparticles. A carboxylic acid end group of PLGA was conjugated to a primary hydroxyl group of doxorubicin. The primary amine group of doxorubicin was protected during the conjugation process and then deprotected. The nanoparticles containing the conjugate exhibited sustained doxorubicin release profiles over a 1-month period, whereas those containing unconjugated free doxorubicin showed a rapid doxorubicin release within 5 days. Doxorubicin release patterns could be controlled by conjugating doxorubicin to PLGA having different molecular weights. The conjugated doxorubicin nanoparticles showed increased uptake within a HepG2 cell line, which was quantitated by a flow cytometry and visualized by confocal microscopy. The nanoparticles exhibited slightly lower IC(50) value against the HepG2 cell line compared to that of free doxorubicin. In vivo anti-tumor activity assay also showed that a single injection of the nanoparticles had comparable activity to that of free doxorubicin administered by daily injection. The conjugation approach of doxorubicin to PLGA was potentially useful for the formulation of nanoparticles that requires targeting for cancer cells as well as sustained release at the site.

Protein-fatty acid complex for enhanced loading and stability within biodegradable nanoparticles.

Lysozyme was hydrophobically modified with a fatty acid, sodium oleate, via an ion-pairing mechanism. Ionic binding between an anionic carboxylic group of sodium oleate and basic amino groups in lysozyme was primarily utilized to form lysozyme-oleate complex. The complex formation was pH dependent. The lysozyme-oleate complex dissolved in an organic solvent exhibited much higher conformational stability at elevated temperature compared with free lysozyme in the same solvent. The complex was formulated into biodegradable nanoparticles by a spontaneous emulsion and solvent diffusion method. The resultant formulation showed near 100% encapsulation efficiency of lysozyme within nanoparticles with < 100 nm in diameter with a narrow size distribution. Lysozyme could be loaded into the nanoparticles up to 18.6% (w/w) with concomitantly increased particle sizes. This study demonstrates a new formulation method of biodegradable nanoparticles with highly efficient encapsulation of proteins, which are potentially useful for oral protein delivery including mucosal vaccination.

Biodegradable nanoparticles containing doxorubicin-PLGA conjugate for sustained release.

PURPOSE: Doxorubicin was chemically conjugated to a terminal end group of poly(D,L-lactic-co-glycolic acid) [PLGA] and the doxorubicin-PLGA conjugate was formulated into nanoparticles to sustain the release of doxorubicin. METHODS: A hydroxyl terminal group of PLGA was activated by p-nitrophenyl chloroformate and reacted with a primary amine group of doxorubicin for conjugation. The conjugates were fabricated into ca. 300 nm size nanoparticles by a spontaneous emulsion-solvent diffusion method. The amount of released doxorubicin and its PLGA oligomer conjugates was quantitated as a function of time. The cytotoxicity of the released species was determined using a HepG2 cell line. RESULTS: Loading efficiency and loading percentage of doxorubicin-PLGA conjugate within the nanoparticles were 96.6% and 3.45 (w/w) %, respectively while those for unconjugated doxorubicin were 6.7% and 0.26 (w/w) %, respectively. Both formulation parameters increased dramatically due to the hydrophobically modified doxorubicin by the conjugation of PLGA. The nanoparticles consisting of the conjugate exhibited sustained release over 25 days, whereas those containing unconjugated free doxorubicin showed rapid doxorubicin release in 5 days. A mixture of doxorubicin and its PLGA oligomer conjugates released from the nanoparticles had comparable IC50 value in a HepG2 cell line compared to that of free doxorubicin. Sustained drug release was attributed to the chemical degradation of conjugated PLGA backbone, which permitted water solubilization and subsequent release of doxorubicin conjugated PLGA oligomers into the medium. CONCLUSIONS: The conjugation approach of doxorubicin to PLGA was potentially useful for nanoparticle formulations that require high drug loading and sustained release. The doxorubicin-PLGA oligomer conjugate released in the medium demonstrated a slightly lower cytotoxic activity than free doxorubicin in a HepG2 cell line.

The cerebrovascular response to traditional acupuncture after stroke.

Acupuncture is useful in treating the nausea and vomiting related to chemotherapy, adult postoperative surgery pain and postoperative dental pain. We obtained single-photon emission computed tomography (SPECT) brain perfusion images of six patients with middle cerebral artery occlusion obtained before and after acupuncture and compared the changes in regional cerebral blood flow (rCBF) to those in normal control. Images were obtained before and after acupuncture at six traditional acupoints (LI 4, 10, 11, 15 and 16 and TE5) in the affected arm. The baseline image was subtracted from the postacupuncture image, to produce a subtraction image displaying only voxels with values >2 SD from the mean and those voxels were coregistered to the baseline SPECT or T2-weighted MRI. Similar images were obtained before and after acupuncture of eight normal volunteers. Statistical parametric mapping with a threshold of P =0.001 and a corrected P of 0.05 was performed for group comparison between postacupuncture and baseline SPECT. Focally increased CBF was seen in all patients especially in the hypoperfused zone surrounding the ischaemic lesion, the ipsilateral or contralateral sensorimotor area, or both. Normal subjects showed increased rCBF mainly in the parahippocampal gyrus, premotor area, frontal and temporal areas bilaterally and ipsilateral globus pallidus. Acupuncture stimulation after stroke patients appears to activate perilesional or use-dependent reorganised sites and might be a way of looking at brain reorganisation.