RESUMO
We report the design and adaptation of iron/iron oxide nanoparticle-based optical nanobiosensors for enzymes or cytokine/chemokines that are established biomarkers of lung diseases. These biomarkers comprise ADAM33, granzyme B, MMP-8, neutrophil elastase, arginase, chemokine (C-C motif) ligand 20 and interleukin-6. The synthesis of nanobiosensors for these seven biomarkers, their calibration with commercially available enzymes and cytokines/chemokines, as well as their validation using bronchoalveolar lavage (BAL) obtained from a mouse model of TLR3-mediated inflammation are discussed here. Exhaled Breath Condensate (EBC) is a minimally invasive approach for sampling airway fluid in the diagnosis and management of various lung diseases in humans (e.g., asthma, COPD and viral infections). We report the proof-of-concept of using human EBC in conjunction with nanobiosensors for diagnosis/monitoring airway inflammation. These findings suggest that, with nanosensor technology, human EBC can be utilized as a liquid biopsy to monitor inflammation/remodeling in lung disease.
Assuntos
Asma , Pneumopatias , Animais , Biomarcadores , Testes Respiratórios , Inflamação/diagnóstico , CamundongosRESUMO
Peptide nanosponges of low polydispersity are spontaneously formed from trigonal supramolecular building blocks in aqueous buffers, which feature cationic and/or anionic oligopeptides (n = 5-20) and a hydrophobic unit. In contrast to classical liposomes/vesicles, nanosponges feature interwoven hydrophilic and hydrophobic nanodomains and are readily taken up by mammalian cells. Perillyl alcohol is known to be a simple, but effective small molecule drug against glioma multiforme. However, its efficacy is limited by a poor bioavailability. In order to make perillyl alcohol bioavailable, two nanosponges consisting of 10 aspartates, to which perillyl alcohol is attached by means of an ester bond, and 20 lysines or arginines (type (D-POH)10K20 and (D-POH)10R20) were synthesized, purified, and characterized by dynamic light scattering (DLS) and atomic force microscopy (AFM). These nanosponges were then tested in cell cultures of murine glioma cells (GL26) and murine neural progenitor cells (NPC) because the latter was previously utilized in cell-based cancer therapy. The two nanosponges exhibited significantly different biophysical properties (size distribution and ζ potentials). Consequently, different efficacies in killing GL26 and NPC were observed in serum-containing culture media. The results from these experiments confirmed that the type (D-POH)10K20 nanosponge is a promising candidate for the (cell-mediated) cytotherapy of glioblastoma.
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Numerous proteases, such as matrix metalloproteinases (MMPs), cathepsins (CTS), and urokinase plasminogen activator (UpA), are dysfunctional (that is, over- or under-expressed) in solid tumors, when compared to healthy human subjects. This offers the opportunity to detect early tumors by liquid biopsies. This approach is of particular advantage for the early detection of pancreatic cancer, which is a "silent killer". We have developed fluorescence nanobiosensors for ultrasensitive (sub-femtomolar) arginase and protease detection, consisting of water-dispersible Fe/Fe3O4 core/shell nanoparticles and two tethered fluorescent dyes: TCPP (Tetrakis(4-carboxyphenyl)porphyrin) and cyanine 5.5. Upon posttranslational modification or enzymatic cleavage, the fluorescence of TCPP increases, which enables the detection of proteases at sub-femtomolar activities utilizing conventional plate readers. We have identified an enzymatic signature for the detection of pancreatic adenocarcinomas in serum, consisting of arginase, matrix metalloproteinase-1, -3, and -â¯9, cathepsin-B and -E, urokinase plasminogen activator, and neutrophil elastase, which is a potential game-changer.
Assuntos
Técnicas Biossensoriais , Carcinoma Ductal Pancreático/diagnóstico , Detecção Precoce de Câncer/métodos , Corantes Fluorescentes/química , Nanopartículas/química , Neoplasias Pancreáticas/diagnóstico , Estudos de Casos e Controles , Feminino , Humanos , Biópsia Líquida , MasculinoRESUMO
The structure of novel binary nanosponges consisting of (cholesterol-(K/D) n DEVDGC)3-trimaleimide units possessing a trigonal maleimide linker, to which either lysine (K)20 or aspartic acid (D)20 are tethered, has been elucidated by means of TEM. A high degree of agreement between these findings and structure predictions through explicit solvent and then coarse-grained molecular dynamics (MD) simulations has been found. Based on the nanosponges' structure and dynamics, caspase-6 mediated release of the model drug 5(6)-carboxyfluorescein has been demonstrated. Furthermore, the binary (DK20) nanosponges have been found to be virtually non-toxic in cultures of neural progenitor cells. It is of a special importance for the future development of cell-based therapies that DK20 nanosponges were taken up efficiently by leucocytes (WBC) in peripheral blood within 3 h of exposure. The percentage of live cells among the WBC was not significantly decreased by the DK20 nanosponges. In contrast to stem cell or leucocyte cell cultures, which have to be matched to the patient, autologous cells are optimal for cell-mediated therapy. Therefore, the nanosponges hold great promise for effective cell-based tumor targeting.
RESUMO
A novel type of supramolecular aggregate, named a "nanosponge" was synthesized through the interaction of novel supramolecular building blocks with trigonal geometry. The cholesterol-(K/D)nDEVDGC)3-trimaleimide unit consists of a trigonal maleimide linker to which homopeptides (either K or D) of variable lengths (n=5, 10, 15, 20) and a consensus sequence for executioner caspases (DEVDGC) are added via Michael addition. Upon mixing in aqueous buffer cholesterol-(K)nDEVDGC)3-trimaleimides and a 1:1 mixture of cholesterol-(K/D)nDEVDGC)3-trimaleimides form stable nanosponges, whereas cholesterol-(D)nDEVDGC)3-trimaleimide is unable to form supramolecular aggregates with itself. The structure of the novel nanosponges was investigated through explicit solvent and then coarse-grained molecular dynamics (MD) simulations. The nanosponges are between 80 nm and several micrometers in diameters and virtually non-toxic to monocyte/macrophage-like cells.
Assuntos
Colesterol/análogos & derivados , Portadores de Fármacos/química , Nanoestruturas/química , Peptídeos/química , Animais , Antineoplásicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Humanos , Camundongos , Simulação de Dinâmica Molecular , Neoplasias/tratamento farmacológico , Células RAW 264.7RESUMO
Here, we report the synthesis, characterization, and efficacy study of Fe/Fe3O4-nanoparticles that were co-labeled with a tumor-homing and membrane-disrupting oligopeptide and the iron-chelator Dp44mT, which belongs to the group of the thiosemicarbazones. Dp44mT and the peptide sequence PLFAERL(D[KLAKLAKKLAKLAK])CGKRK were tethered to the surface of Fe/Fe3O4 core/shell nanoparticles by utilizing dopamine-anchors. The 26-mer contains two important sequences, which are the tumor targeting peptide CGKRK, and D[KLAKLAK]2, known to disrupt the mitochondrial cell walls and to initiate programmed cell death (apoptosis). It is noteworthy that Fe/Fe3O4 nanoparticles can also be used for MRI imaging purposes in live mammals. In a first step of this endeavor, the efficacy of this nanoplatform has been tested on the highly metastatic 4T1 breast cancer cell line. At the optimal ratio of PLFAERD[KLAKLAK]2CGKRK to Dp44mT of 1 to 3.2 at the surface of the dopamine-coated Fe/Fe3O4-nanocarrier, the IC50 value after 24 h of incubation was found to be 2.2 times lower for murine breast cancer cells (4T1) than for a murine fibroblast cell line used as control. Based on these encouraging results, the reported approach has the potential of leading to a new generation of nanoplatforms for cancer treatment with considerably enhanced selectivity towards tumor cells.
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The objective of this study was to evaluate microwave heating enhancements offered by iron/iron oxide nanoparticles dispersed within tissue-mimicking media for improving efficacy of microwave thermal therapy. The following dopamine-coated magnetic nanoparticles (MNPs) were considered: 10 and 20 nm diameter spherical core/shell Fe/Fe3O4, 20 nm edge-length cubic Fe3O4, and 45 nm edge-length/10 nm height hexagonal Fe3O4. Microwave heating enhancements were experimentally measured with MNPs dissolved in an agar phantom, placed within a rectangular waveguide. Effects of MNP concentration (2.5-20 mg/mL) and microwave frequency (2.0, 2.45 and 2.6 GHz) were evaluated. Further tests with 10 and 20 nm diameter spherical MNPs dispersed within a two-compartment tissue-mimicking phantom were performed with an interstitial dipole antenna radiating 15 W power at 2.45 GHz. Microwave heating of 5 mg/mL MNP-agar phantom mixtures with 10 and 20 nm spherical, and hexagonal MNPs in a waveguide yielded heating rates of 0.78 ± 0.02 °C/s, 0.72 ± 0.01 °C/s and 0.51 ± 0.03 °C/s, respectively, compared to 0.5 ± 0.1 °C/s for control. Greater heating enhancements were observed at 2.0 GHz compared to 2.45 and 2.6 GHz. Heating experiments in two-compartment phantoms with an interstitial dipole antenna demonstrated potential for extending the radial extent of therapeutic heating with 10 and 20 nm diameter spherical MNPs, compared to homogeneous phantoms (i.e., without MNPs). Of the MNPs considered in this study, spherical Fe/Fe3O4 nanoparticles offer the greatest heating enhancement when exposed to microwave radiation. These nanoparticles show strong potential for enhancing the rate of heating and radial extent of heating during microwave hyperthermia and ablation procedures.
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A nanobiosensor for arginase detection was designed and synthesized. It features a central dopamine-coated iron/iron oxide nanoparticle to which sulfonated cyanine 7.0 is tethered via a stable amide bond. Cyanine 5.5 is linked to the N-terminal of the peptide sequence GRRRRRRRG. Arginine (R) reacts to ornithine (O) in the presence of arginase. Based on calibration with commercially obtained arginase II, the limit of detection (LOD) is picomolar. It is noteworthy that the nanobiosensor for arginase detection does not show a fluorescence increase when incubated with the enzyme NO-reductase, which also uses arginase as substrate, but is indicative of an inflammatory response by the host to cancer and infections. Arginase activity was determined in a syngeneic mouse model for aggressive breast cancer (4T1 tumors in BALB/c mice). It was found that the arginase activity is systemically enhanced, but especially pronounced in the active tumor regions.
Assuntos
Arginase/metabolismo , Técnicas Biossensoriais , Nanopartículas Metálicas , Animais , Arginina , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/enzimologia , Camundongos Endogâmicos BALB C , Óxido Nítrico , OrnitinaRESUMO
Proteases, including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins (CTS) exhibit numerous functions in tumor biology. Solid tumors are characterized by changes in protease expression levels by tumor and surrounding tissue. Therefore, monitoring protease levels in tissue samples and liquid biopsies is a vital strategy for early cancer detection. Water-dispersable Fe/Fe3O4-core/shell based nanoplatforms for protease detection are capable of detecting protease activity down to sub-femtomolar limits of detection. They feature one dye (tetrakis(carboxyphenyl)porphyrin (TCPP)) that is tethered to the central nanoparticle by means of a protease-cleavable consensus sequence and a second dye (Cy 5.5) that is directly linked. Based on the protease activities of urokinase plasminogen activator (uPA), MMPs 1, 2, 3, 7, 9, and 13, as well as CTS B and L, human breast cancer can be detected at stage I by means of a simple serum test. By monitoring CTS B and L stage 0 detection may be achieved. This initial study, comprised of 46 breast cancer patients and 20 apparently healthy human subjects, demonstrates the feasibility of protease-activity-based liquid biopsies for early cancer diagnosis.
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The quest for renewable and cleaner energy sources to meet the rapid population and economic growth is more urgent than ever before. Being the most abundant carbon source in the atmosphere of Earth, CO2 can be used as an inexpensive C1 building block in the synthesis of aromatic fuels for internal combustion engines. We designed a process capable of synthesizing benzene, toluene, xylenes and mesitylene from CO2 and H2 at modest temperatures (T = 380 to 540 °C) employing Fe/Fe3O4 nanoparticles as catalyst. The synthesis of the catalyst and the mechanism of CO2-hydrogenation will be discussed, as well as further applications of Fe/Fe3O4 nanoparticles in catalysis.
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Numerous proteases are known to be necessary for cancer development and progression including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins. The goal of this research is to develop an Fe/Fe3O4 nanoparticle-based system for clinical diagnostics, which has the potential to measure the activity of cancer-associated proteases in biospecimens. Nanoparticle-based "light switches" for measuring protease activity consist of fluorescent cyanine dyes and porphyrins that are attached to Fe/Fe3O4 nanoparticles via consensus sequences. These consensus sequences can be cleaved in the presence of the correct protease, thus releasing a fluorescent dye from the Fe/Fe3O4 nanoparticle, resulting in highly sensitive (down to 1 × 10(-16) mol l(-1) for 12 proteases), selective, and fast nanoplatforms (required time: 60 min).
Assuntos
Ensaios Enzimáticos/métodos , Nanopartículas de Magnetita/química , Nanotecnologia/métodos , Neoplasias/enzimologia , Peptídeo Hidrolases/metabolismo , Espectrometria de Fluorescência/métodos , Calibragem , Carbocianinas/química , Sequência Consenso , Transferência Ressonante de Energia de Fluorescência , Metaloproteinase 13 da Matriz/química , Metaloproteinase 13 da Matriz/metabolismo , Peptídeo Hidrolases/química , Porfirinas/química , Reprodutibilidade dos Testes , Propriedades de SuperfícieRESUMO
The targeted delivery of therapeutics to the tumor site is highly desirable in cancer treatment, because it is capable of minimizing collateral damage. Herein, we report the synthesis of a nanoplatform, which is composed of a 15 ± 1 nm diameter core/shell Fe/Fe(3)O(4) magnetic nanoparticles (MNPs) and the topoisomerase I blocker SN38 bound to the surface of the MNPs via a carboxylesterase cleavable linker. This nanoplatform demonstrated high heating ability (SAR = 522 ± 40 W/g) in an AC-magnetic field. For the purpose of targeted delivery, this nanoplatform was loaded into tumor-homing double-stable RAW264.7 cells (mouse monocyte/macrophage-like cells (Mo/Ma)), which have been engineered to express intracellular carboxylesterase (InCE) upon addition of doxycycline by a Tet-On Advanced system. The nanoplatform was taken up efficiently by these tumor-homing cells. They showed low toxicity even at high nanoplatform concentration. SN38 was released successfully by switching on the Tet-On Advanced system. We have demonstrated that this nanoplatform can be potentially used for thermochemotherapy. We will be able to achieve the following goals: (1) Specifically deliver the SN38 prodrug and magnetic nanoparticles to the cancer site as the payload of tumor-homing double-stable RAW264.7 cells; (2) Release of chemotherapeutic SN38 at the cancer site by means of the self-containing Tet-On Advanced system; (3) Provide localized magnetic hyperthermia to enhance the cancer treatment, both by killing cancer cells through magnetic heating and by activating the immune system.
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We have transfected murine neural stem cells (NSCs) and rat umbilical cord matrix-derived stem cells (RUCMSCs) with a plasmid expressing gaussia luciferase (gLuc). These cells are engineered to secrete the luciferase. We have used gLuc containing supernatant from culturing the NSCs to perform in vitro photodynamic therapy of murine melanoma cells (B16F10), and RUCMSCs to perform in vivo PDT of lung melanomas in C57BL/6 mice. The treatment system was comprised of aminolevulic acid as a prodrug for the synthesis of the photosensitizer protoporphyrin IX, gaussia luciferase, and its' substrate coelenterazine. A significant reduction of the number of live melanoma cells in vitro and a borderline significant retardation of tumour growth in vivo was observed after coelenterazine-mediated PDT.
Assuntos
Células-Tronco/metabolismo , Ácido Aminolevulínico/química , Ácido Aminolevulínico/farmacologia , Ácido Aminolevulínico/uso terapêutico , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Sangue Fetal/citologia , Imidazóis/química , Imidazóis/farmacologia , Luciferases/genética , Luciferases/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Oxirredução , Fotoquimioterapia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/uso terapêutico , Fármacos Fotossensibilizantes/toxicidade , Plasmídeos/metabolismo , Protoporfirinas/biossíntese , Protoporfirinas/uso terapêutico , Protoporfirinas/toxicidade , Pirazinas/química , Pirazinas/farmacologia , Ratos , Transplante de Células-Tronco , Células-Tronco/citologia , TransfecçãoRESUMO
Using magnetic nanoparticles to absorb alternating magnetic field energy as a method of generating localized hyperthermia has been shown to be a potential cancer treatment. This report demonstrates a system that uses tumor homing cells to actively carry iron/iron oxide nanoparticles into tumor tissue for alternating magnetic field treatment. Paramagnetic iron/ iron oxide nanoparticles were synthesized and loaded into RAW264.7 cells (mouse monocyte/ macrophage-like cells), which have been shown to be tumor homing cells. A murine model of disseminated peritoneal pancreatic cancer was then generated by intraperitoneal injection of Pan02 cells. After tumor development, monocyte/macrophage-like cells loaded with iron/ iron oxide nanoparticles were injected intraperitoneally and allowed to migrate into the tumor. Three days after injection, mice were exposed to an alternating magnetic field for 20 minutes to cause the cell-delivered nanoparticles to generate heat. This treatment regimen was repeated three times. A survival study demonstrated that this system can significantly increase survival in a murine pancreatic cancer model, with an average post-tumor insertion life expectancy increase of 31%. This system has the potential to become a useful method for specifically and actively delivering nanoparticles for local hyperthermia treatment of cancer.
Assuntos
Compostos Férricos/administração & dosagem , Hipertermia Induzida/métodos , Macrófagos/transplante , Campos Magnéticos , Nanopartículas/uso terapêutico , Neoplasias Pancreáticas/terapia , Transplantes , Animais , Modelos Animais de Doenças , Magnetismo , Camundongos , Taxa de SobrevidaRESUMO
Gene-directed enzyme prodrug therapy (GDEPT) has been investigated as a means of cancer treatment without affecting normal tissues. This system is based on the delivery of a suicide gene, a gene encoding an enzyme which is able to convert its substrate from non-toxic prodrug to cytotoxin. In this experiment, we have developed a targeted suicide gene therapeutic system that is completely contained within tumor-tropic cells and have tested this system for melanoma therapy in a preclinical model. First, we established double stable RAW264.7 monocyte/macrophage-like cells (Mo/Ma) containing a Tet-On® Advanced system for intracellular carboxylesterase (InCE) expression. Second, we loaded a prodrug into the delivery cells, double stable Mo/Ma. Third, we activated the enzyme system to convert the prodrug, irinotecan, to the cytotoxin, SN-38. Our double stable Mo/Ma homed to the lung melanomas after 1 day and successfully delivered the prodrug-activating enzyme/prodrug package to the tumors. We observed that our system significantly reduced tumor weights and numbers as targeted tumor therapy after activation of the InCE. Therefore, we propose that this system may be a useful targeted melanoma therapy system for pulmonary metastatic tumors with minimal side effects, particularly if it is combined with other treatments.
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Antineoplásicos Fitogênicos/uso terapêutico , Transplante de Células/métodos , Portadores de Fármacos/metabolismo , Genes Transgênicos Suicidas/genética , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Células Precursoras de Monócitos e Macrófagos/metabolismo , Pró-Fármacos/uso terapêutico , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/metabolismo , Camptotecina/análogos & derivados , Camptotecina/metabolismo , Carboxilesterase/metabolismo , Primers do DNA/genética , Avaliação Pré-Clínica de Medicamentos , Feminino , Irinotecano , Neoplasias Pulmonares/patologia , Magnetismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas , Pró-Fármacos/administração & dosagem , Pró-Fármacos/metabolismoRESUMO
Localized magnetic hyperthermia as a treatment modality for cancer has generated renewed interest, particularly if it can be targeted to the tumor site. We examined whether tumor-tropic neural progenitor cells (NPCs) could be utilized as cell delivery vehicles for achieving preferential accumulation of core/shell iron/iron oxide magnetic nanoparticles (MNPs) within a mouse model of melanoma. We developed aminosiloxane-porphyrin functionalized MNPs, evaluated cell viability and loading efficiency, and transplanted neural progenitor cells loaded with this cargo into mice with melanoma. NPCs were efficiently loaded with core/shell Fe/Fe(3)O(4) MNPs with minimal cytotoxicity; the MNPs accumulated as aggregates in the cytosol. The NPCs loaded with MNPs could travel to subcutaneous melanomas, and after A/C (alternating current) magnetic field (AMF) exposure, the targeted delivery of MNPs by the cells resulted in a measurable regression of the tumors. The tumor attenuation was significant (p < 0.05) a short time (24 h) after the last of three AMF exposures.
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Condutividade Elétrica , Magnetoterapia/métodos , Melanoma/metabolismo , Melanoma/terapia , Nanopartículas , Sistema Nervoso/citologia , Células-Tronco/metabolismo , Animais , Transporte Biológico , Linhagem Celular Tumoral , Feminino , Compostos Férricos/química , Compostos Férricos/metabolismo , Humanos , Ferro/química , Ferro/metabolismo , Melanoma/patologia , Camundongos , Proteômica , Transplante de Células-Tronco , TemperaturaRESUMO
BACKGROUND: There is renewed interest in magnetic hyperthermia as a treatment modality for cancer, especially when it is combined with other more traditional therapeutic approaches, such as the co-delivery of anticancer drugs or photodynamic therapy. METHODS: The influence of bimagnetic nanoparticles (MNPs) combined with short external alternating magnetic field (AMF) exposure on the growth of subcutaneous mouse melanomas (B16-F10) was evaluated. Bimagnetic Fe/Fe3O4 core/shell nanoparticles were designed for cancer targeting after intratumoral or intravenous administration. Their inorganic center was protected against rapid biocorrosion by organic dopamine-oligoethylene glycol ligands. TCPP (4-tetracarboxyphenyl porphyrin) units were attached to the dopamine-oligoethylene glycol ligands. RESULTS: The magnetic hyperthermia results obtained after intratumoral injection indicated that micromolar concentrations of iron given within the modified core-shell Fe/Fe3O4 nanoparticles caused a significant anti-tumor effect on murine B16-F10 melanoma with three short 10-minute AMF exposures. We also observed a decrease in tumor size after intravenous administration of the MNPs followed by three consecutive days of AMF exposure 24 hrs after the MNPs injection. CONCLUSIONS: These results indicate that intratumoral administration of surface modified MNPs can attenuate mouse melanoma after AMF exposure. Moreover, we have found that after intravenous administration of micromolar concentrations, these MNPs are capable of causing an anti-tumor effect in a mouse melanoma model after only a short AMF exposure time. This is a clear improvement to state of the art.