Increase in tumour permeability following TGF-beta type I receptor-inhibitor treatment observed by dynamic contrast-enhanced MRI.

BACKGROUND: To enhance the success rate of nanocarrier-mediated chemotherapy combined with an anti-angiogenic agent, it is crucial to identify parameters for tumour vasculature that can predict a response to the treatment of the anti-angiogenic agent. METHODS: To apply transforming growth factor (TGF)-beta type I receptor (TbetaR-I) inhibitor, A-83-01, to combined therapy, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was carried out in mice bearing colon 26 cells using gadolinium (Gd)-DTPA and for its liposomal formulation to evaluate changes in tumour microvasculature following A-83-01. Tumour vascular parameters from DCE-MRI were compared with histological assessment and apparent diffusion coefficient of water in tumour generated by diffusion-weighted MRI. RESULTS: Contrary to evaluations reported for anti-angiogenic agents, A-83-01 treatment increased the initial area under the Gd concentration-time curve (IAUGC60), volume transfer constant (K(trans)) and fractional plasma volume (v(p)) significantly within 24 h, that was positively related to alpha-smooth muscle actin-positive pericyte coverage and tumour cell proliferation, and was correlated inversely with the apparent diffusion coefficient. The vascular function of the tumour improved by A-83-01 treatment was well assessed on post-liposomal Gd-DTPA-enhanced MR images, which predicted delivery of a liposomal drug to the tumour. CONCLUSION: These findings suggest that DCE-MRI and, in particular, K(trans) and v(p) quantitation, provide important additional information about tumour vasculature by A-83-01 treatment.

Calcium enhanced delivery of tetraarginine-PEG-lipid-coated DNA/protamine complexes.

As we have previously reported the delivery of plasmid DNA (DNA) complexed with oligoarginine-PEG artificial lipids (oligoarginine/DNA complexes), we focused on tetra- and decaarginine (Arg4, Arg10) to improve transfection efficiency by both the formation of oligoarginine-coated DNA complexed with protamine (PD), and the addition of Ca(2+) after formation of complexes. The efficiency of DNA condensation was determined by gel electrophoresis. Cellular uptake and transfection efficiency were evaluated in human cervical carcinoma HeLa cells using flow cytometry and luciferase assay. Oligoarginine-coated PD enhanced transfection efficiency significantly more than complexes where Arg10 in both vectors exhibited higher transfection efficiency than Arg4. As assessed by gel retardation assay, high gene expression by Arg10 may be explained by Arg4 binding DNA more strongly than Arg10. The addition of Ca(2+) to incubation medium increased transfection efficiency of Arg4-coated PD 70-fold, similar to that of Arg10-coated PD alone without an increase of cellular uptake, suggesting that Ca(2+) induced the release of DNA from complexes in endosomes. Only Arg4 with low cytotoxicity could gain an advantage from Ca(2+) in transfection, but Arg10 with relatively high cytotoxicity could not. The present results demonstrate that Arg4-coated PD with Ca(2+) has great potential as an efficient non-viral vector with low toxicity.

The enhancing effect of nasal absorption of FITC-dextran 4,400 by beta-sitosterol beta-D-glucoside in rabbits.

The effect and mechanism of action of beta-sitosterol beta-D-glucoside (Sit-G) on the in vitro and in vivo nasal absorption of FITC-dextran (molecular weight, 4400; FD-4) in rabbits were studied in comparison with beta-sitosterol (Sit). The FD-4 permeation in the powder dosage form was increased by Sit-G and Sit and related to the uptake of Sit-G and Sit with no changes in the amount of cholesterol in the excised nasal mucosa. The application of Sit and Sit-G increased FD-4 permeation with and without a decrease in transepithelial resistance (TEER), respectively. These results suggested that the mechanism of the enhancement by Sit-G was different from those of Sit and sodium caprate; Sit-G may exert its effects mainly via the transcellular pathway due to perturbation of the mucosal membrane.

Antitumor efficacy in vitro and in vivo of falconensones, a new type of polyene.

Falconensones A and B are new type of yellow pigment isolated from the mycelial extract of ascomycetous fungi, Emericella falconensis. To date, these falconensones and their derivatives, falconensone A p-bromophenylhydrazone and falconensone A dioxime are known to exhibit biological activities, which include growth inhibition and both induction of differentiation and apoptosis of HL60 human leukemia cells. The synthetic derivatives have been shown to be more potent than natural falconensone A and B in eliciting these activities. Herein, we investigate whether falconensones inhibit growth of other cancer cell lines in vitro, and we evaluate their ability to modify survival in C57 BL/6J mice using M5076 murine reticulosarcoma in vivo, which is established as the metastasis model. Falconensone A, falconensone A p-bromophenylhydrazone, and falconensone A dioxime inhibit growth of human myeloid leukemia cell lines, HL60 and HL60R, human hepatoma cell line HepG2, human prostate cancer cell line DU-145, and human breast cancer cell line MCF-7/Adr(R), whereas falconensone B, the 4'-nor-methyl derivative of falconensone A, shows extremely low or no activity. In contrast, all of the falconensones are active in growth inhibition of human breast cancer cell line MCF-7. Survival time of M5076-implanted mice was prolonged by treatment with falconensones, particularly falconensone A dioxime. These results indicate that falconensone A and its derivatives exhibit anticancer efficacy in a broad spectrum of cancer cell lines. These agents may have great potential for clinical use in the treatment of various cancers.

Efficiency of liposomes surface-modified with soybean-derived sterylglucoside as a liver targeting carrier in HepG2 cells.

We investigated the interaction of liposomes surface-modified with soybean-derived sterylglucoside (SG) (SG-liposomes) with HepG2 cells in the point of involvement of asialoglycoprotein receptor (ASGP-R) mediated endocytosis and examined the efficiency of SG-liposomes as drug carriers using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) as a maker of liposome, carboxylated polystyrene microspheres (Fluoresbrite) as a model drug not taken up in cells and doxorubicin (DXR). SG-liposomes were composed of dipalmitoylphosphatidylcholine (DPPC), cholesterol (Ch) and SG (DPPC/Ch/SG=6:3:1, molar ratio) and DiI, Fluoresbrite and DXR were entrapped in SG-liposomes, respectively. Each SG-liposome was incubated with HepG2 cells at 4 or 37 degrees C, and co-incubated with asialofetuin (AF) as a competitor of ASGP-R. The association of DiI, Fluoresbrite or DXR entrapped in SG-liposomes with HepG2 cells at 37 degrees C was significantly higher than that in liposomes containing no SG. That of DiI and Fluoresbrite was reduced significantly by the incubation with AF, but that of DXR was not affected. These findings suggest that Fluoresbrite behaves like the lipid component of SG-liposomes, but DXR in SG-liposomes does not behave similar to the lipid component of SG-liposomes, thus, its drug behavior released from liposomes may be due to its physicochemical properties. SG-liposomes are potentially useful drug carriers to the liver, because the glucose residue may work as a kind of ligand for ASGP-R.

Liver-targeted gene transfer into a human hepatoblastoma cell line and in vivo by sterylglucoside-containing cationic liposomes.

We investigated the transfection efficiency of beta-sitosterol beta-D-glucoside (Sit-G)-containing liposome/DNA complex (Sit-G-liposome/DNA complex) for liver targeting. The Sit-G-liposome/DNA complex was composed of Tfx-20 reagent (Tfx), ie synthetic cationic lipid [N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediammonium iodide] with L-dioleoylphosphatidylethanolamine (DOPE), 3 beta[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol) and Sit-G with plasmid DNA. The in vitro studies were performed in HepG2 cells in serum-containing medium and the in vivo studies were carried out in the mice following intravenous injection. The Sit-G-liposome produced a Sit-G-liposome/DNA complex of relatively small size (100--250 nm). Transfection efficiency of the luciferase marker gene by Sit-G-liposome/DNA complex was increased in the presence of 10% serum in vitro, and was selectively high in the mouse liver reaching expression values up to an average of 14.9 pg luciferase/mg tissue protein, compared with Tfx/DNA complex, which showed approximately three-fold higher gene expression than Sit-G-liposome/DNA complex in vitro. High in vitro transfection efficiency by Sit-G-liposome/DNA complex seemed to be possible even with large lipid precipitates, whereas high in vivo activity seemed to be related to small and dispersed complexes. The interaction of liposome/DNA complexes with serum may be a key point to predict the in vivo efficiency of a liposome vector.

In vitro permeation studies of triamcinolone acetonide mouthwashes.

The effects of vehicle composition, contact time of mouthwash and cosolvent on permeation of triamcinolone acetonide (TA) were investigated in vitro using hamster cheek pouch mucosa and synthetic membranes. Mouthwashes containing 0.1% TA with and without the mucoadhesive carboxyvinyl polymer were formulated. Aqueous suspensions and Orabase were used as control formulations. The contact time of mouthwash was varied from 1 to 5 min. Ethanol was used as a cosolvent in various binary-water mixtures. TA was delivered to a significantly lesser extent to mucosal tissue by the mouthwash than by the aqueous suspension (P < 0.001), but to a higher extent than by the Orabase formulation (P < 0.001). No effects of contact time or the mucoadhesive polymer were observed on amount of TA accumulated in the mucosal membrane. These observations have suggested that the use of carboxyvinyl polymer and a high content of ethanol are not appropriate as vehicles for local drug delivery but are suitable for transmucosal drug carriers.

MODIFIED ETHANOL INJECTION METHOD FOR LIPOSOMES CONTAINING beta-SITOSTEROL beta-D-GLUCOSIDE.

A modified ethanol injection method for liposomes containing soybean phosphatidylcholine (SPC), cholesterol (Ch), beta-sitosterol beta-D-glucoside (Sit-G) and oleic acid (OA) was developed, that can produce homogeneous unilamellar liposomes without the use of sonication and dialysis. In this method, water is poured into a concentrated lipid-ethanol solution and then ethanol is removed in an evaporator. Dilution with water causes spontaneous formation of small and homogenous unilamellar vesicles from micellar aggregate. The size of liposomes can be controlled by the ratio of ethanol to water. OA and Sit-G were distributed at the surface of liposomes and were recognized by Concanavalin A, respectively. This easy and quick method for preparation of liposomes may be applicable in many areas.

Release property of progesterone from a mixed-base suppository consisting of Witepsol W35 and Witepsol E85.

The mechanism of drug release from progesterone suppositories that consist of two types of hard fat (Witepsol W35 and Witepsol E85) was investigated. The strength, the thermodynamic characteristics, the surface structures, the drug release property, methylene blue penetration into suppositories, and change of surface structure after the dissolution test were employed for detecting characteristics of progesterone suppositories. The formulation with a mixing ratio of Witepsol W35 and Witepsol E85 at a 1:1 ratio showed the maximum strength value. The peak temperature of the suppositories showed a tendency to increase with increases in the ratio of Witepsol E85. The maximum height of the profiles measured with laser microscopy, from 20.8 microm to 29.2 microm, reached a maximum after 3 h of the dissolution test. When the suppositories were immersed in pH 7.4 phosphate buffer containing 0.5% methylene blue at 37 degrees C, the penetrating area increased with time. The weight of the suppositories also increased with time. According to these findings, it was suggested that the release of drug from a mixed type of suppository containing progesterone was via the matrix and pores.

In vivo evaluation of doxorubicin carried with long circulating and remote loading proliposome.

Long circulating and remote loading proliposome (LRP-L) was a kind of transparent solution and composed of soybean phosphatidylcholine (SPC), cholesterol, polyethylene glycol derivative of distearoylphosphatidyl ethanolamine (PEG-DSPE) and oleic acid sodium salt. When LRP-L was mixed with 0.9% NaCl aqueous solution containing doxorubicin (DXR), liposomes formed and automatically loaded DXR, in which sonication and extruders were not needed. The average diameter of the liposomal DXR in saline was 129.0+/-1.9 nm and the encapsulation efficiency was 98.1+/-0.6%. The pharmacokinetics, biodistribution, acute toxicity and anticancer effect of DXR carried with LRP-L (LRP-L-DXR) were studied. The plasma concentration-time curves of DXR were best fitted to the triexponential decay curves. The area under the plasma concentration-time curve (AUC) of LRP-L-DXR was 22 and five times of free DXR (F-DXR) and conventional cardiolipin liposomal DXR (CL-DXR), respectively. Following i.v. administration, the biodistribution of LRP-L-DXR in the heart and the liver, unlike that of CL-DXR, was not greater than that of F-DXR. However, the biodistribution of LRP-L-DXR in the spleen was less than that of CL-DXR and greater than that of F-DXR. The acute toxicity of LRP-L-DXR was decreased compared with that of F-DXR. The anticancer effect of LRP-L-DXR was significantly increased compared with that of F-DXR in the ascitic M5076 tumor model of C57BL/6 mice and had no significant difference compared with that of doxorubicin HCl liposome injection (Doxil).

Pharmacokinetics and antitumor effect of doxorubicin carried by stealth and remote loading proliposome.

PURPOSE: The aim of the study was to prepare stealth and remote loading proliposome (SRP-L) to carry doxorubicin (DXR) and evaluate the pharmacokinetics, acute toxicity, and anticancer effect of DXR carried with SRP-L. METHODS: SRP-L was transparent solution. When SRP-L was injected into 0.9%, NaCl aqueous solution containing DXR. liposomes formed and automatically loaded DXR (SRP-L-DXR). The long circulation of SRP-L-DXR was evaluated using the pharmacokinetics of SRP-L-DXR. cardiolipin liposomal DXR (CL-DXR) and free DXR (F-DXR). The acute toxicity and anticancer effect of SRP-L-DXR were evaluated in C57BL/6 mice and murine hystocytoma M5076 tumor model. RESULTS: The average diameter of SRP-L-DXR in pure water was 112.9 +/- 8.6 (nm) and the encapsulation efficiency of SRP-L-DXR was 96.5 +/- 0.2% in pure water, 95.5 +/- 0.1% in 5% glucose and 98.01 +/- 0.6% in 0.9% NaCl. The plasma concentration of SRP-L-DXR was much higher than those of F-DXR and CL-DXR. Compared with that of F-DXR, the SRP-L-DXR had lower acute toxicity and its anticancer effects depended upon the therapeutic treatment. CONCLUSIONS: A novel proliposome (SRP-L) was developed, which could automatically load DXR and form SRP-L-DXR with excellent characteristics. SRP-L-DXR had lower acute toxicity but was not always more effective for the treatment of the ascitic M5076 than F-DXR.

The enhancing effect of soybean-derived sterylglucoside and beta-sitosterol beta-D-glucoside on nasal absorption in rabbits.

The aim of this study was to elucidate the efficiency of soybean-derived sterylglucoside (SG) and its main component beta-sitosterol beta-D-glucoside (Sit-G), as nasal absorption enhancers. Nasal administration of verapamil with SG and Sit-G showed the higher bioavailabilities (60.4 and 90.7%, respectively) than that with lactose (39.8%). It was clear that SG and Sit-G promoted the absorption of verapamil through nasal mucosa. To elucidate the mechanism, we measured the calcein leakage from liposomes by incubation with SG, Sit-G, oleic acid, soybean-derived sterol, and beta-sitosterol to investigate transcellular absorption and measured the changes in intracellular Ca2+ concentrations ([Ca2+]i) by Sit-G to analyze paracellular absorption. The large amount of calcein leakage induced by enhancers was consistent with an enhancement of bioavailability of verapamil and insulin following nasal administration (oleic acid < SG < Sit-G). Moreover, Sit-G increased [Ca2+]i in the medium containing Ca2+, but not in Ca2+ free medium. This result suggested that Sit-G increases the fluidity of the mucosal membrane and facilitates Ca2+ influx from extracellular sources. In conclusion, a possible explanation for SG and Sit-G to promote drug absorption, is that they may affect both paracellular pathway and transcellular pathways caused by pertubation of lipid.

Electron spin resonance studies of dipalmitoylphosphatidylcholine liposomes containing soybean-derived sterylglucoside.

The effects of soybean-derived sterylglucoside (SG) on the fluidity of liposomal membrane composed of dipalmitoylphosphatidylcholine (DPPC) were investigated compared with those of soybean-derived sterol (SS) and cholesterol (Ch) using an electron spin resonance spectrometer. Three kinds of liposomes were prepared in the molar ratio of DPPC/X=7/4, where X is SS, Ch or SG. The fluidity close to the polar head groups increased with an increase of temperature in the DPPC membrane containing SS, Ch and SG in the range 35 to 45 degrees C. Those near the hydrophobic end changed with an increase in temperature in liposomes containing SS, Ch and SG, which had a fluidizing effect on the DPPC membrane below the transition temperature (Tm, 41.9 degrees C) and a condensing effect over the Tm. The fluidizing effects of these compounds around 37 degrees C near the polar head group and the hydrophobic end increased in the following order: Ch < SG < or = SS and SS < Ch < SG, respectively. SG increased the fluidity of liposomal membrane dramatically above the Tm (35.4 degrees C). These results suggest that the high fluidity close to the hydrophobic end of the liposomal membranes around 37 degrees C, the decrease of Tm, and the sigmoidal nature of fluidity vs. temperature are important factors in the effectiveness of liposomes containing SG as a carrier of drugs.

High entrapment of insulin and bovine serum albumin into neutral and positively-charged liposomes by the remote loading method.

Remote loading of insulin and bovine serum albumin (BSA) into neutral or positively-charged liposomes by incubation under a transmembrane pH gradient or non-pH gradient was investigated. Trapping efficiencies in several incubation conditions were compared with those of the conventional reverse-phase evaporation vesicle method (c-REV method). For neutral liposomes, insulin could not be effectively loaded into the liposomes by incubation, regardless of the incubation conditions. The trapping efficiency of insulin into positively-charged liposomes was higher than that of neutral liposomes, especially by the pH-gradient method. Insulin could be loaded into positively-charged liposomes about twofold more efficiently than by the pH-gradient method, compared with the c-REV method. Insulin distributed more on the surface of liposomes by the pH-gradient method than by the c-REV method. BSA showed significantly higher affinity to positively-charged liposomes than to neutral ones by various methods. However, the transmembrane pH-gradient method did not increase BSA loading into liposomes, compared with the c-REV and the non-pH-gradient methods. Our results suggest that the pH-gradient method, combining electrostatic interactions, may be useful for preparation of liposomal insulin and that the high hydrophobicity of BSA may not increase the remote loading of BSA into liposomes by the pH-gradient method.

[Interactions of insulin with dipalmitoylphosphatidylcholine liposomes].

AIM: To study the interactions of insulin with dipalmitoylphosphatidylcholine liposomes. METHODS: The liposomes were prepared by reverse-phase evaporation vesicle method. The entrapped efficiency, size and distribution of the liposomes were determined, and the influences of insulin on entrapped efficiency, size and distribution of the liposomes were investigated. The influences of liposomes on the fluorescence emission spectra of insulin and the calcein leakage from the liposomes entrapped calcein induced by insulin were measured. RESULTS: Insulin has little influence on the size and distribution of the liposomes while the sizes of the liposomes were about 170-190 nm. The insertion of tyrosine of insulin into dipalmitoylphosphatidylcholine liposomes membrane was not deep. The insulin disturbed the liposomes membrane, induced the calcein leakage from the calcein-loaded liposomes. CONCLUSION: Amphiphilic, example insulin, may disturb the intact membrane of liposome through the interaction either hydrophobic or hydrophilic. The attention should be paid to the entrapment process of peptides into liposomes.

The relationship between the rigidity of the liposomal membrane and the absorption of insulin after nasal administration of liposomes modified with an enhancer containing insulin in rabbits.

The relationship between the rigidity of the liposomal membrane and the absorption of insulin after nasal administration of liposomes modified with an enhancer containing insulin was investigated for the nasal delivery of peptide drugs in rabbits. The rigid liposomal membrane makes liposomes stable, protecting insulin from enzymatic degradation. Soybean-derived sterol (SS) or its sterylglucoside (SG) was used as an enhancer. Dipalmitoylphosphatidylcholine (DPPC) liposomes modified with SG had increased fluidity of the hydrophobic group of the liposome bilayer compared with the liposomes modified with cholesterol (Ch) or SS, as shown by measurements of the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5,-hexatriene (DPH); however, the fluidity of the polar group of the liposome bilayer was decreased according to measurements of steady-state fluorescence anisotropy of dansylhexadecylamine (DSHA) at 37 degrees C. These findings suggest that the fluidity of the hydrophobic group of the liposome bilayer is responsible for the increase of liposomal leakage and instability of the liposomes. When insulin was administered nasally to rabbits as a solution, no hypoglycemic effect was observed. The administration of insulin contained in DPPC/SG (7/4, mole) liposomes with high fluidity caused a high glucose reduction of long duration (8 hr). DPPC/SS and DPPC/Ch (7/4) liposomes with low fluidity caused low glucose reductions. These results demonstrated that liposomes modified with SG can be useful as carriers of insulin administered nasally.

Uptake and release of budesonide from mucoadhesive, pH-sensitive copolymers and their application to nasal delivery.

Microparticles of novel, bioadhesive graft copolymers of polymethacrylic acid and polyethylene glycol (P(MAA-g-EG)) were prepared. The aims of this study were to investigate the uptake and release kinetics of budesonide from P(MAA-g-EG) in vitro as well as the pharmacokinetics following nasal administration of the polymer contained budesonide. The loading of budesonide into the pH-sensitive polymers was examined using various ethanol solutions. Ethanol was required for drug solubilization but hindered hydrogel swelling at pH 7.2. Maximum loading of the drug in the polymer was obtained using 25% ethanol solutions. The release of budesonide from the polymer swollen in 25% ethanol solutions obeyed classical Fickian release behavior after an initial rapid drug burst. For nasal administration of budesonide-containing P(MAA-g-EG) the plasma concentration of budesonide was kept constant following a peak concentration of the drug approximately 45 min after administration.

Distribution characteristics of entrapped recombinant human erythropoietin in liposomes and its intestinal absorption in rats.

Recombinant human erythropoietin (Epo) is frequently administered by intravenous (i.v.) injection for the clinical treatment of renal anemia. Oral (per os; p.o.) administration is desired as an alternative route to i.v. administration, and liposomes have been chosen as a drug carrier. We found previously that after a p.o. administration to rats of Epo entrapped in liposomes before gel filtration, the Epo was absorbed, but variability in the number of days of appearance and in the levels of pharmacological effects, i.e. , the peak of circulating reticulocyte counts (RTC), was observed. The purpose of the present study was to examine the distribution characteristics of Epo in liposomes and intestinal absorption of liposomal Epo in rats by using purified Epo entrapped in liposomes after gel filtration (Epo/liposomes). The distribution characteristics of Epo/liposomes were determined by measuring the Epo in liposomes by a radioimmunoassay, high-performance liquid chromatography and zeta potential measurements. We observed that the protein part of Epo was mostly entrapped in liposomes, and was not adsorbed by the liposomal membrane at middle and high Epo p.o. doses, but the zeta potential of the Epo/liposomes increased negatively with the increase in the Epo p.o. doses. These results suggest that the sialic acid part of Epo entrapped in liposomes may project out from liposomes, depending on the entrapped Epo concentration. Little Epo was adsorbed or penetrated into liposomes when it was added to empty liposomes. After the p. o. administration of Epo/liposomes, the peak of RTC appeared at a 2-day delay on day 6, without variation and without dose dependency in comparison with that after i.v. administration. These results suggest that one of the reasons for the variability may be because the non-entrapped Epo and/or Epo/liposomes itself affected the intestinal absorption of Epo/liposomes. In conclusion, Epo/liposomes without nonentrapped Epo may be clinically useful for the oral administration of Epo.

Remote loading of diclofenac, insulin and fluorescein isothiocyanate labeled insulin into liposomes by pH and acetate gradient methods.

Remote loading of the model drugs diclofenac, insulin and fluorescein isothiocyanate labeled insulin (FITC-insulin) into liposomes by formation of transmembrane gradients were examined. A trapping efficiency of almost 100% was obtained for liposomal diclofenac, by the calcium acetate gradient method, whereas liposomes prepared by the conventional reverse-phase evaporation vesicle method had 1-8% trapping efficiencies. Soybean-derived sterol was a better stabilizer of the dipalmitoylphosphatidylcholine bilayer membrane than cholesterol, as shown from trapping efficiencies and drug release. The pH gradient method resulted in a 5-50% of FITC-insulin liposomal trapping efficiency, while insulin could not be loaded by this method. Liposomes released calcein in response to insulin, showing insulin interacts with the liposomal membrane in the presence of a transmembrane gradient. The present work has demonstrated a remote loading method for weak acids such as diclofenac into liposomes by the acetate gradient method. From the result of remote loading of FITC-insulin into liposomes by the pH gradient method, this method may be available for the preparation of liposomal peptides.

Evaluation of shikonin on granulation tissue formation compared with carrageenan.

The effect of shikonin (SK) on granulation tissue formation was biochemically evaluated and the biological effect of SK on cotton pellet induced granulation tissue formation and the induction of hind paw edema was compared with that of lambda-carrageenan (carrageenan) in rats. The dry weight of granulation tissue formed was increased by SK. The amounts of hemoglobin and hydroxyproline in granulation tissue were also increased by SK. These results suggest that SK has an enhancing effect on the formation of granulation tissue, accompanied by the proliferation of capillaries and the increased production of collagen in rats. SK showed mild stimulation toward swelling when injected into the hind paws of rats, as did carrageenan, while it showed a marked enhancing effect on granulation tissue formation compared with carrageenan. These results suggest that the mechanism underlying the biological effect of SK on granulation tissue formation is different from that of carrageenan, though the mild stimulation by SK might still contribute in part to the enhancing effect on granulation tissue formation.