Seo Young Jeong

Cellular uptake mechanism and intracellular fate of hydrophobically modified glycol chitosan nanoparticles

Polymeric nanoparticle-based carriers are promising agents for the targeted delivery of therapeutics to the intracellular site of action. To optimize the efficacy in delivery, often the tuning of physicochemical properties (i.e., particle size, shape, surface charge, lipophilicity, etc.) is necessary, in a manner specific to each type of nanoparticle. Recent studies showed an efficient tumor targeting by hydrophobically modified glycol chitosan (HGC) nanoparticles through the enhanced permeability and retention (EPR) effect. As a continued effort, here the investigations on the cellular uptake mechanism and the intracellular fate of the HGC nanoparticles are reported. The HGC nanoparticle, prepared by a partial derivatization of the free amino groups of glycol chitosan (GC) with 5beta-cholanic acid, had a globular shape with the average diameter of 359 nm and the zeta potential of ca. 22 mV. Interestingly, these nanoparticles showed an enhanced distribution in the whole cells, compared to the parent hydrophilic GC polymers. In vitro experiments with endocytic inhibitors suggested that several distinct uptake pathways (e.g., clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis) are involved in the internalization of HGC. Some HGC nanoparticles were found entrapped in the lysosomes upon entry, as determined by TEM and colocalization studies. Given such favorable properties including low toxicity, biocompatibility, and fast uptake by several nondestructive endocytic pathways, our HGC nanoparticles may serve as a versatile carrier for the intracellular delivery of therapeutic agents.

Self-assembled hyaluronic acid nanoparticles for active tumor targeting

Hyaluronic acid nanoparticles (HA-NPs), which are formed by the self-assembly of hydrophobically modified HA derivatives, were prepared to investigate their physicochemical characteristics and fates in tumor-bearing mice after systemic administration. The particle sizes of HA-NPs were controlled in the range of 237-424 nm by varying the degree of substitution of the hydrophobic moiety. When SCC7 cancer cells over-expressing CD44 (the receptor for HA) were treated with fluorescently labeled Cy5.5-HA-NPs, strong fluorescence signals were observed in the cytosol of these cells, suggesting efficient intracellular uptake of HA-NPs by receptor-mediated endocytosis. In contrast, no significant fluorescence signals were observed when Cy5.5-labeled HA-NPs were incubated with normal fibroblast cells (CV-1) or with excess free-HA treated SCC7 cells. Following systemic administration of Cy5.5-labeled HA-NPs with different particle sizes into a tumor-bearing mouse, their biodistribution was monitored as a function of time using a non-invasive near-infrared fluorescence imaging system. Irrespective of the particle size, significant amounts of HA-NPs circulated for two days in the bloodstream and were selectively accumulated into the tumor site. The smaller HA-NPs were able to reach the tumor site more effectively than larger HA-NPs. Interestingly, the concentration of HA-NPs in the tumor site was dramatically reduced when mice were pretreated with an excess of free-HA. These results imply that HA-NPs can accumulate into the tumor site by a combination of passive and active targeting mechanisms.

Hydrophobically modified glycol chitosan nanoparticles-encapsulated camptothecin enhance the drug stability and tumor targeting in cancer therapy

To prepare a water-insoluble camptothecin (CPT) delivery carrier, hydrophobically modified glycol chitosan (HGC) nanoparticles were constructed by chemical conjugation of hydrophobic 5beta-cholanic acid moieties to the hydrophilic glycol chitosan backbone. Insoluble anticancer drug, CPT, was easily encapsulated into HGC nanoparticles by a dialysis method and the drug loading efficiency was above 80%. CPT-encapsulated HGC (CPT-HGC) nanoparticles formed nano-sized self-aggregates in aqueous media (280-330 nm in diameter) and showed sustained release of CPT for 1 week. Also, HGC nanoparticles effectively protected the active lactone ring of CPT from the hydrolysis under physiological condition, due to the encapsulation of CPT into the hydrophobic cores in the HGC nanoparticles. The CPT-HGC nanoparticles exhibited significant antitumor effects and high tumor targeting ability towards MDA-MB231 human breast cancer xenografts subcutaneously implanted in nude mice. Tumor growth was significantly inhibited after i.v. injection of CPT-HGC nanoparticles at doses of 10 mg/kg and 30 mg/kg, compared to free CPT at dose of 30 mg/kg. The significant antitumor efficacy of CPT-HGC nanoparticles was attributed to the ability of the nanoparticles to show both prolonged blood circulation and high accumulation in tumors, as confirmed by near infrared (NIR) fluorescence imaging systems. Thus, the delivery of CPT to tumor tissues at a high concentration, with the assistance of HGC nanoparticles, exerted a potent therapeutic effect. These results reveal the promising potential of HGC nanoparticles-encapsulated CPT as a stable and effective drug delivery system in cancer therapy.

Antitumor efficacy of cisplatin-loaded glycol chitosan nanoparticles in tumor-bearing mice

To make a tumor targeting nano-sized drug delivery system, biocompatible and biodegradable glycol chitosan (M(w)=250 kDa) was modified with hydrophobic cholanic acid. The resulting hydrophobically modified glycol chitosans (HGCs) that formed nano-sized self-aggregates in an aqueous medium were investigated as an anticancer drug carrier in cancer treatment. Insoluble anticancer drug, cisplatin (CDDP), was easily encapsulated into the hydrophobic cores of HGC nanoparticles by a dialysis method, wherein the drug loading efficiency was about 80%. The CCDP-encapsulated HGC (CDDP-HGC) nanoparticles were well-dispersed in aqueous media and they formed a nanoparticles structure with a mean diameter about 300-500 nm. As a nano-sized drug carrier, the CDDP-HGC nanoparticles released the drug in a sustained manner for a week and they were also less cytotoxic than was free CDDP, probably because of sustained release of CDDP from the HGC nanoparticles. The tumor targeting ability of CDDP-HGC nanoparticles was confirmed by in vivo live animal imaging with near-infrared fluorescence Cy5.5-labeled CDDP-HGC nanoparticles. It was observed that CDDP-HGC nanoparticles were successfully accumulated by tumor tissues in tumor-bearing mice, because of the prolonged circulation and enhanced permeability and retention (EPR) effect of CDDP-HGC nanoparticles in tumor-bearing mice. As expected, the CDDP-HGC nanoparticles showed higher antitumor efficacy and lower toxicity compared to free CDDP, as shown by changes in tumor volumes, body weights, and survival rates, as well as by immunohistological TUNEL assay data. Collectively, the present results indicate that HGC nanoparticles are a promising carrier for the anticancer drug CDDP.

Porous chitosan scaffold containing microspheres loaded with transforming growth factor-β1: Implications for cartilage tissue engineering

Damaged articular cartilage, caused by traumatic injury or degenerative diseases, has a limited regenerative capacity and frequently leads to the onset of osteoarthritis. As a promising strategy for the successful regeneration of long-lasting hyaline cartilage, tissue engineering has received increasing recognition. In this study, we attempted to design a novel type of porous chitosan scaffold, containing transforming growth factor-beta1 (TGF-beta1), to enhance chondrogenesis. First, to achieve a sustained release of TGF-beta1, chitosan microspheres loaded with TGF-beta1 (MS-TGFs) were prepared by the emulsion method, in the presence of tripolyphosphate; with an identical manner, microspheres loaded with BSA, a model protein, were also prepared. Both microspheres containing TGF-beta1 and BSA had spherical shapes with a size ranging from 0.2 to 1.5 microm. From the release experiments, it was found that both proteins were slowly released from the microspheres over 5 days in a PBS solution (pH 7.4), in which the release rate of TGF-beta1 was much lower than that of BSA. Second, MS-TGFs were seeded onto the porous chitosan scaffold, prepared by the freeze-drying method, to observe the effect on the proliferation and differentiation of chondrocytes. It was obviously demonstrated from in vitro tests that, compared to the scaffold without MS-TGF, the scaffold containing MS-TGF significantly augments the cell proliferation and production of extracellular matrix, indicating the role of TGF-beta1 released from the microspheres. These results suggest that the chitosan scaffold containing MS-TGF possesses a promising potential as an implant to treat cartilage defects.

Biodistribution and anti-tumor efficacy of doxorubicin loaded glycol-chitosan nanoaggregates by EPR effect

An in vivo tumor targeting test of glycol-chitosan nanoaggregates was carried out with FITC-conjugated glycol-chitosan nanoaggregates (FTC-GC) and the doxorubicin conjugated glycol-chitosan (GC-DOX). To investigate its biodistribution in tumor-bearing rats, glycol-chitosan was labeled with fluorescein isothiocyanate (FITC), which formed nanoaggregates with a diameter of about 250 nm in aqueous media. GC-DOX nanoaggregates containing acid-sensitive spacers were prepared. The GC-DOX formed micelle-like nanoaggregates spontaneously in aqueous media. GC-DOX nanoaggregates had a narrow and unimodal size distribution, and its hydrodynamic diameter measured by dynamic light scattering ranged from 250 to 300 nm. A loading content of doxorubicin into GC-DOX nanoaggregates as high as 38%, with 97% loading efficiency, could be obtained using a physical entrapment method. A tumor-bearing animal model was developed by inoculating tumor cells into the back of a rat. The FTC-GC nanoaggregates were injected into the tail vein of tumor-bearing rats and their tissue distribution was examined. The FTC-GC nanoaggregates were distributed mainly in kidney, tumor and the liver and were scarcely observed in other tissues. They were maintained at a high level for 8 days and their distribution in tumor tissues increased gradually. This suggests that chitosan nanoaggregates accumulate passively in the tumor tissue due to the enhanced permeability and retention (EPR) effect. Doxorubicin loaded GC-DOX nanoaggregates (DOX/GC-DOX) were injected into the tail vein of tumor-bearing rats and their anti-tumor effect was examined. Tumor growth was suppressed over 10 days.

Polymeric micelles of poly(2-ethyl-2-oxazoline)-block-poly(ε-caprolactone) copolymer as a carrier for paclitaxel

Abstract Polymeric micelles based on amphiphilic block copolymers of poly(2-ethyl-2-oxazoline) (PEtOz) and poly(e-caprolactone) (PCL) were prepared in an aqueous phase. The loading of paclitaxel into PEtOz–PCL micelles was confirmed by 1 H-NMR spectra. Paclitaxel was efficiently loaded into PEtOz–PCL micelles using dialysis method, and the loading content of paclitaxel in micelles was in the range 0.5–7.6 wt.% depending on the block composition of block copolymers, organic solvent used in the dialysis, and feed weight ratio of paclitaxel to block copolymer. The higher the content of hydrophobic block in the block copolymers, the higher the loading efficiency of micelles for paclitaxel. When acetonitrile was used as solvent, a higher drug loading efficiency was obtained than with THF. The loading efficiency decreased with increasing feed weight ratio of paclitaxel to block copolymer from 0.1:1 to 0.2:1. The hydrodynamic diameters of paclitaxel-loaded micelles were in the range 18.3–23.4 nm with narrow size distribution. The hemolysis test of PEtOz–PCL performed in vitro indicated that the toxicity of PEtOz–PCLs to lipid membrane was not significant compared with Tween 80, and was comparable to that observed with Cremophore EL. The proliferation inhibition activity of paclitaxel-loaded micelles for KB human epidermoid carcinoma cells was also evaluated in vitro. Paclitaxel-entrapped polymeric micelles exhibited comparable activity to that observed with Cremophore EL-based paclitaxel formulations in inhibiting the growth of KB cells.

PEGylation of hyaluronic acid nanoparticles improves tumor targetability in vivo

A major drawback of hyaluronic acid (HA)-based drug conjugates or nanoparticles for cancer therapy is their preferential accumulation in the liver after systemic administration. In an attempt to investigate the physicochemical characteristics and in vivo fates of poly(ethylene glycol) (PEG)-conjugated HA nanoparticles (HA-NPs), amphiphilic HA derivatives were prepared by varying the degree of PEGylation. The PEGylated HA-NPs formed self-assembled nanoparticles (217-269 nm in diameter) with the negatively charged surfaces in the physiological condition. Although PEGylation of HA-NPs reduced their cellular uptake in vitro, larger amounts of nanoparticles were taken up by cancer cells over-expressing CD44, an HA receptor, than by normal fibroblast cells. The ex vivo images of the organs using an optical imaging technique after the intravenous injection of Cy5.5-labeled nanoparticles into normal mice demonstrated that PEGylation could effectively reduce the liver uptake of HA-NPs and increase their circulation time in the blood. When the nanoparticles were systemically administered into tumor-bearing mice for in vivo real-time imaging, the strongest fluorescence signals were detected at the tumor site of the mice for the whole period of time studied, indicating their high tumor targetability. Interestingly, PEGylated HA-NPs were more effectively accumulated into the tumor tissue up to 1.6-fold higher than bare HA-NPs. The high tumor targetability of PEGylated HA-NPs was further supported by the intravital tumor imaging, in which their rapid extravasation into the tumor tissue was clearly observed. These results suggest that PEGylated HA-NPs can be useful as a means for cancer therapy and diagnosis.

pH-Tunable Calcium Phosphate Covered Mesoporous Silica Nanocontainers for Intracellular Controlled Release of Guest Drugs†

Mesoporous silica nanoparticles (Si-MPs) have emerged as appealing hosts for the exploitation of novel nanocarriers featuring “on demand” drug delivery. To date, a variety of stimuli-responsive “pore blockers”, such as organic molecules (cyclodextrin, dendrimer), metal (gold) nanoparticles, and supramolecular assemblies (rotaxanes, polyrotaxanes), 8] have been introduced on the surfaces of Si-MPs to control the release of guest molecules in response to external stimuli. Various physical/chemical stimuli, such as pH, photoirradiation, redox potential, and enzymes, have been used as triggers for uncapping the pore blockers and releasing encapsulated guest molecules. Although these Si-MPs hold promise as potential delivery vehicles, many of the pore-blocking species have critical drawbacks for clinical applications because dismantled poreblocking agents, for which body toxicity has not been well defined, can induce toxic side effects. Therefore, the design of Si-MP nanocarriers that use a natural nontoxic component as pore blocker remains a significant challenge. Very recently, Si-MPs with nucleic acids and carbohydrates as molecular valves have been reported for controlled-release devices, and recognized as valuable trials of using organic biomolecules as pore-blocking species. This effort toward the introduction of natural components may offer useful guidance to the practical use of Si-MPs for in vivo biological applications. Herein, we report a novel Si-MP carrier covered with pHcontrolled, absorbable calcium phosphate (CaP) nanocoatings as pore blockers that allow the facilitated release of entrapped drugs within acidifying intracellular compartments such as endosomes and lysosomes. Our major aim is to develop a new, natural pore blocker, especially one based on nontoxic inorganic biominerals. CaP is the main component in bone and plays a key role in the natural bone regeneration process. 18] CaP is widely used as a bioactive osteoconductive coating for bone-regenerative materials, and these coating processes currently rely on time-consuming procedures in simulated body fluid (SBF) for several weeks. Our research has recently centered on a unique feature of CaP; it can be dissolved as nontoxic ions (calcium, phosphate ions) in acidic cellular environments such as endosomes (pH ca. 5.0) and lysosomes (pH ca. 4.5). 21] In a previous report, we demonstrated the feasibility of pH-controlled release of doxorubicin (DOX), an anticancer drug, from mineralized polymer nanoparticles in the endo/lysosomes of breast cancer MCF-7 cells. The key idea is urease functionalization of Si-MP surfaces and subsequent enzyme-mediated surface CaP mineralization in the presence of urea under mild conditions for a short time period. Figure 1a and c illustrate the overall process for surface anchoring of urease, the subsequent CaP coating of SiMP surfaces, and controlled release of DOX within cells. For this purpose, we prepared Si-MP-0 with a mean diameter of 100 nm (Figure 1 b), an average pore diameter of 2.5 nm (Barret–Joyner–Halenda (BJH) analysis), and a specific surface area of 900.5 mg 1 (Brunauer–Emmett–Teller (BET) analysis). The surface of Si-MP-0 was functionalized with primary amine groups by the reaction of 3-aminopropyltriethoxysilane (APTES) to provide Si-MP-NH2, which was further allowed to react with glutaraldehyde to obtain SiMP-COH. Primary amines and aldehydes of Si-MP-NH2 and Si-MP-COH exhibited an N–H bending vibration at 1580 cm 1 and C=O stretching at 1645 cm 1 (Figure S1 in the Supporting Information). To prepare urease-functionalized Si-MPs (Si-MP-UR), urease was covalently conjugated through reaction with surface aldehydes (Schiff s base formation). Transmission electron microscopy (TEM) and powder X-ray diffraction (XRD) analyses revealed that Si-MP-UR exhibits a well-ordered porous structure with a hexagonal arrangement (Figure 2 a and c, and Figure S1b in the Supporting Information). For CaP nanocoatings, Si-MP-UR was placed in a mineralizing acidic solution (pH 4.0) containing hydroxyapatite (HAp, Ca5(PO4)3OH) and urea. It is known that surface-conjugated urease catalyzes urea hydrolysis into ammonium bicarbonate and hydroxide ions in a pH range of 4.0–9.5: OC(NH2)2 + 2H2O!2NH4 + HCO3 + OH . It is well known that the surface silanols of silica present a negative charge at neutral pH, which can lead to the formation of an electric double layer with calcium cations. The presence of PO4 3 and released OH ions by urea decomposition increased the ionic concentration above the [*] H. P. Rim, K. H. Min, Prof. Dr. S. Y. Jeong Department of Life and Nanopharmaceutical Science Kyung Hee University 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701 (Korea) E-mail: syjeong@khu.ac.kr

Tumoral acidic pH-responsive MPEG-poly(β-amino ester) polymeric micelles for cancer targeting therapy

Herein, we evaluated the tumoral low pH targeting characteristics of pH-responsive polymer micelles in cancer targeting therapy. To design the pH-responsive polymeric micelles, hydrophilic methyl ether poly(ethylene glycol) (MPEG) and pH-responsive/biodegradable poly(beta-amino ester) (PAE) were copolymerized using a Michael-type step polymerization, resulting in an MEPG-PAE block copolymer. The amphiphilic MPEG-PAE block copolymer formed polymeric micelles with nano-sized diameter by self-assembly, which showed a sharp pH-dependant micellization/demicellization transition at the tumoral acidic pH value (pH 6.4). For the cancer image and therapy, fluorescence dye, tetramethylrhodamine isothiocyanate (TRITC), or anticancer drug, camptothecin (CPT), was efficiently encapsulated into the pH-responsive polymeric micelles (pH-PMs) by a simple solvent casting method. The TRITC or CPT encapsulated pH-PMs (TRITC-pH-PMs or CPT-pH-PMs) showed rapid release of TRITC or CPT in weakly acidic aqueous (pH 6.4) because they still presented a sharp tumoral acid pH-responsive micellization/demicellization transition. The pH-PMs with 10wt.% of TRITC could deliver substantially more fluorescence dyes to the target tumor tissue in MDA-MB231 human breast tumor-bearing mice, compared to the control polymeric micelles of PEG-poly(l-lactic acid) (PEG-PLLA). Importantly, CPT-pH-PMs exhibited significantly increased therapeutic efficacy with minimum side effects by other tissues in breast tumor-bearing mice, compared to free CPT and CPT encapsulated PEG-PLLA micelles. The tumoral acidic pH-responsive polymeric micelles are highly useful for cancer targeting therapy.