Multicolor spectral photon counting CT monitors and quantifies therapeutic cells and their encapsulating scaffold in a model of brain damage.

Rationale & aim: Various types of cell therapies are currently under investigation for the treatment of ischemic stroke patients. To bridge the gap between cell administration and therapeutic outcome, there is a need for non-invasive monitoring of these innovative therapeutic approaches. Spectral photon counting computed tomography (SPCCT) is a new imaging modality that may be suitable for cell tracking. SPCCT is the next generation of clinical CT that allows the selective visualization and quantification of multiple contrast agents. The aims of this study are: (i) to demonstrate the feasibility of using SPCCT to longitudinally monitor and quantify therapeutic cells, i.e. bone marrow-derived M2-polarized macrophages transplanted in rats with brain damage; and (ii) to evaluate the potential of this approach to discriminate M2-polarized macrophages from their encapsulating scaffold. Methods: Twenty one rats received an intralesional transplantation of bone marrow-derived M2-polarized macrophages. In the first set of experiments, cells were labeled with gold nanoparticles and tracked for up to two weeks post-injection in a monocolor study via gold K-edge imaging. In the second set of experiments, the same protocol was repeated for a bicolor study, in which the labeled cells are embedded in iodine nanoparticle-labeled scaffold. The amount of gold in the brain was longitudinally quantified using gold K-edge images reconstructed from SPCCT acquisition. Animals were sacrificed at different time points post-injection, and ICP-OES was used to validate the accuracy of gold quantification from SPCCT imaging. Results: The feasibility of therapeutic cell tracking was successfully demonstrated in brain-damaged rats with SPCCT imaging. The imaging modality enabled cell monitoring for up to 2 weeks post-injection, in a specific and quantitative manner. Differentiation of labeled cells and their embedding scaffold was also feasible with SPCCT imaging, with a detection limit as low as 5,000 cells in a voxel of 250 × 250 × 250 µm in dimension in vivo. Conclusion: Multicolor SPCCT is an innovative translational imaging tool that allows monitoring and quantification of therapeutic cells and their encapsulating scaffold transplanted in the damaged rat brain.

Effect of Gold Nanoparticle Size on Their Properties as Contrast Agents for Computed Tomography.

Computed tomography (CT) is one of the most commonly used clinical imaging modalities. There have recently been many reports of novel contrast agents for CT imaging. In particular, the development of gold nanoparticles (AuNP) as CT contrast agents is a topic of intense interest. AuNP have favorable characteristics for this application such as high payloads of contrast generating material, strong X-ray attenuation, excellent biocompatibility, tailorable surface chemistry, and tunable sizes and shapes. However, there have been conflicting reports on the role of AuNP size on their contrast generation for CT. We therefore sought to extensively investigate the AuNP size-CT contrast relationship. In order to do this, we synthesized AuNP with sizes ranging from 4 to 152 nm and capped them with 5 kDa m-PEG. The contrast generation of AuNP of different sizes was investigated with three clinical CT, a spectral photon counting CT (SPCCT) and two micro CT systems. X-ray attenuation was quantified as attenuation rate in Hounsfield units per unit concentration (HU/mM). No statistically significant difference in CT contrast generation was found among different AuNP sizes via phantom imaging with any of the systems tested. Furthermore, in vivo imaging was performed in mice to provide insight into the effect of AuNP size on animal biodistribution at CT dose levels, which has not previously been explored. Both in vivo imaging and ex vivo analysis with inductively coupled plasma optical emission spectroscopy (ICP-OES) indicated that AuNP that are 15 nm or smaller have long blood circulation times, while larger AuNP accumulated in the liver and spleen more rapidly. Therefore, while we observed no AuNP size effect on CT contrast generation, there is a significant effect of size on AuNP diagnostic utility.

Wulff in a cage gold nanoparticles as contrast agents for computed tomography and photoacoustic imaging.

Nanostructures have potential for use in biomedical applications such as sensing, imaging, therapeutics, and drug delivery. Among nanomaterials, gold nanostructures are of considerable interest for biomedical research, owing to their bio-inertness, controllable surface chemistry, X-ray opacity, and optical properties. Gold nanocages are particularly attractive for imaging and therapeutic applications, because they strongly absorb light in the near infra-red region which has high light transmission in tissue. However, the X-ray attenuation of nanocages is relatively low due to their hollow structure. In this study, for the first time, we sought to combine the attractive optical properties of nanoshells with the high payloads of solid nanoparticles and investigated their biomedical applications. Here, we report the engineering of Wulff in a cage nanoparticles via converting gold Wulff-shaped seeds into gold-silver core-shell structures and then performing a galvanic replacement reaction. The structure of these nanoparticles was determined using transition electron microscopy. This morphological transformation of gold nanoparticles shaped as truncated octahedrons into a complex Wulff in a cage nanoparticles during the reaction resulted in extensive changes in their optical properties that made these unique structures a potential contrast agent for photoacoustic imaging. We found that the Wulff in a cage nanoparticles had no adverse effects on the viabilities of J774A.1, Renca, and HepG2 cells at any of the concentrations tested. In vitro and in vivo experiments showed robust signals in both photoacoustic imaging and computed tomography. To the best of our knowledge, this is the first report of Wulff in a cage nanoparticles serving as a platform for multiple imaging modalities. This unique multifunctional nanostructure, which integrates the competencies of both core and shell structures, allows their use as contrast agents for photoacoustic imaging, computed tomography and as a potential agent for photothermal therapy.

An all-in-one nanoparticle (AION) contrast agent for breast cancer screening with DEM-CT-MRI-NIRF imaging.

Conventional X-ray mammography has low diagnostic sensitivity for women with dense breasts. As a result, alternative contrast-enhanced screening tools such as dual energy mammography (DEM), computed tomography (CT), magnetic resonance imaging (MRI), and near-infrared fluorescence (NIRF) imaging are being used or investigated for these women. However, currently available contrast agents are non-ideal, have safety issues, and each imaging technique requires a different contrast agent. We therefore sought to develop a multimodal contrast agent that is functional for each breast imaging modality to simplify the diagnosis process and address the issues of existing contrast agents. Herein, we present a novel "all-in-one" nanoparticle (AION) multimodal imaging probe that has potent DEM, CT, MRI, and NIRF contrast properties and improved biocompatibility. AION were formed by co-encapsulation of a near-infrared fluorophore (DiR), silver sulfide nanoparticles (Ag2S-NP), and iron oxide nanoparticles (IO-NP) in PEGylated micelles. AION showed negligible cytotoxicity, which was in agreement with its minimal silver ion release profiles. AION generated strong contrast with all imaging modalities as demonstrated in phantom imaging. AION allowed in vivo tumor imaging as evidenced by the increase in contrast after injection. This study indicates the potential of AION as an effective multimodal contrast agent for breast cancer diagnosis with a range of imaging methods.

CD163+ macrophages promote angiogenesis and vascular permeability accompanied by inflammation in atherosclerosis.

Intake of hemoglobin by the hemoglobin-haptoglobin receptor CD163 leads to a distinct alternative non-foam cell antiinflammatory macrophage phenotype that was previously considered atheroprotective. Here, we reveal an unexpected but important pathogenic role for these macrophages in atherosclerosis. Using human atherosclerotic samples, cultured cells, and a mouse model of advanced atherosclerosis, we investigated the role of intraplaque hemorrhage on macrophage function with respect to angiogenesis, vascular permeability, inflammation, and plaque progression. In human atherosclerotic lesions, CD163+ macrophages were associated with plaque progression, microvascularity, and a high level of HIF1α and VEGF-A expression. We observed irregular vascular endothelial cadherin in intraplaque microvessels surrounded by CD163+ macrophages. Within these cells, activation of HIF1α via inhibition of prolyl hydroxylases promoted VEGF-mediated increases in intraplaque angiogenesis, vascular permeability, and inflammatory cell recruitment. CD163+ macrophages increased intraplaque endothelial VCAM expression and plaque inflammation. Subjects with homozygous minor alleles of the SNP rs7136716 had elevated microvessel density, increased expression of CD163 in ruptured coronary plaques, and a higher risk of myocardial infarction and coronary heart disease in population cohorts. Thus, our findings highlight a nonlipid-driven mechanism by which alternative macrophages promote plaque angiogenesis, leakiness, inflammation, and progression via the CD163/HIF1α/VEGF-A pathway.

Use of Nanoparticle Contrast Agents for Cell Tracking with Computed Tomography.

Efforts to develop novel cell-based therapies originated with the first bone marrow transplant on a leukemia patient in 1956. Preclinical and clinical examples of cell-based treatment strategies have shown promising results across many disciplines in medicine, with recent advances in immune cell therapies for cancer producing remarkable response rates, even in patients with multiple treatment failures. However, cell-based therapies suffer from inconsistent outcomes, motivating the search for tools that allow monitoring of cell delivery and behavior in vivo. Noninvasive cell imaging techniques, also known as cell tracking, have been developed to address this issue. These tools can allow real-time, quantitative, and long-term monitoring of transplanted cells in the recipient, providing insight on cell migration, distribution, viability, differentiation, and fate, all of which play crucial roles in treatment efficacy. Understanding these parameters allows the optimization of cell choice, delivery route, and dosage for therapy and advances cell-based therapy for specific clinical uses. To date, most cell tracking work has centered on imaging modalities such as MRI, radionuclide imaging, and optical imaging. However, X-ray computed tomography (CT) is an emerging method for cell tracking that has several strengths such as high spatial and temporal resolution, and excellent quantitative capabilities. The advantages of CT for cell tracking are enhanced by its wide availability and cost effectiveness, allowing CT to become one of the most popular clinical imaging modalities and a key asset in disease diagnosis. In this review, we will discuss recent advances in cell tracking methods using X-ray CT in various applications, in addition to predictions on how the field will progress.

Effect of Gold Nanoparticle Size and Coating on Labeling Monocytes for CT Tracking.

With advances in cell therapies, interest in cell tracking techniques to monitor the migration, localization, and viability of these cells continues to grow. X-ray computed tomography (CT) is a cornerstone of medical imaging but has been limited in cell tracking applications due to its low sensitivity toward contrast media. In this study, we investigate the role of size and surface functionality of gold nanoparticles for monocyte uptake to optimize the labeling of these cells for tracking in CT. We synthesized gold nanoparticles (AuNP) that range from 15 to 150 nm in diameter and examined several capping ligands, generating 44 distinct AuNP formulations. In vitro cytotoxicity and uptake experiments were performed with the RAW 264.7 monocyte cell line. The majority of formulations at each size were found to be biocompatible, with only certain 150 nm PEG functionalized particles reducing viability at high concentrations. High uptake of AuNP was found using small capping ligands with distal carboxylic acids (11-MUA and 16-MHA). Similar uptake values were found with intermediate sizes (50 and 75 nm) of AuNP when coated with 2000 MW poly(ethylene-glycol) carboxylic acid ligands (PCOOH). Low uptake values were observed with 15, 25, 100, and 150 nm PCOOH AuNP, revealing interplay between size and surface functionality. Transmission electron microscopy (TEM) and CT performed on cells revealed similar patterns of high gold uptake for 50 nm PCOOH and 75 nm PCOOH AuNP. These results demonstrate that highly negatively charged carboxylic acid coatings for AuNP provide the greatest internalization of AuNP in monocytes, with a complex dependency on size.

Gold silver alloy nanoparticles (GSAN): an imaging probe for breast cancer screening with dual-energy mammography or computed tomography.

Earlier detection of breast cancer reduces mortality from this disease. As a result, the development of better screening techniques is a topic of intense interest. Contrast-enhanced dual-energy mammography (DEM) is a novel technique that has improved sensitivity for cancer detection. However, the development of contrast agents for this technique is in its infancy. We herein report gold-silver alloy nanoparticles (GSAN) that have potent DEM contrast properties and improved biocompatibility. GSAN formulations containing a range of gold : silver ratios and capped with m-PEG were synthesized and characterized using various analytical methods. DEM and computed tomography (CT) phantom imaging showed that GSAN produced robust contrast that was comparable to silver alone. Cell viability, reactive oxygen species generation and DNA damage results revealed that the formulations with 30% or higher gold content are cytocompatible to Hep G2 and J774A.1 cells. In vivo imaging was performed in mice with and without breast tumors. The results showed that GSAN produce strong DEM and CT contrast and accumulated in tumors. Furthermore, both in vivo imaging and ex vivo analysis indicated the excretion of GSAN via both urine and feces. In summary, GSAN produce strong DEM and CT contrast, and has potential for both blood pool imaging and for breast cancer screening.

Tunable, biodegradable gold nanoparticles as contrast agents for computed tomography and photoacoustic imaging.

Gold nanoparticles (AuNP) have been proposed for many applications in medicine. Although large AuNP (>5.5 nm) are desirable for their longer blood circulation and accumulation in diseased tissues, small AuNP (<5.5 nm) are required for excretion via the kidneys. We present a novel platform where small, excretable AuNP are encapsulated into biodegradable poly di(carboxylatophenoxy)phosphazene (PCPP) nanospheres. These larger nanoparticles (Au-PCPP) can perform their function as contrast agents, then subsequently break down into harmless byproducts and release the AuNP for swift excretion. Homogeneous Au-PCPP were synthesized using a microfluidic device. The size of the Au-PCPP can be controlled by the amount of polyethylene glycol-polylysine (PEG-PLL) block co-polymer in the formulation. Synthesis of Au-PCPP nanoparticles and encapsulation of AuNP in PCPP were evaluated using transmission electron microscopy and their biocompatibility and biodegradability confirmed in vitro. The Au-PCPP nanoparticles were found to produce strong computed tomography contrast. The UV-Vis absorption peak of Au-PCPP can be tuned into the near infrared region via inclusion of varying amounts of AuNP and controlling the nanoparticle size. In vitro and in vivo experiments demonstrated the potential of Au-PCPP as contrast agents for photoacoustic imaging. Therefore, Au-PCPP nanoparticles have high potency as contrast agents for two imaging modalities, as well as being biocompatible and biodegradable, and thus represent a platform with potential for translation into the clinic.

Labeling monocytes with gold nanoparticles to track their recruitment in atherosclerosis with computed tomography.

Monocytes are actively recruited from the circulation into developing atherosclerotic plaques. In the plaque, monocytes differentiate into macrophages and eventually form foam cells. Continued accumulation of foam cells can lead to plaque rupture and subsequent myocardial infarction. X-ray computed tomography (CT) is the best modality to image the coronary arteries non-invasively, therefore we have sought to track the accumulation of monocytes into atherosclerotic plaques using CT. Gold nanoparticles were synthesized and stabilized with a variety of ligands. Select formulations were incubated with an immortalized monocyte cell line in vitro and evaluated for cytotoxicity, effects on cytokine release, and cell uptake. These data identified a lead formulation, 11-MUDA capped gold nanoparticles, to test for labeling primary monocytes. The formulation did not the affect the viability or cytokine release of primary monocytes and was highly taken up by these cells. Gold labeled primary monocytes were injected into apolipoprotein E deficient mice kept on Western diet for 10 weeks. Imaging was done with a microCT scanner. A significant increase in attenuation was measured in the aorta of mice receiving the gold labeled cells as compared to control animals. Following the experiment, the biodistribution of gold was evaluated in major organs. Additionally, plaques were sectioned and examined with electron microscopy. The results showed that gold nanoparticles were present inside monocytes located within plaques. This study demonstrates the feasibility of using gold nanoparticles as effective cell labeling contrast agents for non-invasive imaging of monocyte accumulation within plaques with CT.

Nanoparticle Loaded Polymeric Microbubbles as Contrast Agents for Multimodal Imaging.

Ultrasound contrast agents are typically microbubbles (MB) with a gas core that is stabilized by a shell made of lipids, proteins, or polymers. The high impedance mismatch between the gas core and an aqueous environment produces strong contrast in ultrasound (US). Poly(lactic acid) (PLA) MB, previously developed in our laboratory, have been shown to be highly echogenic both in vitro and in vivo. Combining US with other imaging modalities such as fluorescence, magnetic resonance imaging (MRI), or computerized tomography (CT) could improve the accuracy of many US applications and provide more comprehensive diagnostic information. Furthermore, our MB have the capacity to house a drug in the PLA shell and create drug-loaded nanoparticles in situ when passing through an ultrasound beam. To create multimodal contrast agents, we hypothesized that the polymer shell of our PLA MB platform could accommodate additional payloads. In this study, we therefore modified our current MB by encapsulating nanoparticles including aqueous or organic quantum dots (QD), magnetic iron oxide nanoparticles (MNP), or gold nanoparticles (AuNP) to create bimodality platforms in a manner that minimally compromised the performance of each individual imaging technique.

Systematic in vitro toxicological screening of gold nanoparticles designed for nanomedicine applications.

Gold nanoparticles (AuNP) are increasingly being applied in the biomedical field as therapeutics, contrast agents, and in diagnostic systems, motivating investigations of their toxicity that might arise from accidental exposure. While other work has investigated the toxicological response to gold nanoparticles for industrial purposes, here we have surveyed formulations that have been developed for biomedical use, are in clinical trials or have been FDA-approved. The AuNP library tested contains a range of shapes, including spheres, rods and shells, that possess a range of coatings, such as silica, citrate, lipoprotein, polymaleic acid, polyethylene glycol, DNA and others. Good cytocompatibility for all formulations was observed after 1 h of incubation. However after 24 h exposure, a nanorod and a spherical DNA coated formulation resulted in toxicity. The coating material was the only factor that influenced toxicity. AuNP exposure seemed to have no effect on cell cytoskeleton deformation and cell spreading. Cell uptake, as measured by computed tomography and ICP-OES, as well as TEM images of cells, confirmed strong AuNP uptake for certain formulations, but there was no correlation with toxicity. No glove translocation occurred, therefore, nitrile gloves are an adequate safety precaution for working with the AuNP studied. In conclusion, the majority of AuNP formulations tested have very low adverse effects.

Dextran coated bismuth-iron oxide nanohybrid contrast agents for computed tomography and magnetic resonance imaging.

Bismuth nanoparticles have been proposed as a novel CT contrast agent, however few syntheses of biocompatible bismuth nanoparticles have been achieved. We herein report the synthesis of composite bismuth-iron oxide nanoparticles (BION) that are based on a clinically approved, dextran-coated iron oxide formulation; the particles have the advantage of acting as contrast agents for both CT and MRI. BION were synthesized and characterized using various analytical methods. BION CT phantom images revealed that the X-ray attenuation of the different formulations was dependent upon the amount of bismuth present in the nanoparticle, while T2-weighted MRI contrast decreased with increasing bismuth content. No cytotoxicity was observed in Hep G2 and BJ5ta cells after 24 hours incubation with BION. The above properties, as well as the yield of synthesis and bismuth inclusion efficiency, led us to select the Bi-30 formulation for in vivo experiments, performed in mice using a micro-CT and a 9.4 T MRI system. X-ray contrast was observed in the heart and blood vessels over a 2 hour period, indicating that Bi-30 has a prolonged circulation half-life. Considerable signal loss in T2-weighted MR images was observed in the liver compared to pre-injection scans. Evaluation of the biodistribution of Bi-30 revealed that bismuth is excreted via the urine, with significant concentrations found in the kidneys and urine. In vitro experiments confirmed the degradability of Bi-30. In summary, dextran coated BION are biocompatible, biodegradable, possess strong X-ray attenuation properties and also can be used as T2-weighted MR contrast agents.

Nanodisco balls: control over surface versus core loading of diagnostically active nanocrystals into polymer nanoparticles.

Nanoparticles of complex architectures can have unique properties. Self-assembly of spherical nanocrystals is a high yielding route to such systems. In this study, we report the self-assembly of a polymer and nanocrystals into aggregates, where the location of the nanocrystals can be controlled to be either at the surface or in the core. These nanospheres, when surface decorated with nanocrystals, resemble disco balls, thus the term nanodisco balls. We studied the mechanism of this surface loading phenomenon and found it to be Ca(2+) dependent. We also investigated whether excess phospholipids could prevent nanocrystal adherence. We found surface loading to occur with a variety of nanocrystal types including iron oxide nanoparticles, quantum dots, and nanophosphors, as well as sizes (10-30 nm) and shapes. Additionally, surface loading occurred over a range of polymer molecular weights (∼30-3000 kDa) and phospholipid carbon tail length. We also show that nanocrystals remain diagnostically active after loading onto the polymer nanospheres, i.e., providing contrast in the case of magnetic resonance imaging for iron oxide nanoparticles and fluorescence for quantum dots. Last, we demonstrated that a fluorescently labeled protein model drug can be delivered by surface loaded nanospheres. We present a platform for contrast media delivery, with the unusual feature that the payload can be controllably localized to the core or the surface.

Cyclic stretch-induced oxidative stress increases pulmonary alveolar epithelial permeability.

Mechanical ventilation with high tidal volumes has been associated with pulmonary alveolar flooding. Understanding the mechanisms underlying cyclic stretch-induced increases in alveolar epithelial permeability may be important in designing preventive measures for acute lung injury. In this work, we assessed whether cyclic stretch leads to the generation of reactive oxygen species in type I-like alveolar epithelial cells, which increase monolayer permeability via activation of NF-κB and extracellular signal-regulated kinase (ERK). We cyclically stretched type I-like rat primary alveolar epithelial cells at magnitudes of 12, 25, and 37% change in surface area (ΔSA) for 10 to 120 minutes. High levels of reactive oxygen species and of superoxide and NO specifically were detected in cells stretched at 37% ΔSA for 10 to 120 minutes. Exogenous superoxide and NO stimulation increased epithelial permeability in unstretched cells, which was preventable by the NF-κB inhibitor MG132. The cyclic stretch-induced increase in permeability was decreased by the superoxide scavenger tiron and by MG132. Furthermore, tiron had a dramatic protective effect on in vivo lung permeability under mechanical ventilation conditions. Cyclic stretch increased the activation of the NF-κB signaling pathway, which was significantly decreased with the ERK inhibitor U0126. Altogether, our in vitro and in vivo data demonstrate the sensitivity of permeability to stretch- and ventilation-induced superoxide production, suggesting that using antioxidants may be helpful in the prevention and treatment of ventilator-induced lung injury.