The efficacy of new colchicine derivatives and viability of the T-Lymphoblastoid cells in three-dimensional culture using F MRI and HPLC-UV ex vivo

The paper describes ex vivo applications of colchicine derivatives for the treatment of human T-Lymphoblastoid (CEM) cells. Moreover, the role of the substitutions of ring A at C-1 and C-7 side chain of colchicine analogues was probed by the synthesis and examination of their effects on the three-dimensional (3-D) CEM cells’ growth. The CEM cells were cultured in the hollow fiber bioreactor (HFB) device. We used ¹H and ¹⁹F magnetic resonance imaging (MRI) to monitor changes in 3-D CEM cell culture. ¹⁹F MRI was used for visualization of the cellular uptake of new fluorine derivatives. Before and after treatment CEM cells profile was investigated with high performance liquid chromatography (HPLC-UV).     

The application of F magnetic resonance ex vivo imaging of three-dimensional cultured breast cancer cells to study the effect of δ-tocopherol

The aim of this study was to determine the role of δ-tocopherol in breast cancer cell growth ex vivo. Human gland mammary adenocarcinoma (MCF-7) and human T-lymphoblastoid (CEM) cells were cultured in the presence of δ-tocopherol at various concentrations (0-750 μM) for 5 days. We have grown 3D ex vivo breast cancer cell cultures in the hollow fiber bioreactor (HFBR). ¹⁹F magnetic resonance imaging (MRI) was used to evaluate oxygen concentration in the cell suspension and thus its viability.     

Application of F magnetic resonance to study the efficacy of fluorine labeled drugs in the three-dimensional cultured breast cancer cells

The cellular monitoring of tumor response to treatments is important for drug discovery and drug development in cancer therapy. We studied efficacy of Herceptin, a common breast cancer drug conjugated with a fluorine organic compound, perfluoro-15-crown-5-ether (PFCE) which easily forms biocompatible emulsions. Three new pharmaceutical forms of Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink were synthesized for the ex vivo study of their efficacy in breast cancer treatment. The emulsions were administered to 10⁹ cells mL⁻¹ of HER-2 positive human adenocarcinoma (MCF-7) cells and the same amount of human mammary epithelial cells (HMEC) cultured in three-dimensional (3D) geometry using hollow fiber bioreactor (HFB) device. Following drugs administration ex vivo, fluorine-19 magnetic resonance imaging (¹⁹F MRI) was applied for cells imaging to measure their viability and to study drug efficacy over 72 h. To ensure optimum drug tracking, HydraLink was used to provide stable binding affinity of emulsified Herceptin to receptor while cationic lipid (Lipofectamine) was used to enhance lipophilicity of the emulsions.After 72 h of treatment with Herceptin, Herceptin/PFCE, Herceptin/PFCE/Lipoplex and Herceptin/PFCE/HydraLink the viability of cells was 54 ± 2%, 49 ± 3%, 43 ± 5% and 42 ± 1%, respectively, as compared with control 93 ± 2%. The efficacy (EC₅₀) of Herceptin conjugated with emulsions was found to be 970 ± 13 μg mL⁻¹ for Herceptin/PFCE, 645 ± 11 μg mL⁻¹ for Herceptin/PFCE/Lipoplex, 678 ± 7 μg mL⁻¹ for Herceptin/PFCE/HydraLink and 1000 ± 3 μg mL⁻¹ for Herceptin. The results show that fluorine emulsions improved the efficacy of Herceptin and ¹⁹F signal intensity changes validated drug efficiency. The significant correlations between duration of treatments and MCF-7 cells viability were observed. While we studied breast cancer cells, the fluorine emulsions could be applied for treatment of other cancer cells overexpressing HER-2.     

In vivo imaging of stepwise vessel occlusion in cerebral photothrombosis of mice by 19F MRI

Background

¹⁹F magnetic resonance imaging (MRI) was recently introduced as a promising technique for in vivo cell tracking. In the present study we compared ¹⁹F MRI with iron-enhanced MRI in mice with photothrombosis (PT) at 7 Tesla. PT represents a model of focal cerebral ischemia exhibiting acute vessel occlusion and delayed neuroinflammation.

Methods/Principal Findings

Perfluorocarbons (PFC) or superparamagnetic iron oxide particles (SPIO) were injected intravenously at different time points after photothrombotic infarction. While administration of PFC directly after PT induction led to a strong ¹⁹F signal throughout the entire lesion, two hours delayed application resulted in a rim-like ¹⁹F signal at the outer edge of the lesion. These findings closely resembled the distribution of signal loss on T2-weighted MRI seen after SPIO injection reflecting intravascular accumulation of iron particles trapped in vessel thrombi as confirmed histologically. By sequential administration of two chemically shifted PFC compounds 0 and 2 hours after illumination the different spatial distribution of the ¹⁹F markers (infarct core/rim) could be visualized in the same animal. When PFC were applied at day 6 the fluorine marker was only detected after long acquisition times ex vivo. SPIO-enhanced MRI showed slight signal loss in vivo which was much more prominent ex vivo indicative for neuroinflammation at this late lesion stage.

Conclusion

Our study shows that vessel occlusion can be followed in vivo by ¹⁹F and SPIO-enhanced high-field MRI while in vivo imaging of neuroinflammation remains challenging. The timing of contrast agent application was the major determinant of the underlying processes depicted by both imaging techniques. Importantly, sequential application of different PFC compounds allowed depiction of ongoing vessel occlusion from the core to the margin of the ischemic lesions in a single MRI measurement.     

Terbium, a fluorescent probe for investigation of siderophore pyochelin interactions with its outer membrane transporter FptA

Pyochelin (Pch) is a siderophore and FptA is its outer membrane transporter produced by Pseudomonas aeruginosa to import iron. The fluorescence of the element terbium is affected by coordinated ligands and it can therefore be used as a probe to investigate the pyochelin-iron uptake pathway in P. aeruginosa. At pH 8.0, terbium fluorescence is greatly enhanced in the presence of pyochelin indicating chelation of the metal by the siderophore. Titration curves showed a 2:1 (Pch:Tb³⁺) stoichiometry and an affinity of K =( 2 ± - 1 )× 10¹¹ M^− 2 was determined. Pch-Tb interaction with the transporter FptA could be followed in vitro and in vivo in P. aeruginosa cells, by Fluorescence Resonance Energy Transfer (FRET) between three partners: the tryptophans of FptA (donor), Pch (acceptor for the Trps and donor for Tb³⁺) and Tb³⁺ (acceptor). Pch-Tb binds to the Pch-Fe outer membrane transporter FptA with a dissociation constant (K_d) of 4.6 μM. This three-partner FRET is a potentially valuable tool for investigation of the interactions between FptA and its siderophore Pch.     

In vitro and in vivo pharmacology of CP-945,598, a potent and selective cannabinoid CB1 receptor antagonist for the management of obesity

Cannabinoid CB₁ receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB₁ receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB₁ receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB₁ receptors in both binding (K_i = 0.7 nM) and functional assays (K_i = 0.2 nM). The compound has low affinity (K_i = 7600 nM) for human CB₂ receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10 mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB₁ receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB₁ receptor competitive antagonist that may further our understanding of the endocannabinoid system.     

Preparation, characterization and in vivo assessment of Gd-albumin and Gd-dendrimer conjugates as intravascular contrast-enhancing agents for MRI

We report in vivo and in vitro MRI properties of six gadolinium-dendrimer and gadolinium-albumin conjugates of derivatized acyclic diethylenetriamine-N,N′,N′,N″, N″-pentaacetic acid (1B4M) and macrocyclic 1,4,7,10-tetraazacyclododecane-N,N′,N″,N?-tetraacetic acid (C-DOTA). The three albumin-based agents have comparable protein to chelate ratios (1:16-18) as well as molar relaxivity (8.8-10.4 mM^− 1 s^− 1). The three dendrimer based agents have blood clearance half-lives ranging from 17 to 66 min while that of the three albumin-based agents are comparable to one another (40-47 min). The dynamic image obtained from use of the albumin conjugate based on the macrocycle (C-DOTA) showed a higher contrast compared to the remaining two albumin based agents. Our conclusion from all of the results is that the macrocyclic-based (DOTA) agents are more suitable than the acyclic-based (1B4M) agent for in vivo use based on their MRI properties combined with the kinetic inertness property associated with the more stable Gd(III) DOTA complex.     

Synthesis and evaluation of novel N-fluoropyridyl derivatives of tropane as potential PET imaging agents for the dopamine transporter

A series of novel N-fluoropyridyl-containing tropane derivatives were synthesized and their binding affinities for the dopamine transporter (DAT), serotonin transporter (SERT) and norepinephrine (NET) were determined via competitive radioligand binding assays. Among these derivatives, compound 6d showed the highest binding affinity to DAT (K_i = 4.1 nM), and selectivity for DAT over SERT (5-fold) and NET (16-fold). Compound 6d was radiolabeled with Fluorine-18 in two steps. Regional brain distribution and ex vivo autoradiography studies of [¹⁸F]6d demonstrated that the ligand was selectively localized in the striatum region, where DAT binding sites are highly expressed. [¹⁸F]6d may be useful as a potential radioligand for imaging DATs with PET.     

Quantitative assessment of neurochemical changes in a rat model of long-term alcohol consumption as detected by in vivo and ex vivo proton nuclear magnetic resonance spectroscopy

The aim of present study was to quantitatively investigate the neurochemical profile of the frontal cortex region in a rat model of long-term alcohol consumption, by using in vivo proton magnetic resonance spectroscopy (¹H-MRS) at 4.7 T and ex vivo¹H high-resolution magic angle spinning (HR-MAS) technique at 11.7 T. Twenty male rats were divided into two groups and fed a liquid diet for 10 weeks. After 10 weeks, in vivo¹H MRS spectra were acquired from the frontal cortex brain region. After in vivo¹H MRS experiments, all animals were sacrificed and 20 frontal cortex tissue samples were harvested. All tissue examinations were performed with the 11.7 T HR-MAS spectrometer and high-resolution spectra were acquired. The in vivo and ex vivo spectra were quantified as absolute metabolite concentrations and normalized ratios of total signal-intensity (i.e., metabolites_Norm), respectively. The absolute quantifications of in vivo spectra showed significantly higher glycerophosphocholine plus phosphocholine (GPC + PCh) and lower myo-inositol (mIns) concentrations in ethanol-treated rats compared to controls. The quantifications of ex vivo spectra showed significantly higher PCh_Norm, Cho_Norm and tCho_Norm, and lower GPC_Norm and mIns_Norm ratio levels in ethanol-treated rats compared to controls. Our findings suggest that reduced mIns concentrations caused by the long-term alcohol consumption may lead to hypo-osmolarity syndrome and astrocyte hyponatremia. In addition, increased choline-containing compound concentrations may reflect an increased cell turnover rate of phosphatidylcholine and other phospholipids, indicating an adaptive mechanism. Therefore, these results might be utilized as key markers in chronic alcohol intoxication metabolism.     

Osteogenic effects of flavonoid aglycones from an osteoprotective fraction of Drynaria fortunei—An in vitro efficacy study

Drynaria fortunei (Kunze) J. Sm. is a traditional Chinese herb used for the treatment of osteoporosis and other bone metabolic disorders. Previous studies demonstrated that “small polar active fraction in Drynaria fortunei (SDF)”exerted osteoprotective effects in ovariectomized (OVX) mice. This study aims to investigate the constituents in SDF and systemically evaluate their osteogenic effects in vitro. Five flavonoid aglycones, naringenin, kurarinone, kushennol F, xanthogalenol, and sophoraflavanone G were identified in SDF. All the compounds did not show effects on proliferation of osteoblastic UMR 106 cells at the concentrations of 0.1-1000 nM, but significantly increased the ALP activity of the cells at most of the concentrations from 10 nm to 1000 nM. Xanthogalenol at the concentration of 100 nM significantly increased concentration of acid-solubilized calcium. ICI 182,780, antagonist of estrogen receptor (ER), diminished the effect of kushennol F on ALP activity and the effect of xanthogalenol on acid-solubilized calcium. In conclusion, flavonoid aglycones in SDF could promote differentiation and mineralization of osteoblastic UMR 106 cells in vitro, which was explained by activation of ER signaling pathway. This study provides scientific evidences for the conduction of in vivo experiments to confirm potential effects of flavonoid aglycones on preventing OVX-induced osteoporosis.     

Binding kinetics,potency, and selectivity of the hepatitis C virus NS3 protease inhibitors GS-9256 and vedroprevir

Background

GS-9256 and vedroprevir are inhibitors of the hepatitis C virus NS3 protease enzyme, an important drug target. The potency, selectivity, and binding kinetics of the two compounds were determined using in vitro biochemical assays.

Methods

Potency of the compounds against NS3 protease and selectivity against a panel of mammalian proteases were determined through steady-state enzyme kinetics. Binding kinetics were determined using stopped-flow techniques. Dissociation rates were measured using dilution methods.

Results

GS-9256 and vedroprevir had measured K_i values of 89 pM and 410 pM, respectively, against genotype 1b NS3 protease; K_i values were higher against genotype 2a (2.8 nM and 39 nM) and genotype 3 proteases (104 nM and 319 nM) for GS-9256 and vedroprevir, respectively. Selectivity of GS-9256 and vedroprevir was > 10,000-fold against all tested off-target proteases. Association rate constants of 4 × 10⁵ M^− 1 s^− 1 and 1 × 10⁶ M^− 1 s^− 1, respectively, were measured, and dissociation rate constants of 4.8 × 10^− 5 s^− 1 and 2.6 × 10^− 4 s^− 1 were determined.

Conclusions

GS-9256 and vedroprevir are potent inhibitors of NS3 protease with high selectivity against off-target proteases. They have rapid association kinetics and slow dissociation kinetics.

General Significance

The NS3 protease is a key drug target for the treatment of hepatitis C. The potency, selectivity, and binding kinetics of GS-9256 and vedroprevir constitute a biochemical profile that supports the evaluation of these compounds in combination with other direct-acting antivirals in clinical trials for hepatitis C.     

Microbial tolerance in secondary peritonitis is dose dependent

Local microbial tolerance was investigated in a murine model of peritonitis. Peritoneal bacterial burden and inflammatory cytokine concentrations were determined at different times, within 48 h after infection. Peritoneal macrophages were harvested from naïve mice or from mice 48 h after infection and underwent ex vivo stimulation with different concentrations of Klebsiella. Cytokine secretion was determined in the supernatants. Peritoneal bacteria concentrations, remained relatively steady between 24 h (median: 5.04 log CFU) and 48 h (median: 5.19 log CFU) after infection. Peritoneal cytokine concentrations peaked early but were already diminished at 48 h after infection, despite persistent high bacteria levels. Macrophages, harvested from naïve mice responded vigorously to ex vivo stimulation with 10⁵ CFU and 2 × 10⁸ CFU Klebsiella. Cells harvested from animals 48 h after infection, were unresponsive to an ex vivo stimulation with 10⁵ CFU Klebsiella, but fully responded to 10⁸ CFU. Persistent intraabdominal bacterial infection induced dose dependent microbial tolerance in peritoneal macrophages.     

Butane-2,3-dionethiosemicarbazone: an oxime with antioxidant properties

Oximes are compounds generally used to reverse the acetylcholinesterase (AChE) inhibition caused by organophosphates (OPs). The aim of this study was to examine the capacity of the butane-2,3-dionethiosemicarbazone oxime to scavenge different forms of reactive species (RS) in vitro, as well as counteract their formation. The potential antioxidant and toxic activity of the oxime was assayed both in vitro and ex vivo. The obtained results indicate a significant hydrogen peroxide (H₂O₂), nitric oxide (NO) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity at 0.275, 0.5 and 5 μM of oxime, respectively (p ≤ 0.05). The oxime exhibited a powerful inhibitory effect on dihydroxybenzoate formation (25 μM) (p ≤ 0.05) and also decreased deoxyribose degradation induced by Fe²⁺ and via Fenton reaction (0.44 and 0.66 mM, respectively) (p ≤ 0.05). The oxime showed a significant inhibitory effect on σ-phenantroline reaction with Fe²⁺ (0.4 mM) suggesting a possible interaction between the oxime and iron. A significant decrease in the basal and pro-oxidant-induced lipid peroxidation in brain, liver, and kidney of mice was observed both in vitro and ex vivo (p ≤ 0.05). In addition, in our ex vivo experiments the oxime did not depict any significant changes in thiol levels of liver, kidney and brain as well as did not modify the δ-aminolevulinate dehydratase (δ-ALA-D) activity in these tissues. Taken together our results indicate an in vitro and ex vivo antioxidant activity of the oxime possibly due to its scavenging activity toward different RS and a significant iron interaction.     

Helicobacter pylori hydrogenase accessory protein HypA and urease accessory protein UreG compete with each other for UreE recognition

Background

The gastric pathogen Helicobacter pylori relies on nickel-containing urease and hydrogenase enzymes in order to colonize the host. Incorporation of Ni²⁺ into urease is essential for the function of the enzyme and requires the action of several accessory proteins, including the hydrogenase accessory proteins HypA and HypB and the urease accessory proteins UreE, UreF, UreG and UreH.

Methods

Optical biosensing methods (biolayer interferometry and plasmon surface resonance) were used to screen for interactions between HypA, HypB, UreE and UreG.

Results

Using both methods, affinity constants were found to be 5 nM and 13 nM for HypA–UreE and 8 μM and 14 μM for UreG-UreE. Neither Zn²⁺ nor Ni²⁺ had an effect on the kinetics or stability of the HypA–UreE complex. By contrast, addition of Zn²⁺, but not Ni²⁺, altered the kinetics and greatly increased the stability of the UreE–UreG complex, likely due in part to Zn²⁺-mediated oligomerization of UreE. Finally our results unambiguously show that HypA, UreE and UreG cannot form a heterotrimeric protein complex in vitro; instead, HypA and UreG compete with each other for UreE recognition.

General significance

Factors influencing the pathogen's nickel budget are important to understand pathogenesis and for future drug design.     

Structure and chain conformation of five water-soluble derivatives of a β-d-glucan isolated from Ganoderma lucidum

A linear water-insoluble (1→3)-β-d-glucan, coded as GL-IV-I, was isolated from the fruit body of Ganoderma lucidum by extracting with NaOH solution. Its derivatives were prepared by using sulfation, carboxymethylation, hydroxyethylation, hydroxypropylation, and methylation, respectively, and these were labeled as S-GL, CM-GL, HE-GL, HP-GL and M-GL. Five derivatives exhibited good water solubility. Their structures and chain conformations were investigated with infrared spectroscopy, elemental analysis (EA), one- and two-dimensional NMR spectroscopy, laser light scattering (LLS), and size-exclusion chromatography combined with LLS (SEC-LLS). The reactivity of the hydroxyl group of GL-IV-I was ordered as C-6 > C-4 > C-2 for the five derivatives. The degree of substitution (DS) of the derivatives was calculated from EA and NMR spectroscopy to be from 0.32 to 1.18. The weight-average molecular mass (M_w) of GL-IV-I, S-GL, CM-GL, HE-GL, HP-GL, and M-GL was 13.3 × 10⁴, 10.1 × 10⁴, 6.3 × 10⁴, 7.2 × 10⁴, 5.1 × 10⁴, and 14.1 × 10⁴, respectively. The conformation analysis studies revealed that GL-IV-I exists as a compact coil in dimethyl sulfoxide, whereas the five derivatives are slightly expanded flexible chains in 0.9% aqueous NaCl solution.     

Marinobufagenin is an upstream modulator of Gadd45a stress signaling in preeclampsia

Preeclampsia (PE) is a hypertensive disorder of pregnancy, in which marinobufagenin (MBG), a circulating cardiotonic steroid, is increased. The Gadd45a stress sensor protein is an upstream modulator of the pathophysiological changes observed in PE. However, the effects of MBG on Gadd45a stress signaling remain unknown. We examined the expression of Gadd45a, the sFlt-1 receptor, and p38, as well as caspase 3 and 8 activities in placental samples from four groups of rats. These were: normal pregnant (NP, n = 8); pregnant rats which received weekly injections of desoxycorticosterone acetate and 0.9% saline as their drinking water (PDS, n = 9); normal pregnant rats injected with MBG (NPM, n = 8); and PDS rats injected with resibufogenin (RBG), an in vivo antagonist of MBG (PDSR, n = 8). Utilizing human cytotrophoblast (CTB) cells, we examined the effect of MBG on these stress signaling proteins in vitro. Placental Gadd45a expression, caspase 3 and 8 activities, sFlt-1 concentrations, and sFlt-1 receptor expression were significantly higher in PDS and NPM compared to NP and PDSR rats. Gadd45a protein was significantly upregulated in the CTB cells when MBG was present in concentrations ≥1 nM. Treatment with MBG (≥ 1 nM) also significantly arrested cell cycle progression and activated the expression of the Gadd45a-mediated stress signaling proteins. Inhibition of Gadd45a through RNAi-mediation attenuated MBG-induced CTB cell stress signaling. In conclusion, MBG is involved in the alteration in Gadd45a stress signaling both in vivo and in vitro and RBG prevents these changes when administered in vivo.     

In vivo tracking of human neural stem cells with 19F magnetic resonance imaging

Background

Magnetic resonance imaging (MRI) is a promising tool for monitoring stem cell-based therapy. Conventionally, cells loaded with ironoxide nanoparticles appear hypointense on MR images. However, the contrast generated by ironoxide labeled cells is neither specific due to ambiguous background nor quantitative. A strategy to overcome these drawbacks is ¹⁹F MRI of cells labeled with perfluorocarbons. We show here for the first time that human neural stem cells (NSCs), a promising candidate for clinical translation of stem cell-based therapy of the brain, can be labeled with ¹⁹F as well as detected and quantified in vitro and after brain implantation.

Methodology/Principal Findings

Human NSCs were labeled with perfluoropolyether (PFPE). Labeling efficacy was assessed with ¹⁹F MR spectroscopy, influence of the label on cell phenotypes studied by immunocytochemistry. For in vitro MRI, NSCs were suspended in gelatin at varying densities. For in vivo experiments, labeled NSCs were implanted into the striatum of mice. A decrease of cell viability was observed directly after incubation with PFPE, which re-normalized after 7 days in culture of the replated cells. No label-related changes in the numbers of Ki67, nestin, GFAP, or βIII-tubulin+ cells were detected, both in vitro and on histological sections. We found that 1,000 NSCs were needed to accumulate in one image voxel to generate significant signal-to-noise ratio in vitro. A detection limit of ∼10,000 cells was found in vivo. The location and density of human cells (hunu+) on histological sections correlated well with observations in the ¹⁹F MR images.

Conclusion/Significance

Our results show that NSCs can be efficiently labeled with ¹⁹F with little effects on viability or proliferation and differentiation capacity. We show for the first time that ¹⁹F MRI can be utilized for tracking human NSCs in brain implantation studies, which ultimately aim for restoring loss of function after acute and neurodegenerative disorders.     

Errata

The binding of aflatoxin B₁ to intact DNA has been studied by Optical Detection of Magnetic Resonance. It is demonstrated that this method which does not require degradation of the DNA, has the requisite sensitivity and resolution for investigating the properties of the intact carcinogen-DNA adduct. The results for aflatoxin are consistent with binding at the furofuran ring for both in vitro and in vivo samples.     

PEGylation of protein-based MRI contrast agents improves relaxivities and biocompatibilities

Magnetic resonance imaging (MRI) has emerged as a leading diagnostic technique in clinical and preclinical settings. However, the application of MRI to assess specific disease markers for diagnosis and monitoring drug effect has been severely hampered by the lack of desired contrast agents with high relaxivities, and optimized in vivo retention time. We have reported the development of protein-based MRI contrast agents (ProCA1) by rational design of Gd^3 + binding sites into a stable protein resulting in significantly increased longitudinal (r₁) and transverse (r₂) relaxivities compared to Gd-DTPA. Here, we report a further improvement of protein contrast agents ProCA1 for in vivo imaging by protein modification with various sizes of polyethylene glycol (PEG) chain. PEGylation results in significant increases of both r₁ and r₂ relaxivities (up to 200%), and these high relaxivities persist even at field strengths up to 9.4 T. In addition, our experimental results demonstrate that modified contrast agents have significant improvement of in vivo MR imaging and biocompatibilities including dose efficiency, protein solubility, blood retention time and decreased immunogenicity. Such improvement can be important to the animal imaging and pre-clinical research at high or ultra-high field where there is an urgent need for molecular imaging probes and optimized contrast agent.