Formulation Development of Doxycycline-Loaded Lipid Nanocarriers using Microfluidics by QbD Approach.

Liposomes have been used to improve therapeutic efficacy of drugs by increasing their bioavailability and altering biodistribution. The loading capacity of small molecules in liposomes remains a critical issue. Besides, the manufacturing process of liposomes requires multi-step procedures which hinders the clinical development. In this study, we developed a promising lipid-based nanocarriers (LN) delivery system for hydrophilic charged compounds using doxycycline (Doxy) as a model drug. This Doxy-loaded lipid nanocarrier (LN-Doxy) was fabricated by microfluidic technology. Design of experiments (DoE) was constructed to outline the interactions among the critical attributes of formulation, the parameters of microfluidic systems and excipient compositions. Response surface methodology (RSM) was furthered used for the optimization of LN-Doxy formulation. The LN-Doxy developed in this study showed high drug to lipid ratio and uniform distribution of particle size. Compared to Doxy solution, this LN-Doxy has reduced in vitro cellular toxicity and significant therapeutic efficacy which was verified in a peritonitis animal model. These results show the feasibility of using microfluidic technology combined with QbD approach to develop the LN formulation with high loading efficiency for ionizable hydrophilic drugs.

Fabrication of Doxorubicin-Loaded Lipid-Based Nanocarriers by Microfluidic Rapid Mixing.

Doxorubicin (Dox) is a widely known chemotherapeutic drug that has been encapsulated into liposomes for clinical use, such as Doxil® and Myocet®. Both of these are prepared via remote loading methods, which require multistep procedures. Additionally, their antitumor efficacy is hindered due to the poor drug release from PEGylated liposomes in the tumor microenvironment. In this study, we aimed to develop doxorubicin-loaded lipid-based nanocarriers (LNC-Dox) based on electrostatic interaction using microfluidic technology. The resulting LNC-Dox showed high loading capacity, with a drug-to-lipid ratio (D/L ratio) greater than 0.2, and high efficacy of drug release in an acidic environment. Different lipid compositions were selected based on critical packing parameters and further studied to outline their effects on the physicochemical characteristics of LNC-Dox. Design of experiments was implemented for formulation optimization. The optimized LNC-Dox showed preferred release in acidic environments and better therapeutic efficacy compared to PEGylated liposomal Dox in vivo. Thus, this study provides a feasible approach to efficiently encapsulate doxorubicin into lipid-based nanocarriers fabricated by microfluidic rapid mixing.

Oleic Acid-Based Self Micro-Emulsifying Delivery System for Enhancing Antifungal Activities of Clotrimazole.

Due to the increasing rate of drug resistance in Candida spp., higher doses of antifungal agents are being used resulting in toxicity. Drug delivery systems have been shown to provide an effective approach to enhance the efficacy and reduce the toxicity of antifungal agents. Oleic acid was revealed to effectively inhibit biofilm formation, hence reducing the virulence of Candida albicans. In this study, oleic acid-based self micro-emulsifying delivery systems (OA-SMEDDS) were developed for delivering clotrimazole (CLT). Based on the pseudo-ternary phase diagram and loading capacity test, the optimal ratio of OA-SMEDDS with CLT was selected. CLT-loaded OA-SMEDDS not only bears a higher drug loading capacity but also maintains good storage stability. The minimum inhibitory concentration (MIC50) of CLT-loaded OA-SMEDDS (0.01 µg/mL) in Candida albicans was significantly lower than that of CLT dissolved in DMSO (0.04 µg/mL). Moreover, we showed CLT-loaded OA-SMEDDS could effectively prevent biofilm formation and destroy the intact biofilm structure of Candida albicans. Furthermore, a CLT-loaded OA-SMEDDS gel was developed and evaluated for its antifungal properties. Disk diffusion assay indicated that both CLT-loaded OA-SMEDDS and CLT-loaded OA-SMEDDS gels were more effective than commercially available products in inhibiting the wild-type and drug-resistant species of Candida.

A Novel Treatment Modality for Malignant Peripheral Nerve Sheath Tumor Using a Dual-Effect Liposome to Combine Photodynamic Therapy and Chemotherapy.

Neurofibromatosis type 1 (NF1) is an inherited neurological disorder. Approximately 5-13% of NF1 patients may develop a malignant peripheral nerve sheath tumor (MPNST), which is a neurofibrosarcoma transformed from the plexiform neurofibroma or schwannoma. Given the large size and easy metastasis of MPNST, it remains difficult to be cured by either surgical or conventional chemotherapy. In this study, we investigated the possibility of combining photodynamic therapy (PDT) and chemotherapy to treat MPNST by using a dual-effect liposome (named as PL-cDDP-Ce6), in which a chemotherapeutic agent, cisplatin (cDDP), and photosensitizer, chlorine e6 (Ce6) were encapsulated in the same carrier. The cytotoxic effect of PL-cDDP-Ce6 against MPNST cells was significantly higher than cells treated with liposomal cDDP or Ce6 alone or in combination after light irradiation. Treatment with the dual-effect liposomes in mice bearing xenograft MPNST tumor reveals a significant increase in survival rate compared to those treated with liposomal cDDP and Ce6 in combination. Moreover, there is no weight loss or derangements of serum biochemistry. In conclusion, this study demonstrates the clinical potential and advantage of using this liposomal drug for the treatment of MPNST.

Co-Encapsulation of Chlorin e6 and Chemotherapeutic Drugs in a PEGylated Liposome Enhance the Efficacy of Tumor Treatment: Pharmacokinetics and Therapeutic Efficacy.

Long-circulating PEG-modified liposome has been shown to improve pharmacokinetic properties and reduce systemic toxicity in cancer treatment. However, drug bioavailability from liposome remains a major challenge to the improvement of its therapeutic efficacy. Previously, we designed a PEGylated dual-effect liposome (named as PL-Dox-Ce6) with chlorin e6 incorporated in the lipid bilayer and Doxorubicin encapsulated in the interior. In this study, another dual-effect liposome with cisplatin encapsulated in the interior was further developed. The pharmacokinetics of these two dual-effect liposomes were studied in tumor-bearing mice. Based on the kinetic data of tumor and plasma, light irradiation was applied onto the tumors at different time points after drug administration to compare the therapeutic efficacy. We demonstrated that a single dose of the dual-effect liposomes combined with two doses of light irradiation can completely eradicate over 90% of the tumor in mice alone with significant survival rate and no toxicity. Thus, this study established a platform that utilizes the dual-effect liposome which combines photodynamic therapy and chemotherapy to improve the therapeutic outcomes of tumors.

Doxycycline potentiates antitumor effect of 5-aminolevulinic acid-mediated photodynamic therapy in malignant peripheral nerve sheath tumor cells.

Neurofibromatosis type 1 (NF1) is one of the most common neurocutaneous disorders. Some NF1 patients develop benign large plexiform neurofibroma(s) at birth, which can then transform into a malignant peripheral nerve sheath tumor (MPNST). There is no curative treatment for this rapidly progressive and easily metastatic neurofibrosarcoma. Photodynamic therapy (PDT) has been developed as an anti-cancer treatment, and 5-aminolevulinic (ALA) mediated PDT (ALA-PDT) has been used to treat cutaneous skin and oral neoplasms. Doxycycline, a tetracycline derivative, can substantially reduce the tumor burden in human and animal models, in addition to its antimicrobial effects. The purpose of this study was to evaluate the effect and to investigate the mechanism of action of combined doxycycline and ALA-PDT treatment of MPNST cells. An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the combination of ALA-PDT and doxycycline significantly reduce MPNST survival rate, compared to cells treated with each therapy alone. Isobologram analysis showed that the combined treatment had a synergistic effect. The increased cytotoxic activity could be seen by an increase in cellular protoporphyrin IX (PpIX) accumulation. Furthermore, we found that the higher retention of PpIX was mainly due to increasing ALA uptake, rather than activity changes of the enzymes porphobilinogen deaminase and ferrochelatase. The combined treatment inhibited tumor growth in different tumor cell lines, but not in normal human Schwann cells or fibroblasts. Similarly, a synergistic interaction was also found in cells treated with ALA-PDT combined with minocycline, but not tetracycline. In summary, doxycycline can potentiate the effect of ALA-PDT to kill tumor cells. This increased potency allows for a dose reduction of doxycycline and photodynamic radiation, reducing the occurrence of toxic side effects in vivo.

Assessment of Photodynamic Inactivation against Periodontal Bacteria Mediated by a Chitosan Hydrogel in a 3D Gingival Model.

Chitosan hydrogels containing hydroxypropyl methylcellulose (HPMC) and toluidine blue O were prepared and assessed for their mucoadhesive property and antimicrobial efficacy of photodynamic inactivation (PDI). Increased HPMC content in the hydrogels resulted in increased mucoadhesiveness. Furthermore, we developed a simple In Vitro 3D gingival model resembling the oral periodontal pocket to culture the biofilms of Staphylococcus aureus (S. aureus), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), and Porphyromonas gingivalis (P. gingivalis). The PDI efficacy of chitosan hydrogel was examined against periodontal biofilms cultured in this 3D gingival model. We found that the PDI effectiveness was limited due to leaving some of the innermost bacteria alive at the non-illuminated site. Using this 3D gingival model, we further optimized PDI procedures with various adjustments of light energy and irradiation sites. The PDI efficacy of the chitosan hydrogel against periodontal biofilms can significantly improve via four sides of irradiation. In conclusion, this study not only showed the clinical applicability of this chitosan hydrogel but also the importance of the light irradiation pattern in performing PDI for periodontal disease.

Protective effect of magnolol-loaded polyketal microparticles on lipopolysaccharide-induced acute lung injury in rats.

Magnolol has shown inhibitory effects on NO production and TNF-alpha production in lipopolysaccharide (LPS)-activated macrophages and LPS-induced acute lung injury; however, the poor solubility of magnolol has hindered its clinical success. In this study, magnolol-loaded microparticles were prepared via single emulsion method from a polyketal polymer, termed PK3. The particle sizes of magnolol-loaded PK3 microparticle is 3.73 ± 0.41 µm, and was suitable for phagocytosis by macrophages and pulmonary drug delivery. PK3 microparticles exhibited excellent biocompatibility both in vitro and in vivo. More importantly, intratracheal delivery of these magnolol-loaded microparticles significantly reduced the lung inflammatory responses at low dosage of magnolol (0.5 mg/kg), and have great clinical potential in treating acute lung injury.

Optimization and Evaluation of a Chitosan/Hydroxypropyl Methylcellulose Hydrogel Containing Toluidine Blue O for Antimicrobial Photodynamic Inactivation.

Photodynamic inactivation (PDI) combined with chitosan has been shown as a promising antimicrobial approach. The purpose of this study was to develop a chitosan hydrogel containing hydroxypropyl methylcellulose (HPMC), chitosan and toluidine blue O (TBO) to improve the bactericidal efficacy for topical application in clinics. The PDI efficacy of hydrogel was examined in vitro against the biofilms of Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). Confocal scanning laser microscopy (CSLM) was performed to investigate the penetration level of TBO into viable S. aureus biofilms. Incorporation of HMPC could increase the physicochemical properties of chitosan hydrogel including the hardness, viscosity as well as bioadhesion; however, higher HMPC concentration also resulted in reduced antimicrobial effect. CSLM analysis further demonstrated that higher HPMC concentration constrained TBO diffusion into the biofilm. The incubation of biofilm and hydrogel was further performed at an angle of 90 degrees. After light irradiation, compared to the mixture of TBO and chitosan, the hydrogel treated sample showed increased PDI efficacy indicated that incorporation of HPMC did improve antimicrobial effect. Finally, the bactericidal efficacy could be significantly augmented by prolonged retention of hydrogel in the biofilm as well as in the animal model of rat skin burn wounds after light irradiation.

Histone acetyltransferase p300 is induced by p38MAPK after photodynamic therapy: the therapeutic response is increased by the p300HAT inhibitor anacardic acid.

Oxidative stress mediated by photodynamic therapy (PDT) mediates the tumoricidal effect, but has also been shown to induce the expression of prosurvival molecules, such as cyclooxygenase-2 (COX-2), which is involved in tumor recurrences after PDT. However, the molecular mechanism is still not fully understood. In this study, we found that activated p38MAPK could significantly up-regulate the activity and expression of histone acetyltransferase p300 (p300HAT) in A375 and C26 cells treated with ALA-and chlorin e6 (Ce6)-mediated photodynamic treatment. A colony-formation assay showed that PDT-induced cytotoxicity was dramatically elevated in the presence of the p300HAT inhibitor anacardic acid (AA). Further studies showed that increased p300HAT acetylates histone H3 and NF-κB p65 subunit to up-regulate the COX-2 expression, which was reduced by AA or p300HAT shRNA. Using chromatin immunoprecipitation analysis, we found that the augmented acetylation of histone H3 and NF-κB increases their binding to the COX-2 promoter region. These in vitro findings were further verified in mice bearing murine C26 and human A375 tumors treated with liposomal Ce6 mediated PDT. Meanwhile, the combination of PDT and AA resulted in greater tumor regression in BALB/c mice bearing C26 tumors, compared with PDT only or combined with COX-2 inhibitor. Finally, we demonstrated that suppression of the PDT-induced p300HAT activity also resulted in the decreased expression of survivin, restoring caspase-3 activity and sensitizing PDT-treated cells from autophagy to apoptosis due to the Becline-1 cleavage. This study demonstrates for the first time the molecular mechanisms involved in histone modification induced by PDT-mediated oxidative stress, suggesting that HAT inhibitors may provide a novel therapeutic approach for improving PDT response.

Dual-effect liposomes encapsulated with doxorubicin and chlorin e6 augment the therapeutic effect of tumor treatment.

BACKGROUND AND OBJECTIVE: Long circulating doxorubicin (Dox)-loaded PEGylated liposomes are clinically safer than the free form due to the significant reduction of cardiac toxicity. However, the therapeutic efficacy of the PEGylated liposome could further be improved if poor diffusivity and slow drug release of the liposome in tumor interstitium can be overcome. In this study, a dual-effect liposome triggered by photodynamic effect was developed to improve the therapeutic efficacy of Dox-loaded PEGylated liposomes. MATERIALS AND METHODS: Dox and chlorin e6 (Ce6) were co-encapsulated in PEGylated liposomes (named as PL-Dox-Ce6). To induce the drug release, photodynamic effect was triggered by the light irradiation of a 662 nm diode laser. The cellular distribution of Dox and Ce6 was examined under confocal microscope. The in vitro and in vivo cytotoxicity of PL-Dox-Ce6 was determined via the colony formation assay and the synergistic C26 tumor model, respectively. RESULTS: The cellular distribution of PL-Dox-Ce6 was in the cytoplasmic area; while under light irradiation, Dox was co-localized with nuclear staining positive signals. The cellular cytotoxicity of PL-Dox-Ce6 was significantly higher than the controls including liposomes encapsulating either Dox (PL-Dox) or Ce6 (PL-Ce6). The in vivo treatment efficacy of PL-Dox-Ce6 determined in the C26 tumor model reveals a significant therapeutic effect compared to that of PL-Ce6 and PL-Dox alone or in combination. CONCLUSION: This study indicates that this dual-effect PEGylated liposome could provide clinical advantages in the combination regimen of photodynamic therapy and chemotherapy.

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.

The use of Chitosan to enhance photodynamic inactivation against Candida albicans and its drug-resistant clinical isolates.

Drug-resistant Candida infection is a major health concern among immunocompromised patients. Antimicrobial photodynamic inactivation (PDI) was introduced as an alternative treatment for local infections. Although Candida (C.) has demonstrated susceptibility to PDI, high doses of photosensitizer (PS) and light energy are required, which may be harmful to eukaryotic human cells. This study explores the capacity of chitosan, a polycationic biopolymer, to increase the efficacy of PDI against C. albicans, as well as fluconazole-resistant clinical isolates in planktonic or biofilm states. Chitosan was shown to effectively augment the effect of PDI mediated by toluidine blue O (TBO) against C. albicans that were incubated with chitosan for 30 min following PDI. Chitosan at concentrations as low as 0.25% eradicated C. albicans; however, without PDI treatment, chitosan alone did not demonstrate significant antimicrobial activity within the 30 min of incubation. These results suggest that chitosan only augmented the fungicidal effect after the cells had been damaged by PDI. Increasing the dosage of chitosan or prolonging the incubation time allowed a reduction in the PDI condition required to completely eradicate C. albicans. These results clearly indicate that combining chitosan with PDI is a promising antimicrobial approach to treat infectious diseases.

Liposome-encapsulated photosensitizers against bacteria.

Photodynamic therapy (PDT), utilizing photosensitizers and light, has received considerable interests for its potential to treat microbial infections. The advantages of antimicrobial PDT include a broad spectrum of action, efficient killing against wild-type as well as drug-resistant pathogens. Therefore, antimicrobial PDT could be valuable to rapidly reduce the microbial burden during the management of local infections, especially for the antibiotic resistance. A variety of photosensitizers have been examined its efficacy against pathogens. To increase the efficacy of photosensitizers, various drug delivery systems have been developed. Among these carrier systems, liposomes showed their PDT efficacy and safety in delivering photosensitizers. This review is focused on the application of liposomes mediated photodynamic inactivation of bacteria along with the discussion of few of recent patents.

Preparation and characterization of cosmeceutical liposomes loaded with avobenzone and arbutin.

The objective of this study was to develop and characterize a liposome delivery system coencapsulating two cosmeceutical ingredients, avobenzone (AVO) and arbutin (AR). Two different liposome preparation methods, that is, thin film hydration and reverse-phase evaporation, were evaluated. To obtain the optimal formulation, various ratios of lipid to AVO or AR were tested. The effects of liposome formulation and preparation method on particle size, entrapment efficiency (EE), and skin permeation rate were studied. The mean particle size of the liposome formulations obtained by the thin film hydration method was smaller than that obtained by the reverse-phase evaporation method. The EE of AR and AVO in liposomes prepared by the thin film method, however, was lower than that prepared by the reverse-phase evaporation method. No differences in membrane permeation were observed between the two preparation methods. A large portion of AR permeated through the membrane into the receptor chamber. On the other hand, AVO remained in the donor chamber or accumulated in the membrane. The results of this study revealed that liposomes are a promising delivery system for coencapsulated AR and AVO. Liposomes may aid in retaining the sunscreen (AVO) at the surface of the skin for sun protection meanwhile facilitating the penetration of the whitening agent (AR) into the deeper layers of the skin for whitening effect.

Photodynamic inactivation of chlorin e6-loaded CTAB-liposomes against Candida albicans.

BACKGROUND AND OBJECTIVES: Antimicrobial photodynamic inactivation (PDI) is a promising therapeutic modality for the treatment of local infections. To increase the efficacy of PDI, chlorine e6 (Ce6) was encapsulated in cationic CTAB-liposomes composed of various ratios of dimyristoyl-sn-glycero-phosphatidylcholine (DMPC) and the cationic surfactant, cetyltrimethyl ammonium bromide (CTAB). The PDI efficacy of the liposomal-Ce6 was assessed in vitro against susceptible and drug-resistant clinical isolates of Candida albicans (C. albicans) as well as in infected burn wounds. STUDY DESIGN/MATERIALS AND METHODS: Ce6 was encapsulated in CTAB-liposomes by the film hydration method. Particle size distribution and zeta potential of the cationic liposomes were measured using a Zetasizer Nano-ZS. UV-visible spectra were used to measure lipid/Ce6 (L/C) ratio and drug entrapment efficiency while differential scanning calorimetry (DSC) was used to study the thermotropic behavior of DMPC liposomes upon CTAB addition. In vivo PDI efficacy was carried out in an infected burn wound using a rat model. RESULTS: The increase in zeta potential and a shift in the phase transition temperature (Tm ) upon CTAB addition confirmed its entrapment within the lipid bilayers of the liposome. Meanwhile, the CTAB addition did not affect the Ce6 entrapment efficiency and physical attributes of the liposomes. In vitro studies showed that the PDI effect of the Ce6-loaded CTAB-liposomes was dependent on the lipid to Ce6 molar ratio (L/C), particle size and the concentration of CTAB in the liposomes. The lower L/C ratio and smaller liposomes exerted significantly higher PDI effects. In addition, an increase in the CTAB to lipid ratio led to a significant increase in the PDI effect of Ce6 against susceptible and drug-resistant clinical isolates of C. albicans after light illumination. CONCLUSIONS: Our results indicate that a low L/C ratio, high positive charge, and small particle size of CTAB-liposomes significantly enhances their PDI efficacy against C. albicans.

Preparation of alginate beads containing a prodrug of diethylenetriaminepentaacetic acid.

A penta-ethyl ester prodrug of the radionuclide decorporation agent diethylenetriaminepentaacetic acid (DTPA), which exists as an oily liquid, was encapsulated in alginate beads by the ionotropic gelation method. An optimal formulation was found by varying initial concentrations of DTPA penta-ethyl ester, alginate polymer, Tween 80 surfactant and calcium chloride. All prepared alginate beads were ~1.6mm in diameter, and the optimal formulation had loading and encapsulation efficiencies of 91.0±1.1 and 72.6±2.2%, respectively, and only 3.2±0.8% water absorption after storage at room temperature in ~80% relative humidity. Moreover, Fourier transform infrared spectroscopy showed that DTPA penta-ethyl ester did not react with excipients during formation of the DTPA penta-ethyl ester-containing alginate beads. Release of prodrug from alginate beads was via anomalous transport, and its stability enhanced by encapsulation. Collectively, these data suggest that this solid dosage form may be suitable for oral administration after radionuclide contamination.

Chloride intracellular channel 4 involves in the reduced invasiveness of cancer cells treated by photodynamic therapy.

BACKGROUND AND OBJECTIVES: The mechanisms of photodynamic therapy (PDT) have been studied on the cellular and tissue levels. However, the cellular behaviors of cancer cells survived from PDT are still not clear. Previously, we have found that PDT-derived variants A375/3A5 and A375/6A5 have reduced invasion ability. This study attempted to further elucidate the possible molecules associated with the altered invasiveness in the PDT-derived variants and cancer cells treated with PDT. STUDY DESIGN/MATERIALS AND METHODS: Scratch wound healing assay and invasion assay were performed to evaluate the migration and invasion ability of human A375 melanoma and MDA-MB-231 breast adenocarcinoma cells. Single colony selection and microarray analysis were performed to examine the differentially expressed transcripts in parental A375 and PDT-derived variants. RT-PCR and Western blots analysis were performed to examine the expression levels of matrix metalloproteinase 9 (MMP9) and chloride intracellular channel 4 (CLIC4). The MMP9 activity was examined by Zymography assay. CLIC4 expressing construct was used to examine the influence on MMP9 expression and invasion ability of cancer cells treated with PDT. RESULTS: Correlated with the reduced invasiveness, we found that A375/3A5 and A375/6A5 cells have decreased production of MMP9. Microarray analysis and RT-PCR showed CLIC4 was down-regulated in the PDT-derived variants. Furthermore, down-regulation of CLIC4 and MMP9 was found in cancer cells treated with PDT. Transfection of surviving cancer cells with a plasmid vector encoding CLIC4 increased MMP9 expression and cell invasion. Furthermore, overexpression of CLIC4 in A375 and MDA-MB-231 cancer cells constrains PDT-induced suppression of invasiveness. CONCLUSION: Our results showed that the reduced expression of CLIC4 could further down-regulate MMP9 and result in the suppression of invasion in cancer cells treated with PDT. These results provide an insight into a new mechanism by which PDT affects the metastatic potential of cancer cells through down-regulation of MMP9 by CLIC4.

Quantification of 5-aminolevulinic acid by CE using dynamic pH junction technique.

An online dynamic pH junction preconcentration method was developed for quantification of 5-aminolevulinic acid (ALA) by CE with the separation time less than 6 min. The optimal dynamic pH junction of ALA was carried out between pH 9.3 borate buffer (BGE, 40 mM) and pH 2.5 phosphate buffer (sample matrix, 40 mM) when 4.1 cm of sample plug was hydrodynamically injected into an uncoated fused-silica capillary (48.5 cm in length, id of 50 µm). If a 24 kV separation voltage was applied, the calibration curve of ALA peak area (200 nm) showed good linearity (R(2) = 0.9991) ranging from 0.01 to 6.5 mg/mL. The reproducibility of this system was excellent with RSDs (n = 10) of 2.5% for peak area response and 0.6% for migration time at ALA concentration of 0.5 mg/mL. The LOD was evaluated as 1.0 µg/mL (S/N > 3). Compared to conventional CE procedure, the sensitivity was successfully improved over 50-fold. The analytical results of pharmaceutical formulations show a good agreement with those by HPLC (r = 0.94).

Chitosan nanoparticles for antimicrobial photodynamic inactivation: characterization and in vitro investigation.

The growing resistance to antibiotics has rendered antimicrobial photodynamic inactivation (PDI) an attractive alternative treatment modality for infectious diseases. Chitosan (CS) was shown to further potentiate the PDI effect of photosensitizers and was therefore used in this study to investigate its ability to potentiate the activity of erythrosine (ER) against bacteria and yeast. CS nanoparticles loaded with ER were prepared by ionic gelation method and tested for their PDI efficacy on planktonic cells and biofilms of Streptococcus mutans, Pseudomonas aeruginosa and Candida albicans. The nanoparticles were characterized for their size, polydispersity index and zeta potential. No toxicity was observed when planktonic cells and biofilms were treated with the nanoparticles in the dark. However, when the cells were exposed to light irradiation after treatment with free ER or ER/CS nanoparticles, a significant phototoxicity was observed. The antimicrobial activity of ER/CS nanoparticles was significantly higher than ER in free form. The particle size and incubation time of the nanoparticles also appeared to be important factors affecting their PDI activity against S. mutans and C. albicans.