Pathogen‐binding nanoparticles to inhibit host cell infection by heparan sulfate and sialic acid dependent viruses and protozoan parasites

Abstract Global health faces an immense burden from infectious diseases caused by viruses and intracellular protozoan parasites such as the coronavirus disease (COVID‐19) and malaria, respectively. These pathogens propagate through the infection of human host cells. The first stage of this host cell infection mechanism is cell attachment, which typically involves interactions between the infectious agent and surface components on the host cell membranes, specifically heparan sulfate (HS) and/or sialic acid (SA). Hence, nanoparticles (NPs) which contain or mimic HS/SA that can directly bind to the pathogen surface and inhibit cell infection are emerging as potential candidates for an alternative anti‐infection therapeutic strategy. These NPs can be prepared from metals, soft matter (lipid, polymer, and dendrimer), DNA, and carbon‐based materials among others and can be designed to include aspects of multivalency, broad‐spectrum activity, biocidal mechanisms, and multifunctionality. This review provides an overview of such anti‐pathogen nanomedicines beyond drug delivery. Nanoscale inhibitors acting against viruses and obligate intracellular protozoan parasites are discussed. In the future, the availability of broadly applicable nanotherapeutics would allow early tackling of existing and upcoming viral diseases. Invasion inhibitory NPs could also provide urgently needed effective treatments for protozoan parasitic infections.


| INTRODUCTION
Infectious diseases represent a major burden on global health.Among these, viruses and intracellular protozoan parasites are two types of pathogens responsible for devastating epidemics and pandemics.The COVID-19 pandemic has highlighted that an emerging viral pathogen can rapidly become a huge threat to the global population, especially in the absence of broad-spectrum antivirals.Indeed, for most viral infections there are no drugs available, which hinders effective control of current and future viral diseases.The development of broadspectrum applicable treatments would be particularly beneficial, as developing specific drugs for each viral disease would be very time-consuming and costly.Broadly active antiviral medicines could help to (i) curb current viral infections, (ii) allow rapid response to infections with mutated strains that evade more specific strategies, such as vaccine-induced antibodies, and (iii) ensure that upcoming diseases caused by new viruses can be counteracted quickly.With respect to (ii, iii), antiviral therapeutics could bridge the time needed for developing, testing, approving, manufacturing, and rolling out efficacious vaccines.
In contrast to viral diseases, protozoan parasitic diseases are caused by unicellular eukaryotic pathogens.Although this diverse group of pathogens also includes extracellular organisms (e.g., Giardia lamblia, Trypanosoma brucei, and Entamoeba histolytica), this review only focuses on parasites that require human host cells for propagation.These obligate intracellular protozoans include vector-borne parasites (Plasmodium spp., Trypanosoma cruzi, and Leishmania spp.) and parasites distributed through the fecal-oral route (Toxoplasma gondii and Cryptosporidium spp.).Malaria caused by Plasmodium spp. is the most impactful protozoan disease.These parasites infect >200 million and kill >600,000 people every year, with children and pregnant women most severely affected. 1Malaria elimination programs are off track, with cases even increasing recently. 1,2This highlights the need for innovation and development of alternative malaria control measures.Infection with T. gondii, sometimes referred to as malaria's neglected cousin, can lead to congenital toxoplasmosis, severe toxoplasmosis in immunocompromised people, and neuropsychiatric disorders. 3Leishmaniasis (Leishmania spp.) and Chagas disease (T.5][6] Finally, Cryptosporidium spp.were recently found to be a major causative agent of diarrheal disease in children, registering >44.8 million episodes of diarrhea and 48,000 deaths annually. 7,8For most protozoan parasite infections, no vaccines and only few, often ineffective treatments are currently available.The high prevalence and severity of infections caused by these different intracellular protozoan pathogens illustrates the urgent necessity to increase investment, develop new approaches, and conduct translational research to eventually reduce the high impact of these devastating diseases on global populations. All human viruses and the obligate intracellular parasites discussed in this review utilize host cells for propagation.For viruses, the main entry pathways encompass ligand-receptor interactions to allow subsequent fusion with the plasma and/or endolysosomal membrane.The mechanisms by which protozoan parasites enter host cells are extremely diverse and complex, multistep processes.They can mainly be categorized into parasite-dominated invasion (Plasmodium spp., T. gondii, Cryptosporidium spp., and T. cruzi) and host cell-mediated entry via phagocytosis (Leishmania spp.). 9Similar to the method used by viruses, host cell infection by parasites is initiated by pathogen ligand binding to host cell membrane receptors.Many specific host cell receptors have been identified for these different pathogens.However, in most cases, the pathogens only engage with these specific entry/ invasion receptors after initiating binding via less specific, near universal attachment moieties.These shared initial attachment receptors are of high interest when developing broad-spectrum anti-infectious strategies targeting pathogen entry.Alternatively, pathogen-tailored approaches can be designed by employing the more specific receptors/ mimics.However, this will only achieve functionality against a single pathogen or pathogen type, which is not the focus of this review.
Glycans on host cell membranes represent these less specific universal attachment receptor moieties.1][12][13] Highly anionic host heparan sulfate (HS) is one of these near universal interaction partners for pathogen ligands.Most viruses mentioned herein utilize HS interaction for host cell infection, 11,[14][15][16] although in some cases it is still debated whether HS should be considered

Key points
� Nanoparticles (NPs) that inhibit heparan sulfate (HS) and sialic acid (SA) dependent viruses and protozoan parasites through pathogen surface-binding are discussed.� For viral diseases, nanotechnological strategies with high inhibitory potency and virucidal mechanisms of action are highlighted.� For protozoan parasites, invasion inhibition with soluble heparin and the few available NPbased formulations are summarized to inspire more research into HS/SA-mimetic NPs against these pathogens.
an actual receptor or not. 17HS on host cells is also involved in the invasion mechanism of all the five obligate intracellular protozoan parasites discussed herein. 11,18A second broad-spectrum example of a receptor moiety is sialic acid (SA).SA is part of many glycoproteins on host cell membranes and can mitigate viral and parasite entry. 10,13,19Consequentially, many anti-infectious strategies targeting viral attachment/entry and parasite invasion have leveraged these HS/SA interactions.Nanotechnology is gaining traction as a tool to develop alternative strategies for infectious diseases important in global health. 20,21The highly successful lipid nanoparticle (LNP)-based RNA vaccines for COVID-19 have recently cemented the standing of nanotechnology as a promising and versatile engineering discipline for establishing infection prophylaxis.However, the potential of nanotechnology goes well beyond vaccines.Targeted delivery of antiviral agents [22][23][24] and nanomedicines with intrinsic antiviral therapeutic properties, as discussed herein, are two other intervention strategies that could expand the viral control arsenal.6][27] In contrast, the field of protozoan parasite invasion inhibitors based on NPs is still in its infancy.9][30][31][32][33][34][35][36] However, the concept of invasion inhibition with NPs could provide important alternative therapeutic and/or prophylactic opportunities for protozoan parasites.The distinctively different mechanism of action of these NPs to current anti-parasitic molecular drugs could represent a key advantage, especially in the context where drug-resistant strains have developed and spread.
This review summarizes recent advances in the development of broad-spectrum applicable viral attachment/entry and protozoan parasite invasion inhibitors based on NPs (Figure 1).First, antiviral strategies will be presented, splitting the information according to the NP type, from metal, soft matter, to other/non-spherical NPs.Next, parasite invasion inhibitory NPs will be discussed in the context of malaria, as most NP research against protozoans up to now has focused on inhibiting Plasmodium spp.Nevertheless, the potential of NP strategies aimed at the extracellular forms of other parasites, such as T. gondii, Cryptosporidium spp., T. cruzi, and Leishmania spp., will then be discussed.Finally, a concluding section will put the recent findings into perspective to inspire more research in this direction.

VIRAL INHIBITORS
In the past, virus inhibitory NPs were mainly used as a research tool to identify new viral attachment and entry receptors. 37However, the overall rise of nanomedicine as a promising approach for various diseases has motivated the development of therapeutic antiviral NPs.The COVID-19 pandemic, together with the high success of RNA-loaded LNP coronavirus vaccines, provided an additional boost for the creation of therapeutic NPs for F I G U R E 1 Schematic of virus and parasite entry/invasion inhibitory NPs.Nanomedicines are designed to mimic host HS and/or SA to inhibit pathogen attachment, entry, or invasion of host cells via direct pathogen surface binding.Possible ligand configurations on variously shaped NP types include short or long linkers, dendritic or polymeric groups in end-on or side-on attachment, all for the multivalent presentation of anionic sulfates/sulfonates or SA.Schematic created with Servier Medical Art website CC-BY.DENV, Dengue virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; HS, heparan sulfate; HSV, herpes simplex virus; IAV, influenza A virus; NPs, nanoparticles; RSV, respiratory syncytial virus; SA, sialic acid; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2. NAJER viral diseases.Here, some key examples and most recent works are highlighted rather than providing an exhaustive list of all the available nanostructures for viral entry inhibition.6][27] Additional information on the history of polysaccharides and polymers for the same application, which set the basis for the NP developments, can also be found elsewhere. 25,38ost broad-spectrum applicable antiviral NPs have been designed by leveraging the HS [39][40][41][42][43] and SA 19,44 interactions of viruses.This necessitates either using HS/ heparin or SA as building blocks of NPs or mimicking these chemical entities by including sulfonates/sulfates or carboxylic acids in the NP design.Various possible ligand configurations have been trialed on different NP types and they include short or long linkers, dendritic or polymeric groups in end-on or side-on attachment, all with the aim of achieving multivalent presentation of anionic sulfates/sulfonates or SA (Figure 1).A key advantage of such anionic broad-spectrum inhibitors is their potential to act against many different viruses.This design also provides higher robustness with respect to virus mutation.In contrast, more specific inhibitors, such as natural or vaccine-induced neutralizing antibodies (NAbs), are prone to viral escape via mutation. 45However, the use of anionic inhibitors has historically been challenging in vivo, as found when aiming to translate polyanionic human immunodeficiency virus (HIV) inhibitors. 46Drawbacks of such inhibitors typically included low potency, reversibility of the interaction, and potential side effects (e.g., unwanted anticoagulation activity).These challenges required a re-think of anionic NP designs.Recent advances in NP technology, as discussed in the next subsections, increased potency, included irreversible virucidal mechanisms and lowered the chance of side effects.These achievements have revitalized the research area of anionic NP-based viral entry inhibitors (summarized in Table 1).Recent examples of HS/SA mimetic NP antiviral designs are discussed next by organizing them based on their core material.

| Metal NP-based viral inhibitors
5][66][67] Here, selected highlights that function by direct virus binding are summarized.These examples are then compared to antiviral NPs made from other materials.In early studies, the multivalency of HS-inspired metal NPs was already identified as the key factor for virus entry inhibition.This is exemplified by studies from Baram-Pinto et al. who demonstrated that soluble monovalent mercaptoethanesulfonate (MES) did not affect herpes simplex virus type 1 (HSV-1) host cell entry, while multivalent MES-coated silver and gold NPs (AgNPs and AuNPs) reduced infectivity. 39,40This same effect was found when testing sulfated AuNPs against human immunodeficiency virus 1 (HIV-1). 41Analogously, changing to multivalent SA-modified AuNPs delivered inhibitors for SA-dependent viruses such as influenza A virus (IAV). 68nother early exploration was around optimizing the NP size for inhibition.Many of the viruses discussed herein are in the size range of around 100 nm in diameter.This length scale is comparable to nanomedicines, highlighting the importance of inhibitor size.Vonnemann et al. revealed that polyvalent virus-sized AuNPs were the most efficient attachment inhibitors compared to smaller or bigger NPs when tested against a model pathogen, vesicular stomatitis virus. 42The virus-sized NPs provided increased activity due to better cross-linking of several virions versus smaller NPs.However, when considering the NP size, the binding strength has to be included in these considerations. 69In addition, the optimal inhibitor size might be virus specific, while other mechanisms of inhibition, for example, damaging the virus irreversibly as discussed below, could require a different optimal NP size.
More recently, Cagno et al. have made a significant contribution to the field of antivirals by incorporating a virucidal mechanism into HS-mimetic AuNPs. 43This was achieved by including long hydrophobic linkers between the Au core and the sulfonate functional groups (Figure 2A).These inhibitors were tested under in vitro conditions that better mimic the in vivo situation where the inhibitor will eventually be diluted over time.They found a potent virucidal effect for their mercaptoundecanesulfonic acid/octanethiol (MUS/MUS:OT)-AuNPs against various viruses, including herpes simplex virus type 2 (HSV-2), human papillomavirus, respiratory syncytial virus (RSV), and Dengue virus (DENV).The activity was confirmed in a mouse RSV model.In contrast, the MES-AuNPs introduced above contained much shorter linkers and provided only virustatic activity.The virucidal mechanism for MUS/MUS:OT-AuNPs was characterized by electron microscopy and revealed virus binding and irreversible viral deformation.This functionality was attributed to the strong multivalent interactions of MUS/MUS:OT-AuNPs with the virus, mediated through the sulfonate groups together with the long hydrophobic linkers.Changing the sulfonated MUS component on these virucidal AuNPs to a multisulfonated complex ligand based on glucose yielded slightly improved virucidal activity against DENV. 49ntriguingly, when exploring the activity of MUS:OT-AuNPs against other viruses that do not use HS interaction for host cell attachment (e.g., IAV), they were still  2B). 47However, Cagno et al. revealed recently that MUS:OT-AuNPs only provided virustatic and not virucidal activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, Figure 2C), the causative agent of the COVID-19 pandemic. 48Although HS was previously identified as an attachment receptor for SARS-CoV-2, 16 others have found no inhibition with heparin 48 or only at very high mg/mL concentration. 55his could point toward an explanation for the as of yet unexplained absence of a virucidal activity of MUS:OT-AuNPs against SARS-CoV-2.More research is required to better understand SARS-CoV-2 inhibition with sulfonated/sulfated compounds and to turn these NPs virucidal against this specific virus.
Protein corona formation on surface-active NPs, same as for all other nanomedicines, 70,71 is one underexplored but highly important aspect toward future clinical translation.One attempt to address this challenge modulated virucidal MUS-AuNPs from anionic to mixed charge to increase their potency in high protein environments. 72ince the virucidal activity of the above MUS/MUS:OT-AuNPs still required relatively high concentrations of inhibitor, also when tested in the virustatic assays, 43 it is important to identify more potent structures.Many groups have been evaluating other types of modifications on AuNPs to achieve higher virustatic activity.Avoiding the hydrophobic linkers could potentially be associated with lower toxicity. 50Conversely, incorporating these long-chain linkers might turn more potent virustatic NPs virucidal.Groß et al. have recently explored the formulation of polystyrene sulfonate (PSS)-coated AuNPs against various viruses (Figure 2D). 50These PSS-AuNPs revealed virustatic activity against many viruses (except IAV that does not rely on HS), while SARS-CoV-2 was again the most challenging to inhibit.Despite these challenges, the activity was confirmed in mouse models of RSV and SARS-CoV-2 infection, respectively.
5][66][67] However, it is often difficult to distinguish between different inhibition mechanisms.For example, direct NP action on the virus or indirect NP activity mediated via induction of host cell changes cannot always be decoupled.A recent study that highlights the issue of interconnected multifunctionality used Au nanoclusters with terminal ammonium groups (TMA-GNCs). 73The inhibition mechanism was found to be virucidal but functioning through multi-fold mechanisms by concurrently attacking and destroying the virus itself, reducing coronavirus protease 3CL pro activity, and activating antiviral immune responses.Similarly, other metal-based NP types can have some effect on viral attachment/entry, but it is often combined with other effects on host cells.A common phenomenon is strong stimulation of the interferon pathway by metal NPs in the host cells. 64,74This can result in an indirect antiviral effect as exemplified by studies developing antiviral graphene oxide-AgNP composites and glutathione-capped silver sulfide nanoclusters. 75,76While this might be of benefit to reduce viral load, it might also cause unwanted side effects.Overstimulation of the host immune system could have a detrimental effect and must be evaluated very carefully. 64Although metal-based NPs have shown potential as viral inhibitors, persistent concerns over potential toxic side effects caused by some of these NPs are reasons for caution. 77This is also why more biocompatible and biodegradable systems, such as soft matter-based NPs, are being sought after.

| Soft matter-based nanoscale viral inhibitors
Soft matter-based NPs, made from lipidic, polymeric, and dendritic components, have unique potential for antiinfectious applications.High biocompatibility and the potential to include biodegradable components for many of these NPs is a key advantage over other NP types.Their often characteristic softness also allows NP deformation upon virus binding.This deformation can increase the number of interaction points between NP and virus, which enhances binding strength, and ultimately improves the inhibition potential. 53Arguably the most researched soft matter-based NPs are liposomes, which are vesicles composed of a lipidic membrane and an aqueous core.For example, heparin octasaccharidemodified liposomes successfully inhibited viral attachment and host cell infection by RSV and HSV-1. 51Using only heparin fragments (octasaccharide) ensured the removal of the unwanted anticoagulation activity characteristic for full-length heparin.Instead of oligomeric heparin fragments on liposomes, sulfated liposomes were created by incorporating cholesteryl sodium sulfate, 52 which achieved SARS-CoV-2 inhibition and inactivation, partially through fusion with virions, although much higher liposome concentrations were necessary compared to heparin octasaccharide-modified liposomes (Table 1).Other glycan-modified liposomes with pendant SA groups were also developed to function against IAV, with activity confirmed in mice with lethal IAV infection. 78,79Liposomes that mimic natural membranes have the advantage of allowing lateral mobility of the inhibitory receptor/mimic.This configuration is more biomimetic and can be one explanation for the high potency of the heparin octasaccharide-modified liposomes versus other NPs (Table 1). 51High biocompatibility of liposomes due to their membrane-mimetic design using natural lipids is another key advantage over other NP systems.As one drawback, liposomes are often associated with limited physicochemical stability, which needs to be considered when moving toward the translation.Especially, taking into account the various local environments encountered by NPs depending on the chosen administration route is important.Using heparin as a NP building block was also explored in the development of an inhalable solution of polyplexes based on chitosan-heparin mixtures, which achieved activity in a mouse model with SARS-CoV-2 infection. 80However, the use of fulllength heparin in the construction of these NPs has to be evaluated very carefully with respect to potential downstream anticoagulation side effects once the NPs start to disassemble.
Most other soft matter-based NP systems for viral inhibition are based on heparin-mimetic structures rather than using heparin.Alternatively, SA units are incorporated for SA-dependent viruses.Nanogels (NGs) with sulfate end groups revealed the benefit of using more flexible versus rigid viral binding structures.HSV-1 was inhibited more efficiently when the NGs were more flexible (Figure 3A). 53When modifying the NGs with SA instead, flattening of the flexible NGs when binding to IAV virions was confirmed by electron microscopy (Figure 3B). 60Again, an inhibitor size similar to the virus was ideal for inhibition, while the ligand density needed to be optimized carefully. 81Utilizing a dendritic system, Kwon et al. showed precisely how the inter-ligand spacing is an important factor determining the activity of SA-NPs against IAV. 61Their lead dendrimer formulation (S3-G4, 3.1 nm ligand spacing) was the most potent and applicability was confirmed in a lethal influenza mouse model.The necessity of choosing the correct type of SA on NPs was realized through findings that avian and human IAV were inhibited better with dendrimers containing 3 0 -sialyllactose (3SL) or 6 0 -sialyllactose (6SL), respectively. 82n attempts to incorporate a virucidal inhibition mechanism into more readily translatable, soft backbone structures, cyclodextrins were evaluated. 83,84Again, long hydrophobic linkers with sulfonate (MUS) or SA end groups were incorporated.Virucidal action was achieved for these modified cyclodextrins against a whole range of viruses (HSV-1/2, RSV, HIV-1, DENV2, Zika virus, and hepatitis C virus); however, the overall potency was lower than for the AuNP core structures. 43In the search for higher potency soft matter-based virucidal structures, the MUS modification was recently transferred to a dendritic system. 54This design created the highest potency virucidal HS-mimetic NPs to date (Table 1, Figure 3C).However, whether the in vitro data directly translate to an in vivo situation has not yet been demonstrated for this specific structure.Switching to micellar polymeric NP systems for viral inhibition, Najer et al. have recently demonstrated that modulating the particle surface chemistry from carboxylates to a mixture with sulfonates can produce highly potent virustatic inhibitors of HSV-2 (Figure 3D). 55The IC 50 values were in the femtomolar region (pg/mL) for the lead NP (aminomethanesulfonic acid [AMSA]-modified poly(D,L-lactide)-block-poly (acrylic acid) (PDLLA-b-PAA) copolymer micelles).The reasons for this high inhibitory potential against HSV-2 are yet unknown.Evaluation of binding affinities, multivalency, and potential activation of host responses, as described in the metal NP part, are potential future works to establish the mechanism.As one caveat, when testing the same inhibitors against SARS-CoV-2 they were much less potent. 55The difficulty of obtaining inhibition with heparin in these latter assays (required mg/mL) represents one possible explanation for this discrepancy.
Besides broad-spectrum applicable soft matter-based NPs, there is also a lot of interest and research ongoing in developing NP versions that include specific receptors.For more details on these inhibitors, which were mostly excluded from this review, the reader is referred to cited papers below and a recent review specific to NPs including ACE2 engineering for SARS-CoV-2. 85Recent specific examples include peptide-modified NPs (IAV), 86 NPs based on molecularly imprinted polymers (MIPs against HIV-1, SARS-CoV-2), 87 receptor presenting viruslike NPs (HIV-1), 88 liposomes (SARS-CoV-2), 89 cellmembrane vesicles (SARS-CoV-2, [90][91][92][93][94][95] hepatitis B virus, 96 HIV-1, 95 IAV, 97,98 HSV-1,2 and pseudorabies virus 99 ), and extracellular vesicles (SARS-CoV-2). 100This field has progressed up to successful trials in non-human primates using the cell-membrane-based nanodecoys presenting ACE2 against SARS-CoV-2. 92,94Most of the NP types discussed up to now were based on a spherical morphology.Recent progress in the design of 2D materials and more complex 3D architectures has given rise to other classes of nanomaterials being investigated for viral attachment/entry inhibition.

| Other nanomaterials for virus inhibition
Employing natural base components, such as bacteriophages, generally produces more morphologically defined structures compared to most human engineered NPs.Monodisperse phage capsids functionalized with SA groups enabled studies on the impact of precise ligand spacing on NPs for IAV inhibition. 62,63Icosahedral bacteriophages Qβ, with SA spacing optimized to fit the distance between individual binding sites on the hemagglutinin trimer (~4.7 nm), potently blocked IAV in vitro, ex vivo, and in vivo. 62Others showed that sialyllactose-conjugated filamentous bacteriophages (~1 μm long) can wrap around IAV to yield high inhibition potential, with the lowest IC 50 at 14 pM. 63nstead of binding NPs to the surface of viruses to protect host cells from infection, so-called virus-traps were established.These traps are defined as nanoscale constructs with cavities to bind the virus inside their pockets.DNA nanotechnology is particularly suited for such designs as it allows precise engineering of 2D-and 3D-architectures.This advantage has recently been leveraged to develop anti-pathogenic strategies, including against viral entry. 101It was shown that incorporating viral target-specific peptides 102 or aptamers [103][104][105] into these DNA nanoconstructs produces specific inhibitors.One broad-spectrum option employing very defined viral traps was designed by Monferrer et al. using DNA origami technology (Figure 4A). 106,107Heparin or HS was immobilized on the inside of these DNA origami shells to encage various types of viruses.The mode of action was proposed to be the same as for other NP-based inhibitors, subsequently preventing interaction of the virus with host cell membranes.Keeping the virus within the trap was proposed to passivate the virus surface better than soluble inhibitors (e.g., free HS).However, efficacy data on reducing virus infectivity, comparing this system to soluble inhibitors and other NP types, has not yet been presented.
Carbon structures such as graphene, carbon dots, nanotubes, and fullerenes have also been explored as antivirals. 108,109Some of these structures have intrinsic antiviral activity or they were modified with non-HS/SA mimetic moieties, such as mannose for Ebola virus inhibition, 64 which are not further discussed herein.In turn, broad-spectrum applicable HS-mimetic sulfonated/ sulfated graphene inhibited host cell attachment of HSV-1, pseudorabies, and African swine fever virus. 110,111ydrophobic chains (≥10 carbons in length) were again included and combined with sulfated dendritic groups on flexible 2D nanomaterials such as graphene (Figure 4B). 57,112This yielded potent activity and a virucidal mechanism of action against HSV-1 and SARS-CoV-2.However, the increased hydrophobicity was also associated with higher toxicity in host cells.Due to the high inhibitory potential with IC 50 in the fM range when tested against HSV-1, application at low concentrations below the toxicity limit could be explored. 112The inhibitory potential is much higher than for most other NP types and similar to the highly potent virustatic AMSAmodified polymer micelles functioning against HSV-2. 55ence, further investigations into mechanism are warranted to push these highly potent inhibitors closer to clinical translation.The same 2D graphene platform was also effective against IAV by substituting the sulfates with SA moieties. 113Another two examples highlight the benefit of combining the hydrophobicity of carbon materials with sulfates for viral inhibition.Firstly, the intrinsic hydrophobicity of fullerene (buckyballs) was combined with anionic charge (polyglycerol sulfates) to block SARS-CoV-2. 58Secondly, a carbonized NG with sulfate surface functionalities (Alg@AS 5.0 ) was active against IAV, including in an in vivo influenza model. 59I G U R E 4 Non-spherical nanodesigns for virus inhibition.(A) Schematic of heparin/HS modified T1 DNA origami shells and corresponding electron micrographs showing successful caging of various virus types (Scale bar, 100 nm).Modified and reprinted under terms of the CC-BY license. 106Copyright 2022, The Authors, published by American Chemical Society.(B) 2D graphene nanosheets with sulfated dendritic groups and hydrophobic chains (≥10 carbons in length) achieved potent SARS-CoV-2 inhibition with a virucidal mechanism (bottom images show plaque assay).***p < 0.001.Modified and reprinted under terms of the CC-BY license. 57Copyright 2021, The Authors, published by John Wiley and Sons.HS, heparan sulfate.NAJER Again, the activity of this sulfated carbonized NG against IAV that does not rely on HS for host cell attachment might broaden the applicability of this material, as it was the case for the MUS:OT AuNPs. 47ther 2D nanocomposites were made from sulfonated transition metal carbides (Ti 3 C 2 -Au-MPS) with activity demonstrated against SARS-CoV-2 pseudovirus but only at relatively high concentrations (lowest 50 μg/ mL). 114A recent report on electronegative 2D CuInP 2 S 6 (CIPS) nanosheets revealed strong antiviral activity against SARS-CoV-2 (EC 50 at 11 pM), which was retained in vivo. 115The high activity was attributed to the strong affinity of the nanosheets to the receptor binding domain of the spike protein (K D < 1 pM).Another NP type with non-spherical shape, hexagonal nanoassemblies made from O-palmitoyl-heparin and αcyclodextrin (OPH1-Hep4) efficiently inhibited a range of HS-dependent viruses, with a high degree of sulfation required to achieve high efficiency. 56Overall, nonspherical shapes that match the target virion or flexible 2D-or 3D-inhibitory structures that can wrap around the virions are upcoming materials with high potential.However, direct comparison to other inhibitory materials is still sparce.Hence, a definitive conclusion on whether they are advantageous over spherical inhibitors remains to be drawn.The focus of this review is now shifted away from viruses to protozoan parasites that utilize the same HS/SA interactions for interaction with host cells.Theoretically, the same NPs described in the preceding sections can be applied against parasites.However, as discussed subsequently, differences in parasite versus virus biology and variations in entry mechanisms requires slightly different anti-parasitic designs for optimal activity.

| NPs TO INHIBIT PROTOZOAN PARASITE INVASION OF HOST CELLS
9][30][31][32][33][34][35][36] Herein, these examples are not discussed in detail.Instead, the focus of this review is on broadspectrum NPs that function as invasion inhibitors by occupying parasite surface ligands.Plasmodium spp., the unicellular organism causing malaria, represents the most impactful protozoan parasite in global health.Hence, most of the nanotherapeutic anti-invasion strategies have been aimed at this parasite to date.A summary of these approaches is given herein.Future work is then proposed to tackle other protozoan parasites with NPs employing the same mechanism of action.

| Malaria parasite invasion inhibition with NPs
Already several decades ago, liposomes with specific host receptors were evaluated as invasion inhibitors of Plasmodium merozoites, with a focus on identifying parasite receptors rather than designing therapeutics, as seen in the virus field. 116,117As the production of NPs, including liposomes, has become much more economical throughout the last decades, the interest in developing NP-based invasion inhibitors has gained more interest.Particularly, designs with low-cost, broad-spectrum applicable receptors, such as HS, or simple receptor mimics are prime candidates to develop anti-parasitic NPs.Encouragingly, red blood cell (RBC)-infecting Plasmodium spp.merozoites use HS interaction to invade RBCs and the process can be inhibited with soluble heparin. 1189][120][121] However, only a few NP types with heparin or heparin-mimetic surfaces have so far been developed and tested for an invasion-inhibitory application (Table 2).
Marques et al. have evaluated side-on attached heparin on liposomes, either through electrostatic 129 or covalent attachment (Figure 5A). 123The latter was shown to reduce the unwanted anticoagulation property of heparin, which is a benefit over soluble heparin and highlights an advantage of the covalent method. 122owever, empty heparin-liposomes with side-on attached heparin were not more efficient invasion inhibitors than soluble heparin and blood circulation time was relatively short.Thus, the authors shifted their focus to drug delivery, using a combined action of heparin-liposomes loaded with a conventional antimalarial drug.Electrostatically conjugating heparin in the side-on configuration on cationic chitosan NPs or dendrimers also caused these NPs to show invasion inhibitory activity against Plasmodium falciparum, although again no improved activity was found when compared to soluble heparin. 122,124To build membranous NPs with higher physicochemical stability and a more biomimetic presentation of heparin, polymer-based vesicles (polymersomes) were employed. 125A block copolymer was synthesized with end-on attached heparin serving as the hydrophilic block, which was subsequently mixed with a vesicle-forming copolymer.These NPs were termed nanomimics due to their nanoscale host cell membrane mimetic structure.Nanomimics were over 100-fold more efficient than soluble heparin in inhibiting P. falciparum merozoite invasion of RBCs.
The mechanism of action of the nanomimic system against merozoites was subsequently analyzed in detail.Giant, micron-scale polymersomes built with the same 10 of 20 nanomimic materials revealed that the end-on attached heparin successfully bound fluorescently labeled P. falciparum (clone 3D7) major surface protein 1-42 (PfMSP1 42 , Figure 5B). 130This parasite ligand was previously identified to be responsible for the heparin interaction. 118However, there are many additional heparin-binding ligands present on merozoites, [131][132][133] providing a multitude of binding-partners for these NPs.Due to the high prevalence of PfMSP1 on the merozoite surface, this ligand remains the most likely binding partner.Indeed, strong interaction of PfMSP1 42 with the nanoscale version (nanomimics) was revealed by fluorescence cross-correlation spectroscopy (K D ~12 nM, Figure 5B). 126The invasion inhibition mechanism was confirmed by fluorescence and electron microscopy that visualized NP accumulation on the surface of egressed merozoites (Figure 5C).Interestingly, the soft nanomimics slightly adapted their structure to the merozoite surface by flattening out, which was likely caused by the strong multivalent interactions with PfMSP1 42 .This provides evidence that NPs that can adapt to the pathogen surface are more potent parasite inhibitors.This is Inhibitory polymer conjugation and orientation.e Plasmodium knowlesi strain A1-H.1 adapted to human RBCs. 128JER in agreement with the viral inhibition studies that showed better performance of soft versus rigid NPs. 53,60owever, in vivo parasite invasion inhibition has not yet been demonstrated with any of the described heparinized vesicle structures.
Inorganic hollow mesoporous ferrite NPs were coated with heparin (HMFN@Hep) to serve as an alternative NP system for application against malaria (Figure 6A). 127erozoite invasion inhibition with HMFN@Hep was more efficacious than soluble heparin, again likely due to multivalent interactions of NPs with the merozoites.However, the side-on configuration of heparin on HMFN@Hep yielded less efficient NPs 127 versus the endon heparinized nanomimics (Table 2). 125Since these HMFN@Hep were also found to interact with late-stage infected RBCs, the authors combined the heparin invasion inhibitory effect with drug release of a conventional antimalarial (artemisinin) from the NP.This combination achieved a higher activity of HMFN@ART@Hep versus HMFN@ART.It was speculated that increasing the local drug concentration improved antimalarial efficacy, although this has not yet been demonstrated in vivo.In a similar application, artesunate was directly coupled to heparin to form NPs without another carrier material, which allowed for controlled drug release and slightly improved drug circulation time. 134ll these previous anti-parasitic nanoscale inhibitors included heparin itself as a building block, which was often associated with the unsolved challenges of low efficacy, danger of potential unwanted anticoagulation activity (e.g., after NP disassembly), short blood circulation half-lives, and applicability in vivo has not yet been tested. 123To tackle these challenges, Najer et al. have recently designed HS/SA-mimetic polymer and lipidpolymer NPs that were identified as a viable alternative to heparinized NPs. 55These NPs were designed with pendant carboxylic/sulfonic acid containing polymers to achieve potent merozoite invasion inhibition (Table 2, Figure 6B).The absence of heparin in the designs reduced the potential for unwanted anticoagulation activity.This was confirmed by antifactor Xa activity tests revealing a negligible activity for these synthetic polymeric NPs (<1% vs. heparin).Various versions of the NPs (carboxylates/ sulfonates) potently inhibited P. falciparum strains with SA-dependent (W2mef) and SA-independent (3D7, D10) invasion mechanisms and even a different species, P. knowlesi (A1-H.1)cultured in human RBCs.Stochastic optical reconstruction microscopy images revealed binding of the NPs to the PfMSP1 outer layer on merozoites (Figure 6B).Interestingly, inhibition of strain W2mef was most efficient, as these parasites require both HS and SA to enter host RBCs.The best polymeric NP inhibitors for parasites included methoxybenzenesulfonate (AMBS) moieties, which were less efficient against viral entry (HSV-2, Figure 3D).The virucidal AuNPs (MUS/OT) 43 tested against P. falciparum parasites showed much lower F I G U R E 5 Plasmodium merozoite invasion inhibition with heparin-nanovesicles. (A) Heparin-coated liposomes (side-on configuration) inhibited merozoite invasion but showed relatively short circulation times.Modified and reprinted under terms of the CC-BY license. 123Copyright 2020, The Authors, published by MDPI.Schematic modified from Servier Medical Art website CC-BY.(B) Giant heparin-polymersomes (ca.15 μm in diameter, red membrane stain) bound fluorescent Plasmodium falciparum parasite ligand PfMSP1 42 -OG488 (green).Modified and reprinted with permission. 130Copyright 2016, Swiss Chemical Society.Nanoscale versions of the same polymersomes revealed high binding strength for this interaction (K D ~12 nM) when analyzed by FCCS.Modified and reprinted with permission. 126Copyright 2015, John Wiley and Sons.(C) Nanomimics from (B) successfully inhibited merozoite invasion and surfacebinding was visualized by fluorescence microscopy (left, one merozoite bound to one RBC) and electron microscopy (right, zoom shows a single polymersome bound on the outer merozoite membrane).Modified and reprinted with permission. 125Copyright 2014, American Chemical Society.FCCS, fluorescence cross-correlation spectroscopy; RBC, red blood cell.
12 of 20 -NAJER inhibition potential than the virustatic polymer NPs (AMSA/AMBS).Hence, virus and malaria parasite inhibition require slightly different designs for optimal activity.A biocidal mechanism might not be required for Plasmodium spp. as merozoites lose their invasive potential within a few minutes. 135,136The non-toxic and potent AMBS-modified copolymer was taken forward and coassembled with lipids, including a PEGylated lipid to yield polymer-lipid nanomimics.This produced the best inhibitor of this series.The incorporation of PEG also increased circulation time when tested in the zebrafish embryo model.Most importantly, these NPs were the first to show malaria parasite invasion inhibition in vivo (Plasmodium berghei, Figure 6B).Nevertheless, further in vivo optimization will be required to find optimal dosing and timing of injection, as well as studying subsequent immune responses.The vision for these NPs is to block merozoites in the blood stream and then deliver these complexes to immune cells.This system is desired to induce a stronger immune response to extracellular merozoites for better protection from subsequent infections.The recent finding that these NPs also inhibit and reverse sequestration, that is, binding of P. falciparum infected RBCs to endothelial cells (e.g., via chondroitin sulfate A or intercellular adhesion molecule 1 host receptors), which is a hallmark of severe malaria forms, encourages further development of this system. 137

| Invasion inhibition of other protozoan parasites with NPs
In terms of inhibiting other protozoan parasites from entering their respective host cells, only very few NP studies have been performed to date.][30][31][32][33][34][35][36] Stearylamine-liposomes were active against a range of parasites as summarized recently, but due to their cationic nature, potential interaction with host cells has to be taken into account. 138Here, some examples of surface-binding NPs and information from tests with soluble heparin are combined.These examples inform on considerations necessary for designing HS/SA containing/mimetic NPs that target protozoan parasites other than Plasmodium spp.
Invasion inhibitory NPs were previously designed against T. gondii host cell invasion but using a specific interaction (Figure 7A). 13920 nm AuNPs functionalized with an anti-T.gondii antibody were shown to bind to T. gondii tachyzoites using fluorescence and electron microscopy.Surface binding inhibited invasion successfully; however, the potency was not improved over soluble antibody.Others also investigated a more generic effect when preincubating tachyzoites with AgNPs. 142Adherence and infection was lowered, but the mechanism remains unclear.NP-mediated tachyzoite deformation and intracellular reactive oxygen species production are likely explanations, as found in other AgNP studies. 143,144oxoplasma gondii tachyzoites also bind heparin, as demonstrated with heparin-functionalized NPs, although only soluble heparin was tested as invasion inhibitor. 145 90% reduction in host cell infection was achieved at a concentration of 10 μg/mL heparin, confirming the involvement of HS in T. gondii tachyzoite host cell invasion.This encourages the development of HS-mimetic NPs for toxoplasmosis.Cryptosporidium parvum sporozoite entry into host cells was potently blocked by soluble heparin (Figure 7B). 18,146The related polysaccharide fucoidan also revealed invasion inhibitory activity in vitro. 18ncouragingly, oral administration of fucoidan in mice prior and after parasite inoculation reduced the number of parasites 5-fold. 147Hence, the theoretical basis for an anti-cryptosporidial invasion inhibitory NP leveraging the HS interaction is given.However, the administration route, ideally through oral dosing, will be a key consideration because NPs will have to act in the intestine where C. parvum replicates.
9][150] Surface interaction of NPs with invasive trypomastigotes is not yet a major avenue being explored.Cationic stearylamine-liposomes interacted with anionic extracellular T. cruzi trypomastigotes, which was leveraged for drug delivery. 148However, the short circulation time of stearylamine-liposomes hinders their application via systemic application.If, alternatively, the NPs enter the host cells after infection, the NPs need to escape the endosome to reach and act against cytosolic T. cruzi.Exiting the endosome after NP uptake is a key challenge in the entire NP-based drug delivery field.Supportive evidence that surface binding alone can reduce the host cell infection by T. cruzi trypomastigotes was provided when testing antibodies. 151Trypanosoma cruzi trypomastigotes and amastigotes were also found to bind heparin/HS, as preincubation with these inhibitors reduced host cell invasion (Figure 7C). 140,152This encourages HS-mimetic nanoscale inhibitor formulation for Chagas disease.
In contrast to the previous examples, a surfacebinding NP strategy might have little effect on protozoan parasites that enter host cells via host cell dominated processes such as phagocytosis.This is the case for macrophage infection with Leishmania spp.promastigotes/amastigotes. 153 Exceptions would be NPs that can directly affect promastigote/amastigote viability.Alternatively, binding drug-loaded NPs to the parasite surface could allow the release of the loaded drug locally after the phagocytosis of the NP-parasite complex.These considerations are the reason why most research studies to date have focused on macrophage-targeted activity of NPs for leishmaniasis (e.g., cytotoxic metal NPs and drug delivery). 30,31There is an ongoing debate on the influence of heparin on Leishmania spp.host cell infection. 154eishmania chagasi promastigote and Leishmania amazonensis amastigote infection of macrophages was previously reduced by heparin.141,155 However, others have shown that heparin can increase macrophage interaction with Leishmania donovani promastigotes.156 Heparin in the context of nanomedicine for Leishmania was evaluated recently as an inhalable heparin/chitosan NP for drug delivery.157 In this example, heparin increased macrophage uptake of drug-loaded NPs.Hence, it remains to be seen whether HS mimetic polymers or NPs have a therapeutic potential for leishmaniasis.

| CONCLUSIONS AND OUTLOOK
NPs which contain or mimic HS/SA have emerged as promising candidates for the development of broadspectrum attachment/entry/invasion inhibitors for various viral and protozoan infections.This concept could provide urgently needed treatment options for these devastating diseases.For NPs that act against viral attachment/entry, a key consideration is the processes that occur after NP-virus binding. 25This was a key learning point from the failure of anionic inhibitors in clinical trials against HIV. 46After administration, the NPs will inevitably be diluted over time due to diffusion, fluid flow, and uptake by immune cells.If the NP-virus interaction is based on a reversible interaction, this dilution could again liberate the infective virus, potentially rendering the inhibition strategy ineffective.This was circumvented recently by formulating HS/SA mimetic virucidal NPs.These virucidal NPs not only bind to viruses but also inactivate them by disrupting the viral structure. 25,43,49,54,57,73Compared to viruses, protozoan parasites are much more complex unicellular eukaryotic pathogens with thousands of proteins involved in their life cycles.However, the involvement of the same HS/SA attachment receptors in host cell invasion of various obligate intracellular protozoan parasites was confirmed as summarized herein. 18,118,140,141,145Recent demonstration that broad-spectrum HS/SA-mimetic NPs are also applicable as malaria parasite invasion inhibitors in vitro and in vivo (Table 2) should further encourage the development of HS/SA-mimetic NPs for various parasitic diseases.
Despite all these advances, there are several outstanding challenges that must be addressed before these NPs can be translated into medicines against viral and parasitic pathogens.The first challenge concerns both types of pathogens.Most viruses and obligate intracellular parasites enter their host cells very rapidly.Hence, the route and timing of administration will be key for any entry/invasion inhibitory NPs.This represents a key difference to conventional drug delivery via NPs, which often functions by delivering the drug to infected cells.Since intracellular pathogen development is a much longer process than entry/invasion, timing for the administration of drug delivery NPs is less important than for NPs inhibiting entry/invasion.However, targeting extracellular pathogens with NPs also comes with the advantage that the challenges characteristic to drug delivery systems, that is, the need to cross various membranes and releasing the cargo compound in the correct compartment, fall away.To deal with the shorter time window for the application of entry/invasion inhibitory NPs, administration pathways that follow the main infection or distribution routes of the pathogen are likely to be most successful to maximize the number of NPs brought together with the extracellular forms of the pathogens.For example, respiratory viruses such as IAV and SARS-CoV-2 are best tackled by administering via the respiratory tract. 19,158In contrast, inhibiting most protozoan infections appears to be more applicable through oral dosing or i.v.injection.NP administration for Cryptosporidium spp.could follow the oral route as this pathogen replicates in the intestines.Injection via the i.v.route could be appropriate for Plasmodium spp.merozoites that exclusively replicate in the bloodstream.Similarly, T. gondii tachyzoites also distribute through the blood in the acute stage, and in visceral leishmaniasis spleen and liver are heavily affected, 32 which is the same destination as for most i.v.applied NPs.However, for diseases most prevalent in lowand middle-income countries, and especially for application in rural settings, i.v.administration is not readily applicable.In these situations, oral dosing would be much more appropriate.However, formulation development to produce stable NPs that are efficiently taken up into the bloodstream after oral administration is still an area for further development.
Another challenge to the translation of these inhibitory NPs, applicable to both viruses and parasites, is the locally encountered conditions after administration, such as highly proteinaceous environments or low pH.These complex environments impact NP stability and potency in vivo.The importance of protein corona formation on nanoscale drug delivery systems has only been realized recently. 70,71This protein corona formation could particularly impact surface-active NPs as discussed herein, which must be studied in more detail.Other important properties that must always be investigated are NP biocompatibility, biodistribution, elimination and degradation.This is especially important for NP designs that were found to have inherent toxic effects on pathogens as they might also negatively affect host cells, which has been a known issue for some types of metal-based NPs, for example. 77ith respect to virus inactivation or elimination after NP binding, other strategies could be evaluated further.Indirect viral inactivation could potentially be incorporated through mimicking natural processes.For example, mechanisms of action of natural or vaccine-induced NAbs could serve as an inspiration.These specific inhibitors can either block viral entry or influence post-entry processes after NAb-virus binding. 159This binding is also reversible, but slowing down viral entry can cause irreversible virus damage through other mechanisms: (i) uptake of NAbvirion complexes by immune cells via Fc receptors leads to endocytosis and lysosomal degradation, (ii) NAbs force cell-membrane fusing viruses into the endocytic pathway, or (iii) NAbs delay viral endolysosomal membrane fusion, which increases the residence time of these viruses in this degrading compartment. 159Therefore, NP designs with a reversible binding mechanism, similar to NAbs, but with "NAb-like" mechanisms to trigger the viral damaging NAJER of 20 mechanisms described above (i-iii) could be interesting future avenues of research.A recent example used 2D nanosheets to capture SARS-CoV-2 which led to macrophage uptake and subsequent lysosomal degradation of the virus. 115Hence, NPs with high virustatic potency as highlighted throughout the review could serve as a promising base platform for the development of such approaches.However, testing these mechanisms will require more sophisticated in vitro co-culture and in vivo analysis.Although there are several hurdles, including translational challenges, left to solve before broad-spectrum antiviral NPs are brought to the market, they have clear potential, which should be explored further to eventually tackle ongoing and future viral diseases. 25,160As one advantage, the broad-spectrum design of therapeutic NPs would allow immediate testing and approval if found effective against an upcoming viral disease, which could provide an urgently needed tool to bridge the time needed for developing, testing, approving, manufacturing, and rolling out efficacious vaccines.
Moving from HS/SA-mimetic NPs to treat viral diseases to using these NPs to inhibit infection by obligate intracellular protozoan parasites brings a different set of challenges to consider.The unavailability of suitable in vitro models for the propagation of several protozoan pathogens for inhibitor testing is one obstacle for some of these diseases, for example, Cryptosporidium spp.Particularly, more realistic models employing "organoids-on-achip" technology, such as "mini-intestines" for C. parvum propagation, 161 could have a big impact on the development of novel treatments.High strain variability, antigenic diversity, antigenic variation, and surface shedding of invasion ligands are other parasite specific challenges for any inhibitor targeting the parasite surface.These parasite properties highlight the difficulty in designing inhibitors based on specific interactions, such as vaccineinduced NAbs, and partly explain the lack of efficacious vaccines for most parasites.Broad-spectrum approaches could address this issue because they can simultaneously target several different parasite ligands and functions against various parasite strains and species.The parasites' more complex structures and life cycles are also associated with some advantages for NP inhibitor design.For example, Plasmodium extracellular forms (merozoites) are extremely short-lived extracellularly (invasive only for a few minutes), 135,136 which could make a biocidal NP inhibition mechanism unnecessary.However, this requires more in vivo evaluation to confirm.
Pathogen-binding NPs could potentially also serve other purposes besides simple attachment/invasion inhibition due to their multifunctionality.NPs can be equipped with further targeting moieties and loaded with immunomodulatory molecules or molecular drugs.For example, delivering NP-pathogen complexes to immune cells could represent a strategy to potentially build up stronger immunity to protect against future infections.In addition, the incorporation of immune cell targeting ligands and/or immunomodulatory compounds in the NP design could provide means to modulate this immune response.However, such applications remain to be demonstrated in practice.If surface-binding of NPs proves to be inefficient in inhibiting some of the various protozoan parasites discussed herein, combinations with molecular drugs should be evaluated further.Drug-loaded NPs bound to the parasite surface might be dragged into the host cell during the invasion, which could increase the local drug concentration after release from the NPs.
In summary, nanomedical approaches utilizing pathogen-binding NPs against various viral and protozoan parasite infections show promise to be established as alternative therapeutic tools.This review aims to inspire more exploratory and translational research into nanotherapeutics against these impactful diseases.It is envisioned that the field of pathogen-binding NPs could eventually provide alternative opportunities for controlling various infectious diseases important in global health.

F
I G U R E 2 Antiviral gold nanoparticles.(A, B) MUS:OT AuNP schematic and virucidal activity against IAV compared to other inhibitors.*p < 0.05, **p < 0.01.Modified and reprinted with permission.47Copyright 2020, American Society for Microbiology.(C) Virucidal activity test of MUS:OT AuNPs against SARS-CoV-2, revealing no virucidal activity but only virustatic activity.Modified and reprinted under terms of the CC-BY license.48Copyright 2020, The Authors, published by MDPI.(D) Schematic of AuNP-PSS, IC 50 example curves for virustatic activity against RSV, and illustration of IC 50 for various AuNP sizes and PSS molecular weights when tested against different viruses.Modified and reprinted under terms of the CC-BY license.50Copyright 2022, The Authors, published by John Wiley and Sons.IAV, influenza A virus; PSS, polystyrene sulfonate.

F I G U R E 6
Inorganic and polymeric nanoparticles for Plasmodium merozoite inhibition.(A) Schematic and corresponding data showing the process of merozoite inhibition after egress using HMFN@Hep and a combined effect when incorporating an additional antimalarial drug (ART = artemisinin).Modified and reprinted with permission.127Copyright 2021, American Chemical Society.(B) STORM image of a single inhibited Plasmodium falciparum merozoite (PfMSP1, red; nucleus, blue) using polymeric NPs (cyan).Coinjection of late-stage iRBCs (Plasmodium berghei) with polymeric NPs and polymer-lipid NPs (PEG-0.3)revealed the functionality of these inhibitors in vivo.**p < 0.01, ***p < 0.001.Modified and reprinted under terms of the CC-BY license.55Copyright 2022, The Authors, published by American Chemical Society.NPs, nanoparticles; STORM, stochastic optical reconstruction microscopy.

F I G U R E 7
Invasion inhibition of various protozoan parasites.(A) Toxoplasma gondii tachyzoites were inhibited by surface-binding AuNPs functionalized with an anti-T.gondii antibody, as demonstrated by electron microscopy (top) and fluorescence imaging (bottom).Modified and reprinted with permission.139Copyright 2009, John Wiley and Sons.(B) Cryptosporidium parvum sporozoite host cell entry was inhibited with soluble heparin.Modified and reprinted under terms of the CC-BY license.18Copyright 2015, The Authors, published by Springer Nature.(C) Trypanosoma cruzi trypomastigotes were hindered from entering host cells by treatment with HS and heparin, while chondroitin sulfate and hyaluronic acid were inactive.Modified and reprinted with permission.140Copyright 1991, Elsevier.(D) Heparin treatment of Leishmania chagasi promastigotes reduced macrophage infection.*p < 0.05, **p < 0.01.Modified and reprinted with permission.141Copyright 2015, Elsevier.All schematics were modified from Servier Medical Art website CC-BY.

4 of 20 - NAJER T A B L E 1
Efficacy comparison of selected antiviral NPs based on HS/SA mimetic structures, including a few historic examples but mostly recent formulations where EC 50 /IC 50 values or curves were available.

NP type and reference Size [nm] a EC 50 /IC 50 (virus type) [μg/mL] b EC 50 /IC 50 (virus type) [nM] c Host cell Mode of action Animal model (virus)
Abbreviations: AMSA, aminomethanesulfonic acid; DENV, Dengue virus; HIV, human immunodeficiency virus; HPV, human papillomavirus; HS, heparan sulfate; HSV, herpes simplex virus; IAV, influenza A virus; NPs, nanoparticles; PGS, polyglycerol sulfates; PSS, polystyrene sulfonate; RSV, respiratory syncytial virus; SA, sialic acid; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; ZIKV, Zika virus.a Hydrodynamic or core diameter given.b >/~are used when no numbers were available but the values were estimated from the EC 50 /IC 50 curves.c Refers to NP concentration.d n.a.refers to "not available," because no information on the mechanism was available.e See Figure 3D for a graphical representation.NAJER functional (Figure Efficacy comparison of malaria parasite invasion inhibitory NPs based on HS.
T A B L E 2Abbreviations: AMSA, aminomethanesulfonic acid; HS, heparan sulfate; NPs, nanoparticles; RBC, red blood cell.aConcentration of inhibitory component.bHost cells for in vitro testing were human RBCs.cRefers to NP concentration.d