Ming-Jium Shieh

Targeting efficiency and biodistribution of biotinylated-EGF-conjugated gelatin nanoparticles administered via aerosol delivery in nude mice with lung cancer

Lung cancer is the most malignant cancer today; in order to develop an effective drug delivery system for lung cancer therapy, gelatin nanoparticles (GPs) were modified with NeutrAvidin(FITC)-biotinylated epidermal growth factor (EGF) to form EGF receptor (EGFR)-seeking nanoparticles (GP-Av-bEGF). Aerosol droplets of the GP-Av-bEGF were generated by using a nebulizer and were delivered to mice model of lung cancer via aerosol delivery. Analysis of the aerosol size revealed that 99% of the nanoparticles after nebulization had a mass median aerodynamic diameter (MMAD) within the suitable range (0.5-5 microm) for lower airway deposition. The safety of inhaled nanoparticles was examined by lung edema and myeloperoxidase (MPO) activity assay. Theres no finding suggestive of acute lung inflammation following inhalation. The fluorescence images obtained from live mice showed that the GP-Av-bEGF could target the cancerous lungs in a more specific manner. Fluorescence analysis of the organs revealed that the GP-Av-bEGF was mainly distributed in cancerous lungs. In contrast, nanoparticle accumulation was lower in normal lungs. The histological results indicated that the fluorescent GP-Av-bEGF was colocalized with the anti-EGFR-immunostain due to EGFR binding. The results of this study revealed that GP-Av-bEGF could target to the EGFR-overexpression cancer cells in vivo and may prove to be beneficial drug carriers when administered by simple aerosol delivery for the treatment of lung cancer.

Aptamer-based tumor-targeted drug delivery for photodynamic therapy.

A specialized G-rich DNA structure, G-quadruplex, has been studied for its special physical characteristics and biological effects. Herein we report a novel strategy of using G-quadruplex as a drug carrier to target cancer cells for photodynamic therapy (PDT). A G-quadruplex forming AS1411 aptamer could be physically conjugated with six molecules of porphyrin derivative, 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP4), to fabricate the apt-TMP complex. The TMPyP4 molecules in the complex were identified to bind tightly to the aptamer by intercalation and outside binding. Because the G-quadruplex structure is known to target the overexpressed nucleolin in cancer cells, in this study, the effect of the G-quadruplex structure as a carrier for the delivery of TMPyP4 into cancer cells by nucleolin-mediated internalization was investigated. The results showed that the apt-TMP complex exhibited a higher TMPyP4 accumulation in MCF7 breast cancer cells than in M10 normal epithelium cells. After treated with light for 180 s, the photodamage in MCF7 cells was larger than in M10 cells. These results indicated that the TMPyP4 delivery and uptake were mediated by the specific interaction of the apt-TMP complex with nucleolin on the cellular surface and that the use of the AS1411 aptamer as a drug carrier may be a potential tactic in cancer therapy.

Folic Acid-Conjugated Chitosan Nanoparticles Enhanced Protoporphyrin IX Accumulation in Colorectal Cancer Cells

Folic acid can be covalently conjugated to chitosan molecules via its gamma-carboxyl moiety and thus retain a high affinity for colorectal cancer cells bearing folate receptor overexpression. Colorectal cancer is one of the leading causes of malignant death and often goes undetected with current colonoscopy practices. Improved methods of detecting dysplasia and tumors during colonoscopy will improve mortality. A folic acid conjugated chitosan nanoparticle as a suitable vehicle for carrying 5-aminolaevulinic acid (5-ALA) is developed to enhance the detection of colorectal cancer cells in vivo after a short-term uptake period. Chitosan can be successfully conjugated with folic acid to produce folic acid-chitosan conjugate, which is then loaded with 5-ALA to create nanoparticles (fCNA). The loading efficiency of 5-ALA in fCNA particles and the z-average diameter were in the range 35-40% and 100 nm, respectively. The zeta-potential for fCNA was 20 mV, enough to keep the nanoparticle stable without aggregation. The fCNA is then incubated with HT29 and Caco-2 colorectal cancer cell lines overexpressing folate receptor on the surface of the cell membrane to determine the rate of accumulation of protoporphyrin IX (PpIX). The results show that fCNA can be taken up more easily by HT29 and Caco-2 cell lines after short-term uptake period, most likely via receptor-mediated endocytosis, and the PpIX accumulates in cancer cells as a function of the folate receptor expression and the folic acid modification. Therefore, the folic acid-chitosan conjugate appears to be an ideal vector for colorectal-specific delivery of 5-ALA for fluorescent endoscopic detection.

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.

Multimodal Image-Guided Photothermal Therapy Mediated by 188Re-Labeled Micelles Containing a Cyanine-Type Photosensitizer

Multifunctional micelles loaded with the near-infrared (NIR) dye and labeled with the radionuclide rhenium-188 ((188)Re) have been developed to provide multimodalities for NIR fluorescence and nuclear imaging and for photothermal therapy (PTT) of cancer. The NIR dye, IR-780 iodide, allowed the micelles to have dual functions in cancer NIR imaging and PTT. The (188)Re-labeled IR-780 micelles enabled imaging by NIR fluorescence and by microSPECT to guide the delivery of drugs and to monitor in real-time the tumor accumulation, intratumoral distribution, and kinetics of drug release, which serve as a basis of specific photothermal injury to the targeted tissue. We also investigated the biodistribution, generation of heat, and photothermal cancer ablation of IR-780 micelles of both in vitro and in vivo xenografts. Histopathology observed irreversible tissue damage, such as necrotic features, decreased cell proliferation, increased apoptosis of cells, and increased expression of heat shock proteins in the PTT-treated tumors. The (188)Re-labeled IR-780 micelles offer multifunctional modalities for NIR fluorescence and nuclear imaging and for PTT of cancer.

The cardiomyogenic differentiation of rat mesenchymal stem cells on silk fibroin-polysaccharide cardiac patches in vitro.

Polysaccharides and proteins profoundly impact the development and growth of tissues in the natural extra-cellular matrix (ECM). To mimic a natural ECM, polysaccharides were incorporated to/or co-sprayed with silk fibroin (SF) to produce SF/chitosan (CS) or SF/CS-hyaluronic acid (SF/CS-HA) microparticles that were further processed by mechanical pressing and genipin cross-linking to produce hybrid cardiac patches. The ATR-FTIR spectra confirm the co-existence of CS or CS-HA and SF in microparticles and patches. For evaluating the cellular responses of rMSCs to the SF/CS and SF/CS-HA cardiac patches, the growth of rMSCs and cardiomyogenic differentiation of 5-aza inducing rMSCs cultured on patches was examined. First, the isolated rMSCs were identified with various positive and negative surface markers such as CD 44 and CD 31 by a flow cytometric technique, respectively. For examining the growth of rMSCs on the patches, MTT viability assay was performed, and the results demonstrated that the growth of rMSCs on SF and SF-hybrid patches significantly exceeded (P<0.001) that on culture wells after seven days of cultivation. Additionally, the relative growth rates of rMSCs on SF/CS and SF/CS-HA hybrid patches were significantly better (P<0.01) than that on SF patches that were also observed by using vimentin stain to the cells. For instance, the relative cell growth rates (%) in cell culture wells, SF, SF/CS and SF/CS-HA patches were 100%, 282.9+/-6.5%, 337.0+/-8.0% and 332.6+/-6.6% (n=6, for all), respectively. For investigating the effects of the hybrid patches on cardiomyogenic differentiation of 5-aza inducing rMSCs, the expressions of specific cardiac genes of cells such as Gata4 and Nkx2.5 were examined by real-time quantitative polymerase chain reaction (real-time PCR) analysis. The results of cardiomyogenic differentiation of induced rMSCs on SF/CS and SF/CS-HA hybrid patches significantly improved the expressions of cardiac genes of Gata4, Nkx2.5, Tnnt2 and Actc1 genes (all, P<0.01 or better, n=3) than those on SF patches and culture wells. Interestingly, the results of cardiac gene expressions of the cells on the SF/CS-HA hybrid patches were the most pronounced in promoting cardiomyogenic differentiations in this investigation. Furthermore, immunofluorescence staining of cardiac proteins such as cardiotin and connexin 43 for induced rMSCs cultured on SF/CS and SF/CS-HA hybrid patches were much pronounced compared with SF patches, indicating the improvements of cardiomyogenic differentiation on the hybrid patches. The results of this study demonstrate that the SF/CS and SF/CS-HA hybrid patches may be promising biomaterials for regenerating infarcted cardiac tissues.

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.

Reversal of Doxorubicin Resistance in Breast Cancer Cells by Photochemical Internalization

Multiple drug resistance (MDR) is a problem that seriously reduces the efficacy of many chemotherapy agents. One mechanism for MDR is increased acidification of endocytic vesicles and increased cytosol pH, so weak base chemotherapeutic agents, including doxorubicin, are trapped in endocytic vesicles and exhibit a drug resistant phenotype. Treatments that selectively reverse this accumulation may therefore reverse the MDR phenotype. Photochemical internalization (PCI) is a novel technology developed for site‐specific enhancement of the therapeutic efficacy of macromolecules by selective photochemical rupture of endocytic vesicles and consequent release of endocytosed macromolecules into the cytosol. This study evaluates PCI for release of doxorubicin from endocytic vesicles in MDR cells. Two breast cancer cell lines, MCF‐7 and MCF‐7/ADR (the latter resistant to doxorubicin), were selected. They were found equally sensitive to photochemical treatment with the photosensitiser TPPS2a (disulfonated meso‐tetraphenylporphine) and light. On exposure to doxorubicin alone, the IC50 (drug concentration for 50% reduction in colony formation) was 0.1 μM for MCF‐7 and 1 μM for MCF‐7/ADR. After PCI (photochemical treatment followed by doxorubicin), the IC50 concentration was 0.1 μM for both cell lines. Comparable changes were seen with assay of cell viability using 3‐(4,5‐dimethyltiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT). On fluorescence microscopy in MCF‐7/ADR cells, doxorubicin localised in granules identified as lysosomes. After PCI, doxorubicin was released into the cytosol and entered cell nuclei, as was seen in MCF‐7 cells without PCI. In conclusion, PCI reversed the MDR phenotype of doxorubicin resistant breast cancer cells by endo‐lysosomal release of the drug. The technique is a promising new approach to tackling the problem of MDR.

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.