Incubation Of Plasma Research Articles

Biotransformation of nanoplastics in human plasma and their permeation through a model in vitro blood-brain barrier: An in-depth quantitative analysis

Challenges in characterizing and quantifying nanoplastics within the human body hinder understanding of their transport, biotransformation, and potential for cellular penetration and barrier crossing. By implementing an innovative analytical workflow, including incorporation of gadolinium (Gd) as a tracer into the polymer matrix of nanoplastics, the fate of nanoplastics relative to an in vitro blood-brain barrier (BBB) model is elucidated in the absence or presence of a biomomolecule corona. The nanoplastics were incubated in human plasma for 5 min, 1 h, 6 h, and 24 h, after which the absorbed proteins and lipids (biocorona) were determined. A total of 268 proteins were identified in the biological coronas on polystyrene (PS) and polyvinyl chloride (PVC) nanoplastics, with the initial compositions being broadly similar on both PS and PVC. Both nanoplastics exhibited a strong affinity for phosphatidylcholines (PC) and lysophosphocholines (LPC) from human plasma. The inherent chemical composition of the nanoplastics plays a pivotal role in the corona’s evolution over time. Human induced pluripotent stem cell (iPSC)-derived endothelial cells (iECs) and astrocytes were exposed for 2 h to 5 µg L−1 of pristine nanoplastics or nanoplastics covered with a biological corona (following incubation in plasma for 6 h). A relatively low concentration of PS and PVC nanoplastics was determined to be present within the cellular layer of the BBB. The number of PVC nanoplastics crossing the BBB was higher than the number of PS nanoplastics. The presence of a biological corona on these particles decreases their uptake and transcytosis. This understanding might further the development of preventive measures or therapeutic strategies to counteract potential nanoplastic-induced neurotoxicity, and provide a foundation for development of in silico models to predict the neurotoxic implications of nanoplastics.

Safety of intraoperative autologous plasma incubation of corneal grafts for reducing endothelial cell loss: a pilot study.

Corneal endothelial cell loss contributes to transplant failure. Autologous plasma products (APP) activate salvaging pathways that can prevent oxidative stress perioperatively. This study aimed to evaluate the safety of intraoperative incubation of full-thickness corneal grafts in platelet-rich plasma (aPRP) and plasma rich in growth factors (PRGF-Endoret) in mitigating postoperative corneal endothelial cell loss (ECL). Pilot study including patients undergoing penetrating keratoplasty (PK) for various indications between June 2021 and December 2022. Patients were randomly assigned to receive either aPRP or PRGF-Endoret incubation, while those who declined intervention served as the control group. Demographic and clinical data were collected, including preoperative and postoperative endothelial cell count, intraocular pressure, pachymetry, and adverse reactions. Thirty individuals who underwent PK completed follow-up: eight from the aPRP group, 10 from the PRGF-Endoret group, and 12 from the control group. No adverse events related to APP treatment were recorded. In the first and third postoperative months, the APP group had significantly lower ECL percentages (37% vs. 25%, p = 0.02, and 44% vs. 33%, p = 0.02, respectively); this trend was maintained in the sixth month. When stratified, the PRGF-Endoret group showed significant differences in ECL reduction compared to controls at both time points (p = 0.03 and p = 0.05, respectively). The aPRP group showed a similar statistically significant outcome exclusively on the third postoperative month (p = 0.04). APP tended to reduce corneal edema faster than controls. Hexagonality was significantly better in the APP groups in the first and third months, particularly in the PRGF-Endoret group (p < 0.005). Preoperative incubation with APP is safe and promotes better endothelial cell quality and quantity in the early postoperative period following PK. These findings suggest a potential clinical benefit in enhancing graft outcomes and warrant further investigation.

Disulfide-adducts with cysteine residues in human serum albumin prove exposure to malodorous mercaptans in vitro

Malodorants are mixtures containing mercaptans, which trigger the flight instinct upon exposure and might thus be deployed in military and civilian defense scenarios. Exposure to mercaptans might lead to unconsciousness, thus representing a possible threat for health. Therefore, we developed and validated a bioanalytical procedure for the simultaneous detection and identification of corresponding biomarkers for the verification of exposure to mercaptans. Disulfide-adducts of ethyl mercaptan (SEt), n-butyl mercaptan (SnBu), tert-butyl mercaptan (StBu) and iso-amyl mercaptan (SiAm) with cysteine (Cys) residues in human serum albumin (HSA) were formed by in vitro incubation of human plasma. After pronase-catalyzed proteolysis, reaction products were identified as adducts of the single amino acid Cys and the dipeptide cysteine-proline (Cys34Pro) detected by a sensitive μLC-ESI MS/MS method working in the scheduled multiple reaction monitoring (sMRM) mode. Dose-response studies showed linearity for the yield of Cys34Pro-adducts in the range from 6 nM to 300 μM of mercaptans in plasma and limits of identification (LOI) were in the range from 60 nM to 6 μM. Cys34-adducts showed stability for at least 6 days in plasma (37 °C). The presented disulfide-biomarkers expand the spectrum for bioanalytical verification procedures and might be helpful to prove exposure to malodorants.

Deciphering the monocyte-targeting mechanisms of PEGylated cationic liposomes by investigating the biomolecular corona

Cationic liposomes specifically target monocytes in blood, rendering them promising drug-delivery tools for cancer immunotherapy, vaccines, and therapies for monocytic leukaemia. The mechanism behind this monocyte targeting ability is, however, not understood, but may involve plasma proteins adsorbed on the liposomal surfaces. To shed light on this, we investigated the biomolecular corona of three different types of PEGylated cationic liposomes, finding all of them to adsorb hyaluronan-associated proteins and proteoglycans upon incubation in human blood plasma. This prompted us to study the role of the TLR4 co-receptors CD44 and CD14, both involved in signalling and uptake pathways of proteoglycans and glycosaminoglycans. We found that separate inhibition of each of these receptors hampered the monocyte uptake of the liposomes in whole human blood. Based on clues from the biomolecular corona, we have thus identified two receptors involved in the targeting and uptake of cationic liposomes in monocytes, in turn suggesting that certain proteoglycans and glycosaminoglycans may serve as monocyte-targeting opsonins. This mechanistic knowledge may pave the way for rational design of future monocyte-targeting drug-delivery platforms.

Abstract 4702: MediLink’s TMALIN ADC linker technology: Tumor microenvironment specific extracellular and intracellular double cleavage mechanism for better efficacy and expanded target space

Abstract Antibody-drug conjugates (ADC) are a type of the fastest growing anticancer drug modalities. However, the traditional ADC technologies constrain on therapeutic development, requiring overexpressed targets, high internalizing antigens and efficient intracellular processing. To this end, MediLink’s TMALIN platform was developed, with an irreversible pyrimidine coupling anchor and a valyl dipropyl-lysyl glycinamide-methylene (-VK*G-NHCH2-O-) cleavable sequence. The TMALIN platform is featured as a high hydrophilicity linker-payload, remarkable stability in circulation, coupled with an efficient payload release mechanism which is achieved through the cleavage both extracellularly in the tumor microenvironment and intracellularly within lysosomes. A site-specific and homogeneous conjugation using TMALIN platform provides highly stable ADC in circulation. It is evidenced by less than 2% payload drop-off after a 21-day incubation in plasma under physiological conditions in vitro. This feature is observed in over 7 ADCs developed using the TMALIN platform. Additionally, the high stability was also illustrated by the pharmacokinetic profiles in NHP studies and clinical trials, with the overlapped curves of ADC and TAb in all TMALIN platform-based ADCs. Apart from the stability, a battery of in vitro and in vivo studies showed the linker-payload efficiently releases the payload in both tumor cells and the tumor microenvironment. Due to the high stability in circulation and efficient cleavage within tumors, the TMALIN platform ADCs have shown promising efficacy and good safety windows in preclinical studies. In xenograft mouse models, including those with low antigen expression, these ADCs have demonstrated tumor regressions and good tolerability. This highlights the potential of the TMALIN platform in improving the efficacy of ADCs and expanding their therapeutic applications. Furthermore, favorable safety profiles have been observed in clinical trials and/or pivotal non-human primate (NHP) studies for ADCs developed using the TMALIN platform. No drug-related adverse effects in organs were identified including the lungs, liver, or kidneys for those ADCs. In summary, the TMALIN platform addresses the unmet need of ADCs by releasing the payload extracellularly in tumors and tumor microenvironments on top of the known intracellular lysosomal cleavage mechanisms. This enables the advancement of ADC development, and has the potential to expand the field of ADC therapy and improve patient outcomes. Citation Format: Tongtong Xue, Liang Xiao, Qigang Liu, Shuai Song, Ailin Shan, Xiaozhou Zhang, Sasha Stann, Jiaqiang Cai. MediLink’s TMALIN ADC linker technology: Tumor microenvironment specific extracellular and intracellular double cleavage mechanism for better efficacy and expanded target space [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 4702.

Abstract 2114: Enhancing therapeutic strategies for osimertinib-resistant EGFR-mutant NSCLC: A HER3 dual-payload ADC (dpADC) with topoisomerase I and EGFR tyrosine kinase inhibitor

Abstract Background: EGFR tyrosine kinase inhibitors (TKIs) have significantly improved the survival rate and the quality of life of the NSCLC patients with EGFR mutation in the first line setting. For example, Osimertinib, a potent third-generation EGFR TKI, has demonstrated exceptional efficacy in metastatic NSCLC with specific EGFR mutations. Despite its success, the emergence of drug resistance, often linked to elevated HER3 protein expression, remains a significant challenge. U3-1402 (HER3-Dxd), an HER3 ADC, demonstrates a promising result in managing Osimertinib-resistant NSCLC. It has been reported that combining U3-1402 with Osimertinib has shown enhanced efficacy in resistant cell lines and mouse models. Here, we explore a novel HER3 dual-payload ADC (HER3 dpADC), uniquely combining a Topoisomerase I inhibitor and an EGFR TKI within a single antibody structure using the enzymatic site-specific conjugation platform developed by GeneQuantum (GQ). This innovative approach demonstrates potent and synergistic anti-tumor effects in vitro and in vivo, presenting an alternative therapeutic strategy for EGFR-mutant NSCLC patients. Results: GQ's dpADC platform technology employs two orthogonal enzymatic site-specific conjugations and stable linker technologies to efficiently generate the dual-payload ADC with high homogeneity and quality. For HER3 dpADC, HER3 Ab was conjugated with a novel Topoisomerase I inhibitor, TopoIx, and an EGFR tyrosine kinase inhibitor. In vitro DAR analysis revealed high linker stability, with minimal free payload release even after 96 hours of plasma incubation. Binding affinity assessments confirmed comparable affinity between HER3 dpADC and HER3 mAb in specific EGFR exon 19 deletion cell lines. Using various NSCLC cell lines in a 3D-spheroid culture system, HER3 dpADC demonstrated significant dose-dependent and synergistic anti-tumor activities. A robust bystander killing efficacy was observed in both HER3+/HER3- HEK293T cell coculture assays. Evaluation in CDX mouse models consistently demonstrated heightened in vivo efficacy by HER3 dpADC, aligning with the promising in vitro data. In NSCLC patient-derived xenograft (PDX) mouse models representing EGFR tyrosine kinase inhibitor-sensitive or resistant phenotypes, HER3 dpADC induced tumor regressions with no obvious toxicity, underscoring its potential as the next-generation HER3 targeting agent. Conclusion: The innovative HER3 dpADC, leveraging an efficient dual enzymatic site-specific conjugation, exhibited robust anti-tumor efficacy in both in vitro and in vivo settings, surpassing single-agent treatments. Its synergistic mechanism of action presents a promising possibility for developing a more potent and the front-line therapeutic solutions in NSCLC patients who have progressed on standard therapies. Citation Format: Lina Wang, Meijun Xiong, Xinju Gao, Chengang Zhou, Yu Han, Yanchun Li, Junhao Wang, Lili Shi, Gang Qin, Paul H. Song. Enhancing therapeutic strategies for osimertinib-resistant EGFR-mutant NSCLC: A HER3 dual-payload ADC (dpADC) with topoisomerase I and EGFR tyrosine kinase inhibitor [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 2114.

Assessing in vitro stability of remdesivir (GS-5734) and conversion to GS-441524 in feline plasma and whole blood

Feline infectious peritonitis (FIP) is a potentially fatal coronavirus-driven disease of cats. Treatment with nucleoside analogue GS-441524 and or prodrug remdesivir (RDV) have produced remission in both experimentally induced and naturally occurring FIP, yet information regarding metabolism of RDV into GS-441524 in cats is scarce. This study assessed possible phase I metabolism of RDV in cats, utilising an in vitro feline microsome model with in vitro t1/2 and in vitro Clint calculated using the substrate depletion method. A previously validated high-performance liquid chromatography (HPLC) fluorescence method was utilised for detection and analysis of RDV and GS-441524. Qualitative yield of RDV and intermediate metabolite GS-441524 were determined following microsome incubation, then compared to whole blood and plasma incubations. In vitro microsome incubation resulted in rapid depletion of RDV, though it did not appear to resemble a conventional phase I-dependent reaction in cats, as it is in humans and dogs. Depletion of RDV into GS-441524 was demonstrated in whole blood in vitro, suggesting cats convert RDV to GS-441524, likely via blood esterases, as observed in mice and rats. RDV metabolism is unlikely to be impacted by impaired liver function in cats. Furthermore, as RDV depletes within minutes, whereas GS-441524 is very stable, whole blood or plasma GS-441524 concentrations, rather than plasma RDV concentrations, are more appropriate for therapeutic drug monitoring (TDM) in cats receiving RDV.

Surface Oxygen Deficiency Enabled Spontaneous Antiprotein Fouling in WO3 Nanosheets for Biosensing in Biological Fluids.

Biofouling deteriorates the performance of sensors operated in biofluids. Protein adsorption is believed to be the first step of biofouling, which also reduces biocompatibility by further inducing cell adhesion, platelet activation, and even inflammation. Current studies of antifouling coatings are focused on polymers and hydrogels, which have succeeded in remaining resistant to protein adsorption, but their application on sensor electrodes is limited due to low conductivity and biocompatibility. Here, we report a spontaneous antibiofouling strategy for sensor electrodes by controlling oxygen vacancies in WO3 nanosheets. Irreversible adsorption of proteins was reduced by 76% in unprocessed human plasma when electrodes were coated with WO3 rich in surface oxygen vacancy. These electrodes maintained 91% of the initial current density after 1 month of incubation in human plasma.

Comparative analysis of hemostasis indices of sheep and lambs obtained taking into account body temperature

This paper shows the results of the study of the effect of temperature on the rate of coagulation processes in the blood plasma of small ruminants. The aim of the work was to compare the results of the study of blood coagulation indicators of sheep and lambs of Romanov and Edilbaev breed, obtained using medical and veterinary coagulometer, taking into account the age of the animal and its body temperature. The study was conducted in the Vologda region. The following parameters of coagulogram were analyzed: thrombin time, prothrombin time, activated partial thromboplastin time, amount and activity of fibrinogen. It was found that at physiologic temperature on veterinary coagulometer in plasma of sheep and lambs there is an increase of prothrombin time by 8,54 s and 15,24 s respectively, at that correlation-regression and one-factor dispersion analysis shows a positive moderate relationship between prothrombin time and plasma incubation temperature (r=0,3). In the plasma of lambs at the study with the setting of physiological temperature on the veterinary coagulometer there is an increase in the level of fibrinogen and its activity by 0.35 g and 4.53 sec, respectively, Correlation-regression and one-factor dispersion analysis showed a reliable high positive relationship between fibrinogen level and plasma incubation temperature (r=0,9) and a reliable high negative relationship between fibrinogen activity and plasma incubation temperature (r= -0,9). In this regard, a veterinary coagulometer with the possibility of setting the necessary temperature for adequate assessment of hemostasiological processes in sheep of different age groups can be recommended.

Dependence of coagulation activity of chicken blood IN VITRO on temperature

The paper presents the results of examine the relationship between blood plasma incubation temperature and the hemostasis parameters in chickens. The purpose of this research was to study of the blood coagulation parameters in chickens during hypo-, normo- and hyperthermia in vitro. In addition, to assess the possibility of using a coagulometer to identify hemostasis activity and the use of hemostatic reactions in chickens as models for human medicine. The Vologda region was where the study was conducted. The coagulogram was examined for the following parameters: thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen activity, and antithrombin III activity (AT-III). It was found that at 46° C activation of blood clotting occurs, manifested as an acceleration of the TT by 29% (13 seconds), PT by 72% (111 seconds), and decrease in the antithrombin III activity by 6% (1.5 seconds) compared to normothermia. The parameters of secondary hemostasis also responded to hypothermia (18 °C) with hypercoagulation. It was an acceleration of PT of 81% (125 sec), an increase in fibrinogen activity by 77% (84 sec) and a decrease in AT-III activity by 55% (14 sec), with the exception of thrombin time, which was extended by 70% (104 seconds). There were no significant changes in the APTT at different temperatures. The correlation-regression and one-factor analysis of variance revealed a significant correlation between the temperature and the TT and the fibrinogen activity, where the coefficients of determination for which were 71% and 39%, respectively. Plasma hemostasis activity measured on a coagulometer (at 37 °C) showed no significant differences from those measured at 43 °C, except for prothrombin time, which was 71.51 seconds (47%) less than that measured at 43 °C.

Abstract 17453: In vitro Aspirin Responsiveness Can Predict Platelet Hyperreactivity and Cardiovascular Risk

Background: While antiplatelet therapy decreases cardiovascular events, insufficient platelet aggregation suppression by antiplatelet therapy is associated with an increased risk of cardiovascular events. In this study, we developed a diagnostic tool to establish an individual’s “aspirin (ASA) responsiveness” in vitro and investigated its association with other metrics of platelet activity and cardiovascular risk. Methods: In 115 subjects off all antiplatelet therapy, platelet aggregation in response to high-dose of arachidonic acid (AA; 1.5 mM) with and without incubation of platelet-rich plasma with ASA (3 mM) was measured by light transmission aggregometry (LTA). ASA responsiveness was defined as ([% aggregation to AA - % aggregation to AA + ASA] / % aggregation to AA) x 100. Next, we correlated ASA responsiveness with other metrics of platelet activity. Finally, we evaluated its clinical significance in a cohort of patients undergoing lower extremity revascularization (LER) followed longitudinally for cardiovascular events. Results: Among 115 participants (mean age 50.3 years old, 65% female, and 43.5% underrepresented minorities, off all antiplatelet therapy), mean % aggregation to high-dose AA was 89.5% ± 3.5. In vitro ASA responsiveness was variable, mean 73.72% ± 15 (range: 13.7% to 97.6%). ASA responsiveness was lower in older (P&lt;0.05) and female participants (P&lt;0.05) and was not associated with race/ethnicity, diabetes, or platelet count. Lower ASA responsiveness was associated with a significant increase in platelet aggregation to epinephrine (0.4 μM, P=0.0004), ADP (1.0 μM, P=8.89x10 -9 ), and collagen (0.2 μg/mL, P=1.16x10 -9 ). In an independent cohort of 67 patients undergoing LER, lower in vitro ASA responsiveness (e.g. a reduced platelet-inhibiting effect), was associated with a significantly increased risk of major adverse cardiac and limb events (1 st tertile-63.6%, 2 nd tertile-50.0% and 3 rd tertile-27.3%; P=0.016 for trend). Conclusions: Reduced ASA responsiveness is associated with increased platelet activity and a higher risk of cardiovascular events. We propose that this rapid, relatively simple assay may be used preclinically for ASA testing and to identify individuals at high cardiovascular risk.

Controlling the protein corona of polymeric nanocapsules: effect of polymer shell on protein adsorption.

Understanding the interactions between nanocarriers and plasma proteins is essential for controlling their biological fate. Based on the reported potential of polymeric nanocapsules (NCs) for the targeted delivery of oncological drugs, the main objective of this work has been to investigate how the surface chemical composition influences their protein corona fingerprint. Thus, we developed six NC prototypes with different polymer shells and physicochemical properties and quantified the amount of protein adsorbed upon incubation in human plasma. Using sequential window acquisition of all theoretical mass spectra (SWATH-MS) and following the Minimum Information about Nanomaterial Biocorona Experiments (MINBE) guidelines, we identified different protein corona patterns. As expected, the presence of polyethylene glycol (PEG) in the polymer shell reduced the protein corona, particularly the adsorption of immunoglobulins. However, by comparing the different prototypes, we concluded that the protein adsorption pattern was not exclusively driven by PEG. In fact, a highly PEGylated prototype exhibited intense apolipoprotein IV adsorption. On the other hand, we also observed that polymeric NCs containing 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) exhibited high adsorption of vitronectin, a protein that is known for enhancing the uptake of nanosystems by lung epithelium and several cancer cells. Overall, the gathered information allowed us to identify promising polymeric NCs with an expected prolonged circulation time, enhanced tumor targeting, liver accumulation, and preferential uptake by the immune system. In this sense, the analyses of the protein corona performed along this work will hopefully contribute to advancing a new generation of rationally designed nanometric drug delivery systems.

The Effect of Cushioned Centrifugation, with and without Enzymatic Reduction of Viscosity, on the Motility Pattern and Kinematic Parameters of Dromedary Camel Bull Spermatozoa.

In order to contribute to the development of semen processing procedures in camelids, the aims of the present study were to evaluate (i) the effect of 35% seminal plasma incubation on dromedary camel epididymal sperm motility and kinematic parameters, (ii) the effects of centrifugation, with cushion fluid and enzymatic reduction of viscosity (Papain + E64) during ejaculate processing, on the motility and kinematic parameters of dromedary camel ejaculates. The incubation with seminal plasma significantly reduced the percentage of progressively motile spermatozoa as well as the proportion of medium progressive spermatozoa whilst increasing the percentage of non-progressive spermatozoa. The centrifugation procedure improved the sperms' kinematic parameters, and the highest values were observed for samples centrifugated with cushion fluid. The samples treated with Papain + E64 showed a significant increase in both total and medium progressive spermatozoa, along with a reduction of non-progressive spermatozoa (p < 0.05). The results of this investigation show that a simple, cheap, and effective procedure, such as cushioned centrifugation, could improve the motility patterns of dromedary camel spermatozoa; in combination with enzymatic reduction of viscosity, this method leads to the best results in terms of recovery rates and sperms' kinematic parameters.

Diclofenac prodrugs nanoparticles: An alternative and efficient treatment for rheumatoid arthritis?

We have synthesized new lipidic prodrugs of diclofenac by grafting aliphatic chains (C10, C12, C16 and C18) to diclofenac through an ester bond. Their molecular formulas were confirmed through HR-MS and the formation of ester bond by FTIR and NMR spectroscopy. Nanoparticles of the different prodrugs were successfully formulated using emulsion evaporation method and DSPE-PEG2000 as the only excipient. All nanoparticles were spherical and had a size between 110 and 150 nm, PdI ≤ 0.2 and negative Zeta potential values from −30 to −50 mV. In addition, they were stable upon storage at 4 °C up to 30–35 days. The encapsulation efficiency of the prodrug was above 90 % independently of the aliphatic chain length grafted. Nanoparticles did not induce any toxicity on LPS-activated THP-1 cells up to a concentration of 100 μg/mL (equivalent diclofenac) whereas diclofenac sodium salt IC50 was around 20 μg/mL. Following incubation of nanoparticles with LPS-activated THP-1 cells, a dose dependent inhibition of TNF-α was observed comparable to standard diclofenac sodium. Based on in vitro studies representative nanoparticles, Prodrug 3 NPs (C16 aliphatic chain) were selected for further in vitro and in vivo studies. Upon incubation in murine plasma, Prodrug 3 NPs underwent an enzymatic cleavage and almost 70 % of diclofenac was released from nanoparticles in 8 h. In vivo studies on a collagen induced arthritis murine model showed contrasted results: on one hand Prodrug 3 NPs led to a significant decrease of arthritis score and of paw volume compared to PBS after the second injection, on the other hand the third injection induced an important hepatic toxicity with the death of half of the mice from the NP group. To promote the reduction of inflammation while avoiding hepatic toxicity using NPs would require to precisely study the No Observable Adverse Effect Level and the schedule of administration in the future.

Identification and characterization of soft protein corona absorbed on iron oxide nanoparticles

Nanomedicines have recently garnered significant attention due to their great potential to improve the efficiency of disease diagnosis and treatment. Nonetheless, a wide gap still exists between innovative research and clinical application because of limited understanding of the protein corona formed on the nanocarrier's surface. When NPs are introduced to living systems, the protein corona rather than the originally expected surface of the nanoparticle is initially recognized, further affecting immune clearance and tumor-specific delivery. This study found that after incubation with human plasma, the protein corona formed on the iron oxide nanoparticle's (IONP) surface significantly increased the uptake of IONPs in the leukemic cell line MOLM13. The formation and thicknesses of the protein coronas were measured by dynamic light scattering, nanoparticle tracking analysis, and flow cytometry, and the hard corona was measured by transmission electron microscopy. The size distribution of IONPs before and after incubation with plasma was measured, and the change in particle size was calculated to derive the thickness of the soft corona (7.58±3.94 nm) by subtracting the thickness of the hard corona from that of the full protein corona. The results can provide an approximation of the soft corona area after plasma incubation and contribute to the understanding of the features of the protein corona of nanoparticles with different physical and chemical properties.

Abstract LB001: Encapsulation of IL-12 with an ultra pH-sensitive nanoparticle platform improves tolerability and promotes antitumor response in mice

Abstract Background: Interleukin-12 is a potent proinflammatory cytokine that proliferates and activates T cells, NK cells and differentiates Th1 cells. Translation of IL-12 for cancer treatment has been hindered by lethal toxicities due to cytokine release syndrome and there are currently no approved IL-12 therapies. To minimize the severe toxicities while maintaining potency, we have developed ON-BOARD, an ultra-pH sensitive nanoparticle platform for masked and targeted delivery of payloads to the acidic tumor microenvironment. The clinical feasibility of ON-BOARD has been demonstrated by high tumor specificity of pegsitacianine in multiple tumor types from the Phase I and II clinical trials. Herein we report encapsulation and masked delivery of IL-12 to tumor-bearing mice using ON-BOARD, demonstrating significantly improved tolerability, anti-tumor efficacy, and potential for clinical translation. Methods: A mouse IL-12 fused with Fc was formulated in ON-BOARD nanoparticles. Particle properties were characterized and lead formulations were identified by in vitro screening to determine pH-mediated bioactivity in reporter and ELISA assays and stability in mouse plasma. In vivo studies were performed to compare the activity of unencapsulated IL-12 to ON-BOARD/IL-12 formulations. PD response was evaluated by measuring systemic cytokine levels in plasma, while clinical chemistry was performed to evaluate liver and kidney functions. Anti-tumor efficacy of ON-BOARD/IL-12 formulations was performed in mice bearing syngeneic MC38 colorectal cancer tumors compared to unencapsulated IL-12. Results: ON-BOARD/IL-12 formulations showed high encapsulation efficiency (&amp;gt;85%) and drug loading up to 20% wt. in uniformly distributed stable particles (Dh&amp;lt;50nm). pH-specific payload release was confirmed in vitro with &amp;gt;100-fold activation window between the acid-activated and intact formulations. Following incubation in mouse plasma the lead ON-BOARD formulations showed stable IL-12 encapsulation by an ELISA assay. In vivo, ON-BOARD/IL-12 formulations demonstrated significantly improved tolerability compared to unencapsulated IL-12. When dosed at 5µg/dose compared to unencapsulated protein at 1 µg/dose, ON-BOARD/IL-12 demonstrated reduced body weight loss (&amp;lt;2% vs 13%) and decreased liver injury markers AST and ALT. Analysis of systemic cytokines (IFNγ, IL-6, IL-10, TNFα, etc) showed significantly lower levels for ON-BOARD formulations including &amp;gt;1,000-fold reduction in plasma IFNγ level which is known to be directly induced by IL-12 signaling. ON-BOARD/IL-12 formulations also demonstrated strong anti-tumor efficacy in MC38 tumor-bearing animals with &amp;gt;95% TGI and complete responders. Conclusions: The ON-BOARD platform demonstrated potential for masking toxicity and facilitating tumor-specific delivery of IL-12 proteins for cancer therapy. Citation Format: Qingtai Su, Stephen Gutowski, Irina Kalashnikova, Austin Burcham, Bhargavi Allu, Zirong Chen, Zhichen Sun, Jinming Gao, Ruolan Han, Jason B. Miller, Tian Zhao. Encapsulation of IL-12 with an ultra pH-sensitive nanoparticle platform improves tolerability and promotes antitumor response in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB001.

A Novel Strategy for Liposomal Drug Separation in Plasma by TiO2 Microspheres and Application in Pharmacokinetics.

Liposomes are nano-scale materials with a biofilm-like structure. They have excellent biocompatibility and are increasingly useful in drug delivery systems. However, the in vivo fate of liposomal drugs is still unclear because existing bioanalytical methods for quantitation of total and liposomal-encapsulated drugs have limits. A novel strategy for liposomal-encapsulated drug separation from plasma was developed via the specific coordinate binding interaction of TiO2 microspheres with the phosphate groups of liposomes. Liposomal-encapsulated docetaxel was separated from plasma by TiO2 microspheres and analyzed by the UPLC-MS/MS method. The amount of TiO2, pH of the dilutions, plasma dilution factors and incubation time were optimized to improve extraction recovery. The characterization of the adsorption of liposome-encapsulated drugs by TiO2 microspheres was observed by electron microscopy. For understanding the mechanism, pseudo-first and the pseudo-second order equations were proposed for the adsorption process. The study fully validated the method for quantitation of liposomal-encapsulated in plasma and the method was applied to the pharmacokinetic study of docetaxel liposomes. The encapsulated docetaxel had a concentration range of 15-4000 ng/mL from the plasma sample using a TiO2 extraction method. Successful method validation proved the method was sensitive, selective and stable, and was suitable for quantitation of docetaxel liposomes in plasma samples. Extraction recovery of this method was higher than that of SPE method. As shown in electron microscopy, the liposomes adsorbed on TiO2 microspheres were intact and there was no drug leakage. The study proposed pseudo-first and the pseudo-second order equations to facilitate the adsorption of liposomal drugs with TiO2 microspheres. The proposed strategy supports the pharmacokinetic study of docetaxel liposomes in rats. TiO2 extraction method was stable, reproducible, and reliable for quantitation of encapsulated docetaxel. Because of versatility of lipids, it is expected to a universal bioanalysis method for the pharmacokinetic study of liposomes.

Endothelial cell activation, Weibel-Palade body secretion, and enhanced angiogenesis in severe COVID-19

BackgroundSevere COVID-19 is associated with marked endothelial cell (EC) activation that plays a key role in immunothrombosis and pulmonary microvascular occlusion. However, the biological mechanisms through which SARS-CoV-2 causes EC activation and damage remain poorly defined. ObjectivesWe investigated EC activation in patients with acute COVID-19, and specifically focused on how proteins stored within Weibel-Palade bodies may impact key aspects of disease pathogenesis. MethodsThirty-nine patients with confirmed COVID-19 were recruited. Weibel-Palade body biomarkers (von Willebrand factor [VWF], angiopoietin-2 [Angpt-2], and osteoprotegerin) and soluble thrombomodulin (sTM) levels were determined. In addition, EC activation and angiogenesis were assessed in the presence or absence of COVID-19 plasma incubation. ResultsMarkedly elevated plasma VWF antigen, Angpt-2, osteoprotegerin, and sTM levels were observed in patients with acute COVID-19. The increased levels of both sTM and Weibel-Palade body components (VWF, osteoprotegerin, and Angpt-2) correlated with COVID-19 severity. Incubation of COVID-19 plasma with ECs triggered enhanced VWF secretion and increased Angpt-2 expression, as well as significantly enhanced in vitro EC tube formation and angiogenesis. ConclusionWe propose that acute SARS-CoV-2 infection leads to a complex and multifactorial EC activation, progressive loss of thrombomodulin, and increased Angpt-2 expression, which collectively serve to promote a local proangiogenic state.

Genome-wide screen in human plasma identifies multifaceted complement evasion of Pseudomonas aeruginosa

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is a leading cause of bacteremia with a high mortality rate. We recently reported that P. aeruginosa forms a persister-like sub-population of evaders in human plasma. Here, using a gain-of-function transposon sequencing (Tn-seq) screen in plasma, we identified and validated previously unknown factors affecting bacterial persistence in plasma. Among them, we identified a small periplasmic protein, named SrgA, whose expression leads to up to a 100-fold increase in resistance to killing. Additionally, mutants in pur and bio genes displayed higher tolerance and persistence, respectively. Analysis of several steps of the complement cascade and exposure to an outer-membrane-impermeable drug, nisin, suggested that the mutants impede membrane attack complex (MAC) activity per se. Electron microscopy combined with energy-dispersive X-ray spectroscopy (EDX) revealed the formation of polyphosphate (polyP) granules upon incubation in plasma of different size in purD and wild-type strains, implying the bacterial response to a stress signal. Indeed, inactivation of ppk genes encoding polyP-generating enzymes lead to significant elimination of persisting bacteria from plasma. Through this study, we shed light on a complex P. aeruginosa response to the plasma conditions and discovered the multifactorial origin of bacterial resilience to MAC-induced killing.

Роль печени в регуляции белково-стероидного взаимодействия у здоровых и облучённых животных

The study of the role of the liver in the regulation of protein-steroid interaction in healthy and irradiated animals allows us to supplement a number of theoretical provisions of radiobiology on factors influencing the pathogenesis of acute radiation sickness. The relevance of the study is due to its theoretical significance in creating a holistic concept of radiobiological patterns of pathogenesis of acute radiation sickness. The aim of the experiments is to study the effect of the liver on the fractional composition of 11-oxycorticosteroids (11-OCS) in the blood plasma of healthy and irradiated animals. The experiments were carried out on 7 mongrel dogs weighing from 22 to 27 kg. In dogs, the total concentration of 11-OCS, the level of free, protein-bound and glucuronic acid-bound corticosteroids in the blood of the femoral, portal and hepatic veins were determined. At the first stage, experiments were conducted on healthy animals. 4 animals were subjected to angiostomy by skin flap method. Blood was obtained in the absence of exposure, 90 minutes after intravenous injection of 40 units of adrenocorticotropic hormone (ACTH), the introduction of hydrocortisone into the stomach at a dose of 50 or 100 mg. The total content of 11-OCS, mcg%, was determined by the fluorimetric method in the author's modification. To determine the free 11-OCS, the gel filtration method was used in the author's modification. The fraction associated with plasma proteins was isolated on a column with sephadex G-25. Glucuronides were determined after plasma incubation with betta-glucuronidase. At the second stage, the tests were carried out on irradiated animals that were exposed to gamma-irradiation at the EGO-2 installation at an average dose rate of 5.75 Gy/min at a dose of 3.5 Gy, causing acute stage IV radiation sickness. The results of the study showed that the liver inactivates free 11-OCS, binding them to glucuronic acid. The degree of delay and inactivation of free corticoids correlate with the concentration of the latter in the blood. The liver does not affect the level of protein-related hormones. Corticosteroids bind to plasma proteins outside the liver. In acute radiation sickness, the ability of the liver to inactivate biologically active corticosteroids decreases.