Today’s topic is time-course for our saporin conjugates. Common questions we get are (1) how long does it takes to see cell death, (2) how long should I wait before performing histology on an animal, or (3) how long before I see behavioral changes in an animal?
In this image from Mantyh et. al. (1997), we are looking at confocal imagery of the binding and internalization of our peptide conjugate SP-SAP to the NK1r receptor in primary cultures of neonatal spinal cord neurons.
As you can see, conjugate binding occurs immediately where within hours, the SP-SAP has recognized and bound to the NK1 receptor. Here we see the areas of concentrated NK1R expression marked by yellow immunofluorescence.
But how long until you see cell death? Here is a cytotoxicity graph of in vitro data of our antibody conjugate 192-IgG-SAP. These are typical data after cells have been treated for 3 days, which is standard protocol.
Waite et al. (1994) used 192-IgG-SAP and showed the appearance of behavioral effects associated with neuronal loss at day four and plateauing at day 7.
This coincides with the time course seen in vitro. At this point, microglia will infiltrate, but stop at 7 days, which is probably the peak day for infiltration. Once there is complete removal of the detritus, microglia down-regulate and at 14 days all debris is cleared and the animal begins to regain normal eating and sleeping habits. This is the idea behind waiting about 2 weeks before performing histology.
ZAP conjugates allow you to screen targeting agents in a quick and cost-efficient way, looking for specificity, functional binding, internalization, and EC50 determination.
We are finishing off our topic of ZAP conjugates with a closer look at the typical in vitro data you can expect to see from a cytotoxicity assay.
In this example, you are looking at the cytotoxicity curves using various secondary conjugates when compared to the direct conjugate, 192-IgG-SAP.
Mab-ZAP, our bivalent secondary antibody that recognizes whole IgG, reacted with primary antibody, produces a similar potency to the directly linked conjugate of saporin to the same antibody.
Interestingly, in this example Fab-ZAP and FabFc-ZAP, which both use a monovalent secondary antibody, reacted with primary antibody produced a cytotoxic effect greater than 12-fold over the direct conjugate.
In this example, you will also see an interesting phenomenon with ZAP secondary conjugates. It may be intuitive to think that using a higher dose of primary antibody induces a higher amount of cell death, but as seen in the example, at the highest concentration of 192-IgG (10 nM = Log (-8)) there is a lessened amount of killing, at a 25-fold lower concentration when compared to the antibody.
The explanation is that at the higher concentrations of primary antibody, there are more unconjugated 192-IgG and fewer 192-IgG plus Fab-ZAP complexes. So, this free 192-IgG can then out-compete the conjugates for cell surface binding sites, which, in turn, decreases the amount of saporin being internalized, hence less cell death.
Our publication in the Journal of Toxins, provides a nice review of this phenomenon.
Du R, Zhang Y, Wu S, Zixing W, Jing W (2025) Progress, opportunity, and perspective on long term preservation of extracellular vesicles. SSRN ssrn.5553381. doi: 10.2139/ssrn.5553381
Objective: To provide an overview of the biology, function, and biomedical application of Extracellular vesicles (EVs) and introduce the method to evaluate the storage stability of EVs.
Summary: While there are multiple methods available for EVs preservation, each approach comes with its advantage and limitation. At present, the optimal storage method for various components in EVs is still unknown. The incorporation of cryoprotectants has proven beneficial in enhancing EV preservation. However, it’s important to acknowledge that the concentration of cryoprotectants significantly influences the cryopreservation outcome. Current assessment to evaluate EVs preservation emphasize the alterations in the morphology, size, particle number, and protein of EVs during preservation, however, the description on EVs preservation efficiency has not be standardized.
Usage: When HeLa cell-derived EVs underwent lyophilization, the structure and particle size remained unchanged, the zeta-potential remained around -10 mV before and after lyophilization. However, when the EVs were engineered with arginine-rich cell-penetrating peptide (R16 peptide) and encapsulated with saporin (SAP), the biological activity of EVs were highly affected after lyophilization (Noguchi et al., 2019).
Leanza MH, Storelli E, D’Arco D, de Leo G, Kleiner G, Arancio L, Capodieci G, Gulino R, Bava A, Leanza G (2025) Cerebellar contributions to spatial learning and memory: Effects of discrete immunotoxic lesions. Int J Mol Sci 26(19):9553. doi: 10.3390/ijms26199553 PMID: 41096819
Objective: To analyze the behavioral and anatomical effects of discrete injections, in different groups of animals, of the same 192 IgG-saporin toxin into the basal forebrain nuclei and/or into the cerebellar vermis and hemispheres.
Summary: Authors administered, 192 IgG-saporin, selectively targeting cholinergic neurons in the basal forebrain and a subpopulation of cerebellar Purkinje cells, to adult rats bilaterally into the basal forebrain nuclei, the cerebellar cortices or both areas combined. The results suggest important functional interactions between the ascending regulatory inputs from the cerebellum and those arising in the basal forebrain nuclei that would act together to modulate the complex sensory–motor and cognitive processes required to control whole body movement in space.
Usage: (i) bilateral intraventricular injection of 192 IgG-saporin (ICV, n = 12); (ii) bilateral injections of 192 IgG-saporin into the basal forebrain nuclei (BF, n = 12); (iii) injections of 192 IgG-saporin into the cerebellar hemispheres and vermis (CBL, n = 12); and (iv) injections of 192 IgG-saporin into both the basal forebrain nuclei and cerebellar hemispheres and vermis (BF/CBL, n = 12).
Herrerias A, Oliverio A, Dvorácskó S, Thyagarajan A, Chedester L, Liu J, Cinar R, Iyer MR, Kunos G, Godlewski G (2025) CB1 receptors on a subset of vagal afferent neurons modulate voluntary ethanol intake in mice. Mol Psychiatry doi: 10.1038/s41380-025-03266-9 PMID: 40975751
Alampi MM, Kozlíková M, Mariangeli M, Civita S, Delcanale P, Mussini A, Diaspro A, Bianchini P, Weyergang A, Skarpen E, Berg K, Viappiani C, Abbruzzetti S, Selbo PK (2025) Light-enhanced cytotoxicity and intracellular trafficking of the PD-L1-targeting photoimmunoconjugate EITC-atezolizumab. Biomed Pharmacother 191:118550. doi: 10.1016/j.biopha.2025.118550 PMID: 40946581
Objective: To optimize the cytotoxic efficacy of a photoimmunoconjugate of eosin-5-isothiocyanate and atezolizumab (EITC-atezolizumab) in NSCLC cells; To study the uptake and intracellular transport of atezolizumab; and to evaluate EITC-atezolizumab as a candidate for photochemical internalization (PCI) of the ribosome-inactivating protein gelonin.
Summary: This is the first documentation demonstrating that atezolizumab is transported to CD63-positive organelles, thereby enhancing our understanding of its intracellular trafficking. The study also strengthens the concept of Photoimmunotherapy (PIT) and atezolizumab-based targeting of PD-L1+NSCLCs.
Usage: The cytotoxic efficacy of the PD-L1-targeting immunotoxin (Anti-PD-L1-SAP) was strongly enhanced in PD-L1-positive breast cancer cells by photochemical internalization (PCI),a low-dose, Photodynamictherapy (PDT)-based intracellular drug delivery method.
Lipari NR (2025) Investigating anxiety-like behaviors and basolateral amygdala dysfunction in a novel rat model of Parkinson’s disease. SUNY Binghamton Thesis.
Objective: To create a unique model of PD with improved face validity, and non-motor symptoms.
Summary: This work helped further characterize motor and non-motor symptoms while providing potential underlying physiological markers for early disease course in a unique animal model of Parkinson’s disease (PD).
Usage: It has been demonstrated that lesioning of the basolateral amygdala with the targeted toxin stable substance P (SSP) saporin, a toxin that selectively lesions neurons which express neurokinin1 receptors, increases anxiety-like behaviors in rats.
Diamantoudis SC, Miliotou AN, Galatou E, Telliou S, Sideris K, Grigoriadis N, Vizirianakis IS (2025) Assessing the hematological cancer stem cell landscape to improve immunotherapy clinical decisions. Biocell doi: 10.32604/biocell.2025.067216
Objective: To combine existing information and clinical evidence to assess and bring to the spotlight targets related to Hematological cancer stem cells (HCSCs) that can be considered for the improvement of therapeutic interventions.
Summary: Targeting HCSCs represents one of the most promising advances toward achieving lasting remission and potential cure in hematologic malignancies. Next-generation immunotherapies—enabled by advances in molecular profiling, synthetic biology, and systems immunology—can shift the paradigm in blood cancers by overcoming current limitations.
Usage: CD117-ADC (carrying streptavidin–saporin) has shown dose-dependent results in mice, with a range from 0.3–1.5 mg/kg, as depletion of stem cells was noted with the subsequent successful engraftment of allogenic transplants.
Kolahdouzan M, Ghazisaeidi S, Tu Y, Muley M, Gambeta E, Salter M (2025) Meningeal macrophages mask incision pain sensitization in male rats. Mol Pain doi: 10.1177/17448069251383593
Objective: To investigate whether CD206+macrophages in the meninges play a role in regulating nociception and pain hypersensitivity.
Summary: The results indicate that while CD206+ meningeal macrophages do not regulate basal nociception in naïve rats, they mask mechanical hypersensitivity in male rats after skin incision injury. Thus, we conclude that in a sex-dependent manner, CD206+ meningeal macrophages prevent the spread of pain hypersensitivity after a minor injury.
Usage: Rats were injected intrathecally (30 μl) with saline, CD206-Saporin (20 μg mannose-receptor antibody and 7 μg of Streptavidin-ZAP in 30 μl), or Rabbit-IgG-Saporin (control).
Yuan M, Zhang L, Zhu H, Xie M (2025) Alteration of BDNF and noradrenergic markers in locus coeruleus in a mouse model of cancer-induced bone pain. PLoS One 20(8):e0330207. doi: 10.1371/journal.pone.0330207 PMID: 40811566
Objective: To examine the expression and localization of BDNF and NE neuron-specific proteins in the locus coeruleus (LC) of mice with cancer-induced bone pain (CIBP).
Summary: CIBP mice exhibited enhanced neuronal activity in the LC, upregulation of noradrenergic markers, and BDNF/TrkB-mediated modulation of noradrenergic neurons. Concurrently, inhibitory signalling was attenuated in the spinal dorsal horn (SDH).
Usage: Selective ablation of noradrenergic neurons via intracerebroventricular anti-DBH-SAP (IT-03) administration reduces mechanical and cold allodynia, suggesting that LC-spinal cord pathway activity is critical for pain modulation.
