Where does the saporin payload release after internalization? For example, does it require trafficking into a late endosome/lysosomal compartment?
Answer:
Thank you for reaching out to us. Hopefully I can help answer some of your questions regarding what happens to saporin after being internalized.
I would first like to refer you to an article we published, titled “Streptavidin-Saporin: Converting Biotinylated Materials into Targeted Toxins”. In it we review the internalization of saporin and include a few references for support. To answer your question in general, yes the conjugate is typically endocytosed and makes its way to the late endosome.
As an overview of this debated topic, the Wensley, H.J. et al 2019 article (ref #4) studied the escape of saporin from the late endosome and examined the endocytic process to quantify the endosomal escape into the cytosol. The Holmes, S.E et al 2015 (ref #5) and Giansanti, F. et al 2018 (ref #6) articles describe additional research examining chemical and genetic strategies used in assisting in saporin’s escape from the endosome. After endocytocis, Vago, R. et al 2005 (ref #7) compared saporin and ricin A chain and other bacterial toxins looking at their different intracellular routes to enter the cytosol. These articles should provide a nice foundation and hopefully better answer any questions.
If you’re interested in visualizing lysosomal trafficking, you might consider our pHast product line. These are secondary pH-dependent fluorescent conjugates, meaning that they only fluoresce once inside the endosomes and lysosomes of cells (which are acidic compared to the cytosol).
Related Products
pHast Conjugates – one of our pHastest tools for quantitative testing.
Boucher A (2024) Unveiling cholera toxin binding and intoxication using enteroids and site-specific mutants. Univ Gothenburg Thesis.
Objective: To investigate the binding site requirements of cholera toxin in the human body.
Summary: The cause of cholera symptoms is cholera toxin secreted by bacteria once in the small intestine. Cholera toxin has multiple binding sites that lead to many different intake mechanisms. By identifying the binding sites responsible, the study seeks to lay the groundwork for better means of treatment.
Usage: Leukocytes were treated with biotinylated Cholera toxin B binding-deficient mutants mixed with Streptavidin-SAP (IT-27) and assessed for cell death.
Matsuda T, Morigaki R, Hayasawa H, Koyama H, Oda T, Miyake K, Takagi Y (2024) Striatal parvalbumin interneurons are activated in a mouse model of cerebellar dystonia. Dis Model Mech 17(5):dmm050338. doi: 10.1242/dmm.050338 PMID: 38616770
Objective: To examine the influence of cerebellar abnormalities on the basal ganglia circuitry to investigate dystonia pathophysiology.
Summary: Dystonia is a disorder characterized by twisting, repetitive movements, and abnormal postures induced by sustained muscle contractions. This study utilized a cerebellar dystonia mouse model to examine the cerebellum’s contribution. The authors found that modulating parvalbumin (PV) interneurons might provide a novel treatment strategy.
Usage: In order to selectively ablate dorsolateral striatal PV interneurons, Streptavidin-ZAP (Cat. #IT-27) was mixed equimolar with biotinylated anti-PV and diluted with PBS by 1:100 and 3 ul injected into the striatum of mice. BIgG-SAP Rabbit (Cat. #IT-75) was used as the control.
Komatsu N, Kosai A, Kuroda M, Hamakubo T, Abe T (2024) Cetuximab-toxin conjugate and npe6 with light enhanced cytotoxic effects in head and neck squamous cell carcinoma in vitro. Biomedicines 12(5):973. doi: 10.3390/biomedicines12050973 PMID: 38790935
Objective: Combine the use of antibody-directed saporin and a photosensitizer to exert directed and improved cytotoxicity towards carcinoma cells as compared to either by itself.
Summary: Photodynamic therapy uses photosensitizers and irradiation to exert cytotoxic effects on cancer. When combined with biotinylated anti-EGFR conjugated to Strep-ZAP (IT-27), an increased cytotoxicity was hypothesized. The method developed enhanced the cytotoxicity of anti-EGFR-Strep-ZAP by the photodynamic effect in Sa3 and HO-1-u-1 cells, which have moderate levels of EGFR expression.
Usage: Cells were seeded on a 96-well plate and delivered 1.34 pM to 4.2 nM of anti-EGFR or anti-EGFR-Strep-ZAP, and cell viability was measured with formazan.
Chen S (2024) Nanocapsule-based prodrugs for targeted treatment of AIDS-associated non-hodgkin lymphoma. Univ California Thesis.
Objective: To propose a novel nanocapsule based platform that encapsulates the native drugs using various monomers and crosslinkers through free radical polymerization.
Summary: This encapsulation technology modifies the surface properties of the encapsulated drugs, enhancing their penetration into deeper tumor tissues and across the blood-brain barrier (BBB). Moreover, it significantly improves the stability of the drugs in vivo, protecting them from rapid degradation or clearance by the immune system. By adjusting the composition of the monomers and crosslinkers, the surface charge, hydrophobicity, and size of the nanocapsules can be finely tuned to maximize their efficacy in reaching and penetrating the target tissues.
Usage: Conjugation of ch128.1Av (anti-TfR1 IgG3-avidin fusion protein) with biotinylated saporin 6 (b-SO6) to eliminate malignant cells, including NHL malignancies. However, safe systemic delivery of ch128.1Av/b-SO6 is limited by its non-specific toxicity to normal cells expressing TfR1.
Kitawi R, Ledger S, Kelleher AD, Ahlenstiel CL (2024) Advances in HIV gene therapy. Int J Mol Sci 25(5):2771. doi: doi.org/10.3390/ijms25052771
Objective: This review highlights the various stages of ex vivo gene therapy, current research developments that have increased the efficiency and safety of this process, and a comprehensive summary of Human Immunodeficiency Virus (HIV) gene therapy studies, the majority of which have employed the ex vivo approach.
Summary: The long-term or permanent expression of anti-HIV genes and the modification of CD4+ and CD34+ cells to render them resistant to infection or to allow the disruption of the HIV life cycle are important strategies in the quest to achieve a HIV cure.
Usage: Authors referenced CD117 antibody conjugated to saporin via streptavidin (IT-27, IT-83), which enabled >99% depletion of endogenous HSCs in NSG mice and non-human primates.
Objective: Use biotinylated CD45.2 antibodies conjugated to Streptavidin to target hematopoietic stem cells (HSCs) for HSC transplantation.
Summary: Hematopoietic stem cell transplantation offers a promising alternative to standard cancer treatments. Using Click Chemistry, different toxic payloads were attached to streptavidin and observed.
Usage: 75 micrograms of CD45.2 delivered to HSCs deriving from B6 mice.
Zhao Q, Zong H, Zhu P, Su C, Tang W, Chen Z, Jin S (2024) Crosstalk between colorectal CSCs and immune cells in tumorigenesis, and strategies for targeting colorectal CSCs. Exp Hematol Oncol 13(1):6. doi: 10.1186/s40164-024-00474-x PMID: 38254219
Summary: Cancer immunotherapy has become a promising strategy in the treatment of colorectal cancer, and relapse after tumor immunotherapy. Cancer stem cells (CSCs) have the capabilities of self-renewal and differentiation and are also resistant to the traditional therapies of radiotherapy and chemotherapy. The authors review strategies for targeting colorectal CSCs, where one method described uses a biotinylated antibody against EpCAM (clone 3-171) conjugated to saporin via Streptavidin-ZAP (IT-27).
Bogen O, Araldi D, Sucher A, Kober K, Ohara PT, Levine JD (2024) Isolectin B4 (IB4)-conjugated streptavidin for the selective knockdown of proteins in IB4-positive (+) nociceptors. Mol Pain doi: 10.1177/17448069241230419 PMID: 38246917
Objective: To address the need for selective transfection methods, the authors covalently linked isolectin B4 (IB4) to streptavidin and analyzed whether it could be used to study protein function in IB4(+)-nociceptors
Summary: Rats treated intrathecally with IB4-conjugated streptavidin complexed with biotinylated antisense oligonucleotides for protein kinase C epsilon (PKCε) mRNA werefound to have: (a) less PKCε in dorsal root ganglia (DRG), (b) reduced PKCε expression in IB4(+) but not IB4(–) DRG neurons, and (c) fewer transcripts of the PKCε gene in the DRG. This knockdown in PKCε expression in IB4(+) DRG neurons is sufficient to reverse hyperalgesic priming, a rodent model of chronic pain that is dependent on PKCε in IB4(+)- nociceptors.
Usage: For each intrathecal injection 2.3μL IB4-streptavidin (30 pmol of conjugate with 120 pmol of biotin binding sites), 1.2μL biotinylated ODN (c = 100 pmol/μl) and 16.5μL PBS were mixed (injection volume = 20μL),
Allred CA, Gormley C, Venugopal I, Li S, McGuire MJ, Brown KC (2023) Tumor-specific intracellular delivery: peptide-guided transport of a catalytic toxin. Commun Biol 6(1):60. doi: 10.1038/s42003-022-04385-7 PMID: 36650239
Objective: The demonstration of a peptide optimized by chemical modifications for tumor specificity to deliver saporin, a catalytic toxin, specifically to cancer cells via both in vitro and in vivo.
Summary: Peptides rival antibodies in affinity and specificity and offer an alternative as cancer-targeting molecules. In comparison to antibodies, peptides have a faster development time and lower production cost. The authors isolated peptide MGS4, derived from a phage-displayed library using a non-small cell lung cancer (NSCLC) cell line as the target. MGS4 was modified to identify the minimal binding domain while also improving affinity and stability. Importantly, the authors provide data showing the peptide delivered saporin both in vitro and in vivo to cancer cells demonstrating anti-tumor efficacy in a mouse model.
Usage: In vitro delivery was performed by reacting biotinylated peptide with Streptavidin-ZAP (Cat. #IT-27) in a 1:1 molar ratio. Cells were treated for 6h at 37C. The drug was removed and replaced with media and after 72 hours, cell viability was measured with CellTiter-GLO. In vivo delivery was performed using biotinylated MGS4 reacted with Streptavidin-ZAP and administered via tail-veil injection (7.5 ug/100 ul) 2x/week for 2.5 weeks for a total of 5 treatments.
