Fast in vivo quantification of T1 and T2 MRI relaxation times in the myocardium based on inversion recovery SSFP with in vitro validation post Gd-based contrast administration.

PURPOSE: A fast clinical imaging technique for quantifying myocardial T1 and T2 relaxation times after Gadolinium (Gd)-based contrast administration within a single breathhold is presented with in vitro validation. MATERIALS AND METHODS: From signal intensity curves in ECG-gated segmented inversion recovery balanced steady state free precession (IR-bSSFP) images, T1 and T2 values were determined for 24 agarose samples made from solutions of Omniscan (0.25-2 mg/mL) and copper-sulfate (0.52-22.17 mg/mL). T1 and T2 were also measured using turbo spin-echo (TSE) acquisitions and compared with IR-bSSFP results. In vivo T1 and T2 values from post-contrast IR-bSSFP images of five healthy volunteers were determined for (I) the left ventricular wall, (II) the interventricular septum (IVS) and (III) the lateral wall of the left ventricle (LV). Spin system simulations were performed for selected T1 and T2 values. RESULTS: Good agreement between TSE and IR-bSSFP for T1 for realistic in vivo post-contrast values (below 1,250 ms, R=0.88) and for T2 (entire range, R=0.97) was found. Spin system simulations were in good agreement with measurements. In vivo average T1 was 546±32 ms and average T2 was 59±9 ms. CONCLUSIONS: A fast imaging protocol for absolute quantification of myocardial T1 and T2 post-contrast is presented, validated in vitro and consecutively applied in vivo in humans.

Interactions of liposome carriers with infectious fungal hyphae reveals the role of ß-glucans.

Relatively little is known about how liposomal formulations modulate drug delivery to fungal pathogens. We compared patterns of hyphal cell wall binding for empty rhodmine-labeled liposomes and the clinically available amphotericin B-containing liposomal formulation (AmBisome) in Aspergillus fumigatus and Candida albicans. Following 0.5 h of coincubation with A. fumigatus , empty liposomes concentrated primarily in fungal septae along at the surface of the cell wall, suggesting that liposome uptake is concentrated in areas of the cell wall where linear glucan is exposed on the cell surface, which was confirmed by aniline blue staining. Consistent with this hypothesis, pretreatment of liposomes with soluble linear glucan (laminarin) decreased liposome binding in both Aspergillus and Candida fungal hyphae, while growth of Aspergillus hyphae in the presence of an agent that increases fungal cell wall surface exposure of linear ß-glucans without cell death (caspofungin) increased liposome uptake throughout the Aspergillus fungal cell wall. Increasing the polyethylene glycol (PEG) concentration in liposomes from 0 to 30% significantly increased fungal uptake of liposomes that was only modestly attenuated when fungal cells were incubated in serum concentrations ranging from 10 to 100%. The presence of ß-glucans on the fungal hyphae cell walls of Aspergillus fumigatus is one of the factors responsible for mediating the binding of liposome carriers to the hyphae and could explain possible synergy reported between liposomal amphotericin B and echinocanins.

Gold-silver alloy nanoshells: a new candidate for nanotherapeutics and diagnostics.

We have developed novel gold-silver alloy nanoshells as magnetic resonance imaging (MRI) dual T1 (positive) and T2 (negative) contrast agents as an alternative to typical gadolinium (Gd)-based contrast agents. Specifically, we have doped iron oxide nanoparticles with Gd ions and sequestered the ions within the core by coating the nanoparticles with an alloy of gold and silver. Thus, these nanoparticles are very innovative and have the potential to overcome toxicities related to renal clearance of contrast agents such as nephrogenic systemic fibrosis. The morphology of the attained nanoparticles was characterized by XRD which demonstrated the successful incorporation of Gd(III) ions into the structure of the magnetite, with no major alterations of the spinel structure, as well as the growth of the gold-silver alloy shells. This was supported by TEM, ICP-AES, and SEM/EDS data. The nanoshells showed a saturation magnetization of 38 emu/g because of the presence of Gd ions within the crystalline structure with r1 and r2 values of 0.0119 and 0.9229 mL mg-1 s-1, respectively (Au:Ag alloy = 1:1). T1- and T2-weighted images of the nanoshells showed that these agents can both increase the surrounding water proton signals in the T1-weighted image and reduce the signal in T2-weighted images. The as-synthesized nanoparticles exhibited strong absorption in the range of 600-800 nm, their optical properties being strongly dependent upon the thickness of the gold-silver alloy shell. Thus, these nanoshells have the potential to be utilized for tumor cell ablation because of their absorption as well as an imaging agent.

Novel reducible linear L-lysine-modified copolymers as efficient nonviral vectors.

The development of biodegradable gene delivery systems with high transfection efficiencies is paramount to the clinical translation of nonviral gene carriers. Therefore, to produce a biocompatible, reducible, effective and non-toxic gene delivery system, we have designed and synthesized novel reducible linear L-lysine modified copolymers (LLCs) as an alternative to high molecular weight poly(L-lysine) (PLL). The molecular weight (MW) of the copolymers was found to be approximately 3.2kDa with a polydispersity index of approximately 1.2. Gel retardation assays showed complete condensation of DNA at N/P ratios greater than 20/1 and exceptional LLC/pDNA polyplex stability during incubation with DNase I. Release of DNA from the polyplexes only occurred in the presence of the reducing agent dithiothreitol (DTT). The particle sizes of LLC/pDNA polyplexes were found to be between 100 and 231 nm with surface charges of 0.8-17 mV respectively. The transfection efficiencies of the polyplexes as determined with a luciferase assay showed that LLC polyplexes produced five times higher transfection efficiencies in HDF cells, three times higher transfection efficiencies in MCF-7 cells, and four times higher transfection efficiencies in MA cells as compared to the optimal PLL control. The LLC/pDNA polyplexes showed significantly lower cytotoxicities as compared to the control in HDF, MCF-7 and MA cells at certain N/P ratios. Therefore, these results suggest that these novel LLCs are efficient, reducible and biocompatible polymers for nonviral gene delivery.

Novel nanomedicine-based MRI contrast agents for gynecological malignancies.

Gynecological cancers result in significant morbidity and mortality in women despite advances in treatment and diagnosis. This is due to detection of the disease in the late stages following metastatic spread in which treatment options become limited and may not result in positive outcomes. In addition, traditional contrast agents are not very effective in detecting primary metastatic tumors and cells due to a lack of specificity and sensitivity of the diagnostic tools, which limits their effectiveness. Recently, the field of nanomedicine-based contrast agents offers a great opportunity to develop highly sophisticated devices that can overcome many traditional hurdles of contrast agents including solubility, cell-specific targeting, toxicities, and immunological responses. These nanomedicine-based contrast agents including liposomes, micelles, dendrimers, multifunctional magnetic polymeric nanohybrids, fullerenes, and nanotubes represent improvements over their traditional counterparts, which can significantly advance the field of molecular imaging.

Thermo-responsive systems for controlled drug delivery.

Controlled drug delivery systems represent advanced systems that can be tightly modulated by stimuli in order to treat diseases in which sustained drug release is undesirable. Among the many different stimuli-sensitive delivery systems, temperature-sensitive drug delivery systems offer great potential over their counterparts due to their versatility in design, tunability of phase transition temperatures, passive targeting ability and in situ phase transitions. Thus, thermosensitive drug delivery systems can overcome many of the hurdles of conventional drug delivery systems in order to increase drug efficacies, drug targeting and decrease drug toxicities. In an effort to further control existing temperature-responsive systems, current innovative applications have combined temperature with other stimuli such as pH and light. The result has been the development of highly sophisticated systems, which demonstrate exquisite control over drug release and represent huge advances in biomedical research.

Temperature-sensitive hydrogels with SiO2-Au nanoshells for controlled drug delivery.

Silica-gold (SiO(2)-Au) nanoshells are a new class of nanoparticles that consist of a silica dielectric core that is surrounded by a gold shell. These nanoshells are unique because their peak extinctions are very easily tunable over a wide range of wavelengths particularly in the near infrared (IR) region of the spectrum. Light in this region is transmitted through tissue with relatively little attenuation due to absorption. In addition, irradiation of SiO(2)-Au nanoshells at their peak extinction coefficient results in the conversion of light to heat energy that produces a local rise in temperature. Thus, to develop a photothermal modulated drug delivery system, we have fabricated nanoshell-composite hydrogels in which SiO(2)-Au nanoshells of varying concentrations have been embedded within temperature-sensitive hydrogels, for the purpose of initiating a temperature change with light. N-isopropylacrylamide-co-acrylamide (NIPAAm-co-AAm) hydrogels are temperature-sensitive hydrogels that were fabricated to exhibit a lower critical solution temperature (LCST) slightly above body temperature. The resulting composite hydrogels had the extinction spectrum of the SiO(2)-Au nanoshells in which the hydrogels collapsed reversibly in response to temperature (50 degrees C) and laser irradiation. The degree of collapse of the hydrogels was controlled by the laser fluence as well as the concentration of SiO(2)-Au nanoshells. Modulated drug delivery profiles for methylene blue, insulin, and lysozyme were achieved by irradiation of the drug-loaded nanoshell-composite hydrogels, which showed that drug release was dependent upon the molecular weight of the therapeutic molecule.

Endochondral bone formation from hydrogel carriers loaded with BMP2-transduced cells.

The success of ex vivo viral gene therapy systems for promoting bone formation could be improved through the development of systems to spatially localize gene expression. Towards this goal, we have encapsulated adenovirus-transduced human diploid fetal lung fibroblasts (MRC-5) expressing bone morphogenetic protein-type 2 (BMP-2) within non-degradable poly(ethylene glycol)-diacrylate (PEG-DA) hydrogels and implanted these intramuscularly to promote endochondral bone formation. To optimize BMP-2 secretion, the molecular weight of the polymers and cell densities were varied. Polymers with molecular weights of 6, 10, and 20 kDa were used to prepare hydrogels containing 1, 5, or 10 million transduced cells. The results showed that 10 million transduced fibroblasts that was the maximum number of cells feasible for encapsulation within PEG-DA 10 and 20 kDa hydrogels produced the highest amount of secreted BMP-2 protein. Encapsulation of MRC-5 and transduced fibroblasts resulted in 71 and 58% cell viability, respectively. The bioactivity of secreted BMP-2 protein from the hydrogels was confirmed with an alkaline phosphatase assay. Micro-CT of the lower limb muscles of NOD/SCID mice following implantation with hydrogels showed 39.5 +/- 25.0 mm3 mineralized tissue and 31.8 +/- 7.8 mm3 for the cell-injected mice, and the bone was localized to the hydrogel surfaces. Histology revealed bone as well as cartilage for both hydrogel implanted and cell-injected animals.

Long-circulating DNA-complexed biodegradable multiblock copolymers for gene delivery: degradation profiles and evidence of dysopsonization.

Biodegradable cationic polymers have become promising alternatives to traditional polycationic gene delivery systems in which the high charge densities of high molecular weight polymers contribute significantly to cellular toxicities. Previous research has shown that biodegradable, multiblock copolymers (MBC), PEG-PLL-g-16% His, are efficient gene carriers with negligible cellular toxicities. The present research was designed to characterize the polymer degradation as well as to determine the biodistribution of the MBC after systemic administration. Polymer degradation was performed in buffer as a function of pH, in serum and within polymer/pDNA complexes. The MBC exhibited exponential decay with a half-life (t1/2) of approximately 14 min at pH 9.0, approximately 5 h at pH 7.4 and approximately 2 h in serum. However, there was little or no degradation observed at pH 4.0 and the MBC within the complexes degraded between 4 and 8 h in serum. Biodistribution data performed with fluorescently labeled polymer and pDNA revealed that intact complexes remained in the blood up to 3 days, which was also reflected in the organs as a function of time. Therefore, the cumulative data suggest that PEG may be sterically stabilizing complexes in vivo via dysopsonization in which serum proteins mask the complexes from elements of the reticuloendothelial system (RES).

Local, non-viral IL-12 gene therapy using a water soluble lipopolymer as carrier system combined with systemic paclitaxel for cancer treatment.

Development of improved gene transfer methods is needed for gene therapy to achieve its clinical potential. The use of biocompatible polymeric gene carriers has shown effectiveness in overcoming the current problems associated with viral vectors in safety, immunogenicity and mutagenesis. Previous work has demonstrated that repeated, local, non-viral interleukin-12 (IL-12) gene delivery successfully slows down tumor progression, while improving immunogenicity. Combining IL-12 gene delivery with systemic paclitaxel (PCT) chemotherapy as a treatment for various subcutaneous mouse mammary carcinomas, we used PCT with either a biodegradable polymeric solubilizer, HySolv or Cremophor EL for systemic treatment and injected water soluble lipopolymer (WSLP)/plasmid-encoding IL-12 gene (p2CMVmIL-12) complexes local once every week. The amount of lung metastases being essential for survival as well as subcutaneous tumor volume were compared against untreated controls. We showed inhibition of tumor growth and decreased lung metastases in the combined WSLP/p2CMVmIL-12/HySolv group compared to the controls and the PCT only treated groups. Compared to Cremophor, HySolv performed better alone or in combination with IL-12. Using polymeric vectors as gene carrier systems in combination with improved systemic therapies provide evidence for the efficacy and feasibility of polymer-based drug delivery systems. Especially local cytokine gene delivery showed augmentation of systemic chemotherapy while reducing the hosts risk for further systemic toxicity.

Combination of local, nonviral IL12 gene therapy and systemic paclitaxel treatment in a metastatic breast cancer model.

Repeated, local, nonviral IL12 (interleukin-12) gene delivery decreased tumor progression and increased immunogenicity. We combined our IL12 gene delivery with systemic paclitaxel chemotherapy as a treatment for paclitaxel (PCT)-resistant 4T1 subcutaneous mouse mammary carcinomas and PCT-sensitive, immunogenic/nonimmunogenic tumors. We mixed PCT with either a biodegradable polymeric solubilizer, HySolv, or Cremophor EL for bimonthly systemic treatments and injected water-soluble lipopolymer (WSLP)/p2CMVmIL-12 (plasmid encoding IL12 gene) complexes locally every week. We compared treated subcutaneous tumor volume and lung metastasis with controls. HySolv alone performed better compared to Cremophor EL in combination with WSLP/p2CMVmIL-12. We showed inhibition of 4T1 tumor growth and lung metastases in the combined WSLP/p2CMVmIL-12/HySolv group compared to the controls and the paclitaxel-only treated groups. In parallel experiments we also demonstrated additive responses for tumor growth and number of lung metastases within other PCT-sensitive mammary tumor models using this combination strategy. Our combination therapy provides evidence for the efficacy and feasibility of improved drug delivery systems. Local cytokine gene delivery can augment local and systemic chemotherapy without placing the host at risk for further systemic toxicity.

Sp1-dependent regulation of the RTP801 promoter and its application to hypoxia-inducible VEGF plasmid for ischemic disease.

PURPOSE: Gene therapy using vascular endothelial growth factor (VEGF) is a new potential treatment of ischemic disease. To be safe and effective, VEGF expression should be enhanced locally in ischemic tissue. In this study, we identified the cis-regulatory element for the hypoxia induction of the RTP801 promoter. In addition, pRTP801-VEGF was evaluated as a therapeutic plasmid in vitro. METHODS: The cis-regulatory element for hypoxia induction was identified by deletion and mutation analyses. Antisense oligonucleotide co-transfection assay was performed to evaluate the role of Sp1. pRTP801-VEGF was constructed by the insertion of the RTP801 promoter into the VEGF plasmid. The hypoxia-inducible expression of VEGF was evaluated by in vitro transfection assay. RESULTS: In luciferase assay, the region between -495 and -446 was responsible for the hypoxia-induced transcription. The mutation of the Sp1 site in this region reduced hypoxia-induced transcription. In addition, co-transfection with antisense Sp1 oligonucleotide suggests that hypoxia induction of the RTP801 promoter is mediated by Sp1. In vitro transfection showed that pRTP801-VEGF had higher VEGF expression than pEpo-SV-VEGF. In addition, VEGF expression by pRTP801-VEGF was induced under hypoxia. CONCLUSIONS: With strong basal promoter activity and induction under hypoxia, pRTP801-VEGF may be useful for gene therapy for ischemic disease.

Electron Spin Relaxation in Chromium-Nitrosyl Complexes.

A new method to prepare Cr(NO)(H(2)O)(5)(2+) from dichromate and NH(2)OH is reported. The chromium nitrosyls Cr(NO)(EHBA)(+) and Cr(NO)(EHBA)(2) (EHBA = 2-ethyl-2-hydoxybutyrate) were prepared by a literature reaction and characterized by continuous wave electron paramagnetic resonance and two-pulse electron spin echo spectroscopy at X-band. The g values are characteristic of a single unpaired electron in a predominantly d(xy)() orbital. In fluid and glassy solutions Cr(NO)(EHBA)(2) is a mixture of cis and trans isomers. Rotation of the methyl groups in the EHBA ligands causes an increased rate of spin echo dephasing at temperatures between 40 and 120 K. For the EHBA complexes echo envelope modulation is observed at temperatures below about 40 K that is attributed to inequivalent coupling to protons of the slowly rotating methyl groups. Both the effect of the methyl rotation on spin echo dephasing and the depth of the proton modulation are dependent on the number of ethyl groups in the ligand, and thus the spin echo experiments provide confirmation of the number of EHBA ligands in the complexes. The spin-lattice relaxation rates for the chromium-nitrosyl complexes at temperatures near 100 K are similar to values reported previously for Cr(V) complexes, which also have a single unpaired electron in a predominantly d(xy)() orbital. For Cr(NO)(H(2)O)(5)(2+), Cr(NO)(EHBA)(+), and Cr(NO)(EHBA)(2) the dominant contribution to spin-lattice relaxation between 12 and 150 K is the Raman process with a Debye temperature, theta(D), of 110-120 K. For Cr(NO)(CN)(5)(3)(-) the data are consistent with a Raman process (theta(D) = 135 K) and a contribution from a local mode, which dominates above about 60 K. The formally low-spin d(5) chromium nitrosyl complexes relax about 5 orders of magnitude more slowly than low-spin d(5) Fe(III) porphyrins, which is attributed to the absence of a low-lying excited state.