Aptamer-targeted gold nanoparticles as molecular-specific contrast agents for reflectance imaging

Targeted metallic nanoparticles have shown potential as a platform for development of molecular-specific contrast agents. Aptamers have recently been demonstrated as ideal candidates for molecular targeting applications. In this study, we investigated the development of aptamer-based gold nanoparticles as contrast agents, using aptamers as targeting agents and gold nanoparticles as imaging agents. We devised a novel conjugation approach using an extended aptamer design where the extension is complementary to an oligonucleotide sequence attached to the surface of the gold nanoparticles. The chemical and optical properties of the aptamer-gold conjugates were characterized using size measurements and oligonucleotide quantitation assays. We demonstrate this conjugation approach to create a contrast agent designed for detection of prostate-specific membrane antigen (PSMA), obtaining reflectance images of PSMA(+) and PSMA(-) cell lines treated with the anti-PSMA aptamer-gold conjugates. This design strategy can easily be modified to incorporate multifunctional agents as part of a multimodal platform for reflectance imaging applications.     

Quasi-cubic magnetite/silica core-shell nanoparticles as enhanced MRI contrast agents for cancer imaging

Development of magnetic resonance imaging (MRI) contrast agents that can be readily applied for imaging of biological tissues under clinical settings is a challenging task. This is predominantly due to the expectation of an ideal MR agent being able to be synthesized in large quantities, possessing longer shelf life, reasonable biocompatibility, tolerance against its aggregation in biological fluids, and high relaxivity, resulting in better contrast during biological imaging. Although a repertoire of reports address various aforementioned issues, the previously reported results are far from optimal, which necessitates further efforts in this area. In this study, we demonstrate facile large-scale synthesis of sub-100 nm quasi-cubic magnetite and magnetite/silica core-shell (Mag@SiO2) nanoparticles and their applicability as a biocompatible T2 contrast agent for MRI of biological tissues. Our study suggests that silica-coated magnetite nanoparticles reported in this study can potentially act as improved MR contrast agents by addressing a number of aforementioned issues, including longer shelf life and stability in biological fluids. Additionally, our in vitro and in vivo studies clearly demonstrate the importance of silica coating towards improved applicability of T2 contrast agents for cancer imaging.     

Molecular mechanisms for the hepatic uptake of magnetic resonance imaging contrast agents

The mechanisms were investigated for the hepatic transport of 4 different gadolinium complexes used as contrast agents for magnetic resonance imaging (MRI). In basolateral rat hepatocyte plasma membrane vesicles, Gd-DTPA uptake was indistinguishable from non-specific binding to vesicles; Gd-BOPTA and Gd-EOB-DTPA entered plasma membrane vesicles following a linear, concentration-dependent mechanism up to 1.5 mM of substrate. By contrast, Gd-B 20790 uptake followed a saturative kinetic with an apparent Km of 92 +/- 15 microM and a Vmax of 143 +/- 42 pmol/mg prot/15 sec, and it occurred into an osmotic-sensitive space. Sulfobromophthalein ant taurocholate, but not unconjugated bilirubin inhibited the uptake rate of Gd-B 20790 but not that of the other three compounds. Injection into Xenopus laevis oocytes of 5 ng of human OATP cRNA resulted, after 3 days, in a >/=2-fold stimulation (p < 0.001) of transport of Gd-B 20790 but not of Gd-BOPTA or Gd-EOB-DTPA. Collectively, these data indicate that the hepatic uptake of the MRI contrast agent Gd-B 20790 is a carrier-mediated mechanism operated by OATP while MRI compounds with other chemical structures enter the hepatocyte by other mechanisms.     

Peptide-based hydrogel nanoparticles as effective drug delivery agents

Peptide-based hydrogel nanoparticles represent a promising alternative to current drug delivery approaches. We have previously demonstrated that the Fmoc-FF aromatic dipeptide building block can self-assemble in aqueous solutions to form nano-scaled ordered hydrogels of remarkable mechanical rigidity. Here, we present a scalable process for the assembly of this peptide into hydrogel nanoparticles (HNPs) aimed to be utilized as potential drug delivery carriers. Fmoc-FF based HNPs were formulated via modified inverse-emulsion method using vitamin E-TPGS as an emulsion stabilizer and high speed homogenization. The formed HNPs exhibited two distinguishable populations with an average size of 21.5 ± 1.3 and 225.9 ± 0.8 nm. Gold nanoparticles were encapsulated within the hydrogel nanoparticles as contrast agents to monitor the formation of the assemblies and their ultrastructural properties. Next, we demonstrated a robust experimental procedure developed and optimized for the formulation, purification, storage and handling procedures of HNPs. Encapsulation of doxorubicin (Dox) and 5-flourouracil (5-Fu) within the HNPs matrix showed release kinetics of the drugs depending on their chemical structure, molecular weight and hydrophobicity. The results clearly indicate that Fmoc-FF based hydrogel nanoparticles have the potential to be used as encapsulation and delivery system of various drugs and bioactive molecules.     

Semiconductor quantum dots as contrast agents for whole animal imaging

Recent developments in quantum dot (QD) technology have paved the way for using QDs as optical contrast agents for in vivo imaging. Pioneering papers showed the use of QDs as luminescent contrast agents for imaging cancer and guiding cancer surgery. The possible future use of QDs for clinical applications is expected to have a significant impact, however many challenges in this field have yet to be overcome.     

Direct translocation as major cellular uptake for CADY self-assembling peptide-based nanoparticles

Cell penetrating peptides constitute a potent approach to overcome the limitations of in vivo siRNA delivery. We recently proposed a peptide-based nanoparticle system, CADY, for efficient delivery of siRNA into numerous cell lines. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA thereby improving both their cellular uptake and biological response. With the aim of understanding the cellular uptake mechanism of CADY:siRNA complexes, we have combined biochemical, confocal and electron microscopy approaches. In the present work, we provide evidence that the major route for CADY:siRNA cellular uptake involves direct translocation through the membrane but not the endosomal pathway. We have demonstrated that CADY:siRNA complexes do not colocalize with most endosomal markers and remain fully active in the presence of inhibitors of the endosomal pathway. Moreover, neither electrostatic interactions with cell surface heparan sulphates nor membrane potential are essential for CADY:siRNA cell entry. In contrast, we have shown that CADY:siRNA complexes clearly induce a transient cell membrane permeabilization, which is rapidly restored by cell membrane fluidity. Therefore, we propose that direct translocation is the major gate for cell entry of CADY:siRNA complexes. Membrane perturbation and uptake are driven mainly by the ability of CADY to interact with phospholipids within the cell membrane, followed by rapid localization of the complex in the cytoplasm, without affecting cell integrity or viability.     

Gas fIIIed lipid bilayers as imaging contrast agents

Abstract

Gas bubbles are highly efficient reflectors of sound and are therefore useful as contrast agents for diagnostic ultrasound. In general, in order to pass through the pulmonary circulation and provide contrast for the systemic circulation the gas bubbles must be of defined diameter and stabilized by a coating material. Our group has developed gas bubbles which are stabilized within lipid bilayers. These bubbles may be formed with stable size and gas content. In vivo studies in rabbit and porcine models show sustained ventricular enhancement, increased arterial doppler signal following intravenous injection and myocardial perfusion enhancement following injection into the aortic root of pigs. Preliminary toxicity studies suggest that the therapeutic index (LD50 dose divided by imaging dose) is greater than 400 to 1. For therapy, these agents may be used to increase the capture of ultrasonic energy for hyperthermia to augment local tissue heating. An exciting new class of agents based upon gas fIIIed lipid bilayers has been developed. Work is presently underway to take these agents into clinical trials.     

Evaluation of microbubbles as contrast agents for ultrasonography and magnetic resonance imaging

Background

Microbubbles (MBs) can serve as an ultrasound contrast agent, and has the potential for magnetic resonance imaging (MRI). Due to the relatively low effect of MBs on MRI, it is necessary to develop new MBs that are more suitable for MRI. In this study, we evaluate the properties of SonoVue® and custom-made Fe₃O₄-nanoparticle-embedded microbubbles (Fe₃O₄-MBs) in terms of contrast agents for ultrsonography (US) and MRI.

Methodology/Principal Findings

A total of 20 HepG2 subcutaneous-tumor-bearing nude mice were randomly assigned to 2 groups (i.e., n = 10 mice each group), one for US test and the other for MRI test. Within each group, two tests were performed for each mouse. The contrast agent for the first test is SonoVue®, and the second is Fe₃O₄-MBs. US was performed using a Technos^MPX US system (Esaote, Italy) with a contrast-tuned imaging (CnTI™) mode. MRI was performed using a 7.0T Micro-MRI (PharmaScan, Bruker Biospin GmbH, Germany) with an EPI-T₂* sequence. The data of signal-to-noise ratio (SNR) from the region-of-interest of each US and MR image was calculated by ImageJ (National Institute of Health, USA). In group 1, enhancement of SonoVue® was significantly higher than Fe₃O₄-MBs on US (P<0.001). In group 2, negative enhancement of Fe₃O₄-MBs was significantly higher than SonoVue® on MRI (P<0.001). The time to peak showed no significant differences between US and MRI, both of which used the same MBs (P>0.05). The SNR analysis of the enhancement process reveals a strong negative correlation in both cases (i.e., SonoVue® r = −0.733, Fe₃O₄-MBs r = −0.903, with P<0.05).

Conclusions

It might be important to change the Fe₃O₄-MBs'' shell structure and/or the imagining strategy of US to improve the imaging quality of Fe₃O₄-MBs on US. As an intriguing prospect that can be detected by US and MRI, MBs are worthy of further study.     

Inorganic nanoparticle-based contrast agents for molecular imaging

Inorganic nanoparticles (NPs) including semiconductor quantum dots (QDs), iron oxide NPs and gold NPs have been developed as contrast agents for diagnostics by molecular imaging. Compared with traditional contrast agents, NPs offer several advantages: their optical and magnetic properties can be tailored by engineering the composition, structure, size and shape; their surfaces can be modified with ligands to target specific biomarkers of disease; the contrast enhancement provided can be equivalent to millions of molecular counterparts; and they can be integrated with a combination of different functions for multimodal imaging. Here, we review recent advances in the development of contrast agents based on inorganic NPs for molecular imaging, and also touch on contrast enhancement, surface modification, tissue targeting, clearance and toxicity. As research efforts intensify, contrast agents based on inorganic NPs that are highly sensitive, target-specific and safe to use are expected to enter clinical applications in the near future.     

Oligonucleotide-coated metallic nanoparticles as a flexible platform for molecular imaging agents

Targeted metallic nanoparticles have shown promise as contrast agents for molecular imaging. To obtain molecular specificity, the nanoparticle surface must be appropriately functionalized with probe molecules that will bind to biomarkers of interest. The aim of this study was to develop and characterize a flexible approach to generate molecular imaging agents based on gold nanoparticles conjugated to a diverse range of probe molecules. We present two complementary oligonucleotide-based approaches to develop gold nanoparticle contrast agents which can be functionalized with a variety of biomolecules ranging from small molecules, to peptides, to antibodies. The size, biocompatibility, and protein concentration per nanoparticle are characterized for the two oligonucleotide-based approaches; the results are compared to contrast agents prepared using adsorption of proteins on gold nanoparticles by electrostatic interaction. Contrast agents prepared from oligonucleotide-functionalized nanoparticles are significantly smaller in size and more stable than contrast agents prepared by adsorption of proteins on gold nanoparticles. We demonstrate the flexibility of the oligonucleotide-based approach by preparing contrast agents conjugated to folate, EGF peptide, and anti-EGFR antibodies. Reflectance images of cancer cell lines labeled with functionalized contrast agents show significantly increased image contrast which is specific for the target biomarker. To demonstrate the modularity of this new bioconjugation approach, we use it to conjugate both fluorophore and anti-EGFR antibodies to metal nanoparticles, yielding a contrast agent which can be probed with multiple imaging modalities. This novel bioconjugation approach can be used to prepare contrast agents targeted with biomolecules that span a diverse range of sizes; at the same time, the bioconjugation method can be adapted to develop multimodal contrast agents for molecular imaging without changing the coating design or material.     

One-pot synthesis of pegylated ultrasmall iron-oxide nanoparticles and their in vivo evaluation as magnetic resonance imaging contrast agents

A well-defined copolymer poly(oligo(ethylene glycol) methacrylate-co-methacrylic acid) P(OEGMA-co-MAA) was studied as a novel water-soluble biocompatible coating for superparamagnetic iron oxide nanoparticles. This copolymer was prepared via a two-step procedure: a well-defined precursor poly(oligo(ethylene glycol) methacrylate-co-tert-butyl methacrylate), P(OEGMA-co-tBMA) (M(n) = 17300 g mol(-1); M(w)/M(n) = 1.22), was first synthesized by atom-transfer radical polymerization in the presence of the catalyst system copper(I) chloride/2,2'-bipyridyl and subsequently selectively hydrolyzed in acidic conditions. The resulting P(OEGMA-co-MAA) was directly utilized as a polymeric stabilizer in the nanoparticle synthesis. Four batches of ultrasmall PEGylated magnetite nanoparticles (i.e., with an average diameter below 30 nm) were prepared via aqueous coprecipitation of iron salts in the presence of variable amounts of P(OEGMA-co-MAA). The diameter of the nanoparticles could be easily tuned in the range 10-25 nm by varying the initial copolymer concentration. Moreover, the formed PEGylated ferrofluids exhibited a long-term colloidal stability in physiological buffer and could therefore be studied in vivo by magnetic resonance (MR) imaging. Intravenous injection into rats showed no detectable signal in the liver within the first 2 h. Maximum liver accumulation was found after 6 h, suggesting a prolongated circulation of the nanoparticles in the bloodstream as compared to conventional MR imaging contrast agents.     

Target-specific cellular uptake of taxol-loaded heparin-PEG-folate nanoparticles

To enhance site-specific intracellular delivery against folate receptor, heparin-PEG-folate (H-PEG-F) containing succinylated-heparin conjugated with folate via PEG 1000/3000 spacers has been prepared. Due to covalent strategy, H-PEG-F displays amphiphilic property, which is capable of entrapping a hydrophobic agent, like taxol, to form heparin-PEG-folate-taxol nanoparticles (H-PEG-F-T NPs) in aqueous solution. Hydrophobic agents can be entrapped within the core, while the H-PEG-F conjugates can stabilize the nanoparticles with exposing folate moieties on the surface. The structure of carrier and naoparticles has been characterized by(1)H NMR, and the content of folate and taxol has been quantitatively analyzed by UV method. The morphology and size of H-PEG-F-T NPs have been measured by field emission scanning electron microscopy (FESEM) and dynamic lighting scatter (DLS). All the NPs are in spherical shape and the sizes are less than 200 nm. The sizes of the NPs increases with increasing PEG segment length. By employing the flow cytomery method, the extent of cellular uptake has been comparatively evaluated under various conditions. The results of cellular uptake demonstrate that the cellular uptake of the carrier and the NPs is exceedingly higher for KB-3-1 cells (folate receptor overexpressing cell line) than for A549 cells (folate receptor deficiency cell line); H-PEG-F-T NPs show far greater extent of cellular uptake than that of H-PEG-F conjugates against A549 cells; when the content of folate is fixed at the same value, the extent of cellular uptake for the carrier and NPs ascends with the increase of PEG chain length against KB-3-1 cells. It suggests folate-receptor-mediated endocytosis and formation of nanoparticle and spacer length are considered to coaffect the cellular uptake efficiency of H-PEG-F-T NPs and H-PEG-F conjugates. Flow cytometry analysis depicts that KB-3-1 cells treated with H-PEG-F-T are arrested in the G(2)/M phase of the cell cycle, which states the similar inhibition mechanism as taxol. The strategy based on the formation of H-PEG-F-T NPs could be potentially applied for cancer cell targeted delivery of various therapeutic agents.     

Albumin-based nanoparticles as magnetic resonance contrast agents: II. Physicochemical characterisation of purified and standardised nanoparticles

We are developing a nanoparticulate histochemical reagent designed for histochemistry in living animals (molecular imaging), which should finally be useful in clinical imaging applications. The iterative development procedure employed involves conceptual design of the reagent, synthesis and testing of the reagent, then redesign based on data from the testing; each cycle of testing and development generates a new generation of nanoparticles, and this report describes the synthesis and testing of the third generation. The nanoparticles are based on human serum albumin and the imaging modality selected is magnetic resonance imaging (MRI). Testing the second particle generation with newly introduced techniques revealed the presence of impurities in the final product, therefore we replaced dialysis with diafiltration. We introduced further testing methods including thin layer chromatography, arsenazo III as chromogenic assay for gadolinium, and several versions of polyacrylamide gel electrophoresis, for physicochemical characterisation of the nanoparticles and intermediate synthesis compounds. The high grade of chemical purity achieved by combined application of these methodologies allowed standardised particle sizes to be achieved (low dispersities), and accurate measurement of critical physicochemical parameters influencing particle size and imaging properties. Regression plots confirmed the high purity and standardisation. The good degree of quantitative physicochemical characterisation aided our understanding of the nanoparticles and allowed a conceptual model of them to be prepared. Toxicological screening demonstrated the extremely low toxicity of the particles. The high magnetic resonance relaxivities and enhanced mechanical stability of the particles make them an excellent platform for the further development of MRI molecular imaging.     

Targeted contrast agents for magnetic resonance imaging and ultrasound

The development of contrast agents that can be localized to a particular tissue or cellular epitope will potentially allow the noninvasive visualization and characterization of a variety of disease states. Recent advances have been made in the field of molecular imaging with magnetic resonance imaging and ultrasound and varied approaches have been devised to overcome the high background tissue signal. The types of agents and applications developed include gadolinium-conjugated targeting molecules for imaging of fibrin, superparamagnetic iron oxide particles for stem-cell tracking, multimodal perfluorocarbon nanoparticles for visualization of angiogenesis, liposomes for targeting atheroma components, and microbubbles for imaging transplant rejection.     

Conformation and structure of polymeric contrast agents for medical imaging

The structure of Gd-DTPA-polylysine, Gd-DOTA-polylysine, Gd-SCN-Bz-DOTA-polylysine, and Gd-DTPA-poly(glu:lys) was investigated with circular dichroism, gel permeation chromatography, low angle light scattering, and proton longitudinal relaxivity. Molecular modeling calculations were performed and predicted helical secondary structure for charged Gd-chelator residues, i.e., Gd-DTPA, when the DTPA conjugation levels reached 90% and higher. This helical secondary structure was observed with circular dichroism. The conformational transition from coiled to extended linear was observed also by gel permeation chromatography and by proton relaxivity measurements. The helical secondary structure was not observed when the chelator was changed to DOTA. The residue charge interactions were eliminated in this case since the Gd-DOTA complex had no net charge. For this construct, the gel permeation and relaxivity measurements indicated a coiled conformation. An extended linear conformation was regained when the chelator complex was changed to Gd-SCN-Bz-DOTA, which had a net negative charge. The functional aspects of these structures were investigated by MR imaging of an animal tumor model. The linear extended polymer constructs gave 10-fold higher tumor signals then the coiled-collapsed constructs, indicating a much higher degree of trans-endothelial transport in the tumors.     

MRI contrast agents for functional molecular imaging of brain activity

Functional imaging with MRI contrast agents is an emerging experimental approach that can combine the specificity of cellular neural recording techniques with noninvasive whole-brain coverage. A variety of contrast agents sensitive to aspects of brain activity have recently been introduced. These include new probes for calcium and other metal ions that offer high sensitivity and membrane permeability, as well as imaging agents for high-resolution pH and metabolic mapping in living animals. Genetically encoded MRI contrast agents have also been described. Several of the new probes have been validated in the brain; in vivo use of other agents remains a challenge. This review outlines advantages and disadvantages of specific molecular imaging approaches and discusses current or potential applications in neurobiology.     

Mn(II)-monosubstituted polyoxometalates as candidates for contrast agents in magnetic resonance imaging

Two mono-substituted manganese polyoxometalates, K(6)MnSiW(11)O(39) (MnSiW(11)) and K(8)MnP(2)W(17)O(61) (MnP(2)W(17)), have been evaluated by in vivo and in vitro experiments as the candidates of potential tissue-specific contrast agents for magnetic resonance imaging (MRI). T1-relaxivities of 12.1mM(-1)s(-1) for MnSiW(11) and 4.7 mM(-1)s(-1) for MnP(2)W(17) (400 MHz, 25 degrees C) were higher than or similar to that of the commercial MRI contrast agent (GdDTPA). Their relaxivities in BSA and hTf solutions were also reported. After administration of MnSiW(11) and MnP(2)W(17) to Wistar rats, MR imaging showed longer and remarkable enhancement in rat liver and favorable renal excretion capability. The signal intensity increased by 74.0+/-4.9% for the liver during the whole imaging period (90 min) and by 67.2+/-5.3% for kidney within 20-70 min after injection at 40+/-3 micromol kg(-1) dose for MnSiW(11). MnP(2)W(17) induced 71.5+/-15.1% enhancement for the liver in 10-45 min range and 73.1+/-3.2% enhancement for kidney within 5-40 min after injection at 39+/-3 micromol kg(-1) dose. In vitro and in vivo study showed MnSiW(11) and MnP(2)W(17) being favorable candidates as the tissue-specific contrast agents for MRI.