Cerebrospinal fluid hypovolemia syndrome mimicking ocular myasthenia gravis: A case report.

Purpose: Cerebrospinal fluid hypovolemia syndrome (CHS) is a rare clinical entity that can be caused by spontaneous cerebrospinal fluid (CSF) leakage. The aim of this study is to report a rare case of CHS after a traffic accident in a patient who presented with diplopia and ptosis with fluctuation and was initially diagnosed with ocular myasthenia gravis. Observeations: A 29-year-old man exhibited fluctuating left ptosis and diplopia after a traffic accident. Although he was suspected of having myasthenia gravis and was treated using oral pyridostigmine bromide, his symptoms did not improve. He also had orthostatic headaches and malaise after the accident. His symptoms were suspected to be associated with traumatic cerebrospinal fluid hypovolemia. After 1000-mL fluid replacement, his diplopia and ptosis improved, and orbital T2-weghted MRI detected a high-signal zone around the optic nerve. We diagnosed him with oculomotor nerve paresis associated with cerebrospinal fluid hypovolemia. The symptoms, including ptosis, diplopia, orthostatic headaches, and malaise, disappeared after epidural blood patch therapy. Conclusions and Importance: When treating patients with fluctuating ocular symptoms, such as diplopia and ptosis, who have a history of trauma and orthostatic headaches, the possibility of CHS should be considered in the differential diagnosis.

Rectal mobilization for laparoscopic pelvic lymphadenectomy of the lower paracervical pathway in patients with uterine cancer.

OBJECTIVE: The pelvic lymphatic drainage system comprises the upper and lower paracervical pathways (LPPs). Lymph node dissection of the LPP, including the cardinal ligament, internal iliac, internal common iliac, and presacral lymph nodes, requires higher surgical skills because of the anatomical limitations of the pelvic cavity and the dissection of vessels while preserving the nerves in the pelvic floor. In this video, we demonstrate rectal mobilization for laparoscopic complete pelvic lymph node dissection of the LPP in patients with uterine cancer. METHODS: Rectal mobilization was performed before complete pelvic lymph node dissection of the LPP. The pararectal space was opened widely and the connective tissue between the presacral fascia and prehypogastric nerve fascia was dissected bilaterally, allowing the rectum to be pulled. RESULTS: This procedure created a wide-open space in the pelvic floor, allowing clear visualization of the nerves and lymph nodes of the LPP. Laparoscopic complete lymph node dissection of the LPP was performed in the open space while preserving the hypogastric and pelvic splanchnic nerves and isolating the extensive network of blood vessels in the pelvic cavity. CONCLUSION: Rectal mobilization enabled the safe execution of laparoscopic complete pelvic lymph node dissection of the LPP in patients with uterine cancer.

Inhibition of MEK pathway enhances the antitumor efficacy of chimeric antigen receptor T cells against neuroblastoma.

Disialoganglioside (GD2)-specific chimeric antigen receptor (CAR)-T cells (GD2-CAR-T cells) have been developed and tested in early clinical trials in patients with relapsed/refractory neuroblastoma. However, the effectiveness of immunotherapy using these cells is limited, and requires improvement. Combined therapy with CAR-T cells and molecular targeted drugs could be a promising strategy to enhance the antitumor efficacy of CAR T cell immunotherapy. Here, we generated GD2-CAR-T cells through piggyBac transposon (PB)-based gene transfer (PB-GD2-CAR-T cells), and analyzed the combined effect of these cells and a MEK inhibitor in vitro and in vivo on neuroblastoma. Trametinib, a MEK inhibitor, ameliorated the killing efficacy of PB-GD2-CAR-T cells in vitro, whereas a combined treatment of the two showed superior antitumor efficacy in a murine xenograft model compared to that of PB-GD2-CAR-T cell monotherapy, regardless of the mutation status of the MAPK pathway in tumor cells. The results presented here provide new insights into the feasibility of combined treatment with CAR-T cells and MEK inhibitors in patients with neuroblastoma.

Development of non-viral, ligand-dependent, EPHB4-specific chimeric antigen receptor T cells for treatment of rhabdomyosarcoma.

Ephrin type-B receptor 4 (EPHB4), expressed in tumors including rhabdomyosarcoma, is a suitable target for chimeric antigen receptor (CAR)-T cells. Ligand-independent activation of EPHB4 causes cell proliferation and malignant transformation in rhabdomyosarcoma, whereas ligand-dependent stimulation of EPHB4 induces apoptosis in rhabdomyosarcoma. Therefore, we hypothesized that ligand-based, EPHB4-specific CAR-T cells may kill rhabdomyosarcoma cells without stimulating downstream cell proliferation mechanisms. We developed novel CAR-T cells by targeting EPHB4 via EPHRIN B2, a natural ligand of EPHB4. The generation of EPHB4-CAR-T cells via piggyBac (PB) transposon-based gene transfer resulted in sufficient T cell expansion and CAR positivity (78.5% ± 5.9%). PB-EPHB4-CAR-T cells displayed a dominant stem cell memory fraction (59.4% ± 7.2%) as well as low PD-1 expression (0.60% ± 0.21%) after 14 days of expansion. The PB-EPHB4-CAR-T cells inhibited EPHB4-positive tumor cells without activating cell proliferation downstream of EPHB4, even after multiple tumor re-challenges and suppressed tumor growth in xenograft-bearing mice. Therefore, PB-EPHB4-CAR-T cells possess a memory-rich fraction without early T cell exhaustion and show potential as promising therapeutic agents for treating rhabdomyosarcoma and other EPHB4-positive tumors.

Autologous antigen-presenting cells efficiently expand piggyBac transposon CAR-T cells with predominant memory phenotype.

The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. The manufacturing process of CAR-T cells should be optimized to prevent early T cell exhaustion during expansion. Activation of T cells by monoclonal antibodies is a critical step for T cell expansion, which may sometimes induce excess stimulation and exhaustion of T cells. Given that piggyBac transposon (PB)-based gene transfer could circumvent the conventional pre-activation of T cells, we established a manufacturing method of PB-mediated HER2-specific CAR-T cells (PB-HER2-CAR-T cells) that maintains their memory phenotype without early T cell exhaustion. Through stimulation of CAR-transduced T cells with autologous peripheral blood mononuclear cell-derived feeder cells expressing both truncated HER2, CD80, and 4-1BBL proteins, we could effectively propagate memory-rich, PD-1-negative PB-HER2-CAR-T cells. PB-HER2-CAR-T cells demonstrated sustained antitumor efficacy in vitro and debulked the HER2-positive tumors in vivo. Mice treated with PB-HER2-CAR-T cells rejected the second tumor establishment owing to the in vivo expansion of PB-HER2-CAR-T cells. Our simple and effective manufacturing process using PB system and genetically modified donor-derived feeder cells is a promising strategy for the use of PB-CAR-T cell therapy.

Optical coherence tomography as a possible tool to monitor and predict disease progression in mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes.

Optical coherence tomography (OCT) is an imaging technique used to obtain three-dimensional information on the retina. In this article, we evaluated the structural neuro-retinal abnormalities, especially the thickness in the ganglion cell complex (GCC), in patients with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). The GCC thickness in MELAS patients was significantly thinner than that in normal controls even when they had no history of transient homonymous hemianopia. There was a negative correlation between GCC thickness and disease duration. In conclusion, OCT may be an effective tool to monitor and predict disease progression in MELAS patients.

Cerebrospinal fluid hypovolemia syndrome after a traffic accident with abnormal eye movements: A case report.

PURPOSE: To describe a rare case of cerebrospinal fluid hypovolemia syndrome after a traffic accident with abnormal eye movements. OBSERVATIONS: A 19-year-old man was referred to our clinic after being hit by a car five months ago while riding a bicycle. After the accident, he sometimes noticed oscillopsia, and had postural headaches and reading difficulties. His eye movement recording revealed square wave jerks during fixation and decreased pursuit gain during horizontal smooth pursuit. MR myelography detected cerebrospinal fluid leakage and the patient was diagnosed with cerebrospinal fluid hypovolemia. After undergoing epidural blood patch therapy, the leakage disappeared, and his postural headaches improved immediately. Square wave jerks and decreased pursuit gain improved, and his oscillopsia and reading difficulty also improved after therapy. CONCLUSIONS AND IMPORTANCE: A patient with cerebrospinal fluid hypovolemia presented with square wave jerks and decreased pursuit gain. Epidural blood patch therapy was effective for the symptoms. When treating patients with oscillopsia and postural headaches, we should consider the possibility of cerebrospinal fluid hypovolemia syndrome in the differential diagnosis.

Myxofibrosarcoma of the heart: A case report with positive pleural effusion cytology.

Primary cardiac sarcoma is rare, and there have been only a few reports on its cytologic findings. Myxofibrosarcoma, a variant of fibrosarcoma of the heart, is an extremely rare entity. We present a case of primary cardiac myxofibrosarcoma in a 63-year-old woman. Pleural fluid cytology and imprint cytology of the resected tumor at operation and autopsy were obtained. Cytologic evaluation with immunocytochemical staining utilizing a cell transfer technique revealed that tumor cells of the resected tumor and autopsy specimen and pleural effusion demonstrated large and pleomorphic cells with irregular, hyperchromatic nuclei and were positive for vimentin. Combination of morphology and immunoprofile of the cells of pleural effusion was compatible with the diagnosis of metastatic myxofibrosarcoma. Diagn. Cytopathol. 2016;44:1112-1116. © 2016 Wiley Periodicals, Inc.

[Echocardiography and Biochemical Examination in the Treatment of Ischemic Heart Disease].

The importance of echocardiography is generally known for acute coronary syndrome. However, assessing cardiac biomarker elevation is important together with echocardiography as poor images are obtained in some cases and patients may show unstable angina or angina on effort without asynergy. We examined the usefulness of high sensitive troponin I (hs-cTnI) in 60 patients from October 2014. We performed hs-cTnI and echocardiography in 10 cases (acute myocardial infarction: 8, unstable angina: 1, angina on effort: 1) among those patients. In the 8 acute myocardial infarction cases, asynergy was noted in all cases on echocardiography, but CK, CK-MB, and H-FABP cardiac biomarkers showed mixed negativity and positivity. Also, the unstable angina and angina on effort did not show asynergy but hs-cTnI was positive, the case of unstable angina showed elevation over time, but the case of angina on effort did not show elevation with time. We suggest that echocardiography and hs-cTnI are useful for acute myocardial infarction, although care may be necessary when assessing ACS patients with no detected asynergy in echocardiographs or non-ACS patients with acutely elevated hs-cTnI). In addition, it was suggested that confirmation of a change over time of hs-cTnI is necessary in angina patients.

Hesperetin glucuronides induce adipocyte differentiation via activation and expression of peroxisome proliferator-activated receptor-γ.

In previous reports, hesperidin, a flavonoid glucoside from citrus fruit, is hydrolyzed to hesperetin, an aglycone of hesperidin, and converted to the hesperetin glucuronides (H7-OG and H3'-OG) in vivo and depresses blood glucose levels. But there are no reports on the activity of hesperetin glucuronides. To determine the activity of hesperetin glucuronides, H7-OG and H3'-OG were synthesized and peroxisome proliferator-activated receptor-γ (PPARγ) agonist activity was observed at 250 µM. These glucuronides accelerated the differentiation of 3T3-L1 cells into adipocytes at 10 µM. Furthermore, H7-OG showed additive effects in reporter gene assays and caused noncompetitive reactions in time-resolved fluorescence resonance energy transfer assays with a thiazolidinedione derivative. Our results indicated that hesperetin glucuronides activated PPARγ, accelerated adipocyte differentiation.

Her2/neu small interfering RNA delivered in culture by a streptavidin nanoparticle.

A three-component nanoparticle consisting of biotinylated Trastuzumab antiHer2 antibody, tat transferring peptide and radiolabeled antisense oligomer, linked together through streptavidin, have shown promise in the delivery to Her2+ tumor in mice following intravenous administration and with evidence of radiotherapeutic efficacy. These results have encouraged us to consider the nanoparticle as a delivery vehicle for RNA interference therapy in which the radiolabeled antisense oligomer is replaced with an unlabeled siRNA duplex. The siRNA stability within the nanoparticle was first confirmed by incubation with RNase A. The interferon responses, that indicate off-target cytotoxicity, were evaluated by quantitative real-time RT-PCR in BT-474 (Her2+) human breast cancer cells by measuring the mRNA expression of 2', 5'-oligoadenylate synthetase (OAS1) and Stat-1, two key interferon-responsive genes. Thereafter the cytotoxicity induced by the siRNA nanoparticle was evaluated by a clonogenic survival assay in BT-474 cells while the Her2 expression of these target cells was evaluated for evidence of specific gene silencing. The siRNA within the three-component anti- Her2/neu siRNA nanoparticle was largely protected from RNase-dependent degradation and did not activate an interferon response. The nanoparticle effectively and significantly inhibited colony formation of the target cells and silenced the Her2 gene expression at 5 nM compared with the identical nanoparticle with a scrambled siRNA. Our delivery nanoparticle, with tumor targeting provided by the antibody and its accumulation without entrapment, possibly due to the transfecting peptide, delivered an siRNA duplex to the proper subcellular localization for specific and effective gene silencing in culture by what appears to be an siRNA mechanism.

Auger-mediated cytotoxicity of cancer cells in culture by an 125I-antisense oligomer delivered as a three-component streptavidin nanoparticle.

We reported recently that a three-component nanoparticle, consisting of a targeting antibody, a transfecting peptide and an 111In-antiRIalpha MORF antisense oligomer, provided Auger electron-mediated, antisense-mediated, cytotoxicity of cells in culture. We have now measured the cytotoxicity of the nanoparticle in culture with the 111In replaced by 125I, another attractive Auger electron emitter. The nanoparticle consisted of streptavidin linking the 125I labeled antiRIalpha mRNA antisense MORF oligomer, the tat transfecting peptide and the anti-Her2 Trastuzumab antibody. Cytotoxicity was evaluated by a clonogenic survival assay in BT-474 (Her2+) human breast cancer cells. In a dose escalation study, as measured by the surviving fraction, the cytotoxicity of tumor cells to the 125I-labeled antisense nanoparticle was significantly higher than that for the identical sense control. When compared with our previous study with 111In as label, a similar level of cytotoxicity was achieved but the observed minimal therapeutic dose for the 125I-labeled nanoparticle in BT-474 cells was lower than that for 111In-labeled nanoparticle in SK-BR-3 cells. Thus, a radiolabeled antisense MORF oligomer delivered into cells by a three-component nanoparticle is an effective vehicle for Auger radiotherapy when radiolabeled with 111In or 125I.

In vivo delivery of antisense MORF oligomer by MORF/carrier streptavidin nanoparticles.

Tumor targeting by oligomers is largely limited by the pharmacokinetics and cell-membrane transport obstacles. In this article, we describe the use of a delivery nanoparticle, in which streptavidin served as a convenient bridge between a biotinylated oligomer and a biotinylated cell-membrane-penetrating peptide, to improve the delivery of an antisense phosphorodiamidate morpholino (MORF) oligomer in vivo. A biotinylated (99m)Tc-radiolabeled MORF oligomer with a base sequence antisense to the RIalpha mRNA and its sense control were incorporated separately into nanoparticles, along with biotinylated tat or polyarginine carrier. The streptavidin nanoparticles were administrated intravenously to both normal and nude mice bearing SUM149 breast tumor xenografts. The biodistributions showed much higher normal tissue levels for the radiolabeled MORFs, independent of antisense or sense or tat or polyarginine, when administered as the nanoparticles, compared to naked. A statistically significant higher accumulation of both antisense nanoparticles, compared to the respective sense control nanoparticles, was observed, along with much higher tumor accumulations, compared to historical naked controls. This study has provided evidence that the in vivo function of an antisense oligomer within the streptavidin nanoparticle is not impeded, and, as such, the MORF/streptavidin/carrier nanoparticles may be suitable for in vivo tumor delivery of antisense MORF and other oligomers.

Development of an ultra-high resolution SPECT system with a CdTe semiconductor detector.

OBJECTIVE: The aim of this work was to evaluate an ultra-high spatial resolution SPECT system with a semiconductor detector and a high-resolution parallel-hole collimator or a pinhole collimator for small animal imaging. METHODS: We evaluated an ultra-high spatial resolution SPECT system with a high-resolution parallel-hole collimator attached to a cadmium telluride (CdTe) semiconductor detector for small animal imaging. The sizes of an active area and a pixel in the semiconductor detector were 44 x 44 and 0.5 x 0.5 mm(2), respectively. In the high-resolution parallel-hole collimator the size of a hole was 0.4 x 0.4 mm(2), the thickness of a septum 0.1 mm, and the hole-length 30 mm. We also used a high-resolution pinhole collimator with a hole size of 0.3 or 0.5 mmvarphi. The physical performance of this SPECT system was evaluated with some experiments with phantoms filled with (99m)Tc-pertechnatate solution. In addition ideal performance and limitations of the system were evaluated with Monte Carlo simulations under the same geometrical conditions as in the experiments. In the evaluation for small animal imaging, we used mice that were administered with (99m)Tc-MDP. We also conducted an ultra-high resolution X-ray CT of the mice to verify the accumulated location of (99m)Tc-MDP using the bone CT images of the mice. RESULTS: The results of the phantom experiments showed that we could resolve 1 mmvarphi hot-channels and 1.6 mmvarphi cold-rods with the high-resolution parallel-hole collimator and pinhole collimators. We could image 0.3 mmvarphi hot-channels with the high-resolution pinhole collimators. The results of the simulations showed that the resolution limit in the pinhole imaging was about 0.6 mm FWHM. And the results of experiments with mice showed that we could reconstruct high-resolution images of (99m)Tc-MDP. Furthermore, the distribution of (99m)Tc-MDP in a mouse was found to correspond closely to the location of the bones of the mouse in reconstructions made with the ultra-high resolution X-ray CT system. CONCLUSIONS: Our results demonstrated that the ultra-high spatial resolution SPECT system was feasible for small animal imaging allowing a relatively long data acquisition time.

Lip vermilion profile patterns and corresponding dentoskeletal forms in female adults.

OBJECTIVES: To objectively classify shapes of the human lip vermilion in the lateral view, and to examine whether any morphologic characteristics of dentoskeletal patterns are specific to each classified lip profile pattern. MATERIALS AND METHODS: Pretreatment, lateral facial photographs of 234 Japanese women were selected. Investigators with expert knowledge of the anatomic traits of the lip vermilion in the lateral view extracted from images of the face a 13-dimensional feature vector that represented lip vermilion profile shapes. The vector quantization technique was applied to the feature vectors to mathematically optimize the number of lip vermilion profile patterns. Dentoskeletal patterns that corresponded to each classified lip shape were compared statistically. RESULTS: Seven patterns were found, and differences between patterns were notably maximized by the flatness of the anterior portion of the lip vermilion for the upper and lower lip, the position of the most protruded point of the lip vermilion, lip fissure inclination, and differences between the horizontal positions of the upper and lower lip vermilions. The dentoskeletal forms showed significant differences between classified lip vermilion profile patterns (P < .01). CONCLUSIONS: (1) Vector quantization revealed that classifying lip vermilion profiles into seven representative patterns was optimal for maximizing differences in the configuration of the lip vermilion. (2) Lip vermilion profile shapes were found to be associated with horizontal lengths of the anterior cranial base, horizontal/vertical positions, inclination and length of the mandible, and horizontal positions and labio-lingual inclinations of the upper and lower incisors.

Optical antisense tumor targeting in vivo with an improved fluorescent DNA duplex probe.

Fluorescent conjugated DNA oligonucleotides for antisense targeting of mRNA has the potential of improving tumor/normal tissue ratios over that achievable by nuclear antisense imaging. By conjugating the Cy5.5 emitter to the 3' equivalent end of a 25 mer phosphorothioate (PS) antisense major DNA and hybridizing with a shorter 18 mer phosphodiester (PO) complementary minor DNA (cDNA) with the Black Hole inhibitor BHQ3 on its 5' end (i.e., PS DNA25-Cy5.5/PO cDNA18-BHQ3), we previously achieved antisense optical imaging in mice as a proof of this concept. In a process of optimization, we have now evaluated the stability of a small series of duplexes with variable-length minor strands. From these results, a new study anti-mdr1 antisense duplex was selected with a 10 mer minor strand (i.e., PS DNA25-Cy5.5/PO cDNA10-BHQ3). The new study duplex shows stability in serum environments at 37 degrees C and provides a dramatically enhanced fluorescence in KB-G2 (pgp++) cells when compared with KB-31 (pgp+/-) as evidence of antisense dissociation at its mdr1 mRNA target. The duplex was also administered to KB-G2 tumor bearing mice, and when compared to the duplex used previously, the fluorescence from the tumor thigh was more obvious and the tumor-to-background fluorescence ratio was improved. In conclusion, by a process designed to optimize the duplex for optical antisense tumor targeting, the fluorescence signal was improved both in cells and in tumored mice.

A convenient thiazole orange fluorescence assay for the evaluation of DNA duplex hybridization stability.

OBJECTIVE: A simple and rapid method for measuring the hybridization stability of duplexes of DNAs and other oligomers in different environments is described. When added to an oligomer duplex, the thiazole orange (TO) dye intercalates and in this state is fluorescent. Therefore, when duplex dissociation occurs, the release of TO results in a detectable change in fluorescence intensity. This assay was developed primarily to screen antisense oligomer duplexes that are stable in serum and in the cytoplasm but unstable in the presence of their target messenger RNA (mRNA). METHODS: The two antisense oligomers of this investigation were both 25 mer phosphorothioate (PS) DNAs, one directed against the RIalpha mRNA and the other directed against the mdr1 mRNA. The former duplex was first used in the solution studies, in most cases duplexed with a 16 mer phosphodiester (PO) complementary DNA (i.e., PS-DNA25/PO-cDNA16). Both duplexes were then tested in a series of cell studies using SK-BR-3 (RIalpha+), KB-G2 (mdr1++), and KB-31 (mdr1+/-) cells. RESULTS: Preliminary measurements in solution showed that maximum fluorescence was achieved when more than ten TO molecules were bound to each duplex. When a 25 mer PO-DNA or PO-RNA with the base sequence of the RIalpha mRNA was added, the dramatic change in fluorescence intensity that followed signified dissociation of the antisense DNA from the study duplex and reassociation with the target DNA. Kinetic measurements showed that this process was completed in about 3 min. Fluorescent measurements of SK-BR-3 (RIalpha+) cells incubated at 37 degrees C with the anti-RIalpha study duplex over time showed a maximum at the point where the loss of fluorescence due to dissociation of the study duplex, probably by an antisense mechanism, began to dominate over the increasing fluorescence due to continuing cellular accumulation. A similar result was observed in the KB-G2 (mdr1+) cells incubated with the anti-mdr1 study duplex. CONCLUSIONS: When study duplexes shown to be stable in serum were incubated with their target cells, the assay successfully detected evidence of dissociation, most likely by an antisense mechanism. Thus, a TO fluorescence assay has been developed that is capable of detecting the dissociation of DNA duplexes.

Auger radiation-induced, antisense-mediated cytotoxicity of tumor cells using a 3-component streptavidin-delivery nanoparticle with 111In.

UNLABELLED: When antisense oligomers are intracellular, they migrate to and are retained in the nucleus of tumor cells and therefore may be used to carry Auger electron-emitting radionuclides such as (111)In for effective tumor radiotherapy. METHODS: Our nanoparticle consists of streptavidin that links 3 biotinylated components: the antiHer2 antibody trastuzumab (to improve pharmacokinetics), the tat peptide (to improve cell membrane transport), and the (111)In-labeled antiRIalpha messenger RNA antisense morpholino (MORF) oligomer. RESULTS: As evidence of unimpaired function, tumor cell and nuclear accumulations were orders of magnitude higher after incubation with (99m)Tc-MORF/tat/trastuzumab than after incubation with free (99m)Tc-MORF and significantly higher with the antisense than with the sense MORF. In mice, tumor and normal-tissue accumulations of the (99m)Tc-MORF/tat/trastuzumab nanoparticle were comparable to those of free (99m)Tc-trastuzumab, confirming the improved pharmacokinetics due to the trastuzumab component. Although kidneys, liver, and other normal tissues also accumulated the nanoparticle, immunohistochemical evaluation of tissue sections in mice receiving the Cy3-MORF/tat/trastuzumab nanoparticle showed evidence of nuclear accumulation only in tumor tissue. In a dose escalation study, as measured by the surviving fraction, the nanoparticle significantly increased the kill of SK-BR-3 breast cancer Her2+/RIalpha+ cells, compared with all controls. CONCLUSION: Significant radiation-induced antisense-mediated cytotoxicity of tumor cells in vitro was achieved using an Auger electron-emitting antisense MORF oligomer administered as a member of a 3-component streptavidin-delivery nanoparticle.

Cationic transfectors increase accumulation in cultured tumor cells of radiolabeled antisense DNAs without entrapment.

While cellular accumulations in culture of oligomers, such as interfering RNA and antisense DNA, are reported to benefit from the addition of transmembrane transfectors (TFs), the extent to which individual TFs improve cellular delivery is usually inferred and rarely measured. The goal of this investigation was to use radioactivity to measure in cells in culture the degree to which accumulations of DNA increased when complexed with TFs and without DNA entrapment in vesicles. The antisense (AS) DNA targeting mdr1 mRNA coding for P-glycoprotein (Pgp) and its sense (S) complement DNA were radiolabeled with 99mTc and mixed with jetPEI, Chariot, or Neophectin over a range of TF/DNA ratios. Thereafter,the radiolabeled DNAs with and without TFs were incubated with KB-G2 (mdr1(+/+)) and KB-31 (mdr(+/-))cells at 37 degrees C in serum or serum-free media for 20-24 hours at a fixed DNA concentration of 13 nM. Cellular accumulations were increased under most incubation conditions and by as much as threefold with jetPEI and eightfold with Neophectin. As evidence against entrapment, the accumulations of AS DNAs were higher than S DNAs in virtually all measurements and higher in virtually all accumulations in the mdr1(+/+) cells, compared to the mdr1(+/-) cells. In conclusion, by using radiolabeled DNAs, definitive evidence was obtained showing that the addition of Neophectin and jet PEI increased cellular accumulations of both AS and S DNA without evidence of vesicle entrapment.

Cell culture and xenograft-bearing animal studies of radiolabeled antisense DNA carrier nanoparticles with streptavidin as a linker.

UNLABELLED: Transmembrane transfectors (carriers) are increasingly being viewed as helpful or even necessary to improve cellular delivery in connection with antisense tumor targeting and other applications requiring cell membrane transport of DNAs, RNAs, and other oligomers. We are investigating streptavidin as a convenient linker for biotinylated carriers and oligomers because it requires only simple mixing for preparation. The goal of this study was to evaluate antisense DNA-streptavidin-carrier nanoparticles for accumulation in cell culture and in xenograft-bearing mice. METHODS: The 3 carriers were cholesterol, a 10-mer Tat peptide, and a 10-mer polyarginine peptide. A 20-mer DNA targeting the mdr1 messenger RNA coding for Pgp expression was used as the phosphodiester (PO) DNA as well as the phosphorothioate (PS) DNA. In all cases, the (99m)Tc radiolabel was on the DNA. The 8 nanoparticles were first tested in mdr1(++) KB-G2 and TCO-1 cells and in mdr1(+/-) KB-31 cells in culture for evidence of improved accumulation and antisense targeting. Thereafter, the PS DNA-streptavidin-Tat, PO DNA-streptavidin-Tat, and PS DNA-streptavidin-cholesterol nanoparticles were administered intravenously to KB-G2 xenograft-bearing mice, and tissue distributions were measured. RESULTS: In culture, the PO nanoparticles showed increased accumulation compared with the corresponding nanoparticles without the carrier in all 3 cell types; in contrast, with the PS nanoparticles, any similar carrier-mediated increase may have been obscured by the much higher protein-binding affinity of PS DNA. As evidence of antisense targeting, the Tat and cholesterol PS nanoparticles showed statistically significant accumulation at 23 h in cells in the descending order TCO-1, KB-G2, and KB-31, although there were no significant differences among the PO nanoparticles. In xenograft-bearing mice, the tissue accumulation of both forms of the PS nanoparticles greatly exceeded that of the PO nanoparticles and, including in the tumor, were similar to that obtained previously for naked PS DNA. CONCLUSION: The presence of the streptavidin linker had no obvious detrimental effect on the functions of the carriers and antisense DNAs. The higher protein-binding affinity of the PS nanoparticles than the PO nanoparticles was still apparent both in vitro and in vivo, the pharmacokinetics of the PS nanoparticles were similar to that of naked PS DNA, and the carriers improved cellular accumulation, at least for the PO nanoparticles. These observations, taken together with the higher accumulation of both forms of the antisense PS nanoparticles in mdr1(++) KB-G2 and TCO-1 cells than in mdr1(+/-) KB-31 cells, suggest that further effort is justified to confirm that the antisense properties of the DNAs were not compromised by the presence of streptavidin.