Stents are recommended in patients with dysphagia caused by esophageal stricture, but an ideal stent does not currently exist. Thus, studies on new esophageal stents are necessary, and suitable animal models are desperately needed for these studies. The aim of this study was to establish a model of malignant esophageal stricture in rabbit for studies on stent innovation.
A total of 38 New Zealand white rabbits were used in this study. Using the endoscopic submucosal injection technique, VX2 fragments were inoculated into the submucosal layer of the rabbit thoracic esophagus, and an endoscopic follow-up was subsequently performed to observe the tumor development and progression. The self-expandable metal stents were randomly deployed in rabbits with severe esophageal stricture to investigate the safety and feasibility of the animal models for stenting.
An endoscopic implantation procedure for VX2 tumors was completed in 34/38 rabbits, and tumor development was confirmed in 30/34 animals. The success rate of the endoscopic implantation and tumor development were 89.4% (95% CI, 79.6% to 99.2%) and 88.2% (95% CI, 76.9% to 99.5%) respectively. During the endoscopic follow-up period, severe esophageal stricture occurred in 22/30 rabbits with a rate of 73.3% (95% CI, 57.5% to 89.1%), and 12/22 models received stent placement. During and after stent implantation, no severe stent-related complication or mortality occurred in the animal models. The rabbits that received stent placement survived longer than those without stent implantation (the mean survival time: 53.9 days versus 40.3 days, P = 0.016).
The endoscopic method is a safe and effective method for establishing a malignant esophagostenosis model in rabbits. This model can simulate the human body environment for stent deployment and is an excellent tool for the study of stent innovation for the treatment of esophageal cancer.
Esophageal squamous cell carcinoma; Animal model; Endoscopic surgical procedure; Rabbit; Stent therapy
Overexpression of vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) indicates poor prognosis for cancer patients in a variety of clinical studies. Our goal is to develop a tracer for positron emission tomography (PET) imaging of VEGFR expression using recombinant human VEGF121 with three lysine residues fused to the N-terminus (denoted as K3-VEGF121), which can facilitate radiolabeling without affecting its VEGFR binding affinity. K3-VEGF121 was conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and labeled with 61Cu (t1/2: 3.3 h; 62% β+). The IC50 value of NOTA-K3-VEGF121 for VEGFR-2 was comparable to K3-VEGF121 (1.50 and 0.65 nM, respectively) based on cell binding assay. 61Cu labeling was achieved with good yield (55 ± 10 %) and specific activity (4.2 GBq/mg). Serial PET imaging showed that the 4T1 tumor uptake of 61Cu-NOTA-K3-VEGF121 was 3.4 ± 0.5, 4.9 ± 1.0, 5.2 ± 1.0, and 4.8 ± 0.8 %ID/g (n = 4) at 0.5, 2, 4, and 8 h post-injection respectively, which was consistent with biodistribution data measured by gamma counting. Blocking experiments and ex vivo histology confirmed VEGFR specificity of 61Cu-NOTA-K3-VEGF121. Extrapolated human dosimetry calculation showed that liver was the organ with the highest radiation dose. The use of 61Cu as the radiolabel is desirable for small proteins like K3-VEGF121, which has much higher β+ branching ratio than the commonly used 64Cu (62% vs. 17%) thereby offering stronger signal intensity and lower tracer dose for PET imaging.
Vascular endothelial growth factor (VEGF); VEGF receptor (VEGFR); 61Cu; Positron emission tomography (PET); Tumor angiogenesis; Molecular imaging
The objective of this study was to characterize the in vitro and in vivo properties of the F(ab')2 fragment of TRC105, a human/murine chimeric IgG1 monoclonal antibody that binds with high avidity to human and murine CD105 (i.e. endoglin), and investigate its potential for positron emission tomography (PET) imaging of tumor angiogenesis after 61/64Cu-labeling. TRC105-F(ab')2 of high purity was produced by pepsin digestion of TRC105, which was confirmed by SDS-PAGE, HPLC analysis, and mass spectrometry. 61/64Cu-labeling of NOTA-TRC105-F(ab')2 (NOTA denotes 1,4,7-triazacyclononane-1,4,7-triacetic acid) was achieved with yields of > 75% (specific activity: ~115 GBq/μmol). PET imaging revealed rapid tumor uptake of 64Cu-NOTA TRC105-F(ab')2 in the 4T1 murine breast cancer model (5.8 ± 0.8, 7.6 ± 0.6, 5.6 ± 0.4, 5.0 ± 0.6, and 3.8 ± 0.7 %ID/g at 0.5, 3, 16, 24, and 48 h post-injection respectively; n = 4). Since tumor uptake peaked at 3 h post-injection, 61Cu-NOTA-TRC105-F(ab')2 also gave good tumor contrast at 3 and 8 h post-injection. CD105 specificity of the tracers was confirmed by blocking studies and histopathology. In conclusion, the use of a F(ab')2 fragment led to more rapid tumor uptake (which peaked at 3 h post-injection) than radiolabeled intact antibody (which often peaked after 24 h post-injection), which may allow for same day immunoPET imaging in future clinical studies.
Positron emission tomography (PET); Tumor angiogenesis; Antibody fragment; F(ab')2; CD105 (endoglin); Breast cancer; 61Cu; 64Cu
99mTc-N4-guanine (99mTc-N4amG) was synthesized and evaluated in this study. Cellular uptake and cellular fraction studies were performed to evaluate the cell penetrating ability. Biodistribution and planar imaging were conducted in breast tumor-bearing rats. Up to 17%ID uptake was observed in cellular uptake study with 40% of 99mTc-N4amG was accumulated in the nucleus. Biodistribution and scintigraphic imaging studies showed increased tumor/muscle count density ratios as a function of time. Our results demonstrate the feasibility of using 99mTc-N4amG in tumor specific imaging.
This study was undertaken to establish a rabbit esophageal tumor model for mimicking human esophageal squamous carcinoma (ESC) by endoscopic and surgical implantation of VX2 tumors.
Fragments of a VX2 tumour were endoscopically implanted in the submucosal layer of the thoracic esophagus of 32 New Zealand white rabbits, while 34 animals received surgical implantation into the muscular layer. Then, the animals were studied endoscopically and pathologically. The safety and efficiency of the two methods and the pathological features of the animal models were analyzed.
Both the endoscopic and the surgical method had a relatively high success rate of tumor implantation [93.7% (30/32) vs. 97.1% (33/34)] and tumor growth [86.7% (26/30) vs. 81.8% (27/33)], and the variation in the results was not statistically significant (P>0.05). Compared with those produced by the surgical method, the models produced by the endoscopic method had a higher rate of severe esophageal stricture [61.5% (16/26) vs. 29.6% (8/27)] and of intra-luminal tumor growth [73.1% (19/26) vs. 37.0% (10/27)], and had a lower rate of tumor invasion of adjacent organs [53.8% (14/26) vs. 81.5% (22/27)]; all of these results were statistically significant (P<0.05). However, the difference in the survival time and the rates of tumor regional/distant metastasis [38.5% (10/26) vs. 51.8% (14/27)] between the two methods were not statistically significant (P>0.05).
The endoscopic and surgical methods are both safe and effective for establishment of VX2 tumors in the rabbit esophagus. The models produced by the two methods have different pathologic features mimicking that of human ESC. We recommend the models for studies on surgical procedures and minimally invasive treatments.
In order to explore the high performance bivalent DNA-based vaccine against schistosomes, SjFABP and Sj26GST were selected and used to construct a vaccine. Two strategies were used to construct the bivalent DNA vaccine. In the first strategy, a plasmid encoding antigen in the secreted form was used, while in the other, a plasmid encoding a truncated form of SjFABP and Sj26GST targeted to the cell surface was used. Various parameters, including antibody and cytokine response, proliferation, histopathological examination, and characterization of T cell subsets were used to evaluate the type of immune response and the level of protection against challenge infection. Injection with secreted pIRES-sjFABP-sj26GST significantly increased the levels of antibody, splenocyte proliferation, and production of IFN-γ, compared with membrane-anchored groups. Analysis of splenic T cell subsets showed that the secreted vaccine significantly increased the percentage of CD3+CD4+ and CD3+CD8+ T cells. Liver immunopathology (size of liver granulomas) was significantly reduced in the secreted group compared with the membrane-anchored groups. Moreover, challenge experiments showed that the worm and egg burdens were significantly reduced in animals immunized with recombinant vaccines. Most importantly, secreted Sj26GST-SjFABP markedly enhanced protection, by reducing worm and egg burdens by 31.8% and 24.78%, respectively, while the membrane-anchored group decreased worm and egg burdens by 24.80% and 18.80%, respectively. Taken together, these findings suggest that the secretory vaccine is more promising than the membrane-anchored vaccine, and provides support for the development and application of this vaccine.
Metastatic breast cancer is incurable. The goal of this study was to develop a positron emission tomography (PET)/near-infrared fluorescent (NIRF) probe for imaging of CD105 expression in breast cancer experimental lung metastasis. TRC105, a chimeric anti-CD105 antibody, was dual-labeled with a NIRF dye (IRDye 800CW) and 89Zr to yield 89Zr-Df-TRC105-800CW. Luciferase-transfected 4T1 murine breast cancer cells were injected intravenously into female mice to establish the tumor model. Bioluminescence imaging (BLI) was carried out to non-invasively monitor the lung tumor burden. PET imaging revealed that 4T1 lung tumor uptake of 89Zr-Df-TRC105-800CW was 8.7±1.4, 10.9±0.5, and 9.7±1.1 %ID/g at 4, 24, and 48 h post-injection (n = 4), with excellent tumor contrast. Biodistribution studies, blocking, control studies with 89Zr-Df-cetuximab-800CW, ex vivo BLI/PET/NIRF imaging, and histology all confirmed CD105 specificity of the tracer. Broad clinical potential of TRC105-based agents was shown in many tumor types, which also enabled early detection of small metastasis and intraoperative guidance for tumor removal.
Breast cancer; lung metastasis; positron emission tomography (PET); near-infrared fluorescence (NIRF); tumor angiogenesis; CD105 (endoglin); 89Zr
Biological changes following myocardial infarction (MI) lead to increased secretion of angiogenic factors that subsequently stimulate the formation of new blood vessels as a compensatory mechanism to reverse ischemia. The goal of this study was to assess the role of CD105 expression during MI-induced angiogenesis by positron emission tomography (PET) imaging using 64Cu-labeled TRC105, an anti-CD105 monoclonal antibody. MI was induced by ligation of the left anterior descending (LAD) artery in female rats. Echocardiography and 18F-fluoro-2-deoxy-D-glucose (18F-FDG) PET scans were performed on post-operative day 3 to confirm the presence of MI in the infarct group and intact heart in the sham group, respectively. Ischemia-induced angiogenesis was non-invasively monitored with 64Cu-NOTA-TRC105 (an extensively validated PET tracer in our previous studies) PET on post-operative days 3, 10, and 17. Tracer uptake in the infarct zone was highest on day 3 following MI, which was significantly higher than that in the sham group (1.41 ± 0.45 %ID/g vs 0.57 ± 0.07 %ID/g; n=3, p<0.05). Subsequently, tracer uptake in the infarct zone decreased over time to the background level on day 17, whereas tracer uptake in the heart of sham rats remained low at all time points examined. Histopathology documented increased CD105 expression following MI, which corroborated in vivo findings. This study indicated that PET imaging of CD105 can be a useful tool for MI-related research, which can potentially improve MI patient management in the future upon clinical translation of the optimized PET tracers.
Angiogenesis; myocardial infarction (MI); positron emission tomography (PET); CD105 (endoglin); molecular imaging; 64Cu
Despite the increasing use of panel surveys, little is known about the differences in data quality across panels.
The aim of this study was to characterize panel survey companies and their respondents based on (1) the timeliness of response by panelists, (2) the reliability of the demographic information they self-report, and (3) the generalizability of the characteristics of panelists to the US general population. A secondary objective was to highlight several issues to consider when selecting a panel vendor.
We recruited a sample of US adults from 7 panel vendors using identical quotas and online surveys. All vendors met prespecified inclusion criteria. Panels were compared on the basis of how long the respondents took to complete the survey from time of initial invitation. To validate respondent identity, this study examined the proportion of consented respondents who failed to meet the technical criteria, failed to complete the screener questions, and provided discordant responses. Finally, characteristics of the respondents were compared to US census data and to the characteristics of other panels.
Across the 7 panel vendors, 2% to 9% of panelists responded within 2 days of invitation; however, approximately 20% of the respondents failed the screener, largely because of the discordance between self-reported birth date and the birth date in panel entry data. Although geographic characteristics largely agreed with US Census estimates, each sample underrepresented adults who did not graduate from high school and/or had annual incomes less than US $15,000. Except for 1 vendor, panel vendor samples overlapped one another by approximately 20% (ie, 1 in 5 respondents participated through 2 or more panel vendors).
The results of this head-to-head comparison provide potential benchmarks in panel quality. The issues to consider when selecting panel vendors include responsiveness, failure to maintain sociodemographic diversity and validated data, and potential overlap between panels.
survey methods; community surveys; sampling bias; selection bias; Internet; data sources
Pancreaticobiliary maljunction (PBM) is an unusual anomalous condition in which the pancreatic duct and bile duct merge outside the duodenal wall and form a long common channel. Pancreas divisum (PD) is a congenital anomaly in which the dorsal and ventral pancreatic ducts fail to fuse. Endoscopic retrograde cholangiopancreatography (ERCP) is the gold standard for diagnosing PD and magnetic resonance cholangiopancreatography (MRCP) is the non-invasive choice. In this study, four cases of patients with unusual PBM in addition to PD are described. The patients presented with abdominal pain, which was caused by distal biliary stricture diagnosed by MRCP. The patients received ERCP and had a good prognosis.
pancreaticobiliary maljunction; pancreas divisum; diagnosis; therapy
Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types. The goal of this study was to develop a positron emission tomography (PET) tracer for imaging of TF expression in pancreatic cancer.
ALT-836, a chimeric anti-human TF monoclonal antibody, was conjugated to 2-S-(4-isothiocyanatobenzyl)-1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid (p-SCN-Bn-NOTA) and labeled with 64Cu. To compare the TF binding affinity of ALT-836 and NOTA-ALT-836, flow cytometry analysis was performed in three pancreatic cancer cell lines with different expression level of TF (from low to high: PANC-1, ASPC-1, and BXPC-3). PET imaging, biodistribution, blocking, and histology studies were performed on pancreatic tumor-bearing mice to evaluate the ability and specificity of 64Cu-NOTA-ALT-836 to target TF in vivo.
There was no difference in TF binding affinity between ALT-836 and NOTA-ALT-836. 64Cu-labeling was achieved with high yield and specific activity. Serial PET imaging revealed that the uptake of 64Cu-NOTA-ALT-836 in BXPC-3 tumors (high TF expression) was 5.7 ± 0.5, 10.3 ± 0.5, and 16.5 ± 2.6 %ID/g at 4, 24, and 48 h post-injection respectively (n = 4), significantly higher than that in the PANC-1 and ASPC-1 tumors. Biodistribution data as measured by gamma counting were consistent with the PET findings. Blocking experiments and histology further confirmed the TF specificity of 64Cu-NOTA-ALT-836.
Herein we report the first successful PET imaging of TF expression. Persistent and TF-specific uptake of 64Cu-NOTA-ALT-836 was observed in pancreatic cancer models.
Tissue factor (TF); Pancreatic cancer; Positron emission tomography (PET); Monoclonal antibody; 64Cu; ImmunoPET
AIM: To investigate the efficacy and potential mechanism of Xiaotan Tongfu granules (XTTF) in stress ulcers.
METHODS: One hundred sixty rats were randomly divided into 4 groups (n = 10) as follows: the model group (MP group), the control group (CP group), the ranitidine group (RP group) and the XTTF granule group (XP group). Rats in the MP group received no drugs, rats in the CP group received 0.2 mL of a 0.9% sodium chloride solution via oral gavage, and rats in the RP and XP groups received the same volume of ranitidine (50 mg/kg) or XTTF granule (4.9 g/kg). The cold-restraint stress model was applied to induce stress ulcers after 7 consecutive days of drug administration. Afterwards, rats were sacrificed at 0, 3, 6 and 24 h. Gastric pH was measured by a precise pH meter; gastric emptying rate (GER) was measured by using a methylcellulose test meal; myeloperoxidase activity (MPO), macrophage migration inhibitory factor (MIF), proliferating cell nuclear antigen (PCNA), and heat shock protein 70 (HSP70) were measured by immunohistochemical staining; and mucosal cell apoptosis was measured by transferase dUTP nick end labeling.
RESULTS: In the cold-restraint stress model, the development of stress ulcers peaked at 3 h and basically regressed after 24 h. Gastric lesions were significantly different in the RP and XP groups at each time point. Interestingly, although this index was much lower in the RP group than in the XP group immediately following stress induction (7.00 ± 1.10 vs 10.00 ± 1.79, P < 0.05. Concerning gastric pH, between the RP and XP groups, we detected a statistically significant difference immediately after stress induction (0 h: 4.56 ± 0.47 vs 3.34 ± 0.28, P < 0.05) but not at any of the subsequent time points. For GER, compared to the RP group, GER was remarkably elevated in the XP group because a statistically significant difference was detected (3 h: 46.84 ± 2.70 vs 61.16 ± 5.12, P < 0.05; 6 h: 60.96 ± 6.71 vs 73.41 ± 6.16, P < 0.05; 24 h: 77.47 ± 3.17 vs 91.31 ± 4.34, P < 0.05). With respect to MPO and MIF, comparisons between the RP and XP groups revealed statistically significant differences at 3 h (MPO: 18.94 ± 1.20 vs 13.51 ± 0.89, P < 0.05; MIF: 150.67 ± 9.85 vs 122.17 ± 5.67, P < 0.05) and 6 h (MPO: 13.22 ± 1.54 vs 8.83 ± 0.65, P < 0.05; MIF: 135.50 ± 9.46 vs 109.83 ± 6.40, P < 0.05). With regard to HSP70, HSP70 expression was significantly increased in the RP and XP groups at 3 and 6 h compared to the MP and CP groups. In addition, comparing the RP and XP groups also showed statistically significant differences at 3 and 6 h. The expression of PCNA was higher in the RP and XP groups 3 h after stress induction. Between these two groups, small but statistically significant differences were observed at all of the time points (3 h: 69.50 ± 21.52 vs 79.33 ± 15.68, P < 0.05; 6 h: 107.83 ± 4.40 vs 121.33 ± 5.71, P < 0.05; 24 h: 125.33 ± 5.65 vs 128.50 ± 14.49, P < 0.05) except 0 h. With regard to apoptosis, the apoptotic activity in the RP and XP groups was significantly different from that in the MP and CP groups. The XP group exhibited a higher inhibition of cell apoptosis than the RP group at 3 h (232.58 ± 24.51 vs 174.46 ± 10.35, P < 0.05) and 6 h (164.74 ± 18.31 vs 117.71 ± 12.08, P < 0.05).
CONCLUSION: The Xiaotan Tongfu granule was demonstrated to be similar to ranitidine in preventing stress ulcers. It exhibited multiple underlying mechanisms and deserves further study.
Stress ulcer; Xiaotan Tongfu granule; Inflammation; Heat shock protein 70; Proliferation and apoptosis; Gastric emptying rate
Many oncology drugs are administered at their maximally tolerated dose without the knowledge of their optimal efficacious dose range. In this study, we describe a multifaceted approach that integrated preclinical and clinical data to identify the optimal dose for an antiangiogenesis agent, anti-EGFL7. EGFL7 is an extracellular matrix–associated protein expressed in activated endothelium. Recombinant EGFL7 protein supported EC adhesion and protected ECs from stress-induced apoptosis. Anti-EGFL7 antibodies inhibited both of these key processes and augmented anti-VEGF–mediated vascular damage in various murine tumor models. In a genetically engineered mouse model of advanced non–small cell lung cancer, we found that anti-EGFL7 enhanced both the progression-free and overall survival benefits derived from anti-VEGF therapy in a dose-dependent manner. In addition, we identified a circulating progenitor cell type that was regulated by EGFL7 and evaluated the response of these cells to anti-EGFL7 treatment in both tumor-bearing mice and cancer patients from a phase I clinical trial. Importantly, these preclinical efficacy and clinical biomarker results enabled rational selection of the anti-EGFL7 dose currently being tested in phase II clinical trials.
A γ-AApeptide-based tracer for positron emission tomography imaging of integrin αvβ3 is reported. Despite its shorter sequence and linear nature, this tracer had comparable integrin αvβ3 binding affinity as the cyclic arginine-glycine-aspartic acid peptide but significantly higher resistance to enzymatic degradation and better stability.
Non-invasive and quantitative imaging of vascular endothelial growth factor receptor-2 (VEGFR-2) expression levels is highly important in cancer diagnosis, prognosis, and patient management. Although various literature reports have investigated the tumor expression levels of VEGFR-2 using imaging techniques such as positron emission tomography, single-photon emission computed tomography, targeted ultrasound, etc., accurate evaluation of the dynamic microdistribution of VEGFR-2 in vivo with good spatial and temporal resolution remains a major challenge. In this issue of the American Journal of Nuclear Medicine and Molecular Imaging, He at al. reported the use of a VEGFR-2 targeted probe for magnetic resonance imaging (MRI) of VEGFR-2 in two glioma models in rats (i.e. C6 and RG2). The heterogeneity of VEGFR-2 expression was non-invasively imaged with MRI and validated with various in vitro, in vivo, and ex vivo experiments. Not only was heterogeneous expression of VEGFR-2 found in different glioma tumors, it was also observed in different regions within the same tumor (e.g. tumor periphery, peri-necrotic area, and tumor interior). This report highlights the complex nature of gliomas, which may offer invaluable insights into tumor heterogeneity and potential clinical management of glioma patients. These patients have dismal clinical outcomes and are in urgent need of better tools to improve brain tumor treatment.
Molecular MRI (mMRI); glioma; tumor angiogenesis; VEGFR-2; molecular imaging
Graphene, with its excellent physical, chemical, and mechanical properties, holds tremendous potential for a wide variety of biomedical applications. As research on graphene-based nanomaterials is still at a nascent stage, due to the very short time span since its initial report in 2004, a focused review on this topic is timely and necessary. In this feature review, we first summarize the results from toxicity studies of graphene and its derivatives. Although literature reports have mixed findings, we emphasize that the key question is not how toxic graphene itself is, but how to modify and functionalize it and its derivatives so that they do not exhibit acute/chronic toxicity, can be cleared from the body over time, and thereby can be best used for biomedical applications. Next, we discuss in detail the exploration of graphene-based nanomaterials for tissue engineering, molecular imaging, and drug/gene delivery applications. The future of graphene-based nanomaterials in biomedicine looks brighter than ever, and it is expected that they will find a wide range of biomedical applications with future research effort and interdisciplinary collaboration.
Graphene; graphene oxide (GO); reduced graphene oxide (rGO); molecular imaging; tissue engineering; drug/gene delivery; positron emission tomography (PET); nanomedicine
Hypertrophic cardiomyopathy (HCM) due to mutations in genes encoding sarcomere proteins is most commonly inherited as an autosomal dominant trait. Since nearly 50% of HCM cases occur in the absence of a family history, a recessive inheritance pattern may be involved.
A pedigree was identified with suspected autosomal recessive transmission of HCM. Twenty-six HCM-related genes were comprehensively screened for mutations in the proband with targeted second generation sequencing, and the identified mutation was confirmed with bi-directional Sanger sequencing in all family members and 376 healthy controls.
A novel missense mutation (c.1469G>T, p.Gly490Val) in exon 17 of MYBPC3 was identified. Two siblings with HCM were homozygous for this mutation, whereas other family members were either heterozygous or wild type. Clinical evaluation showed that both homozygotes manifested a typical HCM presentation, but none of others, including 5 adult heterozygous mutation carriers up to 71 years of age, had any clinical evidence of HCM.
Our data identified a MYBPC3 mutation in HCM, which appeared autosomal recessively inherited in this family. The absence of a family history of clinical HCM may be due to not only a de novo mutation, but also recessive mutations that failed to produce a clinical phenotype in heterozygous family members. Therefore, consideration of recessive mutations leading to HCM is essential for risk stratification and genetic counseling.
The goal of this study was to employ nano-graphene for tumor targeting in an animal tumor model, and quantitatively evaluate the pharmacokinetics and tumor targeting efficacy through positron emission tomography (PET) imaging using 66Ga as the radiolabel. Nano-graphene oxide (GO) sheets with covalently linked, amino group-terminated six-arm branched polyethylene glycol (PEG; 10 kDa) chains were conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid, for 66Ga-labeling) and TRC105 (an antibody that binds to CD105). Flow cytometry analyses, size measurements, and serum stability studies were performed to characterize the GO conjugates before in vivo investigations in 4T1 murine breast tumor-bearing mice, which were further validated by histology. TRC105-conjugated GO was specific for CD105 in cell culture. 66Ga-NOTA-GO-TRC105 and 66Ga-NOTA-GO exhibited excellent stability in complete mouse serum. In 4T1 tumor-bearing mice, these GO conjugates were primarily cleared through the hepatobiliary pathway. 66Ga-NOTA-GO-TRC105 accumulated quickly in the 4T1 tumors and tumor uptake remained stable over time (3.8 ± 0.4, 4.5 ± 0.4, 5.8 ± 0.3, and 4.5 ± 0.4 %ID/g at 0.5, 3, 7, and 24 h post-injection respectively; n = 4). Blocking studies with unconjugated TRC105 confirmed CD105 specificity of 66Ga-NOTA-GO-TRC105, which was corroborated by biodistribution and histology studies. Furthermore, histological examination revealed that targeting of NOTA-GO-TRC105 is tumor vasculature CD105 specific with little extravasation. Successful demonstration of in vivo tumor targeting with GO, along with the versatile chemistry of graphene-based nanomaterials, makes them suitable nanoplatforms for future biomedical research such as cancer theranostics.
Graphene; 66Ga; CD105 (endoglin); positron emission tomography (PET); molecular imaging; tumor angiogenesis
High translocation speed of a DNA strand through a nanopore is a major bottleneck for nanopore detection of DNA molecules. Here, we choose MgCl2 electrolyte as salt solution to control DNA mobility. Experimental results demonstrate that the duration time for straight state translocation events in 1 M MgCl2 solution is about 1.3 ms which is about three times longer than that for the same DNA in 1 M KCl solution. This is because Mg2+ ions can effectively reduce the surface charge density of the negative DNA strands and then lead to the decrease of the DNA electrophoretic speed. It is also found that the Mg2+ ions can induce the DNA molecules binding together and reduce the probability of straight DNA translocation events. The nanopore with small diameter can break off the bound DNA strands and increase the occurrence probability of straight DNA translocation events.
Nanopore; DNA sequencing; Translocation speed
The goal of this study is to employ the HaloTag technology for positron emission tomography (PET), which involves two components: the HaloTag protein (a modified hydrolase which covalently binds to synthetic ligands) and HaloTag ligands (HTLs). 4T1 murine breast cancer cells were stably transfected to express HaloTag protein on the surface (termed as 4T1-HaloTag-ECS, ECS denotes extracellular surface). Two new HTLs were synthesized and termed NOTA-HTL2G-S and NOTA-HTL2G-L (2G indicates second generation, S stands for short, L stands for long, NOTA denotes 1,4,7-triazacyclononane-N,N’N’’-triacetic acid). Microscopy studies confirmed surface expression of HaloTag in 4T1-HaloTag-ECS cells, which specifically bind NOTA-HTL2G-S/L. Uptake of 64Cu-NOTA-HTL2G-L in 4T1-HaloTag-ECS tumors (4.3 ± 0.5, 4.1± 0.2, 4.0 ± 0.2, 2.3 ± 0.1, and 2.2 ± 0.1 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection respectively; n = 4) was significantly higher than that in the 4T1 tumors (3.0 ± 0.3, 3.0± 0.1, 3.0 ± 0.2, 2.0 ± 0.4, and 2.4 ± 0.3 %ID/g at 0.5, 3, 6, 18, and 24 h post-injection respectively; n = 4) at early time points. In comparison, 64Cu-NOTA-HTL2G-S did not demonstrate significant uptake in either 4T1-HaloTag-ECS or 4T1 tumors. Blocking studies and autoradiography of tumor lysates confirmed that 64Cu-NOTA-HTL2G-L binds specifically to HaloTag protein in the 4T1-HaloTag-ECS tumors, corroborated by histology. HaloTag protein-specific targeting and PET imaging in vivo with 64Cu-NOTA-HTL2G-L serves as a proof-of-principle for future non-invasive and sensitive tracking of HaloTag-transfected cells with PET, as well as many other studies of gene/protein/cell function in vivo.
HaloTag; positron emission tomography (PET); reporter gene; 64Cu; cancer; molecular imaging
A multifunctional unimolecular micelle made of a hyperbranched amphiphilic block copolymer was designed, synthesized, and characterized for cancer-targeted drug delivery and non-invasive positron emission tomography (PET) imaging in tumor-bearing mice. The hyperbranched amphiphilic block copolymer, Boltorn® H40-poly(L-glutamate-hydrazone-doxorubicin)-b-poly(ethylene glycol) (i.e., H40-P(LG-Hyd-DOX)-b-PEG), was conjugated with cyclo(Arg-Gly-Asp-D-Phe-Cys) peptides (cRGD, for integrin αvβ3 targeting) and macrocyclic chelators (1,4,7-triazacyclononane-N, N′, N″-triacetic acid [NOTA], for 64Cu-labeling and PET imaging) (i.e., H40-P(LG-Hyd-DOX)-b-PEG-OCH3/cRGD/NOTA, also referred to as H40-DOX-cRGD). The anti-cancer drug, doxorubicin (DOX) was covalently conjugated onto the hydrophobic segments of the amphiphilic block copolymer arms (i.e., PLG) via a pH-labile hydrazone linkage to enable pH-controlled drug release. The unimolecular micelles exhibited a uniform size distribution and pH-sensitive drug release behavior. cRGD-conjugated unimolecular micelles (i.e., H40-DOX-cRGD) exhibited a much higher cellular uptake in U87MG human glioblastoma cells due to integrin αvβ3-mediated endocytosis than non-targeted unimolecular micelles (i.e., H40-DOX), thereby leading to a significantly higher cytotoxicity. In U87MG tumor-bearing mice, H40-DOX-cRGD-64Cu also exhibited a much higher level of tumor accumulation than H40-DOX-64Cu, measured by non-invasive PET imaging and confirmed by biodistribution studies and ex vivo fluorescence imaging. We believe that unimolecular micelles formed by hyperbranched amphiphilic block copolymers that synergistically integrate passive and active tumor-targeting abilities with pH-controlled drug release and PET imaging capabilities provide the basis for future cancer theranostics.
Unimolecular micelles; Drug delivery; Theranostic nanocarriers; Hyperbranched amphiphilic block; copolymer; Positron emission tomography (PET); Cyclic arginine-glycine-aspartic acid (cRGD); peptide
Herein we demonstrate that nano-graphene can be specifically directed to the tumor neovasculature in vivo through targeting of CD105 (i.e. endoglin), a vascular marker for tumor angiogenesis. The covalently functionalized nano-graphene oxide (GO) exhibited excellent stability and target specificity. Pharmacokinetics and tumor targeting efficacy of the GO conjugates were investigated with serial non-invasive positron emission tomography (PET) imaging and biodistribution studies, which were validated by in vitro, in vivo, and ex vivo experiments. The incorporation of an active targeting ligand (TRC105, a monoclonal antibody that binds to CD105) led to significantly improved tumor uptake of functionalized GO, which was specific for the neovasculature with little extravasation, warranting future investigation of these GO conjugates for cancer-targeted drug delivery and/or photothermal therapy to enhance therapeutic efficacy. Since poor extravasation is a major hurdle for nanomaterial-based tumor targeting in vivo, this study also establishes CD105 as a promising vascular target for future cancer nanomedicine.
Nano-graphene; tumor angiogenesis; positron emission tomography (PET); CD105 (endoglin); molecular imaging; 64Cu; cancer
The livertaxis of glycyrrhizic acid-conjugated bovine serum albumin (GL-BSA) has been reported in the literature. Now, in this paper, we describe a novel type of drug-targeted delivery system containing 10-hydroxycamptothecin (HCPT) with liver tumor targeting.
First, GL was coupled to BSA then HCPT was encapsulated in GL-BSA by high-pressure homogenization emulsification. In the experimental design, the influencing variables on particle size and drug loading efficiency were determined to be BSA concentration, volume ratio of water to organic phase, and speed and speed duration of homogenization as well as homogenization pressure and the number of times homogenized at certain pressures. Particle size plays an important role in screening optimal conditions of nanoparticles preparation. Characteristics of 10-hydroxycamptothecin-loaded glycyrrhizic acid-conjugated bovine serum albumin nanoparticles (GL-BSA-HCPT-NPs), such as the drug encapsulation efficiency, drug loading efficiency, and GL-BSA content were studied. In addition, the morphology of the nanoparticles (NPs) and weight loss rate were determined and Fourier transform infrared spectroscopy, X-ray diffraction spectroscopy, and thermal analysis performed.
The average particle size of the sample NPs prepared under optimal conditions was 157.5 nm and the zeta potential was −22.51 ± 0.78 mV; the drug encapsulation efficiency and drug loading efficiency were 93.7% and 10.9%, respectively. The amount of GL coupling to BSA was 98.26 μg/mg. Through physical property study of the samples, we determined that the HCPT had been successfully wrapped in GL-BSA. In vitro drug-release study showed that the nanoparticles could release the drug slowly and continuously. Hemolysis testing showed the safety of GL-BSA as a novel drug delivery system. The targeting properties of GL-BSA-HCPT-NPs were studied in an in vitro cell uptake study and cell proliferation assay. Cells incubated with GL-BSA-HCPT-NPs and labeled with fluorescein isothiocyanate showed more extensive fluorescence spots and stronger fluorescence intensity than samples without GL conjugation. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine the inhibitory rate of the samples. It was found that the inhibitory rate of GL-BSA-HCPT-NPs develops as concentration rises. Further, the inhibitory rate of GL-BSA-HCPT-NPs was higher at the same concentration and had a lower half maximal inhibitory concentration value than the other samples. The half maximal inhibitory concentration values of GL-BSA-HCPT-NPs, BSA-HCPT-NPs, and HCPT sodium were 0.78 ± 0.015, 1.62 ± 0.039, and 7.93 ± 0.255 μg/mL, respectively.
The results of this study show GL-BSA-HCPT to be a promising new vehicle for hepatocellular carcinoma-targeting therapy.
HCPT; BSA; GL; high-pressure homogenization emulsification
CD105 (endoglin) is an independent prognostic marker for poor prognosis in > 10 solid tumor types, including breast cancer. The goal of this study was to develop a CD105-specific agent for both positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging, which can have potential clinical applications in diagnosis and imaged-guided surgery of breast cancer. TRC105, a chimeric anti-CD105 monoclonal antibody, was labeled with both a NIRF dye (i.e. 800CW) and 64Cu to yield 64Cu-NOTA-TRC105-800CW. Flow cytometry analysis revealed no difference in CD105 binding affinity/specificity between TRC105 and NOTA-TRC105-800CW. Serial PET imaging revealed that the 4T1 murine breast tumor uptake of 64Cu-NOTA-TRC105-800CW was 5.2 ± 2.7, 11.0 ± 1.4, and 13.0 ± 0.4 %ID/g at 4, 24, and 48 h post-injection respectively. Tumor uptake as measured by ex vivo NIRF imaging exhibited a good linear correlation with the %ID/g values obtained from PET (R = 0.74). Biodistribution data were consistent with the PET/NIRF findings. Blocking experiments, control studies with dual-labeled cetuximab (an isotype-matched control antibody), and histology confirmed the CD105 specificity of 64Cu-NOTA-TRC105-800CW. Successful PET/NIRF imaging of CD105 expression warrants further investigation and clinical translation of dual-labeled TRC105-based imaging agents.
CD105/endoglin; positron emission tomography (PET); near-infrared fluorescence (NIRF); tumor angiogenesis; TRC105; 64Cu; breast cancer
Bone is a slowly regenerating tissue influenced by various physiological processes, including proliferation, differentiation, and angiogenesis, under the control of growth factors. Shortening this healing time is an important and popular clinical research focus in orthopedics. Negative pressure can stimulate angiogenesis, improve blood circulation, promote granulation tissue growth and accelerate tissue wound healing. We sought to determine whether negative pressure could reduce bone healing time in a rabbit cranial defect model.
Four symmetrical holes (diameter, 3.5 mm) were drilled into the skulls of 42 New Zealand white rabbits, with two holes in each parietal bone. For each rabbit, the two sides were then randomly assigned into experimental and control groups. Using negative pressure suction tubes, experimental holes were treated with −50 kPa for 15 minutes, four times per day, whereas the control holes remained untreated. After 4 weeks, the negative pressure suction tubes were removed. At 2, 4, 6 and 8 weeks, three-dimensional (3D) reconstruction computed tomography (CT), X-ray radiopacity, and two-photon absorptiometry were used to evaluate new bone formation. Histological changes were determined by hematoxylin and eosin (H.E) staining. At weekly intervals until 6 weeks, the mRNA expression levels of vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)-2 were evaluated by RT-PCR. A paired student’s t-test was employed to compare X-ray radiopacity and bone density measurements between the experimental and control groups.
3D-reconstruction CT showed that new bone regeneration in the experimental group was greater than that in the control group at 4 and 6 weeks. At these time points, the experimental group presented with higher X-ray radiopacity and increased bone density (P < 0.05) as compared with the control group. Cartilage islands and new bone were observed by H.E staining at 2 weeks in the experimental group. By 6 weeks, the new bone had matured into lamellar bone in the experimental group. RT-PCR results showed that VEGF and BMP-2 were highly expressed in the experimental group as compared with control.
Intermittent negative pressure can promote the regeneration of bone possibly by enhancing the expression of VEGF and BMP-2.
Negative pressure; Bone regeneration; VEGF; BMP-2; Animal experimental use