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Cell surface-engineering to embed targeting ligands or tracking agents on the cell membrane
Biochemical and Biophysical Research Communications xxx (2016) 1e6
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Cell surface-engineering to embed targeting ligands or tracking agents on the cell membrane Kwang Suk Lim a, 1, Daniel Y. Lee a, b, 1, Gabriel M. Valencia a, Young-Wook Won a, *, David A. Bull a, ** a b
Division of Cardiothoracic Surgery, Department of Surgery, School of Medicine, University of Utah, Salt Lake City, UT, USA Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 November 2016 Accepted 27 November 2016 Available online xxx
The key challenge to improve the efficacy of cell therapy is how to efficiently modify cells with a specific molecule or compound that can guide the cells to the target tissue. To address this, we have developed a cell surface engineering technology to non-invasively modify the cell surface. This technology can embed a wide variety of bioactive molecules on any cell surface and allow for the targeting of a wide range of tissues in a variety of disease states. Using our cell surface engineering technology, mesenchymal stem cells (MSC)s were modified with: 1) a homing peptide or a recombinant protein to facilitate the migration of the cells toward a specific molecular target; or 2) magnetic resonance imaging (MRI) contrast agents to allow for in vivo tracking of the cells. The incorporation of a homing peptide or a targeting ligand on MSCs facilitated the migration of the cells toward their molecular target. MRI contrast agents were successfully embedded on the cell surfaces without adverse effects to the cells and the contrast agent-labeled cells were detectable by MRI. Our technology is a promising method of cell surface engineering that is applicable to a broad range of cell therapies. © 2016 Elsevier Inc. All rights reserved.
Keywords: Cell surface engineering Cell delivery Homing peptide SPION Cell tracking
1. Introduction Cell therapy typically involves the systemic infusion of living cells to exert a direct or indirect effect against human disease [1]. The efficacy of cell therapy can be improved by increasing the injection dose and the graft yield, reducing immune-mediated rejection, enhancing circulation time, and/or facilitating migration toward the target tissues [2]. The modification of cells to present targeting ligands and the encapsulation of cells in delivery vehicles have improved the yield of engraftment to the target tissues [3,4]. Such methods, however, still face one or more of the following limitations: (1) exposure of the cells to harsh conditions; (2) use of viral-gene transfer; (3) complicated processes; and (4) permanent modification [5].
* Corresponding author. Medical Research & Education Bldg, Room: 203, 20 N Medical Dr, Salt Lake City, UT 84132, USA. ** Corresponding author. Room 3C127, 30 North 1900 East, Salt Lake City, UT, 84132, USA. E-mail addresses: [email protected] (Y.-W. Won), [email protected] (D.A. Bull). 1 KS Lim and DY Lee contributed equally to this work.
Cell pre-conditioning requires the ex vivo expansion of cells in the presence of toxic chemicals or under a specific condition [6]. Gene-based approaches to induce expression of a particular ligand against the target receptor are highly versatile; however, the poor transfection efficiency and the uncontrolled gene expression remain critical challenges [7,8]. Conjugation of targeting ligands to proteins or polysaccharides present on the cell membrane can alter the membrane structure permanently, suggesting that the activity of the cells is unpredictable upon modification [9,10]. Electrostatic interaction may be an alternative to chemical conjugation [11]. Since the molecules bound to the cell surface by the charge interaction are able to dissociate and internalize into the cells, this method has the potential to affect cell metabolism and viability [12]. Thus, there is a need for an alternative technology that can non-covalently modify cells without altering cellular viability, metabolism or function. We have developed a cell surface-engineering technology based on hydrophobic interaction, allowing any biological molecule to be non-invasively embedded on the cell membrane, while maintaining the inherent activity of the cells [13]. This technology allows for the homogeneous and rapid modification of cells with a variety of biological molecules such as homing peptides, proteins, antibodies,
http://dx.doi.org/10.1016/j.bbrc.2016.11.155 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: K.S. Lim, et al., Cell surface-engineering to embed targeting ligands or tracking agents on the cell membrane, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.155
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and tracking agents regardless of the cell type. In this study, the applicability of our surface-engineering method to modify various types of cells with a small protein, a homing peptide, or magnetic resonance imaging (MRI) contrast agents was examined. 2. Materials and methods 2.1. Materials 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE)poly(ethylene glycols) (PEGs) (3.4 kDa and 5 kDa) and FITCconjugated SPIONs were purchased from Nanocs, Inc. (New York, NY). All cell culture products, FBS, DPBS, PBS, HBSS, and DMEM, were obtained from Invitrogen (Carlsbad, CA). CRPPR-FITC and CRPPR peptide were synthesized by the University of Utah Peptide Core Facility. Recombinant cysteine-rich protein 2 (CRIP2) was purchased from Sino Biological Inc. (Beijing, China). The anti-SDF-1 antibody and FITC-conjugated secondary antibody were obtained from R&D systems (Minneapolis, MN). 2.2. Cell culture All cells were cultured in DMEM containing 20% FBS and 1% antibiotics at 37 C under 5% CO2 atmosphere. At 90% cell confluence, the cells were detached, washed and suspended at 750,000 cells/ml concentration in HBSS. 2.3. Surface-engineering with a homing peptide DMPE-PEG-maleimide was conjugated to CRPPR or CRPPR-FITC peptide. DMPE-PEG-maleimide dissolved in PBS at the concentration of 1 mg/ml and the 1 mg/ml stock solution of CRPPR in PBS was mixed at the final molar ratio of DMPE-PEG to CRPPR ¼ 15:1. After 1 h of incubation at room temperature, impurities were removed by using Amicon Ultra Centrifugal Filters (Ultracel-3 kDa; Millipore, Billerica, MA). Conjugates were kept at 80 C until use. MSCs were cultured as described above and the DMPE-PEG-CRPPR was added directly to the cell suspension. The modified MSCs were collected at different time-points, washed, and suspended in HBSS for confocal microscopy and FACS analysis. 2.4. Modification of MSCs with SDF-1 To prepare DMPE-PEG-SDF-1, 0.5 mg/ml SDF-1 in PBS was mixed with 15-M excess amount of DMPE-PEG-NHS followed by 2 h of reaction at room temperature. The resulting DMPE-PEG-SDF1 was dialyzed (MWCO: 20 kDa) against PBS for 24 h, sterilized by filtration, and stored at 80 C until use. The DMPE-PEG-SDF-1 was added to MSCs at amounts ranging from 5 to 100 mg of SDF-1. The modified MSCs were labeled with a primary anti-SDF-1 antibody and a secondary FITC-goat anti-mouse antibody. These labeled cells were observed by confocal microscopy to visualize the location of SDF-1 on the cell surface and analyzed by flow cytometry to quantify the percentage of the modified cells. 2.5. Migration assay The CRPPR-modified MSCs were placed in the insert of a transwell system and the outer wells were filled with culture media with or without CRIP2. After 24 h of incubation, MSCs remaining on the topside of the insert were removed and the rMSCs on the bottom of the insert were harvested and counted. To determine the dose effect of the CRPPR on the migration, different amounts of the DMPE-PEG-CRPPR were added to a fixed number of MSCs. The modified MSCs were plated in the transwell system as described
above. The effect of CRIP2 dose on MSC migration was investigated. The MSCs modified with 60 mg of the DMPE-PEG-CRPPR were seeded in the transwell system and then the outer wells were filled with the culture media with increasing concentrations of CRIP2. After 24 h of incubation, the number of migrated MSCs was determined by measuring the viable cells on the bottom of the insert using a MTT assay. To generate SDF-1-embedded MSCs, MSCs were mixed with 100 mg of the DMPE-PEG-SDF-1 and seeded in the inserts of the transwell system at a density of 2 104 cells in complete culture media. The lower reservoirs were filled with complete culture media with 0, 50, 100, 200, or 300 ng/ml CXCR4. After 24 h of incubation, cells remaining on the topside of the insert were removed and the cells migrated on the bottom of the insert were also counted by MTT. 2.6. Tests in various cell lines DMPE-PEG-FITC was added directly to various cell lines including 293T, Jurkat, A549, BT-474, MCF-7, MDA-MB-231, MSCs, and SK-BR-3. After 10 min of incubation, cells were harvested, washed, and suspended in HBSS for confocal microscopy and FACS analysis. The modified cells were seeded on 24-well plates and incubated for 18 h. The cell adhesion and the morphology of the cells were observed using an optical microscope (Olympus; Tokyo, Japan). The cell viability and the proliferation after the modification were determined by a MTT assay and a cell proliferation assay (Molecular Probes; Eugene, OR), respectively. The harvested MSCs after modification were stained with trypan blue and counted to calculate the recovery rate. 2.7. Modification with tracking agents FITC-labeled carboxyl terminated SPIONs were attached to DMPE-PEG-NH2 through NHS/EDC conjugation chemistry. Briefly, 5 mg of EDC/HCl and 10 mg of sulfo-NHS were reacted with 1 ml of SPIONs containing 2 mg of iron in MES buffer (pH 6.0) for 30 min at room temperature. After EDC and sulfo-NHS were removed using a spin column (MWCO: 10 kDa), NHS-activated SPIONs were mixed with 20 mg of DMPE-PEG-NH2 to saturate the NHS-activated sites for 2 h at room temperature. The DMPE-PEG-SPION-FITC was dialyzed (MWCO: 10 kDa) against PBS (pH 7.4) for 24 h. Amounts of the DMPE-PEG-SPION-FITC ranging from 10 to 200 ml were used to modify Jurkat cells using the one-step modification process and analyzed by confocal microscopy and FACS analysis. 2.8. MRI Jurkat cells were modified with 50, 100, or 200 ml of the DMPEPEG-SPION-FITC. One hundred thousand SPION-modified or unmodified Jurkat cells were seeded into a pre-molded agar gel prepared in 8-chamber coverglass with 0.5% (w/v) of low melting agarose in PBS. Before the gel completely solidified, cooled 0.5% agarose solution was added on the top of the seeded cells and kept at 4 C to solidify. Agar phantom containing unmodified or SPIONmodified Jurkat cells were examined with Bruker 7T scanner (Bruker Biospin; Ettlingen, Germany). Samples were analyzed using T2*-weighted sequence (TR/TE/flip-angle ¼ 323 ms/7.5 ms/30 ) with fast low-angle shot (FLASH). Images were taken in 25 slices (slice thickness ¼ 0.5 mm) in plane resolution 0.195 mm 0.195 mm. 2.9. Statistical analysis All data are presented as the mean ± standard deviation (n ¼ 3). Statistical analysis was performed with the Student's t-test. Values
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of #P < 0.01 and were considered statistically significant. 3. Results
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observed between the modified MSCs and the non-modified MSCs (Fig. 1c and d), which means that the modified MSCs retain the ability to proliferate and that our cell surface-engineering technology does not affect the cell growth.
3.1. Two-step modification 3.3. Modification of MSCs with a protein We studied two types of lipid-PEGs and found that the 5 kDa lipid-PEG was more effective for cell surface modification than the 3 kDa lipid-PEG (Supplementary Fig. 1). This may be due to the fact that the cargo molecule attached to the lipid-PEG can diminish the hydrophobic interaction of the lipid moiety with the phospholipid in the cell membrane [14]. The two-step modification method has been optimized to embed the lipid-PEG on the cell surface and then conjugates the target molecule to the lipid-PEG (Supplementary Figs. 2 and 3). 3.2. Modification of MSCs with a homing peptide To determine the minimal amount of the DMPE-PEG-conjugated peptide necessary to achieve homogeneous coating of the cell surface, the DMPE-PEG-conjugated CRPPR-FITC was added incrementally to a constant number of MSCs. The CRPPR-FITC incorporated homogeneously on the surface of the MSCs and the degree of modification was in direct proportion to the amount of the conjugate added up to 30 mg (Fig. 1a and b). When the amount of the DMPE-PEG-CRPPR-FITC was over 60 mg, the fluorescence intensity in the cytosol increased significantly, meaning that internalization of the conjugate occurred. The minimal amount of the DMPE-PEGCRPPR required to lead to the complete modification of 750,000 MSCs can be as little as 5 mg. To determine the effect of the surface modification on cell viability and proliferation, the CRPPR-modified MSCs and nonmodified MSCs were plated and cultured for 48 h. The viabilities and the proliferation rates were determined at 24 h and 48 h postseeding. No difference in cell viability and cell proliferation was
SDF-1 was chosen to modify MSCs through the optimized method because the CXCR4/SDF-1 axis can be easily studied in a migration assay. The MSCs were modified to present SDF-1 on the cell surface in order to verify uniform modification with a protein and re-confirm the enhanced migration that we observed in the prior study [13]. Although 5 mg of the DMPE-PEG-SDF-1 was sufficient to modify the given number of cells, the degree of modification was increased with an increasing amount of the added DMPE-PEG-SDF-1 compared to control and the group of 0 mg (non-modified cells incubated with anti-SDF-1 antibody) (Fig. 2a and b). 3.4. Migration of modified MSCs Migration of MSCs modified with the DMPE-PEG-CRPPR or the DMPE-PEG-SDF-1 toward its complementary homing signal was verified. First, MSCs were modified with increasing amounts of the DMPE-PEG-CRPPR to investigate the effect of dosing on migration. The migration of the modified MSCs with CRPPR was examined in the presence of the recombinant CRIP2 protein, which is the molecular target of the CRPPR peptide. The number of MSCs migrating towards CRIP2 was significantly increased compared to that of the non-modified MSCs (Fig. 1e). The extent of migration was in direct proportion to the amount of the embedded DMPE-PEG-CRPPR on the surface of the MSCs. To demonstrate a CRIP2 gradientdependent increase in migration, MSCs modified with 60 mg of the DMPE-PEG-CRPPR were exposed to increasing concentrations of CRIP2. The migration of the modified MSCs was increased in a
Fig. 1. MSCs modified with a homing peptide by the one-step method. (a) Confocal micrograph showing the modified MSCs with CRPPR. (b) The mean fluorescence intensity (MFI) of the modified MSCs determined by FACS analysis. (c) Cell viabilities and (d) Proliferation rate of the modified MSCs. (e) Migration of the MSCs modified with increasing amounts of DMPE-PEG-CRPPR toward CRIP2 (#P < 0.01). Migration of the MSCs modified with (f) DMPE-PEG-CRPPR toward CRIP2 gradient (#P < 0.01).
Please cite this article in press as: K.S. Lim, et al., Cell surface-engineering to embed targeting ligands or tracking agents on the cell membrane, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.155
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Fig. 2. Confocal micrograph and FACS analysis of the SDF-1-modified MSCs. (a) MSCs presenting SDF-1 on the surface observed by confocal microscopy. (b) The degree of modification determined by the FACS analysis. (c) Migration of the MSCs modified with DMPE-PEG-SDF-1 toward CXCR4 gradient (#P < 0.01).
CRIP2 dose-dependent manner (Fig. 1f). As shown in Fig. 2c, migration of the modified MSCs with the DMPE-PEG-SDF-1 increased toward CXCR4, which is the target receptor for SDF-1. 3.5. Normalization To demonstrate the feasibility of our surface-engineering technology to serve as a platform that can be applied to the modification of a wide variety of cells, 293T, Jurkat T, A549, BT-474, MCF-7, MDA-MB-231, and SK-BR-3 cells were modified with the DMPEPEG-FITC through the optimized method and then observed by confocal microscopy. The DMPE-PEG-FITC was detected on the surface of the cells (Fig. 3a). Although the degree of the surface modification varies by cell type, 100% of the given cell population was modified with the DMPE-PEG-FITC. The modified cells were seeded on a cell culture plate to evaluate the ability of the modified cells to adhere to the plate. There was no difference in cell adhesion between the modified cells and the non-modified parent cells (Fig. 3b). 3.6. Modification with tracking agents To test whether or not our surface-engineering technology can be used to label cells with tracking agents, Jurkat cells were modified with the DMPE-PEG-SPION-FITC. Jurkat cells modified with increasing amounts of the DMPE-PEG-SPION-FITC showed
fluorescent emission on the cell surface, but not in the cytosol (Fig. 4a), while FITC emission was not observed in the non-modified Jurkat cells. As shown in Fig. 4b, Jurkat cells modified with the DMPE-PEG-SPION-FITC were detectable by MRI, displayed a positive MRI signal, and the signal intensity increased as the dosage of SPION increased. The modification of Jurkat cells with the DMPEPEG-SPION has no influence on the viability or the proliferative capacity of the cells (Fig. 4c and d). These results demonstrate that our surface engineering technology can be used to embed tracking agents on the surfaces of cells without the creation of covalent bonds or the internalization of the agents into the cells.
4. Discussion For the embedding of amphiphilic proteins or peptides, the twostep modification method minimizes the formation of micelles and liposomes as well as the self-assembly of a lipid-PEG-conjugated protein or -peptide, each of which can inhibit the interaction of the lipid domain with the cell membrane [15,16]. In the case of large molecules such as antibodies, the molar ratio of the lipid to the antibody may be too low, indicating that the lipid portion may be shielded by the antibody [17]. By our analysis, the two-step modification method is a better means to modify cells with a large and/or an amphiphilic molecule. The one-step modification method requires pre-conjugation of lipid-PEG with the required biologic molecule prior to the
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Fig. 3. Surface modification of a variety of cell types. (a) Confocal micrographs showing the DMPE-PEG-FITC located on the surface of cells regardless of the cell type. MFI: Mean Fluorescence Intensity determined using FACS. (b) Adhesion of the modified cells on a tissue culture plate.
Fig. 4. Characteristics of the Jurkat cells modified with the MRI tracking agent. (a) Confocal micrograph of the modified Jurkat cells. (b) MRI of agar phantoms containing Jurkat cells modified with different amounts of the DMPE-PEG-SPION-FITC. (c) Cell proliferation and (d) viability of Jurkat cells after modification.
incubation with cells. This method can be beneficial for the modification of cell surfaces with membrane-permeable peptides or proteins. This method generates modified cells more quickly, decreases the amount of conjugate required, increases the yield of modified cells, improves cell viability, and minimizes any adverse effects on cellular activity compared to the two-step procedure. The one step method is therefore broadly applicable to many types of cell therapy. The percentage of MSCs engrafted into a target organ following systemic injection is typically very low. For example, generally only 1% of injected MSCs that have been expanded ex vivo reach the acutely infarcted myocardium [18]. Although MSC-based cell therapies have shown promising outcomes in several clinical settings [1,19], the homing of the injected MSCs to the target tissues has to be improved. Toward this end, polymeric scaffolds, microspheres, and hydrogels have been tested. The results, however, have been unsatisfactory [20,21], because of the reduced activity, viability, and differentiation potential of the MSCs following ex-vivo expansion.
This means that tremendous numbers of MSCs have to be administered to achieve a therapeutic benefit [22]. On the other hand, the surface modification of MSCs using a lipid-PEG platform has minimal effect on the intrinsic activity of the cells and a high recovery yield, regardless of the type of bioactive molecules embedded on the cell surface. More importantly, the MSCs modified by this method can migrate toward the gradient of the matching ligand. Consequently, modification of the cell surface of MSCs with homing molecules provides a safe means to improve the migration of MSCs toward their target tissues, resulting in enhanced efficacy of the cell therapy. Given their ability to produce a sharp contrast with MRI, SPIONs have become well-established imaging agents for a variety of biomedical applications [23,24]. SPION-based imaging provides for the non-invasive tracking of cells in real-time to monitor their distribution throughout the living body. There are a number of methods currently in use to label cells with SPIONs. A method using cellular uptake has been the most commonly used labeling
Please cite this article in press as: K.S. Lim, et al., Cell surface-engineering to embed targeting ligands or tracking agents on the cell membrane, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.155
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technique, since at high concentrations, surface-modified SPIONs can easily internalize into cells [25,26]. Even though surface-coated SPIONs are generally regarded as safe, adverse effects associated with SPIONs internalized within cells has been observed. Within a cell, SPIONs are often found in the acidic environments of lysosomes [27,28] that can promote the degradation of both the protective coat and iron oxide core of the SPIONs to release iron ions [25,29], which in turn can disrupt cell homeostasis and lead to cytotoxicity [25,30]. Recent studies suggest that the long-term adverse effects of SPIONs should not be neglected, especially when stem cells or immune cells are labeled and administrated for therapeutic purposes [28,31]. Cationic polyelectrolyte-stabilized SPIONs [32] appear to stay bound to the surface of cells [32]. The cytotoxicity of cationic SPIONs is generally higher than that of neutral or slightly negatively charged SPIONs [33,34]. Thus, a method that can non-invasively and rapidly label cells with nonpositively charged SPIONs would offer significant advantages. Labeling cells with SPIONs using a lipid-PEG platform offers several advantages over the methodologies discussed above. The generation of lipid-PEG-conjugated SPIONs is inexpensive and straightforward. A variety of target cells can be instantly functionalized using lipid-PEG-SPIONs without a complicated modification process. Cell-surface modification with lipid-PEG-SPIONs is biocompatible because the modification is transient, the degree of modification is controllable by adjusting the dosage, cell viability is unaffected, and the internalization of SPIONs is substantially reduced. Hence, cell surface modification using lipid-PEG-SPIONs allows for the efficient tracking of cells without altering their therapeutic function. We have explored the applicability of cell surface-engineering to embed various types of bioactive molecules on the surfaces of a variety of cell types. With this technology, a homing peptide, targeting ligand, or contrast agent can be placed non-invasively and transiently on the surface of a cell membrane without adversely affecting the cell or compromising cell function. This cell surfaceengineering technology is broadly applicable to the field of cell therapy. Conflict of interest The authors declare that there are no conflicts of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.11.155. References [1] J. Ankrum, J.M. Karp, Mesenchymal stem cell therapy: two steps forward, one step back, Trends Mol. Med. 16 (2010) 203e209. [2] J.M. Hare, J.E. Fishman, G. Gerstenblith, D.L.D. Velazquez, J.P. Zambrano, V.Y. Suncion, M. Tracy, E. Ghersin, P.V. Johnston, J.A. Brinker, Comparison of allogeneic vs autologous bone marrowederived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial, Jama 308 (2012) 2369e2379. [3] R.F. Wynn, C.A. Hart, C. Corradi-Perini, L. O'Neill, C.A. Evans, J.E. Wraith, L.J. Fairbairn, I. Bellantuono, A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow, Blood 104 (2004) 2643e2645. [4] J.M. Karp, G.S.L. Teo, Mesenchymal stem cell homing: the devil is in the details, Cell Stem Cell 4 (2009) 206e216. [5] Y. Teramura, Y. Kaneda, T. Totani, H. Iwata, Behavior of synthetic polymers immobilized on a cell membrane, Biomaterials 29 (2008) 1345e1355. [6] T.C. Srijaya, T.S. Ramasamy, N.H. Kasim, Advancing stem cell therapy from bench to bedside: lessons from drug therapies, J. Transl. Med. 12 (2014) 243.
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Please cite this article in press as: K.S. Lim, et al., Cell surface-engineering to embed targeting ligands or tracking agents on the cell membrane, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.155
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Cell surface-engineering to embed targeting ligands or tracking agents on the cell membrane Kwang Suk Lim a, 1, Daniel Y. Lee a, b, 1, Gabriel M. Valencia a, Young-Wook Won a, *, David A. Bull a, ** a b
Division of Cardiothoracic Surgery, Department of Surgery, School of Medicine, University of Utah, Salt Lake City, UT, USA Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 November 2016 Accepted 27 November 2016 Available online xxx
The key challenge to improve the efficacy of cell therapy is how to efficiently modify cells with a specific molecule or compound that can guide the cells to the target tissue. To address this, we have developed a cell surface engineering technology to non-invasively modify the cell surface. This technology can embed a wide variety of bioactive molecules on any cell surface and allow for the targeting of a wide range of tissues in a variety of disease states. Using our cell surface engineering technology, mesenchymal stem cells (MSC)s were modified with: 1) a homing peptide or a recombinant protein to facilitate the migration of the cells toward a specific molecular target; or 2) magnetic resonance imaging (MRI) contrast agents to allow for in vivo tracking of the cells. The incorporation of a homing peptide or a targeting ligand on MSCs facilitated the migration of the cells toward their molecular target. MRI contrast agents were successfully embedded on the cell surfaces without adverse effects to the cells and the contrast agent-labeled cells were detectable by MRI. Our technology is a promising method of cell surface engineering that is applicable to a broad range of cell therapies. © 2016 Elsevier Inc. All rights reserved.
Keywords: Cell surface engineering Cell delivery Homing peptide SPION Cell tracking
1. Introduction Cell therapy typically involves the systemic infusion of living cells to exert a direct or indirect effect against human disease [1]. The efficacy of cell therapy can be improved by increasing the injection dose and the graft yield, reducing immune-mediated rejection, enhancing circulation time, and/or facilitating migration toward the target tissues [2]. The modification of cells to present targeting ligands and the encapsulation of cells in delivery vehicles have improved the yield of engraftment to the target tissues [3,4]. Such methods, however, still face one or more of the following limitations: (1) exposure of the cells to harsh conditions; (2) use of viral-gene transfer; (3) complicated processes; and (4) permanent modification [5].
* Corresponding author. Medical Research & Education Bldg, Room: 203, 20 N Medical Dr, Salt Lake City, UT 84132, USA. ** Corresponding author. Room 3C127, 30 North 1900 East, Salt Lake City, UT, 84132, USA. E-mail addresses: [email protected] (Y.-W. Won), [email protected] (D.A. Bull). 1 KS Lim and DY Lee contributed equally to this work.
Cell pre-conditioning requires the ex vivo expansion of cells in the presence of toxic chemicals or under a specific condition [6]. Gene-based approaches to induce expression of a particular ligand against the target receptor are highly versatile; however, the poor transfection efficiency and the uncontrolled gene expression remain critical challenges [7,8]. Conjugation of targeting ligands to proteins or polysaccharides present on the cell membrane can alter the membrane structure permanently, suggesting that the activity of the cells is unpredictable upon modification [9,10]. Electrostatic interaction may be an alternative to chemical conjugation [11]. Since the molecules bound to the cell surface by the charge interaction are able to dissociate and internalize into the cells, this method has the potential to affect cell metabolism and viability [12]. Thus, there is a need for an alternative technology that can non-covalently modify cells without altering cellular viability, metabolism or function. We have developed a cell surface-engineering technology based on hydrophobic interaction, allowing any biological molecule to be non-invasively embedded on the cell membrane, while maintaining the inherent activity of the cells [13]. This technology allows for the homogeneous and rapid modification of cells with a variety of biological molecules such as homing peptides, proteins, antibodies,
http://dx.doi.org/10.1016/j.bbrc.2016.11.155 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: K.S. Lim, et al., Cell surface-engineering to embed targeting ligands or tracking agents on the cell membrane, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.155
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and tracking agents regardless of the cell type. In this study, the applicability of our surface-engineering method to modify various types of cells with a small protein, a homing peptide, or magnetic resonance imaging (MRI) contrast agents was examined. 2. Materials and methods 2.1. Materials 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE)poly(ethylene glycols) (PEGs) (3.4 kDa and 5 kDa) and FITCconjugated SPIONs were purchased from Nanocs, Inc. (New York, NY). All cell culture products, FBS, DPBS, PBS, HBSS, and DMEM, were obtained from Invitrogen (Carlsbad, CA). CRPPR-FITC and CRPPR peptide were synthesized by the University of Utah Peptide Core Facility. Recombinant cysteine-rich protein 2 (CRIP2) was purchased from Sino Biological Inc. (Beijing, China). The anti-SDF-1 antibody and FITC-conjugated secondary antibody were obtained from R&D systems (Minneapolis, MN). 2.2. Cell culture All cells were cultured in DMEM containing 20% FBS and 1% antibiotics at 37 C under 5% CO2 atmosphere. At 90% cell confluence, the cells were detached, washed and suspended at 750,000 cells/ml concentration in HBSS. 2.3. Surface-engineering with a homing peptide DMPE-PEG-maleimide was conjugated to CRPPR or CRPPR-FITC peptide. DMPE-PEG-maleimide dissolved in PBS at the concentration of 1 mg/ml and the 1 mg/ml stock solution of CRPPR in PBS was mixed at the final molar ratio of DMPE-PEG to CRPPR ¼ 15:1. After 1 h of incubation at room temperature, impurities were removed by using Amicon Ultra Centrifugal Filters (Ultracel-3 kDa; Millipore, Billerica, MA). Conjugates were kept at 80 C until use. MSCs were cultured as described above and the DMPE-PEG-CRPPR was added directly to the cell suspension. The modified MSCs were collected at different time-points, washed, and suspended in HBSS for confocal microscopy and FACS analysis. 2.4. Modification of MSCs with SDF-1 To prepare DMPE-PEG-SDF-1, 0.5 mg/ml SDF-1 in PBS was mixed with 15-M excess amount of DMPE-PEG-NHS followed by 2 h of reaction at room temperature. The resulting DMPE-PEG-SDF1 was dialyzed (MWCO: 20 kDa) against PBS for 24 h, sterilized by filtration, and stored at 80 C until use. The DMPE-PEG-SDF-1 was added to MSCs at amounts ranging from 5 to 100 mg of SDF-1. The modified MSCs were labeled with a primary anti-SDF-1 antibody and a secondary FITC-goat anti-mouse antibody. These labeled cells were observed by confocal microscopy to visualize the location of SDF-1 on the cell surface and analyzed by flow cytometry to quantify the percentage of the modified cells. 2.5. Migration assay The CRPPR-modified MSCs were placed in the insert of a transwell system and the outer wells were filled with culture media with or without CRIP2. After 24 h of incubation, MSCs remaining on the topside of the insert were removed and the rMSCs on the bottom of the insert were harvested and counted. To determine the dose effect of the CRPPR on the migration, different amounts of the DMPE-PEG-CRPPR were added to a fixed number of MSCs. The modified MSCs were plated in the transwell system as described
above. The effect of CRIP2 dose on MSC migration was investigated. The MSCs modified with 60 mg of the DMPE-PEG-CRPPR were seeded in the transwell system and then the outer wells were filled with the culture media with increasing concentrations of CRIP2. After 24 h of incubation, the number of migrated MSCs was determined by measuring the viable cells on the bottom of the insert using a MTT assay. To generate SDF-1-embedded MSCs, MSCs were mixed with 100 mg of the DMPE-PEG-SDF-1 and seeded in the inserts of the transwell system at a density of 2 104 cells in complete culture media. The lower reservoirs were filled with complete culture media with 0, 50, 100, 200, or 300 ng/ml CXCR4. After 24 h of incubation, cells remaining on the topside of the insert were removed and the cells migrated on the bottom of the insert were also counted by MTT. 2.6. Tests in various cell lines DMPE-PEG-FITC was added directly to various cell lines including 293T, Jurkat, A549, BT-474, MCF-7, MDA-MB-231, MSCs, and SK-BR-3. After 10 min of incubation, cells were harvested, washed, and suspended in HBSS for confocal microscopy and FACS analysis. The modified cells were seeded on 24-well plates and incubated for 18 h. The cell adhesion and the morphology of the cells were observed using an optical microscope (Olympus; Tokyo, Japan). The cell viability and the proliferation after the modification were determined by a MTT assay and a cell proliferation assay (Molecular Probes; Eugene, OR), respectively. The harvested MSCs after modification were stained with trypan blue and counted to calculate the recovery rate. 2.7. Modification with tracking agents FITC-labeled carboxyl terminated SPIONs were attached to DMPE-PEG-NH2 through NHS/EDC conjugation chemistry. Briefly, 5 mg of EDC/HCl and 10 mg of sulfo-NHS were reacted with 1 ml of SPIONs containing 2 mg of iron in MES buffer (pH 6.0) for 30 min at room temperature. After EDC and sulfo-NHS were removed using a spin column (MWCO: 10 kDa), NHS-activated SPIONs were mixed with 20 mg of DMPE-PEG-NH2 to saturate the NHS-activated sites for 2 h at room temperature. The DMPE-PEG-SPION-FITC was dialyzed (MWCO: 10 kDa) against PBS (pH 7.4) for 24 h. Amounts of the DMPE-PEG-SPION-FITC ranging from 10 to 200 ml were used to modify Jurkat cells using the one-step modification process and analyzed by confocal microscopy and FACS analysis. 2.8. MRI Jurkat cells were modified with 50, 100, or 200 ml of the DMPEPEG-SPION-FITC. One hundred thousand SPION-modified or unmodified Jurkat cells were seeded into a pre-molded agar gel prepared in 8-chamber coverglass with 0.5% (w/v) of low melting agarose in PBS. Before the gel completely solidified, cooled 0.5% agarose solution was added on the top of the seeded cells and kept at 4 C to solidify. Agar phantom containing unmodified or SPIONmodified Jurkat cells were examined with Bruker 7T scanner (Bruker Biospin; Ettlingen, Germany). Samples were analyzed using T2*-weighted sequence (TR/TE/flip-angle ¼ 323 ms/7.5 ms/30 ) with fast low-angle shot (FLASH). Images were taken in 25 slices (slice thickness ¼ 0.5 mm) in plane resolution 0.195 mm 0.195 mm. 2.9. Statistical analysis All data are presented as the mean ± standard deviation (n ¼ 3). Statistical analysis was performed with the Student's t-test. Values
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of #P < 0.01 and were considered statistically significant. 3. Results
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observed between the modified MSCs and the non-modified MSCs (Fig. 1c and d), which means that the modified MSCs retain the ability to proliferate and that our cell surface-engineering technology does not affect the cell growth.
3.1. Two-step modification 3.3. Modification of MSCs with a protein We studied two types of lipid-PEGs and found that the 5 kDa lipid-PEG was more effective for cell surface modification than the 3 kDa lipid-PEG (Supplementary Fig. 1). This may be due to the fact that the cargo molecule attached to the lipid-PEG can diminish the hydrophobic interaction of the lipid moiety with the phospholipid in the cell membrane [14]. The two-step modification method has been optimized to embed the lipid-PEG on the cell surface and then conjugates the target molecule to the lipid-PEG (Supplementary Figs. 2 and 3). 3.2. Modification of MSCs with a homing peptide To determine the minimal amount of the DMPE-PEG-conjugated peptide necessary to achieve homogeneous coating of the cell surface, the DMPE-PEG-conjugated CRPPR-FITC was added incrementally to a constant number of MSCs. The CRPPR-FITC incorporated homogeneously on the surface of the MSCs and the degree of modification was in direct proportion to the amount of the conjugate added up to 30 mg (Fig. 1a and b). When the amount of the DMPE-PEG-CRPPR-FITC was over 60 mg, the fluorescence intensity in the cytosol increased significantly, meaning that internalization of the conjugate occurred. The minimal amount of the DMPE-PEGCRPPR required to lead to the complete modification of 750,000 MSCs can be as little as 5 mg. To determine the effect of the surface modification on cell viability and proliferation, the CRPPR-modified MSCs and nonmodified MSCs were plated and cultured for 48 h. The viabilities and the proliferation rates were determined at 24 h and 48 h postseeding. No difference in cell viability and cell proliferation was
SDF-1 was chosen to modify MSCs through the optimized method because the CXCR4/SDF-1 axis can be easily studied in a migration assay. The MSCs were modified to present SDF-1 on the cell surface in order to verify uniform modification with a protein and re-confirm the enhanced migration that we observed in the prior study [13]. Although 5 mg of the DMPE-PEG-SDF-1 was sufficient to modify the given number of cells, the degree of modification was increased with an increasing amount of the added DMPE-PEG-SDF-1 compared to control and the group of 0 mg (non-modified cells incubated with anti-SDF-1 antibody) (Fig. 2a and b). 3.4. Migration of modified MSCs Migration of MSCs modified with the DMPE-PEG-CRPPR or the DMPE-PEG-SDF-1 toward its complementary homing signal was verified. First, MSCs were modified with increasing amounts of the DMPE-PEG-CRPPR to investigate the effect of dosing on migration. The migration of the modified MSCs with CRPPR was examined in the presence of the recombinant CRIP2 protein, which is the molecular target of the CRPPR peptide. The number of MSCs migrating towards CRIP2 was significantly increased compared to that of the non-modified MSCs (Fig. 1e). The extent of migration was in direct proportion to the amount of the embedded DMPE-PEG-CRPPR on the surface of the MSCs. To demonstrate a CRIP2 gradientdependent increase in migration, MSCs modified with 60 mg of the DMPE-PEG-CRPPR were exposed to increasing concentrations of CRIP2. The migration of the modified MSCs was increased in a
Fig. 1. MSCs modified with a homing peptide by the one-step method. (a) Confocal micrograph showing the modified MSCs with CRPPR. (b) The mean fluorescence intensity (MFI) of the modified MSCs determined by FACS analysis. (c) Cell viabilities and (d) Proliferation rate of the modified MSCs. (e) Migration of the MSCs modified with increasing amounts of DMPE-PEG-CRPPR toward CRIP2 (#P < 0.01). Migration of the MSCs modified with (f) DMPE-PEG-CRPPR toward CRIP2 gradient (#P < 0.01).
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Fig. 2. Confocal micrograph and FACS analysis of the SDF-1-modified MSCs. (a) MSCs presenting SDF-1 on the surface observed by confocal microscopy. (b) The degree of modification determined by the FACS analysis. (c) Migration of the MSCs modified with DMPE-PEG-SDF-1 toward CXCR4 gradient (#P < 0.01).
CRIP2 dose-dependent manner (Fig. 1f). As shown in Fig. 2c, migration of the modified MSCs with the DMPE-PEG-SDF-1 increased toward CXCR4, which is the target receptor for SDF-1. 3.5. Normalization To demonstrate the feasibility of our surface-engineering technology to serve as a platform that can be applied to the modification of a wide variety of cells, 293T, Jurkat T, A549, BT-474, MCF-7, MDA-MB-231, and SK-BR-3 cells were modified with the DMPEPEG-FITC through the optimized method and then observed by confocal microscopy. The DMPE-PEG-FITC was detected on the surface of the cells (Fig. 3a). Although the degree of the surface modification varies by cell type, 100% of the given cell population was modified with the DMPE-PEG-FITC. The modified cells were seeded on a cell culture plate to evaluate the ability of the modified cells to adhere to the plate. There was no difference in cell adhesion between the modified cells and the non-modified parent cells (Fig. 3b). 3.6. Modification with tracking agents To test whether or not our surface-engineering technology can be used to label cells with tracking agents, Jurkat cells were modified with the DMPE-PEG-SPION-FITC. Jurkat cells modified with increasing amounts of the DMPE-PEG-SPION-FITC showed
fluorescent emission on the cell surface, but not in the cytosol (Fig. 4a), while FITC emission was not observed in the non-modified Jurkat cells. As shown in Fig. 4b, Jurkat cells modified with the DMPE-PEG-SPION-FITC were detectable by MRI, displayed a positive MRI signal, and the signal intensity increased as the dosage of SPION increased. The modification of Jurkat cells with the DMPEPEG-SPION has no influence on the viability or the proliferative capacity of the cells (Fig. 4c and d). These results demonstrate that our surface engineering technology can be used to embed tracking agents on the surfaces of cells without the creation of covalent bonds or the internalization of the agents into the cells.
4. Discussion For the embedding of amphiphilic proteins or peptides, the twostep modification method minimizes the formation of micelles and liposomes as well as the self-assembly of a lipid-PEG-conjugated protein or -peptide, each of which can inhibit the interaction of the lipid domain with the cell membrane [15,16]. In the case of large molecules such as antibodies, the molar ratio of the lipid to the antibody may be too low, indicating that the lipid portion may be shielded by the antibody [17]. By our analysis, the two-step modification method is a better means to modify cells with a large and/or an amphiphilic molecule. The one-step modification method requires pre-conjugation of lipid-PEG with the required biologic molecule prior to the
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Fig. 3. Surface modification of a variety of cell types. (a) Confocal micrographs showing the DMPE-PEG-FITC located on the surface of cells regardless of the cell type. MFI: Mean Fluorescence Intensity determined using FACS. (b) Adhesion of the modified cells on a tissue culture plate.
Fig. 4. Characteristics of the Jurkat cells modified with the MRI tracking agent. (a) Confocal micrograph of the modified Jurkat cells. (b) MRI of agar phantoms containing Jurkat cells modified with different amounts of the DMPE-PEG-SPION-FITC. (c) Cell proliferation and (d) viability of Jurkat cells after modification.
incubation with cells. This method can be beneficial for the modification of cell surfaces with membrane-permeable peptides or proteins. This method generates modified cells more quickly, decreases the amount of conjugate required, increases the yield of modified cells, improves cell viability, and minimizes any adverse effects on cellular activity compared to the two-step procedure. The one step method is therefore broadly applicable to many types of cell therapy. The percentage of MSCs engrafted into a target organ following systemic injection is typically very low. For example, generally only 1% of injected MSCs that have been expanded ex vivo reach the acutely infarcted myocardium [18]. Although MSC-based cell therapies have shown promising outcomes in several clinical settings [1,19], the homing of the injected MSCs to the target tissues has to be improved. Toward this end, polymeric scaffolds, microspheres, and hydrogels have been tested. The results, however, have been unsatisfactory [20,21], because of the reduced activity, viability, and differentiation potential of the MSCs following ex-vivo expansion.
This means that tremendous numbers of MSCs have to be administered to achieve a therapeutic benefit [22]. On the other hand, the surface modification of MSCs using a lipid-PEG platform has minimal effect on the intrinsic activity of the cells and a high recovery yield, regardless of the type of bioactive molecules embedded on the cell surface. More importantly, the MSCs modified by this method can migrate toward the gradient of the matching ligand. Consequently, modification of the cell surface of MSCs with homing molecules provides a safe means to improve the migration of MSCs toward their target tissues, resulting in enhanced efficacy of the cell therapy. Given their ability to produce a sharp contrast with MRI, SPIONs have become well-established imaging agents for a variety of biomedical applications [23,24]. SPION-based imaging provides for the non-invasive tracking of cells in real-time to monitor their distribution throughout the living body. There are a number of methods currently in use to label cells with SPIONs. A method using cellular uptake has been the most commonly used labeling
Please cite this article in press as: K.S. Lim, et al., Cell surface-engineering to embed targeting ligands or tracking agents on the cell membrane, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.155
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technique, since at high concentrations, surface-modified SPIONs can easily internalize into cells [25,26]. Even though surface-coated SPIONs are generally regarded as safe, adverse effects associated with SPIONs internalized within cells has been observed. Within a cell, SPIONs are often found in the acidic environments of lysosomes [27,28] that can promote the degradation of both the protective coat and iron oxide core of the SPIONs to release iron ions [25,29], which in turn can disrupt cell homeostasis and lead to cytotoxicity [25,30]. Recent studies suggest that the long-term adverse effects of SPIONs should not be neglected, especially when stem cells or immune cells are labeled and administrated for therapeutic purposes [28,31]. Cationic polyelectrolyte-stabilized SPIONs [32] appear to stay bound to the surface of cells [32]. The cytotoxicity of cationic SPIONs is generally higher than that of neutral or slightly negatively charged SPIONs [33,34]. Thus, a method that can non-invasively and rapidly label cells with nonpositively charged SPIONs would offer significant advantages. Labeling cells with SPIONs using a lipid-PEG platform offers several advantages over the methodologies discussed above. The generation of lipid-PEG-conjugated SPIONs is inexpensive and straightforward. A variety of target cells can be instantly functionalized using lipid-PEG-SPIONs without a complicated modification process. Cell-surface modification with lipid-PEG-SPIONs is biocompatible because the modification is transient, the degree of modification is controllable by adjusting the dosage, cell viability is unaffected, and the internalization of SPIONs is substantially reduced. Hence, cell surface modification using lipid-PEG-SPIONs allows for the efficient tracking of cells without altering their therapeutic function. We have explored the applicability of cell surface-engineering to embed various types of bioactive molecules on the surfaces of a variety of cell types. With this technology, a homing peptide, targeting ligand, or contrast agent can be placed non-invasively and transiently on the surface of a cell membrane without adversely affecting the cell or compromising cell function. This cell surfaceengineering technology is broadly applicable to the field of cell therapy. Conflict of interest The authors declare that there are no conflicts of interest. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.11.155. References [1] J. Ankrum, J.M. Karp, Mesenchymal stem cell therapy: two steps forward, one step back, Trends Mol. Med. 16 (2010) 203e209. [2] J.M. Hare, J.E. Fishman, G. Gerstenblith, D.L.D. Velazquez, J.P. Zambrano, V.Y. Suncion, M. Tracy, E. Ghersin, P.V. Johnston, J.A. Brinker, Comparison of allogeneic vs autologous bone marrowederived mesenchymal stem cells delivered by transendocardial injection in patients with ischemic cardiomyopathy: the POSEIDON randomized trial, Jama 308 (2012) 2369e2379. [3] R.F. Wynn, C.A. Hart, C. Corradi-Perini, L. O'Neill, C.A. Evans, J.E. Wraith, L.J. Fairbairn, I. Bellantuono, A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow, Blood 104 (2004) 2643e2645. [4] J.M. Karp, G.S.L. Teo, Mesenchymal stem cell homing: the devil is in the details, Cell Stem Cell 4 (2009) 206e216. [5] Y. Teramura, Y. Kaneda, T. Totani, H. Iwata, Behavior of synthetic polymers immobilized on a cell membrane, Biomaterials 29 (2008) 1345e1355. [6] T.C. Srijaya, T.S. Ramasamy, N.H. Kasim, Advancing stem cell therapy from bench to bedside: lessons from drug therapies, J. Transl. Med. 12 (2014) 243.
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Please cite this article in press as: K.S. Lim, et al., Cell surface-engineering to embed targeting ligands or tracking agents on the cell membrane, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.11.155