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Magnetic nanoparticles for efficient cell transduction with Semliki Forest virus
Accepted Manuscript Title: Magnetic nanoparticles for efficient cell transduction with Semliki Forest virus Authors: Baiba Kurena, Aleksandra Veˇza¯ne, Dace Skrastin¸a, Olga Trofimova, Anna Zajakina PII: DOI: Reference:
S0166-0934(17)30089-7 http://dx.doi.org/doi:10.1016/j.jviromet.2017.03.008 VIRMET 13222
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
4-2-2017 12-3-2017 12-3-2017
Please cite this article as: Kurena, Baiba, Veˇza¯ne, Aleksandra, Skrastin¸a, Dace, Trofimova, Olga, Zajakina, Anna, Magnetic nanoparticles for efficient cell transduction with Semliki Forest virus.Journal of Virological Methods http://dx.doi.org/10.1016/j.jviromet.2017.03.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Magnetic nanoparticles for efficient cell transduction with Semliki Forest virus Baiba Kurenaa, Aleksandra Vežānea, Dace Skrastiņaa, Olga Trofimova and Anna Zajakinaa,* a
Latvian Biomedical Research and Study Centre, Ratsupites street 1, LV-1067, Riga,
Latvia *
Corresponding author. Tel.: +371 67808200; E-mail: [email protected]; Postal address: Ratsupites street 1, LV-1067, Riga, Latvia Graphical abstract
Highlights:
Positively charged magnetic particles enhance Semliki Forest virus transduction.
SFV magneto-transduction is efficient in presence of fetal bovine serum.
Magnetic precipitation can be used for concentration of SFV particles.
Abstract
Semliki Forest virus (SFV) is a potential cancer gene therapy vector capable of providing high and transient expression of heterologous proteins in mammalian cells. However, SFV has shown suboptimal transduction levels in several cancer cell types as well as wide biodistribution of SFV has been observed after in vivo applications. Magnetic nanoparticles (MNPs) have been shown to increase cell transduction with several viral vectors in vitro under an external magnetic field and enhance magnetically guided viral vector delivery. Here, we examined a panel of MNPs for enhanced cancer cell transduction with SFV vector. Magneto-transduction using positively charged MNPs increased Semliki Forest virus transduction in TS/A mouse mammary carcinoma cells in vitro in the presence of fetal bovine serum. Positively charged MNPs efficiently captured SFV particles independently of capturing medium, and MNPs-SFV complexes were successfully separated from suspension by magnetic precipitation. These results reveal the potential application of MNPs for enhanced gene delivery by SFV vector as well as proposes magnetic precipitation for efficient concentration of SFV particles from different media.
Abbreviations: SFV, Semliki Forest virus; MNPs, magnetic nanoparticles; MRI, magnetic resonance imaging. Keywords: virus; alphavirus; magnetic nanoparticles; cancer gene therapy; transduction;
1. Introduction The virus particles-based technologies contribute significantly to the progress of vaccine development, gene therapy, cancer treatment, and targeted drug design. During the last decades the alphaviral vectors genetically modified to produce therapeutic recombinant proteins showed the high potential in plethora of preclinical studies and several clinical trials (Zajakina et al, 2015). Semliki Forest virus (SFV) is an enveloped positive-strand RNA virus belonging to the family Togaviridae and genus Alphavirus (Strauss and Strauss, 1994; King et al., 2012). SFV has been adapted for the expression of heterologous proteins in mammalian cells. This transient expression system provides several advantages including a broad range of host cells susceptible to infection, a high level of heterologous protein expression that is independent of the splicing machinery, and the ability to produce high viral titers (Aranda et al., 2011). The replication deficient SFV expression system is based on a modified SFV genome in which the viral structural genes are replaced by the gene of interest (Liljeström and Garoff, 1991). Therefore, the infectious virus capable of infecting the new target cells cannot be produced, thus providing safe conditions for in vitro and in vivo studies. The biological features of alphaviruses make them an ideal tool for gene transfer experiments in vivo. Apart from the high expression of heterologous gene, the additional advantages in using alphaviral vectors in vivo include a low specific immune response against the vector itself (Pushko et al., 1997; Berglund et al., 1999) and an absence of vector preimmunity. There is a special interest in the application of alphaviruses as a vaccine platform because these vectors are able to infect dendritic cells and can target lymph nodes (Lundstrom, 2014; Veen et al., 2012; Ryman and Klimstra, 2008). Furthermore, alphaviral vectors have proven to be highly successful as therapeutic and prophylactic cancer vaccines (Lundstrom, 2015). In support to the use of alphaviral vectors in cancer therapy, tumor tropism has been documented for Sindbis virus-based vectors (Tseng et al., 2003; Unno et al., 2005; Huang et al., 2012). SFV vectors have shown the ability to localize within tumor after systemic delivery although a broad vector distribution in other organs has been observed simultaneously (Madoz et al., 2007; Vasilevska et al., 2012). Owing to the broad tissue tropism, mostly intratumoral delivery of alphaviral vectors has been applied for cancer therapy.
Another limitation includes the suboptimal transduction levels of certain cancer cell types, thus decreasing the therapeutic potential of the vector (Madoz et al., 2007; Li et al., 2009; Vähä-Koskela et al., 2006, Lundstrom, 2002). This limitation can be overcome by the use of high virus titers, although this can lead to untargeted vector distribution and cytotoxic effects. One solution is to reduce the viral dose and at the same time enhance vector targeting and transduction capacity. For this reason, superparamagnetic nanoparticles (MNPs) have been applied to increase the delivery efficiency of viral particles as well as nucleic acids and proteins. It has previously been shown that the external magnetic field generated by a permanent magnet increases the concentration of MNP-virus complexes on the surface of the cells, thus leading to improved transduction efficiency (Pereyra et al., 2016). Magneto-transduction has demonstrated efficiency with several viruses, such as retrovirus (Castellani et al., 2016; Haim et al., 2005; Tai et al., 2003; Scherer et al., 2002), rhabdovirus (Almstatter et al., 2015), parvovirus (Hwang et al., 2011), paramyxovirus (Kadota et al., 2005), and adenovirus (Tresilwised et al., 2010; Almstätter et al., 2015; Sapet et al., 2012, Kim et al., 2011; Bhattarai et al., 2008; Kamei et al., 2009; Scherer et al., 2002). The MNPs-virus complexes that are formed by electrostatic and hydrophobic interactions are easily generated, in contrast to specific ligand-ligand interaction-based complexes, which require chemical modification of the virus and nanoparticles. The site-specific targeting of magnetically labelled viral particles through an externally applied magnetic field gradient has been demonstrated in vivo (Tresilwised et al., 2010, Tresilwised et al., 2012; Sapet et al., 2012; Bhattarai et al., 2008; Pereyra et al., 2016; Hashimoto and Hisano, 2011). Moreover, the accumulation of MNPs can be clearly visualized by magnetic resonance imaging (MRI) (Sun et al., 2008) and therefore allows for additional MNPs-labeled vector tracking possibilities. In this study, a panel of MNPs were examined, and a method for enhanced gene delivery by Semliki Forest virus vector in a TS/A mouse mammary cancer model in vitro was developed. Moreover, the potential application of MNPs for the efficient concentration of the SFV vector from different media was addressed.
2. Materials and Methods 2.1. Magnetic nanoparticles
In this study, the aqueous dispersions of magnetic nanoparticles (MNPs) from different sources were used. The properties of MNPs are described in Table 1.
2.2. Cell culturing
TS/A (mouse mammary carcinoma) cells (ATCC) were grown in DMEM Glutamax medium (Gibco) supplemented with 10 % fetal bovine serum (FBS, Gibco) and 50 mg/l gentamicin (Sopharma). BHK-21 (Baby hamster kidney) cells (ATTC) were propagated in BHK - Glasgow MEM (Gibco) supplemented with 5% FBS, 10% tryptose phosphate broth, 2 mM L-glutamine, 20 mM HEPES, 100 mg/ml streptomycin and 100 U/ml penicillin. Cells were cultured in a humidified 5% CO2 incubator at 37°C.
2.3. Production of SFV vector
The pSFV1/Ds-Red construct carrying Discosoma sp. red fluorescent protein gene (DsRed) was generated as previously described (Zajakina et al., 2014). For the synthesis of infectious but replication deficient vector particles pSFV1/Ds-Red and pSFV-helper1, plasmids were linearized using SpeI restriction enzyme (Thermo Scientific, Vilnius, Lithuania). In vitro RNA transcription was performed using 3 µg of linearized DNA and 40 U of SP6 RNA polymerase (Thermo Scientific) in a 50-µl reaction mixture. The RNA transcripts were capped during the transcription reaction by adding 1 mM m7G(5’)ppp(5’)G cap-analogue (New England Biolabs, Hitchin, UK). The DNA template was removed by digestion using RNase-free DNase (Thermo Scientific). For the packaging of SFV1/Ds-Red RNA into SFV particles, in vitro transcribed RNAs (recRNA and SFV-helper1 RNA, 20 µg each) were co-electroporated into 1×107 BHK cells (850 V, 25 mF, 2 pulses) using a Bio-Rad Gene Pulser apparatus (Bio-Rad, Hercules, CA, USA) without the pulse controller unit. The electroporated cells were resuspended in 15
ml of complete BHK medium containing 1% FBS, transferred into tissue culture flasks (75 cm2) and incubated at 33°C, 5% CO2. After a 48 h incubation SFV1/Ds-Redcontaining medium was harvested, rapidly frozen and subsequently used as a virus stock. The virus stocks did not contain the replication competent wild-type virus as confirmed by cell reinfection. The viral titers expressed in infectious units per ml (IFU/mL) were quantified by infecting BHK-21 cells and TS/A cells with serial dilutions of viral stock and analyzing Ds-Red expression via fluorescence microscopy on a Leica DM IL microscope (Leica Microsystems Wetzlar GmbH, Germany) as previously described (Vasilevska et al, 2012). The viral titre in BHK-21 cells was 6.25 × 108 IFU/mL, which corresponded to 2.57 × 107 IFU/mL when measured after TS/A cell infection under the same conditions.
2.4. Generation of MNP/virus complexes and cell transduction
The MNP/virus complexes were generated by diluting 10 µl of SFV/Ds-Red virus particles (6.25 × 108 IFU/mL as measured by infection of BHK cells) in 90 µL of DMEM Glutamax w/o FBS and antibiotics and mixing the viral suspension with the following volumes of MNPs: 1 µl of Fluid-Mag Amine, Fluid-Mag Chitosan and Fluid-Mag CMX and 5 µl of poly-Mag Neo, in vivo Poly Mag, PEI Mag and in vivo Viro Mag. For increased transduction, the amount of MNPs corresponded to double the indicated volume: 2 µl Fluid-Mag Amine, Fluid-Mag Chitosan and Fluid-Mag CMX and 10 µl of poly-Mag Neo, in vivo Poly Mag, PEI Mag and in vivo Viro Mag. After 30 min incubation at room temperature, 900 µl of complete TS/A cell culture medium was added to the mixture. For infection TS/A cells were plated on a 12-well plate at concentration of 5 × 105 cells per well. Next day cells were washed once with PBS containing Mg2+ and Ca2+ (Invitrogen, UK) and MNPs/virus complexes (1 ml in total volume) were added. The cells were incubated for 40 min on a permanent magnet (Universal Magnet Plate 8×13 cm, IBA, Germany) and 30 min without the magnet at 37°C and 5 % CO2. Then, the medium containing MNPs/virus complexes was replaced by TS/A cultivation medium. As a control (control -), cell transduction under the same conditions but without MNPs
was performed. The standard SFV transduction (control +) under serum free conditions was performed as previously described. Briefly, cells were washed once with PBS containing Mg2+ and Ca2+, and 10 µL of SFV/Ds-Red virus particles (6,25 × 108 IFU/mL in a stock) were resuspended in 1 mL of PBS containing Mg2+ and Ca2+ and added to the cells. After incubation at 37°C and 5 % CO2 for 1 h and 10 min the solution containing virus was replaced with 1 mL of cultivation medium and cells were incubated at 37oC and 5 % CO2 for 24 h when they were harvested for analysis by flow cytometry.
2.5. Flow cytometry
The infected TS/A cells were harvested 24 h after transduction. Cells were detached by 0.05 % trypsin solution, washed and resuspended in 1 mL of PBS, and immediately processed for analysis by flow cytometry. Ds-Red fluorescence was measured using a FACSAria II (Becton Dickinson Biosciences, San Jose, California, USA). The flow cytometry data were analyzed by BD FACSDiva 6.1.2 software. Uninfected cells were used as a negative control.
2.6. Virus capturing by MNPs For virus capturing experiments 10 µl of SFV/Ds-Red virus particles (6.25 × 108 IFU/mL in a stock as measured by infection of BHK cells) that were resuspended in 280 µl of the capturing medium, which did not contain FBS and antibiotics, were mixed with MNPs. The following concentrations of MNPs were used for virus capture: 1 µl of Fluid-Mag Amine and Fluid-Mag Chitosan and 5 µl of poly-Mag Neo, in vivo Poly Mag, PEI Mag and in vivo Viro Mag. Different capturing media were tested for virus capturing by 1 µL of Fluid-Mag Amine: (I) DMEM Glutamax w/o FBS and antibiotics, (II) a solution containing 500 mM NaCl and 10 mM sodium phosphate (pH 7.0), (III) complete BHK medium containing 5% FBS, and (IV) phosphate buffered saline. The complexes of MNPs/virus were allowed to assemble by incubating the mixture at +4°C for 1.5 h on a rotator. Subsequently the formed complexes were precipitated by incubating the solution on a magnetic rack for 10 min. The supernatants were retained to control the efficiency of
virus capture and precipitation. The pellets were resuspended in 300 µL volume of capturing medium to maintain the same amount of capturing medium as in the supernatants. Both the supernatants and the MNPs/virus suspension from the pellet were mixed with 700 µL of PBS containing Mg2+ and Ca2+ and used for cell transduction. TS/A cells were plated on a 12-well plate at a concentration of 5 × 105 cells per well. Next day cells were washed once with PBS containing Mg2+ and Ca2+ (Invitrogen, UK) and 1 mL of MNP/virus or supernatant mixture was added. Cells were incubated for 40 min on a permanent magnet (Universal Magnet Plate 8×13 cm, IBA, Germany) and 30 min without a magnet at 37°C and 5 % CO2. Then, the medium containing MNPs/virus complexes was replaced by TS/A cultivation medium and cells were incubated at 37oC and 5 % CO2 for 24 h when they were harvested for analysis by flow cytometry.
3. Results
3.1. Magneto-transduction using positively charged magnetic nanoparticles increases Semliki Forest virus transduction in mouse mammary carcinoma cells
To investigate whether magnetic nanoparticles could enhance the transduction of SFV vector, we used SFV1/Ds-Red recombinant particles expressing Discosoma sp. red fluorescent protein. Usually, the efficiency of transduction by alphaviral vectors is very high for susceptible cell lines and can reach 50-70 % of the total cells. To demonstrate the differences in SFV infection with and without MNPs, we significantly diluted the vector and used the amount of viral particles corresponding to an MOI=0.19 (according to virus infectious units measured in TS/A cells at standard conditions; 2.57 × 107 IFU/mL) that transduced 16.3 % of cells, as determined by flow cytometry (Fig. 1, positive control without FBS and MNPs). Moreover, we applied fetal bovine serum (FBS) to inhibit viral infection in the cell culture and thus simulate in vivo conditions. Therefore, the transduction efficiency in the presence of FBS decreased to 2.1 % on average (Fig. 1, negative control with FBS without MNPs). To evaluate the effect of magnetofection, SFV1/Ds-Red vector (MOI=0.2) was preincubated with MNPs for 30 min in serum free conditions before being mixed with the cell culture medium containing FBS. Cell incubation for 40 min on a permanent magnet increased the transduction efficiency in the presence of FBS by up to 21.8 % compared with that of the negative control (2.1 %) (Fig. 1A and 1B). Notably, the enhancement of transduction by FluidMag Amine was achieved even without cell incubation on the magnetic plate (Fig. 1B). We concluded that magnetofection with all of the positively charged MNPs significantly increased the transduction efficiency of SFV in the presence of FBS in a TS/A cell culture model. The most significant transduction enhancement at the conditions tested was demonstrated with highly positive FluidMag Amine MNPs functionalized with aminosilane, whereas negatively charged FluidMax CMX MNPs coated with carboxymethyl-dextran did not affect SFV transduction.
3.2. Positively charged MNPs efficiently capture SFV particles independently of the capturing medium
We showed in Fig. 1 that although the absolute values of transduction efficiency were different for each tested MNP-reagent, only the positively charged particles enhanced cell infection with SFV in a dose dependent manner. On the basis of these observations, we speculated that there might be a close electrostatic association between SFV and positively charged magnetic nanoparticles that leads to complex formation and subsequently a significant enhancement of transduction in TS/A cells under a magnetic field. To explore the possible application of positively charged MNPs for capture and isolation of SFV, we allowed MNPs/virus complexes to form in a capturing medium that did not contain either FBS or antibiotics. Then, we precipitated MNPs/SFV complexes by using a magnetic rack and transferred the supernatants into new tubes to control the efficiency of SFV capture. Both the resuspended MNPs/virus complexes and the supernatants were subjected to TS/A cell transduction (Fig. 2). The results showed that most of the tested magnetic particles were able to capture SFV from solution in serum free conditions (Fig. 3). Notably, the amount of MNPs was sufficient to capture almost all of the SFV particles in the case of the FluidMag Chitosan and In vivo ViroMag. The SFV capture by the PEI-Mag reagent under the applied capturing conditions resulted in cell transduction rate 3 times less efficient than that of the corresponding supernatant from which the complexes were precipitated. Finally, the effect of the medium on viral capture by magnetic nanoparticles was investigated. The FluidMag Amine MNPs together with SFV1/Ds-Red viral particles were incubated in different capturing media, including (I) DMEM Glutamax w/o FBS and antibiotics, (II) a solution containing 500 mM sodium chloride and 10 mM sodium phosphate, pH 7.0, (III) complete BHK medium containing 5% FBS, and (IV) phosphate buffered saline. Interestingly, neither FBS nor a high salt concentration affected SFV binding to positively charged MNPs, thus demonstrating that the self-assembly of complexes by electrostatic and hydrophobic interactions is a powerful means of viral binding to magnetic particles (Fig. 4).
4. Discussion
In this study a panel of magnetic nanoparticles (MNPs) was examined in association with Semliki Forest virus (SFV) vector for enhanced gene delivery in a TS/A mouse breast cancer model in vitro by magneto-transduction. Moreover, the potential application of MNPs for efficient concentration of SFV vector from different media was addressed. Magneto-transduction has been successfully applied to enhance cell transduction and transgene expression in vitro with several viral vectors, including adenoviruses (Tresilwised et al., 2010; Almstätter et al., 2015; Sapet et al., 2012, Kim et al., 2011; Bhattarai et al., 2008; Kamei et al., 2009), vesicular stomatitis virus (Almstätter et al., 2015;), adeno-associated virus (Hwang et al., 2011) and measles virus (Kadota et al., 2005). Therefore MNPs-associated SFV transduction into TS/A carcinoma cells under magnetic field in the presence of FBS was determined. By assembling recombinant Semliki Forest virus with several positively charged MNPs, effective cancer cell transduction with SFV vector under a magnetic field was achieved even in the presence of FBS. These results demonstrate that a magnetic field is crucial for enhanced cell transduction with SFV-MNPs complexes for up to 10.1-fold when compared with transduction without an applied magnetic field (Fig. 1B). This finding is in agreement with results from previous studies describing the importance of a magnetic field for the sedimentation of MNPs/viral complexes on the surface of cells and subsequent cellular uptake (Pereyra et al., 2016). The efficiency of cell transduction with MNPs/SFV complexes under FBS conditions is probably affected by the shielding effect of MNPs against opsonization of viral particles by serum factors and stabilized MNPs/virus complexes, as previously reported (Tresilwised et al., 2010; Tresilwised et al., 2012; Almstätter et al., 2015). An explanation for the observed slightly enhanced (3.6- and 1.6-fold) transduction efficiency with two of the MNPs-SFV complexes in the absence of a magnetic field under FBS conditions compared with viral particles alone (Fig. 1B) may relate to the protective MNPs shielding SFV particles from serum factors. However, MNPs/virus complexes can aggregate because of dipole-dipole interactions between MNPs (Tresilwised et al., 2010; Tresilwised et al., 2012; Almstätter et al., 2015;), and a
substantial number of MNPs can associate with only one viral particle or even clusters of several viral particles through MNPs/virus aggregation can form (Tresilwised et al., 2010), thus increasing the hydrodynamic size of such complexes. Similar results with enhanced cell transduction without an applied magnetic field have previously been observed for MNPs/adenovirus (Pereyra et al., 2016, Tresilwised et al., 2012) and MNPs/retrovirus (Castellani et al., 2016) before. We speculate that MNPs/SFV complexes of a larger size sedimentate on the cell monolayer without an applied external magnetic field, and the proximity promotes enhanced cellular uptake of the complexes. The ratio of MNPs to viral particles has also been shown to be important in the aggregation of MNPs/virus complexes (Kim et al., 2011), and further optimization of the ratio between a chosen MNPs-reagent and SFV particles can be conducted to produce even better results for specific cells. The results showed that only the positively charged MNPs noticeably enhanced cell transduction with SFV vector in the presence of a magnetic field; therefore, we suspected that the positive surface charge of MNPs is a crucial aspect for the association with SFV virus into complexes. Most of the tested positively charged MNPs were able to capture SFV from a solution in serum free conditions with a remarkable efficiency in the case of FluidMag Chitosan and In vivo ViroMag reagents under the particular capturing conditions (Fig. 3). These results indicate the high capture-capacity of these particles, because the supernatants contained only a leaky amount of the virus after precipitation. Such association between MNPs coated with cationic compounds and viral particles with negative electrokinetic potential has been previously described and occurs because of electrostatic and hydrophobic interactions (Almstätter et al., 2015; Sapet et al., 2012). The reason for the poor capture of SFV from solution by the positively charged PEI-Mag reagent even under increased MNPs concentrations (data not shown) is unclear. It has previously been shown that for efficient sedimentation of PEI-Mag/adenovirus complexes, a prolonged incubation time of at least 30 min under the influence of a magnetic field is necessary (Tresilwised et al., 2010), thus leaving room for optimizing the protocol of PEI-Mag/SFV precipitation. Finally, the effect of the medium on viral capture by FluidMag Amine MNPs was investigated. Neither FBS at a concentration of a standard virus-production medium
(5%) nor a high salt concentration (0.5 M NaCl) affected SFV association with positively charged MNPs (Fig. 4), thus demonstrating that self-assembly of complexes by electrostatic and hydrophobic interactions is a powerful means of viral binding to magnetic particles. Previous studies have already used the association of MNPs with a wide range of viruses for magnetic concentration (Uchida et al., 2007; Satoh et al., 2003) or with negatively charged viral particles such as poliovirus-1 (Zhan et al., 2014) and measles virus (Kadota et al., 2005) for diagnostic isolation purposes. These results suggest that SFV captured by MNPs could represent a rapid and efficient way to isolate, purify and concentrate SFV particles by magnetic precipitation from different capturing media. MNPs/virus complexes can be precipitated with a magnetic rack and then washed and resuspended in a small volume of respective buffer, thereby providing a significant concentration and purification rate. As a result, MNPs can be used for the production of high SFV titers, thereby allowing improved cell transduction. The efficiency of SFV capture by MNPs and enhanced cell transduction under FBS conditions encourages the use of MNPs/virus complexes in the hampering in vivo conditions. In fact, several studies have shown success in magnetically-guided delivery of MNPs/virus complexes in vivo (Tresilwised et al., 2010, Tresilwised et al., 2012; Sapet et al., 2012; Bhattarai et al., 2008; Pereyra et al., 2016; Hashimoto and Hisano, 2011), and the current study should serve as a foundation for further research on the potential of MNPs/SFV delivery in vivo by magnetic force. Such magnetic targeting would be a promising strategy for improving the safety and efficacy of SFV vectors in preclinical and clinical applications. Moreover, because MNPs are a suitable contrast agent for magnetic resonance imaging (MRI) (Sun et al., 2008), by labeling viral particles with MNPs it is possible to track viral localization in vivo (Almstätter et al., 2015; Yun et al., 2012; Räty et al., 2006) by MRI. Thus, the association between MNPs and SFV allows for the possibility for SFV trafficking simultaneously with therapeutic applications. In conclusion, the enhanced cancer cell transduction by SFV/MNPs complexes under magnetic field holds promise for future applications such as targeted and enhanced SFV gene therapy with simultaneous tracing of therapeutics by MRI. Moreover, this study suggests that SFV capture by positively charged MNPs could represent a rapid and
efficient way to isolate, purify and concentrate SFV particles by magnetic precipitation independently of capturing medium. Conflicts of interest The authors declare no conflict of interest.
Acknowledgements This work was carried out with support from the Latvian National Research Programme Biomedicine for Public Health (Biomedicine) 2014-2017 and by the ERA Net Rus Plus AlphaChit project.
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Figure legends
Figure 1. Magneto-transduction of TS/A mouse mammary carcinoma cells with Semliki Forest virus vector SFV1/Ds-Red. (A) Fluorescence microscopy of TS/A cells 24 h after cell transduction with SFV1/Ds-Red vector in the presence of fetal bovine serum (+FBS). For magneto-transduction, the vector (6.25 × 106 IFU; measured by infection of BHK cells) was pre-incubated with 1 μL FluidMag Amine, FluidMag Chitosan and FluidMag CMX and 5 μL of PolyMag Neo, In vivo PolyMag, PEI-Mag and In vivo ViroMag. The controls demonstrated cell transduction without MNPs (-MNPs) in the presence (+FBS) and the absence (-FBS) of FBS. (B) Quantitative flow cytometry analysis of transduced TS/A cells expressing Ds-Red fluorescent protein. The cells were transduced with complexes of MNPs associated with SFV1/Ds-Red in the presence of FBS. Transduction in the presence of MNPs with cell incubation on the magnetic plate (MNPs with magnet) and transduction in the presence of MNPs without cell incubation on the magnetic plate (MNPs without magnet) is shown. Transduction in the presence of an increased amount of MNPs corresponds to a double concentration of MNPs in the reaction mixture (2x MNPs with magnet). The controls demonstrate cell transduction without MNPs in the presence of FBS (control -) and in the absence of FBS (control +). The error bars indicate the standard error of three experiments.
Figure 2. Schematic representation of the experimental procedure for the capture of SFV1/Ds-Red particles by MNPs from medium. SFV1/Ds-Red viral particles (6.25 × 106 IFU; measured by infection of BHK cells) were incubated with different MNPs and the formed complexes were precipitated by a magnet. The supernatants were aspirated and used for magneto-transduction of TS/A cells to control the capture efficiency. The pellet was resuspended in the same amount of capturing medium and was used for TS/A cell magneto-transduction to test the capture efficiency.
Figure 3. Efficiency of Semliki Forest virus (SFV1/Ds-Red) capture under serum free conditions. SFV1/Ds-Red virus (6.25 × 106 IFU; measured by infection of BHK cells) was incubated with different types of magnetic nanoparticles (MNPs): 1 μL of FluidMag Amine and FluidMag Chitosan and 5 μL of PolyMag Neo, In vivo PolyMag, PEI-Mag and In vivo ViroMag. Then, the MNP/virus complexes were magnetoprecipitated, the supernatants were removed, and the pellet was resuspended in the same amount of capturing medium. Both resuspended complexes and supernatants were used for TS/A cell magneto-transduction. The results were visualized by fluorescence microscopy (A) and quantified by flow cytometry of Ds-Red expressing cells (B). The error bars indicate the standard error of three experiments.
Figure 4. Effect of the capturing medium on Semliki Forest virus (SFV1/Ds-Red) capture and precipitation. SFV1/Ds-Red virus (6.25 × 106 IFU; measured by infection of BHK cells) was incubated with 1 μL FluidMag Amine in different capturing medium: DMEM Glutamax w/o FBS and antibiotics, a solution containing 500 mM sodium chloride and 10 mM sodium phosphate with pH 7.0, complete BHK medium containing 5 % FBS, and phosphate buffered saline. After magneto-precipitation, the supernatants were removed, and the MNP pellet was reconstituted with the same capturing medium. TS/A cells were infected with supernatants (capturing control) and resuspended particles (captured virus). The Ds-Red expressing cells were quantified by flow cytometry. The error bars indicate the standard error of three experiments.
Table 1 Properties of the superparamagnetic nanoparticles used in this study Magnetic nanoparticles (MNPs)
Manufacturer
Core
Coating
Charge
Hydrodinamic diameter
FluidMag Amine
Chemicell (Germany)
magnetite
aminosilane
+
50 nm
FluidMag Chitosan
Chemicell (Germany)
magnetite
chitosan
+
50 nm
FluidMag CMX
Chemicell (Germany)
magnetite
carboxymethyl-dextran
-
50 nm
PolyMag Neo
OZBiosciences (France)
magnetite
cationic polymer*
+
160 - 220 nm
In vivo PolyMag
OZBiosciences (France)
magnetite
cationic polymer*
+
160 - 220 nm
PEI-Mag
OceanNanoTech (USA)
maghemite
polyethylenimine (PIE)
+
30 nm
In vivo ViroMag
OZBiosciences (France)
magnetite
cationic polymer*
+
200 nm
* not disclosed by the manufacturer
Concentration 25 mg/ml 25 mg/ml 25 mg/ml ND* ND* 1 mg/ml ND*
S0166-0934(17)30089-7 http://dx.doi.org/doi:10.1016/j.jviromet.2017.03.008 VIRMET 13222
To appear in:
Journal of Virological Methods
Received date: Revised date: Accepted date:
4-2-2017 12-3-2017 12-3-2017
Please cite this article as: Kurena, Baiba, Veˇza¯ne, Aleksandra, Skrastin¸a, Dace, Trofimova, Olga, Zajakina, Anna, Magnetic nanoparticles for efficient cell transduction with Semliki Forest virus.Journal of Virological Methods http://dx.doi.org/10.1016/j.jviromet.2017.03.008 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Magnetic nanoparticles for efficient cell transduction with Semliki Forest virus Baiba Kurenaa, Aleksandra Vežānea, Dace Skrastiņaa, Olga Trofimova and Anna Zajakinaa,* a
Latvian Biomedical Research and Study Centre, Ratsupites street 1, LV-1067, Riga,
Latvia *
Corresponding author. Tel.: +371 67808200; E-mail: [email protected]; Postal address: Ratsupites street 1, LV-1067, Riga, Latvia Graphical abstract
Highlights:
Positively charged magnetic particles enhance Semliki Forest virus transduction.
SFV magneto-transduction is efficient in presence of fetal bovine serum.
Magnetic precipitation can be used for concentration of SFV particles.
Abstract
Semliki Forest virus (SFV) is a potential cancer gene therapy vector capable of providing high and transient expression of heterologous proteins in mammalian cells. However, SFV has shown suboptimal transduction levels in several cancer cell types as well as wide biodistribution of SFV has been observed after in vivo applications. Magnetic nanoparticles (MNPs) have been shown to increase cell transduction with several viral vectors in vitro under an external magnetic field and enhance magnetically guided viral vector delivery. Here, we examined a panel of MNPs for enhanced cancer cell transduction with SFV vector. Magneto-transduction using positively charged MNPs increased Semliki Forest virus transduction in TS/A mouse mammary carcinoma cells in vitro in the presence of fetal bovine serum. Positively charged MNPs efficiently captured SFV particles independently of capturing medium, and MNPs-SFV complexes were successfully separated from suspension by magnetic precipitation. These results reveal the potential application of MNPs for enhanced gene delivery by SFV vector as well as proposes magnetic precipitation for efficient concentration of SFV particles from different media.
Abbreviations: SFV, Semliki Forest virus; MNPs, magnetic nanoparticles; MRI, magnetic resonance imaging. Keywords: virus; alphavirus; magnetic nanoparticles; cancer gene therapy; transduction;
1. Introduction The virus particles-based technologies contribute significantly to the progress of vaccine development, gene therapy, cancer treatment, and targeted drug design. During the last decades the alphaviral vectors genetically modified to produce therapeutic recombinant proteins showed the high potential in plethora of preclinical studies and several clinical trials (Zajakina et al, 2015). Semliki Forest virus (SFV) is an enveloped positive-strand RNA virus belonging to the family Togaviridae and genus Alphavirus (Strauss and Strauss, 1994; King et al., 2012). SFV has been adapted for the expression of heterologous proteins in mammalian cells. This transient expression system provides several advantages including a broad range of host cells susceptible to infection, a high level of heterologous protein expression that is independent of the splicing machinery, and the ability to produce high viral titers (Aranda et al., 2011). The replication deficient SFV expression system is based on a modified SFV genome in which the viral structural genes are replaced by the gene of interest (Liljeström and Garoff, 1991). Therefore, the infectious virus capable of infecting the new target cells cannot be produced, thus providing safe conditions for in vitro and in vivo studies. The biological features of alphaviruses make them an ideal tool for gene transfer experiments in vivo. Apart from the high expression of heterologous gene, the additional advantages in using alphaviral vectors in vivo include a low specific immune response against the vector itself (Pushko et al., 1997; Berglund et al., 1999) and an absence of vector preimmunity. There is a special interest in the application of alphaviruses as a vaccine platform because these vectors are able to infect dendritic cells and can target lymph nodes (Lundstrom, 2014; Veen et al., 2012; Ryman and Klimstra, 2008). Furthermore, alphaviral vectors have proven to be highly successful as therapeutic and prophylactic cancer vaccines (Lundstrom, 2015). In support to the use of alphaviral vectors in cancer therapy, tumor tropism has been documented for Sindbis virus-based vectors (Tseng et al., 2003; Unno et al., 2005; Huang et al., 2012). SFV vectors have shown the ability to localize within tumor after systemic delivery although a broad vector distribution in other organs has been observed simultaneously (Madoz et al., 2007; Vasilevska et al., 2012). Owing to the broad tissue tropism, mostly intratumoral delivery of alphaviral vectors has been applied for cancer therapy.
Another limitation includes the suboptimal transduction levels of certain cancer cell types, thus decreasing the therapeutic potential of the vector (Madoz et al., 2007; Li et al., 2009; Vähä-Koskela et al., 2006, Lundstrom, 2002). This limitation can be overcome by the use of high virus titers, although this can lead to untargeted vector distribution and cytotoxic effects. One solution is to reduce the viral dose and at the same time enhance vector targeting and transduction capacity. For this reason, superparamagnetic nanoparticles (MNPs) have been applied to increase the delivery efficiency of viral particles as well as nucleic acids and proteins. It has previously been shown that the external magnetic field generated by a permanent magnet increases the concentration of MNP-virus complexes on the surface of the cells, thus leading to improved transduction efficiency (Pereyra et al., 2016). Magneto-transduction has demonstrated efficiency with several viruses, such as retrovirus (Castellani et al., 2016; Haim et al., 2005; Tai et al., 2003; Scherer et al., 2002), rhabdovirus (Almstatter et al., 2015), parvovirus (Hwang et al., 2011), paramyxovirus (Kadota et al., 2005), and adenovirus (Tresilwised et al., 2010; Almstätter et al., 2015; Sapet et al., 2012, Kim et al., 2011; Bhattarai et al., 2008; Kamei et al., 2009; Scherer et al., 2002). The MNPs-virus complexes that are formed by electrostatic and hydrophobic interactions are easily generated, in contrast to specific ligand-ligand interaction-based complexes, which require chemical modification of the virus and nanoparticles. The site-specific targeting of magnetically labelled viral particles through an externally applied magnetic field gradient has been demonstrated in vivo (Tresilwised et al., 2010, Tresilwised et al., 2012; Sapet et al., 2012; Bhattarai et al., 2008; Pereyra et al., 2016; Hashimoto and Hisano, 2011). Moreover, the accumulation of MNPs can be clearly visualized by magnetic resonance imaging (MRI) (Sun et al., 2008) and therefore allows for additional MNPs-labeled vector tracking possibilities. In this study, a panel of MNPs were examined, and a method for enhanced gene delivery by Semliki Forest virus vector in a TS/A mouse mammary cancer model in vitro was developed. Moreover, the potential application of MNPs for the efficient concentration of the SFV vector from different media was addressed.
2. Materials and Methods 2.1. Magnetic nanoparticles
In this study, the aqueous dispersions of magnetic nanoparticles (MNPs) from different sources were used. The properties of MNPs are described in Table 1.
2.2. Cell culturing
TS/A (mouse mammary carcinoma) cells (ATCC) were grown in DMEM Glutamax medium (Gibco) supplemented with 10 % fetal bovine serum (FBS, Gibco) and 50 mg/l gentamicin (Sopharma). BHK-21 (Baby hamster kidney) cells (ATTC) were propagated in BHK - Glasgow MEM (Gibco) supplemented with 5% FBS, 10% tryptose phosphate broth, 2 mM L-glutamine, 20 mM HEPES, 100 mg/ml streptomycin and 100 U/ml penicillin. Cells were cultured in a humidified 5% CO2 incubator at 37°C.
2.3. Production of SFV vector
The pSFV1/Ds-Red construct carrying Discosoma sp. red fluorescent protein gene (DsRed) was generated as previously described (Zajakina et al., 2014). For the synthesis of infectious but replication deficient vector particles pSFV1/Ds-Red and pSFV-helper1, plasmids were linearized using SpeI restriction enzyme (Thermo Scientific, Vilnius, Lithuania). In vitro RNA transcription was performed using 3 µg of linearized DNA and 40 U of SP6 RNA polymerase (Thermo Scientific) in a 50-µl reaction mixture. The RNA transcripts were capped during the transcription reaction by adding 1 mM m7G(5’)ppp(5’)G cap-analogue (New England Biolabs, Hitchin, UK). The DNA template was removed by digestion using RNase-free DNase (Thermo Scientific). For the packaging of SFV1/Ds-Red RNA into SFV particles, in vitro transcribed RNAs (recRNA and SFV-helper1 RNA, 20 µg each) were co-electroporated into 1×107 BHK cells (850 V, 25 mF, 2 pulses) using a Bio-Rad Gene Pulser apparatus (Bio-Rad, Hercules, CA, USA) without the pulse controller unit. The electroporated cells were resuspended in 15
ml of complete BHK medium containing 1% FBS, transferred into tissue culture flasks (75 cm2) and incubated at 33°C, 5% CO2. After a 48 h incubation SFV1/Ds-Redcontaining medium was harvested, rapidly frozen and subsequently used as a virus stock. The virus stocks did not contain the replication competent wild-type virus as confirmed by cell reinfection. The viral titers expressed in infectious units per ml (IFU/mL) were quantified by infecting BHK-21 cells and TS/A cells with serial dilutions of viral stock and analyzing Ds-Red expression via fluorescence microscopy on a Leica DM IL microscope (Leica Microsystems Wetzlar GmbH, Germany) as previously described (Vasilevska et al, 2012). The viral titre in BHK-21 cells was 6.25 × 108 IFU/mL, which corresponded to 2.57 × 107 IFU/mL when measured after TS/A cell infection under the same conditions.
2.4. Generation of MNP/virus complexes and cell transduction
The MNP/virus complexes were generated by diluting 10 µl of SFV/Ds-Red virus particles (6.25 × 108 IFU/mL as measured by infection of BHK cells) in 90 µL of DMEM Glutamax w/o FBS and antibiotics and mixing the viral suspension with the following volumes of MNPs: 1 µl of Fluid-Mag Amine, Fluid-Mag Chitosan and Fluid-Mag CMX and 5 µl of poly-Mag Neo, in vivo Poly Mag, PEI Mag and in vivo Viro Mag. For increased transduction, the amount of MNPs corresponded to double the indicated volume: 2 µl Fluid-Mag Amine, Fluid-Mag Chitosan and Fluid-Mag CMX and 10 µl of poly-Mag Neo, in vivo Poly Mag, PEI Mag and in vivo Viro Mag. After 30 min incubation at room temperature, 900 µl of complete TS/A cell culture medium was added to the mixture. For infection TS/A cells were plated on a 12-well plate at concentration of 5 × 105 cells per well. Next day cells were washed once with PBS containing Mg2+ and Ca2+ (Invitrogen, UK) and MNPs/virus complexes (1 ml in total volume) were added. The cells were incubated for 40 min on a permanent magnet (Universal Magnet Plate 8×13 cm, IBA, Germany) and 30 min without the magnet at 37°C and 5 % CO2. Then, the medium containing MNPs/virus complexes was replaced by TS/A cultivation medium. As a control (control -), cell transduction under the same conditions but without MNPs
was performed. The standard SFV transduction (control +) under serum free conditions was performed as previously described. Briefly, cells were washed once with PBS containing Mg2+ and Ca2+, and 10 µL of SFV/Ds-Red virus particles (6,25 × 108 IFU/mL in a stock) were resuspended in 1 mL of PBS containing Mg2+ and Ca2+ and added to the cells. After incubation at 37°C and 5 % CO2 for 1 h and 10 min the solution containing virus was replaced with 1 mL of cultivation medium and cells were incubated at 37oC and 5 % CO2 for 24 h when they were harvested for analysis by flow cytometry.
2.5. Flow cytometry
The infected TS/A cells were harvested 24 h after transduction. Cells were detached by 0.05 % trypsin solution, washed and resuspended in 1 mL of PBS, and immediately processed for analysis by flow cytometry. Ds-Red fluorescence was measured using a FACSAria II (Becton Dickinson Biosciences, San Jose, California, USA). The flow cytometry data were analyzed by BD FACSDiva 6.1.2 software. Uninfected cells were used as a negative control.
2.6. Virus capturing by MNPs For virus capturing experiments 10 µl of SFV/Ds-Red virus particles (6.25 × 108 IFU/mL in a stock as measured by infection of BHK cells) that were resuspended in 280 µl of the capturing medium, which did not contain FBS and antibiotics, were mixed with MNPs. The following concentrations of MNPs were used for virus capture: 1 µl of Fluid-Mag Amine and Fluid-Mag Chitosan and 5 µl of poly-Mag Neo, in vivo Poly Mag, PEI Mag and in vivo Viro Mag. Different capturing media were tested for virus capturing by 1 µL of Fluid-Mag Amine: (I) DMEM Glutamax w/o FBS and antibiotics, (II) a solution containing 500 mM NaCl and 10 mM sodium phosphate (pH 7.0), (III) complete BHK medium containing 5% FBS, and (IV) phosphate buffered saline. The complexes of MNPs/virus were allowed to assemble by incubating the mixture at +4°C for 1.5 h on a rotator. Subsequently the formed complexes were precipitated by incubating the solution on a magnetic rack for 10 min. The supernatants were retained to control the efficiency of
virus capture and precipitation. The pellets were resuspended in 300 µL volume of capturing medium to maintain the same amount of capturing medium as in the supernatants. Both the supernatants and the MNPs/virus suspension from the pellet were mixed with 700 µL of PBS containing Mg2+ and Ca2+ and used for cell transduction. TS/A cells were plated on a 12-well plate at a concentration of 5 × 105 cells per well. Next day cells were washed once with PBS containing Mg2+ and Ca2+ (Invitrogen, UK) and 1 mL of MNP/virus or supernatant mixture was added. Cells were incubated for 40 min on a permanent magnet (Universal Magnet Plate 8×13 cm, IBA, Germany) and 30 min without a magnet at 37°C and 5 % CO2. Then, the medium containing MNPs/virus complexes was replaced by TS/A cultivation medium and cells were incubated at 37oC and 5 % CO2 for 24 h when they were harvested for analysis by flow cytometry.
3. Results
3.1. Magneto-transduction using positively charged magnetic nanoparticles increases Semliki Forest virus transduction in mouse mammary carcinoma cells
To investigate whether magnetic nanoparticles could enhance the transduction of SFV vector, we used SFV1/Ds-Red recombinant particles expressing Discosoma sp. red fluorescent protein. Usually, the efficiency of transduction by alphaviral vectors is very high for susceptible cell lines and can reach 50-70 % of the total cells. To demonstrate the differences in SFV infection with and without MNPs, we significantly diluted the vector and used the amount of viral particles corresponding to an MOI=0.19 (according to virus infectious units measured in TS/A cells at standard conditions; 2.57 × 107 IFU/mL) that transduced 16.3 % of cells, as determined by flow cytometry (Fig. 1, positive control without FBS and MNPs). Moreover, we applied fetal bovine serum (FBS) to inhibit viral infection in the cell culture and thus simulate in vivo conditions. Therefore, the transduction efficiency in the presence of FBS decreased to 2.1 % on average (Fig. 1, negative control with FBS without MNPs). To evaluate the effect of magnetofection, SFV1/Ds-Red vector (MOI=0.2) was preincubated with MNPs for 30 min in serum free conditions before being mixed with the cell culture medium containing FBS. Cell incubation for 40 min on a permanent magnet increased the transduction efficiency in the presence of FBS by up to 21.8 % compared with that of the negative control (2.1 %) (Fig. 1A and 1B). Notably, the enhancement of transduction by FluidMag Amine was achieved even without cell incubation on the magnetic plate (Fig. 1B). We concluded that magnetofection with all of the positively charged MNPs significantly increased the transduction efficiency of SFV in the presence of FBS in a TS/A cell culture model. The most significant transduction enhancement at the conditions tested was demonstrated with highly positive FluidMag Amine MNPs functionalized with aminosilane, whereas negatively charged FluidMax CMX MNPs coated with carboxymethyl-dextran did not affect SFV transduction.
3.2. Positively charged MNPs efficiently capture SFV particles independently of the capturing medium
We showed in Fig. 1 that although the absolute values of transduction efficiency were different for each tested MNP-reagent, only the positively charged particles enhanced cell infection with SFV in a dose dependent manner. On the basis of these observations, we speculated that there might be a close electrostatic association between SFV and positively charged magnetic nanoparticles that leads to complex formation and subsequently a significant enhancement of transduction in TS/A cells under a magnetic field. To explore the possible application of positively charged MNPs for capture and isolation of SFV, we allowed MNPs/virus complexes to form in a capturing medium that did not contain either FBS or antibiotics. Then, we precipitated MNPs/SFV complexes by using a magnetic rack and transferred the supernatants into new tubes to control the efficiency of SFV capture. Both the resuspended MNPs/virus complexes and the supernatants were subjected to TS/A cell transduction (Fig. 2). The results showed that most of the tested magnetic particles were able to capture SFV from solution in serum free conditions (Fig. 3). Notably, the amount of MNPs was sufficient to capture almost all of the SFV particles in the case of the FluidMag Chitosan and In vivo ViroMag. The SFV capture by the PEI-Mag reagent under the applied capturing conditions resulted in cell transduction rate 3 times less efficient than that of the corresponding supernatant from which the complexes were precipitated. Finally, the effect of the medium on viral capture by magnetic nanoparticles was investigated. The FluidMag Amine MNPs together with SFV1/Ds-Red viral particles were incubated in different capturing media, including (I) DMEM Glutamax w/o FBS and antibiotics, (II) a solution containing 500 mM sodium chloride and 10 mM sodium phosphate, pH 7.0, (III) complete BHK medium containing 5% FBS, and (IV) phosphate buffered saline. Interestingly, neither FBS nor a high salt concentration affected SFV binding to positively charged MNPs, thus demonstrating that the self-assembly of complexes by electrostatic and hydrophobic interactions is a powerful means of viral binding to magnetic particles (Fig. 4).
4. Discussion
In this study a panel of magnetic nanoparticles (MNPs) was examined in association with Semliki Forest virus (SFV) vector for enhanced gene delivery in a TS/A mouse breast cancer model in vitro by magneto-transduction. Moreover, the potential application of MNPs for efficient concentration of SFV vector from different media was addressed. Magneto-transduction has been successfully applied to enhance cell transduction and transgene expression in vitro with several viral vectors, including adenoviruses (Tresilwised et al., 2010; Almstätter et al., 2015; Sapet et al., 2012, Kim et al., 2011; Bhattarai et al., 2008; Kamei et al., 2009), vesicular stomatitis virus (Almstätter et al., 2015;), adeno-associated virus (Hwang et al., 2011) and measles virus (Kadota et al., 2005). Therefore MNPs-associated SFV transduction into TS/A carcinoma cells under magnetic field in the presence of FBS was determined. By assembling recombinant Semliki Forest virus with several positively charged MNPs, effective cancer cell transduction with SFV vector under a magnetic field was achieved even in the presence of FBS. These results demonstrate that a magnetic field is crucial for enhanced cell transduction with SFV-MNPs complexes for up to 10.1-fold when compared with transduction without an applied magnetic field (Fig. 1B). This finding is in agreement with results from previous studies describing the importance of a magnetic field for the sedimentation of MNPs/viral complexes on the surface of cells and subsequent cellular uptake (Pereyra et al., 2016). The efficiency of cell transduction with MNPs/SFV complexes under FBS conditions is probably affected by the shielding effect of MNPs against opsonization of viral particles by serum factors and stabilized MNPs/virus complexes, as previously reported (Tresilwised et al., 2010; Tresilwised et al., 2012; Almstätter et al., 2015). An explanation for the observed slightly enhanced (3.6- and 1.6-fold) transduction efficiency with two of the MNPs-SFV complexes in the absence of a magnetic field under FBS conditions compared with viral particles alone (Fig. 1B) may relate to the protective MNPs shielding SFV particles from serum factors. However, MNPs/virus complexes can aggregate because of dipole-dipole interactions between MNPs (Tresilwised et al., 2010; Tresilwised et al., 2012; Almstätter et al., 2015;), and a
substantial number of MNPs can associate with only one viral particle or even clusters of several viral particles through MNPs/virus aggregation can form (Tresilwised et al., 2010), thus increasing the hydrodynamic size of such complexes. Similar results with enhanced cell transduction without an applied magnetic field have previously been observed for MNPs/adenovirus (Pereyra et al., 2016, Tresilwised et al., 2012) and MNPs/retrovirus (Castellani et al., 2016) before. We speculate that MNPs/SFV complexes of a larger size sedimentate on the cell monolayer without an applied external magnetic field, and the proximity promotes enhanced cellular uptake of the complexes. The ratio of MNPs to viral particles has also been shown to be important in the aggregation of MNPs/virus complexes (Kim et al., 2011), and further optimization of the ratio between a chosen MNPs-reagent and SFV particles can be conducted to produce even better results for specific cells. The results showed that only the positively charged MNPs noticeably enhanced cell transduction with SFV vector in the presence of a magnetic field; therefore, we suspected that the positive surface charge of MNPs is a crucial aspect for the association with SFV virus into complexes. Most of the tested positively charged MNPs were able to capture SFV from a solution in serum free conditions with a remarkable efficiency in the case of FluidMag Chitosan and In vivo ViroMag reagents under the particular capturing conditions (Fig. 3). These results indicate the high capture-capacity of these particles, because the supernatants contained only a leaky amount of the virus after precipitation. Such association between MNPs coated with cationic compounds and viral particles with negative electrokinetic potential has been previously described and occurs because of electrostatic and hydrophobic interactions (Almstätter et al., 2015; Sapet et al., 2012). The reason for the poor capture of SFV from solution by the positively charged PEI-Mag reagent even under increased MNPs concentrations (data not shown) is unclear. It has previously been shown that for efficient sedimentation of PEI-Mag/adenovirus complexes, a prolonged incubation time of at least 30 min under the influence of a magnetic field is necessary (Tresilwised et al., 2010), thus leaving room for optimizing the protocol of PEI-Mag/SFV precipitation. Finally, the effect of the medium on viral capture by FluidMag Amine MNPs was investigated. Neither FBS at a concentration of a standard virus-production medium
(5%) nor a high salt concentration (0.5 M NaCl) affected SFV association with positively charged MNPs (Fig. 4), thus demonstrating that self-assembly of complexes by electrostatic and hydrophobic interactions is a powerful means of viral binding to magnetic particles. Previous studies have already used the association of MNPs with a wide range of viruses for magnetic concentration (Uchida et al., 2007; Satoh et al., 2003) or with negatively charged viral particles such as poliovirus-1 (Zhan et al., 2014) and measles virus (Kadota et al., 2005) for diagnostic isolation purposes. These results suggest that SFV captured by MNPs could represent a rapid and efficient way to isolate, purify and concentrate SFV particles by magnetic precipitation from different capturing media. MNPs/virus complexes can be precipitated with a magnetic rack and then washed and resuspended in a small volume of respective buffer, thereby providing a significant concentration and purification rate. As a result, MNPs can be used for the production of high SFV titers, thereby allowing improved cell transduction. The efficiency of SFV capture by MNPs and enhanced cell transduction under FBS conditions encourages the use of MNPs/virus complexes in the hampering in vivo conditions. In fact, several studies have shown success in magnetically-guided delivery of MNPs/virus complexes in vivo (Tresilwised et al., 2010, Tresilwised et al., 2012; Sapet et al., 2012; Bhattarai et al., 2008; Pereyra et al., 2016; Hashimoto and Hisano, 2011), and the current study should serve as a foundation for further research on the potential of MNPs/SFV delivery in vivo by magnetic force. Such magnetic targeting would be a promising strategy for improving the safety and efficacy of SFV vectors in preclinical and clinical applications. Moreover, because MNPs are a suitable contrast agent for magnetic resonance imaging (MRI) (Sun et al., 2008), by labeling viral particles with MNPs it is possible to track viral localization in vivo (Almstätter et al., 2015; Yun et al., 2012; Räty et al., 2006) by MRI. Thus, the association between MNPs and SFV allows for the possibility for SFV trafficking simultaneously with therapeutic applications. In conclusion, the enhanced cancer cell transduction by SFV/MNPs complexes under magnetic field holds promise for future applications such as targeted and enhanced SFV gene therapy with simultaneous tracing of therapeutics by MRI. Moreover, this study suggests that SFV capture by positively charged MNPs could represent a rapid and
efficient way to isolate, purify and concentrate SFV particles by magnetic precipitation independently of capturing medium. Conflicts of interest The authors declare no conflict of interest.
Acknowledgements This work was carried out with support from the Latvian National Research Programme Biomedicine for Public Health (Biomedicine) 2014-2017 and by the ERA Net Rus Plus AlphaChit project.
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Figure legends
Figure 1. Magneto-transduction of TS/A mouse mammary carcinoma cells with Semliki Forest virus vector SFV1/Ds-Red. (A) Fluorescence microscopy of TS/A cells 24 h after cell transduction with SFV1/Ds-Red vector in the presence of fetal bovine serum (+FBS). For magneto-transduction, the vector (6.25 × 106 IFU; measured by infection of BHK cells) was pre-incubated with 1 μL FluidMag Amine, FluidMag Chitosan and FluidMag CMX and 5 μL of PolyMag Neo, In vivo PolyMag, PEI-Mag and In vivo ViroMag. The controls demonstrated cell transduction without MNPs (-MNPs) in the presence (+FBS) and the absence (-FBS) of FBS. (B) Quantitative flow cytometry analysis of transduced TS/A cells expressing Ds-Red fluorescent protein. The cells were transduced with complexes of MNPs associated with SFV1/Ds-Red in the presence of FBS. Transduction in the presence of MNPs with cell incubation on the magnetic plate (MNPs with magnet) and transduction in the presence of MNPs without cell incubation on the magnetic plate (MNPs without magnet) is shown. Transduction in the presence of an increased amount of MNPs corresponds to a double concentration of MNPs in the reaction mixture (2x MNPs with magnet). The controls demonstrate cell transduction without MNPs in the presence of FBS (control -) and in the absence of FBS (control +). The error bars indicate the standard error of three experiments.
Figure 2. Schematic representation of the experimental procedure for the capture of SFV1/Ds-Red particles by MNPs from medium. SFV1/Ds-Red viral particles (6.25 × 106 IFU; measured by infection of BHK cells) were incubated with different MNPs and the formed complexes were precipitated by a magnet. The supernatants were aspirated and used for magneto-transduction of TS/A cells to control the capture efficiency. The pellet was resuspended in the same amount of capturing medium and was used for TS/A cell magneto-transduction to test the capture efficiency.
Figure 3. Efficiency of Semliki Forest virus (SFV1/Ds-Red) capture under serum free conditions. SFV1/Ds-Red virus (6.25 × 106 IFU; measured by infection of BHK cells) was incubated with different types of magnetic nanoparticles (MNPs): 1 μL of FluidMag Amine and FluidMag Chitosan and 5 μL of PolyMag Neo, In vivo PolyMag, PEI-Mag and In vivo ViroMag. Then, the MNP/virus complexes were magnetoprecipitated, the supernatants were removed, and the pellet was resuspended in the same amount of capturing medium. Both resuspended complexes and supernatants were used for TS/A cell magneto-transduction. The results were visualized by fluorescence microscopy (A) and quantified by flow cytometry of Ds-Red expressing cells (B). The error bars indicate the standard error of three experiments.
Figure 4. Effect of the capturing medium on Semliki Forest virus (SFV1/Ds-Red) capture and precipitation. SFV1/Ds-Red virus (6.25 × 106 IFU; measured by infection of BHK cells) was incubated with 1 μL FluidMag Amine in different capturing medium: DMEM Glutamax w/o FBS and antibiotics, a solution containing 500 mM sodium chloride and 10 mM sodium phosphate with pH 7.0, complete BHK medium containing 5 % FBS, and phosphate buffered saline. After magneto-precipitation, the supernatants were removed, and the MNP pellet was reconstituted with the same capturing medium. TS/A cells were infected with supernatants (capturing control) and resuspended particles (captured virus). The Ds-Red expressing cells were quantified by flow cytometry. The error bars indicate the standard error of three experiments.
Table 1 Properties of the superparamagnetic nanoparticles used in this study Magnetic nanoparticles (MNPs)
Manufacturer
Core
Coating
Charge
Hydrodinamic diameter
FluidMag Amine
Chemicell (Germany)
magnetite
aminosilane
+
50 nm
FluidMag Chitosan
Chemicell (Germany)
magnetite
chitosan
+
50 nm
FluidMag CMX
Chemicell (Germany)
magnetite
carboxymethyl-dextran
-
50 nm
PolyMag Neo
OZBiosciences (France)
magnetite
cationic polymer*
+
160 - 220 nm
In vivo PolyMag
OZBiosciences (France)
magnetite
cationic polymer*
+
160 - 220 nm
PEI-Mag
OceanNanoTech (USA)
maghemite
polyethylenimine (PIE)
+
30 nm
In vivo ViroMag
OZBiosciences (France)
magnetite
cationic polymer*
+
200 nm
* not disclosed by the manufacturer
Concentration 25 mg/ml 25 mg/ml 25 mg/ml ND* ND* 1 mg/ml ND*