Engineering Temperature-Responsive Polymer Nanoparticles that Load and Release Paclitaxel, a Low-Molecular-Weight Anticancer Drug.

Poly(N-isopropylacrylamide) (pNIPAm) undergoes a hydrophilicity/hydrophobicity change around its lower critical solution temperature (LCST). Therefore, pNIPAm-based polymer nanoparticles (NPs) shrink above their LCST and swell below their LCST. Although temperature responsiveness is an important characteristic of synthetic polymers in drug and gene delivery, few studies have investigated the temperature-responsive catch and release of low-molecular-weight drugs (LMWDs) as their affinity to the target changes. Since LMWDs have only a few functional groups, preparation of NPs with high affinity for LMWDs is hard compared with that for peptides and proteins. However, LMWDs such as anticancer drugs often have a stronger effect than peptides and proteins. Therefore, the development of NPs that can load and release LMWDs is needed for drug delivery. Here, we engineered pNIPAm-based NPs that capture paclitaxel (PTX), an anticancer LMWD that inhibits microtubules, above their LCST and release it below their LCST. The swelling transition of the NPs depended on their hydrophobic monomer structure. NPs with swelling ratios (=NP size at 25 °C/NP size at 37 °C) exceeding 1.90 released captured PTX when cooled to below their LCST by changing the affinity for PTX. On the other hand, NPs with a swelling ratio of only 1.14 released melittin. Therefore, optimizing the functional monomers of temperature-responsive NPs is essential for the catch and release of the target in a temperature-dependent manner. These results can guide the design of stimuli-responsive polymers that catch and release their target molecules.

Improvement of LXR-mediated lipid metabolism in nephrotic model kidney accompanied by suppression of inflammation and fibrosis.

Kidney disease affects millions of people worldwide. Chronic kidney diseases, such as diabetic nephropathy, are often accompanied by nephrotic syndrome, which causes a large amount of protein and lipid to leak out into the urine. Leaked lipids are well known to accumulate in the proximal tubules as lipid droplets. However, the role of lipid metabolism in the kidney has not been thoroughly studied, and the relationship between accumulated lipid and pathological progression is often unknown. In this study, we showed that reducing accumulated lipids by exerting an agonistic effect on Liver X receptor, one of the nuclear receptors known to play an important role in lipid metabolism, suppressed the development of pathological conditions, such as inflammation and fibrosis, in a nephrosis model. Until now, many renal disease treatments have focused on suppressing the inflammatory response. However, it is now clear that even if the direct anti-inflammatory response is weak, the spread of inflammation and fibrosis can be suppressed by reducing accumulated lipids. Our results suggest that reducing abnormal lipid accumulation in the kidney could lead to disease treatment.

Increasing the siRNA knockdown efficiency of lipid nanoparticles by morphological transformation with the use of dihydrosphingomyelin as a helper lipid.

Lipid nanoparticles (LNPs), comprising ionizable lipids, helper lipids, cholesterol, and PEG lipids, can act as delivery carriers for nucleic acids and have achieved clinical success in the delivery of siRNA and mRNA. It has been shown that the morphology of LNPs varies depending on their lipid composition, but the influence of their morphology on nucleic acid efficacy has not been fully elucidated. In this study, we used our previously developed novel lipid, dioleoylglycerophosphate-diethylenediamine conjugate (DOP-DEDA), to create pH-responsive LNPs (DOP-DEDA LNPs). We evaluated the morphology of DOP-DEDA LNPs composed of different helper lipids and the knockdown efficiency of small interfering RNA (siRNA). A distinctive difference in morphology was observed between DOP-DEDA LNPs of different helper lipids. Significant differences were also observed in the apparent pKa of DOP-DEDA LNPs and the knockdown efficiency of siRNA, which may be due to the difference in the localization of DOP-DEDA molecules in DOP-DEDA LNPs. These findings suggest that changing helper lipids alters the morphology of the DOP-DEDA LNP system, which affects the apparent pKa and knockdown efficiency of siRNA.

Cooling-induced, localized release of cytotoxic peptides from engineered polymer nanoparticles in living mice for cancer therapy.

Temperature-responsive polymers are often characterized by an abrupt change in the degree of swelling brought about by small changes in temperature. Polymers with a lower critical solution temperature (LCST) in particular, are important as drug and gene delivery vehicles. Drug molecules are taken up by the polymer in their solvent swollen state below their LCST. Increasing the temperature above the LCST, typically physiological temperatures, results in desolvation of polymer chains and microstructure collapse. The trapped drug is released slowly by passive diffusion through the collapsed polymer network. Since diffusion is dependent on many variables, localizing and control of the drug delivery rate can be challenging. Here, we report a fundamentally different approach for the rapid (seconds) tumor-specific delivery of a biomacromolecular drug. A copolymer nanoparticle (NP) was engineered with affinity for melittin, a peptide with potent anti-cancer activity, at physiological temperature. Intravenous injection of the NP-melittin complex results in its accumulation in organs and at the tumor. We demonstrate that by local cooling of the tumor the melittin is rapidly released from the NP-melittin complex. The release occurs only at the cooled tumor site. Importantly, tumor growth was significantly suppressed using this technique demonstrating therapeutically useful quantities of the drug can be delivered. This work reports the first example of an in vivo site-specific release of a macromolecular drug by local cooling for cancer therapy. In view of the increasing number of cryotherapeutic devices for in vivo applications, this work has the potential to stimulate cryotherapy for in vivo drug delivery.

Easy preparation of a liposome-mediated protein delivery system by freeze-thawing a liposome-protein complex.

Homeostasis can be achieved by adding a protein supplement; however, an appropriate vector is required to deliver the protein into the cell because of the low stability of proteins in the blood and low cell membrane permeability. Here we report an easy one-step method of encapsulating proteins into liposomes for delivery. We used negatively charged superoxide dismutase (SOD) and a polycation liposome as protein and liposome models, respectively. Liposome-encapsulated SOD was prepared by freeze-thawing the SOD-liposome complex (lipoplexes). The amount of immobilized SOD within the lipoplex significantly increased on freeze-thawing. Surprisingly, subjecting the single-layered lipoplexes to freeze-thawing produced multilayered liposomes with SOD localized between the lipid layers. The amount of SOD delivered intracellularly significantly increased by freeze-thawing compared with that delivered by lipoplexes without freeze-thawing. SOD, liposomes, and endosomes were separately localized in the cells. The freeze-thawed lipoplex-encapsulated SOD samples were intravenously injected in mice. The SOD biodistribution was dramatically changed compared with the injection of free SOD or lipoplex. SOD was detached from the lipoplex in the bloodstream after the injection of non-freeze-thawed lipoplex, whereas the encapsulation of SOD in the liposomes upon freeze-thawing enabled the stable circulation of SOD with the liposomes in the bloodstream. This work paves the way for the application of the freeze-thawing technology for the easy one-step encapsulation of proteins into liposomes for protein delivery.

siRNA Vehicles for High Endosomal Escapability.

Small interfering RNA (siRNA) is a novel therapeutic modality for the treatment of intractable diseases; however, the development of a useful siRNA delivery vector is imperative for clinical use. Since siRNA works in the cytoplasm, the ability of the carrier to escape destruction in the endosomes is a highly required characteristic for the induction of a high knockdown effect. Here, we describe the step-by-step procedure for the evaluation of high endosomal escapability. The vector that has pH-responsive characteristics at around pH = 6.2-6.5 is important for the high endosomal escape.

Recent advances in siRNA delivery mediated by lipid-based nanoparticles.

Small interfering RNA (siRNA) has been expected to be a unique pharmaceutic for the treatment of broad-spectrum intractable diseases. However, its unfavorable properties such as easy degradation in the blood and negative-charge density are still a formidable barrier for clinical use. For disruption of this barrier, siRNA delivery technology has been significantly advanced in the past two decades. The approval of Patisiran (ONPATTRO™) for the treatment of transthyretin-mediated amyloidosis, the first approved siRNA drug, is a most important milestone. Since lipid-based nanoparticles (LNPs) are used in Patisiran, LNP-based siRNA delivery is now of significant interest for the development of the next siRNA formulation. In this review, we describe the design of LNPs for the improvement of siRNA properties, bioavailability, and pharmacokinetics. Recently, a number of siRNA-encapsulated LNPs were reported for the treatment of intractable diseases such as cancer, viral infection, inflammatory neurological disorder, and genetic diseases. We believe that these contributions address and will promote the development of an effective LNP-based siRNA delivery system and siRNA formulation.

Each liver X receptor (LXR) type has a different purpose in different situations.

LXRs, which are nuclear receptors, have 2 isoforms-LXRα and LXRß. Generally, LXRα is expressed in the liver, kidney, and a limited number of other organs, whereas LXRß is thought to be expressed ubiquitously. Nevertheless, no clear consensus has been reached on the role of each in kidney lipid metabolism. Many researchers have reported that lipids accumulate in renal tubular epithelial cells during nephrosis. The nephrosis model we used showed the presence of urinary protein 4 days after the induction of illness. Additionally, the model maintained high levels of urinary protein from day 7-14. Lipid accumulation was clearly verified at day 4 and extreme accumulation was observed at day 7. We observed increased expression of LXRα from an early stage of nephrosis. To explore the role of increased LXRα in diseased kidney in vitro, NRK52E, normal kidney tubular epithelial cells, were forced to overexpress LXRα. These cells showed significantly lower lipid accumulation than mock cells did. In contrast, LXRß knockdown lead to increased lipid accumulation in mock cells, and constancy in overexpressing cells. In normal kidneys, LXRß is expressed stably to control mainly the intracellular lipids. However, with increasing intracellular lipid accumulation, expression of LXRα and its downstream gene, ABCA1, was upregulated, followed by lipid excretion in an LXRα-dependent manner. This phenomenon strongly suggests the importance of LXRα in lipid metabolism in the diseased kidney.

Reduction of Membrane Protein CRIM1 Decreases E-Cadherin and Increases Claudin-1 and MMPs, Enhancing the Migration and Invasion of Renal Carcinoma Cells.

CRIM1 is a membrane protein that has been reported to be related to cell proliferation. CRIM1 is expressed in renal carcinoma cells, but its involvement in proliferation and malignant transformation remains unclear. We analyzed whether alterations in the characteristics of cancer cells are observed following knockdown of CRIM1. Decreased expression of CRIM1 did not affect proliferation or anchorage-independent growth. The results of wound healing and invasion assays showed that reduced expression of CRIM1 increased cells' migratory and invasive abilities. Expression analysis of factors involved in migration and invasion in CRIM1-knockdown cells revealed that expression of the cell adhesion factor E-cadherin declined and expression of claudin-1, which is upregulated in metastatic cancer cells, increased. In addition, increased expression of matrix metalloproteinase (MMP) 2 and MMP9, protease essential for cancer cell invasiveness, was observed. Furthermore, an increase in phosphorylated focal adhesion kinase (FAK), which increases cell migration, was observed. Increased expression of the E-cadherin transcription repressors Snail, Slug, and ZEB-1 were observed, and mRNA levels of E-cadherin were decreased. Therefore, expression of E-cadherin is thought to be decreased by both suppression of E-cadherin mRNA expression and promotion of degradation of the E-cadherin protein. In addition, expression of CRIM1 was decreased in renal cancer cells undergoing epithelial-mesenchymal transition (EMT) stimulated by tumor necrosis factor alpha (TNF-α). Thus, CRIM1 regulates the expression of several EMT-related factors and appears to play a role in suppressing migration and invasion through control of EMT.

Specific delivery of an immunosuppressive drug to splenic B cells by antigen-modified liposomes and its antiallergic effect.

BACKGROUND: Use of the reverse targeting drug delivery system (RT-DDS) is a new targeting strategy based on the specific delivery of drugs to immune cells in antigen-sensitized animals by using antigen-modified liposomes, and it is expected to be a curative treatment for allergic diseases. PURPOSE: Herein, we prepared ovalbumin (OVA)-modified liposomes encapsulating the immunosuppressive drug FK506 (OVA-LipFK) and aimed to demonstrate the delivery selectivity of the liposomes to splenic B cells, and its antiallergic effect in an OVA-sensitized allergic model mouse. METHODS: Fluorescently labeled OVA-LipFK was intravenously injected into OVA-sensitized mice, and the intrasplenic localization of liposomes was observed. The antiallergic effect of OVA-LipFK in OVA-sensitized mice was examined by measuring the blood levels of OVA-specific IgE and IgG antibodies. RESULTS AND DISCUSSION: OVA-LipFK was co-localized to not only B cells but also germinal centers, in the spleen of OVA-sensitized mice. However, there was no accumulation of unmodified liposomes encapsulating FK506 (LipFK) in the splenic B-cell area. In a therapeutic study, OVA-LipFK significantly suppressed the production of both OVA-specific IgE and IgG antibodies in OVA-sensitized mice after the animals had been boosted with OVA, whereas LipFK showed little antiallergic effect. CONCLUSIONS: The present study suggested that the introduction of RT-DDS for use with immunosuppressive drugs could be useful for the treatment of allergic diseases.

Angiostatin prevents IL-1ß-induced down-regulation of eNOS expression by inhibiting the NF-κB cascade.

This study aimed to elucidate the protective potential of angiostatin in inflamed endothelial cells in culture. We assessed the effect of angiostatin on the expression of ICAM-1 and eNOS. Angiostatin prevented IL-1ß-induced down-regulation of eNOS expression, but produced no significant changes on IL-1ß-induced up-regulation of ICAM-1. We then explored the effect of angiostatin on IL-1ß-mediated inflammatory signaling and found that angiostatin inhibited IL-1ß-mediated nuclear translocation of NF-κB. Thus, our results suggest that angiostatin prevents IL-1ß-induced down-regulation of eNOS expression via inhibition of the NF-κB cascade; this may be the anti-inflammatory mechanism of angiostatin.

Suppression of immune response by antigen-modified liposomes encapsulating model agents: a novel strategy for the treatment of allergy.

A specific antigen-sensitized animal has antigen-specific immune cells that recognize the antigen. Therefore, an antigen-modified drug carrier would be recognized by the immune cells. When such a carrier encapsulates certain drugs, these drugs should be specifically delivered to the immune cells. To examine this strategy, ovalbumin (OVA) was used as model antigen, and mice were presensitized with 100 µg of OVA with Alum. For preparing OVA-modified liposomes (OVA-lipo), OVA was incubated with DSPE-PEG-NHS and resulting DSPE-PEG-OVA was inserted into liposomes. OVA-specific IgG was produced 6-fold higher by intravenous injection of OVA-lipo thrice (10 µg as OVA in each injection) in OVA-sensitized mice, than that by the injection of control liposomes, suggesting that OVA-lipo was recognized by the antigen-specific immune cells. Moreover, intra-splenic accumulation of OVA-lipo was observed in OVA-sensitized mice, but not in naive mice. To achieve the delivery of a drug to specific immune cells, OVA-lipo encapsulated low dose of doxorubicin (DOX) as a model drug (20 µg DOX/mouse, Ca. 1 mg/kg) was injected in the sensitized mice. The injection of OVA-lipo encapsulating DOX suppressed the production of IgE against OVA, suggesting that the specific delivery of the drug to immune cells responsible for OVA recognition was achieved and that these immune cells were removed by the drug treatment. This strategy would be useful for the fundamental treatment of allergy by the use of immunosuppressing agents.

Disappearance of the angiogenic potential of endothelial cells caused by Argonaute2 knockdown.

Argonaute2 (Ago2), a component protein of RNA-induced silencing complex, plays a central role in RNA interference. We focused on the involvement of Ago2 in angiogenesis. Human umbilical vein endothelial cells (HUVECs) stimulated with several growth factors such as vascular endothelial growth factor were used for angiogenesis assays. We applied polycation liposomes for transfection of small interfering RNA (siRNA) to determine the biological effects of siRNA for Ago2 (siAgo2) on HUVECs. The proliferation study indicated that siAgo2 significantly suppressed the growth of HUVECs compared with control siRNA. TUNEL staining showed a certain population of HUVECs treated with siAgo2 underwent apoptosis. Furthermore, the treatment with siAgo2 suppressed the tube formation of HUVECs and significantly reduced the length of the tubes. These present data demonstrate that siAgo2 inhibited indispensable events of angiogenesis in vitro. This is the first report suggesting that Ago2 is required for angiogenesis.

Effective tumor regression by anti-neovascular therapy in hypovascular orthotopic pancreatic tumor model.

Pancreatic cancer is one of the most serious cancers with poor therapeutic results and prognosis. In here, we proposed a novel treatment strategy of pancreatic cancer by injuring limited angiogenic vessels with liposome containing adriamycin. At first, we established an orthotopic tumor model, which has a hypovascular characteristic of pancreatic tumor. In this model, we obtained the enhanced therapeutic efficacy with liposome that modified by polyethylene glycol (PEG) and a peptide, Ala-Pro-Arg-Pro-Gly (APRPG), having an affinity to neovessels. Histochemical analysis suggested the degradation of angiogenic vessels after treatment with APRPG-PEG-liposomal adriamycin. In addition, we observed colocalization of fluorescence-labeled APRPG-PEG-liposome with angiogenic endothelial cells, although the biodistribution of (3)H-labeled liposome did not show the difference in the amount of accumulation between PEG-modified liposome and APRPG-PEG-modified liposome. These results suggested the availability of the anti-neovascular therapy against pancreatic cancer and supply a new sight indication on chemotherapeutics against pancreatic cancer.

Antitumor activity of liposomal naphthoquinone esters isolated from Thai medicinal plant: Rhinacanthus nasutus KURZ.

We previously observed that rhinacanthins-C, -N and -Q, three main naphthoquinone esters isolated from the roots of Thai medicinal plant; Rhinacanthus nasutus KURZ. (Acanthaceae) induced apoptosis of human cervical carcinoma HeLaS3 cells. Since these rhinacanthins showed limited solubility in aqueous medium, we attempted to entrap them into liposomal membrane: Liposomalization enabled injection of the drugs and the drugs were expected to transfer to lipoproteins in the bloodstream. Liposomal formulations of rhinacanthins-C, -N and -Q showed strong antiproliferative activity against HeLaS3 cells with the IC50 values of 32, 17, 70 microM; 19, 17, 52 microM and 2.7, 2.0 and 5.0 microM for the exposure time of 24, 48, and 72 h, respectively. These liposomes suppressed the tumor growth in Meth-A sarcoma-bearing BALB/c mice at the dose of 5.0 mg/kg/d for 10 d. Among rhinacanthins, liposomal rhinacanthin-N significantly suppressed solid tumor growth. Based on these results, our findings demonstrated that rhinacanthin-N suppressed tumor growth in vivo, and suggested that liposomes are useful for preparing injectable formulation of hydrophobic drugs.

Induction of apoptosis in tumor cells by three naphthoquinone esters isolated from Thai medicinal plant: Rhinacanthus nasutus KURZ.

Rhinacanthus nasutus KURZ. (Acanthaceae) has been used as Thai traditional medicine for the treatment of various cancers. Recently, we reported that rhinacanthins, active components of the plant, had antiproliferative activity against human cancer line cells. In the present study, we investigated the growth inhibitory mechanism of rhinacanthins-C, -N and -Q, three main naphthoquinone esters isolated from the roots of R. nasutus KURZ. in human cervical carcinoma (HeLaS3) cells by means of TUNEL staining, DNA fragmentation assay, flow cytometry, and cleavage assay of Asp-Glu-Val-Asp-peptide-nitroanilide, a caspase-3 substrate. After the HeLaS3 cells was exposed with different concentrations of the drugs, rhinacanthins-C, -N and -Q exhibited antiproliferative effects on HeLaS3 cells with the IC50 values of 80, 65, 73 microM; 55, 45, 55 microM; and 1.5, 1.5 and 5.0 microM for 24, 48 and 72 h time points, respectively. Morphological changes showing nuclear fragmentation of rhinacanthins-treated cells were clearly observed after 48 h exposure. Consistent with this observation, the appearance of a ladder formation was also evident with an agarose gel electrophoresis of the extracted DNA. Flow cytometric analysis revealed that rhinacanthin-N caused G2/M arrest of HeLaS3 cells after 24 h incubation, and increased the proportion of sub-G1 hypodiploid cells, apoptotic cells, in the population of HeLaS3 cells after 48 and 72 h incubation. Moreover, the drug treatment markedly elevated the activity of caspase-3. Based on these results, our findings demonstrated for the first time that the inhibitory effects of three main naphthoquinone esters isolated from the roots of R. nasutus KURZ. on the growth of HeLaS3 cells appear to arise from the induction of apoptosis, that might be associated with the activation of caspase-3 pathway.

Enhancement of anticancer activity in antineovascular therapy is based on the intratumoral distribution of the active targeting carrier for anticancer drugs.

We previously observed the enhanced anticancer efficacy of anticancer drugs encapsulated in Ala-Pro-Arg-Pro-Gly-polyethyleneglycol-modified liposome (APRPG-PEG-Lip) in tumor-bearing mice, since APRPG peptide was used as an active targeting tool to angiogenic endothelium. This modality, antineovascular therapy (ANET), aims to eradicate tumor cells indirectly through damaging angiogenic vessels. In the present study, we examined the in vivo trafficking of APRPG-PEG-Lip labeled with [2-(18)F]2-fluoro-2-deoxy-D-glucose ([2-(18)F]FDG) by use of positron emission tomography (PET), and observed that the trafficking of this liposome was quite similar to that of non-targeted long-circulating liposome (PEG-Lip). Then, histochemical analysis of intratumoral distribution of both liposomes was performed by use of fluorescence-labeled liposomes. In contrast to in vivo trafficking, intratumoral distribution of both types of liposomes was quite different: APRPG-PEG-Lip was colocalized with angiogenic endothelial cells that were immunohistochemically stained for CD31, although PEG-Lip was localized around the angiogenic vessels. These results strongly suggest that intratumoral distribution of drug carrier is much more important for therapeutic efficacy than the total accumulation of the anticancer drug in the tumor, and that active delivery of anticancer drugs to angiogenic vessels is useful for cancer treatment.

Antiangiogenic photodynamic therapy (PDT) by using long-circulating liposomes modified with peptide specific to angiogenic vessels.

For the improvement of therapeutic efficacy in photodynamic therapy (PDT) by using a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), we previously prepared polyethylene glycol (PEG)-modified liposomes encapsulating BPD-MA (PEG-Lip BPD-MA). PEGylation of liposomes enhanced the accumulation of BPD-MA in tumor tissue at 3 h after injection of it into Meth-A-sarcoma-bearing mice, but, unexpectedly, decreased the suitability of the drug for PDT when laser irradiation was performed at 3 h after the injection of the liposomal photosensitizer. To improve the bioavailability of PEG-Lip BPD-MA, we endowed the liposomes with active-targeting characteristics by using Ala-Pro-Arg-Pro-Gly (APRPG) pentapeptide, which had earlier been isolated as a peptide specific to angiogenic endothelial cells. APRPG-PEG-modified liposomal BPD-MA (APRPG-PEG-Lip BPD-MA) accumulated in tumor tissue similarly as PEG-Lip BPD-MA and to an approx. 4-fold higher degree than BPD-MA delivered with non-modified liposomes at 3 h after the injection of the drugs into tumor-bearing mice. On the contrary, unlike the treatment with PEG-Lip BPD-MA, APRPG-PEG-Lip BPD-MA treatment strongly suppressed tumor growth after laser irradiation at 3 h after injection. Finally, we observed vasculature damage in the dorsal air sac angiogenesis model by APRPG-PEG-Lip BPD-MA-mediated PDT. The present results suggest that antiangiogenic PDT is an efficient modality for tumor treatment and that tumor neovessel-targeted, long-circulating liposomes are a useful carrier for delivering photosensitizer to angiogenic endothelial cells.

Intracellular target for photosensitization in cancer antiangiogenic photodynamic therapy mediated by polycation liposome.

Previous study indicated that antiangiogenic photodynamic therapy (PDT), laser irradiation at 15 min post-injection of photosensitizer in vivo, is effective for cancer treatment, and a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), encapsulated in polycation liposomes (PCLs), liposomes modified with cetylated polyethylenimine (cetyl-PEI), is more effective than BPD-MA encapsulated in non-modified liposomes [Cancer 97 (2003) 2027]. In the present study, we examined intracellular distribution of BPD-MA. BPD-MA encapsulated in liposomes or in PCLs was incubated with human endothelial cell line ECV304 cells or human umbilical vein endothelial cells (HUVECs), and monitored the intracellular distribution of BPD-MA by confocal laser scan microscopy. BPD-MA was taken up time-dependently into the cells and was distributed in not only cytoplasmic area but also intranuclear region. The enhanced uptake of BPD-MA was observed by the PCL formulation. Intracellular distribution of polycation was monitored by using fluorescein isothiocyanate-labeled cetyl-PEI (cetyl-PEI-FITC) and was colocalized with BPD-MA. Cytoplasmic BPD-MA distribution was partly overlapped with that of rhodamine 123, a mitochondrial fluorostaining probe, suggesting that mitochondrial photosensitization as well as nuclear photosensitization, is involved in the antiangiogenic PDT treatment.

Suppressed permeation of linoleic acid in a liposomal formulation through reconstructed skin tissue.

Since the liposomal formulation of linoleic acid (LA) exhibited an enhanced skin-whitening effect, the influence of liposomalization on the cutaneous absorption of LA was examined using a three-dimensional (3D) reconstructed skin model. Liposome entrapped [(14)C]-LA was applied on the skin model, and the permeation of LA through the skin was monitored. The permeation rate of LA in the liposomal formulation was found to be lower than that in the conventional formulation without liposomes, suggesting the increased retention time of LA in the skin by the liposomal formulation. Next, to investigate the dependence of the LA permeation on melanocyte conditions and intactness of the reconstructed skin model, the effect of UV irradiation on LA permeation was examined. Low-dose UVB irradiation (0.03 J/cm(2) for 3 times), which activated melanocytes in the skin, did not influence the extent of LA permeation, while high-dose irradiation (0.30 J/cm(2) for 3 times) enhanced the permeation of LA in both the conventional and liposomal formulation. The present results suggest the importance of skin intactness for LA permeation and that the 3D reconstructed skin model would be useful for evaluating the characteristics of skin-oriented cosmetics and drugs.