Jiwon Han

Preparation of HIFU-triggered tumor-targeted hyaluronic acid micelles for controlled drug release and enhanced cellular uptake

In this study, a novel type of high intensity focused ultrasound (HIFU)-triggered active tumor-targeting polymeric micelle was prepared and investigated for controlled drug release and enhanced cellular uptake. Amphiphilic hyaluronic acid (HA) conjugates were synthesized to form docetaxel loaded micelles in aqueous conditions with high encapsulation efficiencies of over 80%. The micelle sizes were limited to less than 150nm, and they varied slightly according to the encapsulated drug amount. Modifying the micellar surface modification with polyethylene glycol diamine successfully inhibited premature drug leakage at a certain level, and it can be expected to prolong the circulation time of the particles in blood. In addition, high-intensity focused ultrasound was introduced to control the release of docetaxel from micelles, to which the release behavior of a drug can be tuned. The in-vitro cell cytotoxicity of docetaxel-loaded micelles was verified against CT-26 and MDA-MB-231 cells. The IC50 values of drug-loaded micelles to CT-26 and MDA-MB-231 cells were 1230.2 and 870.9ng/mL, respectively. However, when exposed to HIFU, the values decreased significantly, to 181.9 and 114.3ng/mL, suggesting that HIFU can enhance cell cytotoxicity by triggering the release of a drug from the micelles. Furthermore, cellular uptake tests were conducted via the quantitative analysis of intracellular drug concentration within CT-26 (CD44 negative), MDA-MB-231 (CD44 positive), and MDA-MB-231 (CD44 blocked), and then imaged with coumarin-6 loaded micelles. The results verified that intracellular drug delivery can be enhanced efficiently via the CD44 receptor-mediated endocytosis of HA micelles. Moreover, HIFU enhanced the cellular uptake behavior by altering the permeability of the cell membrane. It was also able to aid with the extravasation of micelles into the interior of tumors, which will be explained in further research. Therefore, the present study demonstrates that the micelles prepared in this study can emerge as promising nanocarriers of chemotherapeutic agents for controlled drug release and tumor targeting in cancer treatment.

Preparation of Engineered Salmonella Typhimurium-Driven Hyaluronic-Acid-Based Microbeads with Both Chemotactic and Biological Targeting Towards Breast Cancer Cells for Enhanced Anticancer Therapy.

In this study, a new type of targeted bacteriobots is prepared and investigated as a therapeutic strategy against solid tumors. Maleimide-functionalized hyaluronic acid (HA) polymer is synthesized and cross-linked with four-arm-thiolated polyethylene glycol (PEG-SH) to form HA microbeads with diameter of 8 μm through the Michael-type addition. Docetaxel (DTX)-loaded nanoparticles are encapsulated in HA-PEG microbeads and sustained in vitro drug-release pattern of the DTX from the HA-PEG microbeads is observed for up to 96 h. Dual-targeted bacteriobots are prepared using CD 44 receptor-targeted HA microbeads synthesized via microfluidics, followed by the attachment of the flagellar bacterium Salmonella typhimurium, which have been genetically engineered for tumor targeting, onto the surface of the HA microbeads by the specific interaction between streptavidin on the HA beads and biotin on the bacteria. After the attachment of bacteria, the bacteriobots show an average velocity of 0.72 μm s(-1) and high chemotactic migration velocity of 0.43 μm s(-1) towards 4T1 cells lysates. CD 44 receptor-specific cellular uptake is verified through flow cytometry analysis and confocal imaging, demonstrating enhanced intracellular uptake in CD 44 receptor positive tumor cells compared to normal cells. Therefore, the present study suggests that these bacteriobots have dual-tumor-targeting abilities displaying their potential for targeted anticancer therapy.

Hybrid-Actuating Macrophage-Based Microrobots for Active Cancer Therapy

Using macrophage recruitment in tumors, we develop active, transportable, cancer theragnostic macrophage-based microrobots as vector to deliver therapeutic agents to tumor regions. The macrophage-based microrobots contain docetaxel (DTX)-loaded poly-lactic-co-glycolic-acid (PLGA) nanoparticles (NPs) for chemotherapy and Fe3O4 magnetic NPs (MNPs) for active targeting using an electromagnetic actuation (EMA) system. And, the macrophage-based microrobots are synthesized through the phagocytosis of the drug NPs and MNPs in the macrophages. The anticancer effects of the microrobots on tumor cell lines (CT-26 and 4T1) are evaluated in vitro by cytotoxic assay. In addition, the active tumor targeting by the EMA system and macrophage recruitment, and the chemotherapeutic effect of the microrobots are evaluated using three-dimensional (3D) tumor spheroids. The microrobots exhibited clear cytotoxicity toward tumor cells, with a low survivability rate (<50%). The 3D tumor spheroid assay showed that the microrobots demonstrated hybrid actuation through active tumor targeting by the EMA system and infiltration into the tumor spheroid by macrophage recruitment, resulting in tumor cell death caused by the delivered antitumor drug. Thus, the active, transportable, macrophage-based theragnostic microrobots can be considered to be biocompatible vectors for cancer therapy.

Nanohybrid magnetic liposome functionalized with hyaluronic acid for enhanced cellular uptake and near-infrared-triggered drug release

The aim of this work is to prepare and evaluate a novel lipid-polymer hybrid liposomal nanoplatform (hyaluronic acid-magnetic nanoparticle-liposomes, HA-MNP-LPs) as a vehicle for targeted delivery and triggered release of an anticancer drug (docetaxel, DTX) in human breast cancer cells. We first synthesize an amphiphilic hyaluronic acid hexadecylamine polymer (HA-C16) to enhance the targeting ability of the hybrid liposome. Next, HA-MNP-LPs are constructed to achieve an average size of 189.93±2.74nm in diameter. In addition, citric acid-coated magnetic nanoparticles (MNPs) are prepared and embedded in the aqueous cores while DTX is encapsulated in the hydrophobic bilayers of the liposomes. Experiments with coumarin 6 loaded hybrid liposomes (C6/HA-MNP-LPs) show that the hybrid liposomes have superior cellular uptake in comparison with the conventional non-targeting liposomes (C6/MNP-LPs), and the result is further confirmed by Prussian blue staining. Under near-infrared laser irradiation (NIR, 808nm), the HA-MNP-LPs aqueous solution can reach 46.7°C in 10min, and the hybrid liposomes released over 20% more drug than the non-irradiated liposomes. Using a combination of photothermal irradiation and chemotherapy, the DTX-loaded hybrid liposomes (DTX/HA-MNP-LPs) significantly enhance therapeutic efficacy, with the IC50 value of 0.69±0.10μg/mL, which is much lower than the values for DTX monotherapy. Consequently, the prepared hybrid nanoplatform may offer a promising drug delivery vehicle with selective targeting and enhanced drug release in treating CD44-overexpressing cancers.

A Magnetically Actuated Microscaffold Containing Mesenchymal Stem Cells for Articular Cartilage Repair

This study proposes a magnetically actuated microscaffold with the capability of targeted mesenchymal stem cell (MSC) delivery for articular cartilage regeneration. The microscaffold, as a 3D porous microbead, is divided into body and surface portions according to its materials and fabrication methods. The microscaffold body, which consists of poly(lactic-co-glycolic acid) (PLGA), is formed through water-in-oil-in-water emulsion templating, and its surface is coated with amine functionalized magnetic nanoparticles (MNPs) via amino bond formation. The porous PLGA structure of the microscaffold can assist in cell adhesion and migration, and the MNPs on the microscaffold can make it possible to steer using an electromagnetic actuation system that provides external magnetic fields for the 3D locomotion of the microscaffold. As a fundamental test of the magnetic response of the microscaffold, it is characterized in terms of the magnetization curve, velocity, and 3D locomotion of a single microscaffold. In addition, its function with a cargo of MSCs for cartilage regeneration is demonstrated from the proliferation, viability, and chondrogenic differentiation of D1 mouse MSCs that are cultured on the microscaffold. For the feasibility tests for cartilage repair, 2D/3D targeting of multiple microscaffolds with the MSCs is performed to demonstrate targeted stem cell delivery using the microscaffolds and their swarm motion.

Modeling of chemotactic steering of bacteria-based microrobot using a population-scale approach

The bacteria-based microrobot (Bacteriobot) is one of the most effective vehicles for drug delivery systems. The bacteriobot consists of a microbead containing therapeutic drugs and bacteria as a sensor and an actuator that can target and guide the bacteriobot to its destination. Many researchers are developing bacteria-based microrobots and establishing the model. In spite of these efforts, a motility model for bacteriobots steered by chemotaxis remains elusive. Because bacterial movement is random and should be described using a stochastic model, bacterial response to the chemo-attractant is difficult to anticipate. In this research, we used a population-scale approach to overcome the main obstacle to the stochastic motion of single bacterium. Also known as Keller-Segels equation in chemotaxis research, the population-scale approach is not new. It is a well-designed model derived from transport theory and adaptable to any chemotaxis experiment. In addition, we have considered the self-propelled Brownian motion of the bacteriobot in order to represent its stochastic properties. From this perspective, we have proposed a new numerical modelling method combining chemotaxis and Brownian motion to create a bacteriobot model steered by chemotaxis. To obtain modeling parameters, we executed motility analyses of microbeads and bacteriobots without chemotactic steering as well as chemotactic steering analysis of the bacteriobots. The resulting proposed model shows sound agreement with experimental data with a confidence level <0.01.

Dual tumor-targeted multifunctional magnetic hyaluronic acid micelles for enhanced MR imaging and combined photothermal-chemotherapy

Multifunctional polymeric micelles were developed as a promising dual tumor-targeted drug delivery platform for magnetic resonance (MR) imaging and combined photothermal-chemotherapy. HA-C16 copolymers were synthesized via peptide formation process with subsequent co-encapsulation of therapeutic agent docetaxel (DTX) and superparamagnetic iron oxide nanoparticles (SPIONs) to form the multifunctional micelles. The micelles exhibited uniform nanosize and remarkable colloidal stability in aqueous solution. The sustained drug release behavior from HA micelles was observed over the test period. Moreover, the specific targeting capability based on CD44 recptor-mediated endocytosis and the enhanced targeting efficacy by in presence of external magnetic field were investigated. The clustered SPIONs within micelles exerted excellent contrast effect with high r2 relaxivity in MR phantom test. Furthermore, the multifunctional micelles could readily convert light to heat to hyperthermia temperature upon near infrared light irradition and induce photothermal ablation to breast cancer cells. The combined photothermal therapy with DTX-mediated chemotherapy of the developed multifunctional polymeric micells could generate a synergistic therapeutic effect. Based on these findings, the resulting multifunctional micelles may provide high potential for multimodality theragnosis of cancer.

Combined photothermal-chemotherapy of breast cancer by near infrared light responsive hyaluronic acid-decorated nanostructured lipid carriers

In this study, a novel type of hyaluronic acid (HA)-decorated nanostructured lipid carrier (NLC) was prepared and investigated as a light-triggered drug release and combined photothermal-chemotherapy for cancer treatment. Polyhedral gold nanoparticles (Au NPs) with an average size of 10 nm were synthesized and co-encapsulated with doxorubicin (DOX) in the matrix of NLCs with a high drug loading efficiency (above 80%). HA decoration was achieved by the electrostatic interaction between HA and CTAB on the NLC surface. A remarkable temperature increase was observed by exposing the Au NP-loaded NLCs to an NIR laser, which heated the samples sufficiently (above 40 °C) to kill tumor cells. The entrapped DOX exhibited a sustained, stepwise NIR laser-triggered drug release pattern. The biocompatibility of the NLCs was investigated by MTT assay and the cell viability was maintained above 85%, even at high concentrations. The intracellular uptake of free DOX and entrapped DOX, observed by confocal microscopy, revealed two distinct uptake mechanisms, i.e. passive diffusion and endocytosis, respectively. In particular, internalization of the HA-Au-DOX-NLCs was more extensively enhanced than the Au-DOX-NLCs, which was attributed to HA-CD44 receptor-mediated endocytosis. Meanwhile, the internalized NLCs successfully escaped from the lysosomes, increasing the intracellular DOX. The HA-Au-DOX-NLCs IC50 value decreased from 2.3 to 0.6 μg ml-1 with NIR irradiation at 72 h, indicating the excellent synergistic antitumor effect of photothermal-chemotherapy. The photothermal ablation was further confirmed by a live/dead cell staining assay. Thus, a combined photothermal-chemotherapy approach has been proposed as a promising strategy for cancer treatment.

Manipulation of tumor targeting cell-based microrobots carrying NIR light sensitive therapeutics using EMA system and chemotaxis

In this study, we prepare and evaluate a novel cellbased micro-platform (microrobot) for active tumor therapy. The microrobots are fabricated utilizing the engulfment activity of immune cells (macrophages) with drug-loaded magnetic liposomes via phagocytosis. First, we synthesize magnetic nanoparticles (MNP) with superparamagnetic properties and high energy absorbance to near-infrared (NIR) light, and load the MNPs to liposomes (MNP-DLs). Then, we prepare the microrobots by incubating the MNP-DLs with the macrophages. After that, we characterize the tumor targeting ability of the microrobots using electromagnetic actuating (EMA) system and a transwell chemotactic assay. Experiment results show that the microrobots can be controlled by an external magnetic field to reach the average velocity of approximately 11 μm/second, and they can cross the membranes mimicking the blood barrier to tumor chemo-attractants with the infiltration rate up to 74%. Therefore, the study proposes an innovative approach for active tumor targeting and NIR light triggered drug delivery using the developed cellular microrobots.

Motility steering of bacteriobots using chemical gradient microchannel

In the recent years, several groups focused on the development of bacteria based microrobots (bacteriobots) using microbeads and flagellar bacteria. The bacteriobots will be a promising cancer therapeutic method in the future with drug encapsulation inside microbeads. However, it remains elusive that how to steer the motion of bacteriobots. In this study, we attempted to steer the motion of bacteriobots with the intrinsic bacterial chemotaxis to particular chemicals. Therefore, a new microfluidic channel was designed and fabricated through micro-molding method of hydrogel patterns, which a sustained chemical gradient was investigated using rhodamine B at various determined time intervals. Thereafter, the bacteriobots solution was injected into the central channel with chemoattractant gradient, then the chemotactic motion of bacteriobots was investigated through a microscope and analyzed with MATLAB program. Moreover, some other chemoattractant chemicals, secreted from tumor cells could also stimulate the tumor targeting ability possible with bacteriobots. Overall, the motion of bacteriobots can be steered through bacterial chemotaxis, and we expect drug embedded bacteriobots to be a new targeted therapy in cancer treatment.