Virus-Based Nanoparticles of Simian Virus 40 in the Field of Nanobiotechnology

REVIEW

Nanobiotechnology www.biotechnology-journal.com Virus-Based Nanoparticles of Simian Virus 40 in the Field of Nanobiotechnology

Wenjing Zhang, Xian-En Zhang,* and Feng Li*

novel functional nanostructures for tar- Biomolecular nanostructures derived from living organisms, such as geted delivery, imaging and sensing, protein cages, fibers, and layers are drawing increasing interests as natural catalysis, immunotherapy, tissue engi- [1–3] biomaterials. The virus-based nanoparticles (VNPs) of simian virus 40 neering, and theranostics. Alarge variety of different viruses, ranging from (SV40), with a cage-like structure assembled from the major capsid protein bacteriophages and plant viruses to animal of SV40, have been developed as a platform for nanobiotechnology in the viruses and human viruses with different recent decade. Foreign nanomaterials (e.g., quantum dots (QDs) and gold shapes, sizes, and compositions, are being nanoparticles (AuNPs)) can be positioned in the inner cavity or on the utilized for nanobiotechnological innova- [4–8] outer surface of SV40 VNPs, through self-assembly by engineering the tions. Here we concentrate on the nanoparticle (NP)-protein interfacial interactions. Construction of these explorations of an animal virus, simian virus 40 (SV40) for materials purposes in hybrid nanostructures has enabled integration of different functionalities. this emerging field. This review briefly summarizes the applications of SV40 VNPs in this SV40 belongs to the polyomavirus multidisciplinary field, including NP encapsulation, templated assembly of family, which has been extensively stud- nanoarchitectures, nanophotonics, and fluorescence imaging. ied as a model virus, so the background is relatively clear. SV40 has a closed circular dsDNA genome of 5.2 kb which com- prises three parts including a non- 1. Introduction translated regulatory region, the early region that encodes the replication proteins, and thelateregionthatencodesthe Viruses are natural materials providing unique nanoscale capsid proteins (the major capsid protein VP1, and the minor scaffolds that bridge molecular biology and nanotechnology. capsid proteins VP2 and VP3) and a maturation protein Viruses possess several advantages for nanomaterials purposes, (agnoprotein).[9] SV40 infects almost all mammalian species – including precise spatial arrangement of their capsid proteins and nucleated cell types.[10 12] Therefore, SV40 was once – into well-defined structures, monodispersity, convenient prep- exploited extensively as a gene delivery vehicle.[12 14] In the aration, and addressable modification. When virus-based past decade, virus-based nanoparticles (VNPs) of SV40, materials are exploited as templates or scaffolds to construct assembled from pentamers of the major capsid protein nanoarchitectures, foreign materials can be positioned in VP1, began to attract attention as a nanomaterial for the inner cavity or/and on the outer surface of their capsid, applications in nanobiotechnology.[15,16] through either direct synthesis or controllable self-assembly. Combined with chemically synthesized nanomaterials of various properties, virus-based materials have been expanding 2. VNPs Derived From SV40 their applications in the field of nanobiotechnology, generating SV40 has a nonenveloped icosahedral capsid with a diameter of about 45 nm. The shell of SV40 virion is composed of 72 VP1 W. Zhang, Prof. F. Li State Key Laboratory of Virology pentamers including twelve 5-coordinated pentamers centered Wuhan Institute of Virology on the strict fivefold axes and sixty 6-coordinated pentamers Chinese Academy of Sciences situated at T ¼ 7 icosahedral surface lattice with only local Wuhan 430071, China fivefold symmetry. The C-terminal arms of VP1 subunits interact E-mail: [email protected] with neighboring pentamers and provide the stability basis of the W. Zhang capsid (Figure 1A and B). The CD-loops, the disulfide bonds, the University of Chinese Academy of Sciences Beijing 101407, China interfaces between strict and local pentamers, and the cation- binding sites also play important roles in the capsid stability. A Prof. X.-E. Zhang National Laboratory of Biomacromolecules single copy of VP2 or VP3 is associated with the inner surface of CAS Center for Excellence in Biomacromolecules the conical opening of each VP1 pentamer, bridging the outer Institute of Biophysics, Chinese Academy of Sciences shell and the internal viral minichromosome.[17,18] SV40 VP1 Beijing 100101, China pentamers are capable of self-assembling into polymorphic E-mail: [email protected] empty capsid-like structures in vitro as influenced by calcium DOI: 10.1002/biot.201700619 ions, pH, and ionic strength. Besides the approximately 40 nm

T¼7 72-pentamer icosahedral particles, VP1 pentamers can also Wenjing Zhang received her BS from T¼3 T¼4 form 25 to 35 nm -or -like icosahedral intermediate Zhenzhou University in 2009. She is particles and approximately 20 nm T¼1 icosahedral tiny [19,20] currently a PhD candidate at Wuhan particles, as well as tube-like assemblies (Figure 1C). The Institute of Virology, Chinese ability of SV40 VP1 pentamers to form polymorphic particles, Academy of Sciences under fl makes them exible in nanobiotechnological applications by Professor Feng Li. Her research providing more possibilities in nanostructure fabrication. interests are focused on protein fi SV40 VNPs can be readily modi ed by genetic engineering or nanostructures and their fi chemical modi cation to introduce required functions, as applications in nanobiotechnology several surface-exposed loops of VP1 provide potential sites to and immunology. display peptides of interest and the N-terminal fragment of VP1 protruding to the inner cavity provides a potential site to install – Xian-En Zhang is a distinguished functional peptide.[21 23] There are a considerable number of professor in the Institute of studies taking advantage of such structural features to equip Biophysics, Chinese Academy of functional motifs on SV40 VNPs. For example, enhanced green Sciences (CAS). He became a full fluorescence protein (EGFP) was fused to the N-terminus of VP1 professor in the Wuhan Institute of to visualize the behavior of SV40 VNPs in living cell;[23] Virology, CAS in 1992, and has therapeutic Hirulog peptide was fused to the N-terminus while published 240 peer-reviewed papers targeting peptide was inserted into the HI loop or the DE loop of and three books in biosensors, VP1 for targeted treatment of atherosclerotic plaques;[22] human nanobiology, and analytical epidermal growth factor (hEGF) was chemically conjugated to microbiology. He serves as a vice the DE loop via the hetero-bifunctional crosslinker SM(PEG) for 2 chair of the Chinese Society of Biotechnology and co-chair targeting cells overexpressing the EGF receptor.[24,25] Also, the of the Division of Nanobiotechnology, Biosensors & internal proteins VP2 and VP3 can be used to fuse cargo peptides Biochips. In 2015, he was awarded an Honorary Doctor of and proteins. By encapsulating fluorescence proteins (mCherry Science Degree by the University of Alberta, Canada. or EGFP) fused to the C-terminus of VP2/3, the uptake of SV40 VNPs by cells was tracked. Using a similar strategy, yeast cytosine deaminase (yCD), a prodrug-modifying enzyme that Feng Li is currently a principal converts 5-fluorocytosine to 5-fluorouracil, was encapsulated investigator at Wuhan Institute of into SV40 VNPs. CV-1 cells became sensitive to 5-fluorocytosine- Virology (WIV), Chinese Academy of induced cell death when challenged by the yCD-encapsulating Sciences (CAS), and the deputy SV40 VNPs.[24,26] Taken together, controllable in vitro self- director of the center for analytical assembly and the capacity to load foreign peptides or proteins microbiology and nanobiology of make SV40 VNPs a useful platform for nanobiotechnological WIV. Prof. Li received his BS from applications. Shandong University in 2004 and PhD from WIV, CAS in 2009. After postdoctoral training at Suzhou 3. SV40 VNPs for Nanobiotechnological Institute of Nano-Tech and Nano-Bionics, CAS, he moved back to WIV and started independent research in 2013. Applications His research interests include integration of protein 3.1. Encapsulation of Diverse Nanoparticles nanostructures (especially virus-based nanoparticles) with chemically synthesized nanomaterials and cutting-edge SV40 VNPs have exhibited good compatibility in encapsulating applications of these hybrid nanostructures and devices nanomaterials with different components, shapes, sizes and for delivery and sensing. surface properties. A series of nanoparticles (NPs) including [22,27–33] [29,34] CdSe@ZnS quantum dots (QDs), Ag2S QDs, gold NPs (AuNPs),[29,35,36] citrate-coated magnetic NPs (MNPs)[25] have been encapsulated by SV40 VNPs through self-assembly, (Figure 2A).[35] MNPs with diameters of 8, 20, and 27 nm can with one NP core inside one VNP (Figure 2). An effort to explore also be encapsulated in SV40 VNPs, leading to 45 nm VNPs the NP-induced SV40 VNP assembly mechanism found that (Figure 2B). The C-terminal region of VP1 was found to be there was a strong affinity between QDs and SV40 VP1 critical for the complete coverage of MNPs by VP1 pentamers ¼ pentamers (KD 2.19E-10 M), which should play an important because loss of this region caused nonspecific attachment of VP1 role in driving QD encapsulation in SV40 VNPs.[31] Meanwhile, pentamers to MNPs and incomplete coverage of MNPs.[25,37] disulfide bonds contributed by cysteine 9 and 104 of SV40 VP1 Other NPs that have been coated by SV40 VP1 include are essential for the robustness of SV40 VNPs.[33] polystyrene beads with diameters of 100 nm and 200 nm, silica SV40 VNPs showed flexibility in encapsulating materials with NPs with diameter of 110 nm, PLGA beads with diameter of various sizes and shapes. AuNPs with diameters from 5 to 30 nm 200 nm, cubic MNPs with diameter of 30 nm (Figure 2B) have been encapsulated by SV40 VNPs, resulting in VNPs and distorted surface structures of liposomes with diameter of with different diameters correlated with the size of AuNPs approximately 100 nm. VP1 pentamers may adjust their

Figure 1. Structure and self-assembly of SV40 VP1 pentamer. A) Structure model of VP1 pentamer (PDB ID: 3bwq). Reproduced with permission.[60] Copyright 2008, National Academy of Sciences. B) Negatively stained TEM image of VP1 pentamers. Scale bar, 50 nm. Reproduced with permission.[27]

C) Negatively stained polymorphic particles formed by VP1 in vitro under 1 M NaCl and 2 mM CaCl2 (pH 7.2) at room temperature. V, Virus like particle; I, intermediate particle; Ti, tiny particle; Tu, tubular structure. Scale bar, 100 nm. Reproduced with permission.[19] Copyright 2003, Microbiology Society. coordination on NPs with different sizes and shapes.[38] It is 3.2. SV40 VNP-Templated Hybrid Nanoarchitectures for interesting that SV40 VP1 is so flexible in coating highly Nanophotonics different types of nanomaterials. However, the VP1-NP and VP1- VP1 interactions as well as the adjustment of VP1 pentamers in Ordered assembly of different nanomaterials is one of the major NP encapsulation by VP1 remain to be further investigated. challenges in the field of nanotechnology. VNPs, which possess rigid Additionally, when encapsulating NPs smaller than 10 nm, SV40 3D structures with well-defined addressability, symmetry and sizes, VP1 prefers to form T¼1 icosahedral VNPs according to a three- can be unique platforms for organization of different nano- [31] – dimensional (3D) reconstruction based on cryo-EM. materials.[39 45] In this part we concentrate on SV40 VNP-templated At the same time, SV40 VNPs also showed good compatibility fabrication of tunable nanoarchitectures for nanophotonic studies. to the surface charges of NPs when encapsulating NPs. In a QD In 2011, based on the structure of VP1 pentamer (Figure 1A), encapsulation study, negatively charged mercaptopropanoic a cysteine was introduced into each VP1 subunit on the outer acid-coated QDs (MPA-QDs) and DNA-coated QDs (DNA- surface of SV40 VNPs through site mutation. Citrate-coated QDs), neutrally charged methoxy-terminated polyethylene 4.2 nm AuNPs were bound onto the outer surface of QD- glycol-coated QDs (mPEG-QDs), and positively charged containing SV40 VNPs (QD@VNPs) through gold-sulfur amine-terminated polyethylene glycol-coated QDs (NH2PEG- bonding. Each VP1 pentamer, which displayed five cysteines QDs) could be encapsulated with comparable efficiencies distributed on a circle of ca. 4 nm in diameter, was able to capture [32] (Figure 2C). Different from the common notion that one 4.2 nm AuNP. By simply tuning the ratio of QD@VNPs to negatively charged surface coatings which mimic viral nucleic AuNPs, discrete ensembles with a QD at the center and a tunable acids would promote the encapsulation process, NP encapsula- number (1, 3, 5, 6, 8, 10, and 12) of AuNPs surrounding were tion by SV40 VNPs does not rely on the surface charges of NPs. obtained (Figure 3A).[30] The distance between QD and AuNP This may be related to the unique structural organization of was fixed by the thickness of the VNP shell (ca. 8 nm). When natural SV40 virion. The interaction between the capsid and the optical nanomaterials are placed close enough, their optical minichromosome of SV40 is mediated by the minor capsid properties would interact with each other, yielding novel [18] proteins, so the encapsulation of viral genome depends much properties different from that of the individual materials. For less on the negative charges of viral genome DNA. The example, fluorescence of a fluorophore may be enhanced or encapsulating flexibility of SV40 VNPs to the surface charges of quenched when placed in the vicinity of a metal NP possessing NPs is especially helpful when NPs modified with specific strong surface plasmon resonance (SPR). The extent of functional molecules need to be encapsulated. fluorescence quenching and/or enhancement is determined Moreover, the NP-containing SV40 VNPs (NP@VNPs) could by the intensity of SPR and the distance between the two work as templates for further fabrication of complicated coupling optical materials.[46,47] Taking these tunable hybrid nanoarchitectures. Recently, using SV40 VNPs containing assemblies of AuNPs and QDs, the SPR coupling of AuNPs and 5 nm AuNPs as the template, a series of homogeneous size- energy transfer between QDs and AuNPs were quantitatively tunable AuNPs and gold@siliver core-shell NPs (Au@AgNPs) studied. The SPR coupling between the 4.2 nm AuNPs was very were synthesized inside SV40 VNPs through seeded growth of weak, due to the small particle size and relatively large inter- the pre-encapsulated 5 nm AuNPs (Figure 2D). This method particle distance (Figure 3B and C). However, the energy transfer circumvents the limitation that only gentle reaction conditions from QD to the surrounding AuNPs increased along with the can be used for seed synthesis inside viral protein cages for increase of the AuNP number, resulting in dramatic fluores- further mineralization to avoid protein impairment. Therefore, cence quenching and shortening of fluorescence life-time of this method will potentially enable fabrication of more hybrid QDs (Figure 3D and E).[30] nanostructures because more kinds of NPs with tailorable To control the number of AuNPs on QD@VNPs more [36] components and structures will be usable as seeds. precisely, a strategy to construct monofunctionalized protein

Figure 2. SV40 VNPs exhibit flexibility in encapsulating nanomaterials with different components, sizes, shapes and surface properties by self- assembly or mineralization. A) Negative-staining TEM images of SV40 VNPs encapsulating AuNPs with different sizes as indicated. Scale bar, 100 nm. Reproduced with permission.[35] Copyright 2011, Royal Society of Chemistry. B) Negative-staining TEM images of SV40 VNPs encapsulating spherical MNPs with different sizes as indicated and 30 nm cubic MNPs. Scale bars, 100 or 25 nm (insets). Reproduced with permission.[25,38] Copyright 2013, Elsevier, Copyright 2015, Elsevier. C) Negative-staining TEM images of SV40 VNPs encapsulating CdSe@ZnS QDs with different surface charges. Scale bar, 100 nm. Reproduced with permission.[32] D) Negative-staining TEM image of SV40 VNPs encapsulating Au@AgNPs (scale bar, 50 nm) and corresponding energy-dispersive X-ray spectrometry elemental mapping images (scale bar, 10 nm).[36] Copyright 2017, Springer Science+Business Media. nanostructures have been established. VP1 was simultaneously quenching effect of AuNPs on QD fluorescence was quantified equipped through genetic modification with a cysteine and a more precisely.[28] polyhistidine tag (His-tag) on the outer surface of VNPs. When On the contrary, the density of AuNPs on SV40 VNPs can also this dual-functional VP1 and nonfunctional wild-type VP1 were be increased by tuning the AuNP-protein interfacial interactions. co-assembled at an optimal ratio in the presence of QDs, the AuNPs passivated with bis(p-sulfonatophenyl)-phenylphosphine monofunctionalized QD@VNPs (QD@mfVNPs) formed and (BSPP) were able to bind to the outer surface of SV40 VNPs very were purified through Ni affinity chromatography in virtue of efficiently due to electrostatic interactions between VNPs and the His-tag. The as-obtained QD@mfVNPs worked well in AuNPs and possibly gold-amine bonding, yielding dense templating the organization of hybrid nanostructure of one-QD- clusters of AuNPs (up to 30 AuNPs per cluster) with a NP one-AuNP with constant inter-particle distance. In this way, the core. Owing to the versatility of SV40 VNPs in packaging NPs,

Figure 3. Photonic interactions between NPs were investigated using SV40 VNPs as templates. A) Scheme for SV40 VNP-templated 3D discrete hybrid AuNP (yellow)/QD (red) nanoarchitectures. B) Absorbance spectra of AuNP clusters compared with those of free AuNPs at the same concentration, showing the absorption redshift gradually increased to 5 nm as the surrounding AuNPs increased from 1 to 12, suggesting weak SPR coupling takes place among the surrounding AuNPs. C) Summary of absorbance peaks for AuNP clusters and free AuNPs. D) Fluorescence intensity of QDs with different numbers of AuNPs, suggesting the energy transfer from QDs to the surrounding AuNPs increased with the number of the surrounding AuNPs, which is similar to theoretical calculations. E) Time-resolved fluorescence measurements on (i) QD@VNPs; (ii–viii) QD@VNPs with AuNPs of 1, 3, 5, 6, 8, 10, and 12; (ix) instrument response. Reproduced with permission.[30] the core NP species are tunable in terms of NP components. SV40 VNPs have been a typical example for templated Complete ﬂuorescence quenching of both visible CdSe@ZnS fabrication of nanoarchitectures. All of the studies mentioned and Ag2S QDs with emission in the second near-infrared above have implied that SV40 VNPs are a versatile platform, window (NIR-II) by the dense clusters of AuNPs were which will open up a number of opportunities for various observed.[29] purposes through integration of different functionalities.

Figure 4. Fluorescence imaging of the behaviors of SV40 VNPs in vitro and in vivo with encapsulated QDs. A) In virtue of encapsulation of CdSe@ZnS QDs, live fluorescence imaging showed that QD@VNPs (red) colocalized with the caveolae marker, caveolin-1 (green), suggesting a [27] caveolae-mediated endocytosis of SV40 VNPs. Scale bar: 10 μm. Reproduced with permission. B) By encapsulating the Ag2S QDs, NIR-II fluorescence imaging clearly distinguished the real-time distribution of the naked and PEGylated SV40 VNPs in living mice at 12hr post injection, showing that the naked SV40 VNPs were accumulated in liver, spleen, and bone marrow, while the PEGylated SV40 VNPs were distributed much less there. Reproduced with permission.[34] Copyright 2015, American Chemical Society.

3.3. Encapsulation of Nano Probes for Biological Imaging in living cells. Dissociation of QDs from VNPs was observed to take place in endocytosis vesicles after 18–24 hr incubation of Seeing is believing. Imaging technologies have been playing QD@VNPs with living cells.[23] Multiple labeling of VNPs may critical roles in driving scientific discovery and innovation in provide more insights for understanding uncoating mecha- biology and medicine.[48] Advancements in nanotechnology have nisms of viruses as well as cargo release of delivery vectors. contributed to the development of biological imaging by In addition to fluoresence imaging by QD encapsulation, providing novel probes and imaging principles. As discussed magnetic resonance imaging (MRI) has been realized by above, SV40 VNPs are versatile in encapsulating different kinds packaging MNPs of iron oxide inside SV40 VNPs. For instance, of NPs, which has enabled the tracking of SV40 VNPs in living citrate-coated iron oxide NPs of 8, 20, and 27 nm in diameter cells and animal models by taking advantages of nano probes. were encapsulated in SV40 VNPs, respectively. The magnetic fl T QDs are well-known as uorescence nano probes due to their SV40 VNPs presented higher performance as a 2 contrast agent prominent optical properties, such as high brightness, high than the commercial ResovistTM. photostability and tunable emission spectra.[49,50] In comparison The several examples in this part have shown how with traditional dyes, QDs make real-time, highly sensitive and nanotechnology expands the vision of biological researches. long-term bioimaging much easier.[51] The first paradigm of This trend will be accelerated as more excellent nanomaterials fluorescenceimagingusingencapsulatedQDswasdemostratedin are accessible to biologists. Meanwhile, from a material science 2009. CdSe@ZnS QDs with fluorescence emission peaked at point of view, NP@VNPs with imaging capacities can serve as 592 nm were encapsulated inside SV40 VNPs by self-assembly. It building blocks for construction of complex or hierachical was found that the QD@VNPs were uptaken by living cells structures to achieve more sophisticated functions, such as through caveolar-mediated endocytosis, then travelled along the disease theranostics[22] and coupled imaging-biosperation.[23] microtubules and accumulated in the endoplasmic reticulum (Figure 4A). This process is similar to the early infection steps of wild-type SV40.[27] Therefore, QD encapsulation was put forward 4. Conclusions and Outlooks asanewstrategy forviruslabelingandtracking.Suchastrategy can eliminate the concern that QDs may affect the molecular By virtue of integrating different functional motifs on SV40 interactions between viruses and hosts when labeling on the VNPs, a variety of ideas have been examined on the SV40 VNP outer surface ofviruses.Thisstrategy has been recently adoptedfor platform, that is, tracking VNP behaviors in vitro and in vivo, labeling and tracking of other virues, such as VSV-G pseudotyped targeted imaging and therapeutics of disease in vivo, and lentivirus and human immunodeficiency virus type 1. QDs were exploring of the photonic interactions between adjacent successfullyencapsulated inside live virusesduring theirassembly plasmonic nanomaterials. All these studies have shown that process of life cycle in living cells and facilitated real-time single SV40 VNPs can be a versatile platform for constructing virus tracking in living cells with high fidelity.[52,53] multifunctional nanostructures for diverse purposes. However, visible fluorescence probes are suffering from limited Ability to rationally design protein structures, in combination tissue penetration depth when tracking the behaviors of VNPs in with development of functional materials synthesis approaches fi vivo as tissues make high absorbance, serious scattering and and protein modi cation chemistry, will yield ever more complex strong autofluorescence in the visible light region.[54] The but controllable formulations that will generate new functions. challenge has been overcome to some extent by the invention of There is also space in regard of enhancing the structure NIR-II probes (emission in the range of 1000–1700 nm), which robustness and introducing environmental responsiveness of exhibit unprecedented penetration depth and spatiotemporal SV40 VNPs when used as scaffolds or carriers. More resolutions.[55,56] By encapsulating a typical kind of these NIR-II importantly, for in vivo biomedicine applications, strategies [57,58] for controlling or regulating in vivo dynamics and biodistribu- probes, Ag2S QDs, the immigration dynamics and distribu- fi tion of SV40 VNPs in living mice were tracked in real time. SV40 tion of SV40 VNPs will greatly bene t the translation of SV40 VNPs werecleared fromthebloodstream andaccumulatedinliver, VNP-based techniques. spleen, and bone marrow within five minutes post injection, but surface PEGylation of VNPs significantly prolonged the blood circulation time and reduced accumulation in the reticuloendo- Abbreviations thelial system (Figure 4B).[34] These findings demonstrated the important relevancy between the surface properties of VNPs and AuNP, gold nanoparticle; Au@AgNP, gold@siliver core-shell nanoparti- theirinvivo behaviors. Theunderlyingmolecular mechanism may cle; BSPP, bis(p-sulfonatophenyl)-phenylphosphine; DNA-QD, DNA- coated quantum dot; EGFP, enhanced green fluorescence protein; hEGF, be investigated in terms of receptor recognition and biomolecule human epidermal growth factor; His-tag, polyhistidine tag; MNP, corona formation in future for guiding the rational design of VNP- magnetic nanoparticle; MPA-QD, mercaptopropanoic acid-coated quan- based devices for biomedical applications. tum dot; mPEG-QD, methoxy-terminated polyethylene glycol-coated Combination of QD encapsulation with classic fluorescence quantum dot; MRI, magnetic resonance imaging; NH2PEG-QD, amine- labeling methods makes multiple labeling of VNPs and enables terminated polyethylene glycol-coated quantum dot; NIR-II, the second more detailed investigation of their intracelluar transportation, near-infrared window; NP, nanoparticle; NP@VNP, NP-containing virus- based nanoparticle of simian virus 40; QD, quantum dot; QD@VNP, QD- for example, dissociation of VNPs and cargo release.[51,59] Dual- fl containing virus-based nanoparticle of simian virus 40; QD@mfVNP, colored SV40 VNPs with a red uorescence QD inside and an monofunctionalized QD@VNP; SPR, surface plasmon resonance; SV40, EGFP fused to the inward-protruding N-terminus of VP1 simian virus 40; 3D, three-dimensional; VNP, virus-based nanoparticle; subunit were constructed to monitor cargo delivery and release yCD, yeast cytosine deaminase.

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