Annexin A5-conjugated quantum dots with a paramagnetic lipidic coating for the multimodal detection of apoptotic cells

Apoptosis, or programmed cell death, plays an important role in the etiology of a variety of diseases, including cancer. Visualization of apoptosis would allow both early detection of therapy efficiency and evaluation of disease progression. To that aim we developed a novel annexin A5-conjugated bimodal nanoparticle. The nanoparticle is composed of a quantum dot that is encapsulated in a paramagnetic micelle to enable its use both for optical imaging and MRI. Multiple recombinant human annexin A5 protein molecules were covalently coupled to the nanoparticle for targeting. In this study the specificity of the annexin A5-conjugated nanoparticles for apoptotic cells was demonstrated both with fluorescence microscopy and MRI, which confirms its potential for the detection of apoptosis with both imaging modalities in vivo.     

Gadolinium-rhodamine nanoparticles for cell labeling and tracking via magnetic resonance and optical imaging

A novel dual-labeled nanoparticle for use in labeling and tracking cells in vivo is described. We report the construction and characterization of these gadolinium-rhodamine nanoparticles. These particles are constructed from lipid monomers with diacetylene bonds that are sonicated and photolyzed to form polymerized nanoparticles. Cells are efficiently labeled with these nanoparticles. We have inoculated labeled tumor cells subcutaneouosly into the flanks of C3H mice and have been able to image these labeled tumor cells via MRI and optical imaging. Furthermore, the labeled tumor cells can be visualized via fluorescent microscopy after tissue biopsy. Our results suggest that these nanoparticles could be used to track cells in vivo. This basic platform can be modified with different fluorophores and targeting agents for studying metastisic cell, stem cell, and immune cell trafficking among other applications.     

Combined bioluminescence and fluorescence imaging visualizing orthotopic lung adenocarcinoma xenograft in vivo

Lung adenocarcinoma is the most common type of lung cancer. A close monitor of in vivo tumor development may help to better understand the pathogenesis and pathological processes of this disease. A bimodal imaging strategy has been developed, which is a very important tool to investigate the growth and metastasis of lung adenocarcinoma. In the present study, we used a combined labeling strategy in p53RE-luc-A549 cells via transfecting the reporter gene EGFP. In order to unambiguously identify the growth and metastasis of transfected A549 tumor cells, we established and observed subcutaneous and orthotopic xenografts in nude mice by in vivo bioluminescence and fluorescence imaging, which was verified by our post-mortem histological analysis. In vivo bioluminescence signal was observed for the progression of both subcutaneous and orthotopic xenografts in EGFP-p53RE-luc-A549 cells; in vivo fluorescence was only observed for the growth of subcutaneous xenograft of EGFP-p53RE-luc-A549 cells. Moreover, EGFP-p53RE-luc-A549 cells allow for the improved identification of implanted cells within host tissue during histological analysis. In conclusion, we presented a combined labeling strategy for bimodal A549 cell imaging which leads to improved detection of cellular grafts.     

Dual probe with fluorescent and magnetic properties for imaging solid tumor xenografts

A dual probe with fluorescent and magnetic reporter groups was constructed by linkage of the near-infrared (NIR) fluorescent transferrin conjugate (Tf(NIR)) on the surface of contrast agent-encapsulated cationic liposome (Lip-CA). This probe was used for magnetic resonance imaging (MRI) and optical imaging of MDA-MB-231-luc breast cancer cells grown as a monolayer in vitro and as solid tumor xenografts in nude mice. Confocal microscopy, optical imaging, and MRI showed a dramatic increase of in vitro cellular uptake of the fluorescent and magnetic reporter groups from the probe compared with the uptake of contrast agent or Lip-CA alone. Pretreatment with transferrin (Tf) blocked uptake of the probe reporters, indicating the importance and specificity of the Tf moiety for targeting. Intravenous administration of the dual probe to nude mice significantly enhanced the tumor contrast in MRI, and preferential accumulation of the fluorescent signal was clearly seen in NIR-based optical images. More interestingly, the contrast enhancement in MRI showed a heterogeneous pattern within tumors, which reflected the tumor's morphologic heterogeneity. These results indicate that the newly developed dual probe enhances the tumor image contrast and is superior to contrast agent alone for identifying the tumor pathologic features on the basis of MRI but also is suitable for NIR-based optical imaging.     

In vivo visualization of murine melanoma cells B16-derived exosomes through magnetic resonance imaging

BackgroundNumerous studies demonstrated that exosomes play a powerful role in mediating intercellular communication to induce a pro-tumoral environment to promote tumor progression, including pre-metastatic niche formation and metastasis. Noninvasive imaging could determine the in vivo kinetics of exosomes in real time to provide better understanding of the mechanisms of the tumor formation, progression and metastasis. Magnetic resonance imaging (MRI) is an ideal technique which provides excellent anatomical resolution, intrinsic soft tissue contrast, unlimited penetration depth and no radiation exposure.MethodsA fusion protein composed of ferritin heavy chain (FTH1) and lactadherin was designed for visualizing exosomes through MRI. FTH1 was served as MRI reporter protein and lactadherin is a membrane-associated protein that is distributed on exosome surface. The characterizations of labeled exosomes were validated through transmission electron microscopy, western blot, nanoparticle tracking analysis and finally visualized in vitro and in vivo through MRI.ResultsMR imaging showed that the labeled exosomes are able to be visualized in vitro and in vivo. Verification of the characterizations of exosomes observed no significant difference between labeled and unlabeled exosomes.ConclusionThe proposed FTH1 labeling method was useful for visualizing exosomes through MRI.General significanceThe present study first reported a novel self-label method for imaging labeled exosomes of tumor cells in vivo through MR with cell endogenous MRI reporter protein. It may be further used as a tool to enhance understanding the role of exosomes in various pathophysiological conditions.     

Liposomes to Target Peripheral Neurons and Schwann Cells

While a wealth of literature for tissue-specific liposomes is emerging, optimal formulations to target the cells of the peripheral nervous system (PNS) are lacking. In this study, we asked whether a novel formulation of phospholipid-based liposomes could be optimized for preferential uptake by microvascular endothelia, peripheral neurons and Schwann cells. Here, we report a unique formulation consisting of a phospholipid, a polymer surfactant and cholesterol that result in enhanced uptake by targeted cells. Using fluorescently labeled liposomes, we followed particle internalization and trafficking through a distinct route from dextran and escape from degradative compartments, such as lysosomes. In cultures of non-myelinating Schwann cells, liposomes associate with the lipid raft marker Cholera toxin, and their internalization is inhibited by disruption of lipid rafts or actin polymerization. In contrast, pharmacological inhibition of clathrin-mediated endocytosis does not significantly impact liposome entry. To evaluate the efficacy of liposome targeting in tissues, we utilized myelinating explant cultures of dorsal root ganglia and isolated diaphragm preparations, both of which contain peripheral neurons and myelinating Schwann cells. In these models, we detected preferential liposome uptake into neurons and glial cells in comparison to surrounding muscle tissue. Furthermore, in vivo liposome administration by intramuscular or intravenous injection confirmed that the particles were delivered to myelinated peripheral nerves. Within the CNS, we detected the liposomes in choroid epithelium, but not in myelinated white matter regions or in brain parenchyma. The described nanoparticles represent a novel neurophilic delivery vehicle for targeting small therapeutic compounds, biological molecules, or imaging reagents into peripheral neurons and Schwann cells, and provide a major advancement toward developing effective therapies for peripheral neuropathies.     

Gadolinium-Loaded Polychelating Polymer-Containing Cancer Cell-Specific Immunoliposomes

Liposome loading with Gd via the membrane-incorporated polychelating amphiphilic polymers (PAPs) significantly increases the Gd content and relaxivity (T1 parameter) of PEGylated liposomes, which can be used as contrast agents for magnetic resonance imaging (MRI). Here, we demonstrate that such Gd-containing liposomes can be additionally modified with the monoclonal anticancer antibody 2C5 (mAb 2C5) possessing the nucleosome(NS)-restricted specificity via the PEG spacer. Liposome-bound antibody preserves its specific activity (ELISA) and such Gd-loaded PEGylated 2C5-immunoliposomes specifically recognize various cancer cells in vitro and target an increased amount of Gd to their surface compared to antibody-free Gd-liposomes or Gd-liposomes modified with tumor nonspecific antibody. Gd-loaded cancer cell-targeted immunoliposomes may represent promising agents for enhanced tumor MRI.     

Gadolinium-loaded polychelating polymer-containing cancer cell-specific immunoliposomes

Liposome loading with Gd via the membrane-incorporated polychelating amphiphilic polymers (PAPs) significantly increases the Gd content and relaxivity (T1 parameter) of PEGylated liposomes, which can be used as contrast agents for magnetic resonance imaging (MRI). Here, we demonstrate that such Gd-containing liposomes can be additionally modified with the monoclonal anticancer antibody 2C5 (mAb 2C5) possessing the nucleosome(NS)-restricted specificity via the PEG spacer. Liposome-bound antibody preserves its specific activity (ELISA) and such Gd-loaded PEGylated 2C5-immunoliposomes specifically recognize various cancer cells in vitro and target an increased amount of Gd to their surface compared to antibody-free Gd-liposomes or Gd-liposomes modified with tumor nonspecific antibody. Gd-loaded cancer cell-targeted immunoliposomes may represent promising agents for enhanced tumor MRI.     

Differential conjugation of tat peptide to superparamagnetic nanoparticles and its effect on cellular uptake

Surface modification of superparamagnetic contrast agents with HIV-1 tat peptide has emerged as a promising means for intracellular magnetic labeling and noninvasive tracking of a large number of cell types with MRI. To achieve efficient intracellular delivery of the nanoparticles, we investigated the effect on cellular uptake of superparamagnetic iron oxide particles by varying the number of attached tat peptides. First, we report here a modified P2T method in measuring the numbers of surface attachments per particle through disulfide linkage. The method was shown to have desirable simplicity and reproducibility. With the P2T method as a tool, conjugates with progressively higher ratios of peptide-to-particle were synthesized. We were able to demonstrate that higher numbers of tat peptide facilitate the cellular uptake of iron oxide nanoparticles in a nonlinear fashion. Cells labeled with these optimized preparations were readily detectable by MR imaging. The increase in sensitivity could allow in vivo tracking of 100-fold lower cell concentration than currently described.     

Labeling of immune cells for in vivo imaging using magnetofluorescent nanoparticles

Observation of immune and stem cells in their native microenvironments requires the development of imaging agents to allow their in vivo tracking. We describe here the synthesis of magnetofluorescent nanoparticles for cell labeling in vitro and for multimodality imaging of administered cells in vivo. MION-47, a prototype monocrystalline iron oxide nanoparticle, was first converted to an intermediate bearing a fluorochrome and amine groups, then reacted with either HIV-Tat peptide or protamine to yield a nanoparticle with membrane-translocating properties. We describe how to assess optimal cell labeling with tests of cell phenotype and function. Synthesis of magnetofluorescent nanoparticles and cell-labeling optimization can be realized in 48 h, whereas nanoparticle uptakes and retention studies may generally take up to 120 h. Labeled cells can be detected by magnetic resonance imaging, fluorescence reflectance imaging, fluorescence-mediated tomography, confocal microscopy and flow cytometry, and can be purified based on their fluorescent or magnetic properties. The present protocol focuses on T-cell labeling but can be used for labeling a variety of circulating cells.     

A tumor-targeted nanodelivery system to improve early MRI detection of cancer

The development of improvements in magnetic resonance imaging (MRI) that would enhance sensitivity, leading to earlier detection of cancer and visualization of metastatic disease, is an area of intense exploration. We have devised a tumor-targeting, liposomal nanodelivery platform for use in gene medicine. This systemically administered nanocomplex has been shown to specifically and efficiently deliver both genes and oligonucleotides to primary and metastatic tumor cells, resulting in significant tumor growth inhibition and even tumor regression. Here we examine the effect on MRI of incorporating conventional MRI contrast agent Magnevist into our anti-transferrin receptor single-chain antibody (TfRscFv) liposomal complex. Both in vitro and in an in vivo orthotopic mouse model of pancreatic cancer, we show increased resolution and image intensity with the complexed Magnevist. Using advanced microscopy techniques (scanning electron microscopy and scanning probe microscopy), we also established that the Magnevist is in fact encapsulated by the liposome in the complex and that the complex still retains its nanodimensional size. These results demonstrate that this TfRscFv-liposome-Magnevist nanocomplex has the potential to become a useful tool in early cancer detection.     

Self-assembling nanocomplexes by combining ferumoxytol, heparin and protamine for cell tracking by magnetic resonance imaging

We report on a new straightforward magnetic cell-labeling approach that combines three US Food and Drug Administration (FDA)-approved drugs--ferumoxytol, heparin and protamine--in serum-free medium to form self-assembling nanocomplexes that effectively label cells for in vivo magnetic resonance imaging (MRI). We observed that the ferumoxytol-heparin-protamine (HPF) nanocomplexes were stable in serum-free cell culture medium. HPF nanocomplexes show a threefold increase in T2 relaxivity compared to ferumoxytol. Electron microscopy showed internalized HPF in endosomes, which we confirmed by Prussian blue staining of labeled cells. There was no long-term effect or toxicity on cellular physiology or function of HPF-labeled hematopoietic stem cells, bone marrow stromal cells, neural stem cells or T cells when compared to controls. In vivo MRI detected 1,000 HPF-labeled cells implanted in rat brains. This HPF labeling method should facilitate the monitoring by MRI of infused or implanted cells in clinical trials.     

转铁蛋白导向脂质体核磁造影剂纳米粒子（TfNIR-LipNBD-Magnevist）——一种肿瘤靶向磁共振造影剂

造影剂辅助的核磁共振成像是目前肿瘤诊断的最吁方法之一。但是由于核磁共振成像内在的低灵敏性以及造影剂的非特异性,导致肿瘤早期诊断较为困难。文章将一种新的肿瘤靶向核磁造影剂纳米粒子应用于早期肿瘤的影像诊断。这种新的肿瘤靶向核磁造影剂纳米粒子由配体转铁蛋白（Tf）、纳米水平的正电脂质体（Lip）载体和临床常用的造影剂Magnevist（Tf^NIR-Lip^NBD-Magnevist）三部分构成。另外转铁蛋白和脂质体粒子上,亦标记了荧光物质用于确定转铁蛋白一脂质体一造影剂纳米粒子的靶向性,以及肿瘤的光学影像诊断。在体外实验中,利用激光共聚焦显微镜和光学影像证明了靶向纳米粒子介导的细胞内吞和特异性结合。在裸鼠肿瘤模型中,造影剂纳米粒子Tf^NIR-Lip^NBD-Magnevist经尾静脉注入后,显著增强了肿瘤内信号与周围组织的对比度。由造影剂纳米粒子介导的肿瘤内信号显著强于单独Magnevist辅助的肿瘤内信号。同时,利用光学影像方法,在肿瘤内检测到特异的荧光信号。其结果进一步支持了转铁蛋白一脂质体一造影利（Tf^NIR-Lip^NBD-Magnevist）纳米粒子的靶向性和肿瘤影像诊断的有效性。     

Magnetic targeting of rhodamine-labeled superparamagnetic liposomes to solid tumors: in vivo tracking by fibered confocal fluorescence microscopy

Polyethylene glycol (PEG)ylated and rhodamine-labeled liposomes loaded with maghemite nanocrystals provide a novel nanoscaled hybrid system for magnetic targeting to solid tumors in possible combination with double in vivo imaging by fluorescence microscopy and magnetic resonance imaging (MRI). Human prostate adenocarcinoma tumors implanted in mice were used as a system model. A magnetic field gradient was produced at the tumor level by external apposition of a magnet. Noninvasive fibered confocal fluorescence microscopy was successfully used to track the liposomes in vivo within organs and tumor blood vessels. Active targeting to the magnet-exposed tumors was clearly shown, in agreement with previous MRI studies. The liposomes were driven and accumulated within the microvasculature through a process that preserved vesicle structure and content.     

Human aortic endothelial cell labeling with positive contrast gadolinium oxide nanoparticles for cellular magnetic resonance imaging at 7 Tesla

Positive T? contrast using gadolinium (Gd) contrast agents can potentially improve detection of labeled cells on magnetic resonance imaging (MRI). Recently, gadolinium oxide (Gd?O?) nanoparticles have shown promise as a sensitive T? agent for cell labeling at clinical field strengths compared to conventional Gd chelates. The objective of this study was to investigate Gado CELLTrack, a commercially available Gd?O? nanoparticle, for cell labeling and MRI at 7 T. Relaxivity measurements yielded r1 = 4.7 s?1 mM?1 and r?/r? = 6.2. Human aortic endothelial cells were labeled with Gd?O? at various concentrations and underwent MRI from 1 to 7 days postlabeling. The magnetic resonance relaxation times T? and T? of labeled cell pellets were measured. Cellular contrast agent uptake was quantified by inductively coupled plasma-atomic emission spectroscopy, which showed very high uptake compared to conventional Gd compounds. MRI demonstrated significant positive T? contrast and stable labeling on cells. Enhancement was optimal at low Gd concentrations, attained in the 0.02 to 0.1 mM incubation concentration range (corresponding cell uptake was 7.26 to 34.1 pg Gd/cell). Cell viability and proliferation were unaffected at the concentrations tested and up to at least 3 days postlabeling. Gd?O? is a promising sensitive and stable positive contrast agent for cellular MRI at 7 T.     

Hematopoietic stem cell (CD34+) uptake of superparamagnetic iron oxide is enhanced by but not dependent on a transfection agent

Background aimsTracking the fate of cells after infusion would be a valuable asset for many stem cell therapies, but very few (cell) labels are approved for human therapeutic use. Superparamagnetic iron oxide particles (SPIO) can be internalized into stem cells in vitro to allow real-time tracking with gradient echo magnetic resonance imaging, but SPIO are approved for (diagnostic) imaging and not for (therapeutic) cell labeling in vivo. In this study, we investigated the possibility of labeling stem cells with an SPIO approved for patient use, albeit in a novel manner by enhancing uptake with the use of a transfection agent, also approved for patient use. Although there are many reports of hematopoietic stem cells being labeled with SPIO, there is some controversy regarding the efficiency of this and whether undifferentiated CD34+ progenitor (stem) cells are able to take up iron in the absence of a transfection agent to enhance the process.MethodsHuman CD34+ cells were treated in vitro as follows: incubation with (i) medium only (control), (ii) ferumoxide (Endorem) and (iii) ferumoxide (Endorem) plus exposure to a transfection agent (protamine sulfate). Cells were incubated for 2, 4 and 24 hours and assessed for viability, differentiation capacity and visualized in vitro with 3-T magnetic resonance imaging. The cells were also analyzed by means of flow cytometry and morphology examined by electron microscopy.ResultsCD34+ hematopoietic progenitor cells can internalize ferumoxide (Endorem) independently of a transfection agent. However, uptake of ferumoxide is enhanced after exposure to protamine sulfate. Iron labeling of CD34+ cells in this manner does not affect cell viability and does not appear to affect the potential of the cells to grow in culture. Iron-labeled CD34+ cells can be visualized in vitro on 3-T magnetic resonance image scanning.ConclusionsEndorem and protamine sulfate can be combined to promote iron oxide nanoparticle uptake by CD34+ cells, and this methodology can potentially be used to track the fate of cells in a clinical trial setting because both compounds are (separately) approved for clinical use.     

Tracking metastatic tumor cell extravasation with quantum dot nanocrystals and fluorescence emission-scanning microscopy

Metastasis is an impediment to the development of effective cancer therapies. Our understanding of metastasis is limited by our inability to follow this process in vivo. Fluorescence microscopy offers the potential to follow cells at high resolution in living animals. Semiconductor nanocrystals, quantum dots (QDs), offer considerable advantages over organic fluorophores for this purpose. We used QDs and emission spectrum scanning multiphoton microscopy to develop a means to study extravasation in vivo. Although QD labeling shows no deleterious effects on cultured cells, concern over their potential toxicity in vivo has caused resistance toward their application to such studies. To test if effects of QD labeling emerge in vivo, tumor cells labeled with QDs were intravenously injected into mice and followed as they extravasated into lung tissue. The behavior of QD-labeled tumor cells in vivo was indistinguishable from that of unlabeled cells. QDs and spectral imaging allowed the simultaneous identification of five different populations of cells using multiphoton laser excitation. Besides establishing the safety of QDs for in vivo studies, our approach permits the study of multicellular interactions in vivo.     

In Vivo Imaging of Transplanted Islets Labeled with a Novel Cationic Nanoparticle

To monitor pancreatic islet transplantation efficiency, reliable noninvasive imaging methods, such as magnetic resonance imaging (MRI) are needed. Although an efficient uptake of MRI contrast agent is required for islet cell labeling, commercially-available magnetic nanoparticles are not efficiently transduced into cells. We herein report the in vivo detection of transplanted islets labeled with a novel cationic nanoparticle that allowed for noninvasive monitoring of islet grafts in diabetic mice in real time. The positively-charged nanoparticles were transduced into a β-cell line, MIN6 cells, and into isolated islets for 1 hr. MRI showed a marked decrease in the signal intensity on T1- and T2-weighted images at the implantation site of the labeled MIN 6 cells or islets in the left kidneys of mice. These data suggest that the novel positively-charged nanoparticle could be useful to detect and monitor islet engraftment, which would greatly aid in the clinical management of islet transplant patients.     

转铁蛋白修饰脂质体的制备及其肝癌靶向性研究

目的：肿瘤的靶向治疗是当前研究的热点,肝肿瘤细胞表面有大量的转铁蛋白受体表达,而正常组织较少,因此本研究制备转铁蛋白（TF）修饰的脂质体（TFLPs）,并对其肝肿瘤靶向性进行研究。方法：采用薄膜分散法制备普通脂质体,考察其形态,粒径,电位。通过体外血清稳定性模拟脂质体进入体内后的稳定性。通过HepG2肿瘤细胞对TFLPs的摄取实验考查脂质体与肝癌细胞的亲和力。构建荷瘤裸鼠模型,考查TFLPs在荷瘤裸鼠体内的分布。结果：所制备的TFLPs平均粒径为108．8±9．5nm,Zeta电位为．1．80±0．73mV。学期稳定性试验结果显示,TFLPs在24h内具有良好的血清稳定性。体外细胞摄取实验表明,HepG2细胞对TFLPs的摄取效率是普通长循环脂质体（LPs）的3．4倍。荷瘤裸鼠肝组织和肿瘤组织切片结果显示,TFLPs比LPs具有更好的肿瘤靶向性。结论：该脂质体制备方法简单,与LPs相比,经转铁蛋白修饰可显著提高肿瘤细胞对脂质体的摄取,TFLPs是一种潜在高效的肝癌靶向给药系统。     

Zn(II)-bis(cyclen) complexes and the imaging of apoptosis/necrosis

In vivo cell-death imaging is still a challenging issue. Until now, only (99m)Tc-labeled HYNIC-rh-annexin A5 has been extensively studied in clinical trials. In the ongoing search for an alternative imaging agent, we synthesized a series of fluorescent zinc-cyclen complexes as annexin A5 mimics and studied structural variations on the uptake behavior of cells undergoing apoptosis/necrosis. The number of cyclen chelators was varied and the spacer separating cyclen from the central scaffold was modified. Five zinc-cyclen complexes were labeled with fluorescein for flow cytometric studies and one was labeled with (18)F for in vivo applications. Jurkat cells were treated with staurosporine to induce apoptosis/necrosis, incubated with the fluorescein-labeled zinc complexes and analyzed them by flow cytometry. Fluorescent annexin A5 and propidium iodide were applied as reference dyes. Flow cytometry revealed greater accumulation of zinc-cyclen complexes in staurosporine treated cells. The uptake was contingent on the presence of zinc and the fluorescence intensity was dependent on the number of zinc-cyclen groups. Confocal laser scanning microscopy showed the {bis[Zn(cyclen)]}(4+) complex distributed throughout the cytosol different to annexin A5. Owing to the structural similarity of the bis-cyclen ligands with CXCR4 binding bis-cyclam derivatives the zinc-cyclen complex uptake was challenged with the meta derivative of AMD3100. Lack of uptake depletion in staurosporine treated cells ruled out measurable CXCR4 interaction. PET imaging using the (18)F labeled zinc-cyclen complex revealed significantly higher uptake in an irradiated Dunning R3327-AT1 prostate tumor as compared to the contralateral control tumor. PET imaging of a HelaMatu tumor model additionally showed an increased uptake after taxol treatment. It could be demonstrated that the fluorescent zinc-cyclen complexes offer potential as new agents for flow cytometry and microscopic imaging of cell death. In addition, the (18)F labeled analogue holds promise for in vivo applications providing informations about cell death after radiation therapy and cytostatic drug treatment.