Solveig Kristensen

Formulation and bacterial phototoxicity of curcumin loaded alginate foams for wound treatment applications: Studies on curcumin and curcuminoides XLII

Curcumin loaded alginate foams are proposed for application in antimicrobial photodynamic therapy of infected wounds. The drug loaded foams were formulated to provide a burst release of the photosensitizer when hydrated. The foams remained intact after hydration and would be possible to remove from the wound prior to irradiation without causing any tissue damage. The characterization of the prepared foams showed that both curcumin loaded and unloaded foams hydrated within 1  min and absorbed from 12 to 16 times their dry weight of a model physiological fluid. Curcumin, the model photosensitizer, has an extremely low solubility in water and may aggregate in aqueous environment. Cyclodextrins (CDs) and polyethylene glycol 400 (PEG 400) were therefore selected as solubilizers of curcumin in the foams to provide a burst release of the photosensitizer. Exposure to the prepared foams in combination with visible light irradiation (∼9.7  J/cm(2)) resulted in >6  log reduction of Entrococcus faecalis cells. However, curcumin mediated photokilling of Escherichia coli was ineffective when CDs were selected as solubilizer of curcumin in the foams. An 81% reduction in viable E. coli cells was detected after treatment with the foam containing PEG 400 as the only solubilizer of curcumin combined with visible light irradiation (∼29  J/cm(2)).

Formulation and characterisation of primaquine loaded liposomes prepared by a pH gradient using experimental design

The effect of different formulation factors (lipid type, cholesterol, charge, internal buffer capacity, drug-to-lipid incubation ratio) on the encapsulation efficiency and size of primaquine liposomes (SUVs) in response to a pH gradient was investigated by a fractional factorial screen ing design. Three of the factors (charge, internal buffer capacity, drug -to-lipid incubation ratio) were further studied in a Box--Behnken optimisation design. The lipid type was the most important parameter followed by the drug-to-lipid incubation ratio, buffer capacity, cholesterol and charge. Several of the interactions wer e important. In the optimisation design a robust region with high encapsulation efficiency (>95%) was obtained for DSPC: 33.33 mol% cholesterol-liposomes at high internal citrate concentration (200 mM) by maintaining the drug-to-lipid incubation ratio below 0.15:1 (mol:mol) and varying the charge incorporation between 2 and 10%. In order to achieve long-term stability and sterility, the liposomes were lyophilised followed by gamma irradiation. The pH gradient was maintained during this treatment with little chemical degradation of the substances. The final preparation consisted of three separate vials with lyophilised liposomes, solid state primaquine and hydration medium.

Photoinactivation of Staphylococcus epidermidis biofilms and suspensions by the hydrophobic photosensitizer curcumin – Effect of selected nanocarrier: Studies on curcumin and curcuminoides XLVII

Solubilization and stabilization from rapid degradation by the use of nanocarriers are necessary to exploit curcumins phototoxic potential towards pathogenic bacteria. However, maintenance of the phototoxicity requires a careful selection of type and amount of nanocarrier. The phototoxicity of an aqueous supersaturated curcumin solution without nanocarrier was compared to that of curcumin solubilized in polyethylene glycol 400 (PEG 400), Pluronic® F 127 (F 127) and hydroxypropyl-γ-cyclodextrin (HPγCD) on Staphylococcus (S.) epidermidis biofilms and suspensions. The nanocarriers stabilized the hydrophobic photosensitizer (PS) towards physical precipitation and hydrolytic degradation; however, photobleaching was pronounced (46-100% degradation) after irradiation with a dose of ≈ 9 J/cm(2) blue light depending on selected nanocarrier. Complete inactivation of S. epidermidis in suspension was achieved after exposure of ≈ 5 J/cm(2) combined with curcumin in 20% PEG 400 and 0.5% HPγCD and less than 1J/cm(2) light in case of a supersaturated curcumin solution. Curcumin in 1.5% F 127 induced phototoxicity towards bacterial biofilms; however, it was not phototoxic towards planktonic S. epidermidis. All curcumin preparations investigated demonstrated significant and similar phototoxicity towards biofilms (13-29% bacterial survival). A ≈ 9 J/cm(2) light dose was not sufficient to eradicate S. epidermidis biofilm completely under the current conditions.

Evaluation of Novel Alginate Foams as Drug Delivery Systems in Antimicrobial Photodynamic Therapy (aPDT) of Infected Wounds—An In Vitro Study: Studies on Curcumin and Curcuminoides XL

The aim of the present study was to incorporate a model water-insoluble photosensitizer, curcumin, in novel alginate foams, further to evaluate the suitability of the curcumin loaded foams in antimicrobial photodynamic therapy of infected wounds. Six foam formulations were prepared and characterized with respect to physical characteristics, in vitro release and storage- and photo-stability of curcumin. One formulation was sterilized (gamma-sterilization). The foams contained hydroxypropyl-beta-cyclodextrins or hydroxypropyl-gamma-cyclodextrins as solubilizers of curcumin. A reference foam without cyclodextrins was prepared with PEG 400 as the solubilizer. At a curcumin load of 0.153% (w/w), the water insoluble photosensitizer was uniformly distributed in the hydrophilic foams matrix. All foams were easy to handle, flexible and hydrated rapidly in a model physiological fluid. Release of curcumin in its monomeric form was demonstrated in vitro and found to be dependent on the type and amount of cyclodextrins in the formulation. Curcumin was stable during storage, but susceptible to photodegradation in the foams, especially when the formulations contain PEG 400 or hydroxypropyl-gamma-cyclodextrins. Curcumin did not degrade after gamma-sterilization, however a decrease in the in vitro release rate of curcumin and changes in the foams physical characteristics were detected.

Solid dispersions for preparation of phototoxic supersaturated solutions for antimicrobial photodynamic therapy (aPDT): Studies on curcumin and curcuminoides L

Curcumin is under investigation as a potential photosensitizer (PS) in antimicrobial photodynamic therapy (aPDT). The therapeutic potential of curcumin as a PS is limited by its low aqueous solubility, susceptibility to hydrolytic and photolytic degradation, and limited phototoxicity toward Gram negative (G-) bacteria. Supersaturated solutions of curcumin have demonstrated high phototoxicity toward several species of Gram positive (G+) bacteria as well as the G-Escherichia (E) coli. Thus, solid dispersions that can form supersaturated solutions of curcumin upon hydration may be beneficial in aPDT. In the present study, solid dispersions of curcumin have been prepared through lyophilization of concentrated solutions obtained from dissolution of hydroxypropyl-β-cyclodextrin (HPβCD)-curcumin co-precipitates. Hydroxypropyl methylcellulose (HPMC) was added to curcumin solutions prior to lyophilization. The resulting lyophilizates were porous, amorphous and hydrated and dissolved rapidly in contact with a model physiological salt solution. The detected drug load of the lyophilizates was in the range 0.5-1.0% (w/w) and was dependent on the selected ratio between HPβCD and curcumin in the co-precipitate. The lyophilizate with the highest drug load could easily be dissolved in aqueous medium to form curcumin solutions of relevant concentrations for aPDT (i.e., 10μM). Selected solutions of the curcumin solid dispersions showed a pronounced decrease in curcumin concentration up to 90% after storage for 168h, which indicated that supersaturated curcumin solutions were initially formed upon dissolution of the lyophilizates. Both freshly prepared and 2days old solutions of one selected curcumin lyophilizate induced significant inactivation of E. coli (∼1% bacterial survival) after exposure to a light dose of only 5J/cm(2).

Photoreactivity of biologically active compounds: VII. Interaction of antimalarial drugs with melanin in vitro as part of phototoxicity screening

The drugs commonly used in the treatment of malaria are photochemically unstable. Several of these compounds accumulate in melanin-rich tissues and cause toxic reactions which may be light induced. As part of the screening of the photochemical properties and phototoxic capabilities of antimalarials, the in vitro interaction of eight antimalarials with melanin was studied. The dissociation constant for the drug-melanin complex and the relative number of binding sites on melanin were estimated for six of the drugs using a curve-fitting program. The reaction rate for the formation of the melanin-drug complex was determined, and the complexes were further characterized by zeta potential measurements.

Studies on curcumin and curcuminoids: XXV. Inhibition of primaquine-induced lysis of human red blood cells by curcumin

Abstract The protective effect of curcumin on primaquine (PQ)-induced oxidative damage to red blood cells (RBC) is reported. The protection by curcumin against cell lysis was reversed upon increasing curcumin concentration above 20 μM. In contrast, curcumin added as a liposome preparation remained protective even at higher concentrations. Direct determination of curcumin in RBC indicated that the liposomes retained most of the curcumin, thus allowing its incorporation into RBC at concentrations providing stable protective effects against PQ-induced lysis.

The influence of Pluronics® on dark cytotoxicity, photocytotoxicity, localization and uptake of curcumin in cancer cells: studies of curcumin and curcuminoids XLIX

In order to apply curcumin as a photosensitizer in photodynamic therapy (PDT) one needs a formulation that can solubilize and stabilize the compound. Pluronics® (Pluronic) are reported to both solubilize and stabilize curcumin against hydrolytic degradation. The aim of the present work was therefore to investigate the influence of Pluronic formulation on the photocytotoxicity of curcumin. Interactions between curcumin and Pluronics were investigated by fluorescence emission and absorption spectroscopy. Cell survival was measured with the MTT assay. The location of curcumin in the cells was investigated with fluorescence microscopy, and the cellular uptake was measured with fluorescence emission spectroscopy. Pluronics P123 and F127 in contrast to Pluronic P85 and PEG 400 may solubilize curcumin under non-cytotoxic conditions. An inverse relationship between the concentration of Pluronic and the photocytotoxicity of curcumin was observed. Curcumin could rapidly translocate across the cell membrane by passive diffusion. The fluorescence from curcumin in the cells (in the cytoplasm) after 1 hour of incubation was lowered by the presence of Pluronics in the formulation. However, the absolute amount of cell-bound curcumin after 1 hour of incubation was independent of the presence of Pluronics. Curcumin was bound more strongly to cells when incubated with formulations without Pluronics compared to cells incubated with curcumin formulations with Pluronics. Incubation of WiDr cells with curcumin for 6 hours resulted in lysosomal accumulation of curcumin independent of the presence of Pluronics. Lysosomally located curcumin could not be observed in HT1080 cells after 6 hours of incubation. The Pluronics P123 and F127 were found to be suitable for solubilizing and stabilizing curcumin, but inhibited photocytotoxic effects of curcumin unless the Pluronic concentration during treatment of the cells was less than 5-10× above the critical micellar concentration.

Entrapment in phospholipid vesicles quenches photoactivity of quantum dots

Quantum dots have emerged with great promise for biological applications as fluorescent markers for immunostaining, labels for intracellular trafficking, and photosensitizers for photodynamic therapy. However, upon entry into a cell, quantum dots are trapped and their fluorescence is quenched in endocytic vesicles such as endosomes and lysosomes. In this study, the photophysical properties of quantum dots were investigated in liposomes as an in vitro vesicle model. Entrapment of quantum dots in liposomes decreases their fluorescence lifetime and intensity. Generation of free radicals by liposomal quantum dots is inhibited compared to that of free quantum dots. Nevertheless, quantum dot fluorescence lifetime and intensity increases due to photolysis of liposomes during irradiation. In addition, protein adsorption on the quantum dot surface and the acidic environment of vesicles also lead to quenching of quantum dot fluorescence, which reappears during irradiation. In conclusion, the in vitro model of phospholipid vesicles has demonstrated that those quantum dots that are fated to be entrapped in endocytic vesicles lose their fluorescence and ability to act as photosensitizers.

The influence of Pluronics nanovehicles on dark cytotoxicity, photocytotoxicity and localization of four model photosensitizers in cancer cells

Many photosensitizers (PSs) for use in photodynamic therapy (PDT) are characterized by poor solubility and a tendency to aggregate in aqueous environments. Nanovehicles of Pluronics block copolymers may be used for drug delivery of antineoplastic agents and may also exert a separate effect in enhancing drug efficiency. The objective of this study was to determine the effects of selected Pluronics (F127, P123, L44 and F68) on the dark cytotoxicity, photocytotoxicity and localization of four model photosensitizers, tetraphenyl porphyrins 4-substituted on the phenyl groups with trimethylamine (TAPP), hydroxyl (THPP), sulfonate (TSPP) and carboxyl (TCPP) in cancer cells. The selected PSs showed a 3 log range in sensitivity to cellular photoinactivation. Pluronics were found to efficiently deaggregate the PSs and improve PS solubility as analyzed by fluorescence spectroscopy and dynamic light scattering. The Pluronics had moderate to profound effects on intracellular localization of the PSs and cellular sensitivity to photoinactivation. Confocal microscopy was used to determine the localization of PSs in colon adenocarcinoma cell line (WiDr), guided by co-staining with nuclear (Hoechst 33342) and endolysosomal (LysoTracker Green DND® 26 and Dextran Alexa Fluor® 488) markers. Of the most significant effects P123 and F127 strongly attenuated the uptake and photocytotoxicity of THPP and redirected the cellular uptake to endocytosis. P123 stimulated translocation of TAPP from endocytic vesicles to a cytosolic and nuclear localization followed by an enhanced phototoxicity. In the absence of Pluronics TCPP was found to localize partly in endocytic vesicles and partly in the cytosol and nucleus, while P123 and F127 lowered the fraction in endocytic vesicles followed by a reduced sensitivity to photoinactivation. F68 had only moderate effects on intracellular localization of the evaluated PSs with the exception of a higher endocytic accumulation of TCPP and lowered photocytotoxicity of TCPP and THPP. In conclusion, Pluronics are attractive solubilizers of porphyrin-based PSs which have in many cases substantial effects on intracellular localization and efficacy of the PSs.