Won Il Choi

Graphene oxide mediated delivery of methylene blue for combined photodynamic and photothermal therapy.

Nano graphene oxide sheet (nanoGO) was non-covalently functionalized with Pluronic block copolymer and complexed with methylene blue, a hydrophilic and positively charged photosensitizer, via electrostatic interaction for combined photodynamic-photothermal therapy of cancer. Pluronic coating of nanoGO ensured its stability in biological fluids. NanoGO plays dual role of a photothermal material as well as a delivery agent for photosensitizer. The release of the photosensitizer from nanoGO surface was pH-dependent and an acidic condition increased the release rate considerably. This nanocomplex showed enhanced uptake by cancer cells than normal cells and in the absence of light it showed no major toxicity towards the cells. In contrast, when irradiated with selective NIR laser lights, it induced significant cell death. Intravenous injection of the complex into tumor bearing mice showed high tumor accumulation, and when the tumors were exposed to NIR lights, it caused total ablation of tumor tissue through the combined action of photodynamic and photothermal effects. This work shows the potential of nanoGO for synergistic combination phototherapy of tumor in vivo.

Photothermal cancer therapy and imaging based on gold nanorods.

Gold nanorods (GNRs), which strongly absorb near-infrared (NIR) light, have shown great potential in fields of biomedical application. These include photothermal therapy, molecular imaging, biosensing, and gene delivery, especially for the treatment of diseased tissues such as cancer. These biomedical applications of GNRs arise from their various useful properties; photothermal (nanoheater) properties, efficient large scale synthesis, easy functionalization, and colloidal stability. In addition, GNRs do not decompose and have an enhanced scattering signal and tunable longitudinal plasmon absorption which allow them to be used as a stable contrast agent. Therefore, GNRs are also promising theranostic agents, combining both tumor diagnosis and treatment. In this review, we discuss the recent progress of invitro and invivo explorations of the diagnostic and therapeutic applications of GNRs as a component of cancer therapy.

Brain-targeted delivery of protein using chitosan- and RVG peptide-conjugated, pluronic-based nano-carrier

Brain-targeted delivery of drug or imaging agent is hard to achieve efficiently due to the infiltrative nature of the blood-brain barrier (BBB). Moreover, delivery of therapeutic proteins to brain tissue is further limited by the size and physic-chemical properties of proteins. In this work, we developed a chitosan-conjugated Pluronic-based nano-carrier with a specific target peptide for the brain (rabies virus glycoprotein; RVG29) and applied for the protein delivery to the brain. The in-vivo brain accumulation of the nano-carrier in mice followed i.v injection was optically monitored with Cy5.5-conjugation to the nano-carrier, and the result showed that the Pluronic-based nano-carrier conjugated with both chitosan and the peptide was very efficient for the accumulation in brain tissue and was remarkably better than the nano-carrier conjugated with the peptide only. β-galactosidase, a model protein, was also delivered and accumulated efficiently in the brain by loading in the nano-carrier, analyzed by the bio-distribution of β-galactosidase. The delivered protein in the brain also maintained its bioactivity. Therefore, RVG29- and chitosan-conjugated Pluronic-based nano-carrier could be potentially useful for the diagnosis and therapy of brain diseases.

Efficient skin permeation of soluble proteins via flexible and functional nano-carrier.

In spite of several intrinsic and distinct advantages, a topical and transdermal administration of drugs has been limited mainly due to very low permeability of drugs across skin. Especially, it is generally regarded that hydrophilic macromolecules such as proteins, peptides, and vaccines cannot penetrate across skin. In this study, we demonstrated that chitosan-conjugated, Pluronic-based nano-carrier (nanogel) can act as an efficient delivery vehicle of hydrophilic proteins across human skin. The functional nano-carrier (<100 nm in size), chemically-crosslinking Pluronic F 127 with chitosan conjugation, is flexible and soft with reservoir characteristics for biomacromolecules. The in-vitro permeation experiments through human cadaver skin revealed remarkable permeability of hydrophilic proteins of various sizes including FITC-BSA (67 kDa) and FITC-Insulin (6 kDa) by direct penetration of the nano-carrier across skin. The bioactivity post-permeation of proteins via the functional nano-carrier was also confirmed by delivering ß-galactosidase. Results presented in this paper suggest the use of chitosan-conjugated flexible nano-carrier as a novel platform for transcutaneous delivery of hydrophilic macromolecules and other drug-delivery applications.

Tumor-targeting nanogel that can function independently for both photodynamic and photothermal therapy and its synergy from the procedure of PDT followed by PTT.

A dual-function nano-system for synergistic photodynamic therapy (PDT) and photothermal therapy (PTT) was constructed. Gold nanorods (GNRs) as a PTT agent and chlorin e6 (Ce6) as a photosensitizer (PS) for PDT were loaded into a chitosan-functionalized, Pluronic-based nanogel that was proven to be an efficient delivery vehicle to the tumor site in vivo. Previously reported combined therapy systems relied on quenching and de-quenching of PS for PDT upon thermo-impact of PTT, thus only PTT followed by PDT procedure was possible. In contrast, the present dual-acting system has no quenching between PS and GNRs by preventing direct contact and self-aggregation of photo-sensitizers, allowing independent PDT or PTT procedure. In both in vitro cell culture and in vivo tumor-bearing mice experiments, a remarkably enhanced tumor ablation compared to the treatment of PDT or PTT only was observed by the treatment of PDT followed by PTT, but not significantly by the treatment of PTT followed by PDT. Thus, the present study demonstrated the synergistic effect of PDT and PTT in a sequence-dependent manner, and our system is a promising dual function nano system to achieve the enhanced phototherapy in vivo.

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.

Enhanced regeneration of the ligament-bone interface using a poly(L-lactide-co-ε-caprolactone) scaffold with local delivery of cells/BMP-2 using a heparin-based hydrogel.

Recently, the ligament-bone (LTB) junction has been emphasized for the effective transmission of mechanical force and the reduction in stress concentration between the soft ligament and hard bone tissue. The aim of this study was to regenerate an integrated LTB interface by inoculating LTB-relevant cells, isolated from fibrocartilage (FC) or ligament (LIG), separately into each designated region in a single porous cylindrical PLCL scaffold. An injectable, heparin-based hydrogel that has proved to be effective in the culture of chondrocytes as well as the sustained release of growth factor was employed to locally deliver fibrochondrocytes and osteoinductive bone morphogenetic protein-2 (BMP-2) into the FC region, to promote FC regeneration. In in vitro experiments the hydrogel-combined FC systems produced significantly larger amounts of calcium and glycosaminoglycans (GAGs), but less collagen and DNA than FC samples without the hydrogel and all LIG samples. After in vivo subcutaneous implantation in mice for 8 weeks the secreted calcium and GAG contents of the hydrogel-containing FC samples were superior or similar to those of the in vitro hydrogel-containing FC samples at 6 weeks. As a result of the enhanced production of calcium and GAG, the in vivo hydrogel-containing FC samples produced the highest compressive modulus among all samples. Histological and immunofluorescence analysis as well as elemental analysis also confirmed a denser and more homogeneous distribution of calcium, GAG, osteocalcin and neovascularization marker in the in vitro/in vivo hydrogel-containing FC systems than those without hydrogel. These results also show the beneficial effects of BMP-2 added using the hydrogel. In summary, the use of a heparin-based hydrogel for the local delivery of fibrochondrocytes and BMP-2 could accelerate the maturation and differentiation of LTB-specific FC tissues, and it was also possible to recreate the unique stratification of calcified FC and LIG tissues in a single porous PLCL scaffold in terms of both biochemical and biomechanical properties.

One pot, single phase synthesis of thermo-sensitive nano-carriers by photo-crosslinking of a diacrylated pluronic

A simple, single phase synthesis method for the preparation of thermo-sensitive pluronic-based nano-carriers was developed. Pluronic-based nano-carriers that exhibit a sharp volume transition over a temperature range of 4–40 °C were prepared by aqueous phase photo-crosslinking of diacrylated pluronic F 127 (DA-PF 127) without using an organic dispersion phase. They showed hydrodynamic diameter variation (460 ± 37 nm at 4 °C, 55 ± 5 nm at 40 °C) while maintaining their structural stability. Target agents were efficiently loaded (>90%) by simple incubation with the nano-carriers at 4 °C. FITC-BSA loaded fluorescence imaging, gold nano-particle loaded TEM imaging, and the high loading capacity (over 200%) of the pluronic-based nano-carriers indicated a reservoir characteristic of the system. The release pattern of the loaded protein could be controlled repeatedly and reversibly by temperature change due to the thermo-sensitive properties of the nano-carriers.

Highly selective in-vivo imaging of tumor as an inflammation site by ROS detection using hydrocyanine-conjugated, functional nano-carriers

Previously, the optical imaging of chitosan-functionalized, Pluronic-based nano-carriers by Cy5.5 conjugation revealed a good tumor targeting characteristic of the nano-carriers in vivo [J. Control. Release, 147 (2010) 109-117]. However, in spite of the relatively strong signal from tumor site, they also showed strong fluorescence signals from other organs, especially liver. Thus, for the detection of pathological sites, the direct use of the Cy5.5-conjugated nano-carriers is limited due to significant background signals associated with non-specific delivery of the probes. To overcome this limitation, in this study, we prepared hydrocyanine-conjugated and chitosan-functionalized Pluronic-based nano-carriers (Hydrocyanine-NC) that can detect ROS in pathological sites. The reduction of cyanine to hydrocyanine of the nano-carriers resulted in complete disappearance of fluorescence emission, and the fluorescence could be recovered by ROS-induced re-oxidization. Hydrocyanine-NC could detect various ROS including superoxide anion (O(2)(-)) and hydroxyl radical (OH(-)) in a dose-dependent manner. Hydrocyanine-NC was also stable in serum-containing media and did not show acute cytotoxicity. Hydrocyanine-NC developed strong fluorescence by the intracellular ROS formation in LPS-stimulated macrophage cells in vitro. As an in-vivo inflammation site imaging, SCC7 tumor-bearing mice were optically monitored after the i.v. injection of the dye-conjugated nano-carriers. When non-reduced, cyanine-conjugated and chitosan-functionalized Pluronic-based nano-carriers (Cyanine-NC) were injected, strong fluorescence emission was observed from the abdominal area as well as from the tumor site, and it remained over 2days. In contrast, in the case of Hydrocyanine-NC, the initially very weak fluorescence emission from the abdominal area disappeared over time whereas the fluorescence emission from the tumor site was similar to that of Cyanine-NC. Therefore, the re-oxidation of Hydrocyanine-NC by ROS in vivo specifically eliminated the background signals from non-specific delivery of the probes, but it produced fluorescence emission strong enough to monitor the target inflammation site selectively.

The effect of mechanical properties of iron oxide nanoparticle-loaded functional nano-carrier on tumor targeting and imaging

To achieve a sufficient targeting efficiency and prolonged half-life in-vivo, the physicochemical parameters including size and surface chemistry of therapeutic and imaging agents should be controlled. In this study, we prepared an iron oxide nanoparticle (IONP)-loaded, functional nano-carrier with different loading contents to modulate the mechanical properties of the system, and compared the characteristics of tumor targeting and imaging in terms of loading contents of IONP. As a functional nano-carrier, chitosan-conjugated, Pluronic-based nano-carrier with useful properties such as long blood circulation, good tumor targeting, and easy loading of macromolecules was used. IONPs were efficiently encapsulated into the nano-carrier (high loading efficiency over 95%) and the mechanical properties of the IONP-loaded nano-carrier were controlled by varying the loading amount of IONP. The IONP-loaded nano-carrier with the higher loading content of IONP (40 wt.%) was significantly more rigid (over 2×) than those with lower loading contents of IONP (5 and 15 wt.%). Although the nano-carrier with the higher loading content of IONP showed more enhanced MR contrast effect with higher T(2) relaxivity and higher intracellular uptake in vitro, characteristics of in-vivo tumor targeting and MR cancer imaging were not good compared to that of the nano-carrrier with the lower loading contents of IONP. Since different loading contents did not affect other characteristics of the system (size, surface chemistry, and surface charge), the present result suggests that the mechanical properties (strength/flexibility) of nano-systems are also important factors to be controlled for targeted delivery and imaging.