Calcium phosphate-based organic-inorganic hybrid nanocarriers with pH-responsive on/off switch for photodynamic therapy.

Photodynamic therapy (PDT) is a promising treatment modality for malignant tumors in a light-selective manner. To improve the PDT efficacy, numerous kinds of nanocarriers have been developed to deliver photosensitizers (PSs) selectively into the tumor through leaky tumor-associated vasculature. However, the corresponding prolonged retention of the nanocarrier in the bloodstream may lead to unfavorable photochemical damage to normal tissues such as skin. Here, we report an organic-inorganic hybrid nanocarrier with a pH-responsive on/off switch of PDT efficacy. This hybrid nanocarrier is constructed by hydrothermal synthesis after simple mixing of calcium/phosphate ions, chlorin e6 (amphiphilic low molecular weight PS), and poly(ethylene glycol)-b-poly(aspartic acid) (PEG-PAsp) copolymers in an aqueous solution. The hybrid nanocarrier possesses a calcium phosphate (CaP) core encapsulating the PSs, which is surrounded by a PEG shielding layer. Under physiological conditions (pH 7.4), the nanocarrier suppressed the photochemical activity of PS by lowering the access of oxygen molecules to the incorporated PS, while PDT efficacy was restored in a pH-responsive manner because of the dissolution of CaP and eventual recovery of access between the oxygen and the PS. Owing to this switch, the nanocarrier reduced the photochemical damage in the bloodstream, while it induced effective PDT efficacy inside the tumor cell in response to the acidic conditions of the endo-/lysosomes.

Three-layered polyplex micelle as a multifunctional nanocarrier platform for light-induced systemic gene transfer.

Nanocarriers responding to light have great potential for pinpoint therapy, and recent studies have revealed promising in vivo activity. However, light-selective gene transfer still remains challenging in the systemic application. Here we report systemic light-responsive nanocarriers for gene delivery developed through the sequential self-assembly of ABC-type triblock copolymer/DNA/dendrimeric photosensitizer, forming polyplex micelles with three-layered functional nanocompartments. The DNA-packaged core is covered by the photosensitizer-incorporated intermediate layer, which is encompassed by an outer shielding shell. This three-layered structure permits multistep photosensitizer and DNA delivery into a solid tumour by a systemic route: the shielding layer minimizes unfavourable interactions with blood components, and the photosensitizer is delivered to endo-/lysosomal membranes to facilitate light-selective cytoplasmic translocation of the micelles, accomplishing DNA delivery into the nucleus to exert gene expression. The polyplex micelles display >100-fold photoenhanced gene expression in cultured cells and exhibit light-induced in vivo gene transfer in solid tumours following systemic administration.

Hydrothermally synthesized PEGylated calcium phosphate nanoparticles incorporating Gd-DTPA for contrast enhanced MRI diagnosis of solid tumors.

Organic-inorganic hybrid nanoparticles with calcium phosphate (CaP) core and PEGylated shell were developed to incorporate magnetic resonance imaging (MRI) contrast agent diethylenetriaminepentaacetic acid gadolinium (III) (Gd-DTPA) for noninvasive diagnosis of solid tumors. A two-step preparation method was applied to elaborate hybrid nanoparticles with a z-average hydrodynamic diameter about 80nm, neutral surface ξ-potential and high colloidal stability in physiological environments by self-assembly of poly(ethylene glycol)-b-poly(aspartic acid) block copolymer, Gd-DTPA, and CaP in aqueous solution, followed with hydrothermal treatment. Incorporation into the hybrid nanoparticles allowed Gd-DTPA to show significant enhanced retention ratio in blood circulation, leading to high accumulation in tumor positions due to enhanced permeability and retention (EPR) effect. Moreover, Gd-DTPA revealed above 6 times increase of relaxivity in the nanoparticle system compared to free form, and eventually, selective and elevated contrast enhancements in the tumor positions were observed. These results indicate the high potential of Gd-DTPA-loaded PEGylated CaP nanoparticles as a novel contrast agent for noninvasive cancer diagnosis.

Effective transgene expression without toxicity by intraperitoneal administration of PEG-detachable polyplex micelles in mice with peritoneal dissemination.

Block copolymer of poly(ethylene glycol)-block-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-P[Asp(DET)]) has been originally introduced as a promising gene carrier by forming a nanomicelle with plasmid DNA. In this study, the polyplex micelle of PEG-SS-P[Asp(DET)], which disulfide linkage (SS) between PEG and cationic polymer can detach the surrounding PEG chains upon intracellular reduction, was firstly evaluated with respect to in vivo transduction efficiency and toxicity in comparison to that of PEG-P[Asp(DET)] in peritoneally disseminated cancer model. Intraperitoneal (i.p.) administration of PEG-SS-P[Asp(DET)] polyplex micelles showed a higher (P<0.05) transgene expression compared with PEG-P[Asp(DET)] in tumors. In contrast, the delivered distribution of the micelles was not different between the two polyplex micelles. PEG-SS-P[Asp(DET)] micelle encapsulating human tumor necrosis factor α (hTNF-α) gene exhibits a higher antitumor efficacy against disseminated cancer compared with PEG-P[Asp(DET)] or saline control. No hepatic and renal toxicities were observed by the administration of polyplex micelles. In conclusion, PEG-detachable polyplex micelles may represent an advantage in gene transduction in vivo over PEG-undetachable polyplex micelles after i.p. administration for peritoneal dissemination of cancer.

Impaired activities of cyclic adenosine monophosphate-responsive element binding protein, protein kinase A and calcium-independent phospholipase A2 are involved in deteriorated regeneration of cirrhotic liver after partial hepatectomy in rats.

AIMS: This study is to elucidate whether cyclic adenosine monophosphate (cAMP)-mediated signal is involved in lower regenerative potential of cirrhotic liver. METHODS: Hepatic cAMP concentration, activities of protein kinase A (PKA), c-AMP responsive element binding protein (CREB) and Ca(2+) -independent phospholipase A(2) (iPLA2) and regeneration rate were compared between rats with thioacetamide-induced cirrhotic and normal livers after two-third hepatectomy. RESULTS: The liver regeneration estimated by the rates of [(3) H]-thymidine incorporation and staining of proliferating cell nuclear antigen was significantly lower in the cirrhotic group. CREB, PKA and iPLA2 activities, assessed by western blots and electromobility shift assay, were significantly impaired after hepatectomy in the cirrhosis group. PKA and iPLA2 silencing by siRNA transfection significantly inhibited CREB activity and cell growth in transformed hepatocytes in vitro. CONCLUSIONS: CREB dysfunction, mediated by PKA and iPLA2 suppression, may be involved in the deteriorated liver regeneration in the cirrhotic rats.

Disulfide crosslinked polyion complex micelles encapsulating dendrimer phthalocyanine directed to improved efficiency of photodynamic therapy.

Dendrimer phthalocyanine (DPc)-loaded polyion complex micelle (DPc/m) has been developed as photosensitizer (PS) formulation in photodynamic therapy (PDT). Incorporation of DPc into the micelle showed significant enhancement in the in vitro photocytotoxicity. Also, introduction of disulfide crosslinking in the micellar core further improved the in vitro PDT effect of DPc/m. Here, we aim to analyze the mechanism of the enhanced photocytotoxicity of DPc/m, particularly focusing on the photochemical reactions during photoirradiation. As a result, DPc/m has been shown to protect DPc from photobleaching induced by the reactions with serum proteins, although DPc were considerably quenched in the micellar core. Furthermore, the introduction of disulfide crosslinking into the micellar core has demonstrated to improve the efficiency of reactive oxygen species (ROS) production by DPc in the micellar core as well as more effectively prevent the photobleaching of DPc. These effects might lead to effective photochemical reactions by DPc/m, which may account for the enhanced photocytotoxicity. Our findings provide useful knowledge in designing PS formulations for effective PDT.

Dual blockade of phosphatidylinositol 3'-kinase and mitogen-activated protein kinase pathways overcomes paclitaxel-resistance in colorectal cancer.

Paclitaxel, one of key drugs to treat a wide range of malignancies, exhibits relative low sensitivity for colorectal cancer. The present study was to examine whether and how phosphatidylinositol 3'-kinase (PI3K) signals affect the sensitivity of colorectal cancer to paclitaxel. Four colorectal cancer cell lines were exposed to paclitaxel in the presence of PI3K signal inhibitors, such as LY294002, siRNA for Akt, or rapamycin, with or without MAPK inhibitor, PD98059. Cell viability and apoptosis were determined by MTT assay, cell cycle analysis in flow cytometer and Hoechst nuclear staining. To analyze the PI3K activity, the expression in phosphorylated Akt and downstream effectors of p70S6 kinase (S6K) were evaluated by Western blot analysis. Paclitaxel alone (5-10 nM) did not induce the apoptosis in all four cell lines. Although LY294002 alone did not affect the cell viability, it suppressed the Akt and S6K activities and induced the sub-G1 arrest/apoptosis when paclitaxel was co-administered, as well as the Akt siRNA and rapamycin did. Simultaneous blockade of PI3K and MAPK pathways more suppressed the S6K activity and further increased the apoptosis. In conclusion, PI3K is involved in low susceptibility of colorectal cancer to paclitaxel and dual PI3K/MAPK targeting agents may evolve a new paclitaxel-based chemotherapy for colorectal cancer.

Visible drug delivery by supramolecular nanocarriers directing to single-platformed diagnosis and therapy of pancreatic tumor model.

Nanoparticle therapeutics are promising platforms for cancer therapy. However, it remains a formidable challenge to assess their distribution and clinical efficacy for therapeutic applications. Here, by using multifunctional polymeric micellar nanocarriers incorporating clinically approved gadolinium (Gd)-based magnetic resonance imaging contrast agents and platinum (Pt) anticancer drugs through reversible metal chelation of Pt, simultaneous imaging and therapy of an orthotopic animal model of intractable human pancreatic tumor was successfully performed without any serious toxicity. The strong tumor contrast enhancement achieved by the micelles correlated with the 24 times increase of r(1) of the Gd chelates, the highest for the formulations using clinically approved Gd chelates reported to date. From the micro-synchrotron radiation X-ray fluorescence spectrometry scanning of the lesions, we confirmed that both the Gd chelates and Pt drugs delivered by the micelles selectively colocalized in the tumor interior. Our study provides new insights for the design of theranostic micelles with high contrast enhancement and site-specific clinical potential.

Enhanced in vivo Magnetic Resonance Imaging of Tumors by PEGylated Iron-Oxide-Gold Core-Shell Nanoparticles with Prolonged Blood Circulation Properties.

High-density poly(ethylene glycol) (PEG)-coated iron-oxide-gold core-shell nanoparticles (AuIONs) were developed as T(2) -weighted magnetic resonance imaging (MRI) contrast agents for cancer imaging. The PEG-coated iron-oxide-gold core-shell nanoparticles (PEG-AuIONs) were approximately 25 nm in diameter with a narrow distribution. Biodistribution experiments in mice bearing a subcutaneous colon cancer model prepared with C26 murine colon adenocarcinoma cells showed high accumulation of the PEG-AuIONs within the tumor mass and low nonspecific accumulation in the liver and spleen, resulting in high specificity to solid tumors. T(2) -weighted MR images following intravenous injection of PEG-AuIONs showed selective negative enhancement of tumor tissue in an orthotopic pancreatic cancer model prepared with MiaPaCa-2 human pancreatic adenocarcinoma cells. These results indicate that PEG-AuIONs are a promising MRI contrast agent for diagnosis of malignant tumors, including pancreatic cancer.

Enhanced magnetic resonance imaging of experimental pancreatic tumor in vivo by block copolymer-coated magnetite nanoparticles with TGF-beta inhibitor.

Early detection of solid tumors, particularly pancreatic cancer, is of substantial importance in clinics. Enhanced magnetic resonance imaging (MRI) with iron oxide nanoparticles is an available way to detect the cancer. The effective and selective accumulation of these nanoparticles in the tumor tissue is needed for improved imaging, and in this regard, their longevity in the blood circulation time is crucial. We developed here block copolymer-coated magnetite nanoparticles for pancreatic cancer imaging, by means of a chelation between the carboxylic acid groups in poly(ethylene glycol)-poly(aspartic acid) block copolymer (PEG-PAsp) and Fe on the surface of the iron oxide nanoparticles. These nanoparticles had considerably narrow distribution, even upon increased ionic strength or in the presence of fetal bovine serum. The PEG-PAsp-coated nanoparticles were further shown to be potent as a contrast agent for enhanced MRI for an experimental pancreatic cancer, xenografts of the human-derived BxPC3 cell line in BALB/c nude mice, with combined administration of TGF-beta inhibitor. Iron staining of tumor tissue confirmed the accumulation of the nanoparticles in tumor tissue. Use of the PEG-PAsp-coated magnetite nanoparticles, combined with the TGF-beta inhibitor, is of promising clinical importance for the detection of intractable solid cancers, including pancreatic cancer.

Enhanced photodynamic cancer treatment by supramolecular nanocarriers charged with dendrimer phthalocyanine.

Photodynamic therapy (PDT) is a promising method for the localized treatment of solid tumors. In order to enhance the efficacy of PDT, we have recently developed a novel class of photosensitizer formulation, i.e., the dendrimer phthalocyanine (DPc)-encapsulated polymeric micelle (DPc/m). The DPc/m induced efficient and unprecedentedly rapid cell death accompanied by characteristic morphological changes such as blebbing of cell membranes, when the cells were photoirradiated using a low power halogen lamp or a high power diode laser. The fluorescent microscopic observation using organelle-specific dyes demonstrated that DPc/m might accumulate in the endo-/lysosomes; however, upon photoirradiation, DPc/m might be promptly released into the cytoplasm and photodamage the mitochondria, which may account for the enhanced photocytotoxicity of DPc/m. This study also demonstrated that DPc/m showed significantly higher in vivo PDT efficacy than clinically used Photofrin (polyhematoporphyrin esters, PHE) in mice bearing human lung adenocarcinoma A549 cells. Furthermore, the DPc/m-treated mice did not show skin phototoxiciy, which was apparently observed for the PHE-treated mice, under the tested conditions. These results strongly suggest the usefulness of DPc/m in clinical PDT.

A photo-activated targeting chemotherapy using glutathione sensitive camptothecin-loaded polymeric micelles.

PURPOSE: A novel photo-activated targeted chemotherapy was developed by photochemical internalization (PCI) of glutathione-sensitive polymeric micelles incorporating camptothecin (CPT) prepared from thiolated CPT (CPT-DP) and thiolated poly(ethylene glycol)-b-poly(glutamic acid) (PEG-b-P(Glu-DP)) METHODS: PEG-b-P(Glu-DP) and CPT-DP were synthesized and characterized by (1)H-NMR and gel permeation chromatography, and then mixed to prepare CPT-loaded polymeric micelles (CPT/m). The CPT release from the micelle was studied by reverse phase liquid chromatography. The PCI-activated cytotoxicity of CPT/m against HeLa cells was studied in combination with a non-toxic concentration of dendrimer phthalocyanine-loaded micelles (DPc/m) as the photosensitizer. RESULTS: The diameter of CPT/m was 96 nm and the drug loading was 20% (w/w). CPT was slowly released under the conditions reproducing the extracellular or endosomal environments. However, under the reductive conditions mimicking the cytosol, CPT was rapidly released achieving approximately 90% of the drug release after 24 h. The cytotoxicity of CPT/m was drastically increased on photoirradiation, whereas the CPT/m were not cytotoxic without PCI. CONCLUSIONS: The CPT/m released the drug responding to reductive conditions. The PCI-induced endosomal escape exposed CPT/m to the cytosol triggering the drug release. Thus, CPT/m in combination with DPc/m will behave as smart nanocarriers activated only at photoirradiated tissues.

A novel contrast medium detects increased permeability of rat injured carotid arteries in magnetic resonance T2 mapping imaging.

AIM: Aim of this study was to directly detect increased permeability of vascular lesions by magnetic resonance imaging. METHODS: A novel contrast medium with a mean hydrodynamic diameter of 100 nm was prepared from monodispersed iron colloids incorporated into micelles of block copolymers composed of polyethylene glycol and polyamino acid. T2 mapping was applied to differentiate the minimal shortening of T2 relaxation time in balloon-injured rat carotid arteries. RESULTS: The novel contrast medium accumulated in deendothelialized arteries. T2 relaxation times of injured and uninjured arteries were 50.6 +/- 9.5 ms and 26.9 +/- 2.4 ms, respectively (the mean +/- SD, p< 0.01, n=5). The novel contrast medium, but not commercially available contrast media, shortened the T2 relaxation time of the injured artery to 35.5 +/- 9.7 ms (p< 0.01, n=4). CONCLUSION: A novel iron contrast medium enhanced the lesions with increased permeability. The contrast medium in combination with T2 mapping may be useful to detect unstable atherosclerotic plaques.

Iron hydroxide nanoparticles coated with poly(ethylene glycol)-poly(aspartic acid) block copolymer as novel magnetic resonance contrast agents for in vivo cancer imaging.

PEG-coated beta-FeOOH nanoparticles were prepared through electrostatic complex formation of iron oxide nanoparticles with poly(ethylene glycol)-poly(aspartic acid) block copolymer [PEG-P(Asp)] in distilled water. By dynamic light scattering (DLS) measurement, the nanopaticle size was determined to be 70 nm with narrow distribution. The FT-IR and zeta potential experimental results proved that PEG-PAsp molecules bound to the surface of the iron oxide nanoparticles via the coordination between the carboxylic acid residues in the PAsp segment of the block copolymer and the surface Fe of the beta-FeOOH nanoparticles. The PEG-coated nanoparticles revealed excellent solubility and stability in aqueous solution as well as in physiological saline. In vivo MRI experiments on tumor-bearing mice demonstrated that the PEG-coated nanoparticles prepared by the current approach achieved an appreciable accumulation into solid tumor, suggesting their potential utility as tumor-selective MRI contrast agents.