Genetically engineering encapsulin protein cage nanoparticle as a SCC-7 cell targeting optical nanoprobe
© Moon et al.; licensee BioMed Central. 2014
Received: 10 October 2014
Accepted: 10 November 2014
Published: 23 December 2014
Protein cage nanoparticles are promising nanoplatform candidates for efficient delivery systems of diagnostics and/or therapeutics because of their uniform size and structure as well as high biocompatibility and biodegradability. Encapsulin protein cage nanoparticle is used to develop a cell-specific targeting optical nanoprobe.
FcBPs are genetically inserted and successfully displayed on the surface of encapsulin to form FcBP-encapsulin. Selectively binding of FcBP-encapsulin to SCC-7 is visualized with fluorescent microscopy.
Encapsulin protein cage nanoparticle is robust enough to maintain their structure at high temperature and easily acquires multifunctions on demand through the combination of genetic and chemical modifications.
Conventional drugs and diagnostic probes tend to diffuse rapidly and get distributed throughout the body easily upon systematic administration . Non-targeted treatments generally cause detrimental side effects in normal cells and tissues and the reduction of the effectiveness of the treatment . The targeted delivery of diagnostic or/and therapeutic reagents to desired sites is a challenging, but promising, task for the early diagnosis of diseases as well as effective and localized treatment of diseases.
A variety of inorganic or organic nanoparticles, including metal nanoparticles [3, 4], micelles [5, 6], polymers [7–9], liposomes [9–11], and protein cages [1, 12], have been investigated as efficient delivery vehicles for diagnostic or/and therapeutic reagents because they have great potential to improve the pharmacological properties of drugs and maximize the localized treatment of diseases . The manipulation of size and surface of nanoparticles increase their accessibility to disease sites and circulation time in the bloodstream due to their enhanced permeability and retention (EPR) effect; this improves the biodistribution of drugs [14, 15].
Protein cages, such as ferritins [12, 16–19], viral capsids [17, 20–24], lumazine synthase [20, 25, 26], and encapsulin , are promising nanoplatform candidates for efficient delivery systems of diagnostics and/or therapeutics because they have uniform size and structure as well as high biocompatibility and biodegradability . Protein cages are spontaneously self-assembled from multiple copies of one or a few types of protein subunits in a precisely controlled manner. In addition, they can be manipulated genetically and chemically to have a desired function, using a rational design based on atomic resolution structural information.
Encapsulin, a novel protein cage nanoparticle isolated from thermophile Thermotoga maritima, 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 . Although the exact function of encapsulin in T. maritima is not clearly understood yet, its crystal structure has been recently solved and its function was postulated 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 [28, 29]. This study implied that encapsulin has a large enough central cavity (20 nm in inner diameter) to encapsulate a large amount of therapeutic and/or diagnostic reagents and we recently constructed a heat stable encapsulin variant through genetic engineering and demonstrated the utility of engineered encapsulin as a versatile drug delivery nanoplatform.
In the present study, we genetically engineered a novel protein cage nanoparticle, encapsulin, to display cell-specific targeting peptides (DCAWHLGELVWCT) onto the surface in a controlled manner and demonstrated its selective binding to Squamous cell carcinoma (SCC-7) cell line exclusively.
Genetic modification of encapsulin and protein cage purification
We started with a genetically modified encapsulin, which has only one cysteine per subunit at position 123. Cell targeting peptide with linker (GGGGGGDCAWHLGELVWCTGGGGG) was inserted into residues between 138 and 139 of encapsulin by an established polymerase chain reaction (PCR) protocol using pET-30b based plasmids containing genes encoding encapsulin .
Peptide insertion was confirmed by DNA sequencing and confirmed DNAs were transformed into the competent E.coli strain BL21 (DE3) and the protein cages were over-expressed 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 12000 g for 1 hr at 4°C. Encapsulin protein cage was purified by size exclusion chromatography (SEC) after heat precipitation for 10 min at 65°C .
Quartz crystal microbalance (QCM) measurements
QCM experiments were performed using Q-Sense E4 and standard gold QCM sensors (Q-Sense, Sweden) as described previously . Briefly, the system was operated in flow mode with a pump and temperature was maintained at 25.0 ± 0.1°C. Each sample solution was introduced to the measurement chamber with a pump and continuously measured for 3 min prior to the subsequent introductions. Protein cages and rabbit IgGs were introduced at concentrations of approximately 100 μg/ml and 50 μg/ml, respectively, in phosphate buffer (50 mM phosphate, 100 mM NaCl, pH 6.5). Resonance frequencies were measured simultaneously at seven harmonics (5, 15, 25, 35, 45, 55 and 65 MHz). For clarity, only the normalized frequency of the third overtone is shown.
Surface plasmon resonance (SPR) analysis
SPR experiments were performed with carboxyl dextran CM-5 gold chips on a Biacore 3000 device (Biacore AB, Sweden) at 25 oC using a PBS buffer as a running solution. Rabbit IgG was coupled to the surface of a CM-5 sensor chip by standard amine-coupling chemistry on the SPR instrument as described previously, with slight modifications . Briefly, a mixture of EDC (0.4 M) and NHS (0.6 M) was injected onto the chip at a flow rate of 10 μl/min to activate carboxyl groups on the sensor surface and subsequently 20 μg/ml of rabbit IgG was added at the same flow rate for 7 min. Excess reactive groups were blocked with 1 M ethanolamine (pH 8.0). Encapsulin capture by rabbit IgG was examined by applying various amounts (1, 5, 10, 25, 50, and 100 nM) of encapsulin (PBS, pH 7.4) to the surface at a flow rate of 30 μl/min.
Mass spectrometry of modified encapsulin protein cage
For ESI-TOF analysis, encapsulin protein cages were loaded onto the MassPREP Micro-desalting 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 .
Cell culture and confocal fluorescence microscopy
All cell lines in this article were obtained from the Korean cell line bank (KCLB) and maintained in a humidified atmosphere of 5% CO2 and 95% air at 37°C. SCC-7 cells were incubated in RPMI 1640 medium with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin. MDA-MB-231 and HepG2 cells were incubated in RPMI 1640 medium with 10% FBS, 1% streptomycin. KB cells were cultured in RPMI1640 medium with L-glutamine (300 mg/L), 10% FBS, 1% streptomycin, 25 mM HEPES and 25 mM NaHCO3. Hela cells were incubated in DMEM medium with 4.5 g/L glucoseand L-glutamine, 10% FBS and 1% streptomycin. Cells were grown on microscope cover glasses (18 mm Ø) in 12-well culture plate (SPL, 30012). The cells were fixed with 4% paraformaldehyde in PBS and washed 2 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 18 hr and blocking buffer was aspirated. Encapsulin and FcBP-encapsulin were treated for 20 hr at 4°C. Before sealing, the cells on the cover glasses were washed 3 times (15 min) and nuclei were stained with DAPI. Images of stained substrates were collected using Olympus Fluoview FV1000 confocal microscope (Olympus, UOBC).
Results and discussions
Construction of FcBP-presenting encapsulin protein cage nanoparticles
Inserted FcBPs are displayed on the surface of encapsulin
Next, we examine whether FcBP-encapsulin can recognize the immobilized targets and selectively bind to them. To do that, we performed surface plasmon resonance (SPR) analysis. In contrast to QCM studies, we first immobilized rabbit IgGs on the surface of an SPR CM-5 sensor chip  and introduced either FcBP-encapsulin or encapsulin at several concentrations. If the inserted FcBPs are exposed on the surface of encapsulin and accessible to the biomolecules, they will selectively bind to the immobilized rabbit IgGs resulting in gradual increases in SPR responses depending on the amounts of introduced FcBP-encapsulin or encapsulin. As we expected, gradual increases in SPR responses (RU) were observed upon introduction of FcBP-encapsulin (Figure 2B, filled arrow), with RU values reaching a plateau at each concentration (Figure 2B, open arrow). Consistent with previous QCM results, apparent dissociation of FcBP-encapsulin from the immobilized rabbit IgG was not observed even after extensive buffer washing (Figure 2B). However, encapsulin did not bind to the immobilized rabbit IgGs at all (Figure 2C and inset) regardless of the amount of introduced encapsulin. These results suggest that the FcBPs displayed on the encapsulin can recognize the immobilized target, rabbit IgG, and allow to selectively bind to them and multiple FcBPs on the surface of FcBP-encapsulin may cooperatively capture the immobilized rabbit IgGs resulting in extremely strong binding (Figure 2B).
Specific binding of FcBP-encapsulin to the SCC-7 cells
FcBP-encapsulin has only one cysteine per subunit at position 123 (60 cysteines per cage) which is known to be chemically active. To attach fluorescent probes to the FcBP-encapsulin, we treated FcBP-encapsulin with activated fluorescein-5-maleimide (F5M). F5M-conjugated FcBP-encapsulin (fFcBP-encapsulin) was separated from unreacted F5M by SEC, and we confirmed that every subunit is labeled with one F5M using MS (data not shown).
In this study, we engineered encapsulin protein cage nanoparticle as a SCC-7 cell targeting optical nanoprobe. Fc-binding peptide with linker (GGGGGGDCAWHLGELVWCTGGGGG, FcBP) was introduced onto the surface loop region of encapsulin. Insertion of FcBPs and integrity of FcBP-encapsulin were confirmed by various biophysical methods, including MS, SEC, and TEM. QCM and SPR analyses demonstrated that FcBP-encapsulin indeed polyvalently displayed FcBPs on the surface of encapsulin by monitoring specific binding of it to rabbit IgG. Fluorescently labeled FcBP-encapsulin selectively bound to the SCC-7, but not to the Hela, HepG2, MDA-MB-231 or KB cells as shown by fluorescence imaging. Since FcBP-encapsulin is robust and acquire multifunctions on demand, it can be used as a nanoplatform for developing a multifunctional theranostic system.
This work was supported by the year of 2014 research fund (1.140028.01) of UNIST.
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