Article pubs.acs.org/Biomac
Developing Genetically Engineered Encapsulin Protein Cage Nanoparticles as a Targeted Delivery Nanoplatform Hyojin Moon, Jisu Lee, Junseon Min, and Sebyung Kang* Department of Biological Sciences, School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, Korea S Supporting Information *
ABSTRACT: Protein cage nanoparticles are excellent candidates for use as multifunctional delivery nanoplatforms because they are built from biomaterials and have a well-defined structure. A novel protein cage nanoparticle, encapsulin, isolated from thermophilic bacteria Thermotoga maritima, is prepared and developed as a versatile template for targeted delivery nanoplatforms through both chemical and genetic engineering. It is pivotal for multifunctional delivery nanoplatforms to have functional plasticity and versatility to acquire targeting ligands, diagnostic probes, and drugs simultaneously. Encapsulin is genetically engineered to have unusual heat stability and to acquire multiple functionalities in a precisely controlled manner. Hepatocellular carcinoma (HCC) cell binding peptide (SP94-peptide, SFSIIHTPILPL) is chosen as a targeting ligand and displayed on the surface of engineered encapsulin (Encap_loophis42C123) through either chemical conjugation or genetic insertion. The effective and selective targeted delivery of SP94-peptide displaying encapsulin (SP94-Encap_loophis42C123) to HepG2 cells is confirmed by fluorescent microscopy imaging. Aldoxorubicin (AlDox), an anticancer prodrug, is chemically loaded to SP94-Encap_loophis42C123 via thiol-maleimide Michael-type addition, and the efficacy of the delivered drugs is evaluated with a cell viability assay. SP94-Encap_loophis42C123-AlDox shows comparable killing efficacy with that of free drugs without the platform’s own cytotoxicity. Functional plasticity and versatility of the engineered encapsulin allow us to introduce targeting ligands, diagnostic probes, and therapeutic reagents simultaneously, providing opportunities to develop multifunctional delivery nanoplatforms.
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INTRODUCTION Over the past few decades, a wide range of nanoscale delivery platforms, including liposomes,1−3 micelles,4,5 inorganic and polymeric nanoparticles,3,6−9 and protein cage nanoparticles,10,11 has been developed. The nanometer-range size and surface manipulation of such delivery nanoplatforms generally result in enhanced permeability and retention (EPR) effects that allow deep penetration of delivered cargoes, such as therapeutics and diagnostics, and a long circulation time in the bloodstream.12,13 For the localized treatment of diseases, minimizing side-effects, and target-specific diagnosis of symptoms, various types of targeting ligands, such as peptides, chemicals, and antibodies, have been widely employed.8,13 The nanoscale delivery platforms mentioned above are generally prepared from synthetic polymers and/or inorganic materials, and their chemical complexity and heterogeneity often make it difficult to control their size, shape, and composition in a precise way. However, protein cage nanoparticles have welldefined architectures designed and built by nature.10,11 They are made of biomaterials, proteins, and composed of multiple copies of one or a few identical subunits having a highly uniform size and symmetric structure.11 Furthermore, the atomic resolution crystal structures of many protein cage nanoparticles have been © 2014 American Chemical Society
solved, and their atomic details allow us to control the numbers and positions of encapsulating cargo molecules such as drugs and diagnostic probes at the molecular level. Therefore, protein cage nanoparticles are considered to be excellent candidates for multifunctional delivery nanoplatforms. A variety of protein cage nanoparticles, such as ferritin protein cage,14−20 small Heat shock protein (sHsp),21−25 and virus-like particles (VLPs),26−40 have been used for biomedical applications over the past few decades. Ferritin is one of the most widely used protein cage nanoparticles for biomedical applications. Ferritins have been used as nanoreactors for biomineralizing iron oxides18,19 as well as nanocontainers for carrying diagnostic and/ or therapeutic cargo molecules.14−17 Their applications for MR imaging and the targeted delivery of cargo materials have been extensively studied.14,18,19 Another group of extensively studied protein cage nanoparticles are the VLPs, which are generally derived from viral capsids, especially bacterial viruses. VLPs have been applied as the targeted delivery vehicles of imaging probes Received: July 22, 2014 Revised: August 28, 2014 Published: September 2, 2014 3794
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Scheme 1. Schematic Representation of Encapsulin Utilized as a Versatile Modular Nanoplatform for the Targeted Delivery of Drugs and Fluorescent Probesa
a The position to introduce the SP94-peptide is indicated in green. SP94-peptide (blue) with linker (yellow) was genetically or chemically introduced onto the exterior surface of the assembled encapsulin to target surface markers of HCC cells. The corresponding anticancer drug aldoxorubicin (AlDox) was chemically attached to Encap_loophis42C123 and delivered to the target cells.
cancer cells was evaluated using a cell viability assay. A novel encapsulin-based delivery nanoplatform was established for the first time. This platform is as robust as ferritin and genome-free, unlike other VLPs, such as Qβ and CMV capsids, and even has a large enough cavity to easily encapsulate cargo molecules. The genetic and chemical plasticity of encapsulin allows us to incorporate active cell-specific targeting ligands and diagnostic and/or therapeutic molecules simultaneously.
and therapeutic drugs as well as for vaccine development as antigen delivery platforms.34,35 A novel protein cage nanoparticle, encapsulin, was found in the thermophilic bacteria, Thermotoga maritima, and this crystal structure has been recently solved.41 Encapsulin is assembled from 60 copies of identical 31 kDa monomers having a thin and icosahedral T = 1 symmetric cage structure with interior and exterior diameters of 20 and 24 nm, respectively.41 Although the exact function of encapsulin in T. maritima has not yet been clearly elucidated, a recent study reported that encapsulin serves as a cellular compartment that encapsulates proteins such as DyP (dye decolorizing peroxidase) and Flp (ferritin like protein), which are involved in oxidative stress responses.41,42 The encapsulated proteins have a specific peptide tag at their Ctermini, and it was postulated that this peptide tag leads them to bind to the interior surface of encapsulin. This study implied that encapsulin has a large enough central cavity (20 nm inner diameter) to encapsulate a large amount of therapeutic and/or diagnostic reagents, indicating its potential to be developed as a robust cargo delivery nanoplatform.41,43 In the present study, a heat stable encapsulin variant was constructed through genetic engineering, and the utility of the engineered encapsulin as a versatile drug delivery platform was demonstrated (Scheme 1). Hepatocellular carcinoma (HCC)targeting peptide (SP94-peptide, SFSIIHTPILPL)24,25 was displayed as a targeting ligand on the surface of encapsulin through both chemical and genetic manipulations. Subsequently, fluorescent probes and prodrug molecules such as doxorubicin (AlDox) were chemically attached through thiol-maleimide Michael-type addition and used to treat HepG2 cells. Doxorubicin was released in a pH-dependent manner from delivered encapsulin in the acidic environment of the tumor cells. The targeted delivery of engineered encapsulin was verified with in vitro cell imaging, and the efficacy of the delivered cages to the
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MATERIALS AND METHODS
Genetic Modification of WT Encapsulin and Protein Cage Purification. Hexahistidine with a linker (GGGGGGHHHHHHGGGGG) was inserted between residues 42 and 43 of WT encapsulin (Encap_loophis42) by an established polymerase chain reaction (PCR) protocol using primers (forward, 5′-agctagcggaggaggaggaggatatggctgggaatatgctgcacaccca-3′; reverse, 5′-agctagcgtggtggtggtggtggtgtcctcctcctcctccgggaccttctacgtcaacgaatttacgt-3′) containing extra nucleotides and pET-30b based plasmids containing genes encoding WT encapsulin.44 The cysteine residue at position 197 of Encap_loophis42 was substituted with serine (Encap_loophis42C123). The SP94-peptide (SFSIIHTPTLPL) was inserted between residues 138 and 139 of Encap_loophis42C123 by an established PCR protocol (SP94Encap_loophis42C123) using primers (forward, 5′-aaactagtagctttagcattattcataccccgattctgccgctgggaggaattgagagtggaagcactccgaaagac-3′; reverse, 5′-aaactagtacctcccttccgttcttcaaaggacagcaagcc-3′). Peptide insertion and site-directed mutagenesis were confirmed by DNA sequencing. Encap_loophis42C123 and SP94-Encap_loophis42C123 DNAs were transformed into competent Escherichia coli strain BL21 (DE3), and the protein cages were overexpressed in E. coli. The pelleted E. coli cells from 1.0 L of culture were resuspended in 35 mL of phosphate buffer (50 mM sodium phosphate and 100 mM sodium chloride, pH 6.5). Lysozyme was added, and the solution was incubated for 30 min at 4 °C. The suspension was sonicated for 10 min in 30 s intervals and subsequently centrifuged at 12 000g for 1 h at 4 °C. SP94-Encap_loophis42C123 protein cage was purified by size exclusion chromatography (SEC) after heat precipitation for 10 min at 65 °C.44 3795
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Figure 1. Characterization of WT encapsulin, Encap_loophis42C123, and SP94-Encap_loophis42C123. (A) Molecular mass measurements of dissociated subunits of WT encapsulin (bottom), Encap_loophis42C123 (middle), and SP94-Encap_loophis42C123 (top). Calculated and observed molecular masses are indicated. (B) Transmission electron microscopy images of negatively stained WT encapsulin (bottom), Encap_loophis42C123 (middle), and SP94-Encap_loophis42C123 (top) with 1.5% uranyl acetate. (C) Size exclusion elution profiles of WT encapsulin (bottom), Encap_loophis42C123 (middle), and SP94-Encap_loophis42C123 (top). (D) Dynamic light scattering measurements of WT encapsulin (bottom), Encap_loophis42C123 (middle), and SP94-Encap_loophis42C123 (top). Chemical Modifications of Encap_loophis42C123 and SP94Encap_loophis42C123. Encap_loophis42C123 and SP94-loophis42C123 were incubated with 5 mol equivalents of fluorescein-5maleimide (F5M) at room temperature with vigorous shaking
overnight. Reactions were dialyzed against phosphate buffer (50 mM sodium phosphate and 100 mM sodium chloride, pH 7.5) overnight to remove unreacted F5M. For SP94-peptide conjugation, Encap_loophis42C123 was incubated with 10 mol equivalents of SMCC chemical 3796
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cross-linker at room temperature with vigorous shaking for 3 h, and the reaction was dialyzed against phosphate buffer (50 mM sodium phosphate and 100 mM sodium chloride, pH 6.5) overnight. Subsequently, 10 mol equivalents of SP94-peptide was incubated with Encap_loophis42C123-SMCC with vigorous shaking overnight and dialyzed against the same buffer (50 mM sodium phosphate and 100 mM sodium chloride, pH 6.5) overnight. To conjugate aldoxorubicin (AlDox, INNO-206, CytRx) with SP94Encap_loophis42C123, SP94-Encap_loophis42C123 was dialyzed against HEPES buffer (50 mM HEPES, pH 9.0) overnight to exchange the buffer and incubated with 5 mol equivalents of AlDox for 3 h at room temperature with vigorous shaking. Unreacted AlDox was removed using a spin column (Bio-Rad). Mass Spectrometry of Modified Encapsulin Protein Cages. For ESI-TOF analysis, WT encapsulin, Encap_loophis42, and SP94Encap_loophis42 protein cages were loaded onto a MassPREP microdesalting column (Waters) and eluted with a gradient of 5−95% (v/v) acetonitrile containing 0.1% formic acid at a flow rate of 500 μL/ min. The molecular masses of each species can be determined from the charges and the observed mass-to-charge (m/z) ratio values. Mass spectra were acquired in the range of m/z 500−3000 and deconvoluted using MaxEnt1 from MassLynx version 4.1 to obtain the average mass from multiple charge state distributions.44 Cell Culture and Confocal Fluorescence Microscopy. HepG2 cells were incubated in RPMI 1640 medium with 10% fetal bovine serum (FBS) and 1% streptomycin in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Cells were grown on microscope cover glass (18 mm Ø) in a 12-well culture plate (SPL, 30012). The cells were fixed with 4% paraformaldehyde in PBS and washed two times with PBS containing 0.1% Tween-20. The fixed cells were blocked with 5% BSA, 5% FBS, and 0.5% Tween-20 in PBS at 4 °C for 12 h, and blocking buffer was aspirated. SP94-fEncap_loophis42C123 and fEncap_loophis42C123SMCC-SP94 were treated for 18 h at 4 °C. In the same way, fEncap_loophis42C123 was treated as a negative control (final concentration 200 nM). Before sealing, the cells on the cover glass were washed three times (15 min), and nuclei were stained with DAPI. Images of stained substrates were collected using an Olympus Fluoview FV1000 confocal microscope (Olympus, UOBC). MTT Assay. The cytotoxicity of SP94-Encap_loophis42C123, AlDox-SP94-Encap_loophis42C123, and free AlDox was evaluated with HepG2 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability. Cells were seeded into 96-well plates with an initial cell density of 2.5 × 104 cells/well and grown in medium (10% FBS and 1% streptomycin in RPMI 1640) for 2 days at 37 °C. After adding SP94-Encap_loophis42C123, AlDox-SP94-Encap_loophis42C123, and free AlDox to cells, the cells were incubated at 37 °C for 5 h and washed with 200 μL of fresh medium for 36 h, and the cells were treated for 4 h in 200 μL of medium containing 0.5 mg/mL of MTT. Then, the medium was replaced by 200 μL of dimethyl sulfoxide (DMSO) to dissolve the formazan crystals formed by viable cells. Absorbance was measured at 595 nm using a multiscanner (TECAN). SP94-Encap_loophis42C123 was used as a negative control.
confirmed by TEM images (Figure 1B, bottom). However, WT encapsulin was eluted at void volume in SEC, which is much earlier than was expected (Figure 1C, bottom). In addition, the ratio of absorbance at 260 and 280 nm was much higher than expected (260 nm/280 nm = 1.20−1.30). DLS measurements confirmed a significantly large hydrodynamic diameter of 182 nm (Figure 1D, bottom), which was expected to be 24 nm based on a previous crystallographic study.41 These data imply that nucleic acids may randomly attach to the exterior surface of WT encapsulin resulting in a huge hydrodynamic diameter. Although encapsulin was isolated from the thermophilic microorganism T. maritima, the purified WT encapsulin was not stable (or did not maintain its cage architecture) at high temperatures (55 °C or higher) (Figure S1A). To remove randomly associated nucleic acids, various purification methods, such as ion exchange chromatography and protamine sulfate treatment, were carried out. However, nucleic acids associated with WT encapsulin could not be clearly removed. As an alternative approach to purify encapsulin, six consecutive histidine residues (His-tag) were genetically added to the C-terminus of WT encapsulin, which is known to be exposed to the exterior surface,41 and immobilized metal affinity chromatography (IMAC) was performed. Unfortunately, the C-terminal His-tags introduced into WT encapsulin did not interact with the Ni-NTA column, indicating that the C-terminus of encapsulin is not fully exposed to the exterior surface. N-terminal and several other loop regions (loops between β strands) were alternatively selected as candidates to insert His-tags based on the atomic structural information. Among these candidates, encapsulin that has six consecutive histidines with linker residues (GGGGGHHHHHHGGGGG) at position 42 (Encap_loophis42) or 138 (Encap_loophis138) exhibited unusual heat stability (Figure S1B,C). Loop regions containing residue 42 or 138 were located within the interior surface or exposed on the exterior surface (Figure S2A,B). Encap_loophis42 became stable even at temperatures as high as 90 °C (Figure S3). The heat stability of the Encap_loophis42 template allows us not only to readily increase the purity of encapsulin but also to use it as a metal-binding template for functional nanomaterials synthesis, like ferritin and other heatstable protein cages.18,19 Encap_loophis42 was used as a template hereafter for developing a drug delivery nanoplatform instead of WT encapsulin. Encapsulin subunits have two intrinsic cysteines, residues 123 and 197, and cysteine residues of encapsulin can be used as sites for selectively conjugating diagnostic probes like fluorescent dyes or/and therapeutic molecules such as drugs. The cysteine residue at position 197, which is known to be located at the intersubunit interface but does not participate in disulfide bond formation because the interfacial cysteine residue could make the cage architecture unstable upon chemical modifications, was genetically substituted with serine (C197S, Encap_loophis42C123). The other cysteine, Cys123, is located on the exterior surface of encapsulin. Amino acid substitution was confirmed by measuring the molecular mass of the dissociated subunit (Figure 1A, middle). As a result, Encap_loophis42C123 has one cysteine residue per subunit and has the same heat stability. Although there was no significant size change or dissociation observed by TEM (Figure 1B, middle), purified Encap_loophis42C123 eluted much later than that of WT encapsulin in SEC (Figure 1C, bottom and middle), demonstrating that the hydrodynamic size of Encap_loophis42C123 becomes much smaller than that of WT encapsulin. The ratio of absorbance at 260 and 280 nm was
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RESULTS AND DISCUSSION To utilize encapsulin as a multifunctional delivery nanoplatform, we prepared a gene encoding wild-type encapsulin (WT encapsulin) and subcloned it into the IPTG-inducible pET-30b expression vector. The overexpressed WT encapsulin was purified using an ultracentrifugation protocol described in a recent paper by Sutter et al.41 The biophysical properties of purified WT encapsulin were examined using electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS), size exclusion chromatography (SEC), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The subunit molecular mass of WT encapsulin was measured to be 30463.0 Da, which is in good agreement with the corresponding calculated value of 30461.7 Da (Figure 1A, bottom), and their uniform size distribution with diameters of 23−24 nm was 3797
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Figure 2. Fluorescent microscopy images of HepG2 cells treated with fEncap_loophis42C123 (A−C), fEncap_loophis42C123-SMCC-SP94 (D−F), and SP94-fEncap_loophis42C123 (G−I). DAPI (left rows), fluorescein (middle rows), and merged (right rows) images are presented. fEncap_loophis42C123, fEncap_loophis42C123-SMCC-SP94, and SP94-fEncap_loophis42C123 are visualized as green, and nuclei are shown in blue.
fluorescein-5-maleimide (F5M) or prodrug, Aldoxorubicin (AlDox), to block the thiols of Encap_loophis42C123 (C123). In this study, F5M was used as a fluorescent probe for confocal microscopy imaging, and the same cysteine sites were also used for attaching prodrugs (AlDox) (see below). After chemical conjugation, the degree of modification and shell integrity of the chemically modified Encap_loophis42C123 were examined with ESI-TOF MS, SEC, and TEM. The molecular mass of dissociated subunits of fluorescein-attached Encap_loophis42C123 (fEncap_loophis42C123) was measured to be 32426.0 Da (calc. 32424.6 Da, Figure S4A), adding the additional mass of F5M (427.4 Da) to the subunit mass of Encap_loophis42C123 (31998.0 Da, Figure 1A, middle). Although SMCCSP94 was subsequently attached onto the surface of fEncap_loophis42C123, the exact amount of conjugations could not be determined due to the heterogeneity and complexity of multistep chemical conjugations. In SEC, fEncap_loophis42C123-SMCC-SP94 eluted at the same position as that of Encap_loophis42C123 (Figure S4B), demonstrating that the serial chemical conjugations of targeting peptide SP94 and F5M do not alter the architecture of the encapsulin or induce the aggregation or dissociation of encapsulin. The TEM image of negatively stained fEncap_loophis42C123-SMCCSP94 also confirmed their intactness and uniform size distribution (23−24 nm diameter) (Figure S4C). To assess the capability of targeted delivery of fEncap_loophis42C123SMCC-SP94, we used it to treat HepG2 cells and monitored the cells with confocal fluorescence microscopy.24,25 While fEncap_loophis42C123-SMCC-SP94 efficiently bound to HepG2 cells, fEncap_loophis42C123 without targeting SP94peptide did not bind (Figure 2). This result indicates that the
significantly decreased, implying that there is no apparent nucleic acid association with encapsulin (Figure 1C, middle). DLS data showed a hydrodynamic diameter of Encap_loophis42C123 of 22.1 nm, consistent with SEC and crystallographic studies (Figure 1D, middle).41 SEC and DLS data indicated that the hydrodynamic properties of Encap_loophis42C123 were remarkably different from those of WT encapsulin (Figure 1C, D, middle). These results suggest that the heating procedure effectively removed randomly associated nucleic acids from Encap_loophis42C123 or that the insertion of consecutive histidine residues changed the surface properties of encapsulin so that nucleic acids were prevented from associating with the surface of encapsulin. To give encapsulin targeting capability, SP94-peptide was chemically synthesized with an N-terminal cysteine (CGGSFSIIHTPTLPL), which is well-known as a hepatocellular carcinoma (HCC) binding peptide,24,25 and it was then covalently attached to the exterior surface of Encap_loophis42C123 as a targeting ligand via a chemical cross-linker, SMCC. SMCC is a popular heterobifunctional cross-linker that contains N-hydroxysuccinimide (NHS) ester at one end and a maleimide moiety at the other end.45 NHS esters react with primary amines at pH 7.0−9.0 to form amide bonds, whereas maleimides react with thiol groups at pH 6.5−7.5. Therefore, SMCC was first linked to lysine residues on the surface of Encap_loophis42C123 through NHS ester/amine interactions to present maleimide functional groups, and SP94-peptide with an N-terminal cysteine was subsequently attached to the surface of Encap_loophis42C123 through thiol-maleimide Michael-type addition (Encap_loophis42C123-SMCC-SP94). Prior to SMCC conjugation, Encap_loophis42C123 was treated with either 3798
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Figure 3. In vitro assay for the binding and release of drug molecules. (A) UV/vis absorption spectra of AlDox-SP94-Encap_loophis42 (red, circles) and SP94-Encap_loophis42C123 only (black, squares) at pH 9.0. A standard absorption curve of concentration-dependent AlDox is plotted (inset). (B) Time-dependent release profile of AlDox from SP94-Encap_loophis42C123 at pH 5.5.
(Figure 2), suggesting that the genetically introduced SP94peptide is successfully presented on the surface of encapsulin and allowed them to bind to the target cells specifically. These data demonstrate that encapsulin can be utilized as a nanoplatform that can acquire specific targeting and imaging functionalities simultaneously without complex multistep chemical conjugations. Although both chemical and genetic approaches for targeting ligand presentations exhibited similar levels of targeting capability, the genetic approach was much simpler for further use and it was easy to control the number and position of the targeting ligand, SP94-peptide, resulting in the reproducible formation of a HCC-targeting delivery nanoplatform. Therefore, we focused on investigating the efficacy of drug delivery with genetically engineered encapsulin (SP94-Encap_loophis42C123). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed using SP94Encap_loophis42C123 as a targeted delivery nanoplatform to test whether drugs delivered by encapsulin can effectively kill the target cells. The 6-maleimidocaproyl hydrazone prodrug of doxorubicin (AlDox)46 was chemically loaded onto the cysteine residues (C123) of SP94-Encap_loophis42C123 (AlDox-SP94-Encap_loophis42C123) through thiol-maleimide Michael-type addition and used to treat HepG2 cells. To quantify the amount of AlDox per protein subunit, the absorbance of AlDox-conjugated SP94-Encap_loophis42 (AlDox-SP94-Encap_loophis42) was measured at 495 nm, and the absorbance was compared to that of a standard curve of free AlDox (Figure 3A). UV/vis analysis revealed that the AlDox content was one per protein subunit, translating to 60 AlDox molecules per cage (60 subunits). All available cysteines were labeled with AlDox. Although hydrazine linkage is known to be quite stable at neutral pH, it is acidsensitive (pH 4.5−5.5) and quickly cleaved under an acidic environment.46 Doxorubicin could be released from the delivered encapsulin under the acidic environment of the tumor cells because it is anchored through an acid-sensitive hydrazone linker. A time-dependent release study of AlDox from AlDox-SP94-Encap_loophis42 at pH 5.5 showed that approximately 60% of AlDox was released within 8 h (Figure 3B).46
surface SP94-peptide presented on fEncap_loophis42C123SMCC-SP94 facilitates the recognition of cell surface targets and leads them to effectively bind to the target cells. These data demonstrate that both cell targeting and imaging functionalities can be simultaneously incorporated into the template encapsulin through a series of chemical conjugations, allowing them to be utilized as target-specific diagnostic probes. Although the chemical conjugation of SP94-peptide demonstrated the potential use of encapsulin as a targeted cargo delivery vehicle, the number and position of targeting ligands, SP94peptide, could not be tightly controlled or determined owing to the use of multistep chemical reactions and the heterogeneous nature of the chemical modifications. To overcome this problem, we attempted to genetically insert SP94-peptide on the surface of Encap_loophis42C123. We introduced SP94-peptide with extra amino acids (GGTSSFSIIHTPILPLGG) at the position between residues 138 and 139, which is the middle of the exterior loop. This region was already confirmed to be tolerated for peptide insertion and located at the exterior surface by inserting a His-tag and subsequent purification using IMAC (Figure S2B). This construct was also stable at high temperature (Figure S1D). Genetic insertion of SP94-peptide into the Encap_loophis42C123 template was confirmed by DNA sequencing. The subunit molecular mass of SP94-peptide-inserted Encap_loophis42C123 (SP94-Encap_loophis42C123) was measured to be 33732.0 Da, which was almost identical to the calculated value of 33733.2 Da (Figure 1A, top). The SEC elution profile of SP94Encap-loophis42C123 was same as that of Encap-loophis42C123 (Figure 1C, middle and top). The TEM image also indicated their intact cage architecture with a uniform size of approximately 23 nm in diameter (Figure 1B, top). The DLS measurement of SP94-Encap_loophis42C123 verified a hydrodynamic diameter of 29.1 nm, slightly larger than that of Encap_loophis42C123 (22.1 nm), probably due to the inserted SP94-peptide on the surface (Figure 1D, top). The same elution profile was observed even after F5M labeling (Figure S4D). To evaluate the capability of targeted delivery of SP94-fEncap_loophis42C123, the same method was applied. Although SP94fEncap_loophis42C123 efficiently bound to HepG2 cells, fEncap_loophis42C123 without SP94-peptide did not bind 3799
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AlDox in a dose-dependent manner similar to that of free Dox. SP94-Encap_loophis42C123 without AlDox had no significant effect on cell viability. We clearly demonstrated that the engineered encapsulin can be utilized as a multifunctional theranostic nanoplatform that can acquire specific cell target ligands, diagnostic probes, and therapeutic reagents simultaneously.
HepG2 cells were treated with SP94-Encap_loophis42C123AlDox for 5 h and then washed with fresh medium for 36 h. Free AlDox and empty SP94-Encap_loophis42C123 treatments were performed in parallel as controls. The cytotoxicity of HepG2 cells treated with SP94-Encap_loophis42C123-AlDox increased in a dose-dependent manner, as was also observed with free AlDox (Figure 4). In contrast, SP94-Encap_loophis42C123 without
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ASSOCIATED CONTENT
S Supporting Information *
Solubility and heat stability tests of encapsulin protein cages, IMAC of Encap_loophis42C123 and Encap_loophis138C123, size exclusion elution profiles of Encap_loophis42C123, molecular mass measurements of dissociated subunits of fEncap_loophis42C123, size exclusion elution profiles of fEncap_loophis42C123-SMCC-SP94, transmission electron microscopy images of negatively stained fEncap_loophis42C123-SMCC-SP94, and size exclusion elution profiles of fEncap_loophis42C123 and SP94-fEncap_loophis42C123. This material is available free of charge via the Internet at http://pubs. acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +82-52-217-5325. Fax: +82-52-217-5309. E-mail:
[email protected].
Figure 4. MTT cell viability assay. Dose-dependent cytotoxicity profiles of AlDox-SP94-Encap_loophis42 (red, circles), free AlDox (green, triangles), and SP94-Encap_loophis42 (black, squares) toward HepG2 cells. Three independent experiments were performed.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (NRF2013R1A1A1008228).
AlDox had no significant effect on cell viability. These results suggest that encapsulin itself does not alter cell viability significantly but that it delivers loaded AlDox and effectively releases drugs into target cells, resulting in cytotoxicity toward the target cells. Although SP94-Encap_loophis42C123-AlDox showed similar cytotoxicity to that of free Dox in an in vitro cell viability test, SP94-Encap_loophis42C123 may improve the solubility of AlDox and allow target-specific delivery of drugs, reducing the side effects of the treatment, which is critical for in vivo applications. Thus, the combined incorporation of target SP94-peptide and anticancer prodrug AlDox on encapsulin may show clinical potential for improving the systemic treatment of hepatocellular carcinoma.
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REFERENCES
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CONCLUSIONS In this study, we developed a novel protein cage, encapsulin, as a nanoscale platform for the targeted delivery of therapeutic molecules and/or diagnostic probes using genetic and chemical modifications. Insertion of six consecutive histidines with extra residues between residues 42 and 43 of WT encapsulin (Encap_loophis42C123) provided unusual heat stability and allowed us to readily purify it in a large scale. Hepatocellular carcinoma (HCC) cell-specific cell-binding peptide (SP94peptide) was presented on the exterior surface of Encap_loophis42C123 through either chemical conjugation or genetic insertion. SP94-peptide-displaying Encap_loophis42C123 exhibited specific binding capability to HepG2 cells and an ability to carry imaging probes or prodrug molecules such as fluorescein-5-maleimide (F5M) or aldoxorubicin (AlDox). The MTT cell viability assay confirmed the cytotoxic effect of doxorubicin (Dox) released from SP94-Encap_loophis42C1233800
dx.doi.org/10.1021/bm501066m | Biomacromolecules 2014, 15, 3794−3801
Biomacromolecules
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dx.doi.org/10.1021/bm501066m | Biomacromolecules 2014, 15, 3794−3801
