Adsorption of Salmon Calcitonin to PLGA Microspheres

Purpose. The interaction of salmon calcitonin (sCT) and poly (d,l-lactide-co-glycolide) was detected during preparation and evaluation of microspheres. The purpose of this study was to quantitate the extent and nature of the interaction. Methods. Blank microspheres were prepared by an aqueous emulsification solvent extraction technique. Adsorption studies were carried out at six concentrations of sCT and three concentrations of microspheres. Adsorption isotherms were constructed using the Langmuir and Freundlich treatments. Results. Adsorption at 1 mg/ml sCT concentration resulted in almost complete depletion of the peptide from the adsorption medium with the time to reach maximum adsorption decreasing with increasing microsphere concentration. At sCT concentrations below 100 µg/ml, a true equilibrium occurred in 1 hour or less while at higher concentrations (up to 350 µg/ml), a transient equilibrium was reached in 1 to 2 hours, followed by further adsorption of the peptide. The adsorption followed the Langmuir isotherm at concentrations below 200 µg/ml, indicating formation of a monolayer. Multilayer interaction, described by the Freundlich isotherm, occurred at higher concentrations and resulted in complete depletion of sCT from the adsorption medium. The affinity constant during monolayer formation was 0.09 and the plateau surface concentration was 5.1 µg/mg. The multilayer peptide-peptide adsorption showed a lower affinity (0.025) but higher capacity (24 µg/mg) than the monolayer peptide-polymer adsorption. Conclusions. The results show that poly (d,l-lactide-co-glycolide) microspheres have a high adsorption capacity for sCT which must be considered in formulating a controlled delivery product of this peptide.     

Successful treatment of oral verrucous hyperplasia with topical 5-aminolevulinic acid-mediated photodynamic therapy

Previous studies have shown a selective accumulation of 5-aminolevulinic acid (ALA)-derived protoporphrin IX (PpIX) in oral premalignant and malignant tissues. This provides a biologic rationale for the clinical use of ALA-mediated PDT (ALA-PDT) for oral premalignant and malignant lesions. In this study, five patients with oral verrucous hyperplasia (OVH) were treated with a new protocol of ALA-PDT composed of multiple 3-min irradiations with a light emitting diode (LED) red light at 635+/-5 nm separated with several 3-min rests for a total of 1000 s (fluence rate, 100 mW/cm(2); light exposure dose, 100 J/cm(2)) after topical application of 20% ALA for 1.5 or 2 h. Topical ALA-PDT was repeated once a week until the complete regression of the lesion. Complete regression of all OVH lesions was observed after 1-3 treatments (average, 2 treatments) of topical ALA-PDT. At an average follow-up of 5.6 months (range, 3-11 months), all the five OVH patients were free of tumor recurrence. We conclude that topical ALA-PDT with fractionated irradiations by an LED red light at 635+/-5 nm is an effective and successful treatment modality for OVH.     

In vivo autofluorescence spectroscopy of oral premalignant and malignant lesions: distortion of fluorescence intensity by submucous fibrosis

BACKGROUND AND OBJECTIVES: To test whether autofluorescence spectroscopy can be used for the diagnosis of oral neoplasia in a high-risk population, we characterized the in vivo autofluorescence spectra from oral submucous fibrosis (OSF) lesions and oral premalignant and malignant lesions in both OSF and non-OSF patients. STUDY DESIGN/MATERIALS AND METHODS: Autofluorescence emission spectra were measured under the excitation wavelength of 330 nm, using a Xenon lamp-based fluorospectrometer coupled to a handheld optical fiber probe. Autofluorescence spectroscopies were analyzed among patients with OSF lesions, and oral lesions of epithelial hyperkeratosis (EH), epithelial dysplasia (ED), and squamous cell carcinomas (SCC) and normal oral mucosa (NOM) of healthy volunteers. RESULTS: We found that the most intensely autofluorescence emission peaks occurred at 380 nm and 460 nm. For comparing the spectral patterns among different groups of oral lesions and NOM, ratios of the area under the spectrum of 460+/-10 nm to that under the spectrum of 380+/-10 nm (denoted as A(460+/-10nm)/A(380+/-10nm)) were calculated. The mean ratio values increased gradually from OSF to NOM, to EH and ED, and to SCC. The ANOVA test showed significant differences in the ratio value among all categories of samples (P<0.01). On the other hand, we found that EH, ED, and SCC lesions on OSF patients had distorted autofluorescence intensity. The mean ratio values of EH, ED, and SCC between non-OSF and OSF patients show significant differences. Furthermore, an ANOVA test showed NOM is not distinguishable from EH and ED lesions on oral fibrotic mucosa (P>0.05). CONCLUSIONS: Autofluorescence spectroscopy can be used to diagnose EH, ED, and SCC lesions in non-OSF patients but not in OSF patients.     

PLS-ANN based classification model for oral submucous fibrosis and oral carcinogenesis

BACKGROUND AND OBJECTIVES: For effective management of oral neoplasia, autofluorescence spectroscopy was conducted on patients with different characteristics of oral lesions in vivo. This study tested the possibility of using a multivariate statistical algorithm to differentiate human oral premalignant and malignant lesions from benign lesions or normal oral mucosa. STUDY DESIGN/MATERIALS AND METHODS: A fiber optics-based fluorospectrometer was used to measure the autofluorescence spectra from healthy volunteers (NOM) and patients with oral lesions of submucous fibrosis (OSF), epithelial hyperkeratosis (EH), epithelial dysplasia (ED), and squamous cell carcinoma (SCC). A partial least-squares and artificial neural network (PLS-ANN) classification algorithm was used to characterize these oral lesions to discriminate premalignant (ED) and malignant (SCC) tissues from "benign" (NOM, OSF, and EH) tissues. RESULTS: The normalized and centerized spectra of the different kinds of samples showed similar but divergent patterns. Our PLS-ANN classification algorithm could differentiate "premalignant and malignant" tissues from "benign" tissues with a sensitivity of 81%, a specificity of 96%, and a positive predictive value of 88%. CONCLUSIONS: We conclude that the PLS-ANN classification algorithm based on autofluorescence spectroscopy at 330-nm excitation is useful for in vivo diagnosis of OSF as well as oral premalignant and malignant lesions.     

Chitosan augments photodynamic inactivation of gram-positive and gram-negative bacteria

Antimicrobial photodynamic inactivation (PDI) was shown to be a promising treatment modality for microbial infections. This study explores the effect of chitosan, a polycationic biopolymer, in increasing the PDI efficacy against Gram-positive bacteria, including Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, and methicillin-resistant S. aureus (MRSA), as well as the Gram-negative bacteria Pseudomonas aeruginosa and Acinetobacter baumannii. Chitosan at <0.1% was included in the antibacterial process either by coincubation with hematoporphyrin (Hp) and subjection to light exposure to induce the PDI effect or by addition after PDI and further incubation for 30 min. Under conditions in which Hp-PDI killed the microbe on a 2- to 4-log scale, treatment with chitosan at concentrations of as low as 0.025% for a further 30 min completely eradicated the bacteria (which were originally at ～10(8) CFU/ml). Similar results were also found with toluidine blue O (TBO)-mediated PDI in planktonic and biofilm cells. However, without PDI treatment, chitosan alone did not exert significant antimicrobial activity with 30 min of incubation, suggesting that the potentiated effect of chitosan worked after the bacterial damage induced by PDI. Further studies indicated that the potentiated PDI effect of chitosan was related to the level of PDI damage and the deacetylation level of the chitosan. These results indicate that the combination of PDI and chitosan is quite promising for eradicating microbial infections.     

Photodynamic therapy outcome for oral verrucous hyperplasia depends on the clinical appearance, size, color, epithelial dysplasia, and surface keratin thickness of the lesion

Our previous studies showed that oral verrucous hyperplasia (OVH) lesions can be successfully treated with a topical 5-aminolevulinic acid-mediated photodynamic therapy (topical ALA-PDT) protocol using a 635-nm light-emitting diode light source. In this study, we report the clinical outcomes of 36 OVH lesions treated by this protocol and assess what clinicopathological parameters of OVH lesions could influence PDT treatment outcomes. We found that all the 36 OVH lesions showed complete response (CR) after an average of 3.8 (range, 1-6) treatments of topical ALA-PDT. OVH lesions with an clinical appearance of a mass, with the greatest diameter <1.5 cm, with the pink color, with epithelial dysplasia, or with the surface keratin layer < or =40 microm needed significantly less mean treatment numbers of PDT to achieve a CR than OVH lesions with an outer appearance of a plaque or a combination type of peripheral plaque and central mass (p=0.000), with the greatest diameter > or =1.5 cm (p=0.011), with the white color (p=0.000), without epithelial dysplasia (p=0.043), or with the surface keratin layer > 40 microm(p=0.003), respectively. Multivariate analysis showed that only the clinical appearance of OVH lesions was the independent factor (p=0.0069). We conclude that complete regression of OVH lesions can be achieved by less than seven treatments of topical ALA-PDT once a week. The PDT treatment outcome for OVH depends on the outer appearance, size, color, epithelial dysplasia, and surface keratin thickness of the lesion.     

Spray-Dried Microparticles Containing Polymeric Micelles Encapsulating Hematoporphyrin

The purpose of this study was to examine the properties of a new pulmonary delivery platform of microparticles containing micelles in which a therapeutic photosensitizing drug, hematoporphyrin (Hp), was encapsulated. Different poloxamers were used to form micellar Hp, and one of these, Pluronic L122-Hp, was subsequently incorporated into lactose microparticles by spray-drying. Spectral and morphological analyses were performed on both micellar Hp, and lactose microparticles containing micellar Hp (lactose-micellar Hp) before and after dissolution of the microparticles in water. Photodynamic activity of the various Hp samples were evaluated in human lung epithelial carcinoma A549 cells using a light-emitting diode (LED) device at a wavelength of 630 ± 5 nm. No significant difference was observed between micellar Hp and lactose-micellar Hp regarding the generation of singlet oxygen. The mean particle size of the microparticles was 2.3 ± 0.7 µm which is within the size range for potential lung delivery. The cellular uptake of micellar Hp and lactose-micellar Hp measured on A549 cells was at least twofold higher than those obtained with the Hp at equivalent concentrations. Micellar Hp exhibited higher cytotoxicity than Hp due to reduced formation of Hp aggregates and increased cellar uptake. The spectral properties as well as the photodynamic activity of the micellar Hp was retained when formulated into microparticles by spray-drying. Microparticles containing micelles have the potential for delivering micelle-encapsulated hydrophobic drugs in targeted therapy of pulmonary diseases.Key words: micelle, microparticle, photosensitizer, pulmonary, spray-drying     

5-Aminolevulinic acid induced photodynamic inactivation on Staphylococcus aureus and Pseudomonas aeruginosa

The aim of the present study was to develop a simple and fast screening technique to directly evaluate the bactericidal effects of 5-aminolevulinic acid (ALA)-mediated photodynamic inactivation (PDI) and to determine the optimal antibacterial conditions of ALA concentrations and the total dosage of light in vitro. The effects of PDI on Staphylococcus aureus and Pseudomonas aeruginosa in the presence of various concentrations of ALA (1.0 mM, 2.5 mM, 5.0 mM, 10.0 mM) were examined. All bacterial strains were exponentially grown in the culture medium at room temperature in the dark for 60 minutes and subsequently irradiated with 630 ± 5 nm using a light-emitting diode (LED) red light device for accumulating the light doses up to 216 J/cm². Both bacterial species were susceptible to the ALA-induced PDI. Photosensitization using 1.0 mM ALA with 162 J/cm² light dose was able to completely reduce the viable counts of S. aureus. A significant decrease in the bacterial viabilities was observed for P. aeruginosa, where 5.0 mM ALA was photosensitized by accumulating the light dose of 162 J/cm². We demonstrated that the use of microplate-based assays—by measuring the apparent optical density of bacterial colonies at 595 nm—was able to provide a simple and reliable approach for quickly choosing the parameters of ALA-mediated PDI in the cell suspensions.