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The purpose of this study was to characterize the properties in vitro, i.e. release, degradation, hemolytic potential and anticancer activity, and in vivo disposition of all-trans-retinoic acid (ATRA) in rats after administration of ATRA-loaded micelle-like nanoparticles. The amphiphilic block copolymers consisted of a micellar shell-forming mPEG block and a core-forming PLA block. The mPEG-PLA nanoparticles prepared by an acetone volatilization dialysis procedure were identified as having core-shell structure by (1)H NMR spectroscopy. Critical association concentration, drug contents, loading efficiency, particle size and xi potential were evaluated. The release of ATRA from the nanoparticles and the degradation of PLA were found to be mostly associated with the compositions of the nanoparticles. ATRA release was faster at smaller molecular weight of copolymer and lower drug contents. In vitro, the incorporation of ATRA in mPEG-PLA nanoparticles reduced the hemolytic potential of ATRA. Furthermore, anticancer activity of ATRA against HepG2 cell was increased by encapsulation, which showed an enhancement of tumor treatment of ATRA. In vivo, after intravenous injection to rats, the levels of ATRA in the blood stream and the bioavailability were higher for ATRA-loaded mPEG-PLA nanoparticles than those for ATRA solution. In conclusion, the structure of the mPEG-PLA diblock copolymer could be modulated to fit the demand of in vitro and in vivo characterizations of nanoparticles. The mPEG-PLA nanoparticles' loading ATRA have a promising future for injection administration.

As reported previously, cationic liposomes formulated with dioleoylphosphatidylethanolamine (DOPE) and N,N-methyl hydroxyethyl aminopropane carbamoyl cholesterol (MHAPC-liposomes) achieved efficient gene transfection in the mouse lung following intratracheal injection. We have studied here the role of surfactants, mannosylerythritol lipid-A (MEL-A) and polysorbate 80 (Tween 80), in affecting gene transfection of MHAPC-lipoplexes (complex with pCMV-luc DNA) in A549 cells and in the mouse lung. MEL-A increased gene transfection of MHAPC-lipoplexes significantly in vitro and slightly in the mouse lung, while Tween 80 decreased it both in vitro and in vivo. As assessed by confocal laser scanning microscopy and fluorescence imaging, MEL-A might faciliate gene dissociation from MHAPC-lipoplexes with fluorescein-labeled oligodeoxynucleotide (FITC-ODN) after internalization into the cells and retained the lipoplexes in the mouse lung for prolonged time, while Tween 80 was inefficient to deliver foreign gene into target cells and in the lung. These results demonstrated that MEL-A is advantageous to Tween 80 in the modification of cationic liposomes as gene delivery vectors in the lung.

The surface properties of cationic liposomes and lipoplexes largely determine the cellular association and gene transfection efficiency. In this study, we measured the surface properties, such as zeta potentials, surface pH and hydration levels of MHAPC- and OH-Chol-lipoplexes and their cellular association, without and with the modification of biosurfactant mannosylerythritol lipid-A (MEL-A) or Tween 80 (MHAPC=N,N-methyl hydroxyethyl aminopropane carbamoyl cholesterol; OH-Chol=cholesteryl-3beta-carboxyamindoethylene-N-hydroxyethylamine). Compared to OH-Chol-lipoplexes, the higher cellular association of MHAPC-lipoplexes correlated with the significantly higher zeta potentials, lower surface pH levels and "drier" surface, as evaluated by the generalized polarization of laurdan. Both MEL-A and Tween 80 modification of MHAPC-lipoplexes did not significantly change zeta potentials and surface pH levels, while MEL-A modification of OH-Chol-lipoplexes seriously decreased them. MEL-A hydrated the liposomal surface of MHAPC-lipoplexes but dehydrated that of OH-Chol-lipoplexes, while Tween 80 hydrated those of MHAPC- and OH-Chol-lipoplexes. In all, cationic liposomes composed of lipids with secondary and tertiary amine exhibited different surface properties and cellular associations of lipoplexes, and modification with surfactants further enlarged their difference. The strong hydration ability of Tween 80 may relate to the low cellular association of lipoplexes, while the dehydration of MEL-A-modified OH-Chol-lipoplexes seemed to compensate the negative zeta potential for the cellular association of lipoplexes.

The efficacy of folate-targeted liposomal drug delivery has not been fully achieved in part because of the slow release of the encapsulated drugs following uptake of the liposomes by target cells. Since liposomal mitoxantrone (MXN) composed of lipids with high fluidity was reported to achieve strong anticancer effects in vivo, we hypothesized that folate-targeted liposomal MXN uptake via folate receptor (FR)-mediated endocytosis could effectively release drugs into the endosomal compartment. Folate-targeted liposomal MXN was prepared using two lipids with different fluidities. MXN was released slowly from all types of liposome into PBS, indicating that the cellular uptake of MXN was considered to be in the liposomal form. Folate-targeted liposomes with high fluidity exhibited lower cellular uptake of loaded FITC-labeled dextran into FR (+) KB cells, but, when MXN was loaded, higher cytotoxicity than liposomes with lower fluidity. On the other hand, the cellular uptake of non-folate liposomes was not affected by the membrane fluidity, but higher cytotoxicity was observed in liposomal MXN with high fluidity, which suggested a higher rate of release of the drug from the liposomes. High levels of cytotoxic activity were achieved with folate-targeted liposomal MXN though the cellular uptake rate was restricted by selecting liposomes with higher lipid membrane fluidity. This finding provides a new insight into folate-targeted carrier drug delivery.

Purpose: Tumor cell targeting is a promising strategy for enhancing the therapeutic potential of chemotherapy agents. Polyethylene glycol (PEG)-coated (sterically stabilized) liposomes show enhanced accumulation on the surface of tumors, but steric hindrance by PEGylation reduces the association of the liposome-bound ligand with its receptor. To increase folate receptor (FR) targeting, we optimized the concentration and PEG spacer length of folate-PEG-lipid in liposomes., Experimental Design: Three types of folate-linked liposomal doxorubicin were designed and prepared by optimizing the concentration and PEG spacer length of folate-PEG-lipid in PEGylated or non-PEGylated liposomes and by masking folate-linked liposomes where the folate ligand is "masked" by adjacent PEG spacers. The liposome targeting efficacy was evaluated in vitro and in vivo., Results: In human oral carcinoma KB cells, which overexpress FR, modification with sufficiently long PEG spacer and a high concentration of folate ligand to non-PEGylated liposomes increased the FR-mediated association and cytotoxicity more than with PEGylated and masked folate-linked liposomes. On the contrary, in mice bearing murine lung carcinoma M109, modification with the folate ligand in PEGylated and masked folate-linked liposomes showed significantly higher antitumor effect than with non-PEGylated liposomes irrespective of the length of time in the circulation after intravenous injection., Conclusions: The results of this study will be beneficial for the design and preparation of ligand-targeting carriers for cancer treatment.

The RNA interference (RNAi) effect is an alternative technology to antisense DNA as an experimental method of down-regulating a specific target protein. Optimal gene therapy for tumors must deliver synthetic small interfering RNA (siRNA) to tumor cells with high efficiency and minimal toxicity. Previously, we reported that cationic nanoparticles composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 (NP-OH) could deliver plasmid DNA with high transfection efficiency. In this study, to apply NP-OH for siRNA transfection, we optimized the charge ratio (+/-) of NP-OH/siRNA and nanoplex-forming solution, and evaluated the transfection efficiency into PC-3 cells. Positively charged nanoplex prepared in the presence of sodium chloride exhibited efficient siRNA transfer. The distribution of siRNA after transfection was strongly detected both in the cytoplasm and nucleus. Furthermore, we demonstrated that NP-OH nanoplex of bcl-2 siRNA significantly inhibited tumor growth compared with control siRNA, and the efficacy was comparable to commercial products. The results of the experiments showed that NP-OH nanoparticles have potential as a viable vector candidate for synthetic siRNA delivery.

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by synthetic small interfering RNA (siRNA), and is utilized in a wide range of fields including cancer gene therapy by down-regulating a specific target protein. In this study, for tumor-targeted siRNA delivery, we developed a folate-linked nanoparticle (NP-F), and evaluated the potential of NP-F-mediated tumor gene therapy in human nasopharyngeal KB cells, which overexpressed folate receptor (FR). NP-F was composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine (OH-Chol), Tween 80 and folate-poly(ethylene glycol)-distearoylphosphatidylethanolamine conjugate (f-PEG(2000)-DSPE), and NP-P was substituted f-PEG(2000)-DSPE in NP-F PEG(2000)-DSPE for a non-targeting nanoparticle. The NP-F and siRNA complex (nanoplex) formed at a charge ratio (+/-) of 2/1 in the presence of 5mM NaCl was injectable size and increased transfection efficiency in the cells. NP-F showed a significantly higher intracellular amount of siRNA and stronger localization of siRNA in the cytoplasm than NP-P. When Her-2 siRNA was transfected into cells by NP-F and NP-P, NP-F significantly inhibited tumor growth, and selectively suppressed Her-2 protein expression more than NP-P. In in vivo gene therapy, a NP-F nanoplex of Her-2 siRNA by intratumoral injection significantly inhibited tumor growth of KB xenografts compared with control siRNA, but a NP-P nanoplex did not. These results of the experiments have provided optimal conditions to form folate-linked nanoparticle complexes with siRNA for folate-targeted gene therapy.

To investigate the use of folate-targeted nanoemulsion-loaded aclacinomycin A (ACM) to folate receptor (FR)-positive cells, we attempted to optimize the targeting ability of nanoemulsions by modifying the chain length and amount of the folate-PEG linker. Folate-linked, nanoemulsion-loaded ACM were formulated with 0.24 mol% of folate-poly (ethylene glycol)(3400)- (folate-PEG(3400)-) and folate-PEG(5000)-distearoylphosphatidylethanolamine (DSPE), and 0.03 mol% of folate-PEG(5000)-DSPE in nanoemulsions. Selective FR-mediated uptake was achieved in a human nasopharyngeal tumor cell line, KB, which overexpresses FR, but not in a human hepatoblastoma cell line, (FR(-)) HepG2. At the same amount of folate modification, the association with KB cells was increased with increasing the PEG-chain length. The association of 0.03 and 0.24 mol% folate-PEG(5000)-linked nanoemulsions with cells was 5- and 3.3-fold higher than that of non-folate nanoemulsion, respectively, while their cytotoxicity was similar. Both 0.03 and 0.24 mol% folate-PEG(5000)-linked nanoemulsions and non-folate nanoemulsion following intravenous injection inhibited tumor growth more significantly than ACM solution on day 24 following tumor inoculation (p < 0.01). This study demonstrates that a folate-linked nanoemulsion is feasible for tumor-targeted ACM delivery, and that folate modification with a sufficiently long PEG-chain and a small amount of nanoemulsion is an effective way of targeting nanoemulsion to tumor cells.

We investigated the effect of amido- (NP-OH) and carbamate linkers (NP-HAPC) in nanoparticles composed of hydroxyethylated cationic cholesterol on siRNA transfection. The presence of NaCl in forming a NP-OH nanoplex increased the suppressive effect of gene expression by increasing the size of the nanoplex and changing the cellular uptake mechanism from membrane fusion and clathrin-mediated endocytosis to clathrin- and caveolae-mediated endocytosis.

One obstacle to effective gene therapy lies in low transfection efficiency by non-viral vectors. To meet this challenge, we developed cell-penetrating peptide-based gene delivery vectors. A novel oligoarginine lipid ((Arg)n-B, n=4, 10) conjugated to 3,5-bis(dodecyloxy)benzamide (BDB) lipid with a poly(ethylene glycol) (PEG) spacer was synthesized. Oligoarginine lipid-coated vector was prepared by the addition of (Arg)n-B to DNA/protamine complex (PD) ((Arg)n-B-PD). Transfection efficiency of (Arg)n-B-PD was compared with that of (Arg)n-B/DNA complex ((Arg)n-B/D) for in vitro and in xenograft tumor of human cervical carcinoma HeLa by intratumoral injection. Transfection efficiency in tumors and in vitro greatly depended on the charge ratios of (Arg)n-B to DNA and the length of Arg residues. In vitro, positively charged Arg10-B-PD showed the highest transfection efficiency. In contrast, in tumor transfection, negatively charged Arg4-B-PD showed the highest transfection efficiency, about 2-, 16- and 23-fold higher than PD alone, Arg10-B-PD and a commercial gene transfection reagent, respectively. This result suggests that negatively charged tetraarginine-conjugated-PEG lipid-coated PD is a promising gene delivery vector for intratumoral injection.

A novel technique was developed for the formation of ligand-targeted polymeric micelles that can be applicable to various ligands. For tumor-specific drug delivery, camptothecin (CPT)-loaded polymeric micelles were modified by folate to produce a folate-receptor-targeted drug carrier. Folate-linked PEG5000-distearoylphosphatidylethanolamine (folate-PEG5000-DSPE) was added when preparations of drug-loaded polymeric micelles, resulting in folate ligands exposed to the surface. Folate-modified CPT-loaded polymeric micelles (F-micelle) were evaluated by measuring cellular uptake using a flow cytometer, fluorescence microscopy, and confocal laser scanning microscopy, and by cytotoxicity measurement. The results revealed that F-micelle showed higher cellular uptake in KB cells overexpressing folate receptor (FR) and higher cytotoxicity compared with non-folate modified CPT-loaded polymeric micelles (plain micelles) in KB cells, but not in FR-negative HepG2 cells. This result indicated that polymeric micelles were successfully modified by the folate-linked lipid.

Cationic lipid-based gene delivery systems have shown promise in transfecting cells in vitro and in vivo. However, liposome/DNA complexes tend to form aggregates after preparation. Lyophilization of these systems, therefore, has become of increasing interest. In this study, we investigated the feasibility of preserving complexes as a dried preparation using a modified dehydration rehydration vesicle (DRV) method as a convenient and reliable procedure. We also studied storage stability of a lyophilized novel cationic gene delivery system incorporating sucrose, isomaltose and isomaltotriose. Liposomes were composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and L-dioleoylphosphatidylethanolamine (DOPE), plus sucrose, isomaltose or isomaltotriose. Lyophilized liposome/DNA complexes were stored at -20, 25, 40 and 50 degrees C and their stability was followed for 50 days. Liposome/DNA complexes with sucrose could be stored even at 50 degrees C without large loss of transfection efficiency. The transfection efficiency of formulations stored at various temperatures indicated that the stabilizing effect of sugars on plasmid DNA was higher in the following order: isomaltotriose

Camptothecin (CPT) is a strong antitumor agent, but its use limited by its low solubility and the instability of the active lactone form. To overcome these difficulties, liposomes incorporating CPT (CPT liposomes) were designed and tested. CPT liposomes were formulated by the addition of 3,5-bis(dodecyloxy)benzoic acid (DB) to polyethylene glycol-containing liposomes, and by coating the surface of the liposomes with human serum albumin (HSA, HSA-DB-L). HSA-DB-L successfully entrapped CPT with about 80% efficiency and with a particle size of about 150 nm. HSA-DB-L showed attenuated drug release and storage stability. Pharmacokinetics studies in mice showed that i.v. injection of HSA-DB-L (2.5 mg/kg) led to prolonged circulation in the plasma; the area under the curve was 22-fold higher than that of CPT solution. The tumor growth in mice with subcutaneous transplantation of colon 26 tumor cells was significantly inhibited after a single i.v. injection of HSA-DB-L at a dose of 15 mg/kg without any significant body weight loss. HSA-DB-L increased the accumulation of CPT in tumor tissue significantly (9.6-fold) more efficiently than CPT solution 24 h after i.v. injection. These findings suggest that HSA-DB-L could increase the stability and the antitumor effect of CPT. CPT delivery by novel liposome formulations is a potential approach for effective treatment of cancer.

Camptothecin (CPT) has anticancer activity. While only the lactone form of CPT is biologically active, this form exhibits poor aqueous solubility. Pharmaceutical formulation of CPT incorporated in liposomes is of significant importance to develop the therapeutic utilization of CPT. The aim of this study was to increase incorporation efficiency and stability of CPT in liposomes composed of hydrogenated soybean phosphatidylcholine, cholesterol, and oleic acid (7 : 3 : 1, molar ratio), by incorporating three kinds of artificial lipids (DBs) (DB-liposome); 4-n-(M12B), 3,5-bis(B12B) and 3,4,5-tris(dodecyloxy)benzoic acid (T12B). The interaction of CPT with DB in the state of liposomes, was examined. In DB-liposomes presenting mean diameters of 150 nm, incorporation efficiency of CPT up to 55% and final drug to lipid molar ratio up to 0.07 were obtained when the liposomes were prepared at a feeding ratio of 1/30 (w/w) CPT/total lipid. However, in the optimal formulations, incorporated DB mol% was different; T12B and D12B were incorporated about one third and half mol% of M12B, respectively. Moreover, we demonstrated that T12B stabilized CPT in liposomes significantly compared with other DBs as measured by CPT release, and by steady state fluorescence polarization degree of CPT using intrinsic fluorescence of CPT. These findings suggested that in addition of contribution of phenyl group of DB, dodecyloxy group may interact strongly with lactone ring of CPT. The capacity to contain CPT interacted with DBs may be limited in liposomes. T12B may be incorporated in the interior of the bilayers, resulting in increase of incorporation stability of CPT. This finding demonstrates a potential application of the novel liposome formulation of CPT in drug delivery.

Oligoarginine conjugates are highly efficient vectors for the delivery of plasmid DNA into cells. Decaarginine-conjugated lipid (Arg10-PEG-lipid) was synthesized and the effects of Arg10-PEG-lipid concentration at a fixed DNA concentration on transfection efficiency and the structure of the complexes were studied below and above critical micelle concentration (CMC), and at the lipid nitrogen/DNA phosphate (N/P) ratio corresponding to transfection, respectively. Arg10-PEG-lipid at the concentration below CMC showed stronger interaction with DNA by fluorescence intensity distribution analysis, and significantly higher luciferase and green fluorescent protein expression than that above CMC. A phase-contrast cryo-transmission electron microscope (cryo-TEM) experiment showed that the morphology of the complexes depended on the N/P ratio. At a low N/P ratio corresponding to that in transfection at a lipid concentration below CMC, a net-like structure developed in which plasmid DNA was involved. A further increase in the N/P ratio, a large fibrous nanostructure of complexes, was also observed. Without DNA, these structures were not obtained. The cellular uptake mechanism of complexes using flow cytometry with inhibitors suggested that complexes with two different morphologies showed similar cellular uptake and uptake mechanism, macropinocytosis. Differences in transfection efficiency of the complexes may be explained by a large fibrous nanostructure inhibiting the cellular internalization of complexes or the release of DNA from macropinosomes into cytoplasm. Arg10-PEG-lipid/DNA complexes formed a favorable nanostructure for gene delivery, depending on the N/P ratio in water.

The objectives of this study were to describe the pharmacokinetics and tissue distribution of superparamagnetic iron oxide nanoparticle (SPIO) stabilized with alginate (SPIO-alginate), and investigate its potential in detecting liver cancers as a newly developed magnetic resonance (MR) contrast agent. Pharmacokinetics and tissue distribution of SPIO-alginate were investigated in Sprague-Dawley rats. The results showed that SPIO-alginate was eliminated rapidly from serum with the half-life of 0.27 h at 109.5 micromol Fe/kg and accumulated dominantly in liver and spleen with a total percentage of more than 90% of dose after intravenous injection. The studies of pharmacokinetics and distribution of SPIO-alginate in rats indicated the MR contrast agent, based on SPIO, mainly accumulating in targeting organs that contain phagocytosing cells, i.e. liver and spleen. The efficacies in detecting hepatocellular carcinoma (HCC) of rat with primary liver cancer and xenograft liver cancers of rabbit were investigated before and after injection of SPIO-alginate. The signal intensity of liver parenchyma in rabbit with VX2 tumor after injection of SPIO-alginate was reduced sharply resulting in a significant contrast between liver parenchyma and tumor. Detection of the HCC in rat model was also demonstrated. The present study provides evidence that SPIO-alginate might have the ability to improve the detection of liver tumors as an MR contrast agent, and the efficacy is associated with the SPIO specifically located in Kupffer cells in hepatic sinusoid.

Three cationic cholesterol derivatives (CCDs), which differ in their types of amine and bear a hydroxyethyl group at the amine group, were synthesized and formulated into liposomes and nanoparticles as gene delivery vectors. In vitro transfection into A549 cells proved that liposomes formulated with CCDs and dioleoylphosphatidylethanolamine (DOPE) of 1/2 molar ratio were more effective than the corresponding nanoparticles with CCDs and Tween 80 at charge ratios (+/-) of 1/2, 3/1 and 5/1. Among the liposomal formulations, non-hydroxyethylated CCDs were more effective than hydroxyethylated ones in vitro. However, gene transfection in the lung through intratracheal injection showed opposite results to those in vitro, with liposomes containing hydroxyethylated CCDs being more potent than those containing non-hydroxyethylated CCDs. Transfection by liposomes with N,N-methyl hydroxyethyl aminopropane carbamoyl cholesterol iodide (MHAPC) showed the highest luciferase activity, resulting in 2- and 60-fold higher gene expression than jet-PEI and naked DNA, respectively. The distribution of MHAPC lipoplex after intratracheal injection was heterogeneous, and luciferase was expressed in epithelial cells lining the bronchi and bronchioles. All the lipoplexes led to higher TNF-alpha levels in the lung compared to the nanoplex and jet-PEI, but our findings suggested that modification of the cationic cholesterol with a hydroxyethyl group at the tertiary amine terminal, MHAPC, promoted gene expression in the lung without increasing the toxicity compared with other CCDs. This work firstly proved that liposomes containing hydroxyethylated CCDs could promote gene expression in the lung through intratracheal injection.

The aim of the present study was to investigate whether direct activation of protein kinase C (PKC) in the spinal cord could change brain activation using a functional magnetic resonance imaging (fMRI) analysis in mice that lack the PKCgamma gene. The activation of spinal PKC by intrathecal (i.t.) injection with phorbol 12,13-dibutyrate (PDBu), a specific PKC activator, caused a time-dependent decrease in paw-withdrawal latency to the heat thermal stimulus. In contrast, i.t. injection of PDBu failed to cause thermal hyperalgesia in mice which lacked the PKCgamma gene. We found that the i.t. injection with PDBu caused a remarkable increase in the activity of several brain regions in wild-type mice compared with vehicle injection. In the somatosensory cortex and lateral and medial thalamus, i.t. injection of PDBu produced a dramatic and time-dependent increase in signal intensity at 1-6h after i.t. PDBu injection. In contrast, i.t. injection of PDBu produced a delayed but significant increase in signal intensity at 3-6h in the cingulate cortex, at 4-6h in the nucleus accumbens and at 3-6h in the ventral tegmental area. In addition, all effects of PDBu were abolished in mice that lacked the PKCgamma gene. These results suggest that the activation of spinal PKCgamma associated with the activation of ascending pain transmission may be an important factor in chronic pain-like hyperalgesia with changes in emotionality.

Ferrofluids are attractive candidates for magnetic targeting system because of their fluidity and magnetism. The magnetic nanoparticles in ferrofluids should have combined properties of superparamagnetic behavior, target localization, and biocompatibility. The magnetic targeting and biocompatibility of superparamagnetic iron oxide nanoparticles stabilized by alginate (SPION-alginate) was investigated in vitro and in vivo. The localization of SPION-alginate by an external magnetic field in vitro was quantitatively evaluated by determining the iron content, and the results revealed that the localization ratio of SPION-alginate was 56%. Magnetic targeting of the SPION-alginate after femoral artery administration with the magnetic field in rats was quantitatively investigated by iron content and qualitatively confirmed by histological evaluation and magnetic resonance imaging. The ratio of iron content between the target site and the nontarget site were 8.88 at 0.5 h and 7.50 at 2 h, respectively. The viability of RAW264.7 cells and L929 cells was apparently unaltered upon exposure to SPION-alginate. The incubation with erythrocytes indicated that the SPION-alginate did not induce erythrocytes hemolysis and aggregation. In conclusions, the SPION-alginate had magnetic targeting with an external magnetic field and did not be detained at the injection site without the magnetic field. The SPION-alginate was generally considered to be biocompatible in cytotoxicity and hemolysis aspects., ((c) 2007 Wiley Periodicals, Inc.)

Fenretinide, N-(4-hydroxyphenyl)retinamide (4-HPR) is an aminophenol-containing synthetic retinoid derivative of all-trans-retinoic acid, which is a potent chemopreventive and antiproliferative agent against various cancers. Clinical studies of 4-HPR have shown side effects consisting of night blindness and ocular toxicity. To maintain potent anticancer activity without side effects, p-dodecylaminophenol (p-DDAP) was designed based on structure-activity relationships of 4-HPR. In our study, we investigate whether p-DDAP shows anticancer activity against human prostate cancer cell line PC-3 when compared with 4-HPR. p-DDAP inhibited PC-3 cell growth progressively from low to high concentration in a dose-dependent manner. p-DDAP was the most potent antiproliferative agent in vitro among 6 p-alkylaminophenols and 3 4-hydroxyphenyl analogs examined including 4-HPR. Cells treated with p-DDAP were shown to undergo apoptosis, based on condensation nuclei, cytofluorimetric analysis, propidium iodide staining and the expression of bcl-2 and caspase 3. p-DDAP arrested the S phase of the cell cycle, while 4-HPR arrested the G(0)/G(1) phase. In addition, both the i.v. and i.p. administration of p-DDAP suppressed tumor growth in PC-3-implanted mice in vivo. p-DDAP showed no effects on blood retinol concentrations, in contrast to reductions after 4-HPR administration. These results indicate that p-DDAP exhibits excellent anticancer efficacy against hormonal independent prostate cancer in vitro and in vivo, and it may have great potential for clinical use in the treatment of prostate cancer with reduced side effects., ((c) 2007 Wiley-Liss, Inc.)

Background: Adiponectin (Adipo), an adipocyte hormone involved in the regulation of glucose and lipid metabolism, has already been identified as a potential therapeutic target for the treatment of diabetes. However, successful delivery of Adipo to the receptors is difficult due to their peptide characteristics. Receptors for Adipo are abundantly expressed in the liver and skeletal muscle., Methods: Uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) in hepatoblastoma HepG2 cells expressing Adipo was examined. Adipo-expressing plasmid DNA (10-50 microg) in saline solution (0.1 ml/g body weight) was rapidly injected into the tail vein of 4-week-old diabetic mice after 4-6 weeks of treatment with streptozotocin (STZ). Uptake of glucose in diabetic mice also was measured using a planar positron imaging system featuring 18-fluorodeoxyglucose., Results: HepG2 cells expressing Adipo exhibited significantly increased 2-NBDG uptake compared with cells transfected with control plasmid even in the absence of insulin. STZ-induced diabetic mice showed decreased serum Adipo levels compared with non-diabetic mice. A single hydrodynamic injection of 10-50 microg Adipo-expressing plasmid DNA into diabetic mice led to approximately 10-15-fold elevation in serum Adipo levels, and resulted in decreased serum levels of glucose and triglyceride. As well as exhibiting higher levels of Adipo expression, diabetic mice also had higher hepatic glucose uptake than similar mice injected with control plasmid., Conclusions: We report that STZ-induced diabetic mice exhibited decreased Adipo levels and hyperglycemia which may be alleviated by hydrodynamic injection of the Adipo gene. This type of gene delivery system to the liver offers a different approach in developing novel treatments for type 1 and 2 diabetes., (2007 John Wiley & Sons, Ltd)

Partially benzyl-esterified poly(ethylene glycol)-b-poly(aspartic acid) (PEG-P(Asp(Bzl))) having different hydrophobic inner-core structure were synthesized and analyzed. We obtained two types of the block copolymers for formation of polymeric micelle drug carriers; one had an amide-bond ratio of 1:3 (alpha/beta) in the poly(aspartic acid) residues through alkaline hydrolysis, and the other one had 100% of the alpha-amide through acid hydrolysis. Subsequently, we prepared partially benzyl-esterified block copolymers with an esterification degree of 40 to 100% in the aspartic acid residue. Regarding camptothecin (CPT) incorporation into polymeric micelles, we evaluated effects that block copolymers' inner hydrophobic block structures have on CPT behavior. Regarding CPT-incorporation stability, PEG-P(alpha,beta-Asp(Bzl) block copolymers with the alpha and beta-amides were found to exhibit higher CPT-incorporation stability. Using fluorescent probes, we evaluated the properties of inner-core blocks such as hydrophobicity and mobility/rigidity, and the findings implied that stable CPT incorporation could be obtained by an adequate balance between the micelle inner core's hydrophobicity and the micelle inner core's rigidity or between the micelle inner core's hydrophobicity and steric configuration of the hydrophobic block chain.

Cationic liposomes composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposome, molar ratio, 1:1 or 3:2) prepared by the dry-film method have been often used as non-viral gene delivery vectors. The formulation and preparation of DC-Chol/DOPE liposomes, as well as the formation of their lipoplexes were investigated in an attempt to improve transfection efficiency in vitro. A more efficient transfection in medium with serum was achieved using DC-Chol/DOPE liposomes (molar ratio, 1:2) than those (3:2), and preparation method by a modified ethanol injection than the dry-film. The most efficient DC-Chol/DOPE liposome for gene transfer was molar ratio (1:2) and prepared by a modified ethanol injection method. The enhanced transfection might be related to an increase in the release of DNA in the cytoplasm by the large lipoplex during incubation in optiMEM, not to an increased cellular association with the lipoplex. The use of a modified ethanol injection method might enhance the role of DOPE that is aid in destabilization of the plasma membrane and/or endosome. These findings suggested that cationic liposomes rich in DOPE prepared by a modified ethanol injection method will help to improve the efficacy of liposome vector systems for gene delivery.

Both polyethylenimine (PEI) polymers and cationic nanoparticles have been widely used for non-viral DNA transfection. Previously, we reported that cationic nanoparticles composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80 (NP-OH) could deliver plasmid DNA (pDNA) with high transfection efficiency. To increase the transfection activity of NP-OH, we investigated the potential synergism of PEI and NP-OH for the transfection of DNA into human prostate tumor PC-3, human cervices tumor Hela, and human lung adenocarcinoma A549 cells. The transfection efficiency with low-molecular PEI (MW 600) was low, but that with a combination of NP-OH and PEI was higher than with NP-OH alone, being comparable to commercially available lipofectamine 2,000 and lipofectamine LTX, with very low cytotoxicity. Low-molecular weight PEI could not compact pDNA in size, but rather might help to dissociate pDNA from the complex and release pDNA from the endosome to cytoplasm by the proton sponge effect. Therefore, the combination of cationic cholesterol-based nanoparticles and a low-molecular PEI has potential as a non-viral DNA vector for gene delivery.

Optimal gene therapy for tumors must deliver DNA to tumor cells with high efficiency and minimal toxicity. It has been reported that in non-viral gene delivery, the hydroxyethyl group at the amino terminal in cationic lipid was important for high transfection efficiency. Therefore, in this study, we developed new cationic nanoparticles (NP-OH) composed of cholesteryl-3beta-carboxyamidoethylene-N-hydroxyethylamine and Tween 80, and optimized in vitro and in vivo transfections for potential use as a non-viral DNA vector into human prostate tumor PC-3 cells and xenografts. In vitro transfection resulted in efficient DNA transfer when positive-charged nanoplex was prepared in the presence of sodium chloride (NaCl). In in vivo transfection, negative-charged nanoplex formed in water strongly induced the gene expression compared with positive-charged nanoplex when directly transfected into xenografts. These transfection efficiencies in vitro and in vivo were comparable to each commercial product. Furthermore, NP-OH nanoplexes displayed no induction of tumor necrosis factor (TNF)-alpha when administered by intravenous injection. The results of the experiments provided optimal conditions to form NP-OH nanoplex for gene delivery in vitro and in vivo. NP-OH is a potential non-viral DNA vector for the local treatment of tumor and in vitro.

To enhance the efficacy of ganciclovir/herpes simplex virus thymidine kinase (GCV/HSV-TK) suicide gene therapy for nasopharyngeal cancer KB, we developed long-circulating liposome-encapsulated GCV, and evaluated cytotoxicity in vitro and in vivo. PEGylated liposome-encapsulated GCV (PEG-GCV-lipo) was prepared by the freeze-thawing method. In vitro experiments demonstrated that GCV from liposomes was gradually released over a period of 3 days. The in vitro cytotoxicity of PEG-GCV-lipo was similar to that of GCV solution in human cervical carcinoma HeLa cells expressing HSV-TK. Pharmacokinetics studies in mice showed that, compared with GCV solution, intravenous and intraperitoneal injection of PEG-GCV-lipo (10 mg/kg) led to long circulation in plasma; the area under the curve was 36-fold or 32-fold higher than that of GCV solution, respectively. In GCV/HSV-TK suicide gene therapy, the HSV-TK gene complexed with nanoparticle vector was directly injected into KB xenografts, and PEG-GCV-lipo or GCV solution was injected intravenously in mice once a day (25 mg/kg/day every 2nd day, 4 times). PEG-GCV-lipo was significantly 3-fold more effective than GCV solution in inhibiting tumor growth and produced durable complete tumor remissions on day 11 after injection. These findings demonstrate that long-circulating liposome-encapsulated GCV is a new approach to drug carriers to enhance the efficacy of suicide gene therapy.

Dysregulation of connexin expression is believed to have a role in carcinogenesis, because levels of connexin are reduced in various tumors. We examined the role of connexin 43 (Cx43) alone and combined with a histone deactylase (HDAC) inhibitor in tumor growth inhibition. The transfection of Cx43 plasmid DNA (pCMV-Cx43) into human nasopharyngeal cancer KB cells using folate-linked nanoparticles induced inhibition of cell growth. Cx43 induced a tumor suppressive effect via a gap junctional intercellular communication-independent mechanism. The transfection of pCMV- Cx43 along with an HDAC inhibitor, 4-phenylbutyrate (4-PB), enhanced Cx43 expression greatly in vitro, and inhibited significantly the tumor growth of KB cells and xenografts compared with that of pCMV-Cx43 alone. 4-PB induced increased expression of genes of DNA damage checkpoints and of apoptosis via the down-regulation of anti-apoptotic bcl-2 mRNA expression and up-regulation of the activity of the apoptosis-associated enzyme caspase-3/7. Thus, the amplified Cx43 expression by an antitumor agent, an HDAC inhibitor, may have great potential as a growth inhibitor for nasopharyngeal tumors.

SPION with appropriate surface chemistry have been widely used experimentally for numerous in vivo applications. In this study, SPION stabilized by alginate (SPION-alginate) were prepared by a modified coprecipitation method. The structure, size, morphology, magnetic property and relaxivity of the SPION-alginate were characterized systematically by means of XRD, TEM, ESEM, AFM, DLS, SQUID magnetometer and MRI, respectively, and the interaction between alginate and iron oxide (Fe(3)O(4)) was characterized by FT-IR and AFM. The results revealed that typical iron oxide nanoparticles were Fe(3)O(4) with a core diameter of 5-10 nm and SPION-alginate had a hydrodynamic diameter of 193.8-483.2 nm. From the magnetization curve, the Ms of a suspension of SPION-alginate was 40 emu/g, corresponding to 73% of that of solid SPION-alginate. This high Ms may be due to the binding of Fe(3)O(4) nanoparticles to alginate macromolecule strands as visually confirmed by AFM. SPION-alginate of several hundred nanometers was stable in size for 12 months at 4 degrees C. Moreover, T1 relaxivity and T2 relaxivity of SPION-alginate in saline (1.5 T, 20 degrees C) were 7.86+/-0.20 s(-1) mM(-1) and 281.2+/-26.4 s(-1) mM(-1), respectively.

Previously we have described the development and applications of lipid-based nanoparticles for gene delivery vector. In an attempt to improve transfection efficiency using the cell adhesion of extracellular matrix (ECM) to DNA/lipid complex (nanoplex), the mRNA expression of integrin alpha2beta1 and CD44 in prostate cancer cells was detected as adhesion molecules for fibronectin (Fn), collagen I (Col) and laminin (Lam) using a commercially available cDNA array (GEArray) system. These ECM proteins could enhance DNA transfection activity in cells when coated on the nanoplex. Among the ECM proteins, Fn-coating nanoplexes significantly increased transfection activity 2-fold in prostate cancer PC-3 cells, and exhibited higher DNA transfection activities to PC-3 xenografts, compared with commercially available cationic polymer in vivo jetPEI. These results indicated that Fn-coating nanoplexes could facilitate efficient transfection of prostate tumor cells.

In order to deliver antisense oligonucleotides (asODN) into hepatocytes orientedly in the treatment of hepatitis B virus (HBV) infection, the liver-targeting cationic liposomes was developed as a gene carrier, which was co-modified with the ligand of the asialoglycoprotein receptor (ASGPR), beta-sitosterol-beta-d-glucoside (sito-G) and the nonionic surfactant, Brij 35. Flow cytometry (FCM) analysis and enzyme-linked immunosorbent assay (ELISA) showed that the asODN-encapsulating cationic liposomes exhibited high transfection efficiency and strong antigens inhibition effect in primary rat hepatocytes and HepG2.2.15 cells, respectively. With the help of several inhibitors acting on different steps during the targeting lipofection, the cellular uptake mechanisms of the co-modified liver-targeting cationic liposomes were investigated through antigens inhibition effect assay and confocal laser scanning microscopy (CLSM) analysis. The cellular uptake with high transfection efficiency seemed to involve both endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance ASGPR-mediated endocytosis, the nonionic surfactant Brij 35 seemed to be able to facilitate membrane fusion, and the co-modification resulted in the most efficient transfection but no enhanced cytotoxicity. These results suggested that the co-modified liver-targeting cationic liposomes would be a specific and effective carrier to transfer asODN into hepatocytes infected with HBV orientedly.

Docetaxel (DTX) is used for the treatment of advanced hormone refractory prostate cancer. Connexin 43 (Cx43) is a tumor suppressor gene, and transfection of the Cx43 gene increases sensitivity to several chemotherapeutic agents. The objective of this study was to evaluate the effectiveness of combination therapy of Cx43-expressing plasmid DNA (pCMV-Cx43) and DTX both in vitro and in vivo using a non-viral vector in human prostate cancer PC-3 cells. Transfection of pCMV-Cx43 into the cells neither inhibited tumor growth nor increased gap junctional intercellular communication; however, combination therapy of pCMV-Cx43 and DTX significantly inhibited cell growth. Forced expression of Cx43 in the cells induced apoptotic cells by down-regulation of Bcl-2 expression and significantly more up-regulation of caspase-3 activity than either treatment alone. The combination of repeated intratumoral injection of pCMV-Cx43 (10 microg/tumor) with non-viral vector and a single intravenous injection of DTX (15 mg/kg) was compared with a repeated injection of Cx43 alone and a single injection of DTX alone on PC-3 tumor xenografts. Significant antitumoral effects were observed in mice receiving combined treatment, compared with DTX alone. The data presented here provide a rational strategy for treating patients with advanced hormone refractory prostate cancer.

A two-step transcriptional amplification system (TSTA) was used to enhance the efficacy of suicide gene therapy for treatment of prostate cancer. We designed a TSTA system and constructed two types of plasmid: one containing GAL4-VP16 fusion protein under the control of a tumor-specific promoter, the other containing luciferase or herpes simplex virus thymidine kinase (HSV-tk) under the control of a synthetic promoter. The TSTA systems using nanoparticles based on lipids were evaluated by measuring the amount of induced luciferase activity as a function of prostate-specific membrane antigen (PSMA) and midkine (Mk) promoters, specific for LNCaP and PC-3 prostate cancer cells, respectively. In LNCaP cells that were PSMA-positive, the TSTA system featuring the PSMA enhancer and promoter exhibited activity that was 640-fold greater than a system consisting of one-step transcription with the PSMA promoter. In contrast, this difference in activity did not occur in PSMA-negative PC-3 cells. In Mk-positive PC-3 cells, the TSTA system with the Mk promoter exhibited a five-fold increase in activity over one-step transcription, but such activity was not induced in Mk-negative LNCaP cells. When using HSV-tk for suicide gene therapy, TSTA systems featuring the PSMA or Mk promoter inhibited in vitro cell growth in the presence of ganciclovir. Furthermore, the TSTA system featuring the Mk promoter suppressed in vivo growth of PC-3 tumor xenografts to a greater extent than one-step transcription. These findings show that TSTA systems can enhance PSMA and Mk promoter activities and selectively inhibit PC-3 cell growth in tumors. This suggests that TSTA systems featuring tumor-specific promoters are suitable for cancer treatment by gene therapy.

Background: The most extensively investigated strategy of suicide gene therapy for treatment of cancer is the transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene followed by administration of antiviral prodrugs such as acyclovir (ACV) and ganciclovir (GCV). The choice of the agent that can stimulate HSV-TK enzymatic activity is one of the determinants of the usefulness of this strategy. Previously, we found that a diterpenoid, scopadulciol (SDC), produced a significant increase in the active metabolite of ACV. This suggests that SDC may play a role in the HSV-TK/prodrug administration system., Methods: The anticancer effect of SDC was evaluated in HSV-TK-expressing (TK+) cancer cells and nude mice bearing TK+ tumors. In vitro and in vivo enzyme assays were performed using TK+ cells and tumors. The phosphorylation of ACV monophosphate (ACV-MP) was measured in TK- cell lysates. The pharmacokinetics of prodrugs was evaluated by calculating area-under-the-concentration-time-curve values., Results: SDC stimulated HSV-TK activity in TK+ cells and tumors, and increased GCV-TP levels, while no effect of SDC was observed on the phosphorylation of ACV-MP to ACV-TP by cellular kinases. The SDC/prodrug combination altered the pharmacokinetics of the prodrugs. In accord with these findings, SDC enhanced significantly the cell-killing activity of prodrugs. The bystander effect was also significantly augmented by the combined treatment of ACV/GCV and SDC., Conclusions: SDC was shown to be effective in the HSV-TK/prodrug administration system and improved the efficiency of the bystander effect of ACV and GCV. The findings will be considerably valuable with respect to the use of GCV in lower doses and less toxic ACV. This novel strategy of drug combination could provide benefit to HSV-TK/prodrug gene therapy., (2006 John Wiley & Sons, Ltd.)

Activated macrophages are the key effector cells in rheumatoid arthritis (RA) and secrete multiple mediators of inflammation including proinflammatory cytokines. We investigated delivery of a nuclear factor kappa B (NFkappaB) decoy by folate-linked lipid-based nanoparticles (NP-F) into murine macrophages. The expression of folate receptor (FR) in RAW264.7 cells activated by lipopolysaccaride was confirmed by strong expression of FR mRNA, and association of FITC-labeled folate-BSA conjugate. When transfected via NP-F, the NFkappaB decoy was strongly detected in the cytoplasm, and an inhibitory effect on the translocation of NFkappaB into the nucleus was observed at 0.03 microM of the decoy, suggesting that NP-F effectively delivered the NFkappaB decoy into the cytoplasm. This information is of value for the design of NFkappaB decoy carrier systems targeting FR in activated macrophages in gene therapy for autoimmune diseases such as RA.

The intracellular delivery of proteins using cell-penetrating peptides (CPPs) including oligoarginine (oligo-Arg) carriers raises the possibility of establishing novel therapeutic methods. We compared the effect of the length of oligo-Arg in modified liposomes ((Arg)(n)-L; n = 4, 6, 8, 10) on the delivery of proteins by flow cytometry, fluorescence microscopy, and spectrofluorimetry. As a free liposome, Arg4-modified liposome Arg4-L was most efficiently internalized in cells. The efficiency decreased depending on the length of oligo-Arg. For the intracellular delivery of proteins, (Arg)(n)-L was physically associated with proteins. Concerning the effect of oligo-Arg length, liposome/protein complexes showed a different behavior. Arg4-L carried bovine serum albumin (BSA, 66 kDa) and beta-galactosidase (beta-Gal, 120 kDa) 6-fold higher than free BSA and free beta-Gal. Arg10-L showed similar performance for these two proteins to Arg4-L. The enzymatic activity of beta-Gal in the cells showed that proteins were transported as a biologically active form. Arg10-L carried 100-fold more immunoglobulin G (IgG, 150 kDa) than free IgG, and 3-fold more than Arg4-L into cells. Shorter oligo-Arg chain on liposomes may be enough for liposomes alone to be taken up in cells, but more Arg residues may be needed to form a complex with high molecular weight proteins and deliver them into cells. This information will aid in the design of (Arg)(n)-L as a carrier for delivering proteins into cells.

Beta-sitosterol 3-beta-D-glucoside (Sit-G), an absorption enhancer, was incorporated into ultra-deformable vesicles containing bleomycin to attenuate drug toxicity in human keratinocytes. The presence of Sit-G increased drug entrapment and improved in vitro stability of ultra-deformable vesicles. Confocal laser scanning microscopy revealed the extent to which Sit-G facilitated the penetration of ultra-deformable vesicles containing fluorescent probes into rat skin upon non-occlusive topical application. Furthermore, treatment with preparations incorporating Sit-G resulted in elevated epidermal and dermal concentrations of bleomycin. Ultra-deformable formulation contained Sit-G maintained flexibility for penetration through the skin, increased entrapment efficiency of bleomycin and stability in vitro, and significantly increased distribution of bleomycin in epidermis and dermis compared with those without Sit-G.

The design, synthesis, and evaluation of in vitro gene delivery efficacy of a novel series of oligo-Arg-lipid conjugates are described. 3,5-Bis(dodecyloxy)benzamide (BDB) was employed as the lipid component, and a poly(ethylene glycol) (PEG) spacer was introduced between the C-terminal of oligo-Arg and the amide group of BDB. Four derivatives with various oligo-Arg lengths (ArgN-PEG-BDB; N = 4, 6, 8, 10: the number of arginine residues) were prepared, and the effect of oligo-Arg length on the gene transfection was investigated in HeLa cells. Transfection efficiency increased as the number of arginine residues increased. Arg10-PEG-BDB showed the highest transfection efficiency, without severe toxicity to cells. These findings well corresponded to the cellular association of the Arg-PEG-BDB/DNA complex determined by flow cytometry. Even in the presence of serum, Arg10-PEG-BDB achieved appreciable cellular association and attained high gene expression. Thus, Arg10-PEG-BDB is potentially a simple and useful gene delivery tool, because one need only to mix it with plasmid DNA and apply the complexes to the cells even in a serum-containing medium.

A water-insoluble antitumor agent, camptothecin (CPT) was successfully incorporated into polymeric micelles formed from poly(ethylene glycol)-poly(benzyl aspartate) block copolymers (CPT-loaded polymeric micelles). Antitumor effects and biodistribution of CPT-loaded micelles were evaluated in mice subcutaneously transplanted by colon 26 tumor cells. Tumor growth was significantly inhibited after a single i.v. injection of CPT-loaded polymeric micelles at doses of either 15 or 30 mg/kg. Efficacy of a single high-dose injection was comparable to low dose multiple injections. CPT loaded in polymeric micelles showed prolonged blood circulation and higher accumulation in tumors compared with CPT in solution. Polymeric micelle systems offer a stable and effective platform for cancer chemotherapy with CPT.

Mannnosylerythritol lipid A (MEL-A), a biosurfactant produced by microorganisms, has many biological activities. To enhance the gene transfection efficiency of a cationic liposome, we prepared a MEL-liposome (MEL-L) composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), dioleoyl phosphatidylethanolamine (DOPE) and MEL-A, and investigated its transfection efficiency in human cervix carcinoma Hela cells. MEL-L was about 40 nm in size, and the MEL-L/plasmid DNA complex (MEL-lipoplex) remained an injectable size (169 nm). MEL-A induced a significantly higher level of gene expression, compared to commercially available Tfx20 and the liposome without MEL-A (Cont-L). Analysis of flow cytometric profiles clearly indicated that the amount of DNA associated with the cells was rapidly increased and sustained by addition of MEL-A to the liposome. Confocal microscopic observation indicated that the MEL-lipoplex distributed widely in the cytoplasm, and the DNA was detected strongly in the cytoplasm and around the nucleus, compared with Cont-L. These results suggested that MEL-A increased gene expression by enhancing the association of the lipoplexes with the cells in serum. MEL-L might prove a remarkable non-viral vector for gene transfection and gene therapy.

The objective of the study was to investigate the feasibility of a single-dose hepatitis B vaccine based on three kinds of poly (D, L)-lactide-co-glicolide acid (PLGA) microspheres. PLGA microspheres loaded with recombinant hepatitis B surface antigen (HBsAg) were formulated using a double emulsion microencapsulation technique. The pharmaceutical characteristics of size, surface morphology, protein loading efficiency, antigen integrity, release of HBsAg-loaded PLGA microspheres and degradation of the polymer in vitro were evaluated. The degradation of the polymer corresponded with the composition of the polymer (lactide/glycolide ratio), molecular weight of the polymer (viscosity) and morphology of the microspheres. These PLGA microspheres were able to continuously release antigen under conditions that mimic the environment in vivo. The single subcutaneous injection of HBsAg-loaded PLGA50/50 microspheres, PLGA75/25 microspheres and a mixture of PLGA50/50, PLGA75/25, and PLGA50/50-COOH microspheres in mice resulted in comparable serum antibody titers to those of three injections of the conventional aluminum adjuvant formulated HBsAg vaccine. Based on these findings in vitro and in vivo, it was concluded that HBsAg was successfully loaded into the PLGA microspheres, which can auto-boost an immune response, and the HBsAg-loaded PLGA microsphere is a promising candidate for the controlled delivery of a vaccine.

To improve its aqueous solubility and stability in biological fluid, CPT was physically loaded in polymeric micelles. Polymeric micelles were composed of various poly(ethylene glycol)-poly(aspartate ester) block copolymers (PEG-P(Asp(R))). The incorporation and circulation stability of CPT micelles were evaluated by measuring the CPT in micelle using gel-permeation chromatography and by CPT concentration measurement after intravenous injection using HPLC, respectively, in terms of chemical structure of block copolymers. The stability of CPT-loaded micelles in vivo depended on the amount of benzyl esters, and length of PEG in the polymers to a greater degree than it did in vitro. A stable formulation of CPT-loaded micelles was obtained using PEG-P(Asp) with PEG of 5,000 (MW), 27 Asp units, and 57-75% benzyl esterification of Asp residue. This CPT-loaded micelles showed about a 17-fold lower blood clearance value than unstable micelles. The CPT-loaded micelles are potentially delivered to tumor sites owing to an extended circulation in the blood stream.

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as beta-sitosterol beta-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.

A novel cationic liposome modified with soybean sterylglucoside (SG) and polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE) as a carrier of antisense oligodeoxynucleotide (ODN) for hepatitis B virus (HBV) therapy was constructed. Characteristics of the cationic liposomes modified with SG and PEG (SG/PEG-CL) and their complexes with 15-mer phosphorothioate ODN (SG/PEG-CL-ODN complex) were investigated by incorporation efficiency, morphology, electrophoresis, zeta potentials, and size analysis. Antisense activity of the liposomes and ODN complexes was determined as hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in HepG2 2.2.15 cells by ELISA. Their tissue and intrahepatic distribution were evaluated following intravenous injection in mice. The complexes gained high incorporation efficiency and intact vesicular structure with mean size at approximately 200 nm. The SG/PEG-CL-ODN complexes enhanced the inhibition of both HBsAg and HBeAg expression in the cultured HepG2 2.2.15 cells relative to free ODN. The uptake of SG/PEG-CL and nonmodified cationic liposomes (CL) was primarily by liver, spleen, and lung. Furthermore, the concentration of SG/PEG-CL was significant higher than that of CL in hepatocytes at 0.5 hr postinjection. The biodistribution of SG/DSPE-CL-ODN complex compare with free ODN showed that liposomes enhanced the accumulation of ODN in the liver and spleen, while decreasing its blood concentration. SG/PEG-CL-mediated ODN transfer to the liver is an effective gene delivery method for cell-specific targeting, which has a potential for gene therapy of HBV infections. SG and PEG-modified cationic liposomes have proven to be an alternative carrier for hepatocyte-selective drug targeting.

For targeted gene delivery to human prostate cancer LNCaP and PC-3 cells and nasopharyngeal cancer KB cells, we developed a folate-linked nanoparticle (NP-F), and evaluated the potential of NP-F-mediated suicide gene therapy in the cells and xenografts with herpes simplex virus thymidine kinase (HSV-tk) and connexin 43 (Cx43). An NP-F-plasmid DNA complex (NP-F nanoplex) showed high DNA transfection efficiency in KB, LNCaP and PC-3 cells. Cell growth inhibition in the presence of ganciclovir (GCV) was enhanced with HSV-tk and Cx43 genes in LNCaP cells. In suicide gene therapy, the tumor growths of KB and LNCaP xenografts were significantly inhibited when an NP-F nanoplex of the HSV-tk gene, and HSV-tk and Cx43 genes, respectively, was injected intratumorally and GCV was administered intraperitoneally. These findings suggested that the NP-F is a potential target vector in prostate and nasopharyngeal cancer for suicide gene therapy.

Formulations of ultra-deformable liposomes containing bleomycin (Bleosome) have previously been described and proposed for topical treatment of skin cancer [Lau, K.G., Chopra, S., Maitani, Y., 2003. Entrapment of bleomycin in ultra-deformable liposomes. S. T. P. Pharm. Sci. 13, 237-239]. In this study, the stability of various Bleosome formulations was characterised and a purification process was established to isolate Bleosome for testing on cultures of either human cutaneous keratinocytes (NEB-1) immortalised by human papilloma virus (HPV)-type 16, or a spontaneously immortalised human squamous cell carcinoma (SCC) from a primary tumour. Bleosome facilitated entrapment of high concentrations of active bleomycin and samples purified by gel-filtration chromatography remained stable during 7 days of storage at 4 degrees C or at room temperature. Serially-diluted samples of this purified, high-strength product, 'high dose' were applied onto keratinocyte cell cultures to elucidate Bleosome LD50 profiles. In vitro data revealed that the LD50 of bleomycin encapsulated in Bleosome was approximately three-fold higher than free bleomycin solution for SCC cells, and nearly 30 times higher for NEB-1 cells. However, Bleosome containing 30 microg/ml of active bleomycin killed more than twice as many SCC cells than NEB-1 cells. At that concentration, the potency of liposomal bleomycin on causing cell death of SCC cells was found to be similar to that of free bleomycin solution. This effect was not seen on NEB-1 cells. It seems that SCC cells were particularly susceptible to Bleosome containing high levels of bleomycin. Results from these experiments promote the development of a novel product for the topical treatment of skin cancer.

In the present study, cholesteryl hemisuccinate (CHEMS) was evaluated for use as a membrane stabilizer in dipalmitoylphosphatidylcholine (DPPC) liposomes. Differential scanning calorimetry (DSC) and a calcein release study showed that CHEMS was more effective than cholesterol (CHOL) in increasing DPPC membrane stability. The findings of Fourier transform infrared spectroscopy (FT-IR) also suggested that CHEMS interacts with DPPC via both hydrogen bonding and electrostatic interaction. More importantly, CHEMS did not interact with saikosaponin-d (SSD), a triterpene saponin from Bupleurum species, unlike CHOL. SSD-containing liposomes with DPPC, CHEMS and DSPE-PEG could greatly decrease the hemolytic activity of SSD. This study demonstrated that CHEMS has more stabilization ability than CHOL since CHEMS may exhibit both hydrogen bond interaction and electrostatic interaction with DPPC membrane while CHOL only has hydrogen bond interaction, resulting in stable and low-hemolytic SSD-liposomes.

Cancer gene therapy has been intensively developed using non-viral vectors, among which cationic liposomes and nanoparticles are the most thoroughly investigated. For targeted delivery to tumors, vitamin folic acid has been utilized for folate receptor (FR)-mediated drug delivery, since FR is frequently overexpressed on many types of human tumors. Liposomes conjugated to folate ligand have been used as carriers of chemotherapeutic agents and DNA to receptor-bearing tumor cells in vitro. As an alternative treatment for prostate cancer, suicide gene therapy by local injection using an adenoviral vector has been reported, but not that using non-viral vectors. The folate-linked, lipid-based nanoparticles which we developed could deliver genes extensively to FR-negative LNCaP and PC-3 cells, as well as FR-positive KB and Hela cells. In this review, we outline folate-linked liposomes and nanoparticles, and show the effectiveness of folate-linked, lipid-based nanoparticles as a vector for DNA transfection and for suicide gene therapy, to treat human nasopharyngeal and prostate tumors.

Stability of CPT free drug and CPT-loaded polymeric micelles forming from poly (ethylene glycol)-poly (benzyl aspartate-69) block copolymer in the presence of serum and purified serum albumins were investigated by reverse-phase HPLC and GPC. The hydrolysis of CPT and CPT-loaded micelles follows pseudo-first-order kinetics. The observed hydrolysis rate constants for CPT and CPT-loaded micelles were 7.4x10(-3)min(-1) and 0.7x10(-3)h(-1), corresponding to an increase in half-life of CPT from 94 min to 990 h, respectively. The half-lives of CPT lactone hydrolysis of CPT-loaded micelles in the presence of BSA were significantly longer than the control whereas in the presence of HSA and serum was shorter than the control, and the similar results were obtained from GPC analyzed for micelles stability. This result suggested that the stability of CPT-loaded micelles was significantly decreased only in the presence of human albumin and serum. These were corresponded to the results of CPT free drug observed in the presence of albumins or serum. BSA significantly retarded the CPT lactone ring opening as compared with the control. On the other hand, HSA and serum showed rapid CPT lactone ring opening. This was probably due to preferential HSA binding to the carboxylate form resulting in a change in the lactone-carboxylate equilibrium, whereas, BSA did not bind to the lactone form, but might promote the self-aggregation of CPT and binding to the hydrophobic inner core of the micelles, resulting in enhanced stability of CPT-loaded micelles. MSA did not affect the stability of micelles.

Purpose: To establish a novel formulation tumor-targeted drug carrier of lipophilic antitumor antibiotics, aclacinomycin A (ACM), folate-linked microemulsions were prepared and investigated both in vitro and in vivo., Experimental Design: Three kinds of folate-linked microemulsions with different polyethylene glycol (PEG) chain lengths loading ACM were formulated with 0.24 mol% folate-PEG(2000)-distearoylphosphatidylethanolamine (DSPE), folate-PEG(5000)-DSPE, and folate-lipid (without PEG linker) in microemulsions. In vitro studies were done in a human nasopharyngeal cell line, KB, which overexpresses the folate receptor (FR), and a human hepatoblastoma cell line, [FR(-)] HepG2. In vivo experiments were done in a KB xenograft by systemic administration of folate-linked microemulsions loading ACM., Results: The association of folate-linked microemulsions to KB cells could be blocked by 2 mmol/L free folic acid. Selective FR-mediated cytotoxicity of folate-linked microemulsions loading ACM was obtained in KB but not in HepG2 cells. The association of the folate-PEG(5000)-linked microemulsion and folate-PEG(2000)-linked microemulsion with the cells was 200- and 4-fold higher, whereas their cytotoxicity was 90- and 3.5-fold higher than those of nonfolate microemulsion, respectively. The folate-PEG(5000)-linked microemulsions showed 2.6-fold higher accumulation in solid tumors 24 hours after i.v. injection and greater tumor growth inhibition than free ACM., Conclusion: These findings suggest that a folate-linked microemulsion is feasible for tumor-targeted ACM delivery. This study shows that folate modification with a sufficiently long PEG chain on emulsions is an effective way of targeting emulsion to tumor cells.

Recent advances in biotechnology have promoted biomolecular targeting of drugs, peptides and genes in the treatment and management of major diseases and infections. Therapeutic development of drugs and delivery systems may have various objectives: Systemic drugs require optimal delivery and uptake at target sites; peptide drugs require alternative routes of administration, such as nasal or intestinal absorption; gene medicines need to be delivered efficiently, safely and selectively to diseased areas. The propensity of ligand-modified liposomes to carry drugs and genes to desirable sites has been extensively examined and current reports show considerable progress in this field. Sterylglucoside (SG) is a novel absorption-enhancer of peptide drugs across nasal and intestinal mucosae. Physico-chemical properties and biodistribution of liposomes incorporating SG were studied and compared against the profiles of aglycon and sitosterol derivatives of SG. It was shown that SG particles aided colon drug delivery and increased bioavailability of peptide drugs after nasal and intestinal administration. In addition, they were able to enhance anticancer effects in liver cancer chemotherapy. Biological fate and interaction of SG with hepatocytes support the novel proposition of liver-targeting SG-liposomes.