Yoshie Maitani

Block copolymer design for camptothecin incorporation into polymeric micelles for passive tumor targeting.

AbstractPurpose. Polymeric micelles were designed for targeting of a water-insoluble anticancer agent, camptothecin (CPT). Chemical structures of inner core segment were optimized to achieve high incorporation efficiency and stable CPT-loaded micelles. Methods. Poly(ethylene glycol)-poly(β-benzyl L-aspartate) block copolymer (PEG-PBLA) was synthesized. The PBLA chain was modified by alkaline hydrolysis of its benzyl group followed by esterification with benzyl, n-butyl, and lauryl groups. Incorporation of CPT into micelles was carried out by an evaporation method. The stability of drug-loaded micelles was studied by gel-permeation chromatography (GPC), and their in vitro release behaviors were analyzed. Results. CPT was incorporated into polymeric micelles constructed by various block copolymers. Among the esterified groups, block copolymers with high benzyl ester contents showed high CPT loading efficiency and stable CPT-loaded micelles. In chain lengths, 5-27 Bz-69 showed the highest incorporation efficiency. In contrast, 5-52 Bz-67, which had a longer hydrophobic chain, showed low incorporation efficiency. Release of CPT from the micelles was dependent on the benzyl contents and chain lengths. Sustained release was obtained when the benzyl content was high. Conclusions. CPT was successfully incorporated into polymeric micelles with high efficiency and stability by optimizing chemical structures of the inner core segment.

Novel Chitosan Particles and Chitosan-Coated Emulsions Inducing Immune Response via Intranasal Vaccine Delivery

AbstractPurpose. The aim of this study was to prepare a novel vaccine carrier particulate system (nanoparticles and emulsions) with chitosan and to evaluate the effect of this system on the immune response for intranasal delivery. Methods. Chitosan nanoparticles (NP) and chitosan-coated emulsions (CC-Emul) were prepared by improvement of the method previously reported and by modified ethanol injection methods, respectively. The rats were immunized with the particles adsorbed with ovalbumin (OVA) and cholera toxin (CT) by intranasal (i.n.) and intraperitoneal (i.p.) administration. Results. NP and CC-Emul could be prepared with particle diameter from about 0.4 μm to 3 μm. IgG induced by i.n. of NP was comparable with that by i.p., and IgA induced by i.n. of 0.4-μm- and 1-μm-size NP was significantly higher than control (OVA and CT). IgG and IgA induced by i.n. of 2-μm-size CC-Emul were significantly higher than those with control. Conclusions. The novel chitosan particles used simple preparation methods showed high OVA adsorption. When administered intranasally, NP and CC-Emul induced systemic immune response in rats. These findings suggested that CC-Emul and the smaller-size (0.4 μm) NP are effective for targeting to nasal-associated lymphoid tissues (NALTs) in nasal vaccine delivery.

Design of Folate-Linked Liposomal Doxorubicin to its Antitumor Effect in Mice

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.

Preparation and in vivo imaging of PEG-poly(L-lysine)-based polymeric micelle MRI contrast agents.

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.

Superparamagnetic iron oxide nanoparticles stabilized by alginate: Pharmacokinetics, tissue distribution, and applications in detecting liver cancers

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.

Effect of Polyethylene Glycol Linker Chain Length of Folate-Linked Microemulsions Loading Aclacinomycin A on Targeting Ability and Antitumor Effect In vitro and In vivo

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-PEG2000-distearoylphosphatidylethanolamine (DSPE), folate-PEG5000-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-PEG5000-linked microemulsion and folate-PEG2000-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-PEG5000-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.

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

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

Polymer Design and Incorporation Methods for Polymeric Micelle Carrier System Containing Water-insoluble Anti-cancer Agent Camptothecin

A water-insoluble anti-cancer agent, camptothecin (CPT) was incorporated to a polymeric micelle carrier system forming from poly(ethylene glycol)–poly(aspartate) block copolymers. Incorporation efficiency and stability were analyzed in correlation with chemical structures of the inner core-forming hydrophobic blocks as well as with incorporation methods. Among three incorporation methods (dialysis, emulsion and evaporation methods), an evaporation method brought about much higher CPT yields with less aggregation than the other two methods. By the evaporation method, CPT was incorporated to polymeric micelles in considerably high yields and with high stability using block copolymers possessing high contents of benzyl and methylnaphtyl ester groups as hydrophobic moieties. This indicates importance of molecular design of the hydrophobic block chain to obtain targeting using polymeric micelle carriers as well as importance of the drug incorporation method.

Folate-linked lipid-based nanoparticles for synthetic siRNA delivery in KB tumor xenografts

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.

Folate-linked nanoparticle-mediated suicide gene therapy in human prostate cancer and nasopharyngeal cancer with herpes simplex virus thymidine kinase.

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.