Blood Clearance Research Articles

Advances in extracellular vesicle functionalization strategies for tissue regeneration.

Extracellular vesicles (EVs) are nano-scale vesicles derived by cell secretion with unique advantages such as promoting cell proliferation, anti-inflammation, promoting blood vessels and regulating cell differentiation, which benefit their wide applications in regenerative medicine. However, the in vivo therapeutic effect of EVs still greatly restricted by several obstacles, including the off-targetability, rapid blood clearance, and undesired release. To address these issues, biomedical engineering techniques are vastly explored. This review summarizes different strategies to enhance EV functions from the perspective of drug loading, modification, and combination of biomaterials, and emphatically introduces the latest developments of functionalized EV-loaded biomaterials in different diseases, including cardio-vascular system diseases, osteochondral disorders, wound healing, nerve injuries. Challenges and future directions of EVs are also discussed.

Immuno-PET of colorectal cancer with a CEA-targeted [68Ga]Ga-nanobody: from bench to bedside.

An accurate diagnosis of colorectal carcinoma (CRC) can assist physicians in developing reasonable therapeutic regimens, thereby significantly improving the patient's prognosis. Carcinoembryonic antigen (CEA)-targeted PET imaging shows great potential for this purpose. Despite showing remarkable abilities to detect primary and metastatic CRC, previously reported CEA-specific antibody radiotracers or pretargeted imaging are not suitable for clinical use due to poor pharmacokinetics and complicated imaging procedures. In contrast, radiolabeled nanobodies exhibit ideal characteristics for PET imaging, for instance, rapid clearance rates and excellent distribution profiles, allowing same-day imaging with sufficient contrast. In this study, we developed a novel CEA-targeted nanobody radiotracer, [68Ga]Ga-HNI01, and assessed its tumor imaging ability and biodistribution profile in preclinical xenografts and patients with primary and metastatic CRC. The novel nanobody HNI01 was acquired by immunizing the llama with CEA proteins. [68Ga]Ga-HNI01 was synthesized by site-specifically conjugating [68Ga]Ga with tris(hydroxypyridinone) (THP). Small-animal PET imaging and biodistribution studies were performed in CEA-overexpressed LS174T and CEA-low-expressed HT-29 tumor models. Following successful preclinical assessment, a phase I study was conducted on 9 patients with primary and metastatic CRC. Study participants received 151.21 ± 25.25MBq of intravenous [68Ga]Ga-HNI01 and underwent PET/CT scans at 1h and 2h post injection. Patients 01-03 also underwent whole-body dynamic PET imaging within 0-40min p.i. All patients underwent [18F]F-FDG PET/CT imaging within 1week after [68Ga]Ga-HNI01 imaging. Tracer distribution, pharmacokinetics, and radiation dosimetry were calculated. [68Ga]Ga-HNI01 was successfully synthesized within 10min under mild conditions, and the radiochemical purity was more than 98% without purification. Micro-PET imaging with [68Ga]Ga-HNI01 revealed clear visualization of LS174T tumors, while signals from HT-29 tumors were significantly lower. Biodistribution studies indicated that uptake of [68Ga]Ga-HNI01 in LS174T and HT-29 was 8.83 ± 3.02%ID/g and 1.81 ± 0.87%ID/g, respectively, at 2h p.i. No adverse events occurred in all clinical participants after the injection of [68Ga]Ga-HNI01. A fast blood clearance and low background uptake were observed, and CRC lesions could be visualized with high contrast as early as 30min after injection. [68Ga]Ga-HNI01 PET could clearly detect metastatic lesions in the liver, lung, and pancreas and showed superior ability in detecting small metastases. A significant accumulation of radioactivity was observed in the kidney, and normal tissues physiologically expressing CEA receptors showed slight uptakes of [68Ga]Ga-HNI01. An interesting finding was that strong uptake of [68Ga]Ga-HNI01 was found in non-malignant colorectal tissues adjacent to the primary tumor in some patients, suggesting abnormal CEA expression in these healthy tissues. [68Ga]Ga-HNI01 is a novel CEA-targeted PET imaging radiotracer with excellent pharmacokinetics and favorable dosimetry profiles. [68Ga]Ga-HNI01 PET is an effective and convenient imaging tool for detecting CRC lesions, particularly for identifying small metastases. Furthermore, its high specificity for CEA in vivo makes it an ideal tool for selecting patients for anti-CEA therapy.

Cleavable-Branched Polymer-Modified Liposomes Reduce Accelerated Blood Clearance and Enhance Photothermal Therapy.

In recent years, cationic liposomes have been successfully used as delivery platforms for mRNA vaccines. Poly(ethylene glycol) (PEG)-lipid derivatives are widely used to enhance the stability and reduce the toxicity of cationic liposomes. However, these derivatives are often immunogenic, triggering the rise of anti-PEG antibodies. Understanding the role and impact of PEG-lipid derivatives on PEGylated cationic liposomes is key to solving the PEG dilemma. In this study, we designed linear, branched, and cleavable-branched cationic liposomes modified with PEG-lipid derivatives and investigated the effect of the liposome-induced accelerated blood clearance (ABC) phenomenon on photothermal therapy. Our study indicated that the linear PEG-lipid derivatives mediated the effect of photothermal therapy by stimulating splenic marginal zone (MZ) B cells to secrete anti-PEG antibodies and increasing the level of IgM expression in the follicular region of the spleen. However, the cleavable-branched and branched PEG-lipid derivatives did not activate the complement system and avoided the ABC phenomenon by inducing noticeably lower levels of anti-PEG antibodies. The cleavable-branched PEGylated cationic liposomes improved the effect of photothermal therapy by reversing the charge on the liposome surface. This detailed study of PEG-lipid derivatives contributes to the further development and clinical application of PEGylated cationic liposomes.

Multimodality PET and Near-Infrared Fluorescence Intraoperative Imaging of CEA-Positive Colorectal Cancer

PurposeMolecular imaging is a major diagnostic component for cancer management, enabling detection, staging of disease, targeting therapy, and monitoring the therapeutic response. The coordination of multimodality imaging techniques further enhances tumor localization. The development of a single agent for real-time non-invasive targeted positron emission tomography (PET) imaging and fluorescence guided surgery (FGS) will provide the next generation tool in the surgical management of cancer.ProceduresThe humanized anti-CEA M5A-IR800 “sidewinder” (M5A-IR800-SW) antibody-dye conjugate was designed with a NIR 800 nm dye incorporated into a PEGylated linker and conjugated with the metal chelate p-SCN-Bn-deferoxamine (DFO) for zirconium-89 PET imaging (89Zr, half-life 78.4 h). The dual-labeled 89Zr-DFO-M5A-SW-IR800 was evaluated for near infrared (NIR) fluorescence imaging, PET/MRI imaging, terminal tissue biodistribution, and blood clearance in a human colorectal cancer LS174T xenograft mouse model.ResultsThe 89Zr-DFO-M5A-SW-IR800 NIR fluorescence imaging showed high tumor targeting with normal liver uptake. Serial PET/MRI imaging was performed at 24 h, 48 h, and 72 h and showed tumor localization visible at 24 h that persisted throughout the experiment. However, the PET scans showed higher activity for the liver than the tumor, compared to the NIR fluorescence imaging. This difference is an important finding as it quantifies the expected difference due to the sensitivity and depth of penetration between the 2 modalities.ConclusionsThis study demonstrates the potential of a pegylated anti-CEA M5A-IR800-Sidewinder for NIR fluorescence/PET/MR multimodality imaging for intraoperative fluorescence guided surgery.

Self‐Immolative Amphiphilic Poly(ferrocenes) for Synergistic Amplification of Oxidative Stress in Tumor Therapy

AbstractAmphiphilic self‐immolative polymers (SIPs) can achieve complete degradation solely through one triggerable event, which potentially optimize the blood clearance and uncontrollable/inert degradability for therapeutic nanoparticles. Herein, we report self‐immolative amphiphilic poly(ferrocenes), BPnbs‐Fc, composed by self‐immolative backbone and aminoferrocene (AFc) side chains as well as end‐capping poly(ethylene glycol) monomethyl ether. Upon triggering by tumor acidic milieu, the BPnbs‐Fc nanoparticles readily degrade to release azaquinone methide (AQM) moieties, which can rapidly deplete intracellular glutathione (GSH) to cascade release AFc. Furthermore, both AFc and its product Fe2+ can catalyze intracellular hydrogen peroxide (H2O2) into highly reactive hydroxyl radicals (⋅OH), thus amplifying the oxidative stress of tumor cells. Rational synergy of GSH depletion and ⋅OH burst can efficiently inhibit tumor growth by the SIPs in vitro and in vivo. This work provides an elegant design to adopt innate tumor milieu‐triggerable SIPs degradation to boost cellular oxidative stress, which is a promising candidate for precision medicine.

Self-Immolative Amphiphilic Poly(ferrocenes) for Synergistic Amplification of Oxidative Stress in Tumor Therapy.

Amphiphilic self-immolative polymers (SIPs) can achieve complete degradation solely through one triggerable event, which potentially optimize the blood clearance and uncontrollable/inert degradability for therapeutic nanoparticles. Herein, we report self-immolative amphiphilic poly(ferrocenes), BPnbs-Fc, composed by self-immolative backbone and aminoferrocene (AFc) side chains as well as end-capping poly(ethylene glycol) monomethyl ether. Upon triggering by tumor acidic milieu, the BPnbs-Fc nanoparticles readily degrade to release azaquinone methide (AQM) moieties, which can rapidly deplete intracellular glutathione (GSH) to cascade release AFc. Furthermore, both AFc and its product Fe2+ can catalyze intracellular hydrogen peroxide (H2O2) into highly reactive hydroxyl radicals (·OH), thus amplifying the oxidative stress of tumor cells. Rational synergy of GSH depletion and ·OH burst can efficiently inhibit tumor growth by the SIPs in vitro and in vivo. This work provides an elegant design to adopt innate tumor milieu-triggerable SIPs degradation to boost cellular oxidative stress, which is a promising candidate for precision medicine.

Synergy effects of copper ion in doxorubicin-based chelate prodrug for cancer chemo-chemodynamic combination therapy

Doxorubicin (DOX) is a commonly studied chemotherapeutic agent for the treatment of solid tumors, but the severe side effects limit its clinical application. It is shown that DOX-metal chelate has lower in vitro cytotoxicity compared with DOX, as the anthracyclines of DOX can form coordinative interaction with transition metal ions. In addition, the transition metal ions could catalyze the production of hydroxyl radicals (·OH) via Fenton/Fenton-like reactions to achieve antitumor chemodynamic therapy (CDT). In this study, copper ions (Cu2+) were applied to obtain DOX/Cu(II) prodrug, and a liposomal formulation was used to avoid the rapid blood clearance and optimize the biodistribution of this prodrug. In vitro and in vivo antitumor results demonstrated that this pH sensitive Cu-chelating prodrug can reduce adverse effects of DOX but improve the antitumor efficiency due to the combination of chemotherapy and chemodynamic therapy. Our study provided a facile and effective approach of metal-chelating prodrug strategy for combination cancer therapy strategy.

Demonstration of the Early Cardiac Bioavailability of a Non-Specific Cell-Targeted Peptide Using Radionuclide-Based Imaging In Vivo.

The cardiac bioavailability of peptide drugs that inhibit harmful intracellular protein-protein interactions in cardiovascular diseases remains a challenging task in drug development. This study investigates whether a non-specific cell-targeted peptide drug is available in a timely manner at its intended biological destination, the heart, using a combined stepwise nuclear molecular imaging approach. An octapeptide (heart8P) was covalently coupled with the trans-activator of transcription (TAT) protein transduction domain residues 48-59 of human immunodeficiency virus-1 (TAT-heart8P) for efficient internalization into mammalian cells. The pharmacokinetics of TAT-heart8P were evaluated in dogs and rats. The cellular internalization of TAT-heart8P-Cy(5.5) was examined on cardiomyocytes. The real-time cardiac delivery of 68Ga-NODAGA-TAT-heart8P was tested in mice under physiological and pathological conditions. Pharmacokinetic studies of TAT-heart8P in dogs and rats revealed a fast blood clearance, high tissue distribution, and high extraction by the liver. TAT-heart-8P-Cy(5.5) was rapidly internalized in mouse and human cardiomyocytes. Correspondingly, organ uptake of hydrophilic 68Ga-NODAGA-TAT-heart8P occurred rapidly after injection with an initial cardiac bioavailability already 10 min post-injection. The saturable cardiac uptake was revailed by the pre-injection of the unlabeled compound. The cardiac uptake of 68Ga-NODAGA-TAT-heart8P did not change in a model of cell membrane toxicity. This study provides a sequential stepwise workflow to evaluate the cardiac delivery of a hydrophilic, non-specific cell-targeting peptide. 68Ga-NODAGA-TAT-heart8P showed rapid accumulation in the target tissue early after injection. The implementation of PET/CT radionuclide-based imaging methodology as a means to assess effective and temporal cardiac uptake represents a useful and critical application in drug development and pharmacological research and can be extended to the evaluation of comparable drug candidates.

“Y-type” PEG modified liposomes could eliminate the accelerated blood clearance (ABC) phenomenon and improved tumor therapy

Poly(ethylene glycol) (PEG) is widely applied to decorate nanocarriers due to its “long circulation” characteristics. However, the applications of linear PEG-modified nanocarriers have been hindered by severe adverse effects due to the accelerated blood clearance (ABC) phenomenon. It was universally known that anti-PEG antibodies (APAs) were main culprits in ABC phenomenon which induced the significant change in pharmacokinetics, biological distributions of the second injection and triggered complement activation-related pseudoallergies (CARPA). Recent studies have illustrated that APAs triggered the ABC phenomenon of PEGylated protein drug and even related to the CARPA of COVID-19 vaccine. Therefore, it is urgent to inhibit the generation of APAs and eliminate the ABC phenomenon. Here, “Y-type” PEG was chosen to replace linear PEG due to its weak immunogenicity. “Y-type” PEG-lipid derivatives [DSPE-mPEG2,n (n = 2, 10, and 20 kDa)]-modified doxorubicin liposomes (DOX-PL2,n) and topotecan liposomes (TP-PL2,n) induced lower levels of APAs and could avoid activating complement system. In further research, we found that liposomes decorated with DSPE-mPEG2,n could avoid the ABC phenomenon after duplicate injections. Furthermore, pharmacodynamic tests indicated that DOX-PL2,n and TP-PL2,n improved the curative effect of S180 tumor than DOX-PL2k and TP-PL2k (linear PEGylated liposomes). For the first time, DOX-PL2,n and TP-PL2,n were used for in vivo pharmacokinetic and pharmacodynamic experiments. Liposomes ornamented with “Y-type” PEG may provide new approaches to maintaining long blood circulation time, eliminating the ABC phenomenon of encapsulated active compounds, and also could weaken CARPA and improve tumor therapeutic effect. Our research aims to promote the research and development of “Y-type” PEG-decorated nanocarriers and provide a substantial academic basis for its clinical application.

Discrete polyethylene glycol derivatives as a potent impetus for next-generation biomedicines

PEGylation, which covalently modifies small-molecule drugs, peptides and proteins with polyethylene glycol (PEG) to improve their pharmacokinetics and efficacy, has become one of the most widely used drug modification techniques. However, PEG and its derivatives have been verified to be immunogenic and antigenic, which directly lead to side effects such as accelerated blood clearance (ABC) phenomenon and allergic reactions to PEGylated drugs or PEG excipients. Recent development of single molecular weight discrete PEG synthesis methodology have led to continuous and extensive focus on the difference in biological activity between conventional polydisperse PEG and single molecular weight precise PEG. Discrete PEG has demonstrated obvious advantages in resisting protein adsorption, cell adhesion and enhancing drug efficacy. These preliminary results not only confirm the significant impact of PEG chain polydispersity on its biological effect, but also reveal the possibility and potential advantages of using discrete PEG to reduce the immunogenicity and antigenicity. Hopefully, single chemical structure and molecular weight ensure the batch repeatability of newly developed discrete PEG products, and facilitate the understanding of their physicochemical properties and biological effects that promoting the clinical conversion of precise PEG drugs.

Alternative Isotope Options for Amyloidosis Imaging: A Technologist’s Perspective

The recent pyrophosphate shortages can limit the availability of 99mTc-pyrophosphate scans for cardiac amyloidosis. However, another radiotracer is available: 99mTc-hydroxymethylene diphosphonate (HMDP). 99mTc-HMDP, widely available in the United States for bone scanning, has effectively been used in Europe to diagnose transthyretin amyloidosis. 99mTc-HMDP and 99mTc-pyrophosphate have comparable blood clearance and sensitivity. The imaging protocols for 99mTc-HMDP and 99mTc-pyrophosphate are similar, except 99mTc-HMDP is imaged 2-3 h after injection and whole-body imaging is optional. The interpretation is also essentially the same; however, caution is needed because of the high soft-tissue uptake with 99mTc-HMDP, which can affect heart-to-contralateral-lung ratios.

Red blood cell membrane-coated functionalized Au nanocage as a biomimetic platform for improved MicroRNA delivery in hepatocellular carcinoma

Dysregulation of microRNAs (miRNAs) expression is closely related to cancers and managing miRNA expression holds great promise for cancer therapy. However, their wide clinical application has been hampered by their poor stability, short half-life and non-specific biodistribution in vivo. Herein, a novel biomimetic platform designated as RHAuNCs-miRNA for improved miRNA delivery was prepared through wrapping miRNA-loaded functionalized Au nanocages (AuNCs) with red blood cell (RBC) membrane. RHAuNCs-miRNA not only successfully loaded miRNAs but also effectively protected them from enzymatic degradation. With good stability, RHAuNCs-miRNA had the characteristics of photothermal conversion and sustained release. Cellular uptake of RHAuNCs-miRNA by SMMC-7721 cells was in a time-dependent manner via clathrin- and caveolin-mediated endocytosis. The uptake of RHAuNCs-miRNAs was affected by cell types and improved by mild near infrared (NIR) laser irradiation. More importantly, RHAuNCs-miRNA exhibited a prolonged circulation time without the occurrence of accelerated blood clearance (ABC) in vivo, resulting in efficient delivery to tumor tissues. This study may demonstrate the great potential of RHAuNCs-miRNA for improved miRNAs delivery.

Polyglycerol-Decorated Hollow Mesoporous Ruthenium Nanoparticles for Long Circulation and Antitumor Effect

Hollow mesoporous ruthenium nanoparticles (HMRu NPs) are especially beneficial for drug delivery and photothermal therapy due to their distinguished specific surface area and photothermal properties. However, poor circulation performance in vivo means that the NPs are subject to rapid clearance before reaching the target tissue. Polyethylene glycol (PEG) modified nanoparticles have become the classical standard to extend circulation by reducing protein adsorption and escaping macrophage phagocytosis, but PEGylation tends to have an accelerated blood clearance effect after repeated use. Considering that the hyperbranched polymers exhibit excellent performance in protein shielding, that makes it possible for them to have the potential to replace PEG. Thus, HMRu NPs were decorated by polyglycerol (PG) in this work compared to PEGylation. The antiprotein adsorption capacity and macrophage escape were assayed by the bicinchoninic acid method and confocal microscopy, suggesting that PG-coating could avoid macrophage phagocytosis more efficiently than PEGylation. Finally, the resultant HMRu-PG could be used as drug carrier for synergistic chemotherapy and photothermal therapy in tumor treatment. This work provides a promising multimodal and versatile nanocarrier in tumor therapy.

PEGylated Lipid Nanoparticle Formulations: Immunological Safety and Efficiency Perspective.

Lipid nanoparticles (LNPs) have been recognized as efficient vehicles to transport a large variety of therapeutics. Currently in the spotlight as important constituents of the COVID-19 mRNA vaccines, LNPs play a significant role in protecting and transporting mRNA to cells. As one of their key constituents, polyethylene glycol (PEG)-lipid conjugates are important in defining LNP physicochemical characteristics and biological activity. PEGylation has proven particularly efficient in conferring longer systemic circulation of LNPs, thus greatly improving their pharmacokinetics and efficiency. Along with revealing the benefits of PEG conjugates, studies have revealed unexpected immune reactions against PEGylated nanocarriers such as accelerated blood clearance (ABC), involving the production of anti-PEG antibodies at initial injection, which initiates accelerated blood clearance upon subsequent injections, as well as a hypersensitivity reaction referred to as complement activation-related pseudoallergy (CARPA). Further, data have been accumulated indicating consistent yet sometimes controversial correlations between various structural parameters of the PEG-lipids, the properties of the PEGylated LNPs, and the magnitude of the observed adverse effects. Detailed knowledge and comprehension of such correlations are of foremost importance in the efforts to diminish and eliminate the undesirable immune reactions and improve the safety and efficiency of the PEGylated medicines. Here, we present an overview based on analysis of data from the CAS Content Collection regarding the PEGylated LNP immunogenicity and overall safety concerns. A comprehensive summary has been compiled outlining how various structural parameters of the PEG-lipids affect the immune responses and activities of the LNPs, with regards to their efficiency in drug delivery. This Review is thus intended to serve as a helpful resource in understanding the current knowledge in the field, in an effort to further solve the remaining challenges and to achieve full potential.

68Ga-Labeled TMTP1 Derivatives with Moderate Hydrophilicity for Positron Emission Tomography of Hepatocellular Carcinoma in High Contrast.

The reported specific positron emission tomography (PET) probes for the diagnosis of highly metastatic hepatocellular carcinoma (HCC) suffer from excessively high background uptake and fast blood clearance. Herein, five 68Ga-labeled polyethylene glycol (PEG)-modified derivatives of the TMTP1 peptide were synthesized. The log D values decreased from -1.70 (non-PEGylated) to -1.97 to -2.94 corresponding to the increase of PEG chain length. Subnanomolar and nanomolar affinities comparable to the non-PEGylated TMTP1 derivative were revealed by the IC50 values in SMMC-7721 cells. [68Ga]Ga-NOTA-PEG2-TMTP1 presented a significantly higher tumor/liver ratio (4.19 ± 0.54, at 30 min post intravenous injection) and tumor/muscle ratio (2.14 ± 0.17) compared to the others and the previously radiolabeled TMTP1 derivatives. Small HCC lesions (<2 mm) in situ were detected with high tumor/liver ratio and low tumor/muscle ratio. The improved pharmacokinetics and blood clearance rate of 68Ga-labeled TMTP1 derivatives indicated that moderate hydrophilicity due to PEGylation contributed to high-contrast PET of HCC.

Physiologically-based pharmacokinetic (PBPK) modeling of sodium fluorescein (SF) blood clearance for assessing hepatic ischemia-reperfusion injury (IRI)

Rationale: Quantitative analysis of bile formation machinery has potential to assess the viability of a liver for transplantation. Recently, we demonstrated the utility of the fluorescent dye SF by showing that the bile-to-plasma fluorescence ratio inversely correlated with warm ischemia time in rats and associated this finding with IRI-induced intracellular internalization of the MRP2 transporter (PMID: 35700191). However, it is unclear how much SF is cleared by active MRP2 in relation to other contributing factors associated with complex in vivo physiology. We hypothesize that PBPK modeling can account for dominant vascular, tissue, and biochemical processes contributing to SF clearance, including MRP2 activity, and simulate diminished liver function in IRI leading to decreased disposition of SF in the bile. Methods: We considered previously published data of SF fluorescence intensity in blood, plasma, and bile over time in rats under control and IRI conditions. To model these data, we performed SF calibration experiments and converted these data from fluorescence to concentration using standard curves generated from nonlinear regression. We fit an empirical triple-exponential function to the calibrated control blood SF concentration data and used this function as an input for a mechanistic liver-centric PBPK model. The PBPK model accounts for three regions in the liver (blood, hepatocytes, bile) and major physiological processes, including kinetics of basolateral and canalicular transporters governing SF dynamics in different liver regions. We fit this model to the control SF concentration data in the bile using a pseudo-Monte Carlo strategy. Model parameters relating to maximal velocity for the transporters were allowed to vary, while all others were fixed to physiologic values. Results: The PBPK model was able to provide a good fit to control bile SF concentration data. The input model for blood SF concentration was able to predict plasma SF concentration related by a variable partition coefficient, which led to a large difference between plasma and blood SF concentrations at high SF concentrations but diminished at lower concentrations. We simulated the IRI condition by decreasing MRP2 activity, which showed that SF blood clearance through hepatocytes into bile is highly sensitive to changes in the activity of MRP2, consistent with our hypothesis that SF clearance data and PBPK modeling could quantify the effect of IRI on MRP2 activity. These simulations showed a larger decrease in SF concentration in bile relative to the increase in blood resulting in a lower bile-to-blood SF ratio. Conclusions: Based on our experimental data, we developed a liver-centric PBPK model for the blood clearance of SF through hepatocytes and bile and used this model to simulate the contribution of MRP2 to diminished secretion of SF into the bile as measured following IRI. MCW/BME-FP00022381 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

69 The equine blood fatty acid profile and clearance rate of supplemented fatty acids

An optimal equine blood fatty acid (FA) profile has not been defined, nor has a general baseline profile been established. Further, there is little information on the clearance rate of FAs in plasma and red blood cells (RBCs) after supplementation ends. Data from 4 studies were compiled to evaluate the baseline FA profile in horses fed varied diets without FA supplementation for at least 6 mo (n = 68). Sample handling and processing were performed identically in all studies and 24 FAs were quantified using gas chromatography. A subset of data (n = 18) was used to evaluate how eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) affects the FA profile during and after 3 mo of supplementation (4.3–8.7 g EPA+DHA/d). Data were analyzed for mixed-effects. Baseline plasma FA profile included primarily linoleic acid (LA; 49.49% mean; 95% CI [48.32, 50.65]), stearic acid (15.90%; [15.61, 16.19]), palmitic acid (13.99%; [13.59, 14.38]), and oleic acid (10.66%; [10.12, 11.20]). Other FAs included arachidonic acid (AA; 0.94%; [0.87, 1.01]), EPA (0.14%; [0.12, 0.16]), and DHA (0.16%; [0.14, 0.19]). Baseline RBC profile differed slightly, primarily made up of LA (37.47%; [36.76, 38.17]), oleic acid (20.20%; [19.92, 20.47]), stearic acid (17.40%; [17.09, 17.71]), and palmitic acid (14.86%; [14.53, 15.20]). Other FAs included AA (1.23%; [1.18, 1.29]), EPA (0.19%; [0.16, 0.22]),and DHA (0.21%; [0.18, 0.24]). Plasma EPA and DHA increased 277% (0.41%; [0.29, 0.54]; P < 0.001) and 223% (0.56%; [0.40, 0.73]; P < 0.001), respectively, within 1 mo of supplementation and returned to baseline levels (P = 0.98) by 1 mo post-supplementation. RBC EPA and DHA increased 178% (0.37%; [0.29, 0.44]; P < 0.0001) and 102% (0.37%; [0.31, 0.44]; P < 0.0001), respectively, within the first month of supplementation and continued to climb 313% (0.63%; [0.46, 0.79]; P < 0.001) and 197% (0.70%; [0.55, 0.85]; P < 0.001) by the third month, respectively. RBC EPA returned to baseline (P = 0.21) by 3 mo post-supplementation, but RBC DHA had still not returned (P < 0.05). Compared with plasma, RBCs showed gradual changes over time and required significantly more time to appropriately wash out. Additionally, the clearance rate may differ between fatty acids. Plasma values are more commonly reported than values for RBC or other tissues. These data suggest RBC values may be important to consider and that appropriate washout durations from previous FA supplementation may be longer than expected. Further research is needed to evaluate other tissue affected by FA supplementation and how long the FA profile may be altered.

Subarachnoid Blood Clearance and Aneurysmal Subarachnoid Hemorrhage Outcomes: A Retrospective Review.

Delayed cerebral ischemia (DCI) continues to be a significant contributor to morbidity and mortality following aneurysmal subarachnoid hemorrhage (aSAH). Subarachnoid blood and its degradation products have been implicated in DCI, and faster blood clearance has been hypothesized to confer better outcomes. This study evaluates the relationship between blood volume and its clearance on DCI (primary outcome) and location at 30days (secondary outcome) after aSAH. This is a retrospective review of adult patients presenting with aSAH. Hijdra sum scores (HSS) were assessed independently for each computed tomography (CT) scan of patients with available scans on post-bleed days 0-1 and 2-10. This cohort was used to evaluate the course of subarachnoid blood clearance (group 1). A subset of patients in the first cohort with available CT scans on both post-bleed days 0-1 and post-bleed days 3-4 composed the second cohort (group 2). This group was used to evaluate the association between initial subarachnoid blood (measured via HSS post-bleed days 0-1) and its clearance (measured via percentage reduction [HSS %Reduction] and absolute reduction [HSS-Abs-Reduction] in HSS between days 0-1 and 3-4) on outcomes. Univariable and multivariable logistic regression models were used to identify outcome predictors. One hundred fifty-six patients were in group 1, and 72 patients were in group 2. In this cohort, HSS %Reduction was associated with decreased risk of DCI in univariate (odds ratio [OR] = 0.700 [0.527-0.923], p = 0.011) and multivariable (OR = 0.700 [0.527-0.923], p = 0.012) analyses. Higher HSS %Reduction was significantly more likely to have better outcomes at 30days in the multivariable analysis (OR = 0.703 [0.507-0.980], p = 0.036). Initial subarachnoid blood volume was associated with outcome location at 30days (OR = 1.331 [1.040-1.701], p = 0.023) but not DCI (OR = 0.945 [0.780-1.145], p = 0.567). Early blood clearance after aSAH was associated with DCI (univariable and multivariable analyses) and outcome location at 30days (multivariable analysis). Methods facilitating subarachnoid blood clearance warrant further investigation.

One to one comparison of cell-free synthesized erythropoietin conjugates modified with linear polyglycerol and polyethylene glycol

With more than 20 Food and Drug Administration (FDA)-approved poly (ethylene glycol) (PEG) modified drugs on the market, PEG is the gold standard polymer in bioconjugation. The coupling improves stability, efficiency and can prolong blood circulation time of therapeutic proteins. Even though PEGylation is described as non-toxic and non-immunogenic, reports accumulate with data showing allergic reactions to PEG. Since PEG is not only applied in therapeutics, but can also be found in foods and cosmetics, anti-PEG-antibodies can occur even without a medical treatment. Hypersensitivity to PEG thereby can lead to a reduced drug efficiency, fast blood clearance and in rare cases anaphylactic reactions. Therefore, finding alternatives for PEG is crucial. In this study, we present linear polyglycerol (LPG) for bioconjugation as an alternative polymer to PEG. We report the conjugation of LPG and PEG by click-chemistry to the glycoprotein erythropoietin (EPO), synthesized in a eukaryotic cell-free protein synthesis system. Furthermore, the influence of the polymers on EPOs stability and activity on a growth hormone dependent cell-line was evaluated. The similar characteristics of both bioconjugates show that LPGylation can be a promising alternative to PEGylation.

Pretargeted Alpha Therapy of Disseminated Cancer Combining Click Chemistry and Astatine-211.

To enhance targeting efficacy in the radioimmunotherapy of disseminated cancer, several pretargeting strategies have been developed. In pretargeted radioimmunotherapy, the tumor is pretargeted with a modified monoclonal antibody that has an affinity for both tumor antigens and radiolabeled carriers. In this work, we aimed to synthesize and evaluate poly-L-lysine-based effector molecules for pretargeting applications based on the tetrazine and trans-cyclooctene reaction using 211At for targeted alpha therapy and 125I as a surrogate for the imaging radionuclides 123, 124I. Poly-L-lysine in two sizes was functionalized with a prosthetic group, for the attachment of both radiohalogens, and tetrazine, to allow binding to the trans-cyclooctene-modified pretargeting agent, maintaining the structural integrity of the polymer. Radiolabeling resulted in a radiochemical yield of over 80% for astatinated poly-L-lysines and a range of 66-91% for iodinated poly-L-lysines. High specific astatine activity was achieved without affecting the stability of the radiopharmaceutical or the binding between tetrazine and transcyclooctene. Two sizes of poly-L-lysine were evaluated, which displayed similar blood clearance profiles in a pilot in vivo study. This work is a first step toward creating a pretargeting system optimized for targeted alpha therapy with 211At.