Kwangmeyung Kim

Targeted delivery of low molecular drugs using chitosan and its derivatives

Chitosan has prompted the continuous impetus for the development of safe and effective drug delivery systems because of its unique physicochemical and biological characteristics. The primary hydroxyl and amine groups located on the backbone of chitosan allow for chemical modification to control its physical properties. When the hydrophobic moiety is conjugated to a chitosan molecule, the resulting amphiphile may form self-assembled nanoparticles that can encapsulate a quantity of drugs and deliver them to a specific site of action. Chemical attachment of the drug to the chitosan throughout the functional linker may produce useful prodrugs, exhibiting the appropriate biological activity at the target site. Mucoadhesive and absorption enhancement properties of chitosan increase the in vivo residence time of the dosage form in the gastrointestinal tract and improve the bioavailability of various drugs. The main objective of this review is to provide an insight into various target-specific carriers, based on chitosan and its derivatives, towards low molecular weight drug delivery. The first part of the review is concerned with the organ-specific delivery of low molecular drugs using chitosan and its derivatives. The subsequent section considers the recent developments of drug delivery carriers for cancer therapy with special focus on various targeting strategies.

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.

A Near‐Infrared‐Fluorescence‐Quenched Gold‐Nanoparticle Imaging Probe for In Vivo Drug Screening and Protease Activity Determination

Nanoscale fluorescence optical imaging probes are paving the way for novel methods to sense and spot live molecular targets. Various probes have been developed, including semiconductor quantum dots, magnetofluorescent nanoparticles, polymer conjugates, nanocomplexes, and gold nanoparticles (AuNPs). The application of conventional fluorescent probes is limited because they generally display only modest fluorescence changes, thus providing insufficient resolution. The limited degree of resolution is mainly attributed to the low fluorescence-quenching efficiency and specificity of the probes. Therefore, a high quenching efficiency and specific recognition properties by the target biomolecules are essential for the development of supersensitive fluorescence-based probes. Among the diverse candidates, biocompatible AuNPs offer a considerable advantage in obtaining optical images through their nearinfrared-fluorescence (NIRF) quenching properties. Chromophores in close proximity to AuNPs experience strong electronic interactions with the surface, which results in donation of excited electrons to the metal nanoparticles and almost perfect quenching of the fluorescence. However, the use of AuNP probes for in vivo visual biomolecular detection and real-time fluorescence tomography remains to be explored. Herein, we describe the development of a proteasesensitive selfand AuNP-quenched NIRF probe. Proteases— or their inhibitors—are mainly involved in cancer, inflammation, and vascular disease. Sensitive, convenient, and accurate protease-detection systems constitute a crucial tool for the development of drug-screening systems and the early diagnosis of diseases, such as cancer. The most common detection method for protease activity is the use of small peptide substrates containing chromophores at their termini. We previously reported proteaseand kinase-activating sensory systems based on the fluorescence resonance energy transfer (FRET) properties of NIRF Cy or isothiocyanate dyes. Although these systems are sensitive, their applications are limited because of the modest fluorescent changes (which are too weak to be used in vivo). Therefore, a decrease in the noise intensity of the quenched state—to an undetectable level—is required to maximize the fluorescent changes and achieve an efficient in vivo detection of small amounts of protease. Herein, we propose an alternative, simple, robust, and one-step optical fluorescence nanoprobe to be used in: 1) inhibitor drug screening, 2) the detection of target proteases, and 3) the early diagnosis of cancer. The system Cy5.5-substrate/AuNP is believed to induce a strong multi-quenched state, because the AuNPs serve as ultra-efficient quenchers of the molecular excitation energy in a chromophore through their surface-energy-transfer properties, and the Cy5.5 dye, loaded onto the AuNP surfaces, can be self-quenched as a result of a combination of the staticquenching and FRET mechanisms. When the target proteases meet functionalized AuNP probes, cleavage of the Cy5.5-substrate occurs as a consequence of the specific substrate recognition by the protease. This cleavage is manifested in the form of a pronounced NIRF signal recovery caused by dequenching of the NIRF dyes (Figure 1A). To demonstrate the utility of our rationale, we developed a matrix metalloprotease (MMP) fluorescence imaging probe based on AuNPs. MMPs are a family of zinc-dependent endopeptidases that play key roles in several biological processes. In particular, because of their significant role in promoting cancer progression, MMPs have become important targets for new drug development and in vivo tumor diagnosis. We prepared AuNPs (20 nm) stabilized with a Cy5.5substrate, namely, Cy5.5-Gly-Pro-Leu-Gly-Val-Arg-Gly-Cys(amide), where the core-specific substrate, that is, Pro-LeuGly-Val-Arg, shows selectivity for MMP (see the Supporting Information). Transmission electron microscopy (TEM) [*] Dr. S. Lee, Dr. K. Park, S.-Y. Lee, Dr. K. Choi, Dr. I. C. Kwon, Dr. K. Kim Biomedical Research Center Korea Institute of Science and Technology 39-1 Hawolgok-dong, Seongbuk-gu, Seoul, 136-791 (Korea) Fax: (+82)2-958-5909 E-mail: kim@kist.re.kr E.-J. Cha, J.-K. Hong, I.-C. Sun, Prof. C.-H. Ahn Department of Materials Science and Engineering Seoul National University San 56-1, Sillim, Gwanak, Seoul 151-744 (Korea) Fax: (+82) 2-883-8197 E-mail: chahn@snu.ac.kr

Smart Nanocarrier Based on PEGylated Hyaluronic Acid for Cancer Therapy

Tumor targetability and site-specific drug release of therapeutic nanoparticles are key factors for effective cancer therapy. In this study, poly(ethylene glycol) (PEG)-conjugated hyaluronic acid nanoparticles (P-HA-NPs) were investigated as carriers for anticancer drugs including doxorubicin and camptothecin (CPT). P-HA-NPs were internalized into cancer cells (SCC7 and MDA-MB-231) via receptor-mediated endocytosis, but were rarely taken up by normal fibroblasts (NIH-3T3). During in vitro drug release tests, P-HA-NPs rapidly released drugs when incubated with cancer cells, extracts of tumor tissues, or the enzyme Hyal-1, which is abundant in the intracellular compartments of cancer cells. CPT-loaded P-HA-NPs (CPT-P-HA-NPs) showed dose-dependent cytotoxicity to cancer cells (MDA-MB-231, SCC7, and HCT 116) and significantly lower cytotoxicity against normal fibroblasts (NIH-3T3) than free CPT. Unexpectedly, high concentrations of CPT-P-HA-NPs demonstrated greater cytotoxicity to cancer cells than free CPT. An in vivo biodistribution study indicated that P-HA-NPs selectively accumulated into tumor sites after systemic administration into tumor-bearing mice, primarily due to prolonged circulation in the blood and binding to a receptor (CD44) that was overexpressed on the cancer cells. In addition, when CPT-P-HA-NPs were systemically administrated into tumor-bearing mice, we saw no significant increases in tumor size for at least 35 days, implying high antitumor activity. Overall, P-HA-NPs showed promising potential as a drug carrier for cancer therapy.

Tumor-homing multifunctional nanoparticles for cancer theragnosis: Simultaneous diagnosis, drug delivery, and therapeutic monitoring.

Theragnostic multifunctional nanoparticles hold great promise in simultaneous diagnosis of disease, targeted drug delivery with minimal toxicity, and monitoring of treatment. One of the current challenges in cancer treatment is enhancing the tumor-specific targeting of both imaging probes and anticancer agents. Herein, we report tumor-homing chitosan-based nanoparticles (CNPs) that simultaneously execute cancer diagnosis and therapy (cancer theragnosis). These CNPs are unique for their three distinctive characteristics, such as stability in serum, deformability, and rapid uptake by tumor cells. These properties are critical in increasing their tumor targeting specificity and reducing their nonspecific uptake by normal tissues. To develop these CNPs into novel theragnostic nanoparticles, we labeled them with Cy5.5, a near-infrared fluorescent (NIRF) dye, for imaging and also loaded them with paclitaxel (PTX-CNPs), an anticancer drug, for cancer treatment. Cy5.5 labeled PTX-CNPs exhibited significantly increased tumor-homing ability with low nonspecific uptake by other tissues in SCC7 tumor-bearing mice. Theragnostic nanoparticles, Cy5.5 labeled PTX-CNPs, are highly useful for simultaneous diagnosis of early-stage cancer and drug delivery.

Self-assembled nanoparticles based on hyaluronic acid-ceramide (HA-CE) and Pluronic® for tumor-targeted delivery of docetaxel

Hyaluronic acid-ceramide (HA-CE)-based self-assembled nanoparticles were developed for intravenous docetaxel (DCT) delivery. In this study, physicochemical properties, cellular uptake efficiency, and in vivo targeting capability of the nanoparticles developed were investigated. DCT-loaded nanoparticles composed of HA-CE and Pluronic 85 (P85) with a mean diameter of 110-140 nm were prepared and their morphological shapes were assessed using transmission electron microscopy (TEM). DCT release from nanoparticle was enhanced with increasing P85 concentrations in our in vitro model. Blank nanoparticles exhibited low cytotoxicity in U87-MG, MCF-7 and MCF-7/ADR cell lines. From cellular uptake studies, the nanoparticles developed enhanced the intracellular DCT uptake in the CD44-overexpressing cell line (MCF-7). The nanoparticles were shown to be taken up by the HA-CD44 interaction according to DCT and coumarin 6 (C6) cellular uptake studies. The multidrug resistance (MDR)-overcoming effects of DCT-loaded HA-CE/P85-based nanoparticles were also observed in cytotoxicity tests in MCF-7/ADR cells. Following the intravenous injection of DCT-loaded cyanine 5.5 (Cy5.5)-conjugated nanoparticles in MCF-7/ADR tumor-bearing mice, its in vivo targeting for CD44-overexpressing tumors was identified by non-invasive near-infrared (NIR) fluorescence imaging. These results indicate that the HA-CE-based nanoparticles prepared may be a promising anti-cancer drug delivery system through passive and active tumor targeting.

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.