Selective attachment and release of a chemotherapeutic agent from the interior of a protein cage architecture

Flenniken, Michelle L.; Liepold, Lars; Crowley, Bridgid E.; Willits, Deborah A.; Young, Mark; Douglas, Trevor

doi:10.1039/b413435d

Cited by 158 publications

(151 citation statements)

References 19 publications

(31 reference statements)

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“…[6][7][8][9][10][11][12][13] However, it is still challenging to manipulate their self-assembly in a controlled way and to analyze their assembled products precisely at the molecular level. 14,15 In this study, we have generated two different individual mutants of a protein cage with functional groups either inside or outside of the cage ( Figure 1A).…”

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confidence: 99%

Controlled Assembly of Bifunctional Chimeric Protein Cages and Composition Analysis Using Noncovalent Mass Spectrometry

et al. 2008

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Protein cage architectures, such as viral capsids, ferritins, and heat shock proteins (Hsp), have been extensively used as model systems to study the self-assembly of macromolecular complexes [1][2][3][4][5] and as nanoreactors for materials synthesis. [6][7][8][9][10][11][12][13] However, it is still challenging to manipulate their self-assembly in a controlled way and to analyze their assembled products precisely at the molecular level. 14,15 In this study, we have generated two different individual mutants of a protein cage with functional groups either inside or outside of the cage ( Figure 1A). We chemically modified different cages and reconstructed chimeric cages with a controlled ratio of two subunit types ( Figure 1B). Using mass spectrometry, we were able to determine the compositions of the ensemble population and also of the individual chimeric cages within the population at the molecular level. A model based on a binomial distribution suggested chimeric cages are assembled by random incorporation of the two individual subunits.Mass spectrometry has been used to monitor multicomponent systems, because it can simultaneously resolve individual molecular masses present in a mixture. [16][17][18] Using a combination of electrospray ionization (ESI) 19 and a time-of-flight (TOF) mass analyzer, it is possible to determine the masses of individual protein components of a noncovalently associated macromolecular complex as well as the mass of the intact macromolecular complex without disturbing the structures. 16,[20][21][22][23] The Dps (DNA binding protein from starved cells) from the Gram-positive bacterium Listeria innocua (Li) is a member of the ferritin superfamily and prevents oxidative damage of DNA by accumulating iron atoms within its central cavity to produce an iron oxide core similar to that of ferritins. 24,25 The LiDps consists of 12 identical 18 kDa subunits that self-assemble into a hollow protein cage having tetrahedral 23 symmetry ( Figure 1A). 24 The LiDps has an outer diameter of 9 nm and an inner cavity diameter of 5 nm with 0.8 nm pores at the 3-fold axis where molecules can pass through to the interior ( Figure 1A). 24 The LiDps has been used as a template for nanomaterials synthesis of metal oxides of iron 26 and cobalt 27 as well as cadmium sulfide 28 and platinum 29 with or without modifications. The small number of subunits, robustness at high temperature, 26,27 and intrinsic biomineralizing capability 25 of the LiDps protein cage make it an attractive modifiable nanoreactor for nanomaterials syntheses. In addition, the defined small cavity size 24 allows synthesis of extremely small nanostructured materials. 29 Two individual cysteine mutants, one exposed on the interior surface and the other on the exterior surface, were generated to adapt the LiDps for selective chemical modifications. The serine residue at position 138 located in the middle of helix E where it is directed toward the inside cavity was substituted with cysteine (S138C) 29 ( Figure 1A, blue). Alternatively, for th...

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Controlled Assembly of Bifunctional Chimeric Protein Cages and Composition Analysis Using Noncovalent Mass Spectrometry

et al. 2008

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“…In particular, macromolecular prodrugs based on polymerdrug [6][7][8][9][10] and protein-drug conjugates 11,12 have received considerable attention. Unlike liposomes and polymeric micelles from which encapsulated drugs tend to be released in the bloodstream before accumulating at their target site through dissociation of thermodynamically self-assembled structures, 13,14 drug-carrier conjugates can be kept in a stable conformation if appropriate linkers are used.…”

Section: Introductionmentioning

confidence: 99%

“…[22][23][24][25] Although Hsp cages have several favorable characteristics making them suitable as drug carriers, to the best of the authors' knowledge, only a few reports have examined this possibility. 11 In this study, an Hsp cage-based prodrug was developed. Doxorubicin (DOX) was used as a model drug because it has been clinically used to treat many cancers for more than four decades.…”

Section: Introductionmentioning

confidence: 99%

“…Unlike liposomes and polymeric micelles from which encapsulated drugs tend to be released in the bloodstream before accumulating at their target site through dissociation of thermodynamically self-assembled structures, 13,14 drug-carrier conjugates can be kept in a stable conformation if appropriate linkers are used. Although the conjugates have relatively stable characteristics, the drugs can be released from the conjugates through sensing unique cellular and subcellular environmental factors, including pH, [6][7][8][9][10][11] redox potential, 9,15 and enzyme activity, 3,12,16 and can then promote cell death. In the context of pH-sensitive prodrug systems, numerous acid-cleavable linkers, including imine, As a drug carrier, this study has focused on the naturally occurring small heat shock protein 16.5 (Hsp), which originated from Methanocaldococcus jannaschii.…”

Section: Introductionmentioning

confidence: 99%

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Biological evaluation of protein nanocapsules containing doxorubicin

Toita¹,

Murata²,

Abe³

et al. 2013

IJN

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This study describes the applications of a naturally occurring small heat shock protein (Hsp) that forms a cage-like structure to act as a drug carrier. Mutant Hsp cages (HspG41C) were expressed in Escherichia coli by substituting glycine 41 located inside the cage with a cysteine residue to allow conjugation with a fluorophore or a drug. The HspG41C cages were taken up by various cancer cell lines, mainly through clathrin-mediated endocytosis. The cages were detected in acidic organelles (endosomes/lysosomes) for at least 48 hours, but none were detected in the mitochondria or nuclei. To generate HspG41C cages carrying doxorubicin (DOX), an anticancer agent, the HspG41C cages and DOX were conjugated using acid-labile hydrazone linkers. The release of DOX from HspG41C cages was accelerated at pH 5.0, but was negligible at pH 7.2. The cytotoxic effects of HspG41C-DOX against Suit-2 and HepG2 cells were slightly weaker than those of free DOX, but the effects were almost identical in Huh-7 cells. Considering the relatively low release of DOX from HspG41C-DOX, HspG41C-DOX exhibited comparable activity towards HepG2 and Suit-2 cells and slightly stronger cytotoxicity towards Huh-7 cells than free DOX. Hsp cages offer good biocompatibility, are easy to prepare, and are easy to modify; these properties facilitate their use as nanoplatforms in drug delivery systems and in other biomedical applications.

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“…Protein cage structures such as those mentioned above have been studied for their potential in materials synthesis [7,12], catalysis [13,14], drug and gene delivery [15,16], bio-imaging [17][18][19], cell targeting [20,21], and vaccine development [11]. VLPs in particular are promising, as they exist in a large range of sizes (tens to hundreds of nanometers), have well-defined, monodisperse structures, can be purified in large quantities, and can be easily modified both genetically and chemically [22][23][24][25][26][27].…”

Section: Introductionmentioning

confidence: 99%

X-ray spatial frequency heterodyne imaging of protein-based nanobubble contrast agents

et al. 2014

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Spatial Frequency Heterodyne Imaging (SFHI) is a novel x-ray scatter imaging technique that utilizes nanoparticle contrast agents. The enhanced sensitivity of this new technique relative to traditional absorptionbased x-ray radiography makes it promising for applications in biomedical and materials imaging. Although previous studies on SFHI have utilized only metal nanoparticle contrast agents, we show that nanomaterials with a much lower electron density are also suitable. We prepared protein-based "nanobubble" contrast agents that are comprised of protein cage architectures filled with gas. Results show that these nanobubbles provide contrast in SFHI comparable to that of gold nanoparticles of similar size.

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Selective attachment and release of a chemotherapeutic agent from the interior of a protein cage architecture

Abstract: The antitumor agent doxorubicin was covalently bound and selectively released in a pH dependent manner from the interior surface of a genetically modified small heat shock protein (Hsp) cage.

Cited by 158 publications

References 19 publications

Controlled Assembly of Bifunctional Chimeric Protein Cages and Composition Analysis Using Noncovalent Mass Spectrometry

Controlled Assembly of Bifunctional Chimeric Protein Cages and Composition Analysis Using Noncovalent Mass Spectrometry

Biological evaluation of protein nanocapsules containing doxorubicin

X-ray spatial frequency heterodyne imaging of protein-based nanobubble contrast agents

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