Delivery of siRNA to the Target Cell Cytoplasm: Photochemical Internalization Facilitates Endosomal Escape and Improves Silencing Efficiency, In Vitro and In Vivo

2008 ◽  
Vol 14 (34) ◽  
pp. 3686-3697 ◽  
Author(s):  
S. Oliveira ◽  
A. Hogset ◽  
G. Storm
Keyword(s):  
Target Cell ◽  
Endosomal Escape ◽  
Cell Cytoplasm ◽  
Photochemical Internalization

scholarly journals Mechanosensation and Mechanotransduction in Natural Killer Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Giorgio Santoni ◽  
Consuelo Amantini ◽  
Matteo Santoni ◽  
Federica Maggi ◽  
Maria Beatrice Morelli ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Tyrosine Phosphatase ◽  
Cell Interaction ◽  
Structural Basis ◽  
Adhesive Interactions

Natural killer (NK) cells are a main subset of innate lymphocytes that contribute to host immune protection against viruses and tumors by mediating target cell killing and secreting a wide array of cytokines. Their functions are finely regulated by a balance between activating and inhibitory receptors and involve also adhesive interactions. Mechanotransduction is the process in which physical forces sensed by mechanosensors are translated into chemical signaling. Herein, we report findings on the involvement of this mechanism that is mainly mediated by actin cytoskeleton, in the regulation of NK cell adhesion, migration, tissue infiltration and functions. Actin represents the structural basis for NK cell immunological synapse (NKIS) and polarization of secretory apparatus. NK-target cell interaction involves the formation of both uropods and membrane nanotubes that allow target cell interaction over long distances. Actin retrograde flow (ARF) regulates NK cell signaling and controls the equilibrium between activation versus inhibition. Activating NKIS is associated with rapid lamellipodial ARF, whereas lower centripetal actin flow is present during inhibitory NKIS where β actin can associate with the tyrosine phosphatase SHP-1. Overall, a better knowledge of mechanotransduction might represent a future challenge: Realization of nanomaterials tailored for NK cells, would be important to translate in vitro studies in in vivo new immunotherapeutic approaches.

scholarly journals Carboxylated branched poly(β-amino ester) nanoparticles enable robust cytosolic protein delivery and CRISPR-Cas9 gene editing

2019 ◽  
Vol 5 (12) ◽  
pp. eaay3255 ◽  
Author(s):  
Yuan Rui ◽  
David R. Wilson ◽  
John Choi ◽  
Mahita Varanasi ◽  
Katie Sanders ◽  
...  
Keyword(s):  
Protein Delivery ◽  
Gene Editing ◽  
Gene Knockout ◽  
Cell Types ◽  
Endosomal Escape ◽  
Biological Research ◽  
Amino Ester ◽  
Cytosolic Protein

Efficient cytosolic protein delivery is necessary to fully realize the potential of protein therapeutics. Current methods of protein delivery often suffer from low serum tolerance and limited in vivo efficacy. Here, we report the synthesis and validation of a previously unreported class of carboxylated branched poly(β-amino ester)s that can self-assemble into nanoparticles for efficient intracellular delivery of a variety of different proteins. In vitro, nanoparticles enabled rapid cellular uptake, efficient endosomal escape, and functional cytosolic protein release into cells in media containing 10% serum. Moreover, nanoparticles encapsulating CRISPR-Cas9 ribonucleoproteins (RNPs) induced robust levels of gene knock-in (4%) and gene knockout (>75%) in several cell types. A single intracranial administration of nanoparticles delivering a low RNP dose (3.5 pmol) induced robust gene editing in mice bearing engineered orthotopic murine glioma tumors. This self-assembled polymeric nanocarrier system enables a versatile protein delivery and gene editing platform for biological research and therapeutic applications.

Stearylated INF7 Peptide Enhances Endosomal Escape and Gene Expression of PEGylated Nanoparticles both In Vitro and In Vivo

2012 ◽  
Vol 101 (2) ◽  
pp. 879-882 ◽  
Author(s):  
Ayman El-Sayed ◽  
Tomoya Masuda ◽  
Hidetaka Akita ◽  
Hideyoshi Harashima
Keyword(s):  
Gene Expression ◽  
Endosomal Escape ◽  
Pegylated Nanoparticles

CD30/CD16A Tandab AFM13-Induced Target Cell Lysis By NK-Cells Is Enhanced By CD137 Co-Stimulation and Blocking PD-1

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2747-2747 ◽  
Author(s):  
Xing Zhao ◽  
Narendiran Rajasekaran ◽  
Uwe Reusch ◽  
Jens-Peter Marschner ◽  
Martin Treder ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Nk Cells ◽  
Target Cell ◽  
Tumor Size ◽  
Target Cells ◽  
In Vivo Models ◽  
Effector Cells ◽  
Cytotoxicity Assays

Abstract Introduction: AFM13 is a CD30/CD16A bispecific tetravalent TandAb antibody that recruits and activates NK-cells by specific binding to CD16A for targeted lysis of CD30+ tumor cells. Given promising clinical activity and safety profile of AFM13 and proof-of-mechanism demonstrating dependence on the immune response, potential synergy of AFM13 and checkpoint modulators was evaluated. Methods: Efficacy of AFM13 alone or in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies was assessed by in vitro cytotoxicity assays with human PBMCs or enriched NK-cells and CD30+ target cells as well as patient-derived xenograft in vivo models with autologous PBMC. To evaluate NK-cell-mediated lysis of CD30+ lymphoma cell lines, 4 hour cytotoxicity assays were performed with PBMCs or enriched NK-cells as effector cells in the presence of suboptimal concentrations of AFM13 alone, and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies. For the in vivo model tumor fragments derived from surgical specimens of newly diagnosed patients with CD30+ Hodgkin Lymphoma were xenografted (PDX) in immuno-deficient mice. After 28 days mice were reconstituted with autologous patient-derived PBMC and treated with AFM13 alone and in combination with anti-CTLA-4, anti-PD-1, or anti-CD137 antibodies weekly for a total of three weeks. Tumor size, tumor-infiltrating human lymphocytes and intra-tumoral cytokines were evaluated on day 58. Results: AFM13 as a single agent at suboptimal concentrations induced effector-to-target cell-dependent lysis of CD30+ lymphoma cells up to 40% using enriched NK-cells as effector cells in a 4 hour in vitro assay. Immune-modulating antibodies alone mediated substantially lower lysis (<25%). However, the addition of anti-PD-1 or anti-CD137 to AFM13 strongly enhanced specific lysis up to 70%, whereas the addition of anti-CTLA-4 to AFM13 showed no beneficial effect. The most impressive increase of efficacy was observed when AFM13 was applied together with a combination of anti-PD-1 and anti-CD137. In vivo, reduction of tumor growth was observed when AFM13 and anti-PD-1 were used as single agents or when AFM13 was combined with anti-CD137. Synergy was most impressive in these PDX models for the combination of AFM13 and anti-PD-1 which led to a very strong reduction of tumor size. Of note, reduction of tumor growth was strongly correlated with infiltrating NK- and T-cells and intra-tumoral cytokines. Conclusions: The combination trials performed with companion intra-tumoral assessment of lymphocytes and cytokines may enhance the efficacy of AFM13 in patients. This may be explained by a potential cross-talk between NK-cells and T-cell which was enhanced when AFM13 was used in combination with checkpoint modulators. Disclosures No relevant conflicts of interest to declare.

Depletion of Normal and Malignant B cells with a CD37-specific SMIP molecule

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 3063-3063
Author(s):  
C. Cerveny ◽  
L. Grosmaire ◽  
E. Espling ◽  
R. Bader ◽  
C. Nilsson ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Target Cell ◽  
Growth Arrest ◽  
Xenograft Model ◽  
B Cell Malignancies ◽  
B Lymphoma ◽  
Anti Tumor Activity

3063 Background: CD37 is a member of the tetraspanin family expressed at high levels by normal mature B cells and by most B cell malignancies. Previously, an antibody to CD37 has been labeled with 131I and tested in clinical trials for therapy of NHL. Treatment with 131I-MB-1, resulted in durable tumor remissions in patients lasting from 4 to 11 months (Press OW, Eary JF, Badger CC, et al. Treatment of refractory non-Hodgkin’s lymphoma with radiolabeled MB-1 (anti-CD37) antibody. J Clin Oncol. 1989;7:1027–1038). Here we assess the functional properties and therapeutic potential of a small modular immunopharmaceutical (SMIP) targeting CD37. Methods: Growth arrest and apoptosis of B lymphoma cell lines was assessed. ADCC activity was evaluated using BJAB targets and human peripheral blood mononuclear cells (PBMC) effectors. Drug-drug interactions were assessed by the Combination Index method. In vivo studies were performed utilizing established human B cell tumor xenografts in nude mice. Results: A CD37-directed SMIP drug candidate mediated growth arrest, apoptosis and ADCC, but not CDC, towards B lymphoma cell lines. The protein showed significant anti-tumor activity in a mouse xenograft model, and selectively depleted normal human B cells in short term cultures of PBMC. When combined with rituximab, the molecule increased apoptosis, C1q binding, and C’ dependent target cell death in vitro, and increased anti-tumor activity in vivo in a xenograft model. Conclusions: In vitro and in vivo characterization of the CD37-targeted SMIP drug suggest a potent capacity to eliminate target cells through combined effects of direct target cell signaling and effector cell recruitment. CD37-mediated growth was synergistic with standard chemotherapies in vitro and showed additive in vivo activity with CD20-targeted therapy. On the basis of these data CD37-directed SMIP therapy is being developed for clinical evaluation against B cell malignancies. No significant financial relationships to disclose.

scholarly journals In Vitro and In Vivo Characterization of CD19/CD3 Tandab AFM11 and CD19/CD16A Tandab AFM12 Targeting NHL

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2763-2763
Author(s):  
Xing Zhao ◽  
Narendiran Rajasekaran ◽  
Uwe Reusch ◽  
Michael Weichel ◽  
Kristina Ellwanger ◽  
...  
Keyword(s):  
T Cell ◽  
Target Cell ◽  
Binding Sites ◽  
Effector Cell ◽  
Nk Cell ◽  
Target Cells ◽  
Tumor Target ◽  
Effector Cells

Abstract Introduction: CD19 is expressed by B cells from early development through differentiation into plasma cells, and represents a validated target for the development of therapeutic antibodies to treat B cell malignancies such as Non Hodgkin Lymphoma (NHL) and acute lymphoblastic leukemia (ALL). Different CD19-targeting T-cell engagers are investigated in clinical studies for the treatment of NHL or ALL, including Affimed's AFM11, a bispecific CD19/CD3 TandAb antibody, which is currently investigated in a phase 1 dose escalation study. Indeed, Affimed's bispecific tetravalent platform comprises not only T-cell engaging TandAbs with two binding sites for CD3, but also NK-cell recruiting TandAbs with two binding sites for CD16A. In the present study, Affimed's AFM11, was characterized and compared in in vitro and in vivo studies with the CD19/CD16A TandAb AFM12. Methods: Analogous to the CD19/CD3 TandAb AFM11, a bispecific tetravalent TandAb AFM12 was constructed with two binding sites for CD19 and two sites for CD16A. Both TandAbs were characterized side by side for their biophysical properties, binding affinities to CD19+ tumor target cells and to their respective effector cells by flow cytometry. Kinetics and dose-response characteristics were evaluated in in vitro cytotoxicity assays. Potency and efficacy of both TandAbs were compared on different CD19+ tumor target cell lines using primary human effector cells. To compare the efficacy of AFM11 and AFM12 a patient-derived tumor xenograft model was developed. Results: AFM12 mediated efficacious target cell lysis with a very fast on-set in vitro. Lysis induced by AFM11 was less efficacious (lower specific lysis than AFM12) but reproducibly more potent (lower EC50 value). In addition to the potency and efficacy of AFM11 and AFM12, different aspects of safety, such as effector cell activation in the presence and absence of target cells were investigated and will be described. Conclusions: Affimed's CD19/CD3 and CD19/CD16A TandAbs with identical anti-CD19 tumor-targeting domains but different effector cell-recruiting domains represent interesting molecules to study T-cell- or NK-cell-based immunotherapeutic approaches. The comparison of AFM11 and AFM12 demonstrated that AFM12-mediated lysis was fast and efficacious, whereas AFM11 showed a higher potency. In summary, the NK-cell recruiting TandAb AFM12 represents an alternative to T-cell recruiting molecules, as it may offer a different side effect profile, comparable to that of AFM13, the first NK-cell TandAb clinically investigated. Disclosures No relevant conflicts of interest to declare.

scholarly journals Interaction kinetics of peptide lipids-mediated gene delivery

2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Yinan Zhao ◽  
Tianyi Zhao ◽  
Yanyan Du ◽  
Yingnan Cao ◽  
Yang Xuan ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Endosomal Escape ◽  
Late Endosomes ◽  
Key Factor ◽  
Interaction Kinetics ◽  
Dna Release

Abstract Background During the course of gene transfection, the interaction kinetics between liposomes and DNA is speculated to play very important role for blood stability, cellular uptake, DNA release and finally transfection efficiency. Results As cationic peptide liposomes exhibited great gene transfer activities both in vitro and in vivo, two peptide lipids, containing a tri-ornithine head (LOrn3) and a mono-ornithine head (LOrn1), were chosen to further clarify the process of liposome-mediated gene delivery in this study. The results show that the electrostatically-driven binding between DNA and liposomes reached nearly 100% at equilibrium, and high affinity of LOrn3 to DNA led to fast binding rate between them. The binding process between LOrn3 and DNA conformed to the kinetics equation: y = 1.663631 × exp (− 0.003427x) + 6.278163. Compared to liposome LOrn1, the liposome LOrn3/DNA lipoplex exhibited a faster and more uniform uptake in HeLa cells, as LOrn3 with a tri-ornithine peptide headgroup had a stronger interaction with the negatively charged cell membrane than LOrn1. The efficient endosomal escape of DNA from LOrn3 lipoplex was facilitated by the acidity in late endosomes, resulting in broken carbamate bonds, as well as the “proton sponge effect” of the lipid. Conclusions The interaction kinetics is a key factor for DNA transfection efficiency. This work provided insights into peptide lipid-mediated DNA delivery that could guide the development of the next generation of delivery systems for gene therapeutics.

Time-lapse film analysis of cytoplasmic streaming during late oogenesis of Drosophila

Development ◽  
1982 ◽  
Vol 67 (1) ◽  
pp. 101-111
Author(s):  
Herwigo Gutzeit ◽  
Roswitha Koppa
Keyword(s):  
Cytoplasmic Streaming ◽  
Nurse Cell ◽  
Time Lapse ◽  
Nurse Cells ◽  
Film Analysis ◽  
Cell Cytoplasm ◽  
Morphological Criteria ◽  
Oocyte Stage

Cytoplasmic streaming in follicles of Drosophila has been analysed in vitro by means of time-lapse films. Late vitellogenic follicles develop normally in vitro as judged by morphological criteria. Furthermore, follicles (stage 10 and younger) which were cultured in vitro for the same length of time as follicles which were filmed, developed normally in vivo after injection into a host fly. The recorded cytoplasmic movements are, therefore, unlikely to be an in vitro artefact. At early vitellogenic stages (up to stage 9; King, 1970) no cytoplasmic streaming can be detected, but at stage 10A cytoplasmic movements are initiated within the oocyte. At stage 10B, when the nurse cells start degenerating, nurse cell cytoplasm can be seen to flow into the growing oocyte. At stage 11 a central stream of nurse-cell cytoplasm reaches the oocyte within a minute. The ooplasmic streaming is most rapid at stage 10B and stage 11 and only an oocyte cortex up to 7 μm thick remains stationary. Once the bulk of the nurse-cell cytoplasm has poured into the oocyte (stage 12) the cytoplasmic movement ceases, first in the nurse cells and later in the ooplasm. In mature oocytes no cytoplasmic streaming can be detected.

The Distribution of Electron-Dense Tracers in Peripheral Nerve Fibres

1971 ◽  
Vol 8 (2) ◽  
pp. 541-555
Author(s):  
SUSAN M. HALL ◽  
P. L. WILLIAMS
Keyword(s):  
Schwann Cell ◽  
Peripheral Nerve ◽  
Myelin Sheath ◽  
External Surface ◽  
Nerve Fibres ◽  
Cell Cytoplasm ◽  
Line Region ◽  
Periaxonal Space

Two electron-dense tracers, ferritin and lanthanum, have been administered to peripheral nerve fibres, and their uptake has been studied ultrastructurally. It was found that the perineurium was an effective barrier to ferritin in vivo, and the tracer was subsequently injected sub-perineurially. Ferritin uptake over a 120-min period was confined to occasional phagocytic vesicles in perineurial and Schwann cells, and to the nodal gap substance and paranodal periaxonal space. No uptake was observed in the myelin sheath, incisural intraperiod line gap, or in the axoplasm. Soaking fibres in ferritin in vitro resulted in a more generalized cytoplasmic and axoplasmic uptake, although the myelin sheath and Schmidt-Lanterman incisures remained devoid of the tracer. Lanthanum nitrate, included in the fixative solution, delineated the patent incisural intraperiod line gap, and outlined the external surface of the terminal loops of nodal Schwann cell cytoplasm, and the paranodal Schwann cell-axolemmal junction. Unlike ferritin, La3+ penetrated the myelin sheath, being usually confined to the intraperiod line region of the outer lamellae, where it was associated with a widening of the lamellar unit, and an apparent splitting of the intraperiod line. The results are discussed with regard to distribution of extracellular space in peripheral nerve fibres.

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