Poly (ethylene glycol) (PEG) is a non-ionic polyether compound and has been widely used in common applications in cosmetics, food products, and pharmaceuticals for its excellent properties as a solvent, plasticizer, surfactant, base, and lubricant (D’souza and Shegokar, 2016). In pharmaceutical applications, PEG has several advantages as it increases the circulation time of the drugs and decreases their immunogenicity. PEG increases the molecular weight (MW) of pharmaceutical products such as proteins or peptides, decreasing the kidney clearance, and protects them from proteolytic degradation, thus altering their pharmacokinetic profile. Moreover, as a consequence of the water cloud surrounding the polymer, PEG improves the solubility of drugs. Finally, the use of PEG for the surface modification of nanoparticles (NPs) has been reported to decrease protein adsorption, thereby reducing recognition by the mononuclear phagocytic system (MPS) (Fishburn, 2008; Zhang et al., 2014).

PEG is the most commonly used “stealth” polymeric agent and is regarded as a safe and non-toxic polymer, gaining the generally recognized as safe (GRAS) status from the Food and Drug Administration (FDA) and the European Medical Agency (EMA), and it is widely used to conjugate drugs. The first example using PEG was reported in 1977, where the authors covalently attached PEG to bovine serum albumin and to liver catalase proteins, reporting changes on its immunogenicity and retaining its activity, respectively (Abuchowski et al., 1977a; Abuchowski et al., 1977b). In 1990, the FDA approved the first PEGylated protein product, Adagen, used for severe combined immunodeficiency disease and, in 1995, the first PEGylated lipid NPs encapsulating doxorubicin was released as Doxil (Supplementary Table S1). As a result of the COVID-19 pandemic (Chan et al., 2020), the scientific community has worked intensively to find an effective and practical solution to combat this infection including an effective vaccine that could be administrated widely and with low side effects. It is notable that two out of four vaccines approved in Europe are based on PEGylated NPs: the Pfizer/BioNTech (BNT162b2) and Moderna (mRNA-1273) COVID-19 formulations for delivery of mRNA vaccines. The EMA thus authorized the marketing of these vaccines in December 2020 and January 2021, respectively (Cavaleri et al., 2021), and in December 2020, the FDA approved the emergency use authorization for both vaccines (FDA, 2020a; FDA, 2020b). Only in late 2021, the EMA approved a protein-based vaccine (Novavax). These lipid nanoparticle (LNP) formulations, with a size less than 100 nm (Schoenmaker et al., 2021), encapsulate and protect the mRNA which codifies the spike protein of the SARS-CoV-2 virus. The carrier brings the mRNA into the cells, and the mRNA released in the host cell synthesizes the spike protein, which, in turn, is presented to immune cells to elicit a response against SARS-CoV-2 (Teijaro and Farber, 2021; Hou et al., 2021) (Figure 1). The vaccines are comprised of, amongst other entities, PEGylated lipids (Buschmann et al., 2021; Dolgin, 2021; EMA, 2021; FDA, 2021; Hatziantoniou et al., 2021). While these vaccines have been widely referred to as “new generation” vaccines, for those working in the field of nanomedicine, similar formulations are not novel. In fact, liposomes were first described by Bangham et al., in 1965 (Bangham et al., 1965), and were later used for the delivery of enzymes or drugs (Gregoriadis and Ryman, 1971) and, almost 30 years ago, in 1993, their use as vaccines was reported in a preclinical study. This mRNA vaccine was able to induce virus-specific cytotoxic T cell responses against the influenza virus, and became a quite attractive formulation to tackle the uncontrolled viral infection globally (Martinon et al., 1993). For an overview of the currently available COVID-19 vaccines, see (Kisby et al., 2021).

PEG provides several advantages with respect to the pharmacological profile of drugs/NPs. However, some drawbacks have been also reported. Although widely considered non-immunogenic and biocompatible (Richter and Åkerblom, 1983; Richter and Åkerblom, 1984), several studies have suggested the presence of pre-existing anti-PEG antibodies in healthy individuals after repetitive exposure (Armstrong et al., 2007; Garay et al., 2012; Hsieh et al., 2018; Mohamed et al., 2019). For example, Yang and Lai reported significant concentrations of anti-PEG antibodies in a substantial 42% of patients who had not previously undergone any treatment involving PEGylated therapies (Yang and Lai, 2015). Nevertheless, after receipt of the first doses of the Pfizer/BioNTech and Moderna COVID-19 vaccines in the United States, the Advisory Committee on Immunization Practices (ACIP) detected 21 and 10 cases of an anaphylactic reaction (11.1 and 2.5 per million doses administered), respectively, but with the highest incidence in patients that had already an established allergy to drugs (CDC, 2020; CDC, 2021). The anaphylactic symptoms were speculated to be due to CARPA (complement activation-related pseudoallergy), elicited by the application of PEGylated LNPs (CDC, 2020; CDC, 2021; Greenhawt et al., 2021; Iguchi et al., 2021). CARPA has been described as a non-IgE-mediated (pseudo)allergy related to complement activation, which occurs upon first contact, without prior sensitization (Szebeni, 2014). CARPA was previously described in relation to hypersensitivity reactions observed after the administration of Doxil (Chanan-Khan et al., 2003; Szebeni, 2005; Szebeni et al., 2011). The serious adverse events due to the administration of mRNA vaccines are rare and it still remains to be proven whether PEG could be main the cause of CARPA (Bigini et al., 2021). Here, we provide a general overview of the use of PEGylated NPs for pharmaceutical applications, with the main focus on the role of PEGylation for the improvement of the biological performance of the NPs, and we describe complement activation caused by PEGylated nanomedicines for a better comprehension of immunological adverse reactions, e.g., CARPA. We also touch on other factors which may drive the adverse effects.

Poly (ethylene glycols) (PEG) (HO(CH₂CH₂O)_nH) are hydrophilic and non-ionic polymers synthesised via the polymerization of ethylene oxide under alkaline catalysis. PEG exists in a broad range of different chain lengths and MW, with a linear or branched structure (D’souza and Shegokar, 2016). The term “PEG” is often in combination with a numerical value, which refers to the average number of ethylene oxide units (n) or the average MW (g/mol) of a given PEG. The latter term is commonly used in the pharmaceutical industry. PEGylation is the term used for the conjugation of PEG to molecules, such as drugs or NPs, to enhance their biocompatibility and biodistribution (Estephan et al., 2011; Fam et al., 2020). Commercial PEGs are available in a MW range from 200–35,000 g/mol, with a narrow polydispersity (measure of the heterogeneity of sizes of PEG molecules). They are easy to produce and can be modified with different chemistry functionalities (D’souza and Shegokar, 2016) making them versatile for a wide range of bioconjugation options. PEG ranging from 20 to 50 kDa has been used for the conjugation of small molecules, such as siRNA. On the other hand, PEGs with lower MW (1–5 kDa) are preferable for the conjugation of antibodies or NPs (Knop et al., 2010). PEGylation strategies have progressed since non-specific conjugations, known as “first generation” PEGylation, which resulted in the modification of different amino groups of lysines, mixtures of isomers, impurities, or unstable and irreversible bonds; until the most recent conjugations, the “second generation” PEGylation, mainly characterized by the binding of PEG to an N-terminal or thiol in a selective and reversible way (Gupta et al., 2019; Harris and Chess, 2003; Turecek et al., 2016). Some of the most used PEGs for drug/NP modification are the N-hydroxysuccinimide esters PEG, which react with amine groups to form stable amides and the use of PEG-maleimide for the PEGylation of cysteine residues (Gupta et al., 2019). Heterobifunctional PEGs, with two functionalities in the terminal groups, allow the conjugation of ligand-macromolecules to surfaces, forming a bridge between the target ligand and the NPs (Lo et al., 2020; Huang et al., 2020). For instance, carboxylic-molecules can be attached to gold NPs using thiol-PEG-amine (Zamora-Justo et al., 2019), whereas silane-PEG-carboxylic, used for the PEGylation of silica nanoparticles, allows for the conjugation with amine groups (von Baeckmann et al., 2021) (Figure 2). Although the synthesis of PEG has improved over the years, the products of polymerization still show a complex mixture of different length oligomers, which could lead to variability in the results and/or misunderstanding in the relation between the PEG structure and the activity, bringing problems for their applications. Thus, significant efforts were made to improve the synthesis of monodisperse PEGs, although some of these approaches still present challenges regarding the low yield, complicated purification, applicability only to some MW, or expensive reagents, as well as tedious chemistry (Niculescu-Duvaz et al., 2008; Knop et al., 2010; Zhang et al., 2015; Kinbara, 2018).

One of the greatest advantages of PEGylation for biomedical applications is the high solubility in aqueous medium provided by the PEG chains (D’souza and Shegokar, 2016; Knop et al., 2010; Turecek et al., 2016). It has been reported that an ethylene oxide unit can bind two to three water molecules (Branca et al., 2002; Oelmeier et al., 2012; Kolate et al., 2014; Turecek et al., 2016). This solubility creates a “conformational cloud”, generated by the flexible polymer chains, which hides the charges and provides a steric hindrance, preventing the interaction with other PEGylated NPs, reducing aggregation and providing a high chemical-physical stability (Knop et al., 2010; Gupta et al., 2019). At the same time, the conformational freedom of the PEG chains makes the interaction with other matter thermodynamically unfavourable, decreasing the interactions of the PEGylated products with other biomolecules (Pino et al., 2014; Yang and Lai, 2015; Suk et al., 2016; Fam et al., 2020). In this regard, it has been well established that NPs in contact with the biological environment adsorb biomolecules, such as lipids or proteins, forming the so-called bio-corona. This biomolecular coating dictates the biological “identity” of the NPs, affecting their activity, biodistribution and clearance (Cedervall et al., 2007; Nel et al., 2009; Monopoli et al., 2011; Monopoli et al., 2012; Clemente et al., 2022). When these proteins are opsonins, the NPs are recognized by the MPS, and they are eliminated. However, proteins in the corona may also act as dysopsonins (Schöttler et al., 2016). The MPS is a network of cells descending from monocytes, including tissue-resident macrophages in the liver and spleen, that engulf foreign substances (particles and pathogens). The role of the MPS (also referred as the reticuloendothelial system or RES) for the clearance of NPs with a bio-corona of proteins has been discussed extensively in previous reviews (Gustafson et al., 2015; Moghimi et al., 2019).

The MW and surface density of PEG chains coating the NPs’ surface play an important role in the biologic performance of PEGylated NPs by regulating their biochemical (i.e., protein adsorption) and physical (i.e., aggregation level) properties. Increasing both PEG MW and surface density has been described to improve the NP biologic performance. Higher PEG MW provides more hydrophilicity and flexibility and a higher effect of a steric hindrance than short PEG chains, resulting in a thicker protective layer and avoiding the interactions with biomolecules (Lee and Larson, 2016). Nevertheless, there exists a limit for the MW above which the effect of PEG is negligible and, therefore, the protein adsorption cannot be completely eliminated (Gref et al., 2000). PEG density and conformation also play an important role. It has been proposed that grafted PEG can assume two different conformations, “mushroom” and “brush”, related to the PEG surface density. When the surface density is low, PEG adopts a “mushroom” configuration, where the chains are separated among them and located close to the surface. However, for higher values of density, PEG chains are closer and acquire a straighter (brush-like) conformation thus forming a thicker hydrophilic barrier that might lead to a lower protein adsorption (Walkey et al., 2012). PEG conformation can be described by Flory radius (R_F), which defines the minimum distance required between grafted polymer chains to assume a mushroom conformation: R_F = α×n^ν, where n is the number of monomers in the polymer chain, α is the length of the polymer monomer, and ν is Flory exponent, which is related to the solvent. Different topologies of polymer configuration have been described depending on the relationship between the grafting density and the Flory radius (Figure 3). The influence of PEG properties, including the length, packing density and the conformation assumed after binding to the NP surface has been widely studied. For instance, it was found that the density of PEG chains on the surface of single-walled carbon nanotubes mediates the adsorption of plasma proteins on the nanotubes which, in turn, affects their pharmacokinetic profile (Sacchetti et al., 2013). A change in PEG conformation from mushroom to brush transition for ovalbumin NPs affected the competitive adsorption of plasma proteins on the NPs’ surface, leading to a higher adsorption of clusterin (apolipoprotein J) and a lower adsorption of serum albumin. Thus, the brush conformation led to a shorter phagocytic uptake (Li et al., 2021a). In a similar study using PEGylated gold NPs and PEG of different MW, the authors observed that the protein corona composition for the different NPs was critical to enhance tumour targeting (Cui et al., 2021). Finally, it is worth noting that PEGylation should be carefully optimized if targeting agents are added on the NPs surface to improve the accumulation in a specific tissue and/or cell sub-population. PEG chains may cover the targeting agents and hamper their interaction with tissue/cell receptors. This problem has been tackled by coating the NPs’ surface with PEG chains of different MWs: the shorter ones were used to improve the stability of the NPs in aqueous solutions and decrease the adsorption of biomacromolecules, while the longer ones were used to bind targeting agents at their terminal end, thus allowing them to protrude from the NPs’ surface thereby avoiding the masking by other biomolecules (Cruz et al., 2011).

PEGylated lipid NPs for drug or vaccine delivery in vivo are created by a simple self-assembly mechanism which normally consists of the covalent conjugation of a lipid and the chosen PEG molecule (Nosova et al., 2019). This molecule together with three other lipids (one of which is usually cholesterol) plus the active RNA ingredient will self-assemble into a lipid bilayer nanocapsule (liposome) entrapping the short interfering or messenger RNA therapeutic ingredient inside (Figure 4). The lipid bilayer is held together by hydrogen bonds between the ionic ends of the lipids. The inner and outer surfaces of the lipids are non-polar. Within this arrangement, the PEG functionalized lipid is on the outside of the lipid layer and has its polar end in the centre of the lipid bilayer. A successful and recently developed liposome production technique is microfluidic hydrodynamic focusing, in which a stream of lipid in alcohol solution is forced to flow in the central channel of a device, intersected, and sheathed by coaxial stream(s) of an aqueous phase. Reciprocal diffusion of alcohol and water across the focused alcohol/water interface causes the lipid to precipitate and self-assemble into liposomes (Tenchov et al., 2021). Controlled particle size 70 nm liposomes are achieved by pressurized reverse osmosis filtration through ceramic membranes by batch or continuous processes. The ingredients and formulations for both the Pfizer/BioNTech (BNT162b2) and Moderna (mRNA-1273) vaccines registered with EMA and the FDA are presented in Table 1 (Buschmann et al., 2021; Dolgin, 2021; EMA, 2021; FDA, 2021; Hatziantoniou et al., 2021). There are some differences in lipids and buffers used which may determine the differences observed for the stability and storage of the vaccines. However, both use PEG 2000 as the external “stealth” moiety on the surface of the liposome. Moreover, the observed albeit low incidence of pseudoanaphylaxis of the two vaccines is very similar.

It is notable that other constituents of the mRNA vaccines may also trigger allergic reactions. Unlike the Pfizer/BioNTech vaccine, the Moderna vaccine contains Tris, or tris(hydroxymethyl)aminomethane (tromethamine/trometamol), a compound that has been reported to cause contact dermatitis and rare cases of anaphylaxis in relation to gadolinium-based contrast agents (Klimek et al., 2021). It is thus possible that other excipients might cause hypersensitivity reactions.

PEG as a possible cause of the complement activation and the observed anaphylactic reactions has been widely discussed. However, it is important to consider whether other factors could be involved. For instance, as pointed out by Moghimi (Moghimi, 2021) in a recent commentary, some components of the Pfizer and Moderna vaccines, such as cholesterol and the phospholipid DSCP (1,2-distearoyl-snglycero-3 phosphocholine) (Table 1), may trigger complement activation via classical pathway through the production of natural (or induced) antibodies. Szebeni et al. (Szebeni et al., 2000) reported that the presence of cholesterol in LNPs over a concentration of 45 mol% leads to an increase of the pulmonary arterial pressure as a result of complement activation, whereas formulations containing under 45 mol% had no influence on pulmonary reactions. It was proposed that the interaction between naturally occurring anti-cholesterol antibodies and microcrystal aggregates formed by excess cholesterol trigger complement activation thereby manifesting as increased pulmonary hypertension through the release of anaphylatoxins. Furthermore, other positively charged components or the mRNA have been also identified as possible factors could activate the complement (Dézsi et al., 2022). Notably, the latter study showed that incubation of the Pfizer/BioNTech mRNA vaccine with pig serum ex vivo resulted in significant elevation of C3a anaphylatoxin and sC5b-9, the terminal complement complex.

In another recent study, it was shown that LNPs, alone or complexed with control noncoding poly-cytosine mRNA, are highly inflammatory in mice (Ndeupen et al., 2021). The authors speculated that innate immune memory elicited by the LNPs might form after the first vaccination (in humans) and that this could explain the more robust inflammatory responses upon the second vaccination. However, it is noted that inflammation could also underlie the adjuvant effect of vaccines and that it is not a priori detrimental. LNPs contain ionizable lipids (Table 1), i.e., lipids that are positively charged at acidic pH to condense the RNA into LNPs and neutral at physiological pH to minimize toxicity in comparison to permanently charged lipids (Rietwyk and Peer, 2017). Efforts are under way to develop biodegradable ionizable lipids to reduce any long-term effects (Han et al., 2021).

Complement activation alone (or low-level complement activation) may not explain all instances of observed anaphylactic reactions. Hence, in one study in which Doxil was administrated intravenously to cancer patients, complement activation was measured according to the plasma sC5b-9 levels. The authors found that moderate to severe hypersensitivity occurred in 45% of patients while plasma SC5b-9 at 10 min after infusion was significantly elevated in 92% of reactors versus 56% in the non-reactor group (Chanan-Khan et al., 2003). Thus, it was not possible to establish a direct correlation between released fragments in the complement cascade, such as sC5b-9, and the observed reactions. In this regard, anaphylaxis may be explained due to the interaction of immunoglobulins (IgG) with FcR receptors on the immune cells and the platelet-activating factor (PAF) pathway (Strait et al., 2002; Wibroe et al., 2017; Moghimi, 2018). Immunoglobulins recognizing phospholipids may also trigger anaphylactic reactions through the interaction with FcR receptor on these cells (Moghimi, 2018).

The study by Wibroe et al. (Irvine et al., 2020) is of particular interest as the authors could show, using a set of polystyrene particles of varying geometries, that clearance of nanoparticles by pulmonary intravascular macrophages (PIMs) plays a key role in mediating adverse cardiopulmonary distress in pigs. The study implies that complement assessment alone may not be a sufficiently sensitive approach to predict adverse injection reactions insofar as the adverse reactions may occur in a complement-independent manner. However, PIMs exist in pigs, but not in humans (Irvine et al., 2020).

General procedures for safe-by-design of both nanomedicines and nano-enabled implantable medical devices have been described recently in the frame of the Horizon 2020 project, BIORIMA (Giubilato et al., 2020). The procedures require the evaluation of all nanoscale materials from raw materials, intermediates and final products to be subjected to a range of nanotoxicological tests, e.g., cytotoxicity, genotoxicity, and immunotoxicity tests. In addition to the latter, safe-by-design nanopharmaceuticals requires the development of at-line and online high-functionality characterization and measurement of critical nanomaterial components for ensuring consistency of safe nanointermediates and final nanoscale products. This is known as Process Analytical Control (PAC) and is enshrined in FDA and EMA guidelines from 2003 onwards. Based on our analysis of the FDA and EMA submissions, routine quality assurance and control measures were used in the vaccine manufacturing process, but there was no information presented to indicate whether the PAC procedures were applied. Moreover, lessons learned in the nanosafety field should be considered including the role(s) of bio-corona formation on nanoparticles (Docter et al., 2015). It is also important to further investigate the use of PEG, and possible alternatives to PEG. Studies of biomimetic approaches including cell membrane “cloaking” may also prove very useful. Other critical aspects of the evaluation of nanomedicines including the importance of sterility are discussed elsewhere (Hannon and Prina ‐ Mello, 2021). Finally, given that the vaccination protocols now require repeated booster vaccinations, studies should be initiated to assess potential long-term effects of repeated exposures to the same or similar mRNA vaccines. For instance, whether a progressive build-up of anti-PEG IgG might negate the efficacy of the vaccines.

The authorization and roll-out of the mRNA vaccines manufactured by Pfizer/BioNTech and Moderna has played a significant role in tackling the COVID-19 pandemic. The investigations carried out for decades using PEGylated liposomes as a drug delivery system, together with the evolution of mRNA technology, thus paved the way for the highly anticipated COVID-19 vaccines in a remarkably short time frame while retaining an excellent safety profile and also opens for new opportunities where nanotechnology can tackle other unmet clinical challenges. However, we have learned that after the application of the first dose, rare anaphylactic symptoms may occur, and this adverse effect of the vaccines has been tentatively attributed to the PEGylated LNPs, which may trigger complement activation and, possibly, CARPA. However, as there has been a higher incidence of adverse responses in patients suffering from existing allergies to drugs, they are highly monitored during the vaccine administration offering an excellent risk mitigation strategy. Complement activation by nanomedicines, including PEGylated liposomes, has been previously described and is believed to be associated with protein adsorption on the surface of the nanoparticles. Although the mechanism of these rare adverse reactions is still unknown, complement proteins as well as the presence of anti-PEG antibodies are believed to play an important role. These biomolecules are interconnected in what has been identified as the immune stimulatory vicious cycle (Szebeni, 2016), which produces adverse immunological reactions including CARPA. The anaphylactic reactions associated with COVID-19 vaccines are extremely rare and it is still unclear whether PEG molecules are the main culprit. More studies are needed to further improve on the safety profile of these vaccines as well as other nanomedicines.