Ren L, Zhao Z, Chao Y, Yu P, Mei Z, Du B, Cheng Y (2025) Optimization of lipid nanoparticles with robust efficiency for the delivery of protein therapeutics to augment cancer immunotherapy. Adv Sci (Weinh) e2500844. doi: 10.1002/advs.202500844 PMID: 40056044
Objective: To develop a family of Lipid Nanoparticles (LNPs) with robust high efficiency in addressing the multiple barriers in cytosolic protein delivery by incorporating clinically approved ionizable lipids into traditional cationic LNPs. The authors investigated the in vivo delivery efficacy of the LNPs using saporin as the model protein.
Summary: The combination of cationic and ionizable lipids enables efficient protein binding and endosomal escape. Optimized LNPs efficiently deliver various proteins, including antibodies, enzymes, toxins, and Cas9 into living cells with reserved functions. The optimized LNPs successfully deliver therapeutic proteins such as saporin and interleukin-10 (IL-10) to inhibit tumor growth in several animal models.
Usage: Cell Viability and Apoptosis Assay: For evaluating in vitro saporin delivery efficacy, 143B cells were incubated with free saporin or saporin/LNP at saporin concentrations ranging from 0 to 61 nm. In Vivo Saporin Delivery by LNP: mice were intravenously injected with 100μL saporin or saporin/LNP once every two days. The doses of saporin and LNP in in vivo experiments were fixed at 50μg kg−1 and 2.4 mg lipid/kg, respectively.
Guo S, Xu H, Cheng Z, Wang L, Yang P, Wang R (2025) Efficient cytosolic delivery of protein by preorganized amidiniums on pillar[5]arene. CCL 111022. doi: 10.1016/j.cclet.2025.111022
Objective: To use amidinium functionalized pillar[5]arene (AP5) as a small molecular carrier to facilitate intracellular delivery of proteins with different sizes and isoelectric points.
Summary: The densely preorganized amidinium groups on pillar[5]arene skeleton could not only glue proteins together to form AP5@protein complex through multiple salt-bridges, but also promote cellular internalization AP5@protein complex. The bioactivities of the internalized proteins were well-maintained. This study provides a novel, versatile and macrocyclic-molecule based intracellular protein delivery carrier through the preorganization of amidiniums onpillar[5]arene.
Usage: The AP5@saporin complexes showed significant toxicity toward HeLa cells with an IC50 of 102 nmol/L. However, saporin alone showed minimal toxicity on HeLa cells due to membrane-impermeability of saporin.
Griesgraber MJ, Coolen LM, Onslow KM, Corey JR, Rice RE, Aerts EG, Bowdridge EC, Hardy SL, Lehman MN, Goodman RL, Hileman SM (2025) Critical role of arcuate nucleus kisspeptin and Kiss1R in regulation of the ovine luteinizing hormone surge. J Neuroendocrinol e70010. doi: 10.1111/jne.70010 PMID: 40033679
Objective: To examine the functional role of arcuate nucleus (ARC) dynorphin-containing (KNDy) and kisspeptin (Kiss) 1R-containing neurons in ovine luteinizing hormone (LH) surge secretion via injection of saporin-ligand conjugates (SAP) to ablate these neural populations.
Summary: NKB-SAP injections significantly reduced the percentage of ARC Kiss1 (~65% decrease) cells compared to control animals, and a surge-like increase of LH was prevented in ewes with the greatest degree of Kiss1 cell ablation. Kiss-SAP injections had no effect on Kiss1 cell percentage or ARC Kiss1R cell number compared to controls. However, Kiss-SAP injections consistently and robustly decreased LH surge amplitude, with 80% of Kiss-SAP-treated ewes failing to generate a surge. These results support the conclusion that KNDy neurons contribute significantly to the ovine LH surge, while ARC Kiss1R neurons appear to be necessary for a functional surge to occur in sheep.
Usage: Saporin conjugates, NKB-SAP (IT-63) and Kisspeptin-SAP (IT-102) were injected at 700 ng/μL.
Objective: To determine if local reactivation of antigen-specific oral CD8+ TRM exacerbates ligature-induced periodontitis (LIP) in mice.
Summary: Topical application of virus-mimicking peptides during LIP increased alveolar bone loss, enhanced gingival and cervical lymph node inflammation, and upregulated gingival genes linked to innate immunity and cytotoxicity. Depleting CD103+ CD8+ TRM with αCD103-SAP prior to LIP prevented disease exacerbation, implicating these cells in periodontitis pathology.
Usage: Anti-CD103-SAP (IT-50) was administered in PBS at 5 μg (day -4), 2 μg (day 0), and 2 μg (day +4) relative to LIP induction.
Dib C, Queenan J, Willner H, Swartzrock L, Charlesworth C, Denis M, Davis J, Nakauchi H, Liu DR (2025) Combining hsc base-editing with anti-cd117 antibody conditioning to correct severe combined immunodeficiency disorder in a novel mouse model. Transplantation and Cellular Therapy 31(2):S253-S354. doi: 10.1016/j.jtct.2025.01.385
Objective: To test whether base-edited hematopoietic stem cells (HSPCs) combined with non-genotoxic antibody conditioning can correct severe combined immunodeficiency (SCID) in a novel Rag2 mutant mouse model.
Summary: Base-editing delivered via engineered virus-like particles successfully corrected Rag2 mutations in HSPCs, which restored lymphocyte development following transplantation. Conditioning with an Anti-CD117-Saporin conjugate enabled efficient engraftment without irradiation toxicity, demonstrating a safer strategy for SCID treatment.
Usage: Mice were conditioned with Anti-CD117-SAP (IT-83) at 1.5 mg/kg intravenously prior to transplantation of base-edited or wild-type HSPCs.
Prince C, Kumar D, Chambliss C, Okalava J, Malik S, Doering CB, Spencer HT, Archer D. Chandrakasan S (2025) Clinically relevant non-genotoxic conditioning with cd117 immunotoxin promotes robust donor chimerism and amelioration of sickle cell disease in a murine model. Transplantation and Cellular Therapy 31(2):S178. doi: 10.1016/j.jtct.2025.01.274
Objective: To investigate the efficacy of an Anti-CD117-Saporin conjugate as part of a non-genotoxic HCT strategy in a sickle cell disease (SCD) mouse model.
Summary: An Anti-CD117-SAP, combined with clinically relevant immunosuppression, achieved stable donor chimerism and corrected hematologic abnormalities in SCD mice. This conditioning regimen avoided transfusion requirements, graft-versus-host disease, and transplant-related mortality typical of TBI or busulfan-based approaches.
Usage: HbSS-Townes mice were conditioned with ATG and B cell depletion followed by Anti-CD117-SAP (IT-83, 0.75 µg/g) prior to HCT with HbAA-Townes donor marrow.
Bohl JM, Hassan AR, Sharpe ZJ, Kola M, Shehu A, Beaudoin DL, Ichinose T (2025) Pivotal roles of melanopsin containing retinal ganglion cells in pupillary light reflex in photopic conditions. 19:1547066. doi: 10.3389/fncel.2025.1547066 PMID: 39990971
Objective: To examine the roles of intrinsically photosensitive retinal ganglion cells (ipRGCs) in the pupillary light reflex (PLR) by ablating photoreceptors using N-nitroso-N-methylurea (MNU).
Summary: Results suggest that ipRGCs primarily contribute to the PLR at a high light intensity, which does not agree with the previous results shown by mutant mouse models. The results indicate that the melanopsin response in ipRGCs generate fast and robust PLR when induced by high light.
Usage: Retinal whole mount preparations were fixed using 4% paraformaldehyde and blocked with 10% normal donkey serum and 0.5% Triton-X in PBS (PBS-T). Melanopsin antibody (AB-N39) was used at 1:5000 in PBS-T and was incubated for 3 days at 4°C, followed by Alexa568 donkey-anti-rabbit for 2 h.
Objective: To uncover the role of a sparse but unusual population of genetically-distinct interneurons known as type-I nNOS neurons, using a novel pharmacological strategic to unilaterally ablate these neurons from the somatosensory cortex of mice.
Summary: Region-specific ablation produced changes in both neural activity and vascular dynamics, decreased power in the delta-band of the local field potential, reduced sustained vascular responses to prolonged sensory stimulation, and abolished the post-stimulus undershoot in cerebral blood volume. Coherence between the left and right somatosensory cortex gamma-band power envelope and blood volume at ultra-low frequencies was decreased, suggesting type-1 nNOS neurons integrate long-range coordination of brain signals. Authors also observed decreases in the amplitude of resting-state blood volume oscillations and decreased vasomotion following the ablation of type-I nNOS neurons.
Usage: Type-I nNOS neurons were selectively ablated with saporin conjugated to a substance P analog (SP-SAP, IT-07). Intracortical injection with 4 ng of either SAP conjugate or Blank-SAP (IT-21).
Rahhal N, Rentzsch M, Seiser S, Freystätter C, Elbe-Bürger A, Rademacher C (2025) Targeted delivery of cytotoxic proteins via lipid-based nanoparticles to primary Langerhans cells. Nanoscale doi: 10.1039/d4nr03638g PMID: 39775685
Summary: Saporin was used as a model protein to showcase the potential of delivering intact proteins to Langerhans cells. Authors observed specific killing of cells expressing langerin in vitro, and in primary Langerhans cells isolated from mouse and human skin ex vivo with minimal off target effects.
Zhao Z, Zhang H, Li W, Wang Y, Wang Y, Yang H, Yin L, Liu X (2025) Guanidyl-rich α-helical polypeptide enables efficient cytosolic pro-protein delivery and CRISPR-Cas9 genome editing. J Mater Chem B doi: 10.1039/d4tb02009j PMID: 39760520
Objective: To develop an efficient strategy via cationic alpha-helical polypeptide-mediated anionic proprotein delivery.
Summary: The protein was reversibly modified with adenosine triphosphateviadynamic covalent chemistry to prepare an anionic proprotein (A-protein) with abundant phosphate groups. A guanidyl-decorated a-helical polypeptide (LPP) was employed not only to encapsulate A-protein through electrostatic attraction and hydrogen bonding, forming stable nanocomplexes, but also to enhance cell membrane penetration due to its rigid alpha-helical conformation. Consequently, this strategy mediated the effective delivery of various proteins with different isoelectric points and molecular weights, including a-chymotrypsin, bovine serum albumin, ribonuclease A, cytochromeC, saporin, horseradish peroxidase, b-galactosidase, and anti-phospho-Akt, into cancer cells.
Usage: Cytosolic delivery: Saporin, was employed to evaluate LPP-mediated cytosolic delivery and its cancer cell-killing efficacy. The LPP/A-saporin nocomplexes (NCs) caused significant toxicity, with an IC50 value of 0.4ug/mL. In vivo antitumor efficiency of LPP/A-saporin NCs: LPP/A-saporin NCs were administrated by intratumoral injection in HeLa xenograft tumor-bearing mice and their antitumor efficacy was evaluated. When the tumor volume reached B50 mm3, the mice were randomly divided into three groups (6 mice per group) and intratumorally injected with PBS, free saporin, or LPP/A-saporin NCs at 0.1 mg saporin per kg on days 0 and 2.
