Protein Nanocages for Delivery and Release of Luminescent

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Protein Nanocages for Delivery and Release of Luminescent Ruthenium (II) Polypyridyl Complexes Xiao Li, Yajie Zhang, Hong Chen, Jian Sun, and Fude Feng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b07038 • Publication Date (Web): 22 Aug 2016 Downloaded from http://pubs.acs.org on August 24, 2016

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ACS Applied Materials & Interfaces

Protein Nanocages for Delivery and Release of Luminescent Ruthenium (II) Polypyridyl Complexes Xiao Li,† Yajie Zhang, † Hong Chen, † ,‡ Jian Sun † and Fude Feng*,† †

Department of Polymer Science & Engineering, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing 210023, P. R. China ‡

Lab of Advanced Functional Materials, School of Environmental Science, Nanjing Xiaozhuang University, Nanjing 210013, P. R. China

Email: [email protected]

ACS Paragon Plus Environment


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ABSTRACT：

In this report, non-covalent encapsulation of hydrophobic

ruthenium(II) polyridyl complexes, Ru(bpy)2dppz2+ and Ru(phen)2dppz2+, into apoferritin cavity was achieved with high loading contents by effective prevention of Ru complex-induced protein aggregation, without disruption of protein native architecture. The Ru loaded luminescent nanocomposites have demonstrated improved water-solubility, easy manipulation, reduced cytotoxicity and enhanced cellular uptake as compared to the non-treated Ru complexes.

KEYWORDS: protein nanocage, encapsulation, drug delivery, cell imaging, luminescence

Native protein nanocages formed by self-assembly of peptides have been widely used as drug delivery vehicles due to their robust nanostructures and controllable manipulation.1 As one of the most attractive protein cage architectures, ferritin ubiquitously exists in micro-organisms, plants and animals and plays a key role in iron storage and homeostasis.2 Different from virus-like capsids, ferritin is biocompatible and non-immunogenic,3 safe to be applied as biomineralization scaffolds and magnetic resonance imaging (MRI) agents.4 After de-ironization, the hollow cage termed as apoferritin is available for delivery of various types of molecules that are accommodated into the protein interior cavity and protected from outer environment by the protein shell.2 However, in comparison with heat shock protein (Hsp) nanocage that allows small molecules to diffuse in and out through its


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large 3 nm pores, apoferritin features narrow channels on its shell so that diffuse-controlled cargo loading process is often not favorable particularly when the cargo molecules are larger.4 The channel size of apoferritin shell is not absolutely constant, which makes it possible that small molecules are allowed to diffuse in through the slightly enlarged channels under elevated temperature. By partial unfolding of protein cage structure at 45oC, incorporation of metal complex [Ru(p-cymene)Cl2]2 into ferritin cavity was reported with high loading rate.5 One concern is obvious that heat-sensitive cargoes may be heat deactivated. Alternatively, pretreatment of drug molecules by chelation with metal ions is found effective and convenient. For example, encapsulation of poorly water-soluble zinc-cored phthalocyanine was reported by the Xie group with a loading rate up to ~60 wt%, demonstrating high clinical translation potential in photodynamic therapy (PDT) treatments.6 This strategy was further developed to encapsulate doxorubicin induced by copper with a loading rate up to ~73 wt%.7 These two approaches highlight the essential role of metal ions in helping trap hydrophobic molecules into protein cavity although the mechanism remains to be elucidated. Generally, access of larger molecules into the cage interior is challenging to handle and constructs a hurdle towards broadening bio-applications of native ferritin cage. Most

currently

available

approaches

exploit

the

reversible

cage

assembling-disassembling process which can take place by urea induced protein denaturation/renaturation or pH controlled dissociation/reassociation.8 In the presence of urea, doxorubicin molecules could be encapsulated with a loading number of ~33



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molecules per cage, and the highly pH-dependent stability of the nanocomposite contributed to controllable drug release for effective tumor treatment.8 As an extensively used method, the pH alternation to achieve protein monomeric dissociation at pH 2 and re-association at high pH encounters with bottleneck problems mainly in the low loading efficiencies and intermolecular aggregation issues.9 For example, without metal helper, only one methylene blue molecule retained in the cage shell during pH-induced cage reassembly.10 In the presence of MRI contrast agent Gd, hydrophobic curcumin drug could be encapsulated at a high loading rate up to ~9.5 molecules per subunit, but with observation of protein aggregation10 which might be associated with the property of drug molecules. Ruthenium(II) polypyridyl complexes are well known photosensitizers which have been well reviewed recently

11

and some of them such as [Ru(bpy)2dppz]2+ and

[Ru(phen)2dppz]2+ are readily quenched in luminescence by the presence of water molecules in aqueous media. Recently, [Ru(bpy)2dppz]2+ was developed by the Marti group as a sensor to detect amyloid-β aggregation by its long-life metal-to-ligand charge-transfer (MLCT) luminescence.12 The remarkable “light switch” response upon strong DNA binding also makes them useful for cellular imaging and ROS-induced cancer cell killing studies.13 However, quite a few limitations in relation to their poor water solubility, slow cellular uptake, and considerable cytotoxicity are to be urgently resolved. In the present study, we prepared ruthenium nanoparticles (Ru-NPs) by encapsulation of ruthenium(II) polypyridyl complexes into biocompatible apoferritin.


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The use of apoferritin as a ruthenium complex carrier provides an opportunity to avoid serious aggregation of lipophilic cargoes in aqueous media, enhance cellular uptake and attenuate cytotoxicity. Additional manipulation of exterior surface with targeting ligands such as folic acid is also feasible. Large-sized lipophilic [Ru(bpy)2dppz]2+ (Ru2) and [Ru(phen)2dppz]2+ (Ru3) with PF6 as counter ion are selective cargo molecules with tendency to induce protein aggregation. Hydrophilic [Ru(bpy)3]2+ (Ru1), not so dependent on microenvironment in luminescence as Ru2 and Ru3, is selected for comparison (chemical structures are shown in Figure 1b. See ESI for synthetic details). The n-octanol/water partition coefficients (log P) of ruthenium complexes with Cl or PF6 as counter ion were listed in Table S1.

Figure 1. (a) Encapsulation of Ru(II) complexes Ru complexes into apoferritin. (b) Chemical structures of [Ru(bpy)3]2+ (Ru1), [Ru(bpy)2dppz]2+ (Ru2) and [Ru(phen)2dppz]2+ (Ru3).

Apoferritin was obtained as a colorless solution after de-ironization of horse spleen ferritin pretreated with thioglycolic acid and 2,2’-bipyridine.14 After careful treatments of apoferritin with KMnO4 oxidation and ascorbate reduction, the iron residue concentration was determined using the Fe2+ indicator ferrozine with



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(NH4)2Fe(SO4)2 as standard.15 Protein concentration was available following BCA assay with bull serum albumin as standard (see ESI for standard curves, Figure S2). The average number of Fe atoms per cage was calculated as the concentration ratio between Fe and protein. The results indicated there were less than 2 Fe atoms per apoferritin cage. Encapsulation of large sized ruthenium compounds has been investigated in inorganic and polymer systems but scarcely reported in protein nano platforms. 16-19 Passive diffusion of Ru1~Ru3 into apoferritin even under elevated temperature turned out unfavorable, attributed to the limited pore size of cage shell (Figure 1a). It’s expected that another strategy based on pH alteration induced protein assembly and reassembly would be advantageous since entry barrier through shell channels is bypassed. However, significant precipitation occurred during protein reassembly and particularly dialysis purification processes in the presence of Ru2 or Ru3 (Figure 1a), while no precipitate was observed with Ru1 under the identical procedure. After centrifugation, protein remaining in the pale supernatant was undetectable as checked by BCA assay, indicating full precipitation of protein induced by the Ru compounds. The precipitate formation was also observed if the Ru2 or Ru3 was simply mixed at high concentration with apoferritin at neutral pH or higher, implying that adsorption of Ru compounds may give rise to reversing the hydrophobicity/hydrophilicity property of protein. Under fluorescence microscope, the brownish precipitates emitted intense red luminescence (Figure S3). The Ru2 and Ru3 have been well investigated that they’re non-emissive in the presence of water molecules due to the quenching


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effect by hydrogen bonding with their dppz ligands.20 So it’s reasonably speculated that the hydrophobic feature of Ru compounds plays a critical role in formation of intermolecular aggregates. Considering the widespread use of hydrophobic drug molecules that are potentially causable to such kind of aggregation problem, optimization for encapsulation of large sized hydrophobic molecules, among which Ru2 and Ru3 are eligible model molecules, is urgently desired.

Figure 2. Characterization of Ru-NPs. High resolution transmission electron microscopy images (a) (scale bar = 50 nm, upper insert, 10 nm), energy-dispersive X-ray spectra (b), dynamic light scattering analysis (c) and circular dichroism of apoferritin spectra, Ru1-NPs, Ru2-NPs, and Ru3-NPs (d)

Interestingly, in acetonitrile- or DMSO- containing buffer, the isolated precipitates remained and gradual release of brownish compounds Ru2 and Ru3 into solution took place. This fact suggests that protein in the preformed aggregates was at least partially in the denatured state, and of more importance it also suggests that the denatured protein bound Ru2 and Ru3 could be separated by the presence of organic solvents. Inspired by this observation, we attempted to prepare aggregate-free Ru-NPs by introducing small fraction of organic solvent into both reassembly and



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dialysis processes (Figure 1a). 10% acetonitrile was applied in Tris buffer in order to increase water solubility of Ru compounds and eliminate non-specific adsorption of Ru compounds from protein surface. After removal of excess Ru compounds by dialysis against 10% acetonitrile in Tris buffer, additional dialysis against Tris buffer for acetonitrile displacement was continued, and finally a clear brownish Ru-NP solution without visible precipitates was achieved. The introduction of small amount of acetonitrile in the course of Ru-NP preparation process is beneficial in a bundle of pros: increase in solubility of Ru2 and Ru3, prevention of protein aggregation, clearance of excess Ru2 and Ru3 as well as the high Ru loading capacity. The same treatment was also applied to apoferritin and Ru1. To check the integrity of dialysed Ru-NPs, high resolution transmission electron microscopy (HR-TEM) was performed using uranyl acetate (UA) stained samples. As shown in Figure 2a, intact spheric cage structures were visualized with apoferritin and Ru-NPs. The punctate dense staining in uniformed shape was indicative of UA penetration into cage cavities, in contrast to the nearly unstained shells with minimal UA retardation. There’s no substantial difference in morphology between Ru-NPs and apoferritin. These findings firmly revealed cage refolding as well as Ru encapsulation processed properly. The existence of ruthenium in Ru-NPs was evidenced by the appearance of characteristic K line peak at 2.52 KeV based on energy-dispersive X-ray (EDX) analysis (Figure 2b). In good agreement with TEM results, dynamic light scattering (DLS) analysis displayed monodisperse of each sample (Figure 2c). The hydrodynamic diameters of


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Ru2-NP (13.4 ± 1.2 nm) and Ru3-NP (13.6 ± 1.9 nm) were slightly larger than that of apoferritin (11.7 ± 0.9 nm), owing to the swelling of protein cages after entrapment of abundant cargo molecules. This phenomenon was common in reported literatures.21-22 Consistent with the homogeneous protein dispersion, the ζ-potential values almost remained constant at ~-25 mV (Table 1) before and after Ru encapsulation. In addition, the circular dichroism (CD) spectra of Ru-NPs, which showed characteristic positive peak of β-sheet at 192 nm and negative peaks of α-helix at 208 nm/222 nm,23 were identical to that of apoferritin (Figure 2d), suggesting that the protein structural conformation after encapsulation treatment was in the native state. Above all, it’s safe to use acetonitrile as encapsulation helper to obtain well dispersed Ru-NPs. Table 1 Characterizations of Ru-NPs. NPs Apoferritin

a

Ru atom/protein ε λex λem -1 (M cm ) UV Vis-based ICP OES-based (nm) (nm) -1

a

Φ (%)

ζ potential (mV)

Ru1-NP

-1.46×104

-10.9

-7.53

-440

-608

-2.8

-25.2 -25.9

Ru2-NP

1.57×104

28.9

32.1

450

615

0.97

-26.1

Ru3-NP

4

29.6

39.9

453

612

0.58

-25.2

2.00×10

Φ: determined using [Ru(bpy)3]Cl2 as standard in air-equilibrated water (Φ=0.028).24

Figure 3. (a) UV spectra (—) and luminescence spectra (---) of apoferritin, Ru1-NP, Ru2-NP and Ru3-NP. (b) Plots of retained Ru percentage as a function of time. The curves were fitted in



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mono-exponential decay.

The spectroscopic properties of Ru-NPs were investigated by UV-vis absorption (Figure 3a). A broad intense absorption band showed up at 400~500 nm ranges, distinctly separated from the background signal of apoferritin. Such low-energy absorption band was originated from MLCT transition characteristic for ruthenium(II) polypyridyl complexes.25 Given the known extinction coefficients of MLCT absorption bands.20,

26-27

the Ru concentrations were available based on the

background-corrected MLCT absorption intensities. Accordingly, the Ru loading number could be calculated by dividing CRu by Cprotein, where CRu and Cprotein denote Ru concentration and protein concentration respectively. As shown in Table 1, the Ru loading number up to approx. 30 was achieved for Ru3 encapsulation, almost 3 times the number for Ru1 encapsulation. The interior of protein cage was reported to be hydrophobic,2 in favor of entrapping much more Ru2/Ru3 than Ru1. The Ru loading numbers were comparable to the values provided by ICP-OES based method (Table 1). Note that the Ru3 loading number (approx. 30) from spectrometry-based measurement was underestimated in comparison to the number (approx. 40) based on ICP-OES analysis. For Ru2 or Ru3 without dissociation-reassociation process, UV-Vis spectra (Figure S4a) showed minor MLCT absorption, ruling out the possibility of outside coating of Ru2 or Ru3. The luminescence of Ru-NPs was assayed by MLCT excitation in air-saturated Tris buffer (pH 7.4) at CRu of 10 µM. The appearance of intense MLCT emission bands centered at ~610 nm (Figure 3a) confirmed the “light switch effect” on dppz-bearing


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Ru molecules by the hydrophobic microenvironment of protein inner where Ru2 and Ru3 resided. The luminescence quantum yields (Ф) were measured in the air-saturated assay buffer (Table 1), with [Ru(bpy)3]2+ as reference.28 After 2-week storage of Ru2-NP at 4 oC, the solution remained clear with slightly lowered luminescence intensity (Figure S4b), indicative of a relatively long shelf-life of Ru2-NP under refrigerated condition. Such stability allows for post modification operations on the Ru-NPs with insignificant Ru loss. However, Ru escape from Ru-NPs occurred in the release assay performed at 37 oC. The Ru2-NP solution in dialysis bag underwent a slow decoloring process without emergence of precipitates during incubation in Tris buffer (pH 7.4) at 37 oC, suggestive of Ru moving into the outer solution. The passive diffusional escape of Ru molecules was favoured by two major factors: slight enlargement of cage pores by heat and lack of sufficient Ru binding sites at inner cage surface. To estimate the Ru release kinetics, CRu for retained Ru was determined by the luminescence-based method, assuming that CRu is proportional to the luminescence intensity (I) since I is linearly Ru2-NP concentration dependent (Figure S5). The non-emissive uncaged Ru molecules have negligible effect on I. Thereby the retention percentage is approximately acquired as (I/I0)×100%, where I0 denotes the initial emission intensity. The Ru release kinetics curves for Ru2-NP and Ru3-NP were plotted as shown in Figure 3b and fitted in a pseudo mono-exponential character of decay which afforded half-lives (t1/2) of 6.4 h and 5.9 h for Ru2-NP and Ru3-NP respectively. However, the kinetic curves didn’t approach to zero even at 48 h of incubation time. According to



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the calculation from the fitted curves, there’re fewer than 10 Ru atoms retarded in the cage. To evaluate the effect of Ru encapsulation on cytotoxicity, MTT assay was carried out at the end of 24 h post treatment of MCF-7 cells with 10 µM Ru species or 0.5 ~ 2 µM protein. The apoferritin-MeCN, which was prepared from the same procedure with Ru encapsulation except that Ru compounds were absent, didn’t show detectable difference in cell viability as compared to the control apoferritin without MeCN treatment.

Only

minor

cytotoxicity

was

seen,

which

demonstrated

high

biocompatibility of apoferritin. The FA-apoferritin showed slight higher cytotoxicity. The IC50 values were obtained as 38, 17, and 29 µM for the Ru1, Ru2 and Ru3, respectively (Figure S6). It’s normal for reported Ru complexes that showed cytotoxicity.

29

In comparison, the cell viability for Ru-NP group was improved by

10~20%. Folate-conjugated Ru-NPs (FA-Ru-NPs) were prepared by reaction of folic acid NHS ester with lysine residues on Ru-NP surface (see ESI), and exhibited slightly higher toxicity than folate-free Ru-NPs. These results depicted that Ru encapsulation is effective in reducing cytotoxicity.


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Figure 4. Confocal fluorescent microscopy images. MCF-7 cells were treated with Ru species ([Ru] = 10 µM) for 24 or 36 h (Scale bar = 20 µm).

The intracellular uptake over a period of 24 and 36 h was investigated by confocal fluorescent microscopy (Figure 4). The luminescence intensity increased in the following order: FA-Ru1-NP > Ru1-NP > Ru1, correlated with cellular uptake rate since the Ru1 luminescence was not so environment sensitive. The Ru2 and Ru3 species shared the similar cellular uptake behavior. The uptake of Ru-NP was probably mediated by the ferritin receptors expressed in tumor cell membranes.30 The more efficient cellular uptake of FA-Ru-NP was attributed to the role of folate-receptors overexpressed at the surface of folate-receptor positive MCF-7 cells. This point was supported by the observation that the Ru uptake was less efficient if cells were pre-treated with 500 µM folic acid to block folate receptors prior to incubation with Ru2-NP or Ru3-NP ( Figure S7). Based on the kinetic release assay, it’s definite that over a period of 24 h or 36 h incubation at 37 oC, the Ru2 and Ru3 were largely in the uncaged state regardless of possible intracellular protein degradation. The free Ru2 or Ru3 were luminescent upon binding intracellular biomacromolecules or membrane structures where hydrophobic microenvironments were readily available.2 That’s why luminescence was seen with the uptake of Ru2 and Ru3 species. Over a period of 24 h treatment on MCF-7 cells, the luminescent Ru2 and Ru3 were mostly located in the cytoplasm. After 36 h treatment, however, the Ru3 moved to the nucleus which became brighter than other compartments, as clearly observed with the internalization effective FA-Ru3-NP. In summary, three Ru(II) polypyridyl complexes were successfully encapsulated



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into the cavity of apoferritin in high entrapment efficiencies by harnessing the anti-aggregation effect of acetonitrile. The method is simple, benign and practical, emphasizing

the

following

merits:

firstly,

hydrophobic

molecules-induced

peptide/protein aggregation is effectively prevented; secondly, the loading capacity of large molecules is high, and the prepared Ru-NPs have uniformed intact shell-core structure; thirdly, the Ru-NPs are stable and well dispersed in aqueous media, and allow the entrapped Ru compounds to release in a diffuse-controlled kinetics profile at 37 oC; fourthly, functionalization of the prepared Ru-NPs with recognition ligands such as folic acid is feasible; fifthly, biocompatibility of the prepared Ru-NPs are ensured with lowered cytotoxicity as compared to the Ru complexes alone; and lastly, targeted delivery of large molecules into the tumor cells by promoting cellular uptake is favored. Therefore, the described method is promising for broadening the bio-application of protein nanocages in cell imaging, drug delivery and cell killing studies.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org/. Further experimental details, metal content analysis, measurements of UV/vis absorption and luminescence spectra, TEM analysis, DLS and ζ-potential measurement, CD measurement, Ru release kinetics, MTT assay, fluorescence confocal imaging (PDF).


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AUTHOR INFORMATION Correspondent Author *Email: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We’re grateful to Prof. Wei Wang (Nanjing University) for help with ζ-potential measurements. We thank the National Basic Research Program of China (2015CB856300), 1000 Young Talent Program, Collaborative Innovation Center of Chemistry for Life Sciences, and the Program for Changjiang Scholars and Innovative Research Team in University for financial support.

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(15) Fish, W. W. Rapid Colorimetric for the Quantitation of Complexed Iron in Biological Samples. Methods Enzymol. 1988, 158, 357-364. (16) Frasconi, M.; Liu, Z; Lei, J; Wu, Y.; Strekalova, E.; Malin, D.; Ambrogio, M.W.; Chen, X.; Botros, Y.Y.; Cryns, V.L.; Sauvage, J.; Stoddart, J.F. Photoexpulsion of Surface-Grafted Ruthenium Complexes and Subsequent Release of Cytotoxic Cargos to Cancer Cells from Mesoporous Silica Nanoparticles, J. Am. Chem. Soc. 2013, 135, 11603–11613. (17) Bœuf, G.; Roullin, G.V.; Moreau, J.; Gulick, L.V.; Pineda, N.Z.; Terryn, C.; Ploton, D.; Andry, M.C.; Chuburu, F.; Dukic S.; Molinari, M.; Lemercier, G. Encapsulated Ruthenium(II) Complexes in Biocompatible Poly(d,l-lactide-co-glycolide) Nanoparticles for Application in Photodynamic Therapy. ChemPlusChem 2014, 79, 171-180. (18) Dickerson, M.; Howerton, B.; Bae, Y.; Glazer, E.C. Light-sensitive ruthenium complex-loaded cross-linked polymeric nanoassemblies for the treatment of cancer. J. Mater. Chem. B 2016,4, 394-408. (19) Varpness, Z.; Suci, P. A.; Ensign, D.; Young, M. J.; Douglas, T. Photosensitizer Efficiency in Genetically Modified Protein Cage Architectures. Chem. Commun. 2009, 3726-3728. (20) Hartshorn, R. M.; Barton, J. K. Novel Dipyridophenazine Complexes of Ruthenium(II): Exploring



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Luminescent Reporters of DNA. J. Am. Chem. Soc. 1992, 114, 5919-5925. (21) Yang, M.; Fan, Q.; Zhang, R.; Cheng, K.; Yan, J.; Pan, D.; Ma, X.; Lu, A.; Cheng, Z. Dragon Fruit-Like Biocage as an Iron Trapping Nanoplatform for High Efficiency Targeted Cancer Multimodality Imaging. Biomaterials 2015, 69, 30-37. (22) Fan, R.; Chew, S. W.; Cheong, V. V.; Orner, B. P. Fabrication of Gold Nanoparticles inside Unmodified Horse Spleen Apoferritin. Small 2010, 6, 1483-1487. (23) Huard, D. J. E.; Kane, K. M.; Tezcan, F. A. Re-Engineering Protein Interfaces Yields Copper-Inducible Ferritin Cage Assembly. Nat. Chem. Biol. 2013, 9, 169-176. (24) Ryan, G.J.; Quinn, S.; Gunnlaugsson, T. Highly Effective DNA Photocleavage by Novel “Rigid” Ru(bpy)3-4-nitro- and -4-amino-1,8-naphthalimide Conjugates. Inorg. Chem. 2008, 47, 401-403. (25) Hiort, C.; Lincoln, P.; Norden, B. DNA Binding of ∆- and Λ-[Ru(Phen)2dppz]2+. J. Am. Chem. Soc. 1993, 115, 3448-3454. (26) McKinley, A. W.; Lincoln, P.; Tuite, E. M. Sensitivity of [Ru(phen)2dppz]2+ Light Switch Emission to Ionic Strength, Temperature, and DNA Sequence and Conformation. Dalton Trans. 2013, 42, 4081-4090. (27) Nakamaru, K. Synthesis, Luminescence Quantum Yields, and Lifetimes of Trischelated Ruthenium(II) Mixed-Ligand Complexes Including 3,3'-Dimethyl-2,2'-Bipyridyl. Bull. Chem. Soc. Jpn. 1982, 55, 2697-2705. (28) Latouche, C.; Lanoe, P. H.; Williams, J.A.; Guerchais, V.; Boucekkine, A. and Fillaut, J. L. Switching of Excited States in Cyclometalated Platinum Complexes Incorporating Pyridyl-acetylide Ligands (Pt–CΞC–py): a Combined Experimental and Theoretical Study. New J. Chem. 2011, 35, 2196-2202.


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Table of Contents Graphics


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Fig. 1



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Fig. 2


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Fig. 3



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Fig. 4


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Table 1 NPs

ε (M-1 cm-1)

Ru atom/protein UV Vis-based ICP OES-based

λex

λem

a

Φ

ζ potential

(nm) (nm)

(%)

(mV)

Apoferritin -Ru1-NP 1.46×104

-10.9

-7.53

-440

-608

-2.8

-25.2 -25.9

Ru2-NP

1.57×104

28.9

32.1

450

615

0.97

-26.1

Ru3-NP

4

29.6

39.9

453

612

0.58

-25.2

a

2.00×10

Φ: determined using [Ru(bpy)3]Cl2 as standard in air-equilibrated water (Φ=0.028).24



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TOC image


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Protein Nanocages for Delivery and Release of Luminescent

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