A nanochitosan-D-galactose formulation increases the accumulation of primaquine in the liver.

Primaquine is the mainstream antimalarial drug to prevent Plasmodium vivax relapses. However, this drug can induce hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency. Nanostructure formulations of primaquine loaded with D-galactose were used as a strategy to target the drug to the liver and decrease the hemolytic risks. Nanoemulsion (NE-Pq) and nanochitosan (NQ-Pq) formulations of primaquine diphosphate containing D-galactose were prepared and characterized by their physicochemistry properties. Pharmacokinetic and biodistribution studies were conducted using Swiss Webster mice. A single dose of 10 mg/kg of each nanoformulation or free primaquine solution was administered by gavage to the animals, which were killed at 0.5, 1, 2, 4, 8, and 24 hours. Blood samples and tissues were collected, processed, and analyzed by high-performance liquid chromatography. The nanoformulation showed sizes around 200 nm (NE-Pq) and 400 nm (NQ-Pq) and physicochemical stability for over 30 days. Free primaquine solution achieved higher primaquine Cmax in the liver than NE-Pq or NQ-Pq at 0.5 hours. However, the half-life and mean residence time (MRT) of primaquine in the liver were three times higher with the NQ-Pq formulation than with free primaquine, and the volume distribution was four times higher. Conversely, primaquine's half-life, MRT, and volume distribution in the plasma were lower for NQ-Pq than for free primaquine. NE-Pq, on the other hand, accumulated more in the lungs but not in the liver. Galactose-coated primaquine nanochitosan formulation showed increased drug targeting to the liver compared to free primaquine and may represent a promising strategy for a more efficient and safer radical cure for vivax malaria.

Effect of berberine associated with photodynamic therapy in cell lines.

Cervical cancer is a serious worldwide health problem. In view of the potentially harmful effects of current conventional therapies, photodynamic therapy may be an option as it is a minimally invasive therapy and can promote selective cytotoxic activity for neoplastic cells in the target tissue., Berberine (BBR) as an isolated molecule is a natural compound that has antineoplastic properties and potential action as a photosensitizer agent. The purpose of this study was to evaluate the use of berberine as a photosensitizer in photodynamic therapy (PDT) protocols and observe the effects produced by this association in cervical carcinoma cells and in immortalized keratinocytes. Incubation with 2.5 µM berberine promoted less than 10 % of cellular death in both cell lines studied. In addition, by fluorescence microscopy, we demonstrated that berberine was internalized by the cells, and after a period of 48 h, it was still present in the intracellular environment preferentially localized in the cytoplasm. After photodynamic therapy using berberine as a photosensitizer and visible light activation at 447 (±10) nm, we observed a phototoxic effect, which resulted in 19.84 % cell viability for Caski cells and 47.22 % cell viability for HaCaT. Treatment with berberine associated with photodynamic therapy promoted an increase in the production of reactive species of oxygen (ROS) and caspase-3 activity, indicating a preferential cell death mechanism by caspase-dependent apoptosis. Therefore, we demonstrated that berberine is an efficient photosensitizer and that its association with photodynamic therapy may be a potential anticancer treatment strategy for cervical cancer.

In vivo photodynamic inactivation of Candida albicans using chloro-aluminum phthalocyanine.

This study evaluated the photoinactivation of Candida albicans in a murine model of oral candidiasis using chloro-aluminum phthalocyanine (ClAlP) encapsulated in cationic nanoemulsions (NE) and chloro-aluminum phthalocyanine (ClAlP) diluted in DMSO (DMSO) as photosensitizer (PS). Seventy-five 6-week-old female Swiss mice were immunosuppressed and inoculated with C. albicans to induce oral candidiasis. PDT was performed on the tongue by the application of the photosensitizers and LED light (100 J cm(-2) -660 nm). Twenty-four hours and 7 days after treatments, microbiological evaluation was carried out by recovering C. albicans from the tongue of animals (CFU ml(-1) ). Then, mice were sacrificed and the tongues were surgically removed for histological and biomolecular analysis of pro- and anti-inflammatory cytokines. Data were analyzed by ANOVA followed by Tukey's post hoc test. ClAlP-NE-mediated PDT reduced 2.26 log10 of C. albicans recovered from the tongue when compared with the control group (P-L-) (P < 0.05). PDT did not promote adverse effects on the tongue tissue. Seven days after treatment, all animals were completely healthy. In summary, PDT mediated by chloro-aluminum phthalocyanine entrapped in cationic nanoemulsions was effective in reducing C. albicans recovered from the oral lesions of immunocompromised mice.

Activity and in vivo tracking of Amphotericin B loaded PLGA nanoparticles.

The development of biocompatible polymeric nanoparticles has become an important strategy for optimizing the therapeutic efficacy of many classical drugs, as it may expand their activities, reduce their toxicity, increase their bioactivity and improve biodistribution. In this study, nanoparticles of Amphotericin B entrapped within poly (lactic-co-glycolic) acid and incorporated with dimercaptosuccinic acid (NANO-D-AMB) as a target molecule were evaluated for their physic-chemical characteristics, pharmacokinetics, biocompatibility and antifungal activity. We found high plasma concentrations of Amphotericin B upon treatment with NANO-D-AMB and a high uptake of nanoparticles in the lungs, liver and spleen. NANO-D-AMB exhibited antifungal efficacy against Paracoccidioides brasiliensis and induced much lower cytotoxicity levels compared to D-AMB formulation in vivo and in vitro. Together, these results confirm that NANO-D-AMB improves Amphotericin B delivery and suggest this delivery system as a potential alternative to the use of Amphotericin B sodium deoxycholate.

Chitosan nanoparticles for melanoma cancer treatment by Photodynamic Therapy and electrochemotherapy using aminolevulinic acid derivatives.

For some time Photodynamic Therapy and electrochemotherapy have been used as alternative therapies against skin cancer. The primary aim of this work was to develop, characterize, and evaluate the in vitro cytotoxic activity of new drug delivery systems based on chitosan nanoparticles containing aminolevulinic acid derivatives such as prodrug (5-ALA and its ester derivative 8-ALA). The second goal of this study was to evaluate the synergistic effect of a combination of classical Photodynamic Therapy and electrochemotherapy, which is routinely utilized to modulate and enhance the permeation of photosensitizers, prodrugs, and other active compounds through the skin, improving the efficiency of PDT in the treatment of cutaneous neoplasms.

Photobiostimulation on wound healing treatment by ClAlPc-nanoemulsion from a multiple-wavelength portable light source on a 3D-human stem cell dermal equivalent.

This research evaluated the effect of multiple-wave lasertherapy on the healing process of surgical wounds based on in vitro models denominated stem-dermal equivalents. These human skin models were obtained from a co-culture of dermal cells and bone marrow mesenchymal stem cells. The experimental tests were carried out using a LED portable to multiple waves (operating at 660 nm and 810 nm) at different doses to induce photobiostimulation (10 to 70 mJ.cm-2). Moreover, a photosensitizer drug was employed as a new advanced designed nanomaterial, being a nanoemulsion with biopolymers to obtain an efficient drug delivery system to release lipophilic compounds. The studies were performed considering the light combination application monitoring the kinetic contraction of the dermal equivalent model and the quantification of important macromolecules (as metaloproteases derivatives), related directly with wound healing process. Results showed that an appropriate photomodulation using the combination of both wavelengths (in the red and infrared range) is possible, such that it can contribute to wound healing therapy and/or other pathological skin disease treatment.

Biocompatible magnetic microspheres for Use in PDT and hyperthermia.

Loaded microspheres with a silicon (IV) phthalocyanine derivative (NzPC) acting as a photosensitizer were prepared from polyhydroxybutyrate-co-valerate (PHBHV) and poly(ecaprolactone) (PCL) polymers using the emulsification solvent evaporation method (EE). The aim of our study was to prepare two systems of these biodegradable PHBHV/PCL microspheres. The first one containing only photosensitizer previously incorporated in the PHBHV and poly(ecaprolactone) (PCL) microspheres and the second one with the post magnetization of the DDS with magnetic nanoparticles. Magnetic fluid is successfully used for controlled incorporation of nanosized magnetic particles within the micron-sized template. This is the first time that we could get a successful pos incorporation of nanosized magnetic particles in a previously-prepared polymeric template. This procedure opens a great number of possibilities of post-functionalization of polymeric micro or nanoparticles with different bioactive materials. The NzPC release profile of the systems is ideal for PDT, the zeta potential and the size particle are stable upon aging in time. In vitro studies were evaluated using gingival fibroblastic cell line. The dark citotoxicity, the phototoxicity and the AC magnetic field assays of the as-prepared nanomagnetic composite were evaluated and the cellular viability analyzed by the classical test of MTT.

Photodynamic inactivation of biofilms formed by Candida spp., Trichosporon mucoides, and Kodamaea ohmeri by cationic nanoemulsion of zinc 2,9,16,23-tetrakis(phenylthio)-29H, 31H-phthalocyanine (ZnPc).

The biofilms formed by opportunistic yeasts serve as a persistent reservoir of infection and impair the treatment of fungal diseases. The aim of this study was to evaluate photodynamic inactivation (PDI) of biofilms formed by Candida spp. and the emerging pathogens Trichosporon mucoides and Kodamaea ohmeri by a cationic nanoemulsion of zinc 2,9,16,23-tetrakis(phenylthio)-29H,31H-phthalocyanine (ZnPc). Biofilms formed by yeasts after 48 h in the bottom of 96-well microtiter plates were treated with the photosensitizer (ZnPc) and a GaAlAs laser (26.3 J cm(-2)). The biofilm cells were scraped off the well wall, homogenized, and seeded onto Sabouraud dextrose agar plates that were then incubated at 37°C for 48 h. Efficient PDI of biofilms was verified by counting colony-forming units (CFU/ml), and the data were submitted to analysis of variance and the Tukey test (p < 0.05). All biofilms studied were susceptible to PDI with statistically significant differences. The strains of Candida genus were more resistant to PDI than emerging pathogens T. mucoides and K. ohmeri. A mean reduction of 0.45 log was achieved for Candida spp. biofilms, and a reduction of 0.85 and 0.84, were achieved for biofilms formed by T. mucoides and K. ohmeri, respectively. Therefore, PDI by treatment with nanostructured formulations cationic zinc 2,9,16,23- tetrakis (phenylthio)- 29H, 31H- phthalocyanine (ZnPc) and a laser reduced the number of cells in the biofilms formed by strains of C. albicans and non-Candida albicans as well the emerging pathogens T. mucoides and K. ohmeri.

In vitro evaluation of chloroaluminum phthalocyanine nanoemulsion and low-level laser therapy on human skin dermal equivalents and bone marrow mesenchymal stem cells.

Nanotechnology and tissue engineering are promising scientific fields in the development of advanced materials useful to human health. This article describes the preparation of a nanocarrier for the controlled release of a photosensitizer compound associated with low-level light therapy for skin wound healing treatment and applicable to other skin diseases. A biological model was used as an in vitro skin equivalent based on a three-dimensional culture of fibroblasts and mesenchymal stem cells and denominated by dermal equivalent (DE). Results show that it is possible to use the photomodulation process to control the wound healing in a scratching process and to induce the biomolecules release, both of which are related with the inflammatory wound healing process. In the studies, the MMP-2 and MMP-9 expression from zymography analyses were evaluated. All results showed a dependence on enzymatic activity relating to lowlevel laser applications which indicates a potential application in wound healing processes based on phototherapy and nanotechnology.

Rabbit rubeosis iridis induced by intravitreal latex-derived angiogenic fraction.

PURPOSE: To describe the presence of iris neovascularization in a rabbit-model of retinal neovascularization induced by the intravitreal injection of latex-derived angiogenic fraction microspheres (LAF). MATERIALS AND METHODS: Eight New Zealand rabbits received one intravitreal injection of PLGA (L-lactide-co-glycolide) microspheres with 50 ug of LAF in the right eye (Group A). Microspheres without the LAF (0.1 ml) were injected in controls (Group B; n = 8). Follow-up with clinical evaluation and iris fluorescein angiography was performed after 4 weeks when eyes were processed for light microscopy. RESULTS: All eyes from Group A showed significant vascular dilation, conjunctival hyperemia and neovascularization on the iris surface, after LAF injection. No vascular changes were observed in Group B. CONCLUSIONS: The intravitreal injection of microspheres containing the LAF can induce rubeosis iridis in rabbits and could be used as a simple experimental model for iris neovascularization.

Validated spectrophotometric and spectrofluorimetric methods for determination of chloroaluminum phthalocyanine in nanocarriers.

UV-VIS-Spectrophotometric and spectrofluorimetric methods have been developed and validated allowing the quantification of chloroaluminum phthalocyanine (CIAIPc) in nanocarriers. In order to validate the methods, the linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and selectivity were examined according to USP 30 and ICH guidelines. Linearities range were found between 0.50-3.00 microg x mL(-1) (Y = 0.3829 X [CIAIPc, microg x mL(-1)] + 0.0126; r = 0.9992) for spectrophotometry, and 0.05-1.00 microg x mL(-1) (Y = 2.24 x 10(6) X [CIAIPc, microg x mL(-1)] + 9.74 x 10(4); r = 0.9978) for spectrofluorimetry. In addition, ANOVA and Lack-of-fit tests demonstrated that the regression equations were statistically significant (p<0.05), and the resulting linear model is fully adequate for both analytical methods. The LOD values were 0.09 and 0.01 microg x mL(-1), while the LOQ were 0.27 and 0.04 microg x mL(-1) for spectrophotometric and spectrofluorimetric methods, respectively. Repeatability and intermediate precision for proposed methods showed relative standard deviation (RSD) between 0.58% to 4.80%. The percent recovery ranged from 98.9% to 102.7% for spectrophotometric analyses and from 94.2% to 101.2% for spectrofluorimetry. No interferences from common excipients were detected and both methods were considered specific. Therefore, the methods are accurate, precise, specific, and reproducible and hence can be applied for quantification of CIAIPc in nanoemulsions (NE) and nanocapsules (NC).

Rabbit retinal neovascularization induced by latex angiogenic-derived fraction: an experimental model.

PURPOSE: To create a retinal neovascularization experimental model using intravitreal injection of microspheres loaded with latex-derived angiogenic fraction. METHODS: Thirty-two albino New Zealand rabbits, divided in 4 groups of 8 animals, were enrolled in this study. Rabbits in groups I, II, and III received one intravitreal injection of PLGA (L-lactide-co-glycolide) microspheres with 10, 30, and 50 microg of latex-derived angiogenic fraction into their right eyes, respectively, and group IV received 0.1 ml of microspheres without the angiogenic fraction. Weekly follow-up with ophthalmoscopy and fluorescein angiography was performed; the rabbits were sacrificed in the 4th week and their eyes processed for light microscopy. RESULTS: All eyes from group I demonstrated increased retinal vascular tortuosity, observed from 14 days after injection and maintained for 28 days, otherwise without new vessels detection. All group II eyes showed vascular changes similar to group I. Fifty percent of the eyes from group II rabbits developed retinal neovascularization 21 days after injection. All eyes from group III demonstrated significant vascular tortuosity and retinal new vessels 2 weeks after injection, progressing to fibrovascular proliferation and tractional retinal detachment. No vascular changes or retinal new vessels were observed in group IV eyes. Light microscopy confirmed the existence of new vessels previously seen on fluorescein angiography, in retinal sections adjacent to the optic disc, not observed in sections at the same area in the control group. CONCLUSION: Thirty- and 50-microg microspheres containing latex-derived angiogenic fraction injected into the vitreous cavity induced retinal neovascularization in rabbits.

Long-term follow-up of topical 5-aminolaevulinic acid photodynamic therapy diode laser single session for non-melanoma skin cancer.

Photodynamic therapy (PDT) is based on the association of a light source and light sensitive agents in order to cause the selective death of tumor cells. To evaluate topical 5-aminolaevulinic acid (5-ALA) and diode laser photodynamic single session therapy single session for non-melanoma skin cancer (NMSC), a long-term follow-up was performed. Nineteen Bowen's disease (BD) and 15 basal cell carcinoma (BCC) lesions were submitted to 6-h topical and occlusive 20% 5-ALA plus DMSO and EDTA, and later were exposed to 630 nm diode laser, 100 or 300 J cm(-2) dose. At 3 months tumor-free rate was 91.2% (31/34) whereas at 60 months, 57.7% (15/26), slightly higher in BCC (63.6%; 7/11). The relation between the reduction of the clinical response and the increase of tumor dimension observed at 18 months was lost at 60 months. The sBCC recurrence was earlier compared to the nBCC one. ALA-PDT offered important advantages: it is minimally invasive, an option for patients under risk of surgical complications; clinical feasibility; treatment of multiple lesions in only one session or lesions in poor healing sites and superior esthetical results. However, the recurrence rate increase after ALA-PDT diode laser single session can be observed at long-term follow-up, and the repetitive sessions, an additional advantage of the method, is strongly recommended. The clinical response and recurrence time seem to be related to the laser light dose and NMSC types/sub-types, thickness and dimension, which must be considered for the choice of the ALA-PDT.

In vitro photodynamic therapy on human oral keratinocytes using chloroaluminum-phthalocyanine.

In this study, oral carcinoma cells were used to evaluate chloroaluminum-phthalocyanine encapsulated in liposomes as the photosensitizer agent in support of photodynamic therapy (PDT). The genotoxicity and cytotoxicity behavior of the encapsulated photosensitizer in both dark and under irradiation using the 670-nm laser were investigated with the classical trypan blue cell viability test, the acridine orange/ethidium bromide staining organelles test, micronucleus formation frequency, DNA fragmentation, and cell morphology. The cell morphology investigation was carried out using light and electronic microscopes. Our findings after PDT include reduction in cell viability (95%) associated with morphologic alterations. The neoplastic cell destruction was predominantly started by a necrotic process, according to the assay with acridine orange and ethidium bromide, and this was confirmed by electronic microscopy analysis. Neither the PDT agent nor laser irradiation alone showed cytotoxicity, genotoxicity, or even morphologic alterations. Our results reinforce the efficiency of light-irradiated chloroaluminum-phthalocyanine in inducing a positive effect of PDT.

Dynamic susceptibility investigation of maghemite nanoparticles incorporated in bovine serum albumin template.

Room-temperature measurements of the magnetic susceptibility of Bovine Serum Albumin-based nanocapsules (50 to 300 nm in size) loaded with different amounts of maghemite nanoparticles (7.6 nm average diameter) have been carried out in this study. The field (H) dependence of the imaginary peak susceptibility (fp) of the nanocomposite samples was investigated in the range of 0 to 4 kOe. From the analysis of the fp x H curves the concentration (N) dependence of the effective maghemite magnetocrystalline energy barrier (E) was obtained. Analysis of the E x N data was performed using a modified Mørup-Tronc [Phys. Rev. Lett. 72, 3278 (1994)] model, from which a huge contribution from the magnetocrystalline surface anisotropy was observed.

Experimental selective choriocapillaris photothrombosis using a modified indocyanine green formulation.

BACKGROUND: This in vivo study assessed and compared the effectiveness of an aqueous indocyanine green (ICG) formulation (R-ICG) and a lipid ICG formulation (L-ICG) in occluding the rabbit choriocapillaris, and determined the singlet oxygen quantum yields and aggregation properties of both formulations in vitro. METHODS: Singlet oxygen production and aggregation were compared. The eye fundus of 30 albino rabbits was irradiated 0-15 min after dye injection using an 810 nm diode laser. Fluorescein angiography and light microscopy were used to evaluate the safety and efficacy of R-ICG and L-ICG. RESULTS: L-ICG decreased the dimerisation constant and the tendency of ICG to form aggregates, and increased the efficiency of ICG in generating singlet oxygen (R-ICG, PhiDelta = 0.120 and L-ICG, PhiDelta = 0.210). Using a 10 mg/kg dose, choriocapillaris occlusion was achieved at a light dose of 35.8 J/cm(2) with L-ICG and 71.6 J/cm(2) with R-ICG with minimal damage to the neurosensory retina. CONCLUSION: Restrictions to the use of ICG in aqueous solution, low singlet oxygen quantum yields and high aggregation tendency, were overcome with L-ICG. The lower laser irradiance required to obtain choriocapillaris occlusion may suggest that L-ICG is a more potent and selective photosensitiser than R-ICG.

Evaluation of the binding properties of maghemite nanoparticle surface-coated with meso-2-3-dimercaptosuccinic acid to serum albumin.

In this study the interaction between magnetic nanoparticles (MNPs) surface-coated with meso-2,3-dimercaptosuccinic acid (DMSA) with both bovine serum albumin (BSA) and human serum albumin (HSA) was investigated. The binding of the MNP-DMSA was probed by the fluorescence quenching of the BSA and HSA tryptophan residue. Magnetic resonance and light microscopy analyses were carried out in in vivo tests using female Swiss mice. The binding constants (Kb) and the complex stoichiometries (n) indicate that MNP-DMSA/BSA and MNP-DMSA/HSA complexes have low association profiles. After five minutes following intravenous injection of MNP-DMSA into mice's blood stream we found the lung firstly target by the MNP-DMSA, followed by the liver in a latter stage. This finding suggests that the nanoparticle's DMSA-coating process probably hides the thiol group, through which albumin usually binds. This indicates that biocompatible MNP-DMSA is a very promising material system to be used as a drug delivery system (DDS), primarily for lung cancer treatment.

Analysis of mitochondrial activity related to cell death after PDT with AlPCS(4).

OBJECTIVE: The aim of this study was to report that photodynamic therapy (PDT) with mitochondria-associated chloroaluminum phthalocyanine tetrasulfonate (AlPcS(4)) leads to significant alterations in this organelle. BACKGROUND DATA: PDT is a viable treatment modality for a variety of tumors, as well as for some non-oncologic diseases. The procedure submits cells or tissue to a photosensitizing drug followed by light irradiation of appropriate wavelength, usually in the red area or close to infrared, and compatible with the drug absorption spectrum, inducing the apoptotic process. However, the precise mechanism of PDT-induced apoptosis is not well characterized. Several cellular organelles can be postulated as the target for PDT with different photosensitizers such as plasmatic membrane, nucleus, mitochondria, endoplasmic reticulum, Golgi complex, and others. The mitochondrion is the main target in PDT because it is the main organelle involved in apoptosis. One of the main agents is cytochrome c, a proapoptotic factor that preferentially links itself to the mitochondrial cardiolipin. METHODS: The photosensitizing effects of AlPcS(4) were studied in the mitochondria. Cells were irradiated with a diode laser (670 nm, energy density of 4.5 J/cm(2), and power density of 45 mW/cm(2)). RESULTS: The fluorescent analyses of the mitochondria were performed with MitoTracker and nonyl acridine orange (NAO), and electron microscopy demonstrated that PDT with AlPcS(4) leads to significant alterations in mitochondria, causing membrane potential loss, alteration in cardiolipin distribution and cell death. CONCLUSION: The labels with Mitotracker and NAO demonstrated mitochondrial migration to the perinuclear region, confirmed through electron microscopy, suggesting that intact mitochondria were solicited for possible DNA fragmentation.

Cell toxicity studies of albumin-based nanosized magnetic beads.

The aim of this study was to prepare bovine serum albumin-based beads containing maghemite nanoparticles incorporated via ionic magnetic fluid and to evaluate the cell toxicity of this biocompatible system using the J774-A1 cell line. Transmission electron micrographs obtained from the magnetic fluid sample were used to estimate the average particle diameter around 7.6 nm and diameter dispersion of 0.22. The BSA-based magnetic beads were prepared using the heat protein denaturation route. The nanoparticle concentration in the magnetic fluid sample used for the synthesis of the magnetic beads was in the range of 1.2 x 10(16) to 2.3 x 10(17) particle/ml. The methodology used to investigate the cell toxicity of the magnetic beads was the classical MTT assay. Our observation showed that the toxicity against the J774-A1 cell line depends upon the amount of magnetic material incorporated into the magnetic nanobeads and was found to be 14, 11, 9, 5, and 3% for 2.3 x 10(17), 1.2 x 10(17), 4.6 x 10(16), 2.3 x 10(16), and 1.2 x 10(16) particle/ml, respectively.