Antimicrobial effects of XF drugs against Candida albicans and its biofilms.

Compared with antibiotics for treating bacterial infections, there are a limited number of antifungal agents. This is due to several factors, including the difficulties of identifying suitable antifungals that target the fungal cell without damaging host cells, and the reduced rates of diagnosis of fungal infections compared with those caused by bacteria. The problem of treating fungal infections is exacerbated by an increasing incidence of antifungal resistance among human fungal pathogens. Three XF drugs (XF-73, XF-70, and DPD-207) have previously displayed innate bactericidal effects and a low propensity for microbial resistance, with XF-73 and XF-70 having a second, light-activated mechanism of action [known as photodynamic therapy (PDT)]. In an effort to expand the repertoire of antifungal agents, this research assessed the in vitro activity of XF drugs via both mechanisms of action against six strains of the fungal pathogen Candida albicans in both planktonic and biofilm cultures. In addition, this research examined the effects of XF drug treatment on biofilms of C. albicans in a reconstituted human oral epithelium model. All C. albicans strains tested were susceptible to XF-73 and XF-70, with minimum inhibitory concentrations (MICs) between 0.25 µg/mL and 2 µg/mL; DPD-207 was less potent, with MICs between 4 µg/mL and 16 µg/mL, and light activation did not enhance these MICs. Complete biofilm eradication was not reported at the tested XF drug concentrations. However, live and dead staining of C. albicans cells in biofilms after XF drug treatment demonstrated that XF-73 and XF-70 were active against most Candida biofilms tested from 64 µg/mL; again, light activation did not enhance anti-biofilm activity. Candida biofilms were more resistant to DPD-207, with fungicidal effects occurring from 256 µg/mL. XF-73 and XF-70 reduced penetration of C. albicans biofilm into reconstituted human oral epithelium (RHOE) and resulted in less damage (as determined by reduced lactate dehydrogenase release) than untreated biofilms. Overall, the results highlight the potential of XF drugs as new drugs for the management of topical infections caused by C. albicans. Further studies are warranted on the development of XF drugs as antifungals, particularly for XF-73 and XF-70.

In vivo assessment of the osteointegrative potential of phosphatidylserine-based coatings.

The successful implantation of titanium-based implants for orthopaedic and dental applications is often hindered because of their mobility, which arises because of a lack of direct binding of the metal surface to the mineral phase of the surrounding bone. Ceramic coatings, although ensuring the integration of the implant within the tissue, are unstable and carry risks of delamination and of failure. Recently, a novel biomimetic approach has been developed where porous titanium implants are coated with calcium-binding phospholipids able to catalyse the nucleation of discrete apatite crystals after only 30 min incubation in simulated body fluids. The present work assesses the osteointegrative potential of this new class of coatings in an in vivo rabbit model and compares its performance with those of bare porous titanium and hydroxyapatite-coated titanium. The data obtained show that phosphatidylserine-based coatings, whilst resorbing, drive the growing bone into apposition with the metal surface. This is in contrast to the case of bare titanium.

Photodynamic therapy comes of age.

The concept underlying photodynamic therapy (PDT) is the use of light-absorbing molecules which, when delivered to target cells, and activated by irradiation with light of the appropriate wavelength, produce reactive oxygen species that cause cell death by apoptosis or necrosis. Classically, photodynamic agents have been macrocycles and, as such, application is limited to topical and intravenous administration. In the latter case, reliance has been placed on the characteristic behavior of the photodynamic agents in showing some degree of selectivity for concentrating in the target to minimize non-specific damage to the host tissue. The parameters open to development of improved drugs are: (i) the design of the photodynamic agent molecule as a means of determining the wavelength of light for activation, and for influencing physicochemical characteristics and the pharmacokinetic behavior of the drug; (ii) the delay between administration and activation; (iii) the nature of the activating light source; and, (iv) the duration and intensity of the activating light. Obviously, PDT is attractive for treating disease states in which natural apoptotic mechanisms are compromised, specifically for cancerous states and in cases of uncontrolled cell proliferation. PDT also has immunomodulatory sequelae, including triggering of T-cell mediated activity against residual cancerous cells. The use of PDT is being extended to diverse, related immune and proliferative disease states, and to the inactivation of bacteria and viruses. Increasingly, attention is being given to improving treatment by targeting conjugates and local delivery strategies, as well as by the design of photodynamic agents with closely defined photophysical and physicochemical properties. Progress is being made in challenging indications, such as the treatment of solid and pigmented tumors. Alternative technologies not involving light activation are available for some molecules which may also be used with light activation. Some ex vivo techniques and medical devices have been reported.

Liposome-mediated delivery of photosensitizers: localization of zinc (II)-phthalocyanine within implanted tumors after intravenous administration.

CGP55847, liposomal zinc(II)-phthalocyanine (Zn-Pc), was administered by the intravenous route to Swiss mice bearing intramuscularly implanted Ehrlich carcinomas or to C57/BL6 mice bearing subcutaneously implanted B16 melanomas. Tumors were removed 3 h or 24 h after dosing the intratumoral distribution determined by fluorescence microscopy. Localization of the photosensitizer occurred more rapidly in the Ehrlich carcinoma than in the B16 melanoma; this difference in photosensitizer uptake may be related to a higher degree of vascularization of the carcinoma. The photosensitizer was found in association with blood vessels at 3 h but not 24 h after dosing and appeared to have a greater affinity for areas of tissue necrosis within the tumor compared to viable tumor tissue. Little or no Zn-Pc was detected in the muscle tissue invaded by the Ehrlich carcinoma and was associated with the membranes and the cytosol, but not the nucleus, of cells in both tumors.

Methods for assessing splenic macrophage depletion by liposome encapsulated clodronate.

Small unilamellar vesicles containing clodronate (SUVc) injected intravenously will deplete splenic macrophages and the degree of histological depletion can be assessed by determining the clearance and uptake of monoclonal antibody coated erythrocytes. Splenic Fc dependent clearance, assessed in decomplemented animals, provides a more sensitive index of the effects of large multilamellar liposome encapsulated clodronate (MLVc) and SUVc than does the clearance of complement coated erythrocytes on macrophage depletion in the spleen. MLVc were more efficient than SUVc in inducing a reduction in the number of red pulp macrophages within the spleen. Receptor specific red cell uptake in the spleen could be used as an alternative to histology when assessing splenic macrophage depletion. Encapsulation of clodronate is crucial to its depleting effect since the free drug in saline does not change splenic macrophage number or function.

The effect of free and liposome-encapsulated clodronate on the hepatic mononuclear phagocyte system in the rat.

Clodronate, encapsulated within small unilamellar vesicles (SUVc) will deplete hepatic macrophages after intravenous injection. Functional studies, using probes to evaluate hepatic Fc and C3b uptake, showed a close correlation between the inhibition of receptor-mediated uptake and the depletion of hepatic macrophages. Twenty milligrams of clodronate encapsulated within SUVc produced > or = 90% inhibition of uptake and clearance of Fc- and C3b-coated erythrocytes and a comparable reduction of hepatic macrophage numbers. Inhibition of macrophage receptor-mediated uptake of these erythrocytes was closely related to the reduction in macrophage numbers. Repopulation of macrophages within the liver took place over 2 weeks. At 1 week after depletion, although repopulation was taking place, receptor-mediated function remained suppressed. In a preliminary experiment, treatment of rats with adjuvant arthritis with 20 mg clodronate encapsulated in SUV suppressed the inflammation and reversed the course of the disease, while treatment with 20 mg free clodronate in saline or 20 mg clodronate in multilamellar vesicles (MLVc) did not.

In vitro interaction of zinc(II)-phthalocyanine-containing liposomes and plasma lipoproteins.

We have studied the interaction of small unilamellar liposomes containing zinc(II)-phthalocyanine (Zn-Pc) with human plasma lipoproteins. High-, low- and very low-density lipoproteins (HDL, LDL and VLDL), were purified from plasma and combined in amounts reflecting their natural abundance in plasma. After short periods of incubation at 37 degrees C, the bulk of Zn-Pc was incorporated into HDL and LDL; very little 14C-labelled palmitoyl oleoyl phosphocholine, the most abundant phospholipid in the formulation, was associated with lipoproteins. When liposomes were incubated in pooled plasma, 73%-85% of Zn-Pc and 27%-34% of radiolabelled phospholipid were recovered with HDL and LDL, indicating a possible role for plasma lipid transfer proteins in the incorporation of phospholipid into lipoproteins. Some Zn-Pc was also found in association with VLDL. The buoyant density of Zn-Pc liposomes increased in a dose-dependent fashion when the particles were incubated with plasma, and it is suggested that this was due, at least in part, to opsonization of liposomes by plasma proteins.

Interaction between zinc(II)-phthalocyanine-containing liposomes and human low density lipoprotein.

The interaction of human low density lipoprotein (LDL) and small unilamellar liposomes containing the photosensitiser zinc(II)-phthalocyanine (Zn-Pc) was studied in vitro to determine if Zn-Pc could be directly incorporated into the lipoprotein in the absence of other serum components. Incubation of LDL with increasing concentrations of liposomes resulted in a progressive increase in the net negative charge of LDL as determined by agarose gel electrophoresis and both Zn-Pc and liposomal phospholipid were incorporated into the modified LDL particles. Gel chromatography experiments indicated an increase in the molecular mass of modified LDL and immunoaffinity chromatography provided evidence that apoprotein B epitopes on modified LDL were unable to bind to antibody. The study indicated that the liposomal components could be selectively incorporated into LDL by a process that did not appear to involve either aggregation or fusion of particles.

Efficient clodronate entrapment within multilamellar and unilamellar liposomes.

Clodronate (dichloromethylene bisphosphonate) encapsulated within liposomes and administered intravenously eliminates resident macrophages within the liver and spleen. Macrophage depletion in the rat requires 20 mg of the encapsulated drug, and so far this has only been achieved using large multilamellar vesicles (MLV). Recent studies have shown that small unilamellar vesicles (SUV) when injected intravenously accumulate at inflamed joint sites in both animal models of arthritis and patients with rheumatoid arthritis; multilamellar vesicles were not able to do so. If phagocytic cells, such as macrophages, are responsible for SUV sequestration, then SUV containing clodronate may be targeted to the inflamed joint and may eliminate the macrophage population leading to reduction in the state of inflammation. We have adapted an existing technique to radiolabel clodronate with 99mTechnetium to use as a tracer to determine its encapsulation within liposomes, a technique that has advantages over other current methods. We have achieved a high-encapsulation efficiency of the drug within MLV and produced SUV containing sufficient clodronate to deplete macrophages in rats in a small enough volume to administer it intravenously as a single dose.

Accumulation of polyvinylpyrrolidone within the inflamed paws of adjuvant-induced arthritic rats.

125I-Labelled polyvinylpyrrolidone ([125I]PVP) of a range of molecular weights (mol. wt 10, 40 and 360 kDa) was injected i.v. into adjuvant-induced arthritic and normal rats and the blood clearance and tissue distribution of the polymers determined. The half-life of PVP in the circulation increased with increasing mol. wt; 10, 40 and 360 kDa polymers had mean terminal half-lives of 2.2, 6.9 and 16.4 h, respectively. Tissue uptake was also found to be mol. wt dependent, the largest PVP molecule accumulating to a greater extent in the spleen, liver, lungs and paws in both normal and arthritic rats (P less than 0.01) than the two lower mol. wt polymers. Accumulation of the polymer in inflamed paws (g tissue)-1 greatly exceeded that of normal paws (P less than 0.01). This difference was particularly noticeable with 360 kDa PVP, where arthritic paws amassed 7 times more PVP than normal paws.

Specific accumulation of cholesterol-rich liposomes in the inflammatory tissue of rats with adjuvant arthritis.

High performance liquid chromatography has shown that after intravenous injection cholesterol-poor liposomes (100 nm) are unstable and their phospholipid is redistributed. Under identical conditions cholesterol-rich liposomes remain structurally intact within the circulation. When injected intravenously cholesterol-rich liposomes accumulate within the inflamed paws of rats with adjuvant induced arthritis to the same extent as cholesterol-poor liposomes. Uptake in inflamed tissue of three cholesterol-rich liposome preparations was always significantly greater than the uptake noted in normal tissue. The degree of accumulation in inflamed tissue was found to depend on the size of the liposome, with the greatest uptake, 7% of the injected dose, achieved by the smallest vesicle (100 nm). These results indicate that intact liposomes accumulate at inflamed joint tissue sites. Therefore the passive targeting of anti-inflammatory drugs encapsulated within these liposomes could be contemplated.

High performance liquid chromatographic analysis of liposome stability.

Two techniques have been studied for their suitability for the analysis of the stability of liposomes: (1) High Performance Gel Permeation Liquid Chromatography (HPGPLC), a TSK G5000PW Ultrogel column; (2) Gel Permeation Chromatography (GPC), a Sepharose 4B column. The stability of dual radio-labelled, cholesterol-poor and cholesterol-rich, negatively charged liposomes in vitro (in saline and in serum), and in vivo, have been investigated using these two techniques. The HPGPLC TSK G5000PW column proved to be the superior technique for the analysis of liposome stability with the advantages of rapid run times, increased sample recovery, and smaller sample volumes were required. The results obtained confirm that inclusion of a high ratio of cholesterol into the liposome structure prevents phospholipid loss when exposed to serum, and that the cholesterol-poor liposome structure is dramatically altered under the same conditions. In conclusion, the TSK G5000PW column is ideal for monitoring movement of phospholipid between liposomes and serum proteins and for detecting changes in liposome size.

Specific accumulation of technetium-99m radiolabelled, negative liposomes in the inflamed paws of rats with adjuvant induced arthritis: effect of liposome size.

Technetium-99m labelled, negatively charged liposomes accumulate in the inflamed tissue of rats with adjuvant induced arthritis. Up to 10 times more liposome accumulation was seen in inflamed paws than in paws of control rats, and this represented 5.3% of the injected liposome dose. The accumulation of liposomes in inflamed tissue was directly related to the liposome size, the maximal accumulation occurring with liposomes less than 100 nm in diameter.

Effect of liposome surface charge on the stability of technetium (99mTc) radiolabelled liposomes.

Using liposomes radiolabelled by the 99mTechnetium-stannous chloride technique we have investigated the effect of surface charge on the stability of the isotope in vitro and in vivo. Dialysis of 99mTc-labelled positive, negative and neutral liposomes, which had been incubated in either saline or normal rat serum showed no significant loss of the isotope from the liposome surface with only 2 per cent of the isotope dialysed. A comparison of gel chromatography with dialysis confirmed that most of the isotope remained attached to the liposome surface, but it did reveal greater loss of the isotope, between 15 and 23 per cent. The liposome clearance rates obtained from 125I-egg phosphatidylcholine (EPC) and 99mTc dual-labelled positive or neutral liposomes were significantly different. The 99mTc marker was cleared five times faster from the positive liposomes and twice as fast from the neutral liposomes as the 125I-EPC integral membrane marker. The 99mTc attached to liposomes with a negative surface charge was stable in vivo and had the same clearance rate from the circulation as the 125I-EPC marker. These results indicate that the commonly used in vitro techniques for assessing liposome radiolabel stability are unsuitable for predicting the stability of the 99mTc in vivo.