Cargo-Compatible Encapsulation in Virus-Based Nanoparticles

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Cargo-Compatible Encapsulation in Virus-Based Nanoparticles Lingling Li, Chengchen Xu, Wenjing Zhang, Francesco Secundo, Chunyan Li, Zhi-Ping Zhang, Xian-En Zhang, and Feng Li Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b00679 • Publication Date (Web): 21 Mar 2019 Downloaded from http://pubs.acs.org on March 21, 2019

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Cargo-Compatible Encapsulation in Virus-Based Nanoparticles Lingling Li,†, ‡ Chengchen Xu,† Wenjing Zhang,†, ‡ Francesco Secundo,§ Chunyan Li, Zhi-Ping Zhang,† Xian-En Zhang,*,//and Feng Li*,† †State

Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences,

Wuhan, 430071, China ‡University §Institute

of Chinese Academy of Sciences, Beijing, 100049, China

of Chemistry of Molecular Recognition, National Research Council, Via Mario Bianco

9, Milan, 20131, Italy Key Laboratory of Nano-Bio Interface, Division of Nanobiomedicine and i-Lab, Suzhou Institute of Nano-Tech and Nano-Bionics, CAS, Suzhou, 215123, China //National

Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules,

Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China. *Corresponding authors: Feng Li (Email: [email protected]; phone: +86 27 8719 9279); Xian-En Zhang (Email: [email protected]; phone: +86 10 6488 8148)

ABSTRACT: Molecule encapsulation in virus-based nanoparticles (VNPs) is an emerging bioinspired way to design novel functional nanostructures and devices. Here, we report a general cargo-compatible approach to encapsulate guest materials based on the apparent critical

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assembly concentration (CACapp) of VNPs. Different from the conventional buffer-exchange method, the new method drives the reassembly of VNPs to encapsulate cargoes by simply concentrating an adequately diluted mixture of VNP building blocks and cargoes to a concentration above the CACapp. This method has been proved to work well on different types of cargoes (including inorganic nanoparticles and proteins) and VNPs. The major advantage of this method is that it can maximally preserve cargo stability and activity by providing the freedom to choose cargo-friendly buffer conditions throughout the encapsulation process. This method would benefit the realization of the potentials of VNPs and other protein nanocages as nanomaterials in diverse fields of nanotechnology.

KEYWORDS: encapsulation, cargo stability, apparent critical assembly concentration, virusbased nanoparticles, self-assembly Encapsulation inside protein nanocages is a widely used form of biomolecule organization in living organisms. For example, viral capsids package the genomes for protection against degradation and delivery into host cells;1 bacterial microcompartments (BMCs) encapsulate enzyme cascades to enable unique metabolisms.2 Inspired by nature, encapsulation of foreign cargoes in virus-based nanoparticles (VNPs) has been developed to fabricate new nanostructures. VNPs loaded with various cargoes, including inorganic nanoparticles (NPs), proteins, nucleic acids and small molecule drugs, are receiving increasing interest in catalysis, bioimaging, biosensing, delivery, and theranostics.3-9 However, limited attention has been paid to the issue concerning the stability and activity of cargoes in the encapsulation process. The reassembly of VNP subunits in the presence of cargoes is the main strategy for encapsulation. In general, VNPs are first dissociated into subunit


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oligomers (building blocks) through treatment with appropriate buffers. Then, the oligomers are mixed with cargoes, followed by buffer exchange to drive the reassembly of VNPs along with cargo encapsulation.10, 11 There is a paradox concerning the stability of VNPs: the more robust VNPs are, the better the VNP-based functional nanostructures work in practical applications; on the other hand, the more robust VNPs are, the harsher (thus harmful to the cargo to be encapsulated) the dissociating conditions are. A considerable proportion of VNPs that have attracted broad interest in the field of nanotechnology, such as VNPs derived from bacteriophage MS2, Qbeta, P22, and hepatitis B virus (HBV) are so stable that their dissociation and maintenance of the oligomer forms require extremely acidic pH (1-2) or high concentrations of urea or sodium dodecyl sulfate (SDS) (Table S1). At the initial step of encapsulation, upon mixing with oligomers under such harsh conditions, cargoes such as NPs, peptides and proteins may precipitate or denature, causing encapsulation failure or functional impairment even if encapsulated. To solve this problem, an encapsulation method with a different reassembly control principle, rather than buffer exchange, would be a general and practical solution. Viral capsid assembly has been reported to be a concentration-dependent process in living cells; capsid protein must accumulate to a signature concentration before assembly initiates.12 Adam Zlotnick et al. also published a series of studies on concentration-dependent in vitro selfassembly of viral capsids13 and measured the apparent critical assembly concentrations (CACapp) of several viral capsids.14-16 CACapp is the protein concentration above which higher-order assemblies start to form. By harnessing such a property, we herein report a simple and cargofriendly means to encapsulate guest materials inside VNPs. At a concentration below CACapp, protein oligomers do not assemble even under normal assembling buffer conditions. When the concentration is increased above CACapp, protein oligomers will assemble into VNPs. In this


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First, the CACapp of MS2 CP was determined in the assembly buffer at 4 °C and 1 atm pressure using an ultrafiltration method adapted from the measurement of critical micelle concentration (CMC).19 To this end, a series of MS2 CP dimer solutions with increasing concentrations were dialyzed against the assembly buffer to initiate self-assembly. The initial CP concentration of the input solutions was termed CI. At a very low CI, the protein remains as dimers and flows freely through the ultrafiltration membrane. As the CI increases, the protein begins to self-assemble into VNPs, which are retained by the membrane because of their much larger size. Therefore, the ratio (RFT/L) of the concentration of the CP protein in the flowthrough (FT) fraction (CFT) to that of the CP protein loaded for ultrafiltration (CL) (Figure 1a) is a function of CI, which is similar to the analysis of the CMC of micelles. In particular, the plot of RFT/L as a function of the natural logarithm of CI (lnCI) can be fitted by a decreasing Boltzmanntype sigmoidal curve (Figure 1b) whose inflection point corresponds to the CACapp value. Accordingly, the CACapp of MS2 CP was calculated to be 6.29 O. (CP dimers). We also used size exclusion chromatography (SEC) to measure MS2 CACapp, a method described by Adam Zlotnick et al.,14 resulting in a concentration of 5.75 O. (CP dimers), basically consistent with that obtained by the ultrafiltration method (Figure S1). Similarly, transmission electron microscopy (TEM) observation showed that MS2 CP dimers just began to assemble at a concentration of 5.41 O. forming very few VNPs (Figure S2). Thus, CACapp may be a useful tool for controlling VNP assembly.


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0.4 0.2 CFT

y=0.18437+0.6533/(1+exp((x -1.83924)/0.30266)), R^2=0.94548

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lnCI Figure 1. Measurement of the CACapp of MS2 VNPs using an ultrafiltration method. (a) Schematic of the ultrafiltration process. CL is the CP concentration of the reassembling product loaded for ultrafiltration; CFT is the CP concentration of the fraction that flew through the ultrafiltration membrane. (b) The plot of RFT/L as a function of lnCI, which is fitted by a decreasing Boltzmann-type sigmoidal curve. The inflection point corresponds to the CACapp value of MS2 CP. CI is the molar concentration of MS2 CP initially input for reassembly. RFT/L is the ratio of CFT to CL. To investigate the feasibility of the CACapp method for cargo encapsulation and whether cargo stability or activity can be sustained, we chose three kinds of representative negatively charged cargoes, i.e., Ag2S quantum dots (QDs),20 gold NPs (AuNPs) with different surface coatings,21 and green fluorescent protein with a net charge of -30 (-30GFP),22 since negatively charged cargoes can be preferentially incorporated by MS2 VNPs via CP-cargo electrostatic interaction.23 A brief introduction of these cargoes is given in Table S2. To confirm that the cargoes were able to bind to CP, CP-cargo and CP dimer-dimer interactions were measured using biolayer interferometry (BLI) (Figure S3). As summarized in Table 1, the affinities of dihydrolipoic acid (DHLA)-coated Ag2S QDs, DHLA-coated AuNPs,


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bis(p-sulfonatophenyl) phenyl-phosphine dihydrate dipotassium (BSPP)-coated AuNPs, DNAcoated AuNPs and -30GFP with MS2 CP dimers in the assembly buffer were in all cases very high, with the affinity constant KD values being 7.88E-11 M, 3.21E-11 M, or much less than 1.00E-12 M; the KD of inter-CP dimer interaction was 1.06E-06 M. The much higher CP-cargo affinity than that between CP dimers should be favorable for efficient recruiting of cargoes into MS2 VNPs. Table 1. Binding parameters of MS2 CP dimer alone or with cargoes measured by BLI. Cargo/CP

kon (1/Ms)

kdis (1/s)

KD (M)

DHLA-Ag2S QDs* 4.07E+05

1.00E-6

1.00E-12

DHLA-AuNPs*

5.12E+05

1.00E-6

1.00E-12

BSPP-AuNPs*

1.01E+06

1.00E-6

1.00E-12

DNA-AuNPs

3.14E+05

2.47E-05

7.88E-11

-30GFP

2.87E+06

9.20E-05

3.21E-11

CP dimer

1.04E+03

1.10E+03

1.06E-06

*The kdis and KD are out of the working ranges of the instrument, indicating very tight cargo-CP association.

DHLA-Ag2S QDs 5 nm in diameter were the first cargo to test. DHLA is an excellent ligand for NP coating. It helps Ag2S QDs maintain high fluorescence intensity after water solubilization.20, 24 The carboxyl group of DHLA has a pKa of 4.9; therefore, the starting buffer for encapsulation should have a pH above this value to deprotonate DHLA and thus to provide negative charge repulsion that makes Ag2S QDs stable. When the routine dialysis method was tried to drive encapsulation, Ag2S QDs were added into 10.5 O. CP dimers in 1 mM acetic acid and the mixture was dialyzed against the assembly buffer (see Experimental Procedures in Supporting Information for details). However, QDs


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agglomerated heavily upon addition into the acidic CP dimer solution (Figure 2a, left), resulting in failure of encapsulation but only self-assembly of empty VNPs (Figure 2b, left). Although the recovery efficiency, which is defined as the percentage of CP in the supernatant after reassembly among the CP input, was as high as 90 ± 2% (Table S3), no encapsulation was observed because of nearly complete precipitation of QDs. In another parallel experiment, we also tried an incubation method for QD encapsulation in VNPs. The incubation method went as follows: freshly prepared CP dimers (36 O.B in 1 mM acetic acid were diluted with the assembly buffer to a final concentration of 10.5 O. then mixed with Ag2S QDs and incubated for 48 h to allow reassembly (see Experimental Procedures in Supporting Information for details).18, 23, 25 In this way, the pH of the solution became less acidic, which was expected to be beneficial to the stability of QDs. However, similarly to the dialysis method, nearly complete precipitation of QDs and only empty VNPs were observed (Figure 2a and 2b, middle) with a recovery efficiency of 91 ± 8% (Table S3). Here the failure in QD encapsulation is attributed to the incomplete buffer switch and susceptibility of DHLA-coated QDs to acidic conditions. When it came to the CACapp method (Scheme 1), we diluted CP dimers with the assembly buffer adequately to a concentration of 1.8 O. (lower than the CACapp, 6.29 O.B to keep them dissociated and to achieve the assembly buffer condition. The pH value of the assembly buffer (pH 7.2) is favorable for the stabilization and encapsulation of QDs. Experimentally, the CACapp method proved to be compatible with the DHLA-Ag2S QDs (Figure 2a, right). TEM observation showed that one QD was packaged inside one MS2 VNP in most VNPs using the CACapp method (Figure 2b, right). Based on the TEM images, the VNPs containing Ag2S QDs (QD@VNPs) were morphologically uniform with a narrow size distribution and a mean diameter


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of 27.3 ± 1.9 nm (Figure 2c). Characterization with dynamic light scattering (DLS) also showed the size homogeneity of QD@VNPs with the hydrodynamic diameter peaking at 29.0 nm (Figure 2d). These features agreed well with those reported in the literature.23 The recovery efficiency was measured to be 76 ± 4%; the CP utilization efficiency, which is defined as the percentage of CP in the purified cargo@VNPs among the CP input, was 29 ± 3%; the incorporation efficiency, which is defined as the percentage of cargo-containing VNPs among all VNPs, was calculated to be 91.2% by particle counting (n=197) with TEM images (Table S3). The lower recovery efficiency should have been caused by non-specific absorption of protein on the dialysis membrane during the concentrating process. These results demonstrate that the CACapp method makes it possible to encapsulate sensitive cargoes into MS2 VNPs. As shown in Figure 2e, the fluorescence properties of the encapsulated Ag2S QDs were preserved. Even a fluorescence enhancement was observed, possibly due to better surface protection of the QDs after encapsulation.26 By virtue of the encapsulated Ag2S QDs, real-time monitoring of the in vivo dynamic distribution of MS2 VNPs was achieved with the following advantages of the QD fluorescence in the second near-infrared window (NIR-II): high spatiotemporal resolutions and deep tissue penetration for in vivo imaging.27 The fast accumulation and long-time retention of QD@VNPs in the liver, bone marrow and spleen could be clearly revealed in time course (Figure 2f and 2g). The MS2 QD@VNP structure holds potential for the development of virus-based nanodevices for different purposes, e.g., theranostics. The NIR-II imaging experiment proves that the properties of sensitive cargoes can be preserved very well when encapsulated through the CACapp method and further supports that this method is cargo-compatible.


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TEM data. The average diameter is shown as mean ± SD. (d) DLS measurement of the hydrodynamic diameter of QD@VNPs. (e) Absorbance and fluorescence (FL) spectra of QD@VNPs and free QDs at equivalent QD concentrations. The absorbance spectra of QD@VNPs and free QDs were normalized by dividing the absorbance value at a certain wavelength by that of QDs@VNPs at 400 nm; the fluorescence spectra of QD@VNPs and free QDs were normalized by dividing the fluorescence value at a certain wavelength by that of QDs@VNPs at 1200 nm. (f) NIR-II fluorescence imaging of QD@VNPs in a live mouse at different time points after tail vein injection. (g) Ex vivo bright-field and NIR-II fluorescence imaging of various organs taken from the mouse injected with QD@VNPs at 12 h post-injection. Next, the CACapp method was tested for encapsulation of 5 nm AuNPs with different surface coatings. DHLA-AuNPs, which could not be encapsulated by the dialysis and incubation methods, were successfully encapsulated by the CACapp method, with a recovery efficiency of 81 ± 10%, a CP utilization efficiency of 15 ± 4%, and an incorporation efficiency of 91.9%. (Supplementary Results and Discussion; Figure S4 and Table S3). BSPP-AuNPs could be encapsulated only by the incubation and CACapp methods (Supplementary Results and Discussion; Figure S5 and Table S3), while DNA-AuNPs were stable enough to be encapsulated by all the three methods (Supplementary Results and Discussion; Figure S6 and Table S3). Compared with the other two methods, the CACapp method showed lower recovery efficiency in most cases, but comparable CP utilization efficiency and incorporation efficiency were observed. These results indicate that the CACapp method can be suitable for encapsulation of both sensitive and non-sensitive cargoes. Better compatibility of the CACapp method in terms of NP surface coatings may be meaningful when customizing NP cargoes for specific functions because surface coatings can have profound effects on NP properties.28, 29


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In addition to NPs, protein encapsulation in VNPs also holds great potential in bioimaging, protein delivery, confined catalysis, etc.10,

30-32

Considering the charge dependence of cargo

encapsulation by MS2 VNPs, the negatively charged -30GFP was selected as a model cargo. The -30GFP has been developed by genetic mutation to prevent aggregation and to promote appropriate refolding22 and has been used extensively in bioimaging and biosensing.33, 34 After coassembly of MS2 CP dimers with -30GFP at a CP dimers/-30GFP molar ratio of 1:2 using the CACapp method, sucrose density gradient centrifugation (SDGC) analysis showed that a portion of -30GFP appeared as a fluorescence band in the middle of the SDGC tube, while free -30GFP remained at the top of the SDGC tube (Figure 3a). Analysis of the fractions harvested from the top to the bottom of the SDGC tube by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that MS2 CP and -30GFP colocalized in the fraction of the fluorescence band (Figure 3b). In addition, TEM revealed that the middle fluorescence fraction basically consisted of homogenous VNPs with low electron density inside (Figure 3c), which suggests the encapsulation of -30GFP. SDS-PAGE/densitometry analysis showed that the molar ratio of CP to -30GFP in this sample was 10:1, allowing the estimation that each VNP contained nine -30GFP molecules on average. Measurement of the VNP size on the basis of TEM data showed a narrow distribution with an average diameter of 27.1 ± 1.2 nm (Figure 3d). DLS measurement further evidenced the homogeneity of the sample with the hydrodynamic diameter peaking at 28.8 nm (Figure 3e). To confirm that -30GFP was encapsulated inside and not bound to the outer surface of MS2 VNPs, the coassembled product of CP and -30GFP was also examined by SEC (Figure 3f). A similar peak at an elution volume of approximately 9 mL (peak1) was observed for both empty MS2 VNPs and the coassembled product, indicating that they possess similar hydrodynamic


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volumes. SDS-PAGE analysis of peak 1 from the coassembled product confirmed the presence of both CP and -30GFP (Figure 3g). Due to the polyhistidine tag (His-tag) at the N-terminus of 30GFP, the sample from peak 1 was subjected to a nickel affinity column assay (Figure 3h) to evaluate protein retention. We found that the majority (nearly 80%) of the initially input protein directly flowed through the column and that the ratio of -30GFP to MS2 CP remained unchanged (10:1 prior to and after flowing through the column), supporting that -30GFP was inside MS2 VNPs rather than on the outer surface. In fact, if -30GFP was nonspecifically bound to the outer surface of VNPs, it would have been retained in the column causing a much greater decrease in the total protein content in the FT fraction. To further exclude the possibility of -30GFP binding to the outer surface of VNPs, -30GFP simply mixed with empty MS2 VNPs was analyzed by SEC, in parallel with free -30GFP of the same amount. The elution profiles of -30GFP in the absence and presence of VNPs were identical (Figure 3i), which suggests -30GFP did not bind to the outer surface of VNPs. Taken together, these results corroborate that -30GFP was encapsulated inside MS2 VNPs. For comparison, we also used the dialysis and incubation methods to encapsulate -30GFP (the CP dimers/-30GFP molar ratio=1:2), which appeared stable under the acidic dissociation condition of MS2 CP. Similarly, a series of experiments demonstrated that -30GFP was encapsulated into MS2 VNPs by the two methods (Figure S7). There was no significant difference in morphology and size distribution among the -30GFP@VNPs prepared with the three different methods (Figure 3c-3f and S7c-S7f). The recovery efficiencies of the dialysis and incubation methods were higher than that of the CACapp method, but the CP utilization efficiencies were comparable among the three methods (Table S3).


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F1 F2 F3 F4 F5* F6 F7 F8 F9 F10

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c -30GFP@VNPs-CACapp F1-F2 F3-F4 F5*

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Figure 3. Encapsulation of -30GFP into MS2 VNPs using the CACapp method. (a) Images of tubes of MS2 CP-(-30GFP) coassembled products by the CACapp method in parallel with free 30GFP. Images were taken under excitation with a handheld UV lamp (360 nm) after SDGC. The sample was fractionalized into 10 parts from top to bottom of the tube, F1-10. The asterisk marks the harvested fraction (F5), which was subsequently proved to be -30GFP@VNPs. (b) SDS-PAGE analysis of coassembled products of MS2 CP with -30GFP after separation by


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SDGC. (c) TEM image of the sample from F5, showing MS2 VNPs with low electron density. (d) Size distribution (n=215) of -30GFP@VNPs based on the TEM data. (e) DLS measurement of 30GFP@VNPs. (f) SEC analysis of MS2 CP-(-30GFP) coassembled products, in parallel with purified MS2 VNPs, -30GFP, and MS2 CP dimers. (g) SDS-PAGE analysis of the samples from peak 1 and 2 in (f). (h) Nickel column binding assay of the sample from peak 1 in (f). (i) SEC analysis of -30GFP in the absence and presence of VNPs. Equal amounts of -30GFP were loaded in the two runs. pH is an important factor that influences protein stability and activity.35, 36 An advantage of the CACapp method is that it can avoid the exposure of the cargo protein to harsh buffer conditions. Therefore, to prove that the CACapp method helps to protect cargo activity, the activity (expressed as fluorescence emission) of -30GFP encapsulated in MS2 VNPs using the CACapp method was compared with those obtained by the dialysis and incubation methods. Fluorescence emission comparison was performed by adjusting the average number of -30GFP per VNP to nine in -30GFP@VNPs prepared by all the three methods (Figure S8a). As shown in Figure 4a and 4b, the fluorescence intensity of -30GFP@VNPs prepared by the CACapp method was nearly 20% and 50% higher than those of the counterparts prepared by the incubation and dialysis methods, respectively. Mock samples of free -30GFP treated using the same conditions as the corresponding methods but without MS2 CP (Figure S8b) showed an analogous fluorescence difference (Figure 4c and 4d), supporting the fact that the impaired fluorescence resulted from exposure to the acidic condition of the encapsulation methods. Therefore, the CACapp method made it possible to choose a buffer condition that is compatible with a given cargo to protect its activity.


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1.0

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i s t i o n ap p t ed C a lys a Di a n c u b CA Un t r e I

Figure 4. Effect of encapsulation method on cargo activity (expressed as fluorescence of 30GFP). (a) Representative fluorescence (FL) spectra of -30GFP@VNPs prepared with different methods at equivalent concentrations of -30GFP (7 O 5

B7 The average numbers of -30GFP per

VNP were tuned to 9 in all methods by controlling the molar ratio of CP dimers to -30GFP. (b) Quantitative fluorescence comparison of -30GFP@VNPs prepared with different methods. For normalization, the fluorescence intensity of each sample was divided by the average fluorescence intensity of -30GFP@VNPs prepared with the CACapp methods. (c) As a mock experiment, the fluorescence spectra of free -30GFP (7 O 5

B treated by different methods were compared

likewise. (d) Quantitative fluorescence analysis of free -30GFP treated by different methods. For normalization, the fluorescence intensity of each sample was divided by the average fluorescence intensity of the untreated -30GFP. In (b) and (d), all quantitative experiments were repeated independently three times and calculations were based on the fluorescence intensity at the emission peak. By testing different kinds of cargoes, we have demonstrated that CACapp, as a property of VNPs, can be useful for cargo encapsulation in VNPs in a cargo-compatible manner. It is also interesting to look into the influence of cargo on MS2 CP reassembly by analyzing CACapp. The CACapp of MS2 CP was measured to be 3.24 O. 3.39 O. and 2.92 O. in the presence of DHLA-Ag2S QDs, DHLA-AuNPs and -30GFP, respectively. The cargoes lowered the CACapp of


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MS2 CP reassembly, which is consistent with a theoretical prediction in the literature.37 The lowering of CACapp offers a biochemical explanation for the phenomenon described in the literatures that cargoes could induce or initiate the assembly of VNPs.37,

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In a more general

sense, CACapp can be taken as a parameter for quantitatively characterizing the template-directed assembly of protein nanostructures. Finally, the CACapp method was also tested for encapsulating DHLA-Ag2S QDs in two other VNPs derived from simian virus 40 (SV40) and cowpea chlorotic mosaic virus (CCMV). Typical QD-containing structures were obtained for SV40 VNPs and CCMV VNPs, with the average diameters being 24.2 ± 1.3 nm and 27.2 ± 2.1 nm, respectively (Figure S9). The two kinds of QD@VNPs are similar to the nanostructures prepared with the dialysis method in literatures.39,

40

The results have added to the versatility of the CACapp method. Although a

limited number of VNP and cargo species were tested in this study, the CACapp method, in principle, is a general approach for guest material encapsulation in VNPs and other protein nanocages. In conclusion, we have demonstrated a cargo-friendly approach based on CACapp to encapsulate exogenous materials into VNPs. CACapp, a property of protein assemblies, adds a new dimension to the assembly control of VNPs as well as other protein nanostructures. By testing different kinds of cargoes and VNPs, we have shown that the CACapp method can be a general approach as a substitute for the conventional dialysis and incubation methods. Moreover, compared with the two conventional methods, which often start CP-cargo coassembly in a fixed dissociating buffer, the major merit of the CACapp method lies in that it provides the freedom to choose cargo-compatible buffer conditions according to the properties of a given cargo. In this way, the stability and activity of cargoes can be well preserved, which is critical for both


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successful encapsulation and downstream applications. Different techniques for concentrating proteins (e.g., dialysis against PEG, centrifugal ultrafiltration, and tangential-flow ultrafiltration) are well developed and available to fulfill this purpose. The CACapp method would open up new possibilities in the construction of cargo-loaded VNPs with desirable functions and would benefit the advancement of nanotechnologies based on VNPs and other protein nanocages. ASSOCIATED CONTENT Supporting Information. Additional information regarding results of encapsulation of 5 nm AuNPs, experimental protocols and supplementary figures and tables. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors *[email protected] *[email protected] Author Contributions All authors contributed to the experimental data and writing of the manuscript. All authors have approved the R

version of the manuscript.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT


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This research was supported by the National Natural Science Foundation of China (Nos. 31470931, 31771103 and 91527302), CAS Emergency Project of ASF Research (KJZD-SWL06), Wuhan Huanghe Talents Program of Science and Technology, and Research Foundation of Science and Technology Plan of Guangzhou, China (Nos. 201707010017). We thank Prof. Qiangbin Wang at Suzhou Institute of Nano-Tech and Nano-Bionics, CAS, for helpful discussions. We are grateful to Dr. D. Gao, B. C. Xu, and P. Zhang of the Core Facility and Technical Support, Wuhan Institute of Virology, for their technical support in transmission electron microscopy. We also appreciate the instrumental and technical support in NIR-II imaging from Suzhou NIR-Optics Technologies Co., Ltd. REFERENCES (1) Cao, B. R.; Yang, M. Y.; Mao, C. B. Accounts Chem. Res. 2016, 49, 1111-1120. (2) Kerfeld, C. A.; Aussignargues, C.; Zarzycki, J.; Cai, F.; Sutter, M. Nat. Rev. Microbiol. 2018, 16, 277-290. (3) Comellas-Aragones, M.; Engelkamp, H.; Claessen, V. I.; Sommerdijk, N. A.; Rowan, A. E.; Christianen, P. C.; Maan, J. C.; Verduin, B. J.; Cornelissen, J. J.; Nolte, R. J. Nat. Nanotechnol. 2007, 2, 635-639. (4) Jordan, P. C.; Patterson, D. P.; Saboda, K. N.; Edwards, E. J.; Miettinen, H. M.; Basu, G.; Thielges, M. C.; Douglas, T. Nat. Chem. 2016, 8, 179-185. (5) Liu, A. J.; de Ruiter, M. V.; Zhu, W.; Maassen, S. J.; Yang, L. L.; Cornelissen, J. J. L. M. Adv. Func. Mater. 2018, 201801574. (6) Wen, A. M.; Steinmetz, N. F. Chem. Soc. Rev. 2016, 45, 4074-4126.


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Cargo-Compatible Encapsulation in Virus-Based Nanoparticles

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