Hong Yeol Yoon

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.

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.

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.

Polyethylene glycol-conjugated hyaluronic acid-ceramide self-assembled nanoparticles for targeted delivery of doxorubicin

Polyethylene glycol (PEG)-conjugated hyaluronic acid-ceramide (HACE) was synthesized for the preparation of doxorubicin (DOX)-loaded HACE-PEG-based nanoparticles, 160 nm in mean diameter with a negative surface charge. Greater uptake of DOX from these HACE-PEG-based nanoparticles was observed in the CD44 receptor highly expressed SCC7 cell line, compared to results from the CD44-negative cell line, NIH3T3. A strong fluorescent signal was detected in the tumor region upon intravenous injection of cyanine 5.5-labeled nanoparticles into the SCC7 tumor xenograft mice; the extended circulation time of the HACE-PEG-based nanoparticle was also observed. Pharmacokinetic study in rats showed a 73.0% reduction of the in vivo clearance of DOX compared to the control group. The antitumor efficacy of the DOX-loaded HACE-PEG-based nanoparticles was also verified in a tumor xenograft mouse model. DOX was efficiently delivered to the tumor site by active targeting via HA and CD44 receptor interaction and by passive targeting due to its small mean diameter (<200 nm). Moreover, PEGylation resulted in prolonged nanoparticle circulation and reduced DOX clearance rate in an in vivo model. These results therefore indicate that PEGylated HACE nanoparticles represent a promising anticancer drug delivery system for cancer diagnosis and therapy.

Tumor-targeting hyaluronic acid nanoparticles for photodynamic imaging and therapy

Tumor-targeted imaging and therapy have been the challenging issue in the clinical field. Herein, we report tumor-targeting hyaluronic acid nanoparticles (HANPs) as the carrier of the hydrophobic photosensitizer, chlorin e6 (Ce6) for simultaneous photodynamic imaging and therapy. First, self-assembled HANPs were synthesized by chemical conjugation of aminated 5β-cholanic acid, polyethylene glycol (PEG), and black hole quencher3 (BHQ3) to the HA polymers. Second, Ce6 was readily loaded into the HANPs by a simple dialysis method resulting in Ce6-loaded hyaluronic acid nanoparticles (Ce6-HANPs), wherein in the loading efficiency of Ce6 was higher than 80%. The resulting Ce6-HANPs showed stable nano-structure in aqueous condition and rapid uptake into tumor cells. In particular Ce6-HANPs were rapidly degraded by hyaluronidases abundant in cytosol of tumor cells, which may enable intracellular release of Ce6 at the tumor tissue. After an intravenous injection into the tumor-bearing mice, Ce6-HANPs could efficiently reach the tumor tissue via the passive targeting mechanism and specifically enter tumor cells through the receptor-mediated endocytosis based on the interactions between HA of nanoparticles and CD44, the HA receptor on the surface of tumor cells. Upon laser irradiation, Ce6 which was released from the nanoparticles could generate fluorescence and singlet oxygen inside tumor cells, resulting in effective suppression of tumor growth. Overall, it was demonstrated that Ce6-HANPs could be successfully applied to in vivo photodynamic tumor imaging and therapy simultaneously.

Bioreducible Block Copolymers Based on Poly(Ethylene Glycol) and Poly(γ-Benzyl L-Glutamate) for Intracellular Delivery of Camptothecin

Poly(ethylene glycol)-b-poly(γ-benzyl L-glutamate)s bearing the disulfide bond (PEG-SS-PBLGs), which is specifically cleavable in intracellular compartments, were prepared via a facile synthetic route as a potential carrier of camptothecin (CPT). Diblock copolymers with different lengths of PBLG were synthesized by ring-opening polymerization of benzyl glutamate N-carboxy anhydride in the presence of a PEG macroinitiator (PEG-SS-NH(2)). Owing to their amphiphilic nature, the copolymers formed spherical micelles in an aqueous condition, and their particle sizes (20-125 nm in diameter) were dependent on the block length of PBLG. Critical micelle concentrations of the copolymers were in the range 0.005-0.065 mg/mL, which decreased as the block length of PBLG increased. CPT, chosen as a model anticancer drug, was effectively encapsulated up to 12 wt % into the hydrophobic core of the micelles by the solvent casting method. It was demonstrated by the in vitro optical imaging technique that the fluorescence signal of doxorubicin, quenched in the PEG-SS-PBLG micelles, was highly recovered in the presence of glutathione (GSH), a tripeptide reducing disulfide bonds in the cytoplasm. The micelles released CPT completely within 20 h under 10 mM GSH, whereas only 40% of CPT was released from the micelles in the absence of GSH. From the in vitro cytotoxicity test, it was found that CPT-loaded PEG-SS-PBLG micelles showed higher toxicity to SCC7 cancer cells than CPT-loaded PEG-b-PBLG micelles without the disulfide bond. Microscopic observation demonstrated that the disulfide-containing micelle could effectively deliver the drug into nuclei of SCC7 cells. These results suggest that PEG-SS-PBLG diblock copolymer is a promising carrier for intracellular delivery of CPT.

Hyaluronic acid derivative-coated nanohybrid liposomes for cancer imaging and drug delivery

Nanohybrid liposomes coated with amphiphilic hyaluronic acid-ceramide (HACE) was fabricated for targeted delivery of anticancer drug and in vivo cancer imaging. Nanohybrid liposomes including doxorubicin (DOX) and Magnevist, a contrast agent for magnetic resonance (MR) imaging, with 120-130nm mean diameter and a narrow size distribution were developed. DOX release from the developed formulation was improved at acidic pH (pH5.5 and 6.8) versus physiological pH (pH7.4). Cytotoxicity induced by the blank plain liposome was reduced by coating the outer surface of the nanohybrid liposome with HACE. Cellular uptake of DOX from the nanohybrid liposome was enhanced by HA and CD44 receptor interaction, versus the plain liposome. In vivo contrast-enhancing effects revealed that the nanohybrid liposome can be used as a tumor targeting MR imaging probe for cancer diagnosis. In a pharmacokinetic study in rats, in vivo clearance of DOX was decreased in the order DOX solution, plain liposome (F2), and nanohybrid liposome (F3), indicating prolonged circulation of the drug in the blood stream and improved therapeutic efficacy of the nanohybrid liposome (F3). Based on these findings, the nanohybrid liposomal system may be a useful candidate for real-time cancer diagnosis and therapy.

Hypoxia-responsive polymeric nanoparticles for tumor-targeted drug delivery

Hypoxia is a condition found in various intractable diseases. Here, we report self-assembled nanoparticles which can selectively release the hydrophobic agents under hypoxic conditions. For the preparation of hypoxia-responsive nanoparticles (HR-NPs), a hydrophobically modified 2-nitroimidazole derivative was conjugated to the backbone of the carboxymethyl dextran (CM-Dex). Doxorubicin (DOX), a model drug, was effectively encapsulated into the HR-NPs. The HR-NPs released DOX in a sustained manner under the normoxic condition (physiological condition), whereas the drug release rate remarkably increased under the hypoxic condition. From in vitro cytotoxicity tests, it was found the DOX-loaded HR-NPs showed higher toxicity to hypoxic cells than to normoxic cells. Microscopic observation showed that the HR-NPs could effectively deliver DOX into SCC7 cells under hypoxic conditions. In vivo biodistribution study demonstrated that HR-NPs were selectively accumulated at the hypoxic tumor tissues. As consequence, drug-loaded HR-NPs exhibited high anti-tumor activity in vivo. Overall, the HR-NPs might have a potential as nanocarriers for drug delivery to treat hypoxia-associated diseases.

Theranostic nanoparticles based on PEGylated hyaluronic acid for the diagnosis, therapy and monitoring of colon cancer

Colon cancer is the second leading cause of cancer-related death in the United States. The considerable mortality from colon cancer is due to metastasis to other organs, mainly the liver. In the management of colon cancer, early detection and targeted therapy are crucial. In this study, we aimed to establish a versatile theranostic system for early tumor detection and targeted tumor therapy by using poly(ethylene glycol)-conjugated hyaluronic acid nanoparticles (P-HA-NPs) which can selectively accumulate in tumor tissue. For the diagnostic application, a near-infrared fluorescence (NIRF) imaging dye (Cy 5.5) was chemically conjugated onto the HA backbone of P-HA-NPs. After intravenous injection of Cy5.5-P-HA-NPs into the tumor-bearing mice, small-sized colon tumors as well as liver-implanted colon tumors were effectively visualized using the NIRF imaging technique. For targeted therapy, we physically encapsulated the anticancer drug, irinotecan (IRT), into the hydrophobic cores of P-HA-NPs. Owing to their notable tumor targeting capability, IRT-P-HA-NPs exhibited an excellent antitumor activity while showing a reduction in undesirable systemic toxicity. Importantly, we demonstrated the theranostic application using Cy5.5-P-HA-NPs and IRT-P-HA-NPs in orthotopic colon cancer models. Following the systemic administration of Cy5.5-P-HA-NPs, neoplasia was clearly visualized, and the tumor growth was effectively suppressed by intravenous injection of IRT-P-HA-NPs. It should be emphasized that the therapeutic responses could be simultaneously monitored by Cy5.5-P-HA-NPs. Our results suggest that P-HA-NPs can be used as a versatile theranostic system for the early detection, targeted therapy, and therapeutic monitoring of colon cancer.