key: cord-0813612-7if5b1mc
authors: Figueroa, Sara Maslanka; Fleischmann, Daniel; Goepferich, Achim
title: Biomedical nanoparticle design: What we can learn from viruses
date: 2020-09-30
journal: J Control Release
DOI: 10.1016/j.jconrel.2020.09.045
sha: afe061e0ddfa9144e670bd6db01c9136f470be29
doc_id: 813612
cord_uid: 7if5b1mc

Viruses are nanomaterials with a number of properties that surpass those of many synthetic nanoparticles (NPs) for biomedical applications. They possess a rigorously ordered structure, come in a variety of shapes, and present unique surface elements, such as spikes. These attributes facilitate propitious biodistribution, the crossing of complex biological barriers and a minutely coordinated interaction with cells. Due to the orchestrated sequence of interactions of their stringently arranged particle corona with cellular surface receptors they effectively identify and infect their host cells with utmost specificity, while evading the immune system at the same time. Furthermore, their efficacy is enhanced by their response to stimuli and the ability to spread from cell to cell. Over the years, great efforts have been made to mimic distinct viral traits to improve biomedical nanomaterial performance. However, a closer look at the literature reveals that no comprehensive evaluation of the benefit of virus-mimetic material design on the targeting efficiency of nanomaterials exists. In this review we, therefore, elucidate the impact that viral properties had on fundamental advances in outfitting nanomaterials with the ability to interact specifically with their target cells. We give a comprehensive overview of the diverse design strategies and identify critical steps on the way to reducing them to practice. More so, we discuss the advantages and future perspectives of a virus-mimetic nanomaterial design and try to elucidate if viral mimicry holds the key for better NP targeting.

Virus-based delivery systems that inherit these properties to some extent, such as viral vectors or virus-like particles, also hold great biomedical potential but are frequently associated with considerable safety concerns [8, 9] . Therefore, over the years, research has deeply focused on implementing favorable viral properties into nanomaterials to improve their performance. Thereby, synthetic NPs appear as a safer and more versatile option. They additionally facilitate the incorporation of multiple different cargos for a broad spectrum of applications and have clear advantages regarding production, storage and reproducibility [10] .

In this review we give an overview of the viral traits adopted in targeted nanomaterial design to improve NPs' efficiency. Starting with simple structural characteristics and ligand display, and ending with more complex attributes, such as the sequential interaction with surface receptors, stimuli responsiveness or cell-to-cell spreading. We critically examine the advances that viral mimicry has enabled for targeted nanotherapy and outline pivotal design parameters that must be considered for a rational NP optimization. More so, we discuss the latest trends and outline future perspectives for the virus-like nanomaterial design.

Viruses are nanosized entities of about 20-200 nm (with the 400 nm Mimivirus being the largest one described to date) [11] . There is little research on the effects that size has on the viral life cycle. However, it is known that it is related to the length of the genome the virus is enclosing [12] . This can be also related to nanomaterials, as there are spatial constrictions that determine their cargo loading [13] . However, a nanomaterial´s size is a fundamental parameter determining its suitability for concrete applications. For example, for the extravasation to specific tissues, the NP size must be under a (patho)physiologically determined cutoff [14, 15] . More so, a particles size determines its blood residence, clearance [16] [17] [18] , and interaction with the immune system [19] [20] [21] .

Viruses come in various shapes and forms, such as polyhedral, rod-like, or filamentous, which bestow them with distinct biodistribution and targeting properties [22] . For example, it has been shown that the filamentous form of the Influenza virus has a higher specific infectivity than their spherical virion counterparts [23] . Mimicking this morphology with synthetic filamentous NPs could be a viable option for an efficient drug delivery via inhalation. [24] Due to their rod-shaped form, filamentous NPs would thereby prevent phagocytic internalization and -via paracellular translocation through the air-blood barrierenter blood circulation, where they have been shown to possess a prolonged circulation time as well [25] . A rod shape is also associated with higher virus diffusion rates in tumor tissue [26] , and can be mimicked with synthetic NPs by techniques such as the co-assembly of polyanions and artificial virus capsid proteins [27] , the condensation of DNA and block copolymers [28] , or the seeded growth synthesis of gold NPs [29, 30] . A polyhedral virus shape has been linked to a high targeting specificity [31] , a very sought after property for targeted NPs. from a clathrin-mediated endocytosis for non-functionalized particles, to a combination of caveole-mediated endocytosis and macropinocytosis.

Synthetic spikes can also be constituted by targeting ligands themselves, as it was shown by the formulations developed by Liu et al. [42] and Xu et al. [43] . The former developed lentivirus mimicking NPs displaying Zn-dipicolylamine analogue-spikes, a zinc coordinative ligand with high affinity to phosphate moieties on cell membranes [42] . The latter, imitated coated viruses, such as influenza or herpesvirus (HV), by displaying transferrin (TF) spikes on the surface of their liposome-DNA complexes [43] . In both cases the spikes were able to bind the cell membrane and mediate internalization. Additionally, spikes conferred endosomal escape abilities through membrane destabilization [42] or a higher in vivo stability and gene transfer [43] , respectively, compared to nonfunctionalized particles.

A typical viral characteristic is surface glycosylation [44] . In some cases, such as for the influenza virus, glycosylation is essential to enhance cellular internalization [45] and in others, such as for the human immunodeficiency virus (HIV), it is crucial for the evasion of excessive immune recognition by preventing the attachment of neutralizing antibodies [46] . For synthetic NPs there seems to be contradictory evidence regarding the influence of glycosylation on immune activation, and it is often viewed as an obstacle for the generation of an adequate immune response. However, Tokatlian et. al [47] recently highlighted that adjusting the NP immunogen glycosylation is critical for vaccine design, as they demonstrated that deglycosylation significantly affects antibody response.

Mannose presentation on self-assembling ovalbumin carrying NPs was also J o u r n a l P r e -p r o o f Journal Pre-proof associated with a higher in vivo immune response compared to non-glycosylated NPs [48] . Regarding the efficiency of NP-cell interactions, glycosylation is generally considered an improvement in NP design. Pinnapireddy et al. [49] mimicked enveloped viruses with glycosylated anionic liposomes prepared from lipids found in the envelopes of HIV and herpes simplex virus (HSV). The formulation, intended for gene delivery, achieved an increased particle internalization through lectin receptors [50] . Also, the addition of mannose to cationic albumin NPs allowed for an enhanced brain targeting and in vivo glioma treatment [51] . As for viruses, which ideally possess an optimal glycosylation balance that "shields" from the immune system but still allows efficient receptor binding [52] , virus-mimetic NP approaches likewise need to be manufactured with respect to these factors. While nanoparticular vaccine approaches would generally benefit from an adequate immune response, drug delivery approaches using virus-mimetic nanomaterials could be severely limited by an excessive recognition via the immune system. Especially in the latter case, incorporation of additional surface elements such as glycosylation or also specific targeting sequences to more realistically mimic viral particles always bears the risk of an undesirable immune recognition and should therefore be exactly tailored for each formulation in accordance with its physicochemical characteristics and the intended application.

Once particles enter biological media, they are subjected to protein adsorption and the formation of a protein corona. For viruses, several host factors indispensable for infectivity can attach to their surface, like Apo-E lipoprotein for hepatitis C virus (HCV) [53] . A recent study that evaluated the protein corona J o u r n a l P r e -p r o o f formation on respiratory syncytial virus and HSV type-1 when incubated with different bodily fluids showed that the surface properties of the virus were responsible for determining the enrichment with different corona elements [54] .

Furthermore, the fluid from which the proteins proceeded, determined the corona composition, which in turn affected the viral infectivity. However, viral and NP corona formation may be quite different, due to the diverging surface materials and compositions. Additionally, the attachment of a protein corona to the particle surface is oftentimes followed by an undesirable NP clearance via the mononuclear phagocyte system (MPS), which has been identified as one of the major obstacles for a successful NP targeting [55] . While numerous approaches try to avoid this impediment by manufacturing so-called "stealth" NPs with additional surface coatings such as PEGylation [56] , viral particles may be subjected to a generally lesser protein corona formation. In this regard, Pitek et al. [57] discovered that after plasma incubation, virus particles derived from the tobacco mosaic virus bound 6-times less protein than synthetic NPs. The authors associated this with the display of positive and negative charge patches on the viral particles in combination with hydrophobic and hydrophilic domains.

Recently, our group also showed that zwitterionic polymeric NPs adsorbed less protein when incubated in serum compared to positive, negative or uncharged particles, as it provides less domains allowing for hydrophobic or electrostatic interactions [2] . This is in accordance with previous studies that demonstrated that the protein binding suppression of such materials is due to their ability to electrostatically bind great amounts of water molecules [58] [59] [60] . Therefore, in addition to the typically implemented strategies used to suppress protein corona formation, such as above-mentioned PEG coating, an adjustment of the surface J o u r n a l P r e -p r o o f charge of particles, imitating the charged but net-neutral viral surface, must be considered. Furthermore, a virus-like exploitation of the protein corona may enhance the targeting abilities of NPs, as recently reviewed by Maiolo and coworkers [61] .

Close mimicking of the viral surface has also been extremely useful to achieve mucus-penetration. Surface characteristics that allow viral particles to overcome the mucus barrier are a charged but net-neutral surface and a hydrophilic nature [62] . It has been demonstrated that particles that hold these features are able to overcome the mucus barrier [63] , making them appropriate vehicles for oral [64] and vaginal drug administration [65] . However, the combination of these surface properties with a virus-like active targeting has been shown to further increase the penetrability of nanomaterials, which is especially interesting for the oral insulin delivery. Liu et al. [66] were able to overcome the mucus barrier with polyelectrolyte complexes mimicking the viral envelope, composed of polysaccharides, peptides and lipids, with L-Phenylalanine-functionalized chitosan polymers. The authors found that functionalized polymers yielded a 2fold higher bioavailability than non-modified particles. Zhu et al. [67] demonstrated that active targeting can be used to surmount not only the mucusbut also the epithelial absorption barrier. They functionalized polymeric insulin carriers with poly(ethylene glycol) (PEG)-shielded poly-arginine which was able to mediate epithelial cell penetration and produce hypoglycemia in vivo. They further demonstrated the particle safety [68] and expanded on their mimicry concept implementing the densely charged but net-neutral surface of viruses [69] .

In this manner the NPs displayed the distinct viral attributes needed to overcome not one, but two barriers.

Viruses are meticulously built systems in that every component holds an exact function. This promotes their paramount goal of delivering their genetic content into host cells. More so, the success of this endeavor strictly depends on a perfect interworking of all constituents. Size, shape and surface properties are apparently simple elements of particle design. However, they can have enormous influence on a particle´s cellular interaction and biodistribution [70] . Mimicking the viral morphology and surface characteristics can lead to faster and greater NP-cell interactions and even bestow barrier penetration abilities. Therefore, the structural replica of a virus must be considered the first step when trying to achieve viral traits on synthetic NPs ( Figure 2 ). 

To promote the infection of the host-cell, viruses usually display surface components that bind to specific cell membrane structures. Usually an initial attachment through the interaction of membrane glycoproteins or viral capsid sites with factors, such as heparan J o u r n a l P r e -p r o o f sulphate proteoglycans [7] , is followed by the binding of viral ligands to cell surface receptors that are distinct for each virus [71] . This specific interaction leads either to endocytosis, to the activation of signalling pathways promoting internalization, or to the induction of conformational changes on the virus, which promote fusion and cellular penetration [71] . The targeting of specific receptors has been widely implemented on NPs, not only to achieve the crossing of cellular membranes but also to endow particles with a higher specificity towards distinct cell types.

The multivalent ligand display is one of the viral attributes that has had the most impact on the field of targeted nanomaterials. Viruses present numerous copies of the same ligand on their surface, which allows them to interact with multiple receptors on the cellular surface ( Figure 3 ). It is broadly accepted that mimicking 

A frequently used design approach to mimic the viral targeting principles is the decoration of NPs with natural viral surface "ligands", like attachment factors, cell penetrating peptides, fusion-proteins, and antigen-derived peptides ( Table 1 ). Viral surface antigens tethered to NPs are mainly of interest in the field of vaccine development which will not be addressed here, as they are outside of the scope of this review and have extensively been reviewed elsewhere [75, 76] . In this section, we will focus on systems displaying viral surface molecules seeking an increase in target cell recognition for drug delivery, with diagnostic or therapeutic purposes.

During the initial phases of cell entry, viruses make use of attachment factors that anchor them to the host cell membrane. Despite it being a low-affinity binding, it is of importance in the viral cycle, as it aids receptor recruitment [77] .

Furthermore, its inhibition can suppress the infection process [78] . Almost three decades ago, Rubas et al. [79] discovered that modifying liposomes with the reovirus M cell attachment protein 1 increased their cellular uptake in vitro by 10-fold compared to non-targeted formulations. Also, the recent identification of a new heparin-binding domain, pre-S1 (30) (31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) , of hepatitis B virus (HBV) involved in initial virus attachment enabled the development of virus-mimicking liposomes for the specific identification of human hepatic cells [80] .

Interestingly, particles functionalized with the attachment peptide were able to deliver doxorubicin (DOX) to hepatic cells more efficiently than liposomes functionalized with the peptide associated with viral targeting, i.e., pre-S1(2-47) [81] , corroborating the immensely important role of attachment in viral infectivity.

Viral surface antigens can also be used for the targeting of specific cell types.

Somiya and Kuroda [82] developed a HBV-mimetic nanocapsule using the hepatitis B surface antigen L protein for specific drug delivery to human hepatocytes. To overcome the elicitation of an immune response resulting from the repeated administration of viral antigens on the surface of NPs, they made use of an additional viral trait, which is the ability to mutate [83] . To that end, the Gln-292 and Gly-302 were substituted with Arg, suppressing the immunogenicity of the formulation [84] , which is indispensable for repeated administrations.

Additionally, viral surface ligands can be used to overcome barriers which normally present a huge difficulty for nanocarriers, such as the BBB. In this J o u r n a l P r e -p r o o f regard, a small peptide derived from the rabies virus (RABV) glycoprotein (RVG), RVG29, that interacts with high specificity with the cell entry-mediating neuronal nicotinic acetylcholine receptor, was used by several authors as targeting entity to enable BBB crossing. It was coupled to polymeric PEGpoly(lactic acid) (PLA) NPs to increase the BBB penetration of deferoxamine, an iron chelator used to protect against oxidative damage [85] . A higher BBB crossing of the drug enabled by this formulation prevented neuron damage and neurobehavioral deficits in mice with no systemic adverse effects. RGV29 was also grafted onto calcified calcium carbonate-and DOX-containing polymeric PLGA NPs to address brain tumors [86] . After ligand-mediated uptake, the calcium carbonate contained in the particles generated carbon dioxide gas upon acidification, increasing DOX release, and achieving tumor size suppression in a mouse model. Lee et al. [29] also used RVG29 to develop a nano formulation for the treatment of brain tumors based on silica-coated gold nanorods.

During infection, viruses present different proteins and peptides that aid cell penetration. Several cell penetrating peptides (CPPs) used in active targeting [87] are derived from viral capsids [88] , such as the HIV [89] and the Brome mosaic virus [90] . One of the most commonly used CPPs is the HIV derived transactivating transcriptor (TAT) peptide [91] . It has been used to decorate the surface of several different nanomaterials, such as silica- [92, 93] , magnetic- [94, 95] , Au- [96, 97] , lipid- [98] , and polymeric NPs [99] [100] [101] , amongst many others, to accomplish cell or nucleus penetration and BBB-crossing for diagnostic and therapeutic purposes.

Lastly, ligands inducing viral fusion can also be grafted onto NPs. Gao et al. [102] functionalized parainfluenza virus envelope-mimicking fusiogenic vesicles J o u r n a l P r e -p r o o f with hemagglutinin-neuraminidase protein and the viral fusion protein, which bind sialic acid-containing receptors and initiate fusion, respectively. After fusion, the molecular beacons contained in the NP target miRNAs and generate a quantifiable fluorescence signal, which can be applied in exosome mRNA detection and cancer diagnosis. Wang et al. [38] , took advantage of a phage fusion protein, pVIII, and prepared phage-mimetic rod-shaped NPs selfassembled from Ag@Au nanorods for specific targeting and photothermal therapy. The fusion proteins allowed for a specific colorectal carcinoma cancer cell targeting and ablation. Interestingly, the assembly of the phage proteins on the NP surface was achieved through electrostatic interactions, a coupling which is usually mediated by covalent bonds. However, this allowed the pVIII protein to maintain its natural conformation and orientate itself outwards, facilitating targeting.

Taken all together these results show that by isolating viral ligands and displaying them on NPs we are not only able to enhance important NP characteristics, such as targeting, penetration, and barrier crossing, but also shed some light on their involvement during viral infection. 

To direct nanomaterials towards specific cell types, particles can also be decorated with whichever ligand that targets physio-or pathologically surface receptors on the cell of interest (Table 2) .

Multivalent NPs have been used to target G protein-coupled receptors (GPCRs) [73, 103, 104] , integrins [105, 106] and lectin receptors [107] with ligands such as peptides or proteins, aptamers and small molecules [108] . But, even though the virus-like ligand display seems like an easy enough concept to be reproduced with synthetic materials, it noteworthy that several distinct parameters have an influence on the success of a multivalent NP. One of the most relevant ones is ligand density, which is frequently neglected in nanomaterial design. In their excellent recent review Alkilany et al. [109] calculated the number of ligands J o u r n a l P r e -p r o o f displayed by viruses on their surface. Depending on the virus, they obtained a range between 7-659 ligands per virus particle. Interestingly, this number is usually greatly exceeded in synthetic NPs, where sometimes thousands of molecules are tethered to a single NP. More so, results show that the optimal ligand density is a unique characteristic for each formulation. In some cases, a minimum threshold needs to be surpassed to induce targeting effects. For example, for folate-functionalized NPs, a ligand density over 10% was needed in order to exceed the internalization of non-targeted particles [110] . In other cases, an optimum ligand density may exist, its alteration leading to a decrease in targeting. Fakhari et al. [111] demonstrated that a medium (50%) cLABL density on poly(lactic-co-glycolic acid) (PLGA) NPs achieved the optimum targeting of ICAM-1 expressing cells. Lower or higher ligand densities resulted in a poor particle uptake. Similarly, Elias et al. [112] found that an intermediate ligand density was optimal for targeting purposes using antibodies against HER2/neu, overexpressed in cancer cells. Generally, decreases in targeting with higher grafting densities are explained by a steric hindrance of the ligands and decreased ligand mobility [113] . This may disrupt interaction with the receptors to a point where their internalization is impeded. Another scenario is presented when a targeting plateau is reached above a certain ligand density. Poon et al. [114] showed that low ligand densities (20% folate density) were ideal for their system and higher functionalization did not enhance targeting. Lastly, there are systems where an increase in ligand number results in a continuous enhancement of NP targeting efficiency, which is frequently detected for RGD-ligands [115, 116] .

Therefore, it is essential to carefully and individually adjust the number of ligands on the particle corona for each system.

An additional factor to be considered for the multivalent NP design is the ligand conformation. Viruses often display ligands with defined conformations and regular spacings. This is the case of adenovirus, which presents RGD clusters [117] on five penton base proteins with a 5.7 nm spacing [118] , indispensable for viral infection [119] . RGD ligands are also one of the most used candidates for targeting purposes, as integrins are expressed in both tumor-and tumor endothelial cells [106] . Using adenovirus physical structure as a guide, Ng et al.

[120] studied the influence of the ligand clustering on the targeting efficiency.

RGD ligands were tethered to Au NPs to generate clusters, which were subsequently attached to PEI polyplexes. The cluster-presenting particles achieved a 5.4-or 35-fold increase in gene transfer in cells expressing low and high integrin densities, respectively, compared to non-modified polyplexes,

showing a higher sensitivity to receptor density. This selectivity towards cells expressing high target receptor levels is fundamental for the design of nanomaterials which base their targeting principle on receptor overexpression by specific cells in a diseased state. The clustering principle has also been applied to folate molecules [114, 121] with similar results, demonstrating that it is a promising approach to optimize ligand presentation.

The interactions of ligands with their targets may also be defined by the length of the linkers used to tether them to the particle surface. First, it may increase or decrease the particle size, which highly influences the cellular interaction [14] .

Second, it can alter the ligand disposition, which can be spaced out or tightly grouped by using longer or shorter tethers, respectively [116] . Thirdly, it can influence the ligand mobility on the particle surface, which has a tremendous influence on the cellular internalization of a NP [113] .

It is generally accepted that the virus-like multivalent ligand display enhances the particles overall avidity and targeting capabilities. Nevertheless, it does not increase the nanomaterials overall specificity, which is indispensable for therapeutic applications of such materials. Despite generally addressing diseaserelated overexpressed receptors, they are often also prevalent in "healthy" offtarget tissues, which leads to poor material bioavailabilities and deleterious side effects. One of the elements that can negatively impact the specificity of multivalent NPs is the elevated number of ligands displayed on their corona, which is usually higher than the one presented by viruses [109] . In vivo this may additionally cause a particle stealth loss and increased protein corona formation, which also hinders targeting. More so, a very precise control over NP design features besides ligand density, such as linker length and ligand conformation, is crucial to achieve optimal effects. Altogether, a multivalent ligand display seems to be a prerequisite but not sufficient to effectively imitate the viral target cell recognition. 

The virus-like multivalent display of tethered ligands on NPs highly increases their targeting abilities. However, the cellular interplay of viruses is usually mediated by more than a single ligand. Therefore, as an approach to increase the specificity and targeting capacity of multivalent systems, heteromultivalent NPs, displaying different types of ligands on their surface, were developed ( Figure 3 ).

They more closely mimic viruses, which require several recognition molecules for host-cell identification. For example, the HCV depends on the co-expression of four proteins (SR-B1, CD81, claudin-1, and occludin) to mediate cell entry [71] . This concept´s increase in cell specificity is based on the fact that the probability of more than one cell expressing the same receptors decreases with the number of receptors that are addressed. More so, by using an additional set of ligands, the targeting capacity of heteromultivalent particles should be enhanced compared to multivalent NPs. Heteromultivalently binding NPs have been extensively investigated over the past years [122] . They find mostly application in cancer [123] , and vascular pathologies [124, 125] , characterized by a concomitant spatiotemporal upregulation of several surface receptors, such as the TF receptor (TfR) [126] , folate receptor (FR) [127] , epidermal growth factor receptor (EGFR) [128] , integrins [129] , and selectins [130] . Like for multivalent systems, antibodies, small molecules, and peptides [131] , are mainly used as targeting moieties. An overview of different formulations developed over the past years is depicted in Table 3 .

It is essential to note that as for multivalent systems, there are several factors that can determine the cellular outcome of a heteromultivalent system, such as ligand density, ligand ratio and ligand arrangement. However, for most published formulations there is a lack of information regarding these parameters (Table 3) .

J o u r n a l P r e -p r o o f

As they have enormous influence on the establishment of receptor interactions [109, 132] , it should come as no surprise that heteromultivalent NPs frequently achieve only moderate improvements, regarding targeting and specificity, compared to "simple" multivalent systems [126, 133] . The addition of a second ligand type increases the complexity of the system and does not equal the rise in number of a single ligand. Furthermore, excessive particle functionalization can propitiate targeting ability loss [134] and off-target interactions [135] . A higher functionalization is usually associated with a higher avidity of the particle system. However, a high avidity attachment to the cell surface is not always positive seen from the viral perspective. It can hinder virus diffusion through the cell membrane and reduce the chances to find the cell entry mediating receptor [136] . More so, the number of interactions between viruses and their receptors are limited. Delguste et al. showed that only 2 or 3 simultaneous interactions occurred between HV and glycosaminoglycans [136] . This is something that should be considered when designing functionalized particles.

Ligand arrangement on the particle surface can also determine the enhancement, or lack thereof, in particle internalization through a heteromultivalent system. For instance, HIV initially binds to the CD4 receptor on the membrane of its target cells via the gp120 subunit of its envelope protein (Env). Only then, conformational changes in the Env reveal a variable loop (V3) that subsequently binds a coreceptor and initiates membrane fusion via the fusogenic peptide of the second Env subunit gp41 [137] .

In that regard, dissipative particle dynamics stimulations also showed that length mismatch or interactions between ligands can impede dual ligand binding [138] . The exact definition of the expression patterns of the targeted receptors is also an element that can enable an optimal particle design. Gunawan et al. [130, 144, 145] carried out several studies with immunoliposomes functionalized with antibodies targeting ICAM and E-selectin on activated endothelial cells. The formulation was optimized to achieve a cooperative effect of the two ligands with a 1:1 ratio when lipid rafts were present on the cells [144] . (Table 3) .

Taken all together, these results demonstrate that the virus mimicking heteromultivalency is a promising concept. However, every detail of the particle design (ligand density, ligand ratio, ligand arrangement) and its application (target expression levels, pattern variations) must be systematically studied and combined to achieve the sought-after targeting and specificity goals. 

Ab (1) Ab (2) ICAM E-selectin --- [144] Liposome Peptide (1) Peptide (2) ɑvβ3 integrin Galectin-1 --- [151] Liposome Ab (1) Ab (2) VCAM1 E-selectin 1:1 ligand ratio -- [130] Liposome Peptide (1) Peptide (2) ɑvβ3 integrin Galectin-1 -B16F10 tumor (mouse) - [152] Liposome Ab fragment (1) Ab fragment (2) EGFR CEA --- [157] [146] PEI polyplex

TfR Integrin --- [10] Au NPs Ab (1) Ab (2) FR EGFR --- [127] Au NPs FA Glucose FR GR --DOX [158] Au NPs Peptide (1) Peptide (2) EGFR TfR -U87-MG tumor (mouse)

Phtalocyanine 4 [159] J o u r n a l P r e -p r o o f

Even though (hetero)multivalent display of ligands on a particle surface has been extensively investigated over the past years for the development of targeted nanomaterials, it has yet failed to achieve the desired results. In two widely cited meta-analyses of tumor-targeted NP studies, Wilhelm et al. and Dai et al. concluded, that the vast majority of both non-functionalized and targeted nanomaterials failed to substantially reach their target regions [161, 162] . While the exact numbers of calculated levels for NP delivery efficiency and their implications have since been extensively discussed [163] , it is undoubted, that NP targeting strategies including (hetero)multivalent ligand presentation have so far only shown moderate clinical translation. In that regard, one obstacle indisputably lies in the fact that, despite being virus-inspired, (hetero)multivalent approaches mostly fail to entirely mimic the viral host cell recognition process, which is decidedly more complex [7, 77] . Viruses not only heteromultivalently bind distinct membrane receptors, but they do so in a sequential manner [77] .

Examples for this are the HIV type 1 binding consecutively to the CD4 and the

TfR Endothelium --DOX [133] Polymer NPs Photothermal therapy [160] DNA Nanoclaws

Ab (1) Ab (2) Ab (3) EpCAM EGFR HER-2 --DNA [143] Liposome /Silica NPs - [148] J o u r n a l P r e -p r o o f chemokine receptor [164] or adenoviruses attaching to the coxsackie and adenovirus receptor (CAR) and integrins [165, 166] . If the presentation of ligands by heteromultivalent NPs is simultaneous, any cell expressing either of the target receptors is able to bind the particles. For instance, the independent binding of different tissues by each ligand was demonstrated for RGD and TF targeted polyplexes intended for the treatment of choroidal neovascularization [167] and for p-selectin and v3 integrin targeting NPs used for breast cancer treatment [147] . Overall, this translates into a poor particle specificity and target accumulation. This shortcoming of nanomaterials can be surmounted by a stepwise heteromultivalent approach with a virus-like sequential ligand presentation ( Figure 3 ). In some studies, different ligands are coupled to a NPs surface for its sequential presentation, in that one of them enables cellular targeting, and the second one directs the particle to intracellular compartments, such as mitochondria [168] or the nucleus [169] . Nevertheless, the ligands are ubiquitously present on the particle surface and therefore cannot decrease nonspecific particle accumulation. Our group recently showed, that mimicking the stepwise host cell recognition of influenza A viruses highly increases the target cell specificity of nanomaterials [74] . Influenza A viruses display hemagglutinin on their envelope, which requires activation by an enzyme on the host cell membrane. Activated virus particles can then bind to sialic acid, that triggers cell uptake [170] . To mimic this enzyme-mediated recognition, angiotensin-I (Ang-I) was coupled to biocompatible PEG-PLA block copolymer

NPs with a PLGA-stabilized core [171] . Ang-I probed the target cells for the presence of angiotensin converting enzyme (ACE), which upon binding cleaved the two last amino acids, releasing angiotensin-II (Ang-II). The interaction of

Ang-II with the Gq-coupled receptor angiotensin-II type 1 receptor (AT1R), as an agonist triggered endocytosis [172] . This principle was designed to identify with high specificity mesangial cells, as they play a crucial role in the development of diabetic nephropathy [173] . This renal complication is suffered by 50% of the 425 million diabetic patients worldwide [174] and lacks specific treatment options. The translation of the influenza A recognition principle enabled synthetic NPs to specifically identify mesangial cells in co-culture mixtures where they made up only 10%.

Furthermore, we improved the system incorporating an additional viral cell recognition step, the initial cell attachment [175] . As discussed above, viruses often attach to the host-cell surface to increase the viral concentration and initiate receptor recruitment. It does not mediate internalization of the virus particle, but it has been shown to be a decisive step during the infection process [78] . In order to mimic the attachment while still maintaining specificity, an antagonist for the AT1R, losartan carboxylic acid (EXP3174), was coupled to the particles. EXP3174 decorated NPs were previously shown to attach to the cell membrane, but not mediate internalization [72, 176] . The close mimicking of the viral host cell recognition, through a first attachment, followed by an enzymatic activation and concluded by an agonist-receptor binding ( Figure 4A ) enabled a 5-or 15-fold higher accumulation of particles in mesangial cells in vivo than NPs lacking the attachment principle or any viral traits, respectively ( Figure 4B) . As there are a plethora of ectoenzymes available, this design principle could be expanded to numerous other cell types involved in the development of diseases.

In addition, we decided to even further extend the applicability of the system, namely to cell types lacking suitable ectoenzymes. We therefore introduced a modified recognition concept that was inspired by the cell infiltration strategy of human adenovirus (AdV) J o u r n a l P r e -p r o o f [177] . In order to mimic the stepwise virus-cell interplay of AdV, we implemented a sequential display of two ligands, that was based on the steric shielding of the second, uptake-mediating ligand [178] . While initial cell attachment of the NP was also mediated via binding of EXP3174 to the AT1R, final NP endocytosis was initiated through a cyclic RGD sequence that activated the integrin receptor α v β 3 . However, as the second RGD ligand was attached in closer proximity to the NP core, it could only activate the integrin after a previous binding of the AT1R and a resulting spatial approach of the NP to the cell surface. This two-step ligand display led to a significantly enhanced target cell selectivity of resulting NPs [178] . Additionally, in vivo accumulation in target mesangial cells was comparable to the influenza A mimetic system, thereby proving the potential of both virus-mimetic concepts. 

Viruses have evolved to respond to external stimuli, such as enzymes, reduction, or changes in the pH value, during their infection process [7] . This stimuli-responsiveness has been implemented on the newer generation of nanocarriers as it enables exploiting the specific disease environment to achieve a more effective and specific targeting.

Particles can be designed to switch their surface charge, unmask active targeting ligands or shed their coating in response to pH variations, redox-or enzyme activity (Table 4) .

Following this trend in recent years nanocarriers have been becoming more complex and "smarter" by incorporating multiple stimuli responsive elements. Even though generally not exactly mimicking specific viruses, such systems aim at allowing for a higher control of the particle fate in the organism ( Figure 5) , inspired by the viral cycle.

Despite stimuli-responsive nanomaterials being reviewed in the past [179] , due to the high number of publications in this fields over the last 3 years we will give a brief overview and discuss the newest systems. 

The pathological characteristics of therapeutically relevant tissues can be used to increase the targeting efficiency of nanocarriers. Enzyme-responsive systems that target enzymes linked to a specific disease are an example of this [180] . The enzymes most commonly addressed are the matrix metalloproteinases (MMPs) [181] , which can be found in the extracellular tumor medium. They have been used to unveil PEG-shielded J o u r n a l P r e -p r o o f ligands protected during circulation [182] [183] [184] [185] [186] , or to induce a morphology change [187] [188] [189] , increasing the targeting potential of NPs. Other enzymes, such as legumain, which is upregulated in tumors in correlation with their malignancy [190] , have also been targeted to unveil shielded ligands, like TAT [191] , in a site-specific manner.

Enzymes associated with drug-resistant bacterial strains, such as Penicillin G amidase (PGA) and -lactamase (BLA) [192] or P. aeruginosa elastase [193] , can also be used as targets to achieve a selective delivery of antimicrobial agents. Lastly, the abovementioned influenza A mimetic NP concept of our working group also depends on an enzymatic cleavage of ligand precursor Ang-I in order to initiate final NP uptake via

Ang-II mediated activation of AT1R [74, 175] . More so, enzyme-responsive systems have long been adopted for the controlled delivery of drugs [42, 100, 184, 194] .

As an alternative approach to enzymes, linkers or tethers with pH-responsive functionalities can be introduced in the particle design, to shield ligands during blood circulation, and increase their targeting once an acidic environment is reached [195] [196] [197] [198] . Furthermore, pH-responsiveness can be used to initiate cargo release in acidic intracellular compartments or tumor milieus [199] [200] [201] . It can also be adopted to mimic the viral trait of endosomal escape [202, 203] which is mediated by membrane fusion or disruption, for enveloped and non-enveloped viruses [204] , respectively. The endolysosomal escape of non-enveloped viruses was mimicked by Song et al. [205] with NPs prepared from pH-sensitive hydrazone bond-containing polyurethane. After target-specific cell internalization, upon exposure to acidic pH the particles underwent charge reversal, core exposure and induced endosome rupture. This viral trait has also been implemented in synthetic nanomaterials through additional mechanisms, such as membrane fusion, osmotic disruption, particle swelling and membrane destabilization J o u r n a l P r e -p r o o f [206] or the incorporation of viral and viral-mimetic peptide sequences related to endosomal escape [207, 208] .

Lastly, Redox-responsive systems show great potential as drug delivery systems [209] in disease with elevated reactive oxygen species (ROS), such as cancer [210, 211] or respiratory viral infections [212] .They can be used to induce a site-specific drug release, as shown by Jian et al. [213] . The authors emulated the viral capability of overcoming barriers, such as the BBB, with virus-sized polymerosomes encapsulating the toxin saporin, which is highly degradable in vivo. The particles were functionalized with angiopep-2, a high affinity ligand towards the low-density lipoprotein receptor-related protein-1 (LRP-1). Due to their redox responsiveness, upon reaching intracellular environments the particles were able to release their payload. After administration in glioblastoma-bearing mice, particles were able to accumulate at the target through LRP-1-mediated BBB transcytosis and increase survival rate through tumor inhibition. DOX [195] J o u r n a l P r e -p r o o f "-" PEG copolymer NPs HepG2 tumor (mouse) "-" [198] Charge inversion "-" Silica NPs -"-" [196] Cargo release

Dendritic lipopeptide NPs -DNA [199] Tumor lysosome Polymeric micelles -DOX [200] "-" Dendritic peptide NPs SKOV3/R tumor (mouse) "-" [201] Endosomal escape

Acidic endosome PEG Liposomes -DNA [202] "-" Polymer NPs -Asiatic acid [203] " 

Particles that respond to a single stimulus hold great advantages regarding targeting efficiency. However, in recent years nanomaterials are being designed so that they respond to a combination of stimuli. These multiresponsive systems allow to mimic the continuous changes that viruses undergo during their infection cycle. They also enable targeting a higher variety of diseases with increased specificity. Additionally, they

provide a more precise control of particle fate upon administration.

Some systems present sequential responsiveness to the same type of stimuli, as for example, the multiple targeting of both extracellular and intracellular enzymes. Han et al. [184] used this concept and achieved specific in vivo tumor targeting, through unshielding of a PEGylated ligand by MMP-9 and controlled drug by cathepsin-B, present in the tumor environment and lysosomes, respectively. Another approach is the dual redox-responsiveness, which was employed by He et al. [214] to increase in vivo delivery of nucleic acids to tumor cell with polyplexes that, like viruses, are subject to sequential unshielding and unpacking steps: first after cellular attachment or internalization, and second to enable genome release after endosomal escape.

Other systems are responsive to a combination of different stimuli. The association of MMP-and reactive oxygen species (ROS)-responsiveness enabled Daniel et al. [215] to J o u r n a l P r e -p r o o f use amphiphilic polymer NPs in inflammatory diseases, such as myocardial infarction, arthritis, ischemia and atherosclerosis, where both are upregulated. With this design approach they achieved specific in vivo targeting of ischemic skeletal muscle [216] , avoiding off-targeting of healthy muscle and regulating macrophage internalization [217] .

The increase in NP specificity achieved by multiresponsive systems has the potential to reduce therapy-associated toxicity. Zhang et al. [218] developed a polycaprolacton and Furthermore, due to the myriad of developed materials with transversal application, the therapy options for difficulty targeted diseases are broadened.

A fundament for the perpetuation of the viral cycle is their ability to replicate and spread from cell to cell [220] . After a successful host cell infiltration via above described mechanisms, viruses thereby initially release their genome (RNA/DNA), which is subsequently replicated and translated into further virus components such as protein envelopes, leading to the assembly of numerous new copies of the viral particle ( Figure 1 ). For obvious reasons, synthetic nanoparticles, in contrast, cannot be (to date)

reproduced within the target cell. However, this limitation is only of minor importance since the central aspect of nanomedicine is not the particle reproduction but a drug delivery into or the NP-assisted therapy of targeted cells. In that regard, most approaches discussed in this review (Tables 1-4) present nanoparticles that were loaded with the "usual suspects", i.e. cytotoxic substances such as doxorubicin or paclitaxel to treat severe tumor diseases. Even though therapeutic efficiency was thereby oftentimes

considerably improved compared to the application of a free drug, these classic nanotherapeutic concepts suffer the widely discussed limitation, that generally, only small doses of therapeutic substances can be encapsulated in the currently available nanoparticulate systems. Consequently, most clinically translated NP drug delivery approaches are based on the administration of highly potent chemotherapeutic agents for only a very limited range of mostly malignant tumor diseases [221] . In that regard, the mimicry of viral cargo, i.e. the delivery of DNA or RNA components could offer a substantial expansion of currently treatable diseases as a nucleic acid based therapy generally requires only minor concentrations compared to classical therapy options [222, 223] . Also, above described viral strategies to protect its cargo and deliver it to the necessary intracellular compartments could be a highly promising model to maximize the therapeutic effect of these gene delivery approaches.

As the viral infectivity is to a large extent based on the ability to disseminate from the initial target cell and spread across the surrounding tissue, bestowing NPs with this trait may also allow to lower dosages and enforce valuable properties in applications, such as cancer treatment. Several years ago, Lee et al. [224] developed a virus mimetic delivery vehicle capable of disseminating from cell to cell. It consisted on a core-shell nanogel loaded with DOX. The poly(l-histidine-co-phenylalanine) core was covered by a PEG shell, linked to bovine serum albumin (BSA) molecules. To achieve a specific in vitro targeting to tumor cells, folate molecules were conjugated to the BSA. Upon subjection of the nanogels to different pH values, they were able to shrink and expand due to their pH sensitive core, varying between 55 nm at pH 7.4 and 355 nm at pH 6.4. After "infection" of the target cells, DOX-mediated apoptosis was induced, and the nanogels were released in to the medium ready to target the next cell. This DOX apoptosisderived approach to induce cell spreading was also used by Cui et al. [225] . They additionally enhanced the in vivo penetration in tumors by NP hyaluronidase release under acidic tumor conditions, which is able to cleave HA in the extracellular matrix and reduces the NP size. However, a limitation of this infective mechanism is the amount of DOX loaded in the nanocarriers, since it is responsible for cell death and NP release. Fang et al. [226] developed magnetoresponsive nanocapsules composed of an iron oxide core and a tumor targeting lactoferrin shell. The anti-cancer drug-loaded NPs were able to release therapeutic agents whilst concomitantly producing intense heat after an external high frequency magnetic field was applied. Particles were released after cell death and migrated to neighboring cells. With this dual in vivo treatment mechanism, the need of a drug-induced effect for particle spreading is minimized.

Another option to achieve particle spreading was shown by Zhang et al. who developed a DOX carrier system composed of dendritic peptides, which under acidic pH J o u r n a l P r e -p r o o f conditions revealed arginine rich domains that induced membrane-breaking activity [201] . The problem can be further evaded when the NPs are able to bind to the cell cytoskeleton in order to disseminate to neighboring cells, as was shown recently by Dalmau-Mena et al. [227] . Their in vitro approach was based on the way viruses bind to the microtubule motor to disseminate and replicate. Gold NPs were modified with viral peptides that bind dynein, a microtubule motor which is used for transport by several viruses [228] . Internalized particles were able to move through the cytosol after dynein binding and progress to neighboring cells through cell projections, reducing the particle loss to extracellular compartments.

Overall mimicking the viral transmission shows great potential, especially in the field of oncology where the therapy would enormously benefit from the dose reductions enabled by these technologies. Nevertheless, an improvement of the specificity of the targeting mechanisms would pose a great advantage, as it would facilitate a much wider range of applications. Meanwhile, local therapy seems to be the ideal use of such "infective"

NPs, reducing the risk of off target effects.

A comprehensive examination of the viral characteristics has promoted the design of particles that mimic different aspects of a virus' natural behavior ( Figure 6 ). In that regard, one could argue, that in many cases, viruses do not necessarily have to reach However, even though this fundamental research has considerably advanced our knowledge and has provided the foundations for particle optimization, none of the described concepts has reached clinical translation so far, one impediment that has been found to be critical for the failure of numerous nanomedicine approaches. Therefore, it is crucial to discuss critical parameters for a successful bench-to-bedside transition of promising virus-mimetic concepts. In that regard, Hua et al. excellently reviewed J o u r n a l P r e -p r o o f current challenges for the translational development of nanomedicine approaches in general [221] .

One critical aspect that the authors identified, was that many concepts initially focused too much on a few specific formulation aspects such as novel receptor-specific ligands or other highly developed NP components instead of concentrating on the actual biological target and the entirety of respective structural requirements to reach this site of action. Accordingly, we believe that a holistic approach, i.e. the development of an "artificial virus" instead of merely equipping NPs with single viral traits, may hold more promise for a successful clinical translation of virus-inspired nanomaterials.

In that regard, the combination of structural features, such as shape and surface properties, with ligand mediated cell recognition and stimuli responsiveness has recently led to the development of formulations that are almost an exact synthetic replica of viruses. In publications such as the one by Lee et al. [29] we can see that this provides huge advantages regarding targeting capability. In their work they prepared a synthetic duplicate of the RABV by exactly matching its size, shape and surface glycoprotein. Silica-coated gold nanorods, with elongated morphology equal to the RABV were chosen to increase the cellular receptor interaction. Their close imitation of the RABV properties enabled a virus-like in vivo behavior which could suppress brain tumors after irradiation. Due to their viral surface ligands the particles interact with the virus-used targeted receptors, but the perfect combination with an exact virus-like shape and size potentiates its effect. Also the work published by Mable et al. [229] where the morphology and pH-responsiveness of the Dengue virus (DV) was mimicked with copolymer vesicles points towards the same conclusions. By imitating the framboidal morphology the DV adopts upon transition from the 28ºC mosquito vector to the 37ºC human host [230] and its additional pH-induced transformation that allows for J o u r n a l P r e -p r o o f endosomal escape, they were able to address SR-B1 overexpressing triple-negative breast cancer cells for which there are currently no targeted therapies. More so, their formulation did not accumulate in healthy or SR-B1-negative cells. The authors attributed the in vitro targeting specificity to a combination of the ligand and the Dengue-mimicking rough surface, which was associated with higher targeting efficiency through membrane deformation [229] . These examples support our assessment, that the holistic implementation of viral properties on NPs to create an "artificial virus" may help to increase the therapies´ specificity and effectivity, as individual traits seem insufficient to achieve these goals. We can probably look forward to formulations further implementing multiple viral traits to achieve the perfect "synthetic virus", which may surmount the alarming low specificity and target cell accumulation that nanomaterials achieve up to date [161, 162] .

An important question in this regard is if there will be a single universal "artificial virus formulation" only subjected to slight modifications depending on the application, or if viruses will be mimicked in accordance with their natural targets. The latter is a frequently used approach. For example, for brain targeting the RABV is often used as a model [29, 85] , for its capability of crossing the difficult BBB. Another example is the addressing of hepatic cells with particles imitating the HBV [80] [81] [82] . However, to expand the applicability to therapeutically relevant cells not naturally addressed by viruses the first approach may be a better fit. This is reinforced by the focus on high tunability of the formulations being developed. Indisputably, due to disease individuality an exact tailoring of the formulation is essential.

Apart from the structural considerations for virus-inspired NPs, cargo selection will also play a central role for the feasibility of clinical translation. In that regard, possible drug candidates not only have to possess the necessary physicochemical characteristics for J o u r n a l P r e -p r o o f sufficient encapsulation, but also have a pharmaceutical profile, that would actually benefit from virus-inspired delivery options. As already mentioned above, nucleic acidbased systems thereby rank among the most promising candidates [222, 223] , as they most closely resemble the actual viral cargo.

A widely discussed challenge for sufficient clinical adaptation is also the lack of realistic in vitro and in vivo models. In that regard, we believe that the translation of new nanomaterials including virus-inspired approaches will eventually only be successful, if a central focus is placed on realistically mimicking central physiological aspects during both in vitro and in vivo testing. This includes aspects like the implementation of physiological cell culture and NP incubation conditions [231] or the use of suitable animal models with a realistic pathophysiology. These considerations are particularly important for virus-mimetic concepts, as they are based on a refined adaptation to multiple influencing factors such as immune recognition, the penetration of physiological barriers or the responsiveness to external physicochemical stimuli.

Once virus-inspired materials have proven to be successful, another possible impediment for a clinical and later industrial scale-up will doubtlessly be the considerable structural complexity, that is oftentimes needed to incorporate multiple viral traits. The higher the intricacy of the particle design, the higher the difficulty for its precise characterization, large-scale manufacturing and regulation. In that regard, it is encouraging, that numerous researches have begun to use safe, non-toxic and FDAapproved materials for their developed formulations.

Nevertheless, additional comprehensive research is needed in an exciting field where only the pillars have been set.

J o u r n a l P r e -p r o o f

The implementation of viral features on NPs is a conquerable endeavor that has substantially influenced and improved the design of therapeutic nanomaterials.

Future investigations in the formulation field incorporating viral traits using a more holistic approach may solve the specificity and accumulation problems NPs face up to date. Overall, it is reasonable to say that viruses can be considered as a great source of inspiration for nanomaterial design with enormous potential in the field of targeted drug delivery.

There are no conflicts to declare. Phage fusion protein pVIII "-" Ag@Au nanorods -Photothermal therapy [38] J o u r n a l P r e -p r o o f 

"-" clustered -- [120] J o u r n a l P r e -p r o o f Table 3 . Heteromultivalent NP formulations.

Ref.

Ab (1) Ab (2) CD19 CD20

-Namalwa B-cell lymphoma (mouse) DOX/VIN [154] Liposome Antibody NGR peptides GD 2 AN -SH-SY5Y tumor (mouse) DOX [155] Liposome

Ab (1) Ab (2) CD19 CD20

50% of each ligand -DOX [134] Liposome Ab FA EGFR FR 3 Ab molecules + 200 folate molecules per NP -DOX [156] Liposome

Ab (1) Ab (2) ICAM ELAM 1:1 ligand ratio -- [145] Liposome

Ab (1) Ab (2) ICAM E-selectin --- [144] Liposome

Peptide (1) Peptide (2) ɑvβ3 integrin Galectin-1 --- [151] Liposome

Ab (1) Ab (2) VCAM1 E-selectin 1:1 ligand ratio -- [130] Liposome

Peptide (1) Peptide (2) ɑvβ3 integrin Galectin-1 -B16F10 tumor (mouse) - [152] Liposome Ab fragment (1) Ab fragment (2) EGFR CEA --- [157] Liposome

Peptide (1) Peptide (2) P-selectin ɑvβ3 integrin -MDA-MB-231/4T1 tumor (mouse) - [147] Liposome Peptide cRGDfc EGFR ɑvβ3 integrin 1:1 ligand ratio D2.A1 tumor (mouse) DOX [146] PEI polyplex

TfR Integrin --- [10] Au NPs Ab (1) Ab (2) FR EGFR --- [127] Au NPs FA Glucose FR GR --DOX [158] Au NPs Peptide (1) Peptide (2) EGFR TfR -U87-MG tumor (mouse)

Phtalocyanine 4 [159] PEG-PAMAM

TfR Endothelium --DOX [133] Polymer NPs 

Protein-Nanoparticle Interactions: The Bio-Nano Interface

Interaction of Functionalized Nanoparticles with Serum Proteins and its Impact on Colloidal Stability and Cargo Leaching

Effect of the protein corona on nanoparticles for modulating cytotoxicity and immunotoxicity

Protein corona significantly reduces active targeting yield

Interactions of nanoparticles with plasma proteins: implication on clearance and toxicity of drug delivery systems

Walking the line: The fate of nanomaterials at biological barriers

Virus Entry: Open Sesame

Gene therapy comes of age

Non-viral is superior to viral gene delivery

Dual-targeted polyplexes: one step towards a synthetic virus for cancer gene therapy

The 1.2-megabase genome sequence of Mimivirus

An allometric relationship between the genome length and virion volume of viruses

Spatial and Methodological Considerations for the Manufacturing of Therapeutic Polymer Nanoparticles

The effect of nanoparticle size, shape, and surface chemistry on biological systems

Targeting kidney mesangium by nanoparticles of defined size

Renal Clearance of Quantum Dots

Luminescent gold nanoparticles with efficient renal clearance

Targeting therapeutics to the glomerulus with nanoparticles

Principles of Nanoparticle Design for Overcoming Biological Barriers to Drug Delivery

Vaccine delivery: a matter of size, geometry, kinetics and molecular patterns

Nanoparticles target distinct dendritic cell populations according to their size

A biodistribution study of two differently shaped plant virus nanoparticles reveals new peculiar traits

Host cell dependence of viral morphology

Lung vascular targeting through inhalation delivery: insight from filamentous viruses and other shapes

Shape effects of filaments versus spherical particles in flow and drug delivery

Shape matters: the diffusion rates of TMV rods and CPMV icosahedrons in a spheroid model of extracellular matrix are distinct

Templated co-assembly into nanorods of polyanions and artificial virus capsid proteins

Plasmid-templated shape control of condensed DNA-block copolymer nanoparticles

Rabies Virus-Inspired Silica-Coated Gold Nanorods as a Photothermal Therapeutic Platform for Treating Brain Tumors

Synthesis, characterization and applications

The Structure of Virus Capsids

The importance of nanoparticle shape in cancer drug delivery

Self-folding polymeric containers for encapsulation and delivery of drugs

Structure and physics of viruses: An integrated textbook

Surface properties, more than size, limiting convective distribution of virus-sized particles and viruses in the central nervous system

Viral glycoproteins: biological role and application in diagnosis

Nanoparticles mimicking viral surface topography for enhanced cellular delivery

Bio-mimetic nanostructure self-assembled from Au@Ag heterogeneous nanorods and phage fusion proteins for targeted tumor optical detection and photothermal therapy

Bioreducible Fluorinated Peptide Dendrimers Capable of Circumventing Various Physiological Barriers for Highly Efficient and Safe Gene Delivery

Carrier-free nanodrug-based virus-surface-mimicking nanosystems for efficient drug/gene co-delivery

Facile Synthesis of Uniform Virus-like Mesoporous Silica Nanoparticles for Enhanced Cellular Internalization

Virus Spike and Membrane-Lytic Mimicking Nanoparticles for High Cell Binding and Superior Endosomal Escape

Self-Assembly of a Virus-Mimicking Nanostructure System for Efficient Tumor

Virus glycosylation: role in virulence and immune interactions

Importance of hemagglutinin glycosylation for the biological functions of influenza virus

A role for carbohydrates in immune evasion in AIDS

Innate immune recognition of glycans targets HIV nanoparticle immunogens to germinal centers

Virus Envelope-Like Self-Assembled Nanoparticles Based on α-CD/PEG for Antigens Targeting to Dendritic Cells

Glycosylated Artificial Virus-Like Hybrid Vectors for Advanced Gene Delivery

A review of glycosylated carriers for drug delivery

Doxorubicinloaded nanoparticles consisted of cationic-and mannose-modified-albumins for dual-targeting in brain tumors

Playing hide and seek: how glycosylation of the influenza virus hemagglutinin can modulate the immune response to infection

Human apolipoprotein e is required for infectivity and production of hepatitis C virus in cell culture

The viral protein corona directs viral pathogenesis and amyloid aggregation

Factors Affecting the Clearance and Biodistribution of Polymeric Nanoparticles

Bioinspired Shielding Strategies for Nanoparticle Drug Delivery Applications

The Protein Corona of Plant Virus Nanoparticles Influences their Dispersion Properties, Cellular Interactions, and In Vivo Fates

Probing the Surface Hydration of Nonfouling Zwitterionic and PEG Materials in Contact with Proteins

Zwitterionic surface coating of quantum dots reduces protein adsorption and cellular uptake

Strong resistance of phosphorylcholine self-assembled monolayers to protein adsorption: insights into nonfouling properties of zwitterionic materials

Nanomedicine delivery: does protein corona route to the target or off road?

Selective permeability of mucus barriers

Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus

Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers

Mucus-penetrating nanoparticles for vaginal drug delivery protect against herpes simplex virus

Design of Virus-Mimicking Polyelectrolyte Complexes for Enhanced Oral Insulin Delivery

Sub-50 nm Nanoparticles with Biomimetic Surfaces to Sequentially Overcome the Mucosal Diffusion Barrier and the Epithelial Absorption Barrier

Systematic evaluation of the toxicity and biodistribution of virus mimicking mucus-penetrating DLPC-NPs as oral drug delivery system

Biomimetic Viruslike and Charge Reversible Nanoparticles to Sequentially Overcome Mucus and Epithelial Barriers for Oral Insulin Delivery

Effect of surface properties on nanoparticle-cell interactions

The Cell Biology of Receptor-Mediated Virus Entry

Nanoparticle Multivalency Counterbalances the Ligand Affinity Loss upon PEGylation

G protein-coupled receptors function as logic gates for nanoparticle binding and cell uptake

Influenza A Virus Mimetic Nanoparticles Trigger Selective Cell Uptake

Biomimetic Nanotechnology toward Personalized Vaccines

Nanoparticle Vaccines Adopting Virus-like Features for Enhanced Immune Potentiation

Dynamics of Virus-Receptor Interactions in Virus Binding, Signaling, and Endocytosis

Inhibition of influenza virus infection by a novel antiviral peptide that targets viral attachment to cells

Incorporation of the reovirus M cell attachment protein into small unilamellar vesicles: incorporation efficiency and binding capability to L929 cells in vitro

A hepatitis B virus-derived human hepatic cell-specific heparin-binding peptide: identification and application to a drug delivery system

Hepatitis B virus preS1-derived lipopeptide functionalized liposomes for targeting of hepatic cells

Development of a virus-mimicking nanocarrier for drug delivery systems: The bio-nanocapsule

Anti-immunology: evasion of the host immune system by bacterial and viral pathogens

Low immunogenic bio-nanocapsule based on hepatitis B virus escape mutants

Targeted Brain Delivery of Rabies Virus Glycoprotein 29-Modified Deferoxamine-Loaded Nanoparticles Reverses Functional Deficits in Parkinsonian Mice

Carbon Dioxide-Generating PLG Nanoparticles for Controlled Anti-Cancer Drug Delivery

Cell penetrating peptides: A concise review with emphasis on biomedical applications

Cell-penetrating peptides: breaking through to the other side

Tat-mediated delivery of heterologous proteins into cells

Cell-Penetrating Peptides Derived from Viral Capsid Proteins

Tat peptide-mediated intracellular delivery of pharmaceutical nanocarriers

Nuclear-targeted drug delivery of TAT peptide-conjugated monodisperse mesoporous silica nanoparticles

TAT conjugated, FITC doped silica nanoparticles for bioimaging applications

Tat peptide directs enhanced clearance and hepatic permeability of magnetic nanoparticles

Tat peptidederivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells

HIV Peptide-Mediated Binding Behaviors of Nanoparticles on a Lipid Membrane

Tat Peptide as an Efficient Molecule To Translocate Gold Nanoparticles into the Cell Nucleus

Biologically active core/shell nanoparticles self-assembled from cholesterol-terminated PEG-TAT for drug delivery across the blood-brain barrier

Delivery of Intact Transcription Factor by Using Self-Assembled Supramolecular Nanoparticles

Traceable multifunctional micellar nanocarriers for cancer-targeted codelivery of MDR-1 siRNA and doxorubicin

TAT-conjugated nanoparticles for the CNS delivery of anti-HIV drugs

Rapid Detection of Exosomal MicroRNAs Using Virus-Mimicking Fusogenic Vesicles

Branched Polymer-Drug Conjugates for Multivalent Blockade of Angiotensin II Receptors

Antiandrogen gold nanoparticles dual-target and overcome treatment resistance in hormone-insensitive prostate cancer cells

A review: Integrin alphavbeta3-targeted molecular imaging and therapy in angiogenesis

RGD-based strategies to target alpha(v) beta(3) integrin in cancer therapy and diagnosis

Targeting C-type lectin receptors with multivalent carbohydrate ligands

Active Targeting Strategies Using Biological Ligands for Nanoparticle Drug Delivery Systems

Ligand density on nanoparticles: A parameter with critical impact on nanomedicine

Modular construction of multifunctional bioresponsive cell-targeted nanoparticles for gene delivery

Controlling ligand surface density optimizes nanoparticle binding to ICAM-1

Effect of ligand density, receptor density, and nanoparticle size on cell targeting

The Effect of Ligand Mobility on the Cellular Interaction of Multivalent Nanoparticles

Ligand-clustered "patchy" nanoparticles for modulated cellular uptake and in vivo tumor targeting

Living' PEGylation on gold nanoparticles to optimize cancer cell uptake by controlling targeting ligand and charge densities

Ligand Density and Linker Length are Critical Factors for Multivalent Nanoparticle-Receptor Interactions

Cryo-EM visualization of an exposed RGD epitope on adenovirus that escapes antibody neutralization

Structure of Adenovirus Complexed with Its Internalization Receptor, αvβ5Integrin

Expression of alpha v beta 5 Integrin Is Necessary for Efficient Adenovirus-Mediated Gene Transfer in the

Engineering clustered ligand binding into nonviral vectors: alphavbeta3 targeting as an example

Ligand density and clustering effects on endocytosis of folate modified nanoparticles

Heteromultivalent ligand-decoration for actively targeted nanomedicine

Perspectives on Dual Targeting Delivery Systems for Brain Tumors

Single peptide ligand-functionalized uniform hollow mesoporous silica nanoparticles achieving dual-targeting drug delivery to tumor cells and angiogenic blood vessel cells

Nanomedicine approaches in vascular disease: a review

Anti-tumor activity of paclitaxel through dual-targeting carrier of cyclic RGD and transferrin conjugated hyperbranched copolymer nanoparticles

Efficient delivery of gold nanoparticles by dual receptor targeting

High transfection efficiency of quantum dot-antisense oligonucleotide nanoparticles in cancer cells through dual-receptor synergistic targeting

Dual-ligand α5β1 and α6β4 integrin targeting enhances gene delivery and selectivity to cancer cells

Complementary targeting of liposomes to IL-1α and TNF-α activated endothelial cells via the transient expression of VCAM1 and E-selectin

Selective targeting of cancer cells using synthetic peptides

Multivalency in Drug Delivery-When Is It Too Much of a Good Thing?

PEGylated Poly(amidoamine) dendrimer-based dual-targeting carrier for treating brain tumors

Liposomes targeted via two different antibodies: assay, B-cell binding and cytotoxicity

Exploiting Receptor Competition to Enhance Nanoparticle Binding Selectivity

Multivalent binding of herpesvirus to living cells is tightly regulated during infection

HIV: Cell Binding and Entry, Cold Spring Harbor Perspect

Can dual-ligand targeting enhance cellular uptake of nanoparticles?

Synergetic Combinations of Dual-Targeting Ligands for Enhanced In Vitro and In Vivo Tumor Targeting

Constructing bifunctional nanoparticles for dual targeting: improved grafting and surface recognition assessment of multiple ligand nanoparticles

Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding

Coronavirus membrane fusion mechanism offers a potential target for antiviral development

Virus-Mimicking Cell Capture Using Heterovalency Magnetic DNA Nanoclaws

Immunoliposomes that target endothelium in vitro are dependent on lipid raft formation

The role of antibody synergy and membrane fluidity in the vascular targeting of immunoliposomes

Effective treatment of cancer metastasis using a dual-ligand nanoparticle

Spatiotemporal Targeting of a Dual-Ligand Nanoparticle to Cancer Metastasis

Precise targeting of cancer metastasis using multi-ligand nanoparticles incorporating four different ligands

Multifunctional nanoparticles: cost versus benefit of adding targeting and imaging capabilities

Heteromultivalent targeting of integrin αvβ3 and neuropilin 1 promotes cell survival via the activation of the IGF-1/insulin receptors

Synergistic Targeting of Alphavbeta3 Integrin and Galectin-1 with

Heteromultivalent Paramagnetic Liposomes for Combined MR Imaging and Treatment of Angiogenesis

Dualtargeting of αvβ3 and galectin-1 improves the specificity of paramagnetic/fluorescent liposomes to tumor endothelium in vivo

The effect of dual ligandtargeted micelles on the delivery and efficacy of poorly soluble drug for cancer therapy

Improved Outcome When B-Cell Lymphoma Is Treated with Combinations of Immunoliposomal Anticancer Drugs Targeted to Both the CD19 and CD20 Epitopes

Targeting liposomal chemotherapy via both tumor cell-specific and tumor vasculature-specific ligands potentiates therapeutic efficacy

A dual-ligand approach for enhancing targeting selectivity of therapeutic nanocarriers

Dual Targeting of Tumor Cells with Bispecific Single-Chain Fv-Immunoliposomes

Enhancement of Cell Recognition In Vitro by Dual-Ligand Cancer Targeting Gold Nanoparticles

Dual Receptor-Targeted Theranostic Nanoparticles for Localized Delivery and Activation of Photodynamic Therapy Drug in Glioblastomas

The synergistic effect of folate and RGD dual ligand of nanographene oxide on tumor targeting and photothermal therapy in vivo

Analysis of Nanoparticle Delivery to Tumours

Quantifying the Ligand-Coated Nanoparticle Delivery to Cancer Cells in Solid Tumors

Meta-Analysis of Nanoparticle Delivery to Tumors Using a Physiologically Based Pharmacokinetic Modeling and Simulation Approach

Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease

Role of alpha(v) integrins in adenovirus cell entry and gene delivery

Adenovirus Endocytosis Requires Actin Cytoskeleton Reorganization Mediated by Rho Family GTPases

Intravenous transferrin, RGD peptide and dual-targeted nanoparticles enhance anti-VEGF intraceptor gene delivery to laser-induced CNV

Cancer-Cell-Specific Mitochondria-Targeted Drug Delivery by Dual-Ligand-Functionalized Nanodiamonds Circumvent Drug Resistance

Dual targeting luminescent gold nanoclusters for tumor imaging and deep tissue therapy

Activation of influenza A viruses by host proteases from swine airway epithelium

Polymeric micelle stability

Angiotensin II AT1 receptor internalization, translocation and de novo synthesis modulate cytosolic and nuclear calcium in human vascular smooth muscle cells, Can

The Mesangial Cell in Diabetic Nephropathy

International Diabetes Federation., IDF Diabetes Atlas, IDF Diabetes Atlas

Nanoparticles Mimicking Viral Cell Recognition Strategies Are Superior Transporters into Mesangial Cells

Multivalent Nanoparticles Bind the Retinal and Choroidal Vasculature

Adenovirus-Mimetic Nanoparticles: Sequential Ligand-Receptor Interplay as a Universal Tool for Enhanced In Vitro/In Vivo Cell Identification

Stimuli-Responsive Programmed Specific Targeting in Nanomedicine

MMP-Responsive 'Smart' Drug Delivery and Tumor Targeting

Matrix metalloproteinases (MMPs) in health and disease: an overview

PEGylated self-assembled enzyme-responsive nanoparticles for effective targeted therapy against lung tumors

Dual MMP7-proximity-activated and folate receptor-targeted nanoparticles for siRNA delivery

Dual Enzymatic Reaction-Assisted Gemcitabine Delivery Systems for Programmed Pancreatic Cancer Therapy

Enhanced anticancer activity of nanopreparation containing an MMP2-sensitive PEG-drug conjugate and cell-penetrating moiety

Matrix metalloprotease 2-responsive multifunctional liposomal nanocarrier for enhanced tumor targeting

Tumor Retention of Enzyme-Responsive Pt(II) Drug-Loaded Nanoparticles Imaged by Nanoscale Secondary Ion Mass Spectrometry and Fluorescence Microscopy

Enzyme-Responsive Nanoparticles for Targeted Accumulation and Prolonged Retention in Heart Tissue after Myocardial Infarction

Therapeutic Enzyme-Responsive Nanoparticles for Targeted Delivery and Accumulation in Tumors

Overexpression of Legumain in Tumors Is Significant for Invasion/Metastasis and a Candidate Enzymatic Target for Prodrug Therapy

Legumain protease-activated TAT-liposome cargo for targeting tumours and their microenvironment

Enzyme-Responsive Polymeric Vesicles for Bacterial-Strain-Selective Delivery of Antimicrobial Agents

Enzymeresponsive polyion complex (PIC) nanoparticles for the targeted delivery of antimicrobial polymers

Enzyme-responsive nanomaterials for controlled drug delivery

Virus-Inspired Mimics Based on Dendritic Lipopeptides for Efficient Tumor-Specific Infection and Systemic Drug Delivery

Stepwise-acid-active multifunctional mesoporous silica nanoparticles for tumor-specific nucleusdelivery

Cell-penetrating and cell-targeting peptides in drug delivery

Self-regulated multifunctional collaboration of targeted nanocarriers for enhanced tumor therapy

Dendritic Cells Targeting and pH-Responsive Multilayered Nanocomplexes for Smart Delivery of DNA Vaccines

Viral Capsids Mimicking Based on pH-Sensitive Biodegradable Polymeric Micelles for Efficient Anticancer Drug Delivery

Virion-Like Membrane-Breaking Nanoparticles with Tumor-Activated Cell-and-Tissue Dual-Penetration Conquer Impermeable Cancer

An artificial virus-like nano carrier system: enhanced endosomal escape of nanoparticles via synergistic action of pH-sensitive fusogenic peptide derivatives

A Virus-Mimicking pH-Responsive Acetalated Dextran-Based Membrane-Active Polymeric Nanoparticle for Intracellular Delivery of Antitumor Therapeutics

Learning from the viral journey: how to enter cells and how to overcome intracellular barriers to reach the nucleus

Inspired by nonenveloped viruses escaping from endo-lysosomes: a pH-sensitive polyurethane micelle for effective intracellular trafficking

The Endosomal Escape of Nanoparticles: Toward More Efficient Cellular Delivery

Polycation gene delivery systems: escape from endosomes to cytosol

The Influence of Endosome-disruptive Peptides on Gene Transfer Using Synthetic Virus-like Gene Transfer System

Redox-responsive delivery systems

Advances in redox-responsive drug delivery systems of tumor microenvironment

Production of large amounts of hydrogen peroxide by human tumor cells

Redox Biology of Respiratory Viral Infections

Protein Toxin Chaperoned by LRP-1-Targeted Virus-Mimicking Vesicles Induces High-Efficiency Glioblastoma Therapy In Vivo

Viral mimicking ternary polyplexes: a reductioncontrolled hierarchical unpacking vector for gene delivery

Dual-responsive nanoparticles release cargo upon exposure to matrix metalloproteinase and reactive oxygen species

Enzyme-Targeted Nanoparticles for Delivery to Ischemic Skeletal Muscle

Peptide Brush Polymers and Nanoparticles with Enzyme-Regulated Structure and Charge for Inducing or Evading Macrophage Cell Uptake

Enzyme and Redox Dual-Triggered Intracellular Release from Actively Targeted Polymeric Micelles

Amylase-and Redox-Responsive Nanoparticles for Tumor-Targeted Drug Delivery

Virus cell-to-cell transmission

Current Trends and Challenges in the Clinical Translation of Nanoparticulate Nanomedicines: Pathways for Translational Development and Commercialization

Nanoparticle Delivery Systems for DNA/RNA and their Potential Applications in Nanomedicine

Synthetic materials at the forefront of gene delivery

A Virus-Mimetic Nanogel Vehicle

A Sequential Target-Responsive Nanocarrier with Enhanced Tumor Penetration and Neighboring Effect In Vivo

Magnetoresponsive virus-mimetic nanocapsules with dual heat-triggered sequential-infected multiple drug-delivery approach for combinatorial tumor therapy

Nanoparticles engineered to bind cellular motors for efficient delivery

Recognition of novel viral sequences that associate with the dynein light chain LC8 identified through a pepscan technique

Targeting triple-negative breast cancer cells using Dengue virus-mimicking pH-responsive framboidal triblock copolymer vesicles

Two hosts, two structures

Flow Rate Affects Nanoparticle Uptake into Endothelial Cells

Financial support from the German Research Foundation (DFG), Grant Number GO 565/17-3, is gratefully acknowledged.

DOX [195] "-" PEG copolymer NPs HepG2 tumor (mouse) "-" [198] Charge inversion "-" Silica NPs -"-" [196] Cargo release

Dendritic lipopeptide NPs -DNA [199] Tumor lysosome Polymeric micelles -DOX [200] "-" Dendritic peptide NPs SKOV3/R tumor (mouse) "-" [201] Endosomal escapeAcidic endosome PEG Liposomes -DNA [202] "-" Polymer NPs -Asiatic acid [203] "-" PEG-Polymer NPs SKOV3 tumor (mouse) DOX [205] Redox Cargo release

Polmyerosomes U87-MG tumor (mouse) Saporin [213] J o u r n a l P r e -p r o o f