Investigating Generation of Antibodies against the Lipid Nanoparticle Vector Following COVID-19 Vaccination with an mRNA Vaccine.

Despite the success of mRNA-based vaccines against infectious diseases (including COVID-19), safety concerns have been raised relating to the lipid nanoparticles (LNPs) used to deliver the mRNA cargo. Antibodies against the polyethylene glycol (PEG) coating on these non-viral vectors are present in the general population and can in some instances induce allergic reactions. Furthermore, treatment with PEGylated therapeutics may increase the plasma concentration of such anti-PEG antibodies. The widespread use of PEGylated nanoparticles for mRNA vaccines concerns researchers and clinicians about a potential rise in future cases of allergic reactions against mRNA vaccines and cross-reactions with other PEGylated therapeutics. To determine if vaccination with Comirnaty increased the plasma concentration of antibodies against LNPs, we investigated the blood plasma concentration of anti-LNP antibodies in healthy individuals before and after vaccination with the mRNA-based COVID-19 vaccine Comirnaty (BNT162b2). Blood samples were acquired from 21 healthy adults before vaccination, 3-4 weeks after the first vaccination dose but before the second dose, and 2-6 months after the second (booster) dose. The blood plasma concentration of antibodies recognizing the LNPs was analyzed using a microscopy-based assay capable of measuring antibody-binding to individual authentic LNPs. No significant increase in anti-LNP antibodies was observed after two doses of Comirnaty. The LNPs used for intramuscular delivery of mRNA in the vaccine against COVID-19, Comirnaty, do, therefore, not seem to induce the generation of anti-vector antibodies.

Unravelling Heterogeneities in Complement and Antibody Opsonization of Individual Liposomes as a Function of Surface Architecture.

Coating nanoparticles with poly(ethylene glycol) (PEG) is widely used to achieve long-circulating properties after infusion. While PEG reduces binding of opsonins to the particle surface, immunogenic anti-PEG side-effects show that PEGylated nanoparticles are not truly "stealth" to surface active proteins. A major obstacle for understanding the complex interplay between opsonins and nanoparticles is the averaging effects of the bulk assays that are typically applied to study protein adsorption to nanoparticles. Here, a microscopy-based method for directly quantifying opsonization at the single nanoparticle level is presented. Various surface coatings are investigated on liposomes, including PEG, and show that opsonization by both antibodies and complement C3b is highly dependent on the surface chemistry. It is further demonstrated that this opsonization is heterogeneous, with opsonized and non-opsonized liposomes co-existing in the same ensemble. Surface coatings modify the percentage of opsonized liposomes and/or opsonin surface density on the liposomes, with strikingly different patterns for antibodies and complement. Thus, this assay provides mechanistic details about opsonization at the single nanoparticle level previously inaccessible to established bulk assays.

Pay Attention to Biological Nanoparticles when Studying the Protein Corona on Nanomedicines.

The protein corona of nanoparticles has in recent years received considerable attention, and even been postulated to be the missing link in the translation of nanomedicines from benchtop to bedside. We highlight the different types of biological nanoparticles present in blood that need to be considered in the protein corona research field. We map their size, density, and plasma concentrations, and use this information to stress potential challenges related to the isolation of nanomedicines-with a particular focus on liposomes-when using the traditional isolation methods that separate according to size and density.

Vasomotor function in rat arteries after ex vivo and intragastric exposure to food-grade titanium dioxide and vegetable carbon particles.

BACKGROUND: Humans are continuously exposed to particles in the gastrointestinal tract. Exposure may occur directly through ingestion of particles via food or indirectly by removal of inhaled material from the airways by the mucociliary clearance system. We examined the effects of food-grade particle exposure on vasomotor function and systemic oxidative stress in an ex vivo study and intragastrically exposed rats. METHODS: In an ex vivo study, aorta rings from naïve Sprague-Dawley rats were exposed for 30 min to food-grade TiO2 (E171), benchmark TiO2 (Aeroxide P25), food-grade vegetable carbon (E153) or benchmark carbon black (Printex 90). Subsequently, the vasomotor function was assessed in wire myographs. In an in vivo study, lean Zucker rats were exposed intragastrically once a week for 10 weeks to vehicle, E171 or E153. Doses were comparable to human daily intake. Vasomotor function in the coronary arteries and aorta was assessed using wire myographs. Tetrahydrobiopterin, ascorbate, malondialdehyde and asymmetric dimethylarginine were measured in blood as markers of oxidative stress and vascular function. RESULTS: Direct exposure of E171 to aorta rings ex vivo increased the acetylcholine-induced vasorelaxation and 5-hydroxytryptamine-induced vasocontraction. E153 only increased acetylcholine-induced vasorelaxation, and Printex 90 increased the 5-hydroxytryptamine-induced vasocontraction, whereas Aeroxide P25 did not affect the vasomotor function. In vivo exposure showed similar results as ex vivo exposure; increased acetylcholine-induced vasorelaxation in coronary artery segments of E153 and E171 exposed rats, whereas E171 exposure altered 5-hydroxytryptamine-induced vasocontraction in distal coronary artery segments. Plasma levels of markers of oxidative stress and vascular function showed no differences between groups. CONCLUSION: Gastrointestinal tract exposure to E171 and E153 was associated with modest albeit statistically significant alterations in the vasocontraction and vasorelaxation responses. Direct particle exposure to aorta rings elicited a similar type of response. The vasomotor responses were not related to biomarkers of systemic oxidative stress.

The vast majority of nucleic acid-loaded lipid nanoparticles contain cargo.

Nucleic acid-based therapies are transforming medicine, but rely on an efficient delivery vehicle such as lipid nanoparticles (LNPs). Concerns exists in the nanomedicine field, that a large fraction of the LNPs in the ensemble does not contain any nucleic acid cargo and thus exert no functional effect. Nevertheless, how LNP lipid formulation, the LNP preparation method employed and nucleic acid cargo size correlates with the proportion of empty LNPs remains largely unexplored. Here we employ a well-established single particle based method to study nucleic acid loading heterogeneity in LNPs. We find that only a minor fraction of LNPs are "empty", both for LNPs loaded with siRNA, mRNA and plasmids. For clinically relevant LNPs for mRNA delivery, we never detected more than 16% empty nanoparticles in the ensemble. Thus employing standard LNP lipid-cargo combinations and preparation schemes results in LNPs with the potential to serve their biomedical function.

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.

Studying how administration route and dose regulates antibody generation against LNPs for mRNA delivery with single-particle resolution.

Following the recent approval of both siRNA- and mRNA-based therapeutics, nucleic acid therapies are considered a game changer in medicine. Their envisioned widespread use for many therapeutic applications with an array of cellular target sites means that various administration routes will be employed. Concerns exist regarding adverse reactions against the lipid nanoparticles (LNPs) used for mRNA delivery, as PEG coatings on nanoparticles can induce severe antibody-mediated immune reactions, potentially being boosted by the inherently immunogenic nucleic acid cargo. While exhaustive information is available on how physicochemical features of nanoparticles affects immunogenicity, it remains unexplored how the fundamental choice of administration route regulates anti-particle immunity. Here, we directly compared antibody generation against PEGylated mRNA-carrying LNPs administered by the intravenous, intramuscular, or subcutaneous route, using a novel sophisticated assay capable of measuring antibody binding to authentic LNP surfaces with single-particle resolution. Intramuscular injections in mice were found to generate overall low and dose-independent levels of anti-LNP antibodies, while both intravenous and subcutaneous LNP injections generated substantial and highly dose-dependent levels. These findings demonstrate that before LNP-based mRNA medicines can be safely applied to new therapeutic applications, it will be crucial to carefully consider the choice of administration route.

Comment on "Optimal centrifugal isolating of liposome-protein complexes from human plasma" by L. Digiacomo, F. Giulimondi, A. L. Capriotti, S. Piovesana, C. M. Montone, R. Z. Chiozzi, A. Laganá, M. Mahmoudi, D. Pozzi and G. Caracciolo, Nanoscale Adv., 2021, 3, 3824.

In a recent paper in Nanoscale Advances, Digiacomo et al. conclude that centrifugation should be the method of choice for researchers who want to investigate the protein corona of liposomes for drug delivery in human plasma. In this Comment, we however propose the opposite - that centrifugation, in most cases, is unsuitable for isolating liposomes from human plasma. Our conclusion is based on the bulk literature on this and similar topics, and new experimental data based on formulations and protocols like the ones used by Digiacomo et al.

Accelerated blood clearance and hypersensitivity by PEGylated liposomes containing TLR agonists.

Systemic administration of toll-like receptor (TLR) agonists have demonstrated impressive preclinical results as an anti-cancer therapy due to their potent innate immune-stimulatory properties. The clinical advancement has, however, been hindered by severe adverse effects due to systemic activation of the immune system. Liposomal drug delivery systems may modify biodistribution, cellular uptake, and extend blood circulation, and thus, potentially enable systemic administration of TLR agonists at therapeutic doses. In this study, we investigated potential barriers for the administration of TLR agonists formulated in polyethylene glycosylated (PEGylated) liposomes with regards to liposome formulation, TLR agonist, administration route, administration schedule, biodistribution, blood clearance, and anti-PEG antibodies. We found that administration of TLR agonists formulated in PEGylated liposomes led to high anti-PEG antibody titers, which upon multiple intravenous administrations, resulted in accelerated blood clearance and acute hypersensitivity reactions. The latter was found to be associated with anti-PEG IgG antibody and not anti-PEG IgM antibody opsonization. This study highlights the need to carefully design and evaluate nanoparticle delivery systems for immunotherapy as anti-nanoparticle immune responses may challenge the therapeutic application.

Mechanisms of selective monocyte targeting by liposomes functionalized with a cationic, arginine-rich lipopeptide.

Stimulation of monocytes with immunomodulating agents can harness the immune system to treat a long range of diseases, including cancers, infections and autoimmune diseases. To this end we aimed to develop a monocyte-targeting delivery platform based on cationic liposomes, which can be utilized to deliver immunomodulators and thus induce monocyte-mediated immune responses while avoiding off-target side-effects. The cationic liposome design is based on functionalizing the liposomal membrane with a cholesterol-anchored tri-arginine peptide (TriArg). We demonstrate that TriArg liposomes can target monocytes with high specificity in both human and murine blood and that this targeting is dependent on the content of TriArg in the liposomal membrane. In addition, we show that the mechanism of selective monocyte targeting involves the CD14 co-receptor, and selectivity is compromised when the TriArg content is increased, resulting in complement-mediated off-target uptake in granulocytes. The presented mechanistic findings of uptake by peripheral blood leukocytes may guide the design of future drug delivery systems utilized for immunotherapy. STATEMENT OF SIGNIFICANCE: Monocytes are attractive targets for immunotherapies of cancers, infections and autoimmune diseases. Specific delivery of immunostimulatory drugs to monocytes is typically achieved using ligand-targeted drug delivery systems, but a simpler approach is to target monocytes using cationic liposomes. To achieve this, however, a deep understanding of the mechanisms governing the interactions of cationic liposomes with monocytes and other leukocytes is required. We here investigate these interactions using liposomes incorporating a cationic arginine-rich lipopeptide. We demonstrate that monocyte targeting can be achieved by fine-tuning the lipopeptide content in the liposomes. Additionally, we reveal that the CD14 receptor is involved in the targeting process, whereas the complement system is not. These mechanistic findings are critical for future design of monocyte-targeting liposomal therapies.

Isolation methods commonly used to study the liposomal protein corona suffer from contamination issues.

The liposomal protein corona has been the focus of numerous studies, but there is still no consensus regarding its extent and composition. Rather, the literature is full of conflicting reports on the matter. To elucidate whether there could be a methodological explanation for this, we here scrutinize the efficiency of three commonly used liposome isolation methods at isolating stealth liposomes from human plasma. Firstly, we show that size-exclusion chromatography (SEC) in its standard form is prone to isolating unbound protein material together with the liposomes, but also that the method may be optimized to mitigate this issue. Secondly, we demonstrate that SEC in combination with membrane ultrafiltration is no better at removing the unbound protein material than SEC alone. Thirdly, we show that centrifugation is not able to pellet the liposomes. Overall, our results suggest that previous research on the liposomal protein corona may have suffered from significant methodological problems, in particular related to contaminant proteins interfering with the analysis of the protein corona. We believe that the data presented here may help guide future research around this challenge to reach a converging understanding about the properties of the protein corona on liposomes. STATEMENT OF SIGNIFICANCE: Upon administration into the circulatory system, liposomal drug carriers encounter an environment rich in proteins. These proteins may adsorb to the liposomes to form what is known as the protein corona, potentially governing the interactions of the liposomes with tissues and cells. However, despite decades of intense research efforts, there is currently no clear understanding about the extent and composition of the liposomal protein corona, making it impossible to assess its mechanistic importance. Here we report that the methods commonly used to isolate liposomes from blood plasma or serum to study the protein corona are susceptible to protein contamination. This may be the underlying technical reason for the current confusion about the characteristics of the liposomal protein corona.

Head-to-Head Comparison of the Penetration Efficiency of Lipid-Based Nanoparticles into Tumor Spheroids.

Most tumor-targeted drug delivery systems must overcome a large variety of physiological barriers before reaching the tumor site and diffuse through the tight network of tumor cells. Many studies focus on optimizing the first part, the accumulation of drug carriers at the tumor site, ignoring the penetration efficiency, i.e., a measure of the ability of a drug delivery system to overcome tumor surface adherence and uptake. We used three-dimensional (3D) tumor spheroids in combination with light-sheet fluorescence microscopy in a head-to-head comparison of a variety of commonly used lipid-based nanoparticles, including liposomes, PEGylated liposomes, lipoplexes, and reconstituted high-density lipoproteins (rHDL). Whilst PEGylation of liposomes only had minor effects on the penetration efficiency, we show that lipoplexes are mainly associated with the periphery of tumor spheroids, possibly due to their positive surface charge, leading to fusion with the cells at the spheroid surface or aggregation. Surprisingly, the rHDL showed significantly higher penetration efficiency and high accumulation inside the spheroid. While these findings indeed could be relevant when designing novel drug delivery systems based on lipid-based nanoparticles, we stress that the used platform and the detailed image analysis are a versatile tool for in vitro studies of the penetration efficiency of nanoparticles in tumors.

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes.

Most research employing liposomes as membrane model systems or drug delivery carriers relies on bulk read-out techniques and thus intrinsically assumes all liposomes of the ensemble to be identical. However, new experimental platforms able to observe liposomes at the single-particle level have made it possible to perform highly sophisticated and quantitative studies on protein-membrane interactions or drug carrier properties on individual liposomes, thus avoiding errors from ensemble averaging. Here we present a protocol for preparing, detecting, and analyzing single liposomes using a fluorescence-based microscopy assay, facilitating such single-particle measurements. The setup allows for imaging individual liposomes in a massive parallel manner and is employed to reveal intra-sample size and compositional inhomogeneities. Additionally, the protocol describes the advantages of studying liposomes at the single liposome level, the limitations of the assay, and the important features to be considered when modifying it to study other research questions.

Dissociation of fluorescently labeled lipids from liposomes in biological environments challenges the interpretation of uptake studies.

Within nanomedicine, liposomes are investigated for their ability to deliver drug cargoes specifically into subcellular compartments of target cells. Such studies are often based on flow cytometry or microscopy, where researchers rely on fluorescently labeled lipids (FLLs) incorporated into the liposomal membrane to determine the localization of the liposomes within cells. These studies assume that the FLLs stay embedded in the liposomal membrane throughout the duration of the experiment. Here, we used size exclusion chromatography (SEC) to investigate the validity of this assumption by quantitatively determining the propensity of various widely used FLLs to dissociate from liposomes during incubation in human plasma. For certain commonly used off-the-shelf FLLs, up to 75% of the dye dissociated from the liposomes, while others dissociated less than 10%. To investigate the implications of this finding, we measured the peripheral blood leukocyte uptake of liposomes formulated with different FLLs using flow cytometry, and observed a significant difference in uptake correlating with the FLL's dissociation tendencies. Consequently, the choice of FLL can dramatically influence the conclusions drawn from liposome uptake and localization studies due to uptake of dissociated FLLs. The varying dissociation propensities for the FLLs were not reflected when incubating in buffer, showing that non-biological environments are unsuitable to mimic liposomal stability in a drug delivery context. Overall, our findings suggest that it is crucial for researchers to evaluate the stability of their FLL-labeled liposomes in biological environments, and the simplicity of the SEC assay put forward here makes it very applicable for the purpose.