European Journal of Pharmaceutics and Biopharmaceutics 193 (2023) 227–240

Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics

journal homepage: www.elsevier.com/locate/ejpb

Graphical Review

Activation of the complement system by nanoparticles and strategies for

complement inhibition
Hajira B. Haroon a, b, Elisha Dhillon a, Z. Shadi Farhangrazi c, d, Panagiotis N. Trohopoulos e,
Dmitri Simberg f, g, S. Moein Moghimi a, b, g, *
a
School of Pharmacy, Newcastle University, Newcastle upon Tyne NE1 7RU, UK
b
Translational and Clinical Research Institute, Faculty of Health and Medical Sciences, Newcastle University, Newcastle upon Tyne NE2 4HH, UK
c
S. M. Discovery Group Inc, CO, USA
d
S. M. Discovery Group Ltd, Durham, UK
e
CosmoPHOS Ltd, Thessaloniki, Greece
f
Translational Bio-Nanosciences Laboratory, Department of Pharmaceutical Sciences, Skaggs School of Pharmacy, University of Colorado Anschutz Medical Center,
Aurora, CO, USA
g
Colorado Center for Nanomedicine and Nanosafety, University of Colorado Anschutz Medical Center, Aurora, CO, USA

A R T I C L E I N F O A B S T R A C T

Keywords: The complement system is a multicomponent and multifunctional arm of the innate immune system. Comple­
Complement ment contributes to non-specific host defence and maintains homeostasis through multifaceted processes and
Complement inhibitors pathways, including crosstalk with the adaptive immune system, the contact (coagulation) and the kinin systems,
Complement regulators
and alarmin high-mobility group box 1. Complement is also present intracellularly, orchestrating a wide range of
Complement receptors
Complosome
housekeeping and physiological processes in both immune and nonimmune cells, thus showing its more so­
Nanoparticles phisticated roles beyond innate immunity, but its roles are still controversial. Particulate drug carriers and
Opsonisation nanopharmaceuticals typically present architectures and surface patterns that trigger complement system in
different ways, resulting in both beneficial and adverse responses depending on the extent of complement
activation and regulation as well as pathophysiological circumstances. Here we consider the role of complement
system and complement regulations in host defence and evaluate the mechanisms by which nanoparticles trigger
and modulate complement responses. Effective strategies for the prevention of nanoparticle-mediated comple­
ment activation are introduced and discussed.

1. Introduction accomplished through opsonisation, inflammatory, and lytic processes

1.1. The complement system
The complement system, comprising more than 50 fluid-phase and
membrane-bound proteins that include pattern-recognition molecules,
zymogens, regulators, and inhibitors (Table 1), is among the major ef­
fectors of the innate immune system [1,2]. Complement is present
mainly in the blood, but some complement proteins are also available in
the interstitial and extracellular space, in the lymph, in tear fluid, in the
vitreous, in the cerebrospinal fluid, in serous exudates on mucosal sur­
faces (e.g., oral cavity and the airways) and intracellularly (complo­
some). Complement serves as a functional bridge between innate and
adaptive immunity and its effector functions are predominantly
[1,2]. Thus, complement contributes to robust non-specific host defence
by orchestrating the clearance of immune complexes, cell debris, and
foreign particles by phagocytic cells, and evoking controlled inflam­
matory and cytolytic responses to maintain homeostasis [1,2]. The
broader participation of the complement in homeostasis is reflected in
crosstalk with the coagulation (contact) system, for instance, to resolve
endothelial injury and facilitate clotting, with the kinin system to
amplify inflammation, and alarmin high-mobility group box 1 in alarm
raising and inflammation [3,4]. Intracellularly (within subcellular
compartments and organelles), complosome (cell-autonomous comple­
ment) apparently serves as a regulator of many physiological processes,
including cell metabolism (e.g., glycolysis, mitochondrial respiration),
autophagy, and gene regulation [5]. However, the exact roles of

* Corresponding author at: School of Pharmacy, Newcastle University, Newcastle upon Tyne NE1 7RU, UK.
E-mail address: seyed.moghimi@ncl.ac.uk (S.M. Moghimi).

https://doi.org/10.1016/j.ejpb.2023.11.006
Received 3 October 2023; Received in revised form 31 October 2023; Accepted 6 November 2023
Available online 8 November 2023
0939-6411/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
H.B. Haroon et al. European Journal of Pharmaceutics and Biopharmaceutics 193 (2023) 227–240

Table 1 step process (recognition, proteolytic activation, and expression of

Activation components, regulators and receptors of the complement system with biologic activities). There are three major complement activation
corresponding chromosome location. pathways: classical, lectin, and alternative (Fig. 1) [1,2]. Although each
Activation Chromosome Protein regulators & Chromosome pathway has some unique proteins and enzymes, the activities that
components receptors result are identical for all three pathways, including opsonisation,
C1q A chain 1p36.12 C1 inhibitor 11q12.1 inflammation, chemotaxis, and biomembrane lysis (Fig. 1) [1,2]. Acti­
C1q B chain 1p36.12 C4 binding protein α 1q32.2 vation of the classical pathway is initiated mainly by the binding of
C1q C chain 1p36.12 C4 binding protein β 1q32.1 complement pattern-recognition molecule C1q to immune complexes
C1r 12p13.31 Factor H 1q31.3
(but also to clusters of anionic charge or hydrophobic motifs on particles
C1s 12p13.31 Factor H related-1 1q31.3
C2 6p21.33 Factor H related-2 1q31.3 and cells as well as to surfaces with immobilised C-reactive protein)
C3 19p13.3 Factor H related-3 1q31.3 followed by sequential activation of two zymogens C1r and C1s,
C4A 6p21.33 Factor H related-4 1q31.3 respectively [6]. The lectin pathway is initiated by the binding of
C4B 6p21.33 Factor H related-5 1q31.3 pattern-recognition molecules mannose binding lectin (MBL), ficolins
C5 9q33.2 Factor I 4q25
C6 5p13.1 Clusterin (SP-40) 8p21.1
and collectin-10 and -11 (Fig. 1) to the pathogen/foreign particle-
C7 5p13.1 Vitronectin 17q11.2 associated molecular patterns (e.g., carbohydrate structures and acety­
C8 α chain 1p32.2 CD46 1q32.2 lated residues) as well as to the damage-associated molecular patterns
C8 β chain 1032.2 CD55 (DAF) c 1q32.2 on apoptotic, necrotic and malignant cells, resulting in activation of
C8 γ chain 9q34.3 CD59 (protectin) 11p13
MBL-associated serine proteases (MASPs) [1,2]. MBL is the most studied
C9 5p13.1 CD93 (C1qRp) d 20p11.21
Factor B 6p21.33 CR1 (CD35) e 1q32.2 pattern-recognition molecule of the lectin pathway and the ligand
Factor D 19p13.3 CR2 (CD21) 1q32.2 binding properties and the effector function of MBL is highly dependent
Properdin (P) Xp11.23 CR3A (CD11b) 16p11.2 on its oligomeric state [9,10]. Regardless of the initiation pathway,
MBL-2 a 10q21.1 CD11c 16p11.2 complement activation leads to proteolytic cascades and the assembly of
Ficolin 1 9q34.3 CD18 (LFA-1) f 21q22.3
convertase enzymes that converge toward proteolytic cleavage and
Ficolin 2 9q34.3 CRIg g Xq12
Ficolin 3 1p36.11 C3a receptor 12p13.31 activation of the third complement protein (C3) (Figs. 1 & 2) [1]. In
Collectin-10 8q24.12 C5a receptor 1 19q13.32 contrast to the classical and the lectin pathways, the alternative pathway
Collectin-11 2p25.3 CSMD1 h 8p23.2 maintains constant surveillance through the spontaneous hydrolysis of
MASP-1 b 3q27.3 CSMD2 h 1p35.1
C3 (the “tickover” hypothesis) at a rate of ~1 % of total C3 per hour,
MASP-2 b 1p36.22 CSMD3 h 8q23.3
Elastase 19p13.3 which generates C3b [11]. There are also bypass pathways that directly
Coagulation Factor II 11p11.2 cleave C3 (Fig. 1). Notwithstanding, C3 activation culminates in the
a generation of two highly potent anaphylatoxins (first C3a, and then C5a
MBL = Mannose binding lectin.
b through the subsequent assembly of C5 convertases), opsonisation of
MASP-1 and MASP-2 = MBL-associated serine protease-1 and -2; alternative
splicing of the pre-mRNA encoding MASP-1 generates MASP-3 and MAp44 intruders’ surfaces with C3b and iC3b, which aids their recognition by
(MAP-1). MAp44 competes with MASP-2 for binding to MBL and ficolins, complement receptors (CRs) of phagocytic cells (Fig. 2), and the
resulting in inhibition of complement activation through the lectin pathway. sequential assembly of the lytic membrane attack complex (MAC) [1,2].
c
DAF = Decay accelerating factor. At the core of all the three-complement pathways lies a highly efficient
d
C1qRp = C1q receptor, selectively expressed by cells with a myeloid lineage, C3 amplification loop: a C3 feedback cycle, which enhances amplifica­
endothelial cells, platelets and microglia. tion, and the C3 breakdown cycle, which down-regulates it by gener­
e
CR = Complement receptor. ating iC3b as its primary product (Fig. 1). The main function of the
f
LFA-1 = Lymphocyte function-associated antigen-1. amplification loop is to deposit large quantities of C3b on the surface of
g
CRIg = V-set and immunoglobulin domain-containing 4 (a receptor for C3b
the activating particle/pathogen [1,2].
and iC3b opsonised pathogens).
h
CSMD 1, 2 and 3 = CUB and Sushi multiple domains 1, 2 and 3, respectively
(transmembrane proteins with complement inhibitory properties). 3. Complement regulation and therapeutics

Complement activation is tightly regulated as to prevent uncon­

complosome are still controversial and not universally accepted [5]. All
trolled inflammation and minimising bystander tissue damage [1,2].
in all, complement contributes to protection, tissue development, and
There are many types of soluble and membrane-anchored complement
regeneration, metabolic regulation, and disease pathophysiology.
regulators and inhibitors (Table 1) to ensure complement regulation at
Similar to pathogens, particulate drug carriers and nano­
many steps along complement activation pathways [1,2]. Of particular
pharmaceuticals exhibit diverse architectural arrangements and surface
importance are regulators acting at the level of the convertases (by
patterns that could be sensed by complement, where responses could
affecting their assembly as well as their proteolytic activity) and during
dramatically affect biological and therapeutic performance of adminis­
the assembly of the MAC (Figs. 1 & 2). For example, the fluid-phase
tered nanoparticles [6–8]. On the other hand, if nanoparticles derail
complement regulator factor I cleaves C3b and generates inactive frag­
complement activation this might instigate inflammatory reactions [7].
ments iC3b and C3c, but to prevent non-specific C3b degradation, it
Thus, identification and systematic mapping of physicochemical and
requires cofactor activity for its proteolytic function provided by factor
biological parameters that trigger (or evade) complement activation by
H, membrane cofactor protein (MCP, CD46) and CR1 (Fig. 2) [1,2,12].
therapeutic nanoparticles could open the path to any number of possi­
On the other hand, complement regulators such as decay accelerating
bilities, which in conjunction with artificial intelligence and machine
factor (DAF or CD55) accelerates the decay of C3 and C5 convertases by
learning algorithms, portends to fast-track and new design of nano­
competing with factor B for binding to surface-bound C3b and by dis­
particle libraries with tuneable complement responses. Here we high­
placing Bb from convertases, whereas CD59 interferes with the assembly
light complement activation mechanisms by nanoparticles and discuss
of MAC [1,2,12].
current nanoengineering and therapeutic trends in tuning complement
Notwithstanding, erroneous activation, over-activation, and/or
responses to administered nanoparticles.
insufficient regulation of the complement might turn its destructive
actions against the host [12,13]. Atherosclerosis is a fine, yet, intricate
2. Complement activation
example, where complement exhibits both protective and proathero­
genic properties, and reviewed in detail elsewhere [14]. Here, the role of
Complement activation in the blood may be summarized as a three-
complement is not only driven by plasma-derived complement factors,

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but also there are indications that distinct lesional macrophage subsets
are potential sources of local complement production [14]. Considering
the latter, recently, a complement regulatory axis in atherosclerotic
plaques was realised, where factor H non-redundantly regulates the C3-
dependent efferocytosis capacity of lesional macrophages [15]. In
particular, factor H derived from inflammatory phagocytes exacerbates
atherosclerosis by limiting lesional apoptotic cell clearance [15].
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European Journal of Pharmaceutics and Biopharmaceutics 193 (2023) 227–240
Complement over-activation by enzymatically-modified low-density li­
poproteins and cholesterol crystals has also been suggested to contribute
to pro-inflammatory reactions, macrophage death, and smooth muscle
cell proliferation in atherosclerotic plaques [14]. Cancer is another
example of delicately taking advantage of complement proteins and
complement cleavage products for progression. For instance, intra­
tumoural liberation of C5a (e.g., as a result of intratumoural coagulation
(caption on next page)
H.B. Haroon et al. European Journal of Pharmaceutics and Biopharmaceutics 193 (2023) 227–240

Fig. 1. Complement activation pathways. The scheme shows sequential enzymatic steps in classical, lectin and alternative complement activation pathways, key
fluid-phase complement regulators (e.g., C1-inhibitor, Factor H, Factor I) and selected complement activation products (C4a, C4b, C4d, C3a, C3b, iC3b, C3bc, Bb,
C5a, sC5b-9) that can be measured quantitatively. Antibodies such as IgM, IgG and IgA, depending on their isotype, glycosylation and conformation, can trigger
complement activation through the three pathways. The C-reactive protein (CRP, a pentameric acute-phase protein) on binding to nanoparticles might also trigger
complement activation through the C1 complex arm of the classical pathway. The binding of complement pattern recognition molecules C1q and collectins (e.g.,
MBL, ficolins and collectin-11) to surfaces usually triggers activation of their associated proenzymes (C1r and C1s in the case of C1q; MASPs in the case of collectins),
resulting in complement activation through classical and lectin pathways, respectively. Complement activation through classical and lectin pathways cleaves C4,
resulting in liberation of C4a. Classification of C4a as a complement anaphylatoxin is debatable [95]. Alternative pathway functionality is through autoactivation of
soluble C3 that undergoes slow spontaneous hydrolysis [C3(H2O)], or when nascent C3b undergoes nucleophilic attack for instance, by hydroxyl or amine moieties
on nanoparticle surfaces. The first critical stage in complement cascade is the formation of C3 convertases [C4b2b (formerly called C4b2a) of classical and lectin
pathways and C3bBb of the alternative pathway] that proteolytically cleave C3. This results in the liberation of anaphylatoxin C3a and the opsonic fragments C3b and
iC3b. The next step is the assembly of pathway-specific C5 convertases [C4b2b3b (formerly called C4b2a3b) and C3bBbC3b], which cleaves C5, resulting in liberation
of another anaphylatoxin (C5a) and activation of the terminal pathway of complement. On full complement activation the membrane attack complex C5b-9 is
formed. In soluble form, C5b-9 is bound to vitronectin (sC5b-9). The amplification loop of the alternative pathway is responsible for propagating the responses of all
three-complement pathways by producing large amounts of C3b. Apart from complement convertases, proteases of the coagulation system such as kallikrein and
thrombin can directly cleave C3 and C5 (not shown in the scheme), leading to additional sources of anaphylatoxins C3a and C5a. Similarly, MASP-1 may directly
cleave C3. MASP-3 activates the zymogen factor D of the alternative pathway. The scheme also shows selected natural complement regulators [factors I and H, C1
esterase inhibitor, MAP-1 (MAp44, see Table 1) and small MBL associated protein (sMAP or MAp19)] and sites where they exert their action. The figure and the
legend are reproduced with permission with slight modifications [8].

processes that cleaves C5) helps tumour growth and invasiveness in
many ways; for instance, by promoting immunosuppression, since C5a is
chemoattractant to myeloid-derived suppressor cells [16,17]. Many tu­
mours also overexpress complement proteins such as C1q, which pro­
mote tumourigenesis by favouring angiogenesis and metastasis
independently of complement activation [18]. Complement dysregula­
tion can occur due to genetic mutations (Table 1), as a result of disease
processes, or because of the development of autoantibodies to comple­
ment components [12,13]. A classic example is paroxysmal nocturnal
haemoglobinuria (PNH), caused by a somatic mutation in the phos­
phatidylinositol glycan complementation class A gene in haematopoietic
stem cells [19]. This mutation affects the biosynthesis of glyco­
sylphosphatidylinositol (GPI), where a chronic reduction or absence of
GPI leads to a deficiency in membrane-anchored complement regulators
resulting in inadvertent complement attack on erythrocytes [19].
There are many therapeutic opportunities for inhibiting or modu­
lating the complement system at preclinical and clinical levels
[13,20,21]. For instance, United States FDA-approved complement
therapeutic interventions include a humanised recombinant monoclonal
antibody against C1s, designer biologics such as pegcetacoplan, which
inhibits the central conversion of C3 to C3b and C3a, recombinant
humanised anti-C5 monoclonal antibodies, and a C5a receptor antago­
nist for suppressing anaphylatoxin signalling (Table 2) [21–24]. Inhi­
bition of C3 and its convertases (e.g., using compstatin and its related
family members Cp40 and pegcetacoplan) has the broadest effect, since
this blocks the amplification loop and, in principle down regulates the
terminal activity of the complement pathway. However, strong activa­
tion of calcium-sensitive pathways could still lead to C5 activation in­
dependent of C3 (the so-called C3 bypass pathway) [25,26]. While C3
(and C5) inactivation in humans may increase susceptibility to Menin­
gococcal and other upper respiratory tract infections (e.g., Pneumo­
coccal and Haemophilus influenza type B infections) (Table 2) [27],
prolonged treatment of non-human primates with compstatin neither
weakened immune system nor susceptibility to infection [28]. However,
patients undergoing C5, C3 and C1s complement inhibitor therapies are
recommended for vaccination against encapsulated bacteria (Table 2)
(e.g., Bexsero, Prevnar® and Menitorix vaccines). Other examples of
complement modulators at preclinical or undergoing clinical evaluation
includes soluble form of natural complement regulators (e.g., sCR1,
sDAF, sMCP and sCD59), small molecule factor B and factor D inhibitors
(iptacopan and danicopan, respectively) for oral administration and
anti-factor D antibody fragments, peptide and antibody based inhibitors
of C1s and activated C1s (apart from Enjaymo®, Table 2), anti-MASP3,
anti-C2, anti-C5a, anti-C6 and Factor Bb antibodies, anti-C5 aptamer
and fusion constructs such as CR2–factor H (TT30) and CR2–CR1 (TT32)
[13,20,21,29,30]. For instance, with the latter, the CR2 moiety recog­
nises surface-deposited C3, whereas CR1 blocks C3 convertases [30].
Finally, C1 esterase inhibitor is another complement inhibitor, which is
widely used at experimental levels acting on classical (inactivating C1r
and C1s) and lectin (inactivating MASP-1 and MASP-2) pathways [31].
In addition, C1 esterase inhibitor is the inhibitor of the kinin, fibrino­
lytic, and contact activation pathway of the coagulation cascade [31]. It
is in this context, and not as a complement inhibitor that the substitution
of a natural C1 esterase inhibitor and a recombinant protein C1 esterase
inhibitor has been approved for the inherited disorder hereditary
angioedema in patients deficient in C1 esterase inhibitor (Table 3) [31].
The deficiency of the C1 esterase inhibitor results in dysregulation of the
plasma kallikrein-kinin system and excessive bradykinin production,
resulting in episodes of soft tissue swelling.
4. Complement activation by nanoparticles
Numerous studies have demonstrated complement activation by
different organic and inorganic nanoparticles and regulatory-approved
nanopharmaceuticals in human serum, plasma, and full blood
(reviewed in [6,7]). Generally, complement activation could compro­
mise nanocarrier stability (e.g., liposome and lipid nanoparticles),
causing drug leakage, promoting premature clearance of nanoparticles
by the blood and tissue phagocytic cells and compromising their ther­
apeutic efficacy for intended non-phagocytic cell targets, and when
uncontrolled, might induce adverse reactions and promote disease
progression [6,7].
Regarding the mechanistic issues, recent studies have shown that in a
typical nanoparticle suspension, only a small fraction of nanoparticles
(or their aggregates in plasma) actively trigger complement activation
[32,33]. This is a reflection of nanoparticle population heterogeneity.
While the precise mechanisms are still under investigation, there are
indications that complement activation could proceed on binding of
nascent C3b or C3(H2O) to some nanoparticles, resulting in surface as­
sembly of the alternative pathway C3 convertase (Fig. 3) [32,33]. While
direct binding of C3b or C3(H2O) to pristine surfaces is possible, recent
studies have shown predominant binding to surface-adsorbed proteins
[32,33]. For example, C3b binding to a few surface-deposited antibodies
has been suggested [32], and some antibodies can potentially trigger
complement activation through the alternative pathway [34]. This
“seed” population of nanoparticles with C3 convertases on their surfaces
generate large quantities of C3b molecules from native C3 in plasma (C3
is a highly abundant serum protein with a normal range of 0.8–1.6 mg/
mL) that potentially opsonises bystander nanoparticles in their vicinity.
This, in turn, could accelerate and amplify complement activation
through the assembly of C3 convertases on surfaces of more nano­
particles [33]. However, C3 opsonisation of nanoparticles was shown to
be slow and inefficient, but reversible [33,35]. The latter was ascribed to
the dynamic process of protein adsorption–desorption at the

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(caption on next page)

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Fig. 2. Schematic representation of C3 structure and its sequential fragmentation by complement regulators. C3 is a member of α-2 macroglobulin family of proteins,
which has a 13-domain structure and two polypeptide chains (α and β chains) held together by a single disulfide bond. C3 convertases of classical, lectin and
alternative pathways cleave the α chain of C3 resulting in generation of two fragments C3a and C3b (the α chain of C3b is usually referred to as α′). C3a (a 9 kDa
peptide) is an anaphylatoxin with a short half-life in plasma (it is rapidly processed by carboxypeptidases) exerting proinflammatory functions via its receptor (C3aR)
expressed by a number of immune cells such as monocytes/macrophages, mast cells, neutrophils, basophils, and eosinophils. C3b is an opsonic molecule when is
bound covalently to nanoparticle surfaces aids nanoparticle recognition by cells expressing complement receptors 1 (CR1 or CD35), 3 (CR3 or CD11b/CD18) and 4
(CR4 or CD11c/CD18) and complement receptor of the immunoglobulin family (CRIg). For example, human Kupffer cells express CR1, CR3 and CR4 and human
dendritic cells express CRIg. C3b can also initiate formation of the alternative pathway C3 convertase as well as classical, lectin and alternative pathway C5 con­
vertases (see Fig. 1). Surface bound C3b is rapidly inactivated (due to release of the 2 kDa fragment C3f) by complement regulators such as factor H (FH) and FH like
1 protein in tandem with factor I (FI) to generate inactive C3b (iC3b). Plasma membrane bound complement regulators such as CR1 and CD46 (membrane cofactor
protein) also inactivate C3b bound to cell surfaces, thus protecting cells from complement damage. iC3b is also an opsonic molecule recognised by cells expressing
CR3, CR4 and CRIg. Further processing of iC3b by complement regulators FI, CR1 and other proteases (e.g., elastase) release the fluid phase fragment C3c and the
surface-bound fragment C3dg. C3dg is further processed by proteases (e.g., thrombin) to C3d, which remain bound to surfaces (e.g., nanoparticles). Surface-bound
C3dg/C3d are recognised by CR2 (or CD21). B lymphocytes express CD21; however, the role of B cells in the uptake of complement opsonised nanoparticles has not
received much attention. The lower part of the scheme shows an intramolecular thioester bond in the C3d region of the α chain, which is susceptible to nucleophilic
attack. This becomes the exposed reactive moiety of the metastable binding site of proteolytically produced C3b. The thioester generates a carbonyl group that binds
the C3b irreversibly to reactive pristine groups (e.g., hydroxyl and amine moieties) expressed either on nanoparticles and/or on their surface-bound biomole­
cule corona.

Table 2
Currently approved complement inhibitors by the United States Food and Drug Administration (FDA).
Inhibitor Class Target FDA Current indication
approval
year

Soliris® Recombinant humanised C5 2007 Paroxysmal nocturnal haemoglobinuria (PNH); atypical haemolytic uremic
1
(Eculizumab) monoclonal antibody (IgG2/4 syndrome (aHUS); generalised myasthenia gravis (gMG) in adult patients who are
κ) anti-acetylcholine receptor antibody positive; neuromyelitis optical spectrum
disorder in adult patients who are anti-aquaporin-4 antibody positive
Ultomiris® Recombinant humanised C5 2018 Adult and paediatric patients one month of age and older with PNH or aHUS;
(Ravulizumab) 2 monoclonal antibody (IgG4) patients with gMG who are anti-acetylcholine receptor antibody positive
Tavneos® Small molecule C5aR1 (CD88) 2021 Adjunct treatment of adult patients with severe active anti-neutrophil cytoplasmic
(Avacopan) 3 C33H35F4N3O2 antagonist autoantibody-associated vasculitis
Empaveli® Pegcetacoplan (injection for C3 2021 Adult patients with PNH
(Aspaveli®) 4 subcutaneous use)
Enjaymo® Humanised recombinant C1s 2022 Cold agglutinin disease
(Sutimlimab-jome) monoclonal IgG4 antibody
5
6
Syfovre® Pegcetacoplan (for intravitreal C3 2023 Geographic atrophy secondary to age-related macular degeneration
injection)

Soliris®, Ultomiris®, Empaveli® and Enjaymo®, labels warn on serious infections caused by encapsulated bacteria and recommend appropriate vaccination strategies
prior to the inhibitor administration.
1
https://www.accessdata.fda.gov/drugsatfda_docs/label/2019/125166s431lbl.pdf (accessed 30/10/2023).
2
https://www.accessdata.fda.gov/drugsatfda_docs/label/2022/761108s023lbl.pdf (accessed 30/10/2023).
3
https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/214487s000lbl.pdf (accessed 30/10/2023).
4
https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/215014s000lbl.pdf (accessed 30/10/2023).
5
https://www.accessdata.fda.gov/drugsatfda_docs/label/2022/761164s000lbl.pdf (accessed 30/10/2023).
6
https://www.accessdata.fda.gov/drugsatfda_docs/label/2023/217171s000lbl.pdf (accessed 30/10/2023).

or both [6,7,36]. Here, two interrelated parameters that modulate

Table 3
complement activation are particle size (curvature) and surface archi­
FDA approved C1 esterase inhibitor preparations as inhibitor of the kinin,
tecture (Fig. 4) [6]. The latter includes chemical composition, the
fibrinolytic and contact activation pathway.
spacing arrangement/periodicity of surface functional groups/ligands,
Inhibitor Class FDA Current indication and the structural/conformational state of surface projected macro­
approval
year
molecules (e.g., synthetic polymers, polysaccharides) [6,7,36]. Collec­
tively, these parameters modulate complement activation through
Cinryze Human plasma-derived 2008 Hereditary angioedema
multivalent engagement with and conformational regulation of surface-
concentrate of C1 (HAE) in adults, adolescents
esterase inhibitor and paediatric patients (6 bound antibodies and complement pattern-recognition molecules
years of age and older (Fig. 4). For instance, unbound IgM assumes a planar structure and does
Berinert Human plasma-derived 2009 Acute abdominal, facial, or not activate complement, while bound IgM must be in strained confor­
concentrate of C1 laryngeal HAE in adults and mation to trigger complement activation through the C1 complex
esterase inhibitor paediatric patients
[37,38]. Optimal straining of IgM requires multiple Fab interactions
Ruconest Recombinant protein 2014 Acute attacks in adult and with surface-exposed epitopes. This is best achieved on the surface of
C1 esterase inhibitor adolescent patients with spherical particles in the 100–250 nm size range [38]. A dramatic
HAE decrease or increase in particle size, however, minimises multivalent
engagement of all Fab regions leading to insufficient IgM straining;
nanoparticle-plasma interface [35]. otherwise, high-affinity interactions are needed to sufficiently strain the
Depending on their physicochemical properties, nanoparticles could Cµ2 domain of the immunoglobulin [38]. Thus, curvature- and epitope
also trigger complement activation through classical or lectin pathways, density-dependent variables on nanoparticles could explain inter-

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Fig. 3. A proposed model for complement activation and C3 opsonisation through non-specific plasma protein adsorption on nanoparticles. The scheme shows a
dextran (yellow and yellow arrows) coated superparamagnetic iron oxide nanoworm (the brown region and the white star represent part of the iron core) interacting
with plasma proteins (grey). Experiments have shown that some surface adsorbed plasma proteins and/or plasma proteins intercalated into dextran chains inad­
vertently cause activation of the alternative pathway of the complement system. The hypothesis state that adsorbed and/or intercalated plasma proteins may undergo
conformational changes or form clusters that expose reactive moieties that interact with nascent C3b or C3(H2O) (green). This leads to the formation of the
alternative pathway convertase (C3bBbP, white circle) only on a small fraction of nanoparticles (Bb = red and P = dark blue). These convertases generate additional
C3b molecules that opsonise nearby nanoparticles. Protein-C3b complexes are continuously formed and released (light blue circle) from the surfaces of nanoparticles.
Thus, this turnover might minimise surface deposition of fluid-phase regulators such as factors H and I. Accordingly, nanoparticles in the blood may continuously
activate complement through the alternative pathway turnover. Considering the above, the hypothesis does not disregard surface deposition of IgG–C3b complexes
that are already formed in the fluid phase and with the ability to assemble C3bBbP convertases. P = properdin.

individual differences in the extent of complement activation on IgM
binding. Curvature also plays a role in IgG binding for a given Fc density,
where activation of the classical pathway depends on antigen-driven
multivalent engagement with IgG clusters, particularly IgG hexamers
[39]. The composition of N-glycans in the CH2 domain of IgG also plays
an important role in hexamer assembly through IgG Fc:Fc interactions
[40] and, hence, modulation of complement activation through C1q
binding.
Liposomes and lipid nanoparticles are among the most popular ve­
hicles in drug delivery, and there are many examples of regulatory-
approved lipid-based nanopharmaceuticals and vaccines [41,42].
Many studies have shown that binding of naturally occurring anti-
phospholipid and anti-cholesterol antibodies to liposomes and lipid
nanoparticles could trigger complement activation through classical and
alternative pathways [reviewed in 6,7,36]. Many clinically approved
nanopharmaceuticals also contain poly(ethylene glycol) (PEG)-lipid
conjugates [41,42]. However, the emergence of antibodies against poly
(ethylene glycol) (PEG) [43–46], where the majority exhibit low affinity
for PEG, has raised the question of whether low-affinity anti-PEG anti­
bodies can reach sufficient straining (as in IgM) or form clusters (as in
IgG) on PEGylated surfaces to trigger classical pathway activation [6,7].
While, surface PEG density, conformation and mobility could modulate
the extent of antibody-dependent classical pathway activation, com­
plement activation might prevail on the binding of anti-PEG antibody
sub-classes that trigger the alternative pathway [47].
Ultrastructurally, pattern-recognition molecules such as C1q, fico­
lins, and MBL oligomers sense surface patterns/target motifs spaced at
broad intervals of 2–20 nm (Fig. 4) [9,48–50]. This ensures recognition

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H.B. Haroon et al. European Journal of Pharmaceutics and Biopharmaceutics 193 (2023) 227–240

Fig. 4. Schematic summary showing the role of nanoparticle curvature and surface pattern periodicity on complement activation. Antibody-dependent complement
activation depends on the conformation and strain of the binding antibody (e.g., as a single IgM or clusters of IgG), which, in turn, is modulated by nanoparticle
curvature and spacing/surface density of exposed epitopes (left panel). Structural and geometrical restrictions in pattern recognition molecules such as C1q and MBL
allow these molecules to sense surface patterns with dimensions in the range of 2–20 nm, thereby triggering complement activation through classical and lectin
pathways, respectively (right panel). Surfaces that display pattern periodicity below the nanometer-scale spacing arrangement are expected to escape complement
surveillance by C1q and MBL. This is known as the “angstrom-scale spacing arrangement (ASSA) phenomenon” for complement evasion. However, complement
activation through non-specific protein adsorption could still proceed. Reproduced with permission [6].

of target surfaces with wider and denser display of ligands. For instance,
C1q recognises surface clusters of anionic charge or hydrophobic motifs
[51], whereas MBL oligomers bind to sugars such as D-mannose and N-
acetyl-D-glucosamine [9]. Thus, nanoparticle surface pattern periodicity
and spacing are important in accommodating complement pattern-
recognition molecules. Cardiolipin liposomes [51] and poly(2-methyl-
2-oxazoline)-coated nanoparticles [52] are examples of entities that
trigger human complement activation through C1q sensing. Variable
results, however, have been reported on C1q binding to carbon-based
nanomaterials. While one study reported C1q-dependent complement
activation by carbon nanotubes [53], another study found no comple­
ment activation on C1q binding [54]. These inconsistencies are pre­
sumably related to differences in surface functionality and chemistry of
carbon nanomaterials in modulating C1q conformation. There are also
interesting examples of nanoparticles that lack pristine ligands for
recognition by lectin pathway pattern-recognition molecules, yet they
activate the lectin pathway [55]. One example is poloxamine 908-coated
polystyrene nanoparticles, but surface-projected poloxamine 908 only in
a mushroom-brush intermediate/brush conformation triggers comple­
ment activation through the lectin pathway [55]. Poloxamine 908 is a
star-shaped block copolymer of ethylene oxide and propylene oxide
blocks, generating repetitive patterns of relative polarity and hydro­
phobicity on surface adsorption, which might transiently resemble
structural motifs of natural substrates for MBL and ficolins. Additionally,
selected plasma proteins might intercalate into the loops and trails of
surface-projected copolymer blocks. Thus, an alternative possibility for
complement activation arises if intercalated proteins constitute pattern-
recognition molecules (e.g., MBL and ficolins) with their associated
serine proteases or mannosylated proteins (e.g., apolipoprotein B100)
and glycosylated variants of IgG and IgM that bind MBL [56].
5. Overcoming nanoparticle-mediated complement activation
To date, different strategies have emerged for designing nano­
particles that show little or no complement activation (Fig. 5). The
simplest strategy is nanoparticle surface enrichment and/or function­
alisation with materials that recruit fluid phase complement regulators
such as factor H. Examples include sialic acid, factor H-specific phage
peptides and heparin [57,58]. There are also serendipitous examples of
nanoparticles that do not trigger complement activation in human
serum, such as hexosomes assembled from citrem and glyceryl mono­
oleate [59]. Although the exact mechanisms with this class of hexosomes
were not investigated, it is plausible that the citric acid moiety of citrem
in combination with the glycerol component of glyceryl monooleate
form dynamic platforms resembling factor H binding domains on sialic
acid (factor H bind to glycerol side chain and carboxyl group of sialic
acid [58,60]). In contrast to the above, another strategy is direct grafting
of complement regulators to nanoparticles [61].
With PEGylated liposomes, the anionic phosphate-oxygen of the
PEG-phospholipid conjugate was shown to play a key role in comple­
ment activation and methylation of phosphate-oxygen dramatically
reduced complement activation [62]. However, non-specific protein
binding and protein trapping (including antibodies) in PEG clouds might
still contribute to complement activation through the alternative
pathway [35]. Thus, better strategies are needed to minimise protein
binding/trapping. One such approach is surface functionalization of

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Fig. 5. Schematic representation of main approaches in overcoming complement activation by therapeutic nanoparticles. These approaches include nanoparticle
engineering with components that deter or repel complement pattern-recognition molecules as well as C3 opsonisation such as through polymer-pairing and surface
patterning with angstrom-scale periodicity, or strategies that attract fluid-phase complement regulators to nanoparticle surfaces, or direct functionalization of
nanoparticle surfaces with complement regulators, or co-administration of nanoparticles with designer complement inhibitors for precision surface complement
inhibition (e.g., CR2–CR1 fusion protein).

nanoparticles with combinations of short- and long-chain polymers. For
instance, surface functionalization of nanoparticles with combinations
of PEG-phospholipid conjugates with long and short PEG chains reduced
complement activation, since more rigid short PEG chains caused
stretching of longer PEG chains contributing to more effective protein
exclusion [63]. Alternatively, considering surface sensing by comple­
ment pattern-recognition molecules is restricted to spacing intervals of
2–20 nm [9,48,50], surfaces that display patterns in angstrom-scale
intervals might also escape complement activation. This is referred to
as “angstrom-scale spacing arrangement (ASSA) phenomenon” for
complement evasion and demonstrated with the low generation poly
(amidoamine) (PAMAM) dendrimers of different end-terminal func­
tional moieties (generations 3–5 with dendrimer sizes of 3.2–4.8 nm,
respectively, that display 32–128 periodic surface functional moieties
such as amine, carboxyl and pyrrolidone in a confined space with
angstrom-scale separation from each other) [64].
Typically, the internal thioester bond in the α-chain of nascent C3b
undergoes nucleophilic attack by structures rich in amine and hydroxyl
functionalities; this increases alternative pathway turnover [65,66].
Contrary to this, amine-terminated dendrimers do not directly affect
alternative pathway turnover [64]. Generation 3 PAMAM dendrimers
are open ellipsoids, whereas generations 4 and 5 are “hard spheres”
[67]. Presumably, the presentation of end-terminal amine functional­
ities in ASSA, together with dendrimer shape and the very small size (<5
nm), accounts for the lack of alternative pathway activation. It would be
intriguing if the display of chemically reactive functional groups in ASSA
on engineered larger nanoparticles could minimise C3b binding and
surface assembly of the alternative pathway convertases. However,
particle curvature and non-specific protein adsorption could further play
a role in modulating complement responses. Also, whether larger par­
ticles and macro-scale platforms with surface patterns in the ASSA can
escape sensing by complement pattern-recognition molecules and avoid
complement activation remains to be tested.
The aforementioned complement escape strategies are broadly
applicable to new nanoparticle design. However, universal approaches
are needed to suppress complement activation by existing nano­
pharmaceuticals and follow-on products. One approach is to co-
administer soluble complement inhibitors with nanopharmaceuticals
(Figs. 5 & 6). However, on the one hand, high doses of soluble inhibitors
may be needed due to suboptimal potency. On the other hand, co-

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administration with long-lasting inhibitors (e.g., pegcetacoplan) could
lead to possible adverse effects and immunosuppression. To address
these shortfalls, recently complement activation was successfully avoi­
ded when nanoparticles were co-administered with the short-circulating
fusion construct CR2–CR1 (Fig. 6) [33]. Since, only a small percentage
of nanoparticles randomly trigger complement and C3 opsonised (i.e.,
the “seed” population), CR2–CR1 exclusively targeted C3 fragments and
C3 convertases on nanoparticle surfaces with picomolar to low
nanomolar efficacy, blocked complement opsonisation and overcome
nanoparticle uptake by monocytes/granulocytes in the blood [33]. It is
hypothesised that inhibiting the function of C3 convertases on the “seed”
population of nanoparticles overcomes the subsequent cleavage of
plasma C3 and the opsonisation of bystander nanoparticles [33]. In rats,
CR2–CR1 also prevented lethargy caused by bolus injection of nano­
particles, without inducing long-lasting complement suppression [33].

Fig. 6. Inhibition of complement activation by selected synthetic complement inhibitors. The scheme depicts complement inhibition by two different groups of
designer complement inhibitors: compstatin family of peptides and a fusion peptide assembled from two complement regulators (CR2–CR1). Compstatin is a cyclic
tridecapeptide. Other family members include Cp40 (a 14 amino-acid cyclic peptide) and pegcetacoplan (consisting of two 15 amino-acid cyclic peptides conjugated
via a linear 40 kDa poly(ethylene glycol) molecule). Pegcetacoplan has a half-life of 8–10 days in humans compared with a half-life of 12 h for Cp40 in cynomolgus
monkeys. Compstatin binds native C3 and C3b and sterically interfere with the assembly of the C3 convertase complexes. Thus, unlike other C3-regulatory proteins,
the mechanism of action of compstatin does not involve the destabilisation of the C3 convertase or the accelerated degradation of the C3b. Compstatin neither
interfere with the generation of C3(H2O) nor blocks C3 cleavage by non-C3 convertase proteases. Similar to compstatin, Cp40 also binds to fluid phase and surface-
bound C3 targets. In contrast, pegcetacoplan, has weaker affinities but preferentially targets surface complement inhibition, presumably through an avidity effect
binding two C3b molecules. This mode of action prevents alternative pathway convertase binding to C3 and subsequent cleavage. Fusion constructs such as CR2–CR1
inhibit complement activation with picomolar to low nanomolar efficacy on many nanoparticles in human and rat serum. The CR2 moiety targets surface C3 deposits
(C3b, iC3b, C3c and C3d), but not the fluid phase C3. The CR1 moiety targets C3 convertases. Although being a short-circulating complement regulator (a fast phase
of 2.5 min and a slow phase of 64 min in rats), co-injection of CR2–CR1 with nanoparticles targets activated C3 on nanoparticle surfaces and inhibits complement
activation as well as acute responses in rats.

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6. Conclusions and future directions
The interactions between complement proteins and nanoparticles
have long been realized; however, the importance of complement in
nanomedicine research is still underappreciated [6,7,36]. By virtue of
their structural complexity and pattern display, nanoparticles trigger or
evade complement in different ways; however, there are species differ­
ences in complement responses to nanoparticles and the results cannot
be generalised and extrapolated from one species to another
[7,8,36,52,68]. For example, nanoparticle/antigen adjuvanticity is
improved on C3d/C3dg tagging as this ensures long-term B cell memory
and optimal antibody production in mice [69–71] and, again, this
concept was recently revisited and applied for enhancing the immuno­
genicity of lipid-nanoparticle mRNA vaccines in mice [72]. Importantly,
C3d/C3dg adjuvanticity has only been demonstrated in the murine
model and, apparently, does not apply to human B cells [73]. This is
presumably related to differences between the function of CR1 and CR2
between the two species [73]. Another example is nanoparticles coated
with poly(2-methyl-2-oxazoline), which triggers complement activation
in human but not in murine serum [52]. Here, C3 opsonisation was
essential for nanoparticle recognition by human blood leucocytes and
monocyte-derived macrophages [52]. Notwithstanding, our current
mechanistic understanding of nanoparticle-mediated complement acti­
vation processes in human serum/plasma has opened exciting ap­
proaches for the design and engineering of complement-evading
entities. These developments are important since uncontrolled activa­
tion or over-activation of the complement system by therapeutic nano­
particles could compromise their safety and therapeutic efficacy and
contribute to disease progression, but still, it is overlooked. For example,
intratumoral complement activation by extravasated long circulating
drug-free nanoparticles has promoted tumour growth in syngeneic
immunocompetent mice bearing TC-1 tumour [74]. By considering
inter-individual as well as species differences in complement responses
to nanoparticles [7,8,36,75], we need more integrated studies that map
and correlate materials’ angstrom- and nano-scale parameters to com­
plement responses not only extracellularly, but also intracellularly
(complosome) and depending on microenvironmental conditions (e.g.,
hypoxia can alter complement activity as well as regulation of comple­
ment proteins in many cell types [76]). Therefore, attention must also be
paid to the local and compartmental concentration of immunoglobulins,
complement proteins and regulators as well as non-specific proteins that
could modulate complement responses. For instance, an early study in
the leg lymph of normal men identified much lower levels of immuno­
globulins and complement proteins than in serum, which was consistent
with the capillary sieving mechanism [77]. Thus lymph levels of IgG,
IgM, C1q, C1s, C1 inhibitor, C4, and C9 were found to be 17 %, 7.4 %,
12 %, 21 %, 16 %, 22 %, and 24 % of that of serum, respectively [77].
The total mean complement haemolytic activity in lymph was approx­
imately 7-fold lower than in serum [77]. Another related example is
salivary scavenger and agglutinin (SALSA), also known as gp340, and its
isoforms, which is found at mucosal surfaces of the lungs, oral cavity,
gastrointestinal tract, and vagina as well as in amniotic fluid [78].
SALSA binds to C1q, MBL, ficolins, surfactant proteins A and D, secre­
tory IgA, fibrin, and fibrinogen, which suggests a role in regulating
inflammation and immune responses [78]. Future studies are needed to
assess whether the binding of SALSA to locally administered nano­
particles plays a central role in directing local complement activation.
Nevertheless, studies with immobilised SALSA (SALSA coated onto a
microtiter plate) have demonstrated complement activation, whereas
SALSA in the fluid phase caused a dose-dependent inhibition of the
lectin pathway [79]. Also, not all complement proteins and regulators
are present in different body compartments, and their levels may vary in
disease processes. For example, complement components up to C5 have
been demonstrated in lumen samples obtained from bacterially infected
human intestines, but the MAC was absent from the lumen environment
[80]. Intestinal epithelial cells have been suggested as a source of these
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European Journal of Pharmaceutics and Biopharmaceutics 193 (2023) 227–240
complement proteins in addition to the secretion of complement pro­
teins from the pancreas into the duodenum, but oedema in the mucosa
or bleeding might also provide local complement [81]. Another example
is human tear, where C1q, C3, C4, C5, factor B, C9, and DAF have been
detected in closed-eye tears, whereas only C3, C4, and factor B were
found in open-eye and reflex tears [82]. On the other hand, lactoferrin is
present in all tear types [82], and lactoferrin-derived peptides poten­
tially inhibit the classical pathway [83]. Thus, considering the afore­
mentioned discussion, nanoparticle-mediated complement activation
processes and response magnitude are expected to differ in different
body compartments.
Growing evidence suggests perturbations in complosome activities
could contribute to a wide range of human diseases [5,84]. However, the
impact of nanoparticle-based therapies on cell type-specific complosome
functionality has been overlooked and rarely touched upon. Not only
many types of immune cells such as CD4+ and CD8+ T cells, B cells,
monocytes, macrophages and neutrophils have intracellular pools of C3
and C5 (and some also express intracellular C3a receptor and C5a re­
ceptor 1 that engage with ribosomes, inflammasomes and other intra­
cellular machineries) [5,15,84–88], but also many types of healthy
nonimmune cells such as fibroblasts, hepatocytes, a range of endothelial
and epithelial cells, neurons and pancreatic β-cells have active intra­
cellular complement components [5,84,87]. Although the roles of
complosome are still debatable, nevertheless, it would be interesting to
consider to what extent nanoparticle localisation to nonimmune cells
could perturb complosome. Lipid nanoparticles with cationic or ionis­
able cationic lipids, which are known to activate and fix complement,
serve as good examples, and the currently available cationic and ionis­
able cationic lipids have been shown to induce pro-inflammatory re­
sponses [89–91]. Considering these, cytoplasmic sensing of C3 can
activate mitochondrial antiviral-signalling protein located in the outer
membrane of the mitochondria, peroxisomes, and mitochondrial-
associated endoplasmic reticulum membrane and induce pro-
inflammatory cytokine secretion [92].
Finally, continuous developments in multiparametric and plasmonic
complement assays, multi-omics complement technologies, precision
intracellular complement sensors, and species- and pan-specific com­
plement inhibitors (including cell-permeable inhibitors) together with
artificial intelligence guidance are expected to address and help with the
precision engineering of complement- and complosome-tuneable ther­
apeutic nanoparticulate systems, thus paving the path towards stratified
approaches in nanomedicine therapies. Efforts might extend to more
sophisticated nanoparticle designs compatible with the contact and the
kinin systems. In parallel, advances in “complotype” genetics (the
repertoire of inherited rare or common polymorphisms in genes
encoding complement components) [93], which has an impact on sus­
ceptibility to inflammatory diseases and conditions, might further help
with future stratification strategies in co-administering nanomedicines
with appropriate complement inhibitors.
Last, but not least, apart from their therapeutic potential, nano­
particles could also be utilised as functional tools in clinical research and
investigation. Thus, developments in “pseudo pathogen-like” nano­
particles could substitute for highly virulent pathogens and provide
simpler and safer ways of assessing complement responses. A functional
example is nanoparticles bearing ~70 copies of the recombinant
receptor-binding domain of SARS-CoV-2, which were employed to
monitor complement responses and C3 opsonisation in sera of vacci­
nated, convalescent and naïve individuals [94]. The results demon­
strated the heterogeneity of C3 opsonisation and leukocyte uptake,
which might predict individual responses to clear virions and modulate
the severity of the COVID-19 [94].
CRediT authorship contribution statement
Hajira B. Haroon: Formal analysis, Writing – original draft, Writing
– review & editing. Elisha Dhillon: Writing – original draft. Z. Shadi
H.B. Haroon et al.
Farhangrazi: Formal analysis, Writing – review & editing. Panagiotis
N. Trohopoulos: Formal analysis, Writing – review & editing, Funding
acquisition. Dmitri Simberg: Formal analysis, Writing – review &
editing, Funding acquisition. S. Moein Moghimi: Conceptualization,
Formal analysis, Writing – original draft, Writing – review & editing,
Funding acquisition.
Declaration of Competing Interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
[P.N.T. declares financial interests in CosmoPHOS Ltd (Greece). Z.S.F. &
S.M.M. declare financial interests in S. M. Discovery Inc. and S. M.
Discovery Ltd. H.B.H., E.D. & D.S. declare that they have no competing
financial interests or personal relationships that could have appeared to
influence the work reported in this paper.].
Data availability
No data was used for the research described in the article.
Acknowledgements
S.M.M. acknowledges support by the European Union’s Horizon
2020 programme funded under H2020-EU.1.3. – Excellent Science –
Marie Skłodowska-Curie Actions, grant agreement ID. 956544 (DIR­
NANO: Directing the Immune Response through Designed Nano­
materials). Z.S.F. (S. M. Discovery Group Ltd.) is a partner organisation
in DIRNANO consortium. H.B.H. is an Early Stage Researcher financially
supported by the DIRNANO programme. E.D. is a recipient of a research
scholarship from Newcastle University funded by George Brown
Endowment, George Henderson Endowment and Florence Kirkby
Endowment. P.N.T. & S.M.M. acknowledge support by the European
Union’s Seventh Framework Programme (FP7-NMP-2012-Large-6)
under the grant agreement no. 310337 (CosmoPHOS-nano Large-Scale
Project). D.S. acknowledges support by the National Institute of
Health grants R01AI154959 and R01 CA257958.
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