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Surface PEGylation on nanoparticles has greatly helped prolong their blood circulation half-lives. However, The injection of PEGylated nanoparticles into mice induced poly(ethylene glycol) (PEG)-specific IgM antibodies (anti-PEG IgMs), significantly changing PEG-liposomes' pharmacokinetics. In this study, we used various PEG-conjugates to conduct a mechanistic study of anti-PEG IgMs' binding behavior. The conventional belief has been that anti-PEG IgMs bind to PEG main chains; however, our findings reveal that anti-PEG IgMs did not bind to PEG main chains, whereas anti-PEG IgMs did bind to PEG-hydrophobic polymer blocks. The insertion of a hydrophilic polymer between each PEG chain and each hydrophobic polymer block suppressed anti-PEG IgMs' binding. We prove here that hydrophobic blocks are essential to anti-PEG IgMs' binding, and also that anti-PEG IgMs do not bind to intact PEGs without hydrophobic moiety. These results support our conclusion that anti-PEG IgMs exhibit specificity to PEG; however, the presence of a hydrophobic block at a proximity position from each PEG chain is essential for the binding. Also in the present study, we elucidate relations between anti-PEG IgMs and PEGylated nanoparticles. In one of our previous studies, anti-PEG IgMs scarcely affected the pharmacokinetics of PEG-b-poly(β-benzyl l-aspartate) block copolymer (PEG-PBLA) micelles, whereas anti-PEG IgMs significantly decreased PEG-liposomes' blood circulation half-life. Finally, we found that the ratio of anti-PEG IgM molecules to PEG-liposome particles is critical to these pharmacokinetic changes, and that a 10-fold increase in the number of anti-PEG IgM molecules permitted them to capture the PEG-liposome particles, thus leading to the aforementioned changes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We evaluated structural factors characterizing PEG-b-P(Asp-Bzl) micelles including core size, aggregation number (Nagg), and core surface PEG density by means of small-angle X-ray scattering (SAXS), field flow fractionation with multi-angle light scattering (FFF-MALS) analysis, and DLS. Furthermore, we evaluated the stability of PEG-b-P(Asp-Bzl) micelles by means of GPC. This paper reports the correlation between the evaluated micelles' structural factors and the micelles' behaviors including the micelles' in vivo pharmacokinetic behaviors. One micelle PEG(12)-b-P(Asp-Bzl) (PEG=12,000) exhibited a high core surface density (~0.99 chain/nm(2)). In these circumstances, PEG(12)-b-P(Asp-Bzl) micelles exhibited a highly stretched PEG brush form. However, the evaluated core surface PEG densities could not fully explain the micelles' in vivo pharmacokinetic behaviors. In contrast, GPC will become a strong tool for predicting PEG(12)-b-P(Asp-Bzl) micelles' in vivo behaviors, as well as the micelles' in vitro behaviors. The stability results correlated strongly with the area-under-the-curve (AUC) values of PEG-b-P(Asp-Bzl) micelles' in vivo pharmacokinetics. Finally, we evaluated PEG(12)-b-P(Asp-Bzl) micelles' most effective structural factor for determining the micelles' behaviors, and the micelles' outermost shell surface's PEG density (DOS, PEG) correlated with the micelles' behaviors. We revealed that the evaluated DOS, PEG is the most important factor for understanding PEG(12)-b-P(Asp-Bzl) micelles' behaviors., (Copyright © 2015. Published by Elsevier B.V.)

The development of antiviral agents that have novel mechanisms of action is urgently required in the topical therapy of herpes simplex virus type 2 (HSV-2) infections. We reported previously that topical application of branched 3610-Da polyethylenimine (PEI) exhibited preventative antiviral activity. In this study, to develop therapeutic anti-HSV-2 agents, the most potent PEI combined with ~200 nm-sized liposomes with or without oleic acid (liposomes/PEI) was selected in vitro and further evaluated using in vivo studies. The mechanism of action in vivo was elucidated using PEIs with decreased numbers of primary amine residues, resulting from ethylene carbonate treatment, and polyallylamine, a linear polyamine consisting of primary amines. Cytotoxicity and antiviral activity in vitro, and the appearance of acute herpetic disease and virus yields in mice intravaginally administered with liposomes/PEI were evaluated in cell culture assays and a mouse genital herpes model, respectively. In addition, the cellular association of liposome/PEI was examined by flow cytometry and confocal microscopy. PEI showed higher antiviral activity postinfection than preinfection in vivo. Liposome/PEI and PEI with decreased numbers of primary amine residues at a dose of 0.2 mg PEI/mouse exhibited more potent therapeutic antiviral activity than acyclovir and PEI alone without acute lesion appearance or toxicity pre- or postinfection, but polyallylamine was moderately effective only preinfection. Liposome concentrations were important for the effectiveness of liposome/PEI. This finding suggests that PEI combined with liposomes and with slightly decreased numbers of primary amines may be an effective vaginally administrated antiviral drug, and secondary and tertiary amine residues of PEI may contribute to the inhibitory efficiency against viral infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Injections of poly(ethylene glycol)-modified liposomes (PEG-liposomes) cause rapid clearance of the second dose of PEG-liposomes. This phenomenon is known as the accelerated blood clearance (ABC) phenomenon. Previous studies have suggested that PEG-specific IgM (anti-PEG IgM) can play a major role in the ABC phenomenon. In our previous study, however, a PEG-shell-possessing polymeric micelle with hydrophilic inner core (PEG-P(Lys-DOTA-Gd) micelle) did not induce the ABC phenomenon nor the IgM responses, and exhibited no change in its plasma concentration in PEG-liposome-injected mice. In the present paper, we studied the ABC-phenomenon in more detail by comparing the behaviors between PEG-liposomes, PEG-P(Lys-DOTA-Gd) micelle, and hydrophobic-core-possessing PEG-PBLA micelles. We demonstrated that the PEG-PBLA micelle induced similar IgM responses as observed in PEG-liposome; however, the second dose of PEG-PBLA micelle exhibited no decreases in their plasma concentration, while the second dose of PEG-liposome did exhibit rapid clearances. Furthermore, we did not observe any PEG main chain specific IgM in PEG-liposome injected mice by sandwich ELISA which can measure more specific IgM to the PEG main chain theoretically. These results suggested that the induced IgM recognizes an interface between PEG chain and hydrophobic chain, rather than PEG main chain, and the anti-PEG IgM hypothesis should be re-evaluated., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The current study synthesized macromolecular magnetic resonance imaging (MRI) contrast agents constituted of the poly(ethylene glycol)-b-poly(L-lysine) block copolymer (PEG-P(Lys)). A chelate group, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), was attached to the primary amino group of the block copolymer in desired contents. Gd-DOTA-based macromolecular contrast agents were prepared from PEG-P(Lys) having DOTA (PEG-P(Lys-DOTA) and Gd(III) ions. All of the PEG-P(Lys) block copolymers having gadolinium ions (PEG-P(Lys-DOTA-Gd)) showed higher T(1) relaxivity (per gadolinium), r(1)=5.6-7.3mM(-1)s(-1), than that of a low-molecular-weight gadolinium-chelate, diethylenetriaminepentaacetic acid-gadolinium(III) (Gd-DTPA) at 9.4T. The study prepared the polyion complex (PIC) micelles from the amino groups of the lysine units and an oppositely charged polyanion, poly(methacrylic acid) or dextran sulfate, in an aqueous medium. In contrast, the fully DOTA-attached PEG-P(Lys-DOTA-Gd) formed a PIC with a polycation. Compared with partially DOTA-attached cationic PEG-P(Lys-DOTA-Gd), this PIC micelle yielded a forty percent decrease of r(1). This r(1) decrease was considered to result from a change in the accessibility of water molecules to gadolinium ions in the micelles' inner core. The r(1) was decreased upon formation of the PIC micelle, and this change proved that our concept worked in vitro. Blood-circulation characteristics of PIC micelles were controlled by means of changing the molecular weight of the counter anion. The PIC micelles accumulated in tumor tissues, and MRI study showed T1W image of axial slice of tumor area was significantly enhanced at 24h after the injection., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A polymeric micelle drug carrier system was applied to the targeting of an MRI (magnetic resonance imaging) contrast agent. A block copolymer, PEG-b-poly(L-lysine), was used for conjugation of gadolinium ions through chelating moieties, DOTA. The DOTA moieties were successfully conjugated to all primary amine groups of the lysine residues. The obtained block copolymer, PEG-b-poly(L-lysine-DOTA), formed a polymeric micelle. The polymeric micelle structure was maintained even after partial gadolinium chelation ( approximately 40%) to the DOTA moieties. The prepared polymeric micelle MRI contrast agent was injected into a mouse tail vein at a dose of 0.05 mmol Gd/kg. The polymeric micelle-based MRI contrast agent exhibited stable blood circulation. A considerable amount (6.1+/-0.3% of ID/g of the polymeric micelle) was found to accumulate at solid tumors 24 h after intravenous injection by means of the EPR effect. An MRI analysis revealed that the signal intensity of the tumor was enhanced 2.0-fold by the use of this contrast agent.

Phytic acid (IP-6) is a polyphosphorylated carbohydrate with antitumor activity for many kinds of tumors. In this study, we developed a novel method of loading irinotecan (CPT-11) into liposomes using IP-6, and evaluated its antitumor effect on colon tumors in vivo. Liposomal CPT-11 was prepared by loading CPT-11 to distearoylphosphatidylcholine/cholesterol/methoxy-poly(ethyleneglycol)-distearylphosphatidylethanolamine liposomes prepared in IP-6 solution, CuSO(4) solution and citrate buffer, respectively (IP6-, Cu- and Cit-L). CPT-11 loading efficiency for IP6-L (90-100%) was higher than that for Cit-L (less than 40%), and similar to Cu-L when CPT-11 to total lipid weight ratio was increased from 0.2 to 0.6. Plasma elimination and biodistribution of liposomal CPT-11 and its metabolite SN-38 were measured after intravenous administration. IP6-L following i.v. injection showed 1.3- and 1.7-fold higher plasma area under the curves of CPT-11 and SN-38, respectively, than Cu-L. Finally, therapeutic activity was determined in mouse Colon 26 and human COLO 320DM tumor xenografts in mice. IP6-L significantly exhibited superior anticancer activity to Cu-L and free CPT-11 in Colon 26 tumor. Using IP-6 as a drug-trapping agent in liposome, IP6-L improved CPT-11 pharmacokinetics and increased antitumor activity in colon tumors.

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.

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.

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.

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.

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.

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.

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.

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.

Novel folate-linked, cationic nanoparticles (NPs) were developed and evaluated for potential use for gene delivery to human oral cancer (KB cells) and human prostate cancer (LNCaP cells), which abundantly expressed folate binding proteins. Folate-polyethylenglycol-distearoylphosphatidylethanolamine conjugate (f-PEG-DSPE) was incorporated in NPs composed of 3([N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and Tween 80. NP-0.3FT, -1FT and -1FLT, which contain 0.3 and 1 mol% f-PEG2000-DSPE, and 1 mol% f-PEG5000-DSPE, respectively, showed about 100-200 nm in size. The NP/plasmid DNA complex (nanoplex) remained in an injectable size (230-340 nm) and slightly increased its size in serum. The association of NP-1FT with KB cells was enhanced by f-PEG2000-DSPE and was blocked by co-incubation with free folic acid in medium. In transfection activity, the NP-1FT, but not NP-1FLT, showed high activity into KB and LNCaP cells in the presence of serum. The NP-0.3FT also showed high activity into LNCaP cells, but not KB cells. In RT-PCR analysis, KB cells strongly expressed folate receptors mRNA, but LNCaP cells did not. In contrast, LNCaP cells expressed mRNA of prostate-specific membrane antigen (PSMA), which interacts with the folate substrate. Uptake mechanism of folate-linked NPs in LNCaP cells may be different from that in KB cells. This is the first report that folate-linked NPs selectively deliver the DNA to LNCaP cells, suggesting that such NPs are potentially targeted vectors to prostate cancer for gene delivery., (Coyright 2004 Elsevier B.V.)

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.

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.

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.