Short Half-life In Circulation Research Articles

Enhancing biomedical imaging: the role of nanoparticle-based contrast agents.

Biomedical imaging plays a critical role in early detection, precise diagnosis, treatment planning, and monitoring responses, but traditional methods encounter challenges such as limited sensitivity, specificity, and inability to monitor therapeutic responses due to factors like short circulation half-life and potential toxicity. Nanoparticles are revolutionizing biomedical imaging as contrast agents across modalities like computed tomography (CT), optical, magnetic resonance imaging (MRI), and ultrasound, exploiting unique attributes such as those of metal-based, polymeric, and lipid nanoparticles. They shield imaging agents from immune clearance, extending circulation time, and enhancing bioavailability at tumor sites. This results in improved imaging sensitivity. The study highlights advancements in multifunctional nanoparticles for targeted imaging, tackling concerns regarding toxicity and biocompatibility. Critically evaluating conventional contrast agents, emphasizes the shortcomings that nanoparticles aim to overcome. This review provides insight into the current status of nanoparticle-based contrast agents, illuminating their potential to reshape therapeutic monitoring and precision diagnostics.

MG53 Protein-Mediated biomimetic nanotherapeutics for the treatment of acute myocardial infarction by driving the impaired plasma membrane resealing

Myocardial infarction (MI) remains a leading global cause of mortality. The frequent disruptions of cardiomyocyte plasma membranes underscore the pivotal role played by MG53, a tissue repair protein secreted by skeletal muscle in response to physical exercise, in cardioprotection. However, the clinical application of recombinant human MG53 (rhMG53) is challenged by its short circulation half-life, necessitating frequent administration, and its low cell membrane permeability. Here we introduce an innovative MG53 protein-mediated biomimetic nanotherapeutic (Rm@mSiO2@MG53) to address the poor delivery. Rm@mSiO2@MG53 nanotherapeutics, attributed to the high specific surface area and adjustable pore size of mSiO2 and the expression of CD47 on erythrocyte membranes, enable sustained protein delivery, enhance their ability to evade the mononuclear phagocytic system (MPS), and extend the half-life of rhMG53 proteins in circulation. In a mouse MI model, Rm@mSiO2@MG53 demonstrated superior efficacy in protecting injured cardiomyocytes and preserving heart function post-MI. Our findings developed a novel protein biomimetic nanotherapeutics that mediates heart repair by driving cell membrane resealing, offering the potential to expedite the clinical deployment of rhMG53 as a viable therapeutic for treating MI and other tissue injuries.

Enhanced anti-cancer efficacy of arginine deaminase expressed by tumor-seeking Salmonella Gallinarum.

Amino acid deprivation, particularly of nonessential amino acids that can be synthesized by normal cells but not by cancer cells with specific defects in the biosynthesis pathway, has emerged as a potential strategy in cancer therapeutics. In normal cells, arginine is synthesized from citrulline in two steps via two enzymes: argininosuccinate synthetase (ASS1) and argininosuccinate lyase. Several cancer cells exhibit arginine auxotrophy due to the loss or down-regulation of ASS1. These cells undergo starvation-induced cell death in the presence of arginine-degrading enzymes such as arginine deaminase (ADI). Thus, ADI has emerged as a potential therapeutic in cancer therapy. However, the use of ADI has two major disadvantages: ADI of bacterial origin is strongly antigenic in mammals, and ADI has a short circulation half-life (∼5 h). In this study, we engineered tumor-targeting Salmonella Gallinarum to express and secrete ADI and deployed this strain into mice implanted with ASS1-defective mouse colorectal cancer (CT26) through an intravenous route. A notable antitumor effect was observed, suggesting that the disadvantages were overcome as ADI was expressed constitutively by tumor-targeting bacteria. A combination with chloroquine, which inhibits the induction of autophagy, further enhanced the effect. Anti-cancer effect of Salmonella Gallinarum expressing an arginine deiminase (ADI) on arginine-dependent tumors in situ.

Hypoxia-Inducible Factor-1α-Activated Protein Switch Based on Allosteric Self-Splicing Reduces Nonspecific Cytotoxicity of Pharmaceutical Drugs.

Protein-based therapeutic agents currently used for targeted tumor therapy exhibit limited penetrability, nonspecific toxicity, and a short circulation half-life. Although targeting cell surface receptors improves cancer selectivity, the receptors are also slightly expressed in normal cells; consequently, the nonspecific toxicity of recombinant protein-based therapeutic agents has not been eliminated. In this study, an allosteric-regulated protein switch was designed that achieved cytoplasmic reorganization of engineered immunotoxins in tumor cells via interactions between allosteric self-splicing elements and cancer markers. It can target the accumulated HIF-1α in hypoxic cancer cells and undergo allosteric activation, and the splicing products were present in hypoxic cancer cells but were absent in normoxic cells, selectively killing tumor cells and reducing nonspecific toxicity to normal cells. The engineered pro-protein provides a platform for targeted therapy of tumors while offering a novel universal strategy for combining the activation of therapeutic functions with specific cancer markers. The allosteric self-splicing element is a powerful tool that significantly reduces the nonspecific cytotoxicity of therapeutic proteins.

Degree of PEGylatation of Lumbricus terrestris Hemoglobin Improves Microcirculatory Blood Flow but Increases the Rate of Auto-Oxidation.

The demand for red blood cells (RBCs) is on the rise due to the increasing diagnosis of chronic diseases such as sickle cell anemia, malaria, and thalassemia. Despite many commercial attempts, there are no U.S. FDA-approved artificial RBCs for use in humans. Existing RBC substitutes have employed various strategies to transport oxygen, extend the circulation time, and reduce organ toxicity, but none have replicated the natural protective mechanisms of RBCs, which prevent hemoglobin (Hb) dimerization and heme iron oxidation. Lumbricus terrestris (earthworm) erythrocruorin (LtEc) is a naturally occurring extracellular hemoglobin (Hb) with promising attributes: large molecular diameter (30 nm), high molecular weight (3.6 MDa), low auto-oxidation rate, and limited nitric oxide-scavenging properties. These characteristics make LtEc an ideal candidate as an RBC substitute. However, LtEc has a significant drawback, its short circulatory half-life. To address this issue, we explored thiol-mediated surface PEGylation of LtEc (PEG-LtEc) at varying polyethylene glycol (PEG) surface coverages. Increasing PEG surface coverage beyond 40% destabilizes LtEc into smaller subunits that are 1/12th the size of LtEc. Therefore, we evaluated two PEG surface coverage options: PEG-LtEc-0.2 (20% PEGylation) and PEG-LtEc-1.0 (100% PEGylation). We conducted experiments using golden Syrian hamsters with dorsal window chambers and catheters to assess the efficacy of these solutions. We measured microvascular parameters, organ function, cerebral blood flow, circulation time, mean arterial pressure, heart rate, and blood gases and performed histology to screen for toxicity. Our findings indicate that both PEG-LtEc molecules offer significant benefits in restoring microvascular parameters, organ function, cerebral blood flow, and circulation time compared to LtEc alone. Notably, PEG-LtEc-1.0 showed superior microvascular perfusion, although it exhibited a higher rate of auto-oxidation compared to PEG-LtEc-0.2. These results underscore the advantages of PEGylation in terms of tissue perfusion and organ health while highlighting its limitations.

Hyaluronic acid modified indocyanine green nanoparticles: a novel targeted strategy for NIR-II fluorescence lymphatic imaging.

The lymphatic system, alongside blood circulation, is crucial for maintaining bodily equilibrium and immune surveillance. Despite its importance, lymphatic imaging techniques lag behind those for blood circulation. Fluorescence imaging, particularly in the near-infrared-II (NIR-II) region, offers promising capabilities with centimeter-scale tissue penetration and micron-scale spatial resolution, sparking interest in visualizing the lymphatic system. Although indocyanine green (ICG) has been approved by the Food and Drug Administration (FDA) for use as a near-infrared-I (NIR-I) region fluorescent dye, its limitations include shallow penetration depth and low signal-to-noise ratio. Research suggests that ICG's fluorescence emission tail in the second near-infrared window holds potential for high-quality NIR-II imaging. However, challenges like short circulation half-life and concentration-dependent aggregation hinder its wider application. Here we developed HA@ICG nanoparticles (NPs), a superior ICG-based NIR-II fluorescent probe with excellent biocompatibility, prolonging in vivo imaging, and enhancing photostability compared to ICG alone. Leveraging LYVE-1, a prominent lymphatic endothelial cell receptor that binds specifically to hyaluronic acid (HA), our nanoprobes exhibit exceptional performance in targeting lymphatic system imaging. Moreover, our findings demonstrate the capability of HA@ICG NPs for capillary imaging, offering a means to assess local microcirculatory blood supply. These compelling results underscore the promising potential of HA@ICG NPs for achieving high-resolution bioimaging of nanomedicines in the NIR-II window.

Development and characterization of a novel variant of long-acting bovine follicle-stimulating hormone (brscFSH)

Assisted reproduction is a key aspect of modern animal breeding, providing valuable assistance in improving breeding programs. In this field, the administration of exogenous hormones, such as follicle-stimulating hormone (FSH), plays a crucial role in the induction of multiple ovulations. However, commercial FSH used in veterinary practice has been derived primarily from pituitary glands, obtained mostly from pigs for nearly four decades. Although these hormones have contributed significantly to the advancement of assisted reproductive techniques, they have certain limitations that warrant further improvements. These limitations include contamination with luteinizing hormone (LH), the potential risk of pathogen contamination, the potential to trigger an immune response in non-pig species, and the short half-life in circulation, requiring the implementation of complex 8-dose superovulation schedules. Our research team has developed and characterized a new variant of bovine follicle-stimulating hormone (bscrFSH) to address these limitations. The new hormone is produced recombinantly in CHO cell cultures, with a specific productivity of about 30 pg/cell/day. The bscrFSH can be purified to a high purity of 97 % using a single step of immobilized metal affinity chromatography (IMAC). N-glycan analysis of bscrFSH showed that approximately 74 % of the glycans corresponded to charged structures, including mono-, di-, tri-, and tetra-sialylated glycans. Superovulation trials conducted in cattle revealed that bscrFSH, administered at a total dose of about 0.5 μg per kg of body weight, using a decrescent schedule of 4 doses with 24-h intervals, resulted in an average yield of 8–12 transferable embryos per animal. Further research is required; however, the preliminary findings indicate that bscrFSH, currently packaged under the provisional brand name of Cebitropin B, holds potential as a commercial product for assisted reproduction in ruminants.

Indocyanine green-loaded N-doped carbon quantum dot nanoparticles for effective photodynamic therapy and cell imaging of melanoma cancer: in vitro, ex vivo and in vivo study

Background Indocyanine Green (ICG) as an agent for photodynamic therapy (PDT) of melanoma cancer has low quantum yield, short circulation half-life, poor photo-stability, and tendency to aggregation. Purpose N-doped carbon quantum dot (CQD) nanoparticle was applied to encapsulate ICG and overcome ICG obstacle in PDT with simultaneous cell imaging property. Methods CQD was prepared using hydrothermal method. Cell culture study and In vivo assessments on C57BL/6 mice containing melanoma cancer cells was performed. Results Results showed that CQD size slightly enhanced from 24.55 nm to 42.67 nm after ICG loading. Detection of reactive oxygen species (ROS) demonstrated that CQD improved ICG photo-stability and ROS generation capacity upon laser irradiation. Cell culture study illustrated that ICG@CQD could decrease survival rate of melanoma cancer cells of B16F10 cell line from 48% for pure ICG to 28% for ICG@CQD. Confocal microscopy images approved more cellular uptake and more qualified cell imaging ability of ICG@CQD. In vivo assessments displayed obvious inhibitory effect of tumor growth for ICG@CQD in comparison to free ICG on the C57BL/6 mice. In vivo fluorescence images confirmed that ICG@CQD accumulates remarkably more than free ICG in tumor region. Finally, ICG@CQD was proposed as an innovative nanocarrier for PDT and diagnosis.

Cytokine/Antibody Fusion Protein Design and Evaluation.

Cytokines constitute a class of secreted proteins that activate transmembrane receptors to coordinate a vast array of physiological processes, particularly those related to immune activity. Due to their vital role in immune regulation, cytokines have garnered great interest as potential therapeutic agents. Unfortunately, the clinical success of cytokine drugs has been limited by their multifunctional activities, which hinder therapeutic performance and lead to harmful toxicities. In addition, the strikingly short circulation half-life of cytokines further hampers their efficacy as drugs. To overcome the translational challenges associated with natural cytokines, significant efforts have focused on engineering cytokines to target their activities and improve their pharmacological properties. One such strategy is the design of fusion proteins that tether a cytokine to an anti-cytokine antibody that selectively biases its functions and extends its serum half-life. These cytokine/antibody fusion proteins (termed immunocytokines) assemble intramolecularly to bias cytokine signaling behavior through multi-layered structural and molecular effects. Here, we present a detailed workflow for the design, production, and functional validation of intramolecularly assembled immunocytokines. In-depth procedures are presented for gene manipulation, mammalian cell-based expression and purification, binding analysis via bio-layer interferometry, and interrogation of cytokine signaling activity on human primary cells. In contrast with immunocytokines in which the tethered cytokine and antibody do not bind one another, intramolecularly assembled immunocytokines require special considerations with respect to their production to avoid oligomerization and/or aggregation. The protocol herein was developed based on experience with immunocytokines that incorporate interleukin-2 (IL-2); however, this modular approach can be extended to any cytokine of interest for a broad range of biomedical applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Design and generation of immunocytokine genes Basic Protocol 2: Immunocytokine expression and purification Basic Protocol 3: Validation of immunocytokine assembly and binding by bio-layer interferometry Basic Protocol 4: Analysis of immunocytokine signaling on human primary cells.

Long-Circulating Vasoactive 1,18-Octadecanedioic Acid-Terlipressin Conjugate.

Hepatorenal syndrome (HRS) is a life-threatening complication of end-stage liver disease first reported over a century ago, but its management still poses an unmet challenge. A therapeutic agent found to stabilize the condition is a short cyclic peptide, vasopressin analogue, terlipressin (TP). While TP is commonly prescribed for HRS patients in most parts of the world, it was only recently approved for use in the United States. TP exhibits short circulation half-lives and adverse side effects associated with the dose required. Herein, we present a 1,18-octadecanedioic acid (ODDA) conjugate of the cyclic peptide (ODDA-TP), which enables noncovalent binding to serum albumin via native fatty acid binding modes. ODDA-TP is demonstrated to outperform TP alone in studies including in vitro cellular receptor activation, stability in plasma, pharmacokinetics, and performance in vivo in rats. Specifically, ODDA-TP had an elimination half-life 20 times that of TP alone while exhibiting a superior safety profile.

Enhancing radioprotection: A chitosan-based chelating polymer is a versatile radioprotective agent for prophylactic and therapeutic interventions against radionuclide contamination.

To mitigate the risk of radioactive isotope dissemination, the development of preventative and curative measures is of particular interest. For mass treatment, the developed solution must be easily administered, preferably orally, with effective, nontoxic decorporating properties against a wide range of radioactive isotopes. Currently, most orally administered chelation therapy products are quickly absorbed into the blood circulation, where chelation of the radioactive isotope is a race against time due to the short circulation half-life of the therapeutic. This report presents an alternative therapeutic approach by using a functionalized chitosan (chitosan@DOTAGA) with chelating properties that remains within the gastrointestinal tract and is eliminated in feces, that can protect against ingested radioactive isotopes. The polymer shows important in vitro chelation properties towards different metallic cations of importance, including (Cs(I), Ir(III), Th(IV), Tl(I), Sr(II), U(VI) and Co(II)), at different pH (from 1 to 7) representing the different environments in the gastrointestinal tract. An in vivo proof of concept is presented on a rodent model of uranium contamination following an oral administration of Chitosan@DOTAGA. The polymer partially prevents the accumulation of uranium within the kidneys (providing a protective effect) and completely prevents its uptake by the spleen.

Abstract 500: Liposomal two-tailed topotecan (L-2TT) induces more apoptosis than topotecan hydrochloride on neuroblastoma xenografts

Abstract Background: Neuroblastoma (NB) is the second most common malignancy diagnosed in infants, accounting for 15% of pediatric tumor deaths. High risk NB have a <50% 5-year survival and often resist treatment, with acute and long-term toxicities. Topotecan, part of the standard of care in NB is a Topoisomerase1 inhibitor with a short half-life in circulation (30 minutes). We esterified phospholipid to topotecan at the phenolic hydroxyl group, producing the prodrug 2T-T. 2T-T was loaded onto liposome membranes (termed L-2TT); thus preventing drug leakage. We hypothesized L-2TT would be more efficient at inducing apoptosis than topotecan in NB models (in vitro and in vivo) in part due to its extended circulation time and endocytic uptake. Methods: IC50 was determined on 8 NB cell lines using a WST assay, comparing equal dosages of L-2TT to topotecan (empty liposomes were assessed as controls). NB cells were imaged with DiD-tagged liposomes together with lysotracker green, under confocal microscopy 24 hrs. Non-tumor bearing mice received increasing L-2TT doses, assessing weight, body score over five days. Drug circulation time was estimated by following DiR-liposomes in vivo (IVIS) over 72 hrs. Nude mice received 1 × 10^6 NGP cells (NB cells) intrarenally. When tumors reached 1 gram, xenografts received 10mg/kg L-2TT (containing 3.33mg/kg topotecan) via retroorbital injection, 3.33 mg/kg topotecan-hcl or no drug. Tumors were harvested 24 hrs. later, immunohistochemical analysis (apoptosis marker TUNEL), counting the % of positive cells of total tumor area. Some mice received DiR-liposomes together with L-2TT, fixed in PFA and 50mm sections captured total DiR under confocal microscopy. Finally, we interrogated L-2TT vs. Topotecan-HCl tumors via RNAseq for chemoresistance pathways. Results: IC50 values of L-2TT were lower than topotecan-HCl by almost 3-fold in 7/8 cell lines (empty liposomes at equal concentrations had no effect). DiD-tagged liposomes formed a punctate pattern and colocalized with lysotracker green, confirming liposomes are endocytosed by NB cells in vitro. L-2TT caused no changes adverse effects on mice. Drug circulation time remained stable over 48 hours and was still detectable at 72hrs after liposomal delivery. L-2TT resulted in 3-fold higher tumor apoptosis than Topotecan-hcl (p<0.0001), and itself 3-fold higher than controls (p<0.001, n=5). Mice receiving L-2TT+L-DiR resulted in 2-fold higher DiR signal than L-DiR only (p<0.05, n=3. Finally, RNAseq suggests L-2TT elicits less chemoresistance pathways than topotecan. Conclusions: L-2TT is more potent than topotecan in vitro and in vivo. Mechanisms for these effects could be longer circulation times, different tumor cell uptake, as well as changes in tumor vascular permeability. These drug modifications could provide potent alternative for NB treatment as well as other solid tumor treatment. Citation Format: Sonia L. Hernandez, Isabella Iwanicki, Paula Viza-Gomes, Lydia Wu, Mendi Marquez, Fernando Flores-Guzman, Rachael Sundland, Michaelann Tartis. Liposomal two-tailed topotecan (L-2TT) induces more apoptosis than topotecan hydrochloride on neuroblastoma xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 500.

Abstract 5740: Peptide-functionalized dendrimers for enhanced binding to immune checkpoint proteins

Abstract The human immune system has complex and dynamic mechanisms to distinguish and eliminate malignant cells and defend the body against cancer. Various therapeutic approaches to stimulate the immune system have thus been investigated, demonstrating strong anti-tumor immune responses capable of controlling metastasis and preventing recurrence. Nevertheless, existing cancer immunotherapies suffer from several limitations, including inconsistent benefits across cancer types and patients. In recent years, the utilization of peptides for immunotherapy has attracted a great deal of interest due to their ability to mimic protein functionalities while maintaining a high degree of modularity and synthetic reproducibility in their molecular design. However, the clinical translation of otherwise promising peptide-based immunotherapeutics has been hindered due to their significantly weaker binding to target proteins and short half-life in plasma circulation in comparison to their monoclonal antibody counterparts. Nevertheless, these issues can potentially be addressed by conjugation to nanoparticles, which enhances cell binding/uptake and prolongs circulation. To this end, our group has extensively investigated the use of poly(amidoamine) (PAMAM) dendrimers to improve the therapeutic utility of peptides. These dendrimers can facilitate the simultaneous binding of conjugated peptides to multiple cell receptors, resulting in a significantly enhanced binding interaction through the multivalent binding effect. Here, we report an efficient de novo nanoparticle design strategy that combines organic synthesis, phage display, and molecular dynamics to identify a novel immuno-oncology therapeutic modality. We first employed phage display to identify several highly selective sequences that bind to the mouse ortholog of TIGIT—an inhibitory receptor expressed on lymphocytes that has been identified as a potential therapeutic target to enhance anti-tumor immune responses. Subsequent computational modeling (NAMD with CHARMM force field) revealed several conserved regions of interaction that we hypothesize drive favorable binding interactions and enhance the binding affinity of the peptide inhibitors. We next incorporated experimental kinetic measurements of the identified sequences with adaptive evolution computational modeling to identify optimized mutant analogs for dendrimer conjugation. It was found that the TIGIT targeting DPCs (dendrimer-peptide conjugates) demonstrated increased binding avidity in vitro and anti-tumor efficacy in vivo, most notably when administered in combination with PD-L1 targeting DPCs to a syngeneic mouse model bearing mouse oral carcinoma (MOC1) tumors. Overall, this work demonstrates the potential of rational peptide design and our multi-functional DPC platform for the development of both effective and novel immuno-oncology therapeutics. Citation Format: Piper Anne Rawding, Francesco Coppola, DaWon Kim, Michael Poellmann, Deric Wheeler, Petr Král, Seungpyo Hong. Peptide-functionalized dendrimers for enhanced binding to immune checkpoint proteins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5740.

Abstract 4058: Nanoparticle delivery of interleukin-2 induces anti-tumor immune response and therapeutic efficacy in mouse tumor models

Abstract Immune checkpoint inhibitors (ICIs) have demonstrated robust responses in some solid tumor types. However, the low response rate, which is associated with lack of pre-existing tumor-infiltrating lymphocytes (TILs), remains a significant challenge. Interleukin-2 (IL-2) can effectively expand TILs and has been FDA-approved for metastatic renal cancer and melanoma. However, IL-2 has several drawbacks—including short circulation half-life, dose-limiting toxicities, and counterproductive activation of regulatory T cells (Tregs)—resulting in less than 10% complete response rate. Numerous efforts have been made to enhance IL-2 formulations, but none have yet received FDA approval. In this study, we utilized our proprietary nanoparticle platform (Pdx-NP™) to improve IL-2 formulation. Pdx-NP™ can load IL-2 with near-complete binding, resulting in a 160-nm nanoconstruct (IL2-NP), suitable for both local and intravenous administrations. To evaluate the efficacy, we utilized bilateral melanoma (B16F10) and colon (MC38) tumor models, in which the local tumor was treated intratumorally for 3 doses and the distal tumor was left untreated to assess the systemic anti-tumor immune response. In the B16F10 model, IL2-NP treatment demonstrated favorable immune profiles in local and distal tumors and the draining lymph nodes, such as increased CD8+ T cell proliferation, CD8+ T cells/Treg ratio, and recruitment of dendritic cells and macrophages, compared to saline or free IL-2 counterparts. These positive immune responses translated into significant growth reduction of IL2-NP treated tumors and improvement in survival of the mice compared to free IL-2. In the MC38 tumor model, the IL2-NP treatment resulted in complete regression of both local and distal tumors in 43% of the mice. When combined with ICIs (αPD-1 and αCTLA-4), the treatment achieved a cure rate of 100%, whereas ICIs alone yielded only a 29% cure rate. In conclusion, IL2-NP induces robust proliferation and activation of TILs and is efficacious both as a monotherapy and in combination with ICIs. This is due to the ability of Pdx-NP™ to protect and retain therapeutics within tumors, with an 8-fold increase in tumor accumulation over the free drug counterpart. Our ongoing efforts are focused on the targeted delivery of IL-2 to tumors upon systemic administration, using αPD-L1 antibody-conjugated Pdx-NP™. This antibody-conjugated nanoparticle has an elimination half-life (t1/2) of about 25 hrs in non-human primates. Paired with αPD-L1 for tumor homing, this nanoconstruct is hypothesized to enhance IL-2 accumulation in tumors compared to free IL-2, which has t1/2 of only 1.5 hrs in human. The current clinical landscape of cancer immunotherapy focuses on combinational approaches, and our versatile Pdx-NP™ platform, which is capable of co-delivering different classes of therapeutics, is well suited for this endeavor. Citation Format: Ruijie Wang, Alyssa Wallstrum, Natalie White, Pramod Kumar, Tongchatra Watcharawittayakul, Noah A. Crumrine, Cole Baker, Jeremy Saito, Moataz Reda, Worapol Ngamcherdtrakul, Wassana Yantasee. Nanoparticle delivery of interleukin-2 induces anti-tumor immune response and therapeutic efficacy in mouse tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4058.

Thrombotic microenvironment responsive crosslinking cyclodextrin metal-organic framework nanocarriers for precise targeting and thrombolysis

Global public health is seriously threatened by thrombotic disorders because of their high rates of mortality and disability. Most thrombolytic agents, especially protein-based pharmaceuticals, have a short half-life in circulation, reducing their effectiveness in thrombolysis. The creation of an intelligent drug delivery system that delivers medication precisely and releases it under regulated conditions at nearby thrombus sites is essential for effective thrombolysis. In this article, we present a unique medication delivery system (MCRUA) that selectively targets platelets and releases drugs by stimulation from the thrombus' microenvironment. The thrombolytic enzyme urokinase-type plasminogen-activator (uPA) and the anti-inflammatory medication Aspirin (acetylsalicylic acid, ASA) are both loaded onto pH-sensitive CaCO3/cyclodextrin crosslinking metal-organic frameworks (MC) that make up the MCRUA system. c(RGD) is functionalized on the surface of MC, which is functionalized by RGD to an esterification reaction. Additionally, the thrombus site's acidic microenvironment causes MCRUA to disintegrate to release uPA for thrombolysis and aiding in vessel recanalization. Moreover, cyclodextrin-encapsulated ASA enables the treatment of the inflammatory environment within the thrombus, enhancing the antiplatelet aggregation effects and promoting cooperative thrombolysis therapy. When used for thrombotic disorders, our drug delivery system (MCRUA) promotes thrombolysis, suppresses rethrombosis, and enhances biosafety with fewer hemorrhagic side effects.

Delivering Relaxin Plasmid by Polymeric Metformin Lipid Nanoparticles for Liver Fibrosis Treatment.

Liver fibrosis usually progresses to liver cirrhosis and even results in hepatocellular carcinoma, which accounts for one million deaths annually worldwide. To date, anti-liver fibrosis drugs for clinical treatment have not yet been approved. Nowadays, as a natural regulator, Relaxin (RLX) has received increased attention because the expression of RLX could deactivate the activation of hepatic stellate cells (aHSCs) and resolve liver fibrosis. However, its application in treatment is limited due to the short half-life in circulation and low accumulation within the target organ. To address these problems, a kind of polymeric metformin (PolyMet)-loaded relaxin plasmid (pRLX) core-membrane lipid nanoparticle (PolyMet-pRLX-LNPs, PRLNP) was prepared. Here, PolyMet was used as a carrier to replace the traditional polymer polyethylene diene (PEI), which is of higher toxicity, to prolong the circulation time of pRLX in vivo. Then, the antifibrotic ability of PRLNP to overcome liver fibrosis was carried out in C57BL/6 mice. It is worth mentioning that this is the first time to investigate the potential of PRLNP in carbon tetrachloride-induced liver fibrosis. The results showed that PRLNP effectively downregulated fibrosis-related biomarkers such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Meanwhile, histopathological examinations also showed low collagen accumulation, revealing that PRLNP could histologically and functionally alleviate liver fibrosis. In addition, no significant difference in serum biochemical value between the PRLNP and the normal group, suggesting the safety profile of PRLNP. This research proposed a novel non-toxic treatment method for liver fibrosis with a nanosystem to effectively treat liver fibrosis.

Curcumin Nanocarriers with a Focus on Cellular Uptake Studies – An Innovative Cancer Therapy

Once a dye, curcumin (CUR) has transformed into a versatile therapeutic agent with antioxidative, anti-inflammatory, and anticancer properties. Despite its potent anticancer effects, CUR encounters challenges such as poor solubility and a short circulation half-life. To address this, researchers utilize nanocarriers like nanoparticles, liposomes, and micelles for efficient CUR delivery. With its multifaceted anticancer activity, CUR holds promise as a cancer therapeutic. Recent studies concentrate on crafting nanocarriers tailored for size, charge, and functionalization, offering adaptable tools for combinational cancer therapy. The synergistic combination of CUR with chemotherapy, magnetic nano hyperthermia, or photodynamic therapy amplifies the efficacy of malignancy treatment. The investigation into CUR-loaded nanocarriers, whether used alone or in combination with other modalities, aims to enhance cancer treatment outcomes, highlighting the diverse potential of curcumin in contemporary therapeutic strategies. This research underscores the importance of combinational drug delivery therapies, providing a renewed perspective on the versatile applications of curcumin in modern medicine.

A Modular Albumin-Oligonucleotide Biomolecular Assembly for Delivery of Antisense Therapeutics.

Antisense nucleic acid drugs are susceptible to nuclease degradation, rapid renal clearance, and short circulatory half-life. In this work, we introduce a modular-based recombinant human albumin-oligonucleotide (rHA-cODN) biomolecular assembly that allows incorporation of a chemically stabilized therapeutic gapmer antisense oligonucleotide (ASO) and FcRn-driven endothelial cellular recycling. A phosphodiester ODN linker (cODN) was conjugated to recombinant human albumin (rHA) using maleimide chemistry, after which a complementary gapmer ASO, targeting ADAMTS5 involved in osteoarthritis pathogenesis, was annealed. The rHA-cODN/ASO biomolecular assembly production, fluorescence labeling, and purity were confirmed using polyacrylamide gel electrophoresis. ASO release was triggered by DNase-mediated degradation of the linker strand, reaching 40% in serum after 72 h, with complete release observed following 30 min of incubation with DNase. Cellular internalization and trafficking of the biomolecular assembly using confocal microscopy in C28/I2 cells showed higher uptake and endosomal localization by increasing incubation time from 4 to 24 h. FcRn-mediated cellular recycling of the assembly was demonstrated in FcRn-expressing human microvascular endothelial cells. ADAMTS5 in vitro silencing efficiency reached 40%, which was comparable to free gapmer after 72 h incubation with human osteoarthritis patients' chondrocytes. This work introduces a versatile biomolecular modular-based "Plug-and-Play" platform potentially applicable for albumin-mediated half-life extension for a range of different types of ODN therapeutics.

Bionanoengineered 2D monoelemental selenene for piezothrombolysis

Thrombosis-related diseases represent the leading causes of disability or death worldwide. However, conventional thrombolytic therapies are subjected to narrow therapeutic window, short circulation half-life and bleeding. Herein, we rationally design and develop a safe and efficient nonpharmaceutical thrombolysis strategy based on a specific piezocatalytic effect arising from platelet membrane (PM)-conjugated two-dimensional (2D) piezoelectric selenene, Se-PM nanosheets (NSs). The 2D selenene is fabricated from nonlayered bulk selenium powder by a facile liquid-phase exfoliation method, and the PM conjugation confers selenene with the distinct thrombus-homing feature. Under ultrasonic activation, the piezoelectric characteristic of selenene triggers electrons and holes separation, resulting in generation of reactive oxygen species (ROS) by reacting with surrounding H2O and O2 in the thrombosis microenvironment for thrombolysis. Both systematic in vitro and in vivo assessments demonstrate that the biocompatible Se-PM NSs efficiently degrade erythrocytes, fibrin and artificial blood clots under ultrasound irradiation. Compared to the clinical thrombolytic drug urokinase plasminogen activator, the engineered Se-PM NSs possess excellent thrombolytic efficacy by single treatment in the tail thrombosis animal model without bleeding risk. The engineered Se-PM nanoplatform marks an exciting jumping-off point for research into the application of piezocatalysis in clinical treatment of thrombosis.

VISUALIZATION OF NANOPOROUS SILICA NANOPARTICLE DISTRIBUTION FOR IMPLANT-DIRECTED MAGNETIC DRUG TARGETING BY 68GA-LABELING AND PET/CT

In orthopedic surgery, implant infections are a serious issue and difficult to treat. The aim of this study was to use superparamagnetic nanoporous silica nanoparticles (MNPSNP) as candidates for directed drug delivery. Currently, short blood circulation half-life due to interactions with the host's immune system hinder nanoparticles in general from being clinically used. PEGylation is an approach to reduce these interactions and to enhance blood circulation time. The effect of PEGylation of the used 68Ga-labelled MNPSNP on the distribution and implant accumulation was examined by PET/CT imaging and gamma counting in an implant mouse model.Female Balb/c mice (n=24) received a magnetic implant subcutaneously on the left and a titanium implant on the right hind leg. On day one, 12 of these mice received an additional clodronate®-injection for macrophage depletion. On the second postoperative day, mice were anaesthetized and MNPSNP (native or PEGylated) injected intravenously, followed by a dynamic PET-scan over 60 minutes, a CT- and a static PET-scan at 120 min. As control, 12 mice received only 68Ga-MNPSNP (native or PEGylated). Gamma counting of inner organs, urine, blood and implant area was performed as further final analysis. Although PEGylation of the nanoparticles already resulted in lower liver uptakes, both variants of 68Ga-labeled MNPSNP accumulated in liver and spleen. Combination of PEGylation with clodronate®-injection led to a highly significant effect whereas clodronate®-injection alone could not reveal significant differences. In gamma counting, a significantly higher %I.D./g was found for the tissue surrounding the magnetic implants compared to the titanium control, although in a low range. PEGylation and/or clodronate®-injection revealed no significant differences regarding nanoparticle accumulation at the implantation site. PEGylation increases circulation time, but MNPSNP accumulation at the implant site was still insufficient for treatment of infections. Additional efforts have to further increase circulation time and local accumulation.Acknowledgements: This work is funded by the German Research Foundation (DFG, project number 280642759).