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1.  In vivo and in vitro evaluation of octyl methoxycinnamate liposomes 
Solar radiation causes damage to human skin, and photoprotection is the main way to prevent these harmful effects. The development of sunscreen formulations containing nanosystems is of great interest in the pharmaceutical and cosmetic industries because of the many potential benefits. This study aimed to develop and evaluate an octyl methoxycinnamate (OMC) liposomal nanosystem (liposome/OMC) to obtain a sunscreen formulation with improved safety and efficacy by retaining OMC for longer on the stratum corneum.
Methods
The liposome/OMC nanostructure obtained was tested for enzymatic hydrolysis with lipase from Rhizomucor miehei and biodistribution with liposomes labeled with technetium-99m. The liposome/OMC formulation was then incorporated in a gel formulation and tested for ocular irritation using the hen’s egg test-chorio-allantoic membrane (HET-CAM) assay, in vitro and in vivo sun protection factor, in vitro release profile, skin biometrics, and in vivo tape stripping.
Results
The liposome/OMC nanosystem was not hydrolyzed from R. miehei by lipase. In the biodistribution assay, the liposome/OMC formulation labeled with technetium-99m had mainly deposited in the skin, while for OMC the main organ was the liver, showing that the liposome had higher affinity for the skin than OMC. The liposome/OMC formulation was classified as nonirritating in the HET-CAM test, indicating good histocompatibility. The formulation containing liposome/OMC had a higher in vivo solar photoprotection factor, but did not show increased water resistance. Inclusion in liposomes was able to slow down the release of OMC from the formulation, with a lower steady-state flux (3.9 ± 0.33 μg/cm2/hour) compared with the conventional formulation (6.3 ± 1.21 μg/cm2/hour). The stripping method showed increased uptake of OMC in the stratum corneum, giving an amount of 22.64 ± 7.55 μg/cm2 of OMC, which was higher than the amount found for the conventional formulation (14.57 ± 2.30 μg/cm2).
Conclusion
These results indicate that liposomes are superior carriers for OMC, and confer greater safety and efficacy to sunscreen formulations.
doi:10.2147/IJN.S51383
PMCID: PMC3864883  PMID: 24376350
sunscreen; liposome; tape stripping; technetium-99-m; lipase
2.  Feather keratin hydrolysates obtained from microbial keratinases: effect on hair fiber 
BMC Biotechnology  2013;13:15.
Background
Hair is composed mainly of keratin protein and a small amount of lipid. Protein hydrolysates, in particular those with low molecular weight distribution have been known to protect hair against chemical and environmental damage. Many types of protein hydrolysates from plants and animals have been used in hair and personal care such as keratin hydrolysates obtained from nails, horns and wool. Most of these hydrolysates are obtained by chemical hydrolysis and hydrothermal methods, but recently hydrolyzed hair keratin, feather keratin peptides, and feather meal peptides have been obtained by enzymatic hydrolysis using Bacillus spp in submerged fermentation.
Results
Keratin peptides were obtained by enzymatic hydrolysis of keratinases using Bacillus subtilis AMR. The microorganism was grown on a feather medium, pH 8.0 (1% feathers) and supplemented with 0.01% of yeast extract, for 5 days, at 28°C with agitation. The supernatant containing the hydrolysates was colleted by centrifugation and ultra filtered in an AMICON system using nano–membranes (Millipore – YC05). The Proteins and peptides were analyzed using HPTLC and MALDI-TOF-MS. Commercial preparations of keratin hydrolysates were used as a comparative standard. After five days the feather had been degraded (90-95%) by the peptidases and keratinases of the microorganism. MALDI-TOF mass spectrometry showed multiple peaks that correspond to peptides in the range of 800 to 1079 Daltons and the commercial hydrolysate was in the range of 900 to 1400 Da. HPTLC showed lower molecular mass peptides and amino acids in the enzymatic hydrolysate when compared with the commercial hydrolysate . A mild shampoo and a rinse off conditioner were formulated with the enzymatic hydrolysate and applied to hair fibers to evaluate the hydration, with and without heat, using a Corneometer® CM 825. The hydration was more efficient with heat, suggesting a more complete incorporation of hydrolysates into the fibers. Scanning Electron Microscopy showed deposits of organic matter in the junction of the cuticles that probably collaborates to the sealing of the cuticles, increasing the brightness and softness.
Conclusions
These results show that the enzymatic method to produce keratin peptides for hair care products is an attractive and eco- friendly method with a great potential in the cosmetic industry.
doi:10.1186/1472-6750-13-15
PMCID: PMC3621039  PMID: 23414102
Keratin; Hydrolysate enzymatic; Peptides
3.  Evaluation of octyl p-methoxycinnamate included in liposomes and cyclodextrins in anti-solar preparations: preparations, characterizations and in vitro penetration studies 
Purpose
Awareness of the harmful effects of ultraviolet radiation has led to the increasing use of sunscreens, thus, the development of safe and effective antisolar preparations is important. The inclusion of sunscreen molecules in different release systems, like liposomes (lipo) and cyclodextrins (CD) is therefore required.
Methods
The in vivo sun protection factor (SPF), water resistance, and in vitro transdermal penetration test of octyl p-methoxycinnamate (OMC) in different dispersions, such as OMC encapsulated in liposomes (lipo/OMC), OMC encapsulated in β-cyclodextrins (β-CD/OMC), OMC encapsulated in both release systems (lipo/OMC and β-CD/OMC), and an OMC-free formulation were determined.
Results
Although the formulation containing only the lipo/OMC system revealed high value of in vivo SPF (11.0 ± 1.3) and water resistance (SPF = 10.3 ± 2.2), the formulation containing both release systems (lipo/OMC + β-cyclodextrin/OMC) showed the best result in the in vivo SPF test (11.6 ± 1.6). In the penetration test, the formulation containing the lipo/OMC system had better performance, since a high amount of OMC in the epidermis (18.04 ± 1.17 μg) and a low amount of OMC in the dermis (9.4 ± 2.36 μg) were observed. These results suggest that liposomes interact with the cells of the stratum corneum, promoting retention of OMC in this layer.
Conclusion
According to our study, the lipo/OMC system is the most advantageous release system, due to its ability to both increase the amount of OMC in the epidermis and decrease the risk of percutaneous absorption.
doi:10.2147/IJN.S28550
PMCID: PMC3391002  PMID: 22787399
octyl p-methoxycinnamate; sunscreen; liposomes; β-cyclodextrin; penetration
4.  Nanostructured delivery system for zinc phthalocyanine: preparation, characterization, and phototoxicity study against human lung adenocarcinoma A549 cells 
In this study, zinc phthalocyanine (ZnPc) was loaded onto poly-ɛ-caprolactone (PCL) nanoparticles (NPs) using a solvent emulsification–evaporation method. The process yield and encapsulation efficiency were 74.2% ± 1.2% and 67.1% ± 0.9%, respectively. The NPs had a mean diameter of 187.4 ± 2.1 nm, narrow distribution size with a polydispersity index of 0.096 ± 0.004, zeta potential of −4.85 ± 0.21 mV, and spherical shape. ZnPc has sustained release, following Higuchi’s kinetics. The photobiological activity of the ZnPc-loaded NPs was evaluated on human lung adenocarcinoma A549 cells. Cells were incubated with free ZnPc or ZnPc-loaded NPs for 4 h and then washed with phosphate-buffered saline. Culture medium was added to the wells containing the cells. Finally, the cells were exposed to red light (660 nm) with a light dose of 100 J/cm2. The cellular viability was determined after 24 h of incubation. ZnPc-loaded NPs and free photosensitizer eliminated about 95.9% ± 1.8% and 28.7% ± 2.2% of A549 cells, respectively. The phototoxicity was time dependent up to 4 h and concentration dependent at 0–5 μg ZnPc. The cells viability decreased with the increase of the light dose in the range of 10–100 J/cm2. Intense lysis was observed in the cells incubated with the ZnPcloaded NPs and irradiated with red light. ZnPc-loaded PCL NPs are the release systems that promise photodynamic therapy use.
doi:10.2147/IJN.S15860
PMCID: PMC3075896  PMID: 21499420
photosensitizer; nanoparticles; photodynamic therapy; lung cancer; phototoxicity; biodistribution
5.  Early Changes With Diabetes in Renal Medullary Hemodynamics as Evaluated by Fiberoptic Probes and BOLD Magnetic Resonance Imaging 
Investigative radiology  2007;42(3):157-162.
Objective
We sought to evaluate the influence of streptozotocin (STZ)-induced diabetes on renal outer medullary pO2 and blood flow by invasive microprobes and to demonstrate feasibility that blood oxygenation level-dependent (BOLD) magnetic resonance imaging (MRI) can monitor these changes.
Materials and Methods
A total of 60 Wistar-Furth rats were used. Diabetes was induced by STZ in 48. Animals were divided into OxyLite group (n = 30) and BOLD MRI groups (n = 30) each with a 5 subgroups of 6 animals: control and 2, 5, 14, and 28 days after induction of diabetes. Outer renal medullary oxygen tension and blood flow were measured by the combined OxyLite/OxyFlo probes.
Results
Both OxyLite and BOLD MRI showed a significant increase in the renal hypoxia levels after STZ at all time points. However, no changes were observed in the outer renal medullary oxygen tension and blood flow between diabetic and control groups.
Conclusions
These preliminary results suggest that hypoxic changes can be detected as early as 2 days in rat kidneys with diabetes by BOLD MRI and that these early changes are not dependent on blood flow.
doi:10.1097/01.rli.0000252492.96709.36
PMCID: PMC2904752  PMID: 17287645
kidney; diabetes; oxygenation; BOLD MRI; OxyLite
6.  Evaluation of Genetic Association and Expression Reduction of TRPC1 in the Development of Diabetic Nephropathy 
American journal of nephrology  2008;29(3):244-251.
Background/Aims
The TRPC1 gene on chromosome 3q22–24 resides within the linkage region for diabetic nephropa-thy (DN) in type 1 (T1D) and type 2 diabetes mellitus (T2D). A recent study has demonstrated that TRPC1 expression is reduced in the kidney of diabetic ZDF- and STZ-treated rats. The present study aimed to evaluate the genetic and functional role of TRPC1 in the development of DN.
Methods
Genetic association study was performed with two independent cohorts, including 1,177 T1D European Americans with or without DN from GoKinD population and 850 African-American subjects with T2D-associated end-stage renal disease (ESRD), or with hypertensive (non-diabetic) ESRD, and nondiabetic controls. Seven tag SNP markers derived from HapMap data (phase II) were genotyped. TRPC1 gene expression was examined using real time RT-PCR.
Results
No significant association of TRPC1 DNA polymorphisms with DN or ERSD was found in GoKinD and African-American populations. TRPC1 gene mRNA expression in kidney was found to be trendily reduced in 12-week and significantly in 26-week-old db/db mice.
Conclusions
TRPC1 genetic polymorphism may not fundamentally contribute to the development of DN, while reduction of the gene expression in kidney may be a late phenomenon of DN as seen in diabetic animal models.
doi:10.1159/000157627
PMCID: PMC2698220  PMID: 18802326
TRPC1 gene; Single-nucleotide polymorphism; Diabetic nephropathy; End-stage renal disease; Diabetes types 1 and 2
7.  Evaluation of Genetic Association and Expression Reduction of TRPC1 in the Development of Diabetic Nephropathy 
American Journal of Nephrology  2008;29(3):244-251.
Background/Aims
The TRPC1 gene on chromosome 3q22–24 resides within the linkage region for diabetic nephropathy (DN) in type 1 (T1D) and type 2 diabetes mellitus (T2D). A recent study has demonstrated that TRPC1 expression is reduced in the kidney of diabetic ZDF- and STZ-treated rats. The present study aimed to evaluate the genetic and functional role of TRPC1 in the development of DN.
Methods
Genetic association study was performed with two independent cohorts, including 1,177 T1D European Americans with or without DN from GoKinD population and 850 African-American subjects with T2D-associated end-stage renal disease (ESRD), or with hypertensive (non-diabetic) ESRD, and nondiabetic controls. Seven tag SNP markers derived from HapMap data (phase II) were genotyped. TRPC1 gene expression was examined using real time RT-PCR.
Results
No significant association of TRPC1 DNA polymorphisms with DN or ERSD was found in GoKinD and African-American populations. TRPC1 gene mRNA expression in kidney was found to be trendily reduced in 12-week and significantly in 26-week-old db/db mice.
Conclusions
TRPC1 genetic polymorphism may not fundamentally contribute to the development of DN, while reduction of the gene expression in kidney may be a late phenomenon of DN as seen in diabetic animal models.
doi:10.1159/000157627
PMCID: PMC2698220  PMID: 18802326
TRPC1 gene; Single-nucleotide polymorphism; Diabetic nephropathy; End-stage renal disease; Diabetes types 1 and 2

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