Ping-Shan Lai

Self-assembled star-shaped chlorin-core poly(ɛ-caprolactone)–poly(ethylene glycol) diblock copolymer micelles for dual chemo-photodynamic therapies

Amphiphilic 4-armed star-shaped chlorin-core diblock copolymers based on methoxy poly(ethylene glycol) (mPEG) and poly (epsilon-caprolactone) (PCL) were synthesized and characterized in this study. The synthesized photosensitizer-centered amphiphilic star block copolymer that forms assembled micelle-like structures can be used in a photodynamic therapy (PDT)-functionalized drug delivery system. Moreover, the hydrophobic chemotherapeutic agent, paclitaxel, can be trapped in the hydrophobic inner core of micelles. In our results, the star-polymer-formed micelle exhibited efficient singlet oxygen generation, whereas the hydrophobic photosensitizer failed due to aggregation in aqueous solution. The chlorin-core micelle without paclitaxel loading exhibited obvious phototoxicity in MCF-7 breast cancer cells with 7J/cm2 or 14J/cm2 light irradiation at a chlorin concentration of 125microg/ml. After paclitaxel loading, the size of micelle increased from 71.4nm to 103.2nm. Surprisingly, these micelles were found to improve the cytotoxicity of paclitaxel significantly in MCF-7 cells after irradiation through a synergistic effect evaluated by median effect analysis. This functionalized micellar delivery system is a potential dual carrier for the synergistic combination of photodynamic therapy and chemotherapy for the treatment of cancer.

Dual chemotherapy and photodynamic therapy in an HT-29 human colon cancer xenograft model using SN-38-loaded chlorin-core star block copolymer micelles

Chlorin-core star-shaped block copolymer (CSBC) may self-assemble to form micelles, which act as nanosized photosensitizing agents for photodynamic therapy (PDT) and further encapsulate hydrophobic drugs. This functionalized micellar delivery system is a potential dual carrier for the synergistic combination of photodynamic therapy and chemotherapy for the treatment of cancer. In this study, SN-38 encapsulated CSBC micelles were successfully prepared using a lyophilization-hydration method. Our results show that the prolonged plasma residence time of SN-38/CSBC micelles as compared with free CPT-11 permit increased tumor accumulation and consequently, improved antitumor activity. The combined effects of SN-38/CSBC micelles with PDT were evaluated in an HT-29 human colon cancer xenograft model. Interesting, SN-38/CSBC-mediated PDT synergistically inhibited tumor growth, resulting in up to 60% complete regression of well-established tumors after 3 treatments. These treatments also decreased the microvessel density (MVD) and cell proliferation within the subcutaneous tumors. Therefore, this SN-38/CBSC delivery system has the potential to offer dual therapies for the synergistic combination of PDT and chemotherapy for the treatment of cancer.

Non-toxic phototriggered gene transfection by PAMAM-porphyrin conjugates

Development of controllable and non-toxic gene transfection systems is a core issue in gene therapy. Photochemical internalization, an innovative strategy in cytosolic release, provides us with an opportunity to develop a light-inducible gene delivery system. In this study, a novel photochemical internalization (PCI)-mediated gene delivery system was synthesized by surface modification of polyamidoamine (PAMAM) dendrimers via 5,10,15-tri(4-acetamidophenyl)-20-mono(4-carboxyl-phenyl)porphyrin (TAMCPP) conjugated to the generation 4 PAMAM dendrimer (G4). This water-soluble PAMAM-TAMCPP conjugate was characterized for cell viability, phototoxicity, DNA complexation, and in vitro transfection activity. The results show that TAMCPP conjugation did not increase the cytotoxicity of the PAMAM dendrimer below 20 microM, but significantly induced cell death after suitable irradiation. Under almost non-toxic G4-TAMCPP-mediated PCI treatment, the expression of green fluorescent protein determined by flow cytometry could be markedly enhanced in HeLa cells. Therefore, the G4-TAMCPP conjugate had an inducible and effective gene transfection activity, and showed considerable potential as a bimodal biomaterial for PCI-mediated gene therapy.

Reversal of doxorubicin-resistance by multifunctional nanoparticles in MCF-7/ADR cells

The efficacy of many chemotherapeutic agents is reduced in cells that have developed multiple drug resistance (MDR). To address this important problem, a biodegradable polymer was coupled to a photosensitizer and the resulting photosensitizer-nanoparticles were loaded with the chemotherapeutic agent doxorubicin. The combination of photosensitizer and chemotherapeutic agent had a synergistic action on a doxorubicin-resistant breast cancer MCF-7 cell line. To increase the effectiveness of this combination, d-alpha-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS), an inhibitor of the multidrug transporter overproduced in these resistant cells, was added during the formation of the nanoparticles. The insertion of TPGS decreased the P-glycoprotein activity, increased the intracellular accumulation doxorubicin, and also increased the therapeutic efficacy of the resulting nanoparticles. Both TPGS and irradiation of the photoreactive nanoparticles caused doxorubicin to move from the cytoplasm to the nucleus. This combination of photodynamic activity in a powerful nanocarrier loaded with the chemotherapeutic agent doxorubicin can be used to deliver two types of cancer therapy simultaneously, and the addition of TPGS can further enhance the entry of doxorubicin into the nucleus. Therefore, this innovative delivery system can act as a potential nanomedicine for both drug-sensitive and drug-resistant cancer therapy.

Real-time visualization of pH-responsive PLGA hollow particles containing a gas-generating agent targeted for acidic organelles for overcoming multi-drug resistance

Chemotherapy research highly prioritizes overcoming the multi-drug resistance (MDR) effect in cancer cells. To overcome the drug efflux mediated by P-glycoprotein (P-gp) transporters, we developed pH-responsive poly(D,L-lactic-co-glycolic acid) hollow particles (PLGA HPs), capable of delivering doxorubicin (DOX) into MDR cells (MCF-7/ADR). The shell wall of PLGA HPs contained DiO (a hydrophobic dye), and their aqueous core carried DOX hydrochloride salt and sodium bicarbonate, a gas-generating agent when present in acidic environments. Both DiO and DOX could serve as fluorescence probes to localize HPs and visualize their intracellular drug release in real-time. Real-time confocal images provided visible evidences of the acid-responsive intracellular release of DOX from PLGA HPs in MDR cells. Via the macropinocytosis pathway, PLGA HPs taken up by cells experienced an increasingly acidic environment as they trafficked through the early endosomes and then matured into more acidic late endosomes/lysosomes. The progressive acidification of the internalized particles in the late endosomes/lysosomes generated CO(2) bubbles, leading to the disruption of HPs, prompt release of DOX, its accumulation in the nuclei, and finally the death of MDR cells. Conversely, taken up via a passive diffusion mechanism, free DOX was found mainly at the perimembrane region and barely reached the cell nuclei; therefore, no apparent cytotoxicity was observed. These results suggest that the developed PLGA HPs were less susceptible to the P-gp-mediated drug efflux in MDR cells and is a highly promising approach in chemotherapy.

Bioluminescence resonance energy transfer using luciferase-immobilized quantum dots for self-illuminated photodynamic therapy

Photodynamic therapy (PDT) is an innovative method for cancer treatment that involves the administration of a photosensitizing agent followed by exposure to visible light. An appreciable amount of a particular light source is a key to activate photosensitizers in PDT. However, the external excitation light source is a problem for clinical application because of the limitation of tissue-penetrating properties. Additionally, the wavelength of laser emission should match the absorption wavelength of each photosensitizer for efficient generation of reactive oxygen species and cell killing. In this study, Renilla luciferase-immobilized quantum dots-655 (QD-RLuc8) was used for bioluminescence resonance energy transfer (BRET)-mediated PDT to resolve these problems. The bioluminescent QD-RLuc8 conjugate exhibits self-illumination at 655 nm after coelenterazine addition, which can activate the photosensitizer, Foscan(®)-loaded micelles for PDT. Our results show that BRET-mediated PDT by QD-RLuc8 plus coelenterazine (20 μg/mL) successfully generated reactive oxygen species (40.8%), killed ~ 50% A549 cells at 2 μg/mL equivalent Foscan(®)in vitro and significantly delayed tumor growth in vivo due to cell apoptosis under TUNEL analysis without obvious weight loss. Based on immunohistochemical observations, the proliferating cell nuclear antigen (PCNA)-negative area of tumor sections after BRET-mediated PDT was obviously increased compared to the PDT-untreated groups without an external light source. We conclude that this nanotechnology-based PDT possesses several clinical benefits, such as overcoming light penetration issues and treating deeper lesions that are intractable by PDT alone.

Reduced Skin Photosensitivity with meta-Tetra(hydroxyphenyl)chlorin-Loaded Micelles Based on a Poly(2-ethyl-2-oxazoline)-b-poly(d,l-lactide) Diblock Copolymer in Vivo

Photodynamic therapy (PDT) is a light-induced chemical reaction that produces localized tissue damage for the treatment of cancers and other nonmalignant conditions. The activation of photosensitizers in a target tissue is accomplished with a specific light source in the presence of molecular oxygen. In the clinic, patients treated with PDT should be kept away from direct sunlight or strong indoor lighting to avoid skin phototoxicity. In this study, a photosensitizer encapsulated within a micelle was developed to overcome this problem. The pH-sensitive micelles were successfully incorporated with meta-tetra(hydroxyphenyl)chlorin (m-THPC), and the cytotoxicity and antitumor effects were investigated in vitro and in vivo. Our results demonstrated that PDT with m-THPC-loaded micelles had no significant adverse effects on the body weight of mice in vivo. Furthermore, after an extended delivery time, m-THPC-loaded micelles and free m-THPC had similar antitumor effects, but the m-THPC-loaded micelles had less skin phototoxicity. Thus, this strategy could be used as a potential nanocarrier for PDT-mediated cancer therapy.

Development of pH sensitive 2-(diisopropylamino)ethyl methacrylate based nanoparticles for photodynamic therapy

Photodynamic therapy is an effective treatment for tumors that involves the administration of light-activated photosensitizers. However, most photosensitizers are insoluble and non-specific. To target the acid environment of tumor sites, we synthesized three poly(ethylene glycol) methacrylate-co-2-(diisopropylamino)ethyl methacrylate (PEGMA-co-DPA) copolymers capable of self-assembly to form pH sensitive nanoparticles in an aqueous environment, as a means of encapsulating the water-insoluble photosensitizer, meso-tetra(hydroxyphenyl)chlorin (m-THPC). The critical aggregation pH of the PEGMA-co-DPA polymers was 5.8-6.6 and the critical aggregation concentration was 0.0045-0.0089 wt% at pH 7.4. Using solvent evaporation, m-THPC loaded nanoparticles were prepared with a high drug encapsulation efficiency (approximately 89%). Dynamic light scattering and transmission electron microscopy revealed the spherical shape and 132 nm diameter of the nanoparticles. The in vitro release rate of m-THPC at pH 5.0 was faster than at pH 7.0 (58% versus 10% m-THPC released within 48 h, respectively). The in vitro photodynamic therapy efficiency was tested with the HT-29 cell line. m-THPC loaded PEGMA-co-DPA nanoparticles exhibited obvious phototoxicity in HT-29 colon cancer cells after light irradiation. The results indicate that these pH sensitive nanoparticles are potential carriers for tumor targeting and photodynamic therapy.

Colorectal cancer cell detection by 5-aminolaevulinic acid-loaded chitosan nano-particles

Colorectal cancer is one of the leading causes of malignant death in Taiwan because it often remains undetected until later stages of the disease. In this study, we designed an oral form nano-particle to encapsulate 5-aminolaevulinic acid (5-ALA) to improve the detection of colorectal cancer cells in vivo. The nano-particle should escape from bacteria uptake in the gastrointestinal tract which seriously interferes the results of endoscopic observation. In this study, chitosan was mixed with sodium tripolyphosphate (STPP) and 5-ALA to prepare chitosan nano-particles (CN) and 5-ALA loaded chitosan nano-particles (CNA) by adding different pH values and concentrations of 5-ALA solution. The average particle size and zeta-potential of CN and CNA were measured by the Zetasizer-3000. The results revealed that particle size with different zeta-potential could be manipulated just by 5-ALA concentrations and pH values. CNA particles prepared at pH 7.4 and pH 9 of 5-ALA solutions with a concentration higher than 0.5 mg/ml showed a promising loading efficiency of up to 75% and an optimum average particle size of 100 nm. The zeta-potential for CNA was over 30 mV that kept the nano-particle stable without aggregation when stored in suspension solution. Fluorescence microscope examination showed that CNA could be engulfed by Caco-2 colon cancer cells but showed no evidence of being taken up by Escherichia coli. This result implies that CNA could exclude the influence of normal flora inside the gut and serves as an adequate tool for fluorescent endoscopic detection of colorectal cancer cells in vivo.

Metal nanobullets for multidrug resistant bacteria and biofilms.

Infectious diseases were one of the major causes of mortality until now because drug-resistant bacteria have arisen under broad use and abuse of antibacterial drugs. These multidrug-resistant bacteria pose a major challenge to the effective control of bacterial infections and this threat has prompted the development of alternative strategies to treat bacterial diseases. Recently, use of metallic nanoparticles (NPs) as antibacterial agents is one of the promising strategies against bacterial drug resistance. This review first describes mechanisms of bacterial drug resistance and then focuses on the properties and applications of metallic NPs as antibiotic agents to deal with antibiotic-sensitive and -resistant bacteria. We also provide an overview of metallic NPs as bactericidal agents combating antibiotic-resistant bacteria and their potential in vivo toxicology for further drug development.