Seung Young Lee

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.

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

Polymeric Nanoparticle-Based Activatable Near-Infrared Nanosensor for Protease Determination In Vivo

We report here a new protease activatable strategy based on a polymer nanoparticle platform. This nanosensor delivers chemically labeled matrix metalloproteinase (MMP)-activatable fluorogenic peptides to the specific MMPs of interest in vivo. Intravenous administration of the nanosensor in an MMP-positive SCC-7 xenograft tumor and a colon cancer mouse model verified the enzyme specificity of the nanosensor in vivo. The design platform of the nanosensor is flexible and can be fine-tuned for a wide array of applications such as the detection of biomarkers, early diagnosis of disease, and monitoring therapeutic efficacy.

Tumor-homing glycol chitosan/polyethylenimine nanoparticles for the systemic delivery of siRNA in tumor-bearing mice

Here, we designed a new nano-sized siRNA carrier system composed of biocompatible/biodegradable glycol chitosan polymer (GC) and strongly positively charged polyethylenimine (PEI) polymers. In order to make a stable and tumor-homing nano-sized carrier, each polymer was modified with hydrophobic 5beta-cholanic acid, and they were simply mixed to form self-assembled GC-PEI nanoparticles (GC-PEI NPs), due to the strong hydrophobic interactions of 5beta-cholanic acids in the polymers. The freshly prepared GC-PEI NPs showed a stable nanoparticle structure (350nm) and they presented a strongly positive-charged surface (zeta potential=23.8) that is enough to complex tightly with negatively charged RFP-siRNAs, designed for inhibiting red fluorescent protein (RFP) expression. The siRNA encapsulated nanoparticles (siRNA-GC-PEI NPs) formed more compact and stable nanoparticle structures (250nm) at 1: 5 weight ratio of siRNA to GC-PEI nanoparticles. In vitro RFP expressing B16F10 tumor cell (RFP/B16F10) culture system, the siRNA-GC-PEI NPs presented a rapid time-dependent cellular uptake profile within 1h. Moreover, the internalized siRNA-GC-PEI NPs lead to specific mRNA breaks down. Furthermore, our new formulation of siRNA-GC-PEI NPs presented a significant inhibition of RFP gene expression of RFP/B16F10-bearing mice, due to their higher tumor-targeting ability. These results revealed the promising potential of GC-PEI NPs as a stable and effective nano-sized siRNA delivery system for cancer treatment.

The effect of surface functionalization of PLGA nanoparticles by heparin- or chitosan-conjugated Pluronic on tumor targeting

The poly (lactide-co-glycolide) (PLGA)-based nanoparticles, coated by the heparin- or chitosan-Pluronic conjugate, were used to improve a relatively low tumor-targeting efficiency of the bare PLGA nanoparticles. The prepared nanoparticles were in the size range of 100-150nm, and the surface exposure of the functional moiety (heparin or chitosan) was confirmed by negatively or positively increased zeta potential values, respectively. The viability tests for both normal and tumor cells displayed minimal cytotoxicity of the nanoparticles. The stable surface coating, which was evident from no change in the size distribution profiles in spite of the surface charge changes in serum environment, effectively provided the desired functionalized surface that clearly enhanced the in vitro cellular uptake of the nanoparticles for both heparin and chitosan functionalization. The in vivo tumor model study, which was carried out in SCC7 tumor-bearing athymic mice, demonstrated that there was a limited, but positive effect of surface functionalization, more effective for chitosan functionalization. The accumulation of chitosan-functionalized PLGA nanoparticles in tumor was 2.4 folds higher than that of the control, PLGA nanoparticles coated with bare Pluronic, and the accumulation in liver was lower than the control. In the case of heparin functionalization, the mean value was 2.2 folds higher than that of the control, but the accumulation in liver was similar to that of the control. Therefore, the surface-functionalization by the chitosan- or heparin-conjugated Pluronic may be an effective approach for the hydrophobic nanoparticle systems aiming for the enhanced tumor imaging and therapy.

In-vivo tumor targeting of pluronic-based nano-carriers.

Pluronic-based nano-carriers including bare forms that were composed of Pluronic F 68(NC(PF 68)) or Pluronic F 127(NC(PF 127)), and chitosan-conjugated forms (Chito-NC(PF 68) or Chito-NC(PF 127)) were prepared by photo-polymerizing two kinds of diacrylated Pluronic (F 68 and F 127) and acrylated chitosan to investigate the effect of chitosan conjugation and their physicochemical characteristics (size and hydrophilicity) of Pluronic-based nano-carriers on the tumor targeting efficiency. All of the nano-carriers were stable in serum-containing media without forming any aggregation and did not show any acute cytotoxicity to both normal (NIH3T3 fibroblast) and tumor (SCC7) cells. Chitosan conjugation did not change their sizes or thermo-sensitive properties of the nano-carriers, but significantly increased their in-vitro cellular uptake compared to the corresponding bare forms. The in-vivo tumor accumulation of these nano-carriers was optically monitored by using Cy5.5-attached nano-carriers in SCC7 tumor-bearing mice. For all cases, local accumulation of the injected nano-carriers in liver was not dominant compared to the tumor site, demonstrating good tumor targeting efficacy of the Pluronic-based nano-carriers. Among different samples, chitosan-conjugated nano-carriers showed much better tumor accumulation than bare forms, and mostly remained up to 72h, implying prolonged blood circulation and more efficient tumor accumulation. Between Chito-NC(PF 68) and Chito-NC(PF 127), Chito-NC(PF 68) showed a little better tumor accumulation and retention, suggesting the difference in Pluronic, thus difference in hydrophilicity and the size of the nano-carriers also might affect the tumor targeting. In contrast, bare nano-carriers were initially accumulated well in tumor, but they were excreted from the tumor site relatively rapidly. Therefore, chitosan-functionalization was very effective for improving the tumor targeting efficacy of Pluronic-based nano-carriers.

A Near-Infrared Fluorescence-Based Optical Thermosensor

A polymeric thermosensor composed of the thermo-responsive block copolymer Pluronic F127 (PF127) and the near-infrared (NIR) dye Cy5.5 can simply monitor, image, and analyze temperature changes. The thermoprobe exhibited linear NIR fluorescent emission changes (see figure) over a broad temperature range (0–80 °C).

In vivo NIRF Imaging of Tumor Targetability of Nanosized Liposomes in Tumor‐Bearing Mice

To optimize tumor targetability of nanosized liposomes for application as drug carriers, various liposomes are prepared by incorporating different amounts (10, 30, and 50 wt%) of cationic, anionic, and PEGylated lipids into neutral lipid. In vivo near-infrared fluorescence images reveal that PEG-PE/PC liposomes display high tumor accumulation in tumor-bearing mice, while large amounts of DOTAP/PC liposomes are rapidly captured in the liver, resulting in poor tumor accumulation. These results demonstrate that optimization of the surface properties of liposomes is very important for their tumor targetability, and that in vivo imaging techniques are useful in developing and optimizing nanosized liposome-based drug carriers.