SPIO–RGD nanoparticles as a molecular targeting probe for imaging tumor angiogenesis using synchrotron radiation

Angiogenesis, new blood vessels sprouting from pre‐existing vessels, is essential to tumor growth, invasion and metastasis. It can be used as a biomarker for early stage tumor diagnosis and targeted therapy. To visualize angiogenesis many molecular imaging modalities have been used. In this study a novel X‐ray molecular targeting probe using superparamagnetic iron oxide (SPIO) conjugated with arginine–glycine–aspartic acid (SPIO–RGD) has been developed. Based on the extremely high sensitivity to the iron element of synchrotron radiation X‐ray fluorescence and the superior spatial resolution of third‐generation synchrotron radiation, the feasibility of SPIO–RGD as a promising molecular probe for imaging tumor angiogenesis has been demonstrated.     

Amplified Photoacoustic Imaging of Tumor through In Situ Cycloaddition

Photoacoustic (PA) imaging has received great attention in the field of biomedical applications due to the combination advantages of the high contrast of optical imaging and the high spatial resolution of ultrasound. The limited targeting property of PA contrast agents is restricted to elaborate its advantage. To overcome this point, a pretargeting strategy is developed to amplify the targeting property and PA imaging of a model dye in vivo. As a proof of concept, the dibenzyl cyclootyne (DBCO)‐modified Fe@Fe₃O₄ nanoparticles (NPs) (Fe@Fe₃O₄/DBCO) and azide‐modified Cy7.5 (Cy7.5‐N₃) are adopted as the pretargeting and PA contrast agents, respectively. Fe@Fe₃O₄/DBCO NPs are first targeted into tumors by the enhanced permeability and retention effect, and then Cy7.5‐N₃ is conjugated to the pretargeted Fe@Fe₃O₄/DBCO labeled tumor cells via strain‐promoted alkyne azide cycloaddition reaction after intravenous injection, which results in an obvious increase of the accumulated dose and PA signal of Cy7.5 in tumor, and simultaneously extends its residence time. This signal amplification strategy should have an important guiding significance for the clinical application in cancer theranostics.     

Characterization of peptide immobilization on an acetylene terminated surface via click chemistry

Peptide (A-A-A-A-G-G-G-E-R-G-D)¹ conjugated surfaces were prepared on silicon surfaces through click chemistry. The amino acid sequence RGD is the cellular attachment site of a large number of extracellular matrices such as blood and cell surface proteins. Recent research has focused on developing RGD peptides which mimic cell adhesion proteins and integrins [1], [2].The steps involved the formation of an alkyne-terminated monolayer on Si(111), followed by linking the peptide to 4-azidophenyl isothiocyanate via a specific and gentle reaction. This was followed by the attachment of the azido peptide to the surface-bound alkynes using the Cu (I)-catalyzed Huisgen 1,3-dipolar cycloaddition reaction. The surface structures of the alkyne terminated monolayer and the attached peptide were characterized using high resolution impedance spectroscopy (EIS), X-ray photoelectron spectroscopy (XPS) and Fourier Transform Infrared (ATR-FTIR) Spectroscopy. EIS characterization revealed the alkyne layer and the hydrophobic and polar regions of the attached peptide. XPS analysis showed a high surface coverage of the peptide on the silicon substrates and this was confirmed by FTIR.Our results confirmed a specific covalent attachment of the peptide on the silicon surfaces. This approach offers a versatile, experimentally simple, method for the specific attachment of peptide ligands. This approach would have applications for cell attachment and biosensors.     

Nanosized cancer cell-targeted polymeric immunomicelles loaded with superparamagnetic iron oxide nanoparticles

Stable 30–50 nm polymeric polyethylene glycol–phosphatidylethanolamine (PEG–PE)-based micelles entrapping superparamagnetic iron oxide nanoparticles (SPION) have been prepared. At similar concentrations of SPION, the SPION-micelles had significantly better magnetic resonance imaging (MRI) T2 relaxation signal compared to ‘plain’ SPION. Freeze-fracture electron microscopy confirmed SPION entrapment in the lipid core of the PEG–PE micelles. To enhance the targeting capability of these micelles, their surface was modified with the cancer cell-specific anti-nucleosome monoclonal antibody 2C5 (mAb 2C5). Such mAb 2C5-SPION immunomicelles demonstrated specific binding with cancer cells in vitro and were able to bring more SPION to the cancer cells thus demonstrating the potential to be used as targeted MRI contrast agents for tumor imaging.     

Encapsulation of Photosensitizers and Upconversion Nanocrystals in Lipid Micelles for Photodynamic Therapy

Photodynamic therapy (PDT) is a promising method for cancer therapy. However, it is constrained by limited penetration depth of visible light, hydrophobicity of photosensitizers, and lack of tumor targeting. In this work, the photosensitizer zinc phthalocyanine (ZnPc) and upconversion nanocrystals (UCNs) are encapsulated into OQPGA‐PEG/RGD/TAT lipid micelles. The UCNs acting as a nanotransducer convert deep‐penetrating near‐infrared (NIR) light to visible light for activating the photosensitizer. OQPGA‐PEG/RGD/TAT lipid micelles are used as a carrier for the photosensitizer, with improved biocompatibility and cancer‐targeting ability. The results show that the photosensitizer ZnPc‐ and UCNs‐loaded OQPGA‐PEG/RGD/TAT lipid micelles are nanoparticles with an average size of 25 nm. The lipid micelle nanoparticles are stable in water with low leakage of photosensitizer. The absorption peak of the photosensitizer overlaps with the emission peak of UCNs, so the visible fluorescence emitted from the UCNs upon excitation by the NIR laser at 980 nm can activate the photosensitizer to produce singlet oxygen for PDT. The targeting RGD peptide and cell‐penetrating TAT peptide on the surface help the nanoparticles getting into cancer cells. The OQPGA‐PEG/RGD/TAT lipid micelles encapsulated with both the photosensitizer ZnPc and UCNs could be used for targeted PDT by using deep‐penetrating NIR light as the light source.     

准高斯声束对超声造影剂的声辐射力研究*

超声造影剂的定向输运在超声医学成像领域有着极为重要的意义,而声辐射力作用是实现该过程的关键,相比于高斯声束,准高斯声束是无源亥姆霍兹方程的精确解,可以使用标准波分解法简化计算。因此,本文研究了准高斯声束对超声造影剂的声辐射力作用。文章首先分析了准高斯声束与高斯声束之间的相关性;随后通过数值计算求得了准高斯声束对超声造影剂模型的声辐射力函数与无量纲频率之间的关系;最后,本文研究了不同造影剂气泡情况下的声辐射力。研究结果表明:声辐射力函数随无量纲频率变化将在不同位置出现共振峰,不同的波束宽度值将改变辐射力强度,但不改变共振峰的位置。相关结果可为利用声辐射力定向输运超声造影剂至靶向位置提供理论参考。     

Performances of a Pristine Graphene–Microbubble Hybrid Construct as Dual Imaging Contrast Agent and Assessment of Its Biodistribution by Photoacoustic Imaging

Coupling near‐infrared (NIR) nanoscale absorbing materials with microbubbles (MBs) can generate a multifunctional dual imaging contrast agent. A new approach is presented for a hybrid photoacoustic/ultrasound contrast enhancer where pristine graphene is stably tethered to poly(vinyl alcohol) (PVA)‐based MBs. The main advantages of this approach are i) the preservation of optical and mechanical properties of intact graphene for an efficient photoacoustic (PA) enhancement and ii) the echogenicity and biocompatibility due to the robust anchoring of graphene to the bioinert PVA shell. PVA MBs provide ideal platforms for drug loading and ligand tethering for specific tumor targeting. One of the crucial goals toward this direction is optimizing this system in terms of balance between favorable acoustic/photoacoustic properties, immune shielding, and cytotoxicity. Such a combination strongly depends on the bridging moieties between graphene and the microbubble surface and can be easily tuned by PEGylation. The optimized graphene PVA MBs as contrast agent provide an efficient enhancement in vivo both in ultrasound and photoacoustic modes. The spectrally separable absorbance profile allows to a first demonstration of performing real‐time in vivo multiplexed photoacoustic imaging of graphene PVA MBs, and assessment of their full body biodistribution using a Vevo LAZR‐X photoacoustic imaging system.     

超声分子成像进展

超声分子成像在超声医学成像的基础上,利用靶向超声造影剂为分子探针,以可视化和定量获取活体组织细胞的分子信息为目标的影像术。它不用进行手术活检,不仅可以给出病灶的空间信息,而且能确定它的性质,进行针对性的治疗和对疗效进行评估。本文对现有的核医学分子成像,磁共振分子成像,光学分子成像和光声分子成像技术作了简单介绍,着重讨论了超声分子成像技术和应用的进展。     

Assessment of angiogenesis: implications for ultrasound imaging

In this paper, the fundamentals of tumor angiogenesis and the implications for ultrasound imaging will be described. Twenty-eight athymic nude mice were implanted with the human melanoma cell lines DB-1 or MW-9 (14 mice/group). Ultrasound contrast agents were injected in the tail veins. Power Doppler and pulse inversion harmonic imaging (PI-HI) was performed (in real time and intermittently). Ultrasound results were compared to immunohistochemical stains for endothelial cells (CD31), vascular endothelial growth factor (VEGF), and cyclooxygenase-2 (COX-2). Linear regression analysis indicated statistically significant correlations between percent area stained with COX-2 and with VEGF relative to power Doppler (p<0.05) and intermittent PI-HI (p<0.05) measures of tumor neovascularity in the MW-9 and the DB-1 mice, respectively. Preliminary results from a human trial of the anti-angiogenic drug Angiostatin (Entremed, Rockville, MD) showed tumor volumes increased in two patients, while the vascularity remained virtually unchanged. Conversely, in three patients with diminished tumor volumes vascularity increased by 38%. In conclusion, contrast enhanced ultrasound imaging of tumor neovascularity may provide noninvasive markers of angiogenesis and may become a useful tool for monitoring anti-angiogenic therapies in vivo.     

Delivery by shock waves of active principle embedded in gelatin-based capsules

PURPOSE: Delivering a drug close to the targeted cells improves its benefit versus risk ratio. A possible method for local drug delivery is to encapsulate the drug into solid microscopic carriers and to release it by ultrasound. The objective of this work was to use shock waves for delivering a molecule loaded in polymeric microcapsules. MATERIAL AND METHODS: Ethyl benzoate (EBZ) was encapsulated in spherical gelatin shells by complex coacervation. A piezocomposite shock wave generator (120 mm in diameter, focused at 97 mm, pulse length 1.4 micros) was used for sonicating the capsules and delivering the molecule. Shock parameters (acoustic pressure, number of shocks and shock repetition frequency) were varied in order to measure their influence on EBZ release. A cavitation-inhibitor liquid (Ablasonic) was then used to evaluate the role of cavitation in the capsule disruption. RESULTS: The measurements showed that the mean quantity of released EBZ was proportional to the acoustic pressure of the shock wave (r2 > 0.99), and increased with the number of applied shocks. Up to 88% of encapsulated EBZ could be released within 4 min only (240 shocks, 1 Hz). However, the quantity of released EBZ dropped at high shock rates (above 2Hz). Ultrasound imaging sequences showed that cavitation clouds might form, at high shock rates, along the acoustic axis making the exposure inefficient. Measurements done in Ablasonic showed that cavitation plays a major role in microcapsules disruption. CONCLUSIONS: In this study, we designed polymeric capsules that can be disrupted by shock waves. This type of microcapsule is theoretically a suitable vehicle for carrying hydrophobic drugs. Following these positive results, encapsulation of drugs is considered for further medical applications.     

Multifunctional Small Molecule Fluorophore for Long‐Duration Tumor‐Targeted Monitoring and Dual Modal Phototherapy

In this work, a specific tumor‐targeted small molecular fluorophore for synchronous long‐duration cancer imaging, photodynamic therapy, and photothermal therapy is synthesized. This novel fluorophore exhibits specific targeting ability in certain tumors (U87MG, MDA‐MB‐231, A549, etc.) based on its inherent structure and efficiently generates local hyperthermia and reactive oxygen species simultaneously for imaging‐guided precise cancer therapy combining the photothermic and photodynamic effects under laser irradiation. Meanwhile, compared to traditional near infrared fluorophore, this novel fluorophore with significantly enhanced stability against photobleaching can prolong the time of tumor imaging and improve the phototherapy efficiency. This work presents a potential strategy to develop small‐molecule‐based cancer theranostic agents for simultaneous cancer targeting, imaging, and therapy.     

Characterization of bioactive RGD peptide immobilized onto poly(acrylic acid) thin films by plasma polymerization

Plasma surface modification can be used to improve the surface properties of commercial pure Ti by creating functional groups to produce bioactive materials with different surface topography. In this study, a titanium surface was modified with acrylic acid (AA) using a plasma treatment and immobilized with bioactive arginine-glycine-aspartic acid (RGD) peptide, which may accelerate the tissue integration of bone implants. Both terminals containing the -NH₂ of RGD peptide sequence and -COOH of poly(acrylic acid) (PAA) thin film were combined with a covalent bond in the presence of 1-ethyl-3-3-dimethylaminopropyl carbodiimide (EDC). The chemical structure and morphology of AA film and RGD immobilized surface were investigated by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR), atomic force microscopy (AFM), and scanning electron microscopy (SEM). All chemical analysis showed full coverage of the Ti substrate with the PAA thin film containing COOH groups and the RGD peptide. The MC3T3-E1 cells were cultured on each specimen, and the cell alkaline phosphatase (ALP) activity were examined. The surface-immobilized RGD peptide has a significantly increased the ALP activity of MC3T3-E1 cells. These results suggest that the RGD peptide immobilization on the titanium surface has an effect on osteoblastic differentiation of MC3T3-E1 cells and potential use in osteo-conductive bone implants.     

Pulse sequences for uniform perfluorocarbon droplet vaporization and ultrasound imaging

Phase-change contrast agents (PCCAs) consist of liquid perfluorocarbon droplets that can be vaporized into gas-filled microbubbles by pulsed ultrasound waves at diagnostic pressures and frequencies. These activatable contrast agents provide benefits of longer circulating times and smaller sizes relative to conventional microbubble contrast agents. However, optimizing ultrasound-induced activation of these agents requires coordinated pulse sequences not found on current clinical systems, in order to both initiate droplet vaporization and image the resulting microbubble population. Specifically, the activation process must provide a spatially uniform distribution of microbubbles and needs to occur quickly enough to image the vaporized agents before they migrate out of the imaging field of view. The development and evaluation of protocols for PCCA-enhanced ultrasound imaging using a commercial array transducer are described. The developed pulse sequences consist of three states: (1) initial imaging at sub-activation pressures, (2) activating droplets within a selected region of interest, and (3) imaging the resulting microbubbles. Bubble clouds produced by the vaporization of decafluorobutane and octafluoropropane droplets were characterized as a function of focused pulse parameters and acoustic field location. Pulse sequences were designed to manipulate the geometries of discrete microbubble clouds using electronic steering, and cloud spacing was tailored to build a uniform vaporization field. The complete pulse sequence was demonstrated in the water bath and then in vivo in a rodent kidney. The resulting contrast provided a significant increase (>15 dB) in signal intensity.     

Targeting Upconversion Nanoprobes for Magnetic Resonance Imaging of Early Colon Cancer

Colon cancer (CC) is one of the most common intestinal malignancies and is difficult to detect in its early stage by magnetic resonance imaging (MRI) with currently used contrast agents (CAs). The development of targeted CAs contributes to the early diagnosis of CC and thereby enables early intervention and timely therapy. Considering the outstanding performance of upconversion nanoprobes (UCNPs) in high‐performance MR and fluorescence imaging, a new type of nanoprobes with considerably enhanced imaging performance is developed herein. Carcinoembryonic antigen (CEA) antibody is conjugated onto the surface of UCNPs to achieve the targeted imaging of early CC tumors, which overexpress CEA. Both toxicity tests and histological/hematological examinations demonstrate the excellent biocompatibility of these CC‐targeting nanoprobes, which possess great potential for clinical application in the early diagnosis of CC.     

Sonodynamic therapy

Recently, there have been numerous reports on the application of non-thermal ultrasound energy for treating various diseases in combination with drugs. Furthermore, the introduction of microbubbles and nanobubbles as carriers/enhancers of drugs has added a whole new dimension to therapeutic ultrasound. Non-thermal mechanisms for effects seen include various forms of energy due to cavitation, acoustic streaming, micro jets and radiation force which increases possibilities for targeting tissue with drugs, enhancing drug effectiveness or even chemically activating certain materials. Examples such as enhancement of thrombolytic agents by ultrasound have proven to be beneficial for acute stroke patients and peripheral arterial occlusions. Non-invasive low intensity focused ultrasound in conjunction with anti-cancer drugs may help to reduce tumor size and lessen recurrence while reducing severe drug side effects. Chemical activation of drugs by ultrasound energy for treatment of atherosclerosis and tumors is another new field recently termed as “Sonodynamic therapy”. Lastly, advances in molecular imaging have aroused great expectations in applying ultrasound for both diagnosis and therapy simultaneously. Microbubbles or nanobubbles targeted at the molecular level will allow medical doctors to make a final diagnosis of a disease using ultrasound imaging and then immediately proceed to a therapeutic ultrasound treatment.     

Destruction of polylactic acid microcapsules under ultrasound irradiation

Hollow microcapsules have been considered for potential applications as drug or gene carriers. This paper describes an investigation into the mechanical properties of microcapsules with a biocompatible polylactic acid (PLA) shell that can be destroyed using ultrasound irradiation. The microcapsules had a radius of 1 to 25 μm and a shell thickness of 100 nm to 3 μm, and their response to ultrasound pulses with a center frequency of 700 kHz to 2 MHz was investigated. It was found that approximately 50% of capsules with a radius of 20 μm were destroyed using pulses with a pressure amplitude of 50 kPa and a frequency of 700 kHz, which is close to the resonance frequency of the capsules.     

Quo vadis medical ultrasound?

The last three decades of development in diagnostic ultrasound imaging and technology are briefly reviewed and the impact of the crucial link between the two apparently independent research efforts, which eventually facilitated implementation of harmonic imaging modality is explored. These two efforts included the experiments with piezoelectric PVDF polymer material and studies of the interaction between ultrasound energy and biological tissue. Harmonic imaging and its subsequent improvements revolutionized the diagnostic power of clinical ultrasound and brought along images of unparalleled resolution, close to that of magnetic resonance imaging (MRI) quality. The nonlinear propagation effects and their implications for both diagnostic and therapeutic applications of ultrasound are also briefly addressed. In diagnostic applications, the impact of these effects on image resolution and tissue characterization is reviewed; in therapeutic applications, the influence of nonlinear propagation effects on highly localized tissue ablation and cauterization is examined. Next, the most likely developments and future trends in clinical ultrasound technology, including 3D and 4D imaging, distant palpation, image enhancement using contrast agents, monitoring, and merger of diagnostic and therapeutic applications by e.g. introducing ultrasonically controlled targeted drug delivery are reviewed. Finally, a possible competition from other imaging modalities is discussed.     

Synthesis and bio-applications of targeted magnetic-fluorescent composite nanoparticles

Magnetic-fluorescent nanoparticles have a tremendous potential in biology. As the benefits of these materials gained recognition, increasing attention has been given to the conjugation of magnetic-fluorescent nanoparticles with targeting ligands. The magnetic and fluorescent properties of nanoparticles offer several functionalities, including imaging, separation, and visualization, while the presence of a targeting ligand allows for selective cell and tissue targeting. In this review, methods for the synthesis of targeted magnetic-fluorescent nanoparticles are explored, and recent applications of these nanocomposites to the detection and separation of biomolecules, fluorescent and magnetic resonance imaging, and cancer diagnosis and treatment will be summarized. As these materials are further optimized, targeted magnetic-fluorescent nanoparticles hold great promise for the diagnosis and treatment of some diseases.     

Time evolution of enhanced ultrasonic reflection using a fibrin-targeted nanoparticulate contrast agent

Complex molecular signaling heralds the early stages of pathologies such as angiogenesis, inflammation, unstable atherosclerotic plaques, and areas of remote thrombi. In previous studies, acoustic enhancement of blood clot morphology was demonstrated with the use of a nongaseous, fibrin-targeted acoustic nanoparticle emulsion delivered to areas of thrombosis both in vitro and in vivo. In this study, a system was designed and constructed that allows visualization of the evolution of acoustic contrast enhancement. To evaluate the system, two targets were examined: avidin-complexed nitrocellulose membrane and human plasma clots. The time evolution of enhancement was visualized in 10-min increments for 1 h. A monotonic increase was observed in ultrasonic reflection enhancement from specially treated nitrocellulose membranes for targeted emulsions containing perfluorooctylbromide (1.30+/-0.3 dB) and for perfluorooctane (2.64+/-0.5 dB) within the first 60 min of imaging. In comparison, the inherently nonechogenic plasma clots showed a substantial increase of 12.0+/-0.9 dB when targeted with a perfluoro-octane emulsion. This study demonstrates the concept of molecular imaging and provides the first quantifiable time-evolution report of the binding of a site-targeted ultrasonic contrast agent. Moreover, with the incorporation of specific drug treatments into the nanoparticulate contrast agent, ultrasonic molecular imaging may yield reliable detection and quantification of nascent pathologies and facilitate targeted drug therapy.