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1,179 results on '"Rotem, R"'

2. Specific immunosuppressive role of nanodrugs targeting calcineurin in innate myeloid cells

Author

Colombo, M, Marongiu, L, Mingozzi, F, Marzi, R, Cigni, C, Facchini, F, Rotem, R, Valache, M, Stucchi, G, Rocca, G, Gornati, L, Rizzuto, M, Salvioni, L, Zanoni, I, Gori, A, Prosperi, D, Granucci, F, Colombo M., Marongiu L., Mingozzi F., Marzi R., Cigni C., Facchini F. A., Rotem R., Valache M., Stucchi G., Rocca G., Gornati L., Rizzuto M. A., Salvioni L., Zanoni I., Gori A., Prosperi D., Granucci F., Colombo, M, Marongiu, L, Mingozzi, F, Marzi, R, Cigni, C, Facchini, F, Rotem, R, Valache, M, Stucchi, G, Rocca, G, Gornati, L, Rizzuto, M, Salvioni, L, Zanoni, I, Gori, A, Prosperi, D, Granucci, F, Colombo M., Marongiu L., Mingozzi F., Marzi R., Cigni C., Facchini F. A., Rotem R., Valache M., Stucchi G., Rocca G., Gornati L., Rizzuto M. A., Salvioni L., Zanoni I., Gori A., Prosperi D., and Granucci F.

Abstract

Calcineurin (CN) inhibitors currently used to avoid transplant rejection block the activation of adaptive immune responses but also prevent the development of tolerance toward the graft, by directly inhibiting T cells. CN, through the transcription factors of the NFAT family, plays an important role also in the differentiation dendritic cells (DCs), the main cells responsible for the activation of T lymphocytes. Therefore, we hypothesized that the inhibition of CN only in DCs and not in T cells could be sufficient to prevent T cell responses, while allowing for the development of tolerance. Here, we show that inhibition of CN/NFAT pathway in innate myeloid cells, using a new nanoconjugate capable of selectively targeting phagocytes in vivo, protects against graft rejection and induces a longer graft acceptance compared to common CN inhibitors. We propose a new generation of nanoparticles-based selective immune suppressive agents for a better control of transplant acceptance.

Published
2022

3. Direct quantification of cytosolic delivery of drug nanocarriers using FlAsH-EDT2

Author

Rotem, R, Bertolini, J, Salvioni, L, Barbieri, L, Rizzuto, M, Tinelli, V, Gori, A, Adams, S, Colombo, M, Prosperi, D, Bertolini, JA, Rizzuto, MA, Rotem, R, Bertolini, J, Salvioni, L, Barbieri, L, Rizzuto, M, Tinelli, V, Gori, A, Adams, S, Colombo, M, Prosperi, D, Bertolini, JA, and Rizzuto, MA

Abstract

The delivery of therapeutics across the cell membrane and into the cytoplasm is a major challenge that limits the development of new therapies. This challenge is compounded by the lack of a general assay for cytosolic delivery. Here we develop this assay based on the pro-fluorophore CrAsH-EDT2, and provide cytosolic penetration results for a variety of drug delivery agents (polyethyleneimine, poly-arginine, Ferritin, poly [maleic anhydride-alt-isobutene] grafted with dodecylamine, and cationic liposomes) into HeLa and T98G cells. Our results show that this method can be widely applicable to different cells and drug delivery agents, and yield statistically robust results. We later use this method to optimize and improve a model drug delivery agent's (Ferritin) cytosolic penetration.

Published
2023

4. Conjugation of gold nanoparticles with multidentate surfactants for enhanced stability and biological properties

Author

Rotem, R, Giustra, M, Arrigoni, F, Bertolini, J, Garbujo, S, Rizzuto, M, Salvioni, L, Barbieri, L, Bertini, L, De Gioia, L, Colombo, M, Prosperi, D, Bertolini, JA, Rizzuto, MA, Rotem, R, Giustra, M, Arrigoni, F, Bertolini, J, Garbujo, S, Rizzuto, M, Salvioni, L, Barbieri, L, Bertini, L, De Gioia, L, Colombo, M, Prosperi, D, Bertolini, JA, and Rizzuto, MA

Abstract

This work originated from the need to functionalize surfactant-coated inorganic nanoparticles for biomedical applications, a process that is limited by excess unbound surfactant. These limitations are connected to the bioconjugation of targeting molecules that are often in equilibrium between the free aliquot in solution and that which binds the surface of the nanoparticles. The excess in solution can play a role in the biocompatability in vitro and in vivo of the final nanoparticles stock. For this purpose, we tested the ability of common surfactants - monothiolated polyethylene glycol and amphiphilic polymers - to colloidally stabilize nanoparticles as excess surfactant is removed and compared them to newly appearing multidentate surfactants endowed with high avidity for inorganic nanoparticles. Our results showed that monothiolated polyethylene glycol or amphiphilic polymers have an insufficient affinity to the nanoparticles and as the excess surfactant is removed the colloidal stability is lost, while multidentate high-avidity surfactants excel in the same regard, possibly allowing improvement in an array of nanoparticle applications, especially in those stated.

Published
2023

6. Development of an Effective Tumor-Targeted Contrast Agent for Magnetic Resonance Imaging Based on Mn/H-Ferritin Nanocomplexes

Author

Tullio, C, Salvioni, L, Bellini, M, Degrassi, A, Fiandra, L, D'Arienzo, M, Garbujo, S, Rotem, R, Testa, F, Prosperi, D, Colombo, M, Tullio C., Salvioni L., Bellini M., Degrassi A., Fiandra L., D'Arienzo M., Garbujo S., Rotem R., Testa F., Prosperi D., Colombo M., Tullio, C, Salvioni, L, Bellini, M, Degrassi, A, Fiandra, L, D'Arienzo, M, Garbujo, S, Rotem, R, Testa, F, Prosperi, D, Colombo, M, Tullio C., Salvioni L., Bellini M., Degrassi A., Fiandra L., D'Arienzo M., Garbujo S., Rotem R., Testa F., Prosperi D., and Colombo M.

Abstract

Magnetic resonance imaging (MRI) is one of the most sophisticated diagnostic tools that is routinely used in clinical practice. Contrast agents (CAs) are commonly exploited to afford much clearer images of detectable organs and to reduce the risk of misdiagnosis caused by limited MRI sensitivity. Currently, only a few gadolinium-based CAs are approved for clinical use. Concerns about their toxicity remain, and their administration is approved only under strict controls. Here, we report the synthesis and validation of a manganese-based CA, namely, Mn@HFn-RT. Manganese is an endogenous paramagnetic metal able to produce a positive contrast like gadolinium, but it is thought to result in less toxicity for the human body. Mn ions were efficiently loaded inside the shell of a recombinant H-ferritin (HFn), which is selectively recognized by the majority of human cancer cells through their transferrin receptor 1. Mn@HFn-RT was characterized, showing excellent colloidal stability, superior relaxivity, and a good safety profile. In vitro experiments confirmed the ability of Mn@HFn-RT to efficiently and selectively target breast cancer cells. In vivo, Mn@HFn-RT allowed the direct detection of tumors by positive contrast enhancement in a breast cancer murine model, using very low metal dosages and exhibiting rapid clearance after diagnosis. Hence, Mn@HFn-RT is proposed as a promising CA candidate to be developed for MRI.

Published
2021

8. Modeling the interaction of amphiphilic polymer nanoparticles with biomembranes to Guide rational design of drug delivery systems

Author

Rotem, R, Micale, A, Rizzuto, M, Migliavacca, M, Giustra, M, Salvioni, L, Tasin, F, Prosperi, D, Colombo, M, Rotem R., Micale A., Rizzuto M. A., Migliavacca M., Giustra M., Salvioni L., Tasin F., Prosperi D., Colombo M., Rotem, R, Micale, A, Rizzuto, M, Migliavacca, M, Giustra, M, Salvioni, L, Tasin, F, Prosperi, D, Colombo, M, Rotem R., Micale A., Rizzuto M. A., Migliavacca M., Giustra M., Salvioni L., Tasin F., Prosperi D., and Colombo M.

Abstract

Nanoparticle assisted drug delivery to the cytoplasm is limited by sequestration of nanoparticles in endosomes. Endosomal escape through polymer-induced membrane destabilization is one of a few well characterized mechanisms to overcome it. Aiming to utilize this method in vivo, it is necessary to understand how modulating the structural and chemical features of the polymer and the presence of proteins in biological fluids can affect this activity. Here, as a model for the endosomal membrane, we use the membrane of red blood cells to evaluate the membrane destabilization ability of a model amphiphilic polymer in the presence of blood plasma using a hemolysis assay. This allows determination of red blood cells membrane permeabilization through the quantification of hemoglobin leakage. Our results showed a strong inhibitory effect of plasma, and that hemolytic activity can be improved by chemical modification of the polymeric micelle, reducing its interaction with plasma proteins. Finally, a second mechanism of pH-induced direct diffusion is proposed and tested using an oil/water partitioning model. These results offer a body of knowledge to improve delivery of drugs across biological membranes using carefully designed polymeric nanocarriers.

Published
2020

9. MnO Nanoparticles Embedded in Functional Polymers as T1 Contrast Agents for Magnetic Resonance Imaging

Author

Mauri, M, Collico, V, Morelli, L, Das, P, Garcia, I, Penaranda Avila, J, Bellini, M, Rotem, R, Truffi, M, Corsi, F, Simonutti, R, Liz-Marzan, L, Colombo, M, Prosperi, D, Mauri M., Collico V., Morelli L., Das P., Garcia I., Penaranda Avila J., Bellini M., Rotem R., Truffi M., Corsi F., Simonutti R., Liz-Marzan L. M., Colombo M., Prosperi D., Mauri, M, Collico, V, Morelli, L, Das, P, Garcia, I, Penaranda Avila, J, Bellini, M, Rotem, R, Truffi, M, Corsi, F, Simonutti, R, Liz-Marzan, L, Colombo, M, Prosperi, D, Mauri M., Collico V., Morelli L., Das P., Garcia I., Penaranda Avila J., Bellini M., Rotem R., Truffi M., Corsi F., Simonutti R., Liz-Marzan L. M., Colombo M., and Prosperi D.

Abstract

The design and development of contrast agents (CAs) for magnetic resonance imaging (MRI) in clinical analysis is expected to improve the image spatial resolution and to increase the detection sensitivity, especially regarding neurological disorders and cancer disease. In particular, advanced CAs for T1-weighted images are investigated to achieve the sensitive detection of early-stage primary tumors or brain metastases. In this study, we present a strategy toward diagnostic T1 CAs for MRI, based on polymer-modified MnO nanoparticles (NPs). Two different nanosystems were synthesized, consisting of (1) colloidal MnO nanocrystals wrapped by a multidentate amphiphilic polymer and (2) MnO nanocrystals embedded within poly(lactic-co-glycolic acid) (PLGA) NPs. These nanosystems were compared in terms of their MRI contrast power and biological safety. The latter system combines the excellent biocompatibility of PLGA with the unique magnetic properties of MnO NPs and allows sustained contrast enhancement over time. Longitudinal relaxivities of both MnO composite nanomaterials proved to be higher than those of commercial Gd-based CAs and Teslascan, both in phosphate-buffered saline and in plasma, also exhibiting low cytotoxicity. The high relaxation rates achieved with these contrast enhancers are promising toward future application in in vivo imaging.

Published
2020

10. Targeted delivery of nanoparticles

Author

Parak, WJ, Feliu, N, Rotem, R, Prosperi, D, Colombo, M, Rotem R., Prosperi D., Colombo M., Parak, WJ, Feliu, N, Rotem, R, Prosperi, D, Colombo, M, Rotem R., Prosperi D., and Colombo M.

Abstract

The curative effectiveness of current and new drugs is often limited by poor pharmacokinetics in vivo. Nanoparticles could be the solution to this problem, as they are the right size to infiltrate and interact with biological systems (i.e., size is similar to proteins). They could allow the division of this big problem into two smaller problems: in vivo delivery, achieved by the nanoparticle, and target site interaction, achieved by the drug. This chapter focuses on the first smaller problem, summarizing the nanoparticle properties governing their biodistribution, i.e., size, surface charge, nonspecific interactions, elasticity, geometry, molecular recognition, and triggered response. It discusses current research focused on controlling and characterizing these parameters and the creation of new drug delivery platforms for specific applications.

Published
2020

12. Are nanotechnological approaches the future of treating inflammatory diseases?

Author

Rizzuto, M, Salvioni, L, Rotem, R, Colombo, M, Zanoni, I, Granucci, F, Prosperi, D, Rizzuto M. A., Salvioni L., Rotem R., Colombo M., Zanoni I., Granucci F., Prosperi D., Rizzuto, M, Salvioni, L, Rotem, R, Colombo, M, Zanoni, I, Granucci, F, Prosperi, D, Rizzuto M. A., Salvioni L., Rotem R., Colombo M., Zanoni I., Granucci F., and Prosperi D.

Abstract

The current treatments for chronic inflammatory diseases cause severe side effects due to nonspecific drug accumulation. Nanotechnology opens the way to new therapeutic strategies that exploit the ability of immune cells, and especially of phagocytes, to internalize nanoparticles. The cellular uptake of nanoparticles requires specific interactions and is affected by the chemical and physical properties of the carriers. Therefore, optimizing these properties is crucial for designing nanodrugs for immunotherapy. In perspective, we discuss the nanoparticle-based approaches that have been proposed to induce tolerance in autoimmune disorders and lessen the symptoms of inflammatory diseases.

Published
2019

16. Covid‐19 vaccination during the third trimester of pregnancy: rate of vaccination and maternal and neonatal outcomes, a multicentre retrospective cohort study.

Author

Rottenstreich, M, Sela, HY, Rotem, R, Kadish, E, Wiener‐Well, Y, and Grisaru‐Granovsky, S

Subjects
THIRD trimester of pregnancy, COVID-19 vaccines, IMMUNIZATION of children, COVID-19, VACCINATION, OBSTETRICAL extraction, NATIONAL health insurance
Abstract

Objective: To evaluate the impact of Covid‐19 vaccination (Pfizer–BioNTech BNT162b2) during the third trimester of pregnancy on maternal and neonatal outcomes. Design: A multicentre, retrospective computerised database. Population: Women who gave birth at >24 weeks of gestation in Israel, between January and April 2021, with full records of Covid‐19 disease and vaccination status. Methods: Women who received two doses of the vaccine were compared with unvaccinated women. Women who were recorded as having disease or a positive Covid‐19 polymerase chain reaction (PCR) swab during pregnancy or delivery were excluded from both study groups. Univariate analysis was followed by multivariate logistic regression. Main outcome measures: Composite adverse maternal outcomes. Secondary outcomes were vaccination rate and composite adverse neonatal outcomes. Results: The overall uptake of one or both vaccines was 40.2%; 712 women who received two doses of the Covid‐19 vaccine were compared with 1063 unvaccinated women. Maternal composite outcomes were comparable between the groups; however, women who received the vaccine had higher rates of elective caesarean deliveries (CDs) and lower rates of vacuum deliveries. An adjusted multivariable logistic regression analysis demonstrated that Covid‐19 vaccination was not associated with maternal composite adverse outcome (aOR 0.8, 95% CI 0.61–1.03); a significant reduction in the risk for neonatal composite adverse outcomes was observed (aOR 0.5, 95% CI 0.36–0.74). Conclusions: In a motivated population covered by a National Health Insurance Plan, we found a 40.2% rate of vaccination for the Covid‐19 vaccine during the third trimester of pregnancy, which was not associated with adverse maternal outcomes and, moreover, decreased the risk for neonatal adverse outcomes. Covid‐19 vaccine during pregnancy is safe for both mother and fetus. Covid‐19 vaccine during pregnancy is safe for both mother and fetus. [ABSTRACT FROM AUTHOR]

Published
2022
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19. Association of blood mercury levels with non-melanoma skin cancer in the United States using NHANES data from 2003-2016

Author

Vance T, Rhee J, Cho E, Rotem R, and Qureshi A

Subjects
Oncology, Global and Planetary Change, medicine.medical_specialty, Epidemiology, business.industry, Health, Toxicology and Mutagenesis, Public Health, Environmental and Occupational Health, medicine.disease, Pollution, Blood mercury, Internal medicine, medicine, Skin cancer, business, Non melanoma
Published
2019
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20. Development of Reliable Experimental Models for the Study of the Biological Behavior of Drug Nanocarriers

Author

COLOMBO, MIRIAM, Rotem, R, PROSPERI, DAVIDE, ROTEM, RANY, COLOMBO, MIRIAM, Rotem, R, PROSPERI, DAVIDE, and ROTEM, RANY

Abstract

The curative effectiveness of current and new drugs is often limited by poor pharmacokinetics in-vivo. The use of nanoparticles as drug carriers seems promising in solving this problem. In this work we aimed to further explore and improve common drug delivery components and techniques. Starting with the synthesis of nanoparticles with a controlled number of molecular recognition ligands, we used bulky ligands and gel separation to obtain nanoparticles with a discrete number of chemical functional groups, used later to conjugate the same number of molecular recognition ligands. These nanoparticles later showed substantial difference in the in-vivo behavior. A second project focused on the in-depth characterization of the relationship between hydrophobic inorganic nanoparticles and the polymer surfactants used to enable their water dispersibility, as well enabling their functionalization. This investigation was done through separate quantification of polymer and inorganic nanoparticles and assessment of stability. Our results showed that the removal of excess polymer from such systems can result in loss of colloidal stability. A third project was aimed to describe the mechanism of polymeric nanoparticle’s endosomal escape and provide a platform for qualitative investigation and enhancement of this process. This goal was accomplished through two complementary in-vitro experiments testing two proposed mechanisms of endosomal escape. These results raised a key consideration when matching a particle capable of endosomal escape to a specific cell type as well as methods reduce interaction with serum proteins. A fourth project focused on developing an assay to quantify cytosolic delivery of nanoparticles and theoretically assessed the possibility of using fluorescence resonance energy transfer (FRET) - which was found to be not practical in this case - as well as implementing a pro-fluorophore to generate a measurable signal. Our preliminary results indicate this method mig, The curative effectiveness of current and new drugs is often limited by poor pharmacokinetics in-vivo. The use of nanoparticles as drug carriers seems promising in solving this problem. In this work we aimed to further explore and improve common drug delivery components and techniques. Starting with the synthesis of nanoparticles with a controlled number of molecular recognition ligands, we used bulky ligands and gel separation to obtain nanoparticles with a discrete number of chemical functional groups, used later to conjugate the same number of molecular recognition ligands. These nanoparticles later showed substantial difference in the in-vivo behavior. A second project focused on the in-depth characterization of the relationship between hydrophobic inorganic nanoparticles and the polymer surfactants used to enable their water dispersibility, as well enabling their functionalization. This investigation was done through separate quantification of polymer and inorganic nanoparticles and assessment of stability. Our results showed that the removal of excess polymer from such systems can result in loss of colloidal stability. A third project was aimed to describe the mechanism of polymeric nanoparticle’s endosomal escape and provide a platform for qualitative investigation and enhancement of this process. This goal was accomplished through two complementary in-vitro experiments testing two proposed mechanisms of endosomal escape. These results raised a key consideration when matching a particle capable of endosomal escape to a specific cell type as well as methods reduce interaction with serum proteins. A fourth project focused on developing an assay to quantify cytosolic delivery of nanoparticles and theoretically assessed the possibility of using fluorescence resonance energy transfer (FRET) - which was found to be not practical in this case - as well as implementing a pro-fluorophore to generate a measurable signal. Our preliminary results indicate this method mig

Published
2019

23. Bioengineered Approaches for Site Orientation of Peptide-Based Ligands of Nanomaterials

Author

Avvakumova, S, Colombo, M, Galbiati, E, Mazzucchelli, S, Rotem, R, Prosperi, D, ROTEM, RANY, Avvakumova, S, Colombo, M, Galbiati, E, Mazzucchelli, S, Rotem, R, Prosperi, D, and ROTEM, RANY

Abstract

Successful application of nanoconjugates either in vitro or in vivo does depend on the design strategy used for their development. In this chapter, we focus on the most important factors that need to be taken into account to optimize nanoparticle biofunctionalization and provide an overview of recent approaches yielding the efficient and orientation-controlled immobilization of complex molecules, including peptides and proteins. In addition, we discuss on the elements that cooperate with ligand orientation in affecting the biological activity of nanoconjugates. Indeed, the control of ligand orientation on the nanoparticle surface is of crucial importance in determining the affinity of the immobilized biomolecules toward the biological target. However, the ability of a nanoconjugate to recognize its target is even strongly affected by other important items, including (1) multivalency as a result of ligand density and organization; (2) conformational changes potentially occurring on binding of ligands to the nanoparticle; (3) thermodynamic and kinetic factors related to the interaction of the ligand with the nanoparticle surface; and (4) interaction between nanoparticle and its surrounding environment to form the so-called "protein corona." Altogether, these five issues are the driving force in the specificity and selectivity of designed nanoconjugates toward their target, and, as a consequence, define their potential utility for biomedical application. Therefore, a careful control on the abovementioned "magic five" contributions is needed to improve the quality standard of the next-generation nanomedicines. In addition to the illustrated nanobioconjugation strategies, we emphasize that all these features need to be carefully optimized when assessing the biological effects of synthetic nanoparticles designed for biomedical purposes.

Published
2018

26. Tumour homing and therapeutic effect of colloidal nanoparticles depend on the number of attached antibodies

Author

Colombo, M, Fiandra, L, Alessio, G, Mazzucchelli, S, Nebuloni, M, De Palma, C, Kantner, K, Pelaz, B, Rotem, R, Corsi, F, Parak, W, Prosperi, D, COLOMBO, MIRIAM, ALESSIO, GIULIA, MAZZUCCHELLI, SERENA, ROTEM, RANY, PROSPERI, DAVIDE, Colombo, M, Fiandra, L, Alessio, G, Mazzucchelli, S, Nebuloni, M, De Palma, C, Kantner, K, Pelaz, B, Rotem, R, Corsi, F, Parak, W, Prosperi, D, COLOMBO, MIRIAM, ALESSIO, GIULIA, MAZZUCCHELLI, SERENA, ROTEM, RANY, and PROSPERI, DAVIDE

Abstract

Active targeting of nanoparticles to tumours can be achieved by conjugation with specific antibodies. Specific active targeting of the HER2 receptor is demonstrated in vitro and in vivo with a subcutaneous MCF-7 breast cancer mouse model with trastuzumab-functionalized gold nanoparticles. The number of attached antibodies per nanoparticle was precisely controlled in a way that each nanoparticle was conjugated with either exactly one or exactly two antibodies. As expected, in vitro we found a moderate increase in targeting efficiency of nanoparticles with two instead of just one antibody attached per nanoparticle. However, the in vivo data demonstrate that best effect is obtained for nanoparticles with only exactly one antibody. There is indication that this is based on a size-related effect. These results highlight the importance of precisely controlling the ligand density on the nanoparticle surface for optimizing active targeting, and that less antibodies can exhibit more effect.

Published
2016

29. O04 Fetal Outcome following Dydrogesterone Exposure in Pregnancy

Author

Koren, G, Gilboa, D, Rotem, R, Levy, A, Daniel, S, and Shalev, V

Abstract

BackgroundThe progestin dydrogesterone (DYD) is widely used for threatened and recurrent miscarriages, as well as for dysfunctional bleeding, infertility and other obstetric and gynecological indications. While its apparent efficacy has been compared to other progestins, its fetal safety has not been investigated.ObjectivesTo follow up fetal outcome after gestational exposure to DYD.Patients and methodsUsing a 2.5 million patients database, we compared congenital malformations among babies exposed in uteroto DYD between 1999 and 2016, to a control group not receiving this medication. We adjusted for concomitant exposure to in vitrofertilization (IVF) and to other forms of assisted reproductive technology (ART).ResultsThere were 8508 children exposed in uteoto DYD (4417 males, 4091 females) out of 777,422 live births. After excluding cases with concomitant exposure to IVF and other forms of ART, DYD was associated with increased risk for hypospadias [OR 1.28(95% confidence interval 1.06–1.55)], overall cardiovascular malformations [OR 1.18 (1.06–1.33)], spina bifida [OR 2.29(1.32–3.97] and hydrpcephalus [OR 2.04(1.28–3.25]. In additional analysis, including also those exposed to IVF and other forms of ART, there was also increased risk for cryptorchidism [1.37(1.19–1.58)] and congenital dislocation of the hip [OR 1.58(1.42–1.78)].ConclusionsDYD confers teratogenic effects after exposure to the recommended doses in pregnant women. Some of these adverse fetal effects are further augmented by concomitant use of IVF and other forms of ART. These independent teratogenic effects may have important implication for the child and family.Disclosure(s)Nothing to disclose

Published
2019
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30. Conjugation of gold nanoparticles with multidentate surfactants for enhanced stability and biological properties

Author

Rany Rotem, Marco Giustra, Federica Arrigoni, Jessica A. Bertolini, Stefania Garbujo, Maria A. Rizzuto, Lucia Salvioni, Linda Barbieri, Luca Bertini, Luca De Gioia, Miriam Colombo, Davide Prosperi, Rotem, R, Giustra, M, Arrigoni, F, Bertolini, J, Garbujo, S, Rizzuto, M, Salvioni, L, Barbieri, L, Bertini, L, De Gioia, L, Colombo, M, and Prosperi, D

Subjects
Gold nanoparticle, Polyethylene glycol, Sol, Biomedical application, Medical application, Biomedical Engineering, Amphiphilic polymer, General Chemistry, General Medicine, Surface active agents, Inorganic nanoparticle, Stability propertie, Enhanced stability, Polyethylene, Multidentate, Bio-conjugation, Biological propertie, General Materials Science
Abstract

This work originated from the need to functionalize surfactant-coated inorganic nanoparticles for biomedical applications, a process that is limited by excess unbound surfactant. These limitations are connected to the bioconjugation of targeting molecules that are often in equilibrium between the free aliquot in solution and that which binds the surface of the nanoparticles. The excess in solution can play a role in the biocompatability in vitro and in vivo of the final nanoparticles stock. For this purpose, we tested the ability of common surfactants - monothiolated polyethylene glycol and amphiphilic polymers - to colloidally stabilize nanoparticles as excess surfactant is removed and compared them to newly appearing multidentate surfactants endowed with high avidity for inorganic nanoparticles. Our results showed that monothiolated polyethylene glycol or amphiphilic polymers have an insufficient affinity to the nanoparticles and as the excess surfactant is removed the colloidal stability is lost, while multidentate high-avidity surfactants excel in the same regard, possibly allowing improvement in an array of nanoparticle applications, especially in those stated.

Published
2023
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31. Direct quantification of cytosolic delivery of drug nanocarriers using FlAsH-EDT2

Author

R, Rotem, J A, Bertolini, L, Salvioni, L, Barbieri, M A, Rizzuto, V, Tinelli, A, Gori, S, Adams, M, Colombo, D, Prosperi, Rotem, R, Bertolini, J, Salvioni, L, Barbieri, L, Rizzuto, M, Tinelli, V, Gori, A, Adams, S, Colombo, M, and Prosperi, D

Subjects
Pharmaceutical Preparations, Drug delivery, Biomedical Engineering, Pharmaceutical Science, Molecular Medicine, Medicine (miscellaneous), General Materials Science, Bioengineering, Cell penetration, Flow cytometry, Quantification method, FlAsH-EDT2
Abstract

The delivery of therapeutics across the cell membrane and into the cytoplasm is a major challenge that limits the development of new therapies. This challenge is compounded by the lack of a general assay for cytosolic delivery. Here we develop this assay based on the pro-fluorophore CrAsH-EDT2, and provide cytosolic penetration results for a variety of drug delivery agents (polyethyleneimine, poly-arginine, Ferritin, poly [maleic anhydride-alt-isobutene] grafted with dodecylamine, and cationic liposomes) into HeLa and T98G cells. Our results show that this method can be widely applicable to different cells and drug delivery agents, and yield statistically robust results. We later use this method to optimize and improve a model drug delivery agent's (Ferritin) cytosolic penetration.

Published
2023

32. MnO Nanoparticles Embedded in Functional Polymers as T1 Contrast Agents for Magnetic Resonance Imaging

Author

Davide Prosperi, Michele Mauri, Michela Bellini, Lucia Morelli, Pradip Das, Isabel García, Miriam Colombo, Roberto Simonutti, Fabio Corsi, Rany Rotem, Luis M. Liz-Marzán, Jesus Penaranda Avila, Marta Truffi, Veronica Collico, Mauri, M, Collico, V, Morelli, L, Das, P, Garcia, I, Penaranda Avila, J, Bellini, M, Rotem, R, Truffi, M, Corsi, F, Simonutti, R, Liz-Marzan, L, Colombo, M, and Prosperi, D

Subjects
Materials science, medicine.diagnostic_test, media_common.quotation_subject, Nanoparticle, Magnetic resonance imaging, T1 contrast, plasma effect, contrast agent, Nuclear magnetic resonance, relaxivity, medicine, magnetic resonance imaging, Contrast (vision), manganese oxide nanoparticle, General Materials Science, Functional polymers, active polymer coating, human activities, Image resolution, media_common
Abstract

The design and development of contrast agents (CAs) for magnetic resonance imaging (MRI) in clinical analysis is expected to improve the image spatial resolution and to increase the detection sensitivity, especially regarding neurological disorders and cancer disease. In particular, advanced CAs for T1-weighted images are investigated to achieve the sensitive detection of early-stage primary tumors or brain metastases. In this study, we present a strategy toward diagnostic T1 CAs for MRI, based on polymer-modified MnO nanoparticles (NPs). Two different nanosystems were synthesized, consisting of (1) colloidal MnO nanocrystals wrapped by a multidentate amphiphilic polymer and (2) MnO nanocrystals embedded within poly(lactic-co-glycolic acid) (PLGA) NPs. These nanosystems were compared in terms of their MRI contrast power and biological safety. The latter system combines the excellent biocompatibility of PLGA with the unique magnetic properties of MnO NPs and allows sustained contrast enhancement over time. Longitudinal relaxivities of both MnO composite nanomaterials proved to be higher than those of commercial Gd-based CAs and Teslascan, both in phosphate-buffered saline and in plasma, also exhibiting low cytotoxicity. The high relaxation rates achieved with these contrast enhancers are promising toward future application in in vivo imaging.

Published
2020
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33. Specific immunosuppressive role of nanodrugs targeting calcineurin in innate myeloid cells

Author

Miriam Colombo, Laura Marongiu, Francesca Mingozzi, Roberta Marzi, Clara Cigni, Fabio Alessandro Facchini, Rany Rotem, Mihai Valache, Giulia Stucchi, Giuseppe Rocca, Laura Gornati, Maria Antonietta Rizzuto, Lucia Salvioni, Ivan Zanoni, Alessandro Gori, Davide Prosperi, Francesca Granucci, Colombo, M, Marongiu, L, Mingozzi, F, Marzi, R, Cigni, C, Facchini, F, Rotem, R, Valache, M, Stucchi, G, Rocca, G, Gornati, L, Rizzuto, M, Salvioni, L, Zanoni, I, Gori, A, Prosperi, D, and Granucci, F

Subjects
Multidisciplinary, Health science, Immunology, Drug, Immune response
Abstract

Calcineurin (CN) inhibitors currently used to avoid transplant rejection block the activation of adaptive immune responses but also prevent the development of tolerance toward the graft, by directly inhibiting T cells. CN, through the transcription factors of the NFAT family, plays an important role also in the differentiation dendritic cells (DCs), the main cells responsible for the activation of T lymphocytes. Therefore, we hypothesized that the inhibition of CN only in DCs and not in T cells could be sufficient to prevent T cell responses, while allowing for the development of tolerance. Here, we show that inhibition of CN/NFAT pathway in innate myeloid cells, using a new nanoconjugate capable of selectively targeting phagocytes in vivo, protects against graft rejection and induces a longer graft acceptance compared to common CN inhibitors. We propose a new generation of nanoparticles-based selective immune suppressive agents for a better control of transplant acceptance.

Published
2021

34. Inositol 1,4,5-trisphosphate 3-kinase B promotes Ca2+mobilization and the inflammatory activity of dendritic cells

Author

Laura Marongiu, Reiko Sakaguchi, Takashi Morii, Tiziano Catelani, Miriam Colombo, Davide Prosperi, Francesca Mingozzi, Rany Rotem, Massimiliano Garrè, Dario Parazzoli, Francesca Granucci, Monica Moro, Stéphane Schurmans, Roberta Marzi, Clara Cigni, Laura Sironi, Maddalena Collini, Marco Di Gioia, Mariacristina Crosti, Stephen B. Shears, Ivan Zanoni, Marongiu, L, Mingozzi, F, Cigni, C, Marzi, R, Di Gioia, M, Garre, M, Parazzoli, D, Sironi, L, Collini, M, Sakaguchi, R, Morii, T, Crosti, M, Moro, M, Schurmans, S, Catelani, T, Rotem, R, Colombo, M, Shears, S, Prosperi, D, Zanoni, I, and Granucci, F

Subjects
0303 health sciences, Receptor complex, Dendritic cells Inflammation NFAT, Innate immune system, Phospholipase C, Chemistry, CD14, NFAT, Cell Biology, Inositol trisphosphate receptor, Biochemistry, Cell biology, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, TLR4, Inositol, Molecular Biology, 030304 developmental biology, 030215 immunology
Abstract

Innate immune responses to Gram-negative bacteria depend on the recognition of lipopolysaccharide (LPS) by a receptor complex that includes CD14 and TLR4. In dendritic cells (DCs), CD14 enhances the activation not only of TLR4 but also that of the NFAT family of transcription factors, which suppresses cell survival and promotes the production of inflammatory mediators. NFAT activation requires Ca2+ mobilization. In DCs, Ca2+ mobilization in response to LPS depends on phospholipase C γ2 (PLCγ2), which produces inositol 1,4,5-trisphosphate (IP3). Here, we showed that the IP3 receptor 3 (IP3R3) and ITPKB, a kinase that converts IP3 to inositol 1,3,4,5-tetrakisphosphate (IP4), were both necessary for Ca2+ mobilization and NFAT activation in mouse and human DCs. A pool of IP3R3 was located on the plasma membrane of DCs, where it colocalized with CD14 and ITPKB. Upon LPS binding to CD14, ITPKB was required for Ca2+ mobilization through plasma membrane-localized IP3R3 and for NFAT nuclear translocation. Pharmacological inhibition of ITPKB in mice reduced both LPS-induced tissue swelling and the severity of inflammatory arthritis to a similar extent as that induced by the inhibition of NFAT using nanoparticles that delivered an NFAT-inhibiting peptide specifically to phagocytic cells. Our results suggest that ITPKB may represent a promising target for anti-inflammatory therapies that aim to inhibit specific DC functions.

Published
2021

35. Development of an Effective Tumor-Targeted Contrast Agent for Magnetic Resonance Imaging Based on Mn/H-Ferritin Nanocomplexes

Author

Lucia Salvioni, Miriam Colombo, Anna Degrassi, Massimiliano D’Arienzo, Davide Prosperi, Chiara Tullio, Stefania Garbujo, Michela Bellini, Luisa Fiandra, Filippo Testa, Rany Rotem, Tullio, C, Salvioni, L, Bellini, M, Degrassi, A, Fiandra, L, D'Arienzo, M, Garbujo, S, Rotem, R, Testa, F, Prosperi, D, and Colombo, M

Subjects
diagnostic imaging, Gadolinium, H-ferritin, Biomedical Engineering, chemistry.chemical_element, Contrast Media, Transferrin receptor, Antineoplastic Agents, Breast Neoplasms, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Article, law.invention, Biomaterials, Mice, Breast cancer, In vivo, law, medicine, Animals, Humans, medicine.diagnostic_test, tumor targeting, Biochemistry (medical), Magnetic resonance imaging, General Chemistry, contrast agent, 021001 nanoscience & nanotechnology, medicine.disease, Magnetic Resonance Imaging, In vitro, 3. Good health, 0104 chemical sciences, chemistry, Toxicity, Apoferritins, Cancer research, Recombinant DNA, manganese, Female, 0210 nano-technology, MRI
Abstract

Magnetic resonance imaging (MRI) is one of the most sophisticated diagnostic tools that is routinely used in clinical practice. Contrast agents (CAs) are commonly exploited to afford much clearer images of detectable organs and to reduce the risk of misdiagnosis caused by limited MRI sensitivity. Currently, only a few gadolinium-based CAs are approved for clinical use. Concerns about their toxicity remain, and their administration is approved only under strict controls. Here, we report the synthesis and validation of a manganese-based CA, namely, Mn@HFn-RT. Manganese is an endogenous paramagnetic metal able to produce a positive contrast like gadolinium, but it is thought to result in less toxicity for the human body. Mn ions were efficiently loaded inside the shell of a recombinant H-ferritin (HFn), which is selectively recognized by the majority of human cancer cells through their transferrin receptor 1. Mn@HFn-RT was characterized, showing excellent colloidal stability, superior relaxivity, and a good safety profile. In vitro experiments confirmed the ability of Mn@HFn-RT to efficiently and selectively target breast cancer cells. In vivo, Mn@HFn-RT allowed the direct detection of tumors by positive contrast enhancement in a breast cancer murine model, using very low metal dosages and exhibiting rapid clearance after diagnosis. Hence, Mn@HFn-RT is proposed as a promising CA candidate to be developed for MRI.

Published
2021

36. Modeling the interaction of amphiphilic polymer nanoparticles with biomembranes to Guide rational design of drug delivery systems

Author

Martina Migliavacca, Marco Giustra, Miriam Colombo, Rany Rotem, Angelo Micale, Davide Prosperi, Federico Tasin, Maria Antonietta Rizzuto, Lucia Salvioni, Rotem, R, Micale, A, Rizzuto, M, Migliavacca, M, Giustra, M, Salvioni, L, Tasin, F, Prosperi, D, and Colombo, M

Subjects
Endosome, Polymers, 02 engineering and technology, Endosomes, 01 natural sciences, Micelle, Colloid and Surface Chemistry, Drug Delivery Systems, 0103 physical sciences, medicine, Physical and Theoretical Chemistry, Micelles, Drug Carriers, 010304 chemical physics, Chemistry, Rational design, Amphiphilic polymer, Biological membrane, Surfaces and Interfaces, General Medicine, 021001 nanoscience & nanotechnology, medicine.disease, Endosomal escape, Blood proteins, Hemolysis, Membrane, Drug delivery, Biophysics, Nanoparticles, 0210 nano-technology, Biotechnology
Abstract

Nanoparticle assisted drug delivery to the cytoplasm is limited by sequestration of nanoparticles in endosomes. Endosomal escape through polymer-induced membrane destabilization is one of a few well characterized mechanisms to overcome it. Aiming to utilize this method in vivo, it is necessary to understand how modulating the structural and chemical features of the polymer and the presence of proteins in biological fluids can affect this activity. Here, as a model for the endosomal membrane, we use the membrane of red blood cells to evaluate the membrane destabilization ability of a model amphiphilic polymer in the presence of blood plasma using a hemolysis assay. This allows determination of red blood cells membrane permeabilization through the quantification of hemoglobin leakage. Our results showed a strong inhibitory effect of plasma, and that hemolytic activity can be improved by chemical modification of the polymeric micelle, reducing its interaction with plasma proteins. Finally, a second mechanism of pH-induced direct diffusion is proposed and tested using an oil/water partitioning model. These results offer a body of knowledge to improve delivery of drugs across biological membranes using carefully designed polymeric nanocarriers.

Published
2020

37. Targeted delivery of nanoparticles

Author

Davide Prosperi, Miriam Colombo, Rany Rotem, Rotem, R, Prosperi, D, and Colombo, M

Subjects
Biodistribution, Computer science, Nanoparticle, Drug development, Nanotechnology, Molecular recognition, Target site, In vivo, Drug delivery, Pharmacokinetics, Biotechnology
Abstract

The curative effectiveness of current and new drugs is often limited by poor pharmacokinetics in vivo. Nanoparticles could be the solution to this problem, as they are the right size to infiltrate and interact with biological systems (i.e., size is similar to proteins). They could allow the division of this big problem into two smaller problems: in vivo delivery, achieved by the nanoparticle, and target site interaction, achieved by the drug. This chapter focuses on the first smaller problem, summarizing the nanoparticle properties governing their biodistribution, i.e., size, surface charge, nonspecific interactions, elasticity, geometry, molecular recognition, and triggered response. It discusses current research focused on controlling and characterizing these parameters and the creation of new drug delivery platforms for specific applications.

Published
2020
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38. Development of Reliable Experimental Models for the Study of the Biological Behavior of Drug Nanocarriers

Author

ROTEM, RANY, Rotem, R, and PROSPERI, DAVIDE

Subjects
nanoparticle, drug delivery, CHIM/06 - CHIMICA ORGANICA, phase transfer, endosomal escape, quantification
Abstract

The curative effectiveness of current and new drugs is often limited by poor pharmacokinetics in-vivo. The use of nanoparticles as drug carriers seems promising in solving this problem. In this work we aimed to further explore and improve common drug delivery components and techniques. Starting with the synthesis of nanoparticles with a controlled number of molecular recognition ligands, we used bulky ligands and gel separation to obtain nanoparticles with a discrete number of chemical functional groups, used later to conjugate the same number of molecular recognition ligands. These nanoparticles later showed substantial difference in the in-vivo behavior. A second project focused on the in-depth characterization of the relationship between hydrophobic inorganic nanoparticles and the polymer surfactants used to enable their water dispersibility, as well enabling their functionalization. This investigation was done through separate quantification of polymer and inorganic nanoparticles and assessment of stability. Our results showed that the removal of excess polymer from such systems can result in loss of colloidal stability. A third project was aimed to describe the mechanism of polymeric nanoparticle’s endosomal escape and provide a platform for qualitative investigation and enhancement of this process. This goal was accomplished through two complementary in-vitro experiments testing two proposed mechanisms of endosomal escape. These results raised a key consideration when matching a particle capable of endosomal escape to a specific cell type as well as methods reduce interaction with serum proteins. A fourth project focused on developing an assay to quantify cytosolic delivery of nanoparticles and theoretically assessed the possibility of using fluorescence resonance energy transfer (FRET) - which was found to be not practical in this case - as well as implementing a pro-fluorophore to generate a measurable signal. Our preliminary results indicate this method might indeed be useful for this purpose in the future. The curative effectiveness of current and new drugs is often limited by poor pharmacokinetics in-vivo. The use of nanoparticles as drug carriers seems promising in solving this problem. In this work we aimed to further explore and improve common drug delivery components and techniques. Starting with the synthesis of nanoparticles with a controlled number of molecular recognition ligands, we used bulky ligands and gel separation to obtain nanoparticles with a discrete number of chemical functional groups, used later to conjugate the same number of molecular recognition ligands. These nanoparticles later showed substantial difference in the in-vivo behavior. A second project focused on the in-depth characterization of the relationship between hydrophobic inorganic nanoparticles and the polymer surfactants used to enable their water dispersibility, as well enabling their functionalization. This investigation was done through separate quantification of polymer and inorganic nanoparticles and assessment of stability. Our results showed that the removal of excess polymer from such systems can result in loss of colloidal stability. A third project was aimed to describe the mechanism of polymeric nanoparticle’s endosomal escape and provide a platform for qualitative investigation and enhancement of this process. This goal was accomplished through two complementary in-vitro experiments testing two proposed mechanisms of endosomal escape. These results raised a key consideration when matching a particle capable of endosomal escape to a specific cell type as well as methods reduce interaction with serum proteins. A fourth project focused on developing an assay to quantify cytosolic delivery of nanoparticles and theoretically assessed the possibility of using fluorescence resonance energy transfer (FRET) - which was found to be not practical in this case - as well as implementing a pro-fluorophore to generate a measurable signal. Our preliminary results indicate this method might indeed be useful for this purpose in the future.

Published
2019

39. Are nanotechnological approaches the future of treating inflammatory diseases?

Author

Miriam Colombo, Ivan Zanoni, Lucia Salvioni, Davide Prosperi, Maria Antonietta Rizzuto, Francesca Granucci, Rany Rotem, Rizzuto, M, Salvioni, L, Rotem, R, Colombo, M, Zanoni, I, Granucci, F, and Prosperi, D

Subjects
chronic inflammation, medicine.medical_treatment, Biomedical Engineering, Medicine (miscellaneous), Bioengineering, 02 engineering and technology, Development, Autoimmune Diseases, 03 medical and health sciences, Immune system, Phagocytosis, anti-inflammatory drug, Immune Tolerance, Medicine, Animals, Humans, Nanotechnology, General Materials Science, 030304 developmental biology, Inflammation, 0303 health sciences, tolerance, business.industry, nanoparticle, phagocyte, immunosuppressive treatment, Immunotherapy, Mononuclear phagocyte system, 021001 nanoscience & nanotechnology, Drug accumulation, Nanomedicine, mononuclear phagocytic system, Immunology, Nanoparticles, 0210 nano-technology, business
Abstract

The current treatments for chronic inflammatory diseases cause severe side effects due to nonspecific drug accumulation. Nanotechnology opens the way to new therapeutic strategies that exploit the ability of immune cells, and especially of phagocytes, to internalize nanoparticles. The cellular uptake of nanoparticles requires specific interactions and is affected by the chemical and physical properties of the carriers. Therefore, optimizing these properties is crucial for designing nanodrugs for immunotherapy. In perspective, we discuss the nanoparticle-based approaches that have been proposed to induce tolerance in autoimmune disorders and lessen the symptoms of inflammatory diseases.

Published
2019

40. Bioengineered Approaches for Site Orientation of Peptide-Based Ligands of Nanomaterials

Author

Davide Prosperi, Elisabetta Galbiati, Rany Rotem, Miriam Colombo, Svetlana Avvakumova, Serena Mazzucchelli, Avvakumova, S, Colombo, M, Galbiati, E, Mazzucchelli, S, Rotem, R, and Prosperi, D

Subjects
Nanoparticle, Peptide, Nanotechnology, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Oriented ligation, Nanomaterials, targeting, chemistry.chemical_classification, Ligand immobilization, Chemistry, Ligand, Biomolecule, nanoparticle, biofunctionalization, 021001 nanoscience & nanotechnology, BIO/10 - BIOCHIMICA, peptide, 0104 chemical sciences, antibodie, Biological target, Quality standard, Multivalency, Nanoconjugate, 0210 nano-technology, Nanoconjugates
Abstract

Successful application of nanoconjugates either in vitro or in vivo does depend on the design strategy used for their development. In this chapter, we focus on the most important factors that need to be taken into account to optimize nanoparticle biofunctionalization and provide an overview of recent approaches yielding the efficient and orientation-controlled immobilization of complex molecules, including peptides and proteins. In addition, we discuss on the elements that cooperate with ligand orientation in affecting the biological activity of nanoconjugates. Indeed, the control of ligand orientation on the nanoparticle surface is of crucial importance in determining the affinity of the immobilized biomolecules toward the biological target. However, the ability of a nanoconjugate to recognize its target is even strongly affected by other important items, including (1) multivalency as a result of ligand density and organization; (2) conformational changes potentially occurring on binding of ligands to the nanoparticle; (3) thermodynamic and kinetic factors related to the interaction of the ligand with the nanoparticle surface; and (4) interaction between nanoparticle and its surrounding environment to form the so-called “protein corona.” Altogether, these five issues are the driving force in the specificity and selectivity of designed nanoconjugates toward their target, and, as a consequence, define their potential utility for biomedical application. Therefore, a careful control on the abovementioned “magic five” contributions is needed to improve the quality standard of the next-generation nanomedicines. In addition to the illustrated nanobioconjugation strategies, we emphasize that all these features need to be carefully optimized when assessing the biological effects of synthetic nanoparticles designed for biomedical purposes.

Published
2018

41. Tumour homing and therapeutic effect of colloidal nanoparticles depend on the number of attached antibodies

Author

Giulia Alessio, Clara De Palma, Miriam Colombo, Fabio Corsi, Davide Prosperi, Karsten Kantner, Serena Mazzucchelli, Wolfgang J. Parak, Beatriz Pelaz, Luisa Fiandra, Rany Rotem, Manuela Nebuloni, Colombo, M, Fiandra, L, Alessio, G, Mazzucchelli, S, Nebuloni, M, De Palma, C, Kantner, K, Pelaz, B, Rotem, R, Corsi, F, Parak, W, and Prosperi, D

Subjects
Cell Survival, Science, medicine.medical_treatment, General Physics and Astronomy, Nanoparticle, 02 engineering and technology, Conjugated system, 010402 general chemistry, 01 natural sciences, Antibodies, Article, General Biochemistry, Genetics and Molecular Biology, Mice, Targeted therapies, Drug Delivery Systems, In vivo, Cell Line, Tumor, medicine, Animals, Humans, Colloids, Drug Carriers, Multidisciplinary, biology, Antibodies, Monoclonal, Neoplasms, Experimental, General Chemistry, Immunotherapy, 021001 nanoscience & nanotechnology, Molecular biology, In vitro, 0104 chemical sciences, 3. Good health, Nanoscale biophysic, Colloidal gold, biology.protein, Biophysics, Nanoparticles, Female, Antibody, 0210 nano-technology, Drug carrier
Abstract

Active targeting of nanoparticles to tumours can be achieved by conjugation with specific antibodies. Specific active targeting of the HER2 receptor is demonstrated in vitro and in vivo with a subcutaneous MCF-7 breast cancer mouse model with trastuzumab-functionalized gold nanoparticles. The number of attached antibodies per nanoparticle was precisely controlled in a way that each nanoparticle was conjugated with either exactly one or exactly two antibodies. As expected, in vitro we found a moderate increase in targeting efficiency of nanoparticles with two instead of just one antibody attached per nanoparticle. However, the in vivo data demonstrate that best effect is obtained for nanoparticles with only exactly one antibody. There is indication that this is based on a size-related effect. These results highlight the importance of precisely controlling the ligand density on the nanoparticle surface for optimizing active targeting, and that less antibodies can exhibit more effect., A common strategy to target nanoparticles to tumours is conjugation with specific antibodies, targeting protein expressed preferentially by cancer cells. Here the authors show that the number of antibodies bound to the nanoparticle influences the targeting ability in vitro and in vivo.

Published
2016
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43. Depletion of key gut bacteria predicts disrupted bile acid metabolism in inflammatory bowel disease.

Author

Peterson D, Weidenmaier C, Timberlake S, and Gura Sadovsky R

Subjects
Humans, Animals, Mice, Bacteria classification, Bacteria genetics, Bacteria metabolism, Bacteria isolation & purification, Operon, Metagenomics, Metagenome, Gastrointestinal Microbiome genetics, Bile Acids and Salts metabolism, Inflammatory Bowel Diseases microbiology, Inflammatory Bowel Diseases metabolism, Feces microbiology, Clostridium genetics, Clostridium metabolism, Clostridium isolation & purification
Abstract

The gut microbiome plays a key role in bile acid (BA) metabolism, where a diversity of metabolic products contribute to human health and disease. In particular, Inflammatory Bowel Disease (IBD) is characterized by a low concentration of secondary bile acids (SBAs), whose transformation from primary bile acids (PBAs) is an essential function performed solely by gut bacteria. BA-transformation activity mediated by the bile acid inducible (bai) operon has been functionally characterized in the genus Clostridium , and homologous bai gene sequences have been found in metagenome-assembled genomes (MAGs) belonging to other taxa in the human gut, but it is unclear which species of bai-carrying bacteria perform physiologically significant amounts of bile acid transformation in healthy and sick individuals. Here, we analyzed hundreds of stool samples with paired metagenomic and metabolomic data from IBD patients and controls and found that the abundance of the bai operon in metagenomic samples was highly predictive of that sample's high- or low-SBA metabolic state. We further found that bai genes from the Clostridium species best characterized as BA transformers were more prevalent in IBD patients than in non-IBD controls, while bai genes from uncharacterized taxa known only from MAGs were much more physiologically relevant in non-IBD samples. These un-isolated clades of BA-transforming bacteria merit further research; as beyond their prevalence in the human population, we found some cases in which they engrafted in IBD patients who had undergone fecal microbiota transplantation and experienced a clinical response.IMPORTANCEIn this paper, we identify specific bacteria that perform an important metabolic function in the human gut and demonstrate that in the guts of a large subset of patients with IBD, these bacteria are missing and the function is defective. This is a rare example where the correlation between the absence of specific bacteria and the dysfunction of metabolism is directly observed, not in mice nor in the lab, but in physiologic microbial communities in the human gut. Our results point to a path for studying how a small but important set of bacteria is affected by conditions in the IBD gut and perhaps to the development of interventions to mitigate the loss of these bacteria in IBD., Competing Interests: The authors declare no conflict of interest.

Published
2025
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44. Drug resistance testing at regimen failure in individuals diagnosed with HIV-1 between 2010 and 2018 in Israel.

Author

Wagner T, Levy I, Wieder-Finesod A, Wax M, Gozlan Y, Elbirt D, Kedem E, Olshtain-Pops K, Elinav H, Chowers M, Istomin V, Smolyakov R, Matus N, Girshengorn S, Marom R, Turner D, and Mor O

Abstract

Objective: Assess virological failures, analyze the results of resistance testing (RET), and investigate factors associated with acquired drug resistance mutations (aDRM)., Design: A retrospective longitudinal cohort study., Methods: Virological failures (viral load >50 copies/ml) from a cohort of 1130 individuals, diagnosed with HIV-1 in 2010-2018 and followed up until 2020, were included. Demographic, clinical, and virological data were collected. A piecewise exponential additive mixed model was employed to estimate the association of various factors with aDRM., Results: Only 82 individuals had virological failure, 20/82 had multiple virological failures. The majority of virological failures (77%) were men, 48% were Israeli-born,79% were diagnosed in 2010-2014. Only 18% initiated with second-generation integrase-inhibitor (INI) based regimens. Although no baseline differences were identified between those with single and multiple virological failures, the latter had lower CD4+ levels before first virological failure. NRTI M184IV and INI N155H were identified in more than 10% of the cases. In those with additional failures, INI N155H was more prominent in cases with subtype B compared to those with non-B subtypes (P = 0.039). Diagnoses with CD4+ cell count less than 200 cells/μl and AIDS [hazard ratio = 3.46, 95% confidence interval (95% CI): 1.51-7.92, P = 0.003], second-generation INI at the first virological failure (HR = 0.32, 95% CI: 0.11-0.91, P = 0.033), and RET at baseline (hazard ratio = 0.34, 95% CI: 0.13-0.86, P = 0.022) had a significant and persistent relative effect on aDRM., Conclusion: The risk for aDRM is reduced in those who are treated with second-generation INI-based regimens. Diagnosis with low CD4+ cell counts and AIDS is associated with detection of aDRM., (Copyright © 2025 Wolters Kluwer Health, Inc. All rights reserved.)

Published
2025
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45. ER + HER2- early-stage breast cancer: association of HER2 expression, tumor characteristics, and outcomes.

Author

Goldvaser H, Yerushalmi R, Mutai R, Kuchuk I, Toker M, Paluch-Shimon S, Drumea K, Evron E, Sonnenblick A, Gal-Yam E, Sela GB, Shai A, Merose R, Bareket-Samish A, Soussan-Gutman L, and Stemmer SM

Subjects
Humans, Female, Middle Aged, Aged, Adult, Prognosis, Kaplan-Meier Estimate, Neoplasm Recurrence, Local pathology, Gene Expression Profiling, Receptor, ErbB-2 metabolism, Breast Neoplasms pathology, Breast Neoplasms metabolism, Breast Neoplasms mortality, Breast Neoplasms genetics, Neoplasm Staging, Receptors, Estrogen metabolism, Biomarkers, Tumor metabolism
Abstract

Purpose: To evaluate the association between the HER2 score as provided by the Oncotype DX Recurrence Score (RS) assay, tumor characteristics, and outcomes in early-stage, ER + HER2-negative breast cancer (BC)., Methods: All women insured by the Clalit Health Services, with early-stage, ER + HER2-negative BC who underwent RS testing between 2008 and 2011 were included. Patient/tumor characteristics and Kaplan-Meier estimates for distant recurrence-free survival (DRFS) and overall survival (OS) were compared by HER2 category, based on the HER2 score provided by the RS assay: lower HER2 score group representing the lower third of the HER2 score range (≤ 8.5); higher HER2 score group representing the upper 2 thirds of the HER2 score range (8.6-10.7)., Results: 1535 patients were included (948 node negative, 587 node positive); 330 (21.5%) were categorized as lower HER2 score and 1205 (78.5%) as higher HER2 score. Compared to the higher HER2 score group, the lower score group included a significantly higher proportion of patients with RS ≥ 26 in both node-negative (41% vs. 13.6%, P < .001) and node-positive diseases (36% vs. 19.4%, P < .001). Compared to the higher HER2 score group, the lower score group had significantly lower Oncotype ER and PR scores and lower proportion of lobular disease. Age and tumor size were comparable between the HER2 score groups. Within each RS category, DRFS and OS were not associated with the HER2 score., Conclusion: Lower HER2 score was associated with higher RS results. Further study is desired to elucidate the role and significance of HER2 expression in early-stage, ER + HER2-negative., Competing Interests: Declarations. Competing interests: Author HG reports personal fee from: AstraZeneca (Honorarium), Gilead (Honorarium and consulting), Eli-Lilly (Honorarium and consulting), MSD (Honorarium and consulting), Novartis (Honorarium and consulting), Pfizer (Honorarium and consulting), Roche (Honorarium), Rhenium Oncotest (Honorarium and consulting), all not related to the submitted manuscript. Author RY reports personal fees from: Roche (consulting, invited speaker, Research grant), Pfizer (consulting), Novartis (consulting, invited speaker), Rhenium (consulting), Medison (invited speaker), MSD (consulting, invited speaker), Astra-Zeneca (consulting, invited speaker), Eli Lilly (consulting, invited speaker), Gilead (consulting), Stemline (invited speaker, consulting) all not related to submitted manuscript. Author SPS reports: Roche (consultancy, advisory board, speaker's bureau, travel grant), Novartis (consultancy, advisory board, speaker's bureau), Pfizer (consultancy, advisory board, speaker's bureau, travel grant, Institutional independent research grant), Astra-Zeneca (consultancy, advisory board, speaker's bureau), Gilead (consultancy, advisory board, speaker's bureau, travel grant), Eli Lily (consultancy, advisory board, speaker's bureau), MSD (consultancy, advisory board, speaker's bureau), Stemline (consultancy), all via institutional fees and not related to the submitted manuscript. Author AS reports: Roche(consultancy, advisory board, speaker's bureau, travel grant), Novartis (consultancy, advisory board, speaker's bureau), Pfizer (consultancy, advisory board, speaker's bureau, travel grant, institutional independent research grant), Astra-Zeneca (consultancy, advisory board, speaker's bureau), Gilead (consultancy, advisory board, speaker's bureau, travel grant), Lily (consultancy, advisory board, speaker's bureau), MSD (consultancy, advisory board, speaker's bureau), Stemline (consultancy), all via institutional fees and not related to the submitted manuscript. Author AS reports: Eli Lilly (consulting, advisory board, speakers bureau), Pfizer (consulting, advisory board, speakers bureau), Roche (consulting, advisory board, speakers bureau, research grant), Novartis (consulting, advisory board, speakers bureau, research grant), Gilead (consulting, advisory board), MSD (consulting, advisory board, speakers bureau, travel grant), Astra-Zenca (consulting, advisory board), Progenetics (consulting, advisory board), Rhenium (consulting, advisory board), Neopharm (travel grant), Celgene (travel grant), Medison (travel grant), all not related to the submitted work. Author ABS reports being a consultant for Oncotest Rhenium, and Exact Sciences, related to the submitted manuscript, and to Pfizer, Can-Fite, and MDI, not related to the submitted manuscript. Author SMS reports: research grant from Can-Fite, AstraZeneca, Bioline RX, BMS, Halozyme, Clovis Oncology, CTG Pharma, Exelexis, Geicam, Halozyme, Incyte, Lilly, Moderna, Teva pharmaceuticals, and Roche, and owning stocks and options in CTG Pharma, DocBoxMD, Tyrnovo, VYPE, Cytora, and CAN-FITE, all not related to the submitted manuscript. All other authors have no conflicts of interest. This study was funded by Oncotest-Rhenium. The funder played no role in the design and conduct of the analysis or its interpretation, and the decision to submit the manuscript for publication., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Published
2025
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46. Cognitive-Linguistic Profiles Underlying Reading Difficulties Within the Unique Characteristics of Hebrew Language and Writing System.

Author

Yinon R and Shaul S

Subjects
Humans, Child, Male, Female, Language, Cognition physiology, Israel, Linguistics, Phonetics, Dyslexia physiopathology, Writing, Reading
Abstract

While the multiple cognitive deficits model of reading difficulties (RD) is widely supported, different cognitive-linguistic deficits may manifest differently depending on language and writing system characteristics. This study examined cognitive-linguistic profiles underlying RD in Hebrew, characterised by rich Semitic morphology and two writing versions differing in orthographic consistency-a transparent-pointed version and a deep-unpointed version. A two-step cluster analysis grouped 96 s graders and 81 fourth graders based on their phonological awareness (PA), rapid naming (RAN), orthographic knowledge (OK) and morphological-pattern identification (MPI) abilities. Word-reading accuracy and fluency in both writing versions were also examined. The analysis revealed substantial variation in cognitive-linguistic functioning levels, identifying distinct homogeneous profiles at both ages. PA and RAN deficits were most significant across grades. A distinct OK deficit profile emerged among fourth graders, particularly manifested in Hebrew's deep-unpointed script. Moreover, the findings converge on the conclusion that while the phonological deficit is primary and persists over time, its importance is secondary to the main morphological deficit, reflecting a central independent factor in explaining RD in Hebrew. These results support a hybrid-dynamic perspective, emphasising how the relevance of different cognitive-linguistic deficits underlying RD varies with development, influenced by Hebrew's unique orthographic and morphological characteristics., (© 2025 The Author(s). Dyslexia published by John Wiley & Sons Ltd.)

Published
2025
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47. Treatment of Early Pregnancy Loss With Mifepristone and Misoprostol Compared With Misoprostol Only.

Author

Friedman M, Mor L, Shazar R, Paul N, Kerner R, Keidar R, Sagiv R, and Gluck O

Subjects
Humans, Female, Pregnancy, Retrospective Studies, Adult, Drug Therapy, Combination, Abortion, Spontaneous, Abortifacient Agents, Steroidal administration & dosage, Misoprostol administration & dosage, Misoprostol therapeutic use, Mifepristone administration & dosage, Mifepristone therapeutic use, Abortifacient Agents, Nonsteroidal administration & dosage, Treatment Failure
Abstract

Objective: To compare the rates of treatment failure in cases of early pregnancy loss between mifepristone-misoprostol and misoprostol only., Methods: This retrospective cohort study included patients who received medical treatment for early pregnancy loss between 2016 and 2023 at a single medical center. Patients returned for a follow-up ultrasonogram after 1 week and were treated again with misoprostol if needed. Finally, they were instructed to obtain an ultrasonogram after menstruation and to return for evaluation in case retained product of conception was suspected. We defined treatment failure as needing any surgical intervention because of retained product of conception, including cases when retained product of conception was diagnosed and treated after menstruation. In May 2022, we changed our protocol for treating early pregnancy loss from misoprostol to mifepristone and misoprostol. We compared the failure rate between patients who received mifepristone-misoprostol and those treated with misoprostol only., Results: A total of 999 patients were included: 224 in the mifepristone-misoprostol group and 775 in the misoprostol-only group. The rate of treatment failure was significantly lower in the mifepristone-misoprostol group compared with the misoprostol-only group (17.8% vs 25.1%, P =.002). After multivariant analysis was performed, the use of mifepristone and misoprostol was associated with a reduction of 34% in the odds ratio for treatment failure compared with misoprostol alone (adjusted odds ratio 0.661, 95% CI, 0.44-0.97, P =.038). In addition, prior vaginal delivery was associated with a lower risk for treatment failure, and increasing gestational age (according to ultrasonogram) was correlated with a higher risk for treatment failure., Conclusion: The addition of mifepristone to misoprostol was associated with a significantly lower rate of treatment failure, including late surgical intervention for early pregnancy loss, compared with misoprostol alone., Competing Interests: Financial Disclosure The authors did not report any potential conflicts of interest., (Copyright © 2024 by the American College of Obstetricians and Gynecologists. Published by Wolters Kluwer Health, Inc. All rights reserved.)

Published
2025
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48. TIR signaling activates caspase-like immunity in bacteria.

Author

Rousset F, Osterman I, Scherf T, Falkovich AH, Leavitt A, Amitai G, Shir S, Malitsky S, Itkin M, Savidor A, and Sorek R

Subjects
Bacteriophages physiology, Bacterial Proteins metabolism, Protein Domains, Escherichia coli genetics, Escherichia coli immunology, Immunity, Innate, Caspases metabolism, Caspases genetics, Signal Transduction
Abstract

Caspase family proteases and Toll/interleukin-1 receptor (TIR)-domain proteins have central roles in innate immunity and regulated cell death in humans. We describe a bacterial immune system comprising both a caspase-like protease and a TIR-domain protein. We found that the TIR protein, once it recognizes phage invasion, produces the previously unknown immune signaling molecule adenosine 5'-diphosphate-cyclo[N7:1'']-ribose (N7-cADPR). This molecule specifically activates the bacterial caspase-like protease, which then indiscriminately degrades cellular proteins to halt phage replication. The TIR-caspase defense system, which we denote as type IV Thoeris, is abundant in bacteria and efficiently protects against phage propagation. Our study highlights the diversity of TIR-produced immune signaling molecules and demonstrates that cell death regulated by proteases of the caspase family is an ancient mechanism of innate immunity.

Published
2025
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49. The Polar Saccadic Flow model: Re-modeling the center bias from fixations to saccades.

Author

Mairon R and Ben-Shahar O

Abstract

Research indicates that a significant component of human eye movement behavior constitutes a set of consistent biases independent of visual content, the most well-known of which is the central bias. While all prior art focuses on representing saccadic motion and biases in Cartesian retinotopic coordinates, here we propose the Polar Saccadic Flow model, a novel approach for modeling saccades' space-dependent biases in a polar representation. By breaking saccades into orientation and amplitude, the Polar Saccadic Flow model enables more accurate modeling of these components, leading also to a better understanding of the saccadic bias. Moreover, the polar representation also uncovers hitherto unknown patterns and biases in eye movement data, allowing for a more detailed and nuanced analysis of saccadic behavior. These findings have implications for the study of human visual perception, can help to develop more accurate eye movement models, and also may improve eye tracking technologies., (Copyright © 2025 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Published
2025
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50. CARD domains mediate anti-phage defence in bacterial gasdermin systems.

Author

Wein T, Millman A, Lange K, Yirmiya E, Hadary R, Garb J, Melamed S, Amitai G, Dym O, Steinruecke F, Hill AB, Kranzusch PJ, and Sorek R

Abstract

Caspase recruitment domains (CARDs) and pyrin domains are important facilitators of inflammasome activity and pyroptosis 1 . Following pathogen recognition by nucleotide binding-domain, leucine-rich, repeat-containing (NLR) proteins, CARDs recruit and activate caspases, which, in turn, activate gasdermin pore-forming proteins to induce pyroptotic cell death 2 . Here we show that CARD domains are present in defence systems that protect bacteria against phage. The bacterial CARD domain is essential for protease-mediated activation of certain bacterial gasdermins, which promote cell death once phage infection is recognized. We further show that multiple anti-phage defence systems use CARD domains to activate a variety of cell death effectors, and that CARD domains mediate protein-protein interactions in these systems. We find that these systems are triggered by a conserved immune-evasion protein used by phages to overcome the bacterial defence system RexAB 3 , demonstrating that phage proteins inhibiting one defence system can activate another. Our results suggest that CARD domains represent an ancient component of innate immune systems conserved from bacteria to humans, and that CARD-dependent activation of gasdermins is shared in organisms across the tree of life., Competing Interests: Competing interests: R.S. is a scientific cofounder and advisor of BiomX and Ecophage. The other authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature Limited.)

Published
2025
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