Michihiro Iijima

Endosomal release and intracellular delivery of anticancer drugs using pH-sensitive PEGylated nanogels

A pH-sensitive PEGylated nanogel was prepared by emulsion copolymerization of 2-(N,N-diethylamino)ethyl methacrylate (EAMA) with heterobifunctional poly(ethylene glycol) bearing a 4-vinylbenzyl group at the α-end and a carboxylic acid group at the ω-end (CH2CH–Ph–PEG–COOH; Mn = 8000) in the presence of potassium persulfate and ethylene glycol dimethacrylate (1.0 mol%) as cross-linker. The loading of the anticancer drug doxorubicin (DOX) into the pH-sensitive PEGylated nanogel was carried out by means of a solvent evaporation method, and the amount of DOX loaded into the PEAMA core was found to be 26 wt%. Furthermore, the DOX-loaded, pH-sensitive PEGylated nanogel showed almost no initial burst release of the DOX under physiological pH, whereas significant release of DOX from the pH-sensitive PEGylated nanogel was observed at the endosomal pH. The antitumor activity of the DOX-loaded, pH-sensitive, PEGylated nanogel against the human breast cancer cell line MCF-7 was lower than that of free DOX. On the other hand, the antitumor activity of the DOX-loaded, pH-sensitive, PEGylated nanogel against the human hepatoma cell line HuH-7, which is a natural drug-resistant tumor line, was superior to that of both free DOX and the DOX-loaded, pH-insensitive, PEGylated nanogel. Using fluorescence microscopy, pH-sensitive PEGylated nanogel in HuH-7 cells was found to be initially localized within the endosome and/or lysosome, with subsequent release of DOX from the nanogel in response to the endosomal pH, and ultimately, diffusion via the cytoplasm into the cell nucleus. These findings suggest that the pH-sensitive PEGylated nanogel represents a promising nano-sized carrier for anticancer drug delivery systems in vivo.

Core-stabilized Polymeric Micelle as Potential Drug Carrier: Increased Solubilization of Taxol

ABSTRACTWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWW Poly(ethylene glycol-b-lactide) possessing a methoxygroup at the poly(ethylene glycol) (PEG) chain end anda polymerizable methacryloyl group at the poly(lacticacid) (PLA) chain end (MeO–PEG/PLA–methacryloyl)was prepared by an anionic ring-opening polymerizationof ethylene oxide and DL -lactide in tandem mannerinitiatedwithapotassium2-methoxyethanolate,followedby end-capping with an excess of methacrylic anhydride.The molecular weight of the obtained polymer wascontrolled by the initial monomer/initiator ratio, whichwas confirmed by the combination of gel permeationchromatography and nuclear magnetic resonance ana-lyses. The functionality of the methacryloyl–PLA endwas almost quantitative. The MeO–PEG/PLA–metha-cryloyl (38/35; these numbers in parentheses denote themolecular weights of PEG and PLA segments divided by100, respectively) formed a core–shell type sphericalmicelle in aqueous media obtained by a dialysistechnique, the cumulant diameter of which was ca.30nm with very low polydispersity factor.The methacryloyl group adjacent to the PLA waspolymerized in the PLA core of the micelle. Thepolymerizationproceededthermallywithradicalinitiatorand photochemically with photo-initiator to producecore-polymerized nanoparticles, which was found byspectroscopic and light-scattering techniques. Taxol-incorporated micelles were prepared to entrap Taxol intoMeO–PEG/PLA–methyacryloyl block copolymer mi-celles by the oil/water emulsion method. Copyright O1999 John Wiley & Sons, Ltd.KEYWORDS: polymeric micelle; stable nanoparticle;drugdeliverysystem;PEG/PLAblockcopolymer;micellestability; taxol

Preparation of non-fouling surface through the coating with core-polymerized block copolymer micelles having aldehyde-ended PEG shell

A new type of surface modification with reactive polymeric micelle was carried out for the creation of non-fouling surface. Amphiphilic poly(ethylene glycol)-b-poly(D,L lactide) (PEG/PLA) copolymers possessing acetal group at PEG-end and methacryloyl group at PLA-end were quantitatively synthesized via an anionic polymerization technique. A micelle of narrow distribution was prepared from the block copolymer. Acetal groups on the micelle surface were quantitatively converted into aldehyde group by an acid treatment. The methacryloyl group located in the core of the micelle was polymerized via radical polymerization to form core-polymerized micelle having reactive aldehyde groups on the surface. The core-polymerized reactive micelle was coated to a primary amino-containing polypropylene (PP) plate that was prepared by a plasma treatment. A reductive amination reaction was employed for a conjugation of the reactive core-polymerized micelle on the surface via a covalent linkage. The coating was evaluated by X-ray photoelectron spectroscopy, zeta-potential measurement, and the adsorption of bovine serum albumin, and compared with the PEG-coating under the same condition. The ratio of peak from &Cmacr;&z.sbnd;O bond to C&z.sbnd;&Cmacr;&z.sbnd;C bond indicated that the density of PEG on the surface was higher for the micelle coating than the linear PEG-coating. This is also confirmed by the zeta-potential measurement. By coating the amino-PP surface with micelle, the zeta-potential was remarkably decreased while the PEG-coating under the same condition decreased only appreciably, indicating that micelle coating efficiently masked the surface charge. Further, micelle-covered surface exhibited reduction of protein adsorption. The reduction of protein adsorption along with remarkably masked surface charge implies the high applicability of the micelle coatings to biomedical and bioanalytical applications.

A potassium alcoholate-initiated polymerization of 2-(trialkylsiloxyethyl) methacrylate

Anionic polymerization of 2-(t-butyldimethylsiloxyethyl) methacrylate (ProHEMA) was performed with potassium ethanolate as an initiator in tetrahydrofuran. It was found that the potassium ethanolateinitiated polymerization of ProHEMA proceeds smoothly at ambient temperature to 90% monomer conversion. The polymerization was initiated by the addition of ethanolate at the double bond in ProHEMA. It is considered that complexation of potassium cation with the ProHEMA molecule increased the nucleophilicity of the oxonium anion to increase the initiation ability. Based on the 13C n.m.r, analysis, poly(ProHEMA) obtained with potassium ethanolate was syndiotactic rich in microstructure. This is also explained by the bulky counter cation complexed with monomer molecules, which controls monomer insertion around the growing centre. This polymerization method can be applied to a synthesis of an end-functionalized poly(2-hydroxyethyl methacrylate) (poly(HEMA)).

Polymeric micelles as drug delivery systems: A reactive polymeric micelle carrying aldehyde groups

WWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWW WWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWW WWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWW WWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWWW Nanospheric particles as drug delivery systems are gaining increasing interest in the biomedical field. Nanospheres have been proven as efficient drug delivery systems for intravenous administration because of their comparatively long bloodstream circulation. A novel approach in the field of polymeric drug delivery systems was introduced by the formation of polymeric micelles and subsequently by functionalized polymeric micelles. Functionalized polymeric micelles are expected to find a wide application in the fields of drug delivery and diagnosis since the possibility of coupling to bioactive substances is provided. A large number of densely packed functional groups on the outer shell of the micelle allows an immobilization of biologically active substances at a high density. This is a great advantage for utilizing this particular type of nanosphere in the biomedical field. The possibilities of synthesizing heterobifunctional block copolymers will be demonstrated and the influence of the individual block length on the micelle properties will be discussed. Functionalized polymeric micelles were synthesized from poly(ethylene glycol) (PEG) and poly(lactide) (PLA), and combine the advantages given by the hydrophobic PLA core and the hydrophilic PEG corona. An established quantitative synthetic method for the formation of heterobifunctional PEG was advanced and applied to the block copolymerization. A hetero- bifunctional block copolymer was synthesized, termin- ated by an acetal group at the PEG end and a vinyl group was introduced at the PLA end in a one-pot synthesis. After the micellization the acetal groups on the micelle surface were converted into aldehyde groups by an acidic treatment. Aldehyde groups can react rapidly with primary amines forming Schiff bases, a potential future pathway for the conjugation of functionalized polymeric micelles with proteins. PLA was chosen as core-forming segment since it is a biodegradable, non-toxic polymer that is well established as implant material. Dynamic and static light scattering was applied to determine the micelle size and shape and to study the dependence of the micelle geometry on the block length of the copolymer.

One Pot Synthesis of Poly(Ethylene Oxide) with a Cyano Group at one End and a Hydroxyl Group at the Other End

Well-defined poly(ethylene oxide) with a cyano group at one end and a hydroxyl group at the other terminus was quantitatively synthesized by anionic polymerization of ethylene oxide using cyanomethylpotassium as an initiator in the presence of 18-crown-6. The molecular weight of the poltmers determined from GPC results agreed well with expected values calculated from the monomer/initiator molar ratios. From 13C-NMR analysis, the polymer shows several signals derived from terminal groups together with methylene protons of PEO main chain. The chemical shifts of these signals were in good accordance with those of cyano hydroxyl terminals calculated. In addition, in 1H-NMR spectrum, the peak intensity ratio of OH signal vs. CH 2 CH2CN were 1/2.