Article pubs.acs.org/bc
Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
Clickable Methyltetrazine-Indocarbocyanine Lipids: A Multicolor Tool Kit for Efficient Modifications of Cell Membranes Hanmant Gaikwad,†,‡ Guankui Wang,†,‡,§ Weston J. Smith,† Keisha L. Alexander,∥ Angelo D’Alessandro,∥ Wei Zhang,⊥ Enkhtsetseg Purev,⊥ and Dmitri Simberg*,†,‡,§
Bioconjugate Chem. Downloaded from pubs.acs.org by UNIV AUTONOMA DE COAHUILA on 05/03/19. For personal use only.
†
Translational Bio-Nanosciences Laboratory and ‡Department of Pharmaceutical Sciences, The Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, United States § Colorado Center for Nanomedicine and Nanosafety, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, United States ∥ Department of Biochemistry and Molecular Genetics, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado 80045, United States ⊥ Division of Hematology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, United States S Supporting Information *
ABSTRACT: Cell-based therapeutics are one of the most promising and exciting breakthroughs in modern medicine. Modification of the cell surface with ligands, biologics, drugs, and nanoparticles can further enhance the functionality. Previously, we described the synthesis of a dioctadecyl indocarbocyanine Cy3 analog (aminomethyl-DiI) for efficient and stable modification (painting) of mouse erythrocytes with small molecules, enzymes, and biologics. Here, we synthesized a near-infrared aminomethyl dioctadecyl derivative of Cy7 (aminomethyl-DOCy7) and systematically compared it to aminomethyl-DiI as an anchor for the modification of human erythrocytes, Jurkat cells, and primary T cells with immunoglobulin G. To enable copper-free click chemistry modification of cell membranes, we conjugated a methyltetrazine (MTz) group to the amino-indocyanine lipids via a polyethylene glycol (PEG) linker. DOCy7−PEG3400−MTz showed over 99% modification efficiency of human red blood cells (RBCs) at 25 μM. Reaction of trans-cyclooctene (TCO) modified immunoglobulin G (IgG) with DOCy7−PEG4−MTz-modified RBCs (2-step method) resulted in ∼80,000 IgG molecules per erythrocyte, whereas modification with a preconjugated DOCy7−PEG3400−IgG construct (1-step method) resulted in ∼20,000 IgG molecules per erythrocyte as detected by immuno dot-blot. The number of IgG/RBC was controlled by the concentration of IgG. The incubation of RBCs with DiI−PEG3400−MTz resulted in a similar number of IgG/RBC. Modification of the T-lymphocyte cell line Jurkat with IgG resulted in ∼1 × 106 IgG/cell with the 1-step and 2-step methods, and the efficiency was similar for DOCy7 and DiI constructs. Finally, we used DOCy7 and DiI constructs to demonstrate efficient modification of primary CD3+T cells from healthy donors. In conclusion, click indocarbocyanine conjugates represent a novel multicolor chemical biology tool kit for efficient surface modification of different cells types and can be used for potential imaging and drug delivery applications involving engineered cells.
■
INTRODUCTION
exploit the biology of the underlying pathological process, and engage the target in a highly specific and effective manner. At the same time, there is an attractive notion of combining cells and chemical/biological (cell-free) delivery systems in order to both improve the precision of cell therapies and to utilize potency and mechanisms of action of chemical and biological therapies. Erythrocytes are particularly interesting due to their availability and in vivo longevity,8 and there are many emerging applications for delivery of enzymes9,10 and antigens11,12 and
Cell-based therapies are some of the oldest types of treatments (e.g., blood transfusion, bone marrow transplantation), which at the same time are an emerging frontier in the treatment of many diseases.1−3 As a notable example, chimeric antigen receptor T-cell (CAR-T) therapy received 2 clinical approvals in 2017 and 2018, and many more are in clinical trials for various cancers.3 Other emerging applications are stem cell therapies for neurodegenerative and metabolic diseases4,5 and tumor infiltrating lymphocytes and natural killer cells for cancer.6,7 One of the distinctive advantages of cells versus other types of delivery systems including biologics, polymers, and nanoparticles is the ability to cross biological barriers, © XXXX American Chemical Society
Received: March 18, 2019 Revised: April 25, 2019
A
DOI: 10.1021/acs.bioconjchem.9b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
Figure 1. Modification efficiency of RBCs with phospholipid dyes and indocyanine dyes: A) Structure of DiI and DiR and the corresponding Cy3 and Cy7 distearoylphosphatidylcholine; B) Flow cytometry analysis of modification efficiency of human RBCs shows more efficient membrane incorporation of indocyanine lipids than phospholipids; C) Structure of DOCy7-NH2 and DOCy7−PEG3400−MTz; D) Flow cytometry analysis of modification efficiency of human RBCs with different DOCy7 derivatives; E) Modification efficiency (percent of modified cells) of human RBCs incubated with DOCy7−PEG3400−MTz at various concentrations as determined by flow cytometry.
targeting of diseased sites13 and pathogens.14 There is also an unmet need of stable camouflaging of RBCs to prevent immune clearance after transfusion.15 To this end, several approaches have been explored to modify the surface of CART cells, neutrophils, erythrocytes, and stem cells. For example, adsorption of nanoparticles to erythrocyte membranes can improve nanoparticle circulation properties and reduce the toxicity profile,16,17 whereas binding to neutrophils could improve tissue penetration of nanoparticles.18 Other strategies for the modification of cell membranes include specific affinity ligands (anti-CD45, anti-CD3, anti-CD8, anti-CR1, etc.10,11,19−21), covalent chemistry,22,23 and genetic expression of sortase for subsequent ligation.24 In addition to the above-mentioned modification methods, we and others have shown that cells can be efficiently modified (a.k.a. painted) with phospholipid-conjugated antibodies.25,26 Thus, we demonstrated that anti-CD45 antibody-modified and anti-CD20 antibody-modified RBCs efficiently targeted leukocytes, cancer cells, and leukemic cells in vitro and in vivo.27 Unfortunately, phospholipids were gradually lost from the RBC membrane in vivo with a half-life of ∼48 h.25 Phospholipids are not very stable and undergo transfer to other membranes and lipoproteins.28 However, the indocarbocyanine lipid DiI (dioctadecyl-Cy3 or DiIC18(3)) showed much better retention than phospholipids in the RBC membrane after modification, with 90% of the lipid present in the RBC membrane 48 h postinjection into mice.25 Indocarbocyanine lipids have been used for efficient labeling of cell membranes in vitro and in vivo, which led to their widespread adoption in life
sciences for cell labeling and tracking.29,30 Some of their remarkable stability could be attributed to high lipophilicity and a mild cationic charge on the indole ring, allowing the lipids to deeply embed in the negatively charged bilayer.31 We reasoned that indocyanine lipids could be used as more stable lipid anchors for cell modification. We previously prepared an aminomethyl derivative of DiI and conjugated it to thiolated enzymes and antibodies via Michael addition. The DiIanchored molecules efficiently painted mouse RBCs and showed good in vivo stability.32 Due to the importance of the painting technology for potential cell-based therapies, we set out to further explore the indocyanine chemistries for modification of different cell types in vitro before applying this technology to concrete applications in vivo. In order to expand the repertoire of indocyanine anchors beyond DiI, we synthesized a rigid aminomethyl derivative of dioctadecyl Cy7 (DOCy7). We further tested copper-free Inverse Electron Demand Diels− Alder (IEDDA) bio-orthogonal click chemistry33 involving strained trans-cyclooctene (TCO) and methyl tetrazine (MTz) against Michael (thiol-maleimide) addition as the way to conjugate antibodies to lipids. We show that both DiI and DOCy7 can serve as anchors for fast and efficient incorporation of IgG into the membrane of human erythrocytes, T lymphocyte cell line Jurkat, and primary T cells and also demonstrate efficient click modification of cells postinsertion in the cell membrane. B
DOI: 10.1021/acs.bioconjchem.9b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
Figure 2. A) Bio-orthogonal click chemistry reaction between IgG labeled with trans-cyclooctene (TCO) and DOCy7−PEG3400−MTz. The reaction was performed in PBS at room temperature for 30 min; B) SDS-PAGE analysis of DOCy7−PEG3400−MTz conjugation with IgG−TCO antibody. NIR fluorescence was detected by an Li-COR Odyssey scanner at 800 nm; C) Schematic representation of erythrocyte modification using a 1-step or 2-step method. In the 1-step method, RBCs are incubated with preconjugated IgG construct. In the 2-step method, RBCs are first modified with MTz lipid, followed by reaction with IgG−TCO; D) IgG on human RBCs painted with the 1-step or 2-step method was detected with AlexaFluor 488 goat anti-human IgG (green channel); E) Dot plot of RBCs painted with the 2-step method showing double labeled RBCs. Flow cytometry controls are in Supplemental Figure S3; F) NIR fluorescent microscopy shows DOCy7−PEG−MTz and IgG on RBCs; G) Number of IgG per erythrocyte was determined by dot blot assay using secondary anti-human IgG−IRDye680 as described in Methods and in the extended Supporting Information. The 2-step method results in a larger amount of IgG/RBC than the 1-step method. Means and standard deviation of 3 replicates are shown (2-sided t test).
■
RESULTS AND DISCUSSION In order to compare membrane modification efficiency of dioctadecyl indocyanine dyes with matching distearoyl phospholipid indocyanine dyes, fresh human erythrocytes from healthy donors were painted with dioctadecyl dyes DiR, DiI, or their phospholipid analogs Cy7-distearoylphosphatidylethanolamine and Cy3-distearoylphosphatidylethanolamine, respectively (Figure 1A). According to flow cytometry analysis (Figure 1B), DiR and DiI showed ∼5-fold and ∼20-fold higher mean fluorescence intensity (MFI) than the respective phospholipid indocyanine analogs, suggesting more efficient incorporation of the former into the RBC membrane. We synthesized an aminomethyl dioctadecyl analog of Cy7 (DOCy7-NH2, Figure 1C; all synthesis and characterization data are in Supplemental Methods). The compound showed a shift in the excitation and emission spectra compared to Cy7 (spectrum in Supplemental Figure S1). In order to enable conjugation to ligands via click copper-free addition, DOCy7
was conjugated to MTz (Figure 1C) via polyethylene glycol linkers of different lengths (3400 and 265 Da (PEG4)). DOCy7-NH2 and DOCy7−PEG3400−MTz showed similar RBC modification efficiency at 25 μM, whereas DOCy7− PEG4−MTz did not show any measurable incorporation into the RBC membrane (Figure 1D) and therefore was not used further. DOCy7−PEG3400−MTz showed optimal modification efficiency at 25 μM with over 95% RBCs labeled with the anchor (Figure 1E). Next, we reacted human IgG with a 10-fold excess of TCO− PEG4−NHS which results in approximately 3 TCO groups based on subsequent reaction with Cy3-MTz and spectrophotometric measurement (Supplemental Figure S2 and Methods). IgG−TCO was then reacted with DOCy7−PEG3400− MTz at multiple ratios, from 1:1 to 1:4 (Figure 2A). Near-infrared imaging of reducing SDS-PAGE gel after separation showed mostly the reacted product (Figure 2B), with the main bands around 25 kDa and 50 kDa (light and C
DOI: 10.1021/acs.bioconjchem.9b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
Figure 3. A) Bio-orthogonal conjugate of IgG labeled with TCO and DiI−PEG3400−MTz; B) Human RBCs were first painted with DiI− PEG3400−MTz and then conjugated with human IgG−TCO. IgG was detected with AlexaFluor 488 goat anti-human IgG (green channel), and DiI fluorescence was detected in the yellow channel. Flow cytometry controls are in Supplemental Figure S3; C) Fluorescent microscopy of painted RBCs; D) RBCs were painted with DiI−PEG3400−MTz or DOCy7−PEG3400−MTz and then conjugated with IgG−TCO-IRDye680. For flow analysis, the antibody was detected with AlexaFluor 488 goat anti-human IgG. Both lipids produce similar IgG modification levels; E) The IgG/ RBC ratio was determined by direct dot blot assay of IRDye680-labeled antibody as described in Methods. The ratio is similar to the ratio determined using indirect dot blot using secondary IRDye680-labeled antibody (Figure 2G). Means and standard deviation of 3 replicates are shown. The experiment was repeated twice.
PEG3400−MTz (25 μM)/IgG−TCO (3 μM). Flow cytometry comparison of IgG levels on RBCs painted with the 2-step DiI−PEG3400−MTz/IgG−TCO versus the DOCy7− PEG3400−MTz/IgG−TCO method showed similar IgG levels (MFI 2043 versus 2166, respectively; Figure 3D). Modification of mouse RBCs using 2-step click chemistry also showed similar IgG modification for both anchor types (Supplemental Figure S4). To determine the number of IgG/RBC without secondary antibody detection, we prelabeled IgG−TCO with IRDye 680. Quantification (Figure 3E and additional Supporting Information for raw data) showed the same ratio of IgG/RBC for DOCy7 and DiI anchors (96,000 versus 106,000 at 4 μM IgG, respectively). This is similar to the IgG/RBC ratio determined using a secondary IRDye 680-labeled antibody (Figure 2G), suggesting that direct fluorescence measurements and immuno dot-blot methods produce equivalent results. At the same time, the ratio was lower than what we reported for mouse RBCs using the DiI−PEG3400−IgG construct,32 which could be due to differences in incubation conditions, IgG concentration, chemistry (thiol-maleimide versus MTz−TCO), or antibody type.
heavy chains of the antibody) and higher molecular weight bands consistent with multiple lipids conjugated per antibody.32 Fresh human RBCs were then modified with either the DOCy7−PEG3400−IgG construct (1-step method, Figure 2C, top) or first modified with DOCy7−PEG3400−MTz, washed, and then bio-orthogonally reacted with IgG−TCO (2-step method, Figure 2C, bottom). According to flow cytometry analysis and fluorescent microscopy of RBCs stained with secondary anti-human AlexaFluor 488 labeled antibody (Figure 2D-F, Supplemental Figure S3 for flow cytometry controls), over 95% of RBCs were modified with IgG using either method. Immuno dot-blot analysis of lysates of RBCs showed that the IgG/RBC ratio increases with increasing IgG concentration and that the 2-step method consistently results in a higher IgG/RBC ratio than the 1-step method (Figure 2G). In order to compare the modification efficiency of different indocyanine anchors, we conjugated the previously described aminomethyl dioctadecyl Cy3 (aminomethyl DiI32) to MTz via a PEG3400 linker (Figure 3A). According to flow cytometry and fluorescence microscopy (Figure 3B−C and Supplemental Figure S3 for flow cytometry controls), human RBCs were efficiently painted with DiI− D
DOI: 10.1021/acs.bioconjchem.9b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
Figure 4. A) Lipid modification of Jurkat cells with DOCy7−PEG3400−MTz and DiI−PEG3400−MTz; B) Comparison of IgG levels after modification with DOCy7−PEG3400−IgG and DiI−PEG3400−IgG (1-step). IgG was detected with AlexaFluor 488 goat anti-human IgG (green channel); C) Comparison of 1-step and 2-step methods using DOCy7 anchor. Flow cytometry controls are in Supplemental Figure S5; D) Confocal microscopy image of Jurkat cells painted with DOCy7−PEG3400−IgG (detected with AF488 secondary antibody); E) The number of IgG per Jurkat cell was detected by dot blot assay using secondary anti-human IgG−IRDye680 as described in Methods. Means and standard deviation of 3 replicates are shown (2-sided t test). The experiment was repeated twice.
As seen in Figure 4C, modification with DOCy7−PEG3400− MTz (10 μM)/IgG−TCO (1 μM) was more efficient than DOCy7−PEG3400−IgG (1 μM). According to confocal fluorescence microscopy, the antibody formed a dotted pattern (Figure 4D), similar to that observed on RBCs. Quantitative analysis of the modification efficiency confirmed that the number of IgG/cell was higher for the 2-step approach (except for 1 μM IgG) and was partially IgG concentration-dependent (Figure 4E). Interestingly, the number of antibodies per cell was higher than per RBCs at the same IgG concentration (900,000 versus 30,000), suggesting a much more efficient incorporation of the construct in the nucleated cell membrane. Finally, we tested modification efficiency using CD3+ enriched primary T cells. Both DOCy7−PEG3400−MTz (10 μM) and DOCy7−PEG3400−MTz (10 μM)/IgG−TCO (3 μM) showed 100% of cells positive for DOCy7 and IgG (Figure 5A). Quantitative dot blot assay showed that the IgG/cell ratio was similar for the 1-step and 2-step methods and was ∼7 times higher than for Jurkat cells (Figure 5B). Furthermore, we
Our previous studies demonstrated that RBC modification with lipids does not cause significant hemolysis.25,32 In order to test whether indocyanine lipids cause metabolic perturbations of RBCs, we performed a comprehensive metabolomics study of human RBCs painted with DiI, DiR, DiI−PEG3400−MTZ, and DOCy7−PEG3400−MTZ at 12.5 μM and 25 μM concentration. As shown in Supplemental Figure S5, the lipid modification did not cause changes in metabolomics profile compared to nonpainted RBCs, and there were no changes in glutathione homeostasis, glycolysis, and purine pathways. The data above establish the efficient modification of human RBCs via bio-orthogonal click chemistry. To test the painting of nucleated cells, we used immortalized T-cell lymphoma Jurkat, which is a model cell line for functional T cells.34 First, we found that both DOCy7−PEG3400−MTz and DiI− PEG3400−MTz paint 100% of the cells at 10 μM (Figure 4A). Incubation with 1 μM DiI−PEG3400−IgG showed higher IgG levels than with 1 μM DOCy7−PEG3400−IgG, but both constructs painted over 95% of the cells (Figure 4B). E
DOI: 10.1021/acs.bioconjchem.9b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
Figure 5. A) T-cells were first painted with DOCy7−PEG3400−MTz (10 μM) and then conjugated with human IgG−TCO (3 μM). IgG was detected with AlexaFluor 488 antibody as described above; B) The IgG/cell ratio was determined by dot blot assay using secondary anti-human IgG−IRDye680 as described in Methods. Cells were incubated with 4 μM IgG. Means and standard deviation of 3 replicates are shown; C) T-cells were painted with 10 μM DOCy7−PEG3400−MTz or 10 μM DiI−PEG3400−MTz and then conjugated to polyclonal human Cy5−IgG−TCO (left, center) or Cy5−rituximab−TCO (right). Cy5 fluorescence was detected in the red channel. Flow cytometry controls are in Supplemental Figure S6; D) Confocal microscopy of T-cells painted with DiI−PEG−MTz/Cy5−IgG−TCO.
show better modification than PEG4 conjugates; 2) DiI (DOCy3) and DOCy7 conjugates have similar modification efficiencies (number of IgG per cell); 3) indocyanine lipids can paint different cell types, including human and mouse RBCs, Jurkat cells, and primary T cells; 4) modification of RBCs results in ∼80,000 IgG per cell, whereas modification of Jurkat and T-cells results in over 1 million and 7 million IgG per cell, respectively; 5) cells can be modified directly by lipidconjugated antibodies or in 2 steps by lipid-MTz first, followed by IgG−TCO. The next critical milestone would be to use indocyanine anchors for therapy, imaging, and drug delivery.
compared modification with DOCy7−PEG3400−MTz (10 μM)/Cy5−IgG−TCO (3 μM) and DiI−PEG3400−MTz (10 μM)/Cy5−IgG−TCO (3 μM) and found similar IgG levels (Figure 5C left and center, Supplemental Figure S6 for flow cytometry controls). In order to test modification with a functional therapeutic antibody, we prepared a Cy5-labeled anti-CD20 antibody (rituximab) that is used for therapy of hematological malignancies35 as well as for autoimmune diseases.36 T-cells painted with DiI−PEG3400−MTz (10 μM)/Cy5−rituximab−TCO (3 μM) showed higher levels of rituximab than human IgG (Figure 5C right). Confocal microscopy of T-cells showed colocalization of DiI and the antibody (Figure 5D). Interestingly, the antibody also appeared clustered similarly to the antibody detected with secondary IgG, suggesting that the dotted pattern reflects the true distribution of the constructs in the membrane. In conclusion, we systematically compared click derivatives of two different dioctadecyl indocyanines for decoration of cell surfaces with antibodies. The technology can be utilized for multicolor modification of various cell types with ligands, therapeutics, or reporter molecules for research or cell-based therapies. The main conclusions are 1) PEG-3400 conjugates
■
EXPERIMENTAL PROCEDURES Materials. Anticoagulant Citrate Dextrose (ACD) buffer was obtained from the Colorado Blood Donation Center and was kept sterile before use. Pure human RBCs were obtained from discarded leukodepletion filters after processing donor blood units at the University of Colorado Blood Donation Center. Institutional Review Board approval was not required due to discarded material and anonymous nature. RBCs were used less than 2 h after blood collection. Heparinated mouse blood was collected from female or male BALB/c mice (6−10
F
DOI: 10.1021/acs.bioconjchem.9b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
ACD buffer) at 37 °C for 1 h and then washed three times in ACD buffer. For modification with antibodies in the 1-step method, DOCy7−PEG3400−MTz and DiI−PEG3400−MTz were first conjugated with IgG−TCO at room temperature for 30 min and purified using a 40 kDa cut off Zeba spin column. The construct (0.5−3 μM IgG) was incubated with washed RBCs at 37 °C for 1 h. The RBCs were washed three times in ACD buffer. For the 2-step method, human RBCs (13 billion/ mL) were first painted with DOCy7−PEG3400−MTz or DiI− PEG3400−MTz (both at 25 μM), washed as described above, and then incubated with IgG−TCO (0.5−4 μM) at room temperature for 30 min on a Thermomixer (600 rpm) and washed in ACD buffer. For Jurkat modification, cells (6 million/mL) were combined with DOCy7−PEG3400−MTz (10 μM) or DiI− PEG3400−MTz (10 μM) in RPMI (1% BSA). The mixture was incubated at 37 °C for 1 h, and cells were washed three times with 1% BSA in RPMI. For modification in 2 steps, Jurkat cells from the previous step were incubated with IgG− TCO (1 μM) in RPMI (1% BSA) at room temperature for 30 min on the Thermomixer (600 rpm) and washed three times with RPMI (1% BSA). For the 1-step method, DOCy7− PEG3400−IgG or DiI−PEG3400−IgG (1 μM IgG) constructs were incubated with Jurkat cells (6 million/mL) as desctribed above and washed three times with RPMI (1% BSA). For T cell modification, primary human CD3+ enriched Tcells (6 million/mL) were incubated with DOCy7− PEG3400−MTz (10 μM) in Iscove’s Modified Dulbecco’s Medium (IMDM, supplemented with 2% FBS) at 37 °C for 1 h and then washed three times with IMDM (2% FBS). For the 2-step method, cells were then incubated with IgG−TCO (3 μM, IgG was optionally labeled with Cy5) in IMDM (2% FBS) at room temperature for 30 min on the Thermomixer (600 rpm) and washed three times with IMDM (2% FBS). For the 1-step method, cells were incubated with 3 μM DOCy7− PEG3400−IgG (IgG was labeled with TCO or TCO and Cy5) in IMDM (2% FBS) at room temperature for 30 min on the Thermomixer (600 rpm) and washed three times with IMDM (2% FBS). Flow Cytometry and Fluorescence Microscopy. Cells were analyzed with a Guava EasyCyte HT flow cytometer (Merck KGaA). Cells were resuspended at ∼0.5 million/mL, and 20,000 events were detected. To determine IgG conjugation efficiency, cells were incubated with secondary AlexaFluor 488 goat anti-human IgG in ACD buffer at room temperature for 30 min on the Thermomixer (600 rpm) and washed 3 times. AlexaFluor 488 fluorescence was detected in the Green-B fluorescence channel using the Blue (488 nm) laser. Cy7 fluorescence was detected in the near IR-R fluorescence channel using the Red (647 nm) laser. Cy3 fluorescence was detected in the Yellow-B fluorescence channel using the Blue (488 nm) laser. Cy5 fluorescence was detected in the Red-R fluorescence channel using the Red (647 nm) laser. FSC threshold was set to 2200 in order to exclude debris. The data were analyzed using FlowJo software version V10. For microscopy, labeled RBCs were applied to a slide, covered with a cover glass, and imaged with a Zeiss Axio Observer 5 epifluorescent microscope equipped with an Exelitas near-infrared light source and 5 filter cubes including NIR (760 nm excitation/780 nm emission) and an Axiocam 506 monochromatic camera. Jurkat cells and T-cells were applied to a slide using Shandon cytospin III (ThermoFisher), fixed for 30 min with 10% formalin, and mounted on a slide
weeks of age according to the animal protocol approved by the University of Colorado). RBCs were washed 3 times in ACD buffer at 500g to remove plasma and buffy coats. All cell media were from ThermoFisher. ChromPure human IgG, whole molecule, and AlexaFluor488 conjugated AffiniPure goat antihuman IgG (H+L) secondary antibody were obtained from Jackson ImmunoResearch (West Grove, PA). Anti-CD20 mAb rituximab was obtained from the University of Colorado Hospital Pharmacy. IRDye 680RD goat anti-human secondary antibody and IRDye 680RD NHS were obtained from Li-COR Biosciences (Lincoln, NE). TCO−PEG4−NHS, MTz−Cy3, and MTz−NHS were obtained from Click Chemistry Tools (Scottsdale, AZ). Cy5-NHS was from Lumiprobe Inc. All chemicals for lipid synthesis were from Sigma and Acros Organics, except for NH2−PEG3400−COOH (Laysan Bio, Arab, AL) and DSPE (Avanti Polar Lipids, Alabaster, AL). Methods. Synthesis of IgG−TCO, IRDye 680−IgG−TCO, and Cy5−IgG−TCO. Human polyclonal IgG in PBS (100 μL, 10 mg/mL) was combined with a 10-fold excess of TCO− PEG4−NHS (in 4 μL of DMSO). The reaction mixture was incubated at 4 °C for 12 h and purified using a 7 kDa cutoff Zeba spin column (ThermoFisher). Alternatively, IgG in PBS (100 μL, 10 mg/mL) was combined with a 1:1 equiv of IRDye 680 NHS ester (in 1 μL of DMSO) for 5 min before adding a 10-fold excess of TCO−PEG4−NHS (in 4 μL of DMSO). The reaction mixture was incubated at 4 °C for 12 h and purified using a 7 kDa cutoff Zeba spin column. Conjugation efficiency was about ∼1 IRDye 680/IgG as determined by UV absorbance and the dye extinction coefficient of 165,000 M−1 cm−1. Conjugation efficiency was about ∼3 TCO/IgG as determined by UV absorbance of Sufo-Cy3 after click reaction with Sulfo-Cy3-MTz and based on the dye extinction coefficient of 162,000 M−1 cm−1. Alternatively, IgG in PBS (100 μL, 10 mg/mL) was combined with a 1:1 equiv of Cy5NHS ester (in 1 μL DMSO) for 5 min before adding a 10-fold excess of TCO-PEG4-NHS (in 4 μL DMSO). The reaction mixture was incubated at 4 °C for 12 h and purified using a 7 kDa cutoff Zeba spin column. Conjugation efficiency was about ∼1 Cy5/IgG as determined by UV absorbance and the dye extinction coefficient of 250,000 M−1 cm−1. Cell Culture. Jurkat cells were obtained from ATCC (TIB152), and the cell line was verified using UC Denver Molecular Biology Service Center. Cells were grown in RPMI 1640 media containing 10% FCS and 1% penicillin/streptomycin; the cells had undergone fewer than 10 passages prior to experiments. Primary human T cells were obtained from fresh peripheral blood mononuclear cells (PBMCs) of healthy donors using Ficoll-Paque PREMIUM (GE Healthcare, Chicago, IL)). The University of Colorado Institutional Review Board approved the tissue bank protocol (IRB Protocol 06-0720), and all subjects gave informed consent in accordance with the Declaration of Helsinki. Tissue samples were processed for T cell selection using human CD3MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified CD3+ cells were cultured in X-VIVO15 media (Lonza Group, Basel, Switzerland) with 10% heat-inactivated fetal bovine serum (Corning Inc., Corning, NY), 50 unit/mL IL-2 (PeproTech, NJ), and 0.5 ng/mL IL-15 (PeproTech). Cell Painting. Freshly collected RBCs were washed three times in ACD buffer by centrifugation at 0.8 g to remove the remaining plasma. For modification with lipids, the erythrocyte pellet (13 billion/mL) was incubated with different concentrations of constructs (micromolar cocnetrations of lipids in G
DOI: 10.1021/acs.bioconjchem.9b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry
(5) Phull, P., Sanchorawala, V., Brauneis, D., Sloan, J. M., Siddiqi, O. K., Quillen, K., and Sarosiek, S. (2019) High-dose melphalan and autologous peripheral blood stem cell transplantation in patients with AL amyloidosis and cardiac defibrillators. Bone Marrow Transplant. DOI: 10.1038/s41409-019-0440-5. (6) Bachanova, V., and Miller, J. S. (2014) NK cells in therapy of cancer. Crit. Rev. Oncog. 19, 133−41. (7) Rohaan, M. W., van den Berg, J. H., Kvistborg, P., and Haanen, J. (2018) Adoptive transfer of tumor-infiltrating lymphocytes in melanoma: a viable treatment option. J. Immunother Cancer 6, 102. (8) Muzykantov, V. R. (2010) Drug delivery by red blood cells: vascular carriers designed by mother nature. Expert Opin. Drug Delivery 7, 403−27. (9) Magnani, M., Rossi, L., Fraternale, A., Bianchi, M., Antonelli, A., Crinelli, R., and Chiarantini, L. (2002) Erythrocyte-mediated delivery of drugs, peptides and modified oligonucleotides. Gene Ther. 9, 749− 51. (10) Zaitsev, S., Spitzer, D., Murciano, J. C., Ding, B. S., Tliba, S., Kowalska, M. A., Marcos-Contreras, O. A., Kuo, A., Stepanova, V., Atkinson, et al. (2010) Sustained thromboprophylaxis mediated by an RBC-targeted pro-urokinase zymogen activated at the site of clot formation. Blood 115, 5241−8. (11) Lorentz, K. M., Kontos, S., Diaceri, G., Henry, H., and Hubbell, J. A. (2015) Engineered binding to erythrocytes induces immunological tolerance to E. coli asparaginase. Sci. Adv. 1, e1500112. (12) Kontos, S., Kourtis, I. C., Dane, K. Y., and Hubbell, J. A. (2013) Engineering antigens for in situ erythrocyte binding induces T-cell deletion. Proc. Natl. Acad. Sci. U. S. A. 110, E60−8. (13) Fens, M. H. A. M., Mastrobattista, E., De Graaff, A. M., Flesch, F. M., Ultee, A., Rasmussen, J. T., Molema, G., Storm, G., and Schiffelers, R. M. (2008) Angiogenic endothelium shows lactadherindependent phagocytosis of aged erythrocytes and apoptotic cells. Blood 111, 4542−4550. (14) Wong, C. H., Jenne, C. N., Petri, B., Chrobok, N. L., and Kubes, P. (2013) Nucleation of platelets with blood-borne pathogens on Kupffer cells precedes other innate immunity and contributes to bacterial clearance. Nat. Immunol. 14, 785. (15) Scott, M. D., Murad, K. L., Koumpouras, F., Talbot, M., and Eaton, J. W. (1997) Chemical camouflage of antigenic determinants: stealth erythrocytes. Proc. Natl. Acad. Sci. U. S. A. 94, 7566−71. (16) Anselmo, A. C., Gupta, V., Zern, B. J., Pan, D., Zakrewsky, M., Muzykantov, V., and Mitragotri, S. (2013) Delivering nanoparticles to lungs while avoiding liver and spleen through adsorption on red blood cells. ACS Nano 7, 11129−37. (17) Wibroe, P. P., Anselmo, A. C., Nilsson, P. H., Sarode, A., Gupta, V., Urbanics, R., Szebeni, J., Hunter, A. C., Mitragotri, S., Mollnes, T. E., et al. (2017) Bypassing adverse injection reactions to nanoparticles through shape modification and attachment to erythrocytes. Nat. Nanotechnol. 12, 589−594. (18) Zhang, C., Ling, C. L., Pang, L., Wang, Q., Liu, J. X., Wang, B. S., Liang, J. M., Guo, Y. Z., Qin, J., and Wang, J. X. (2017) Direct Macromolecular Drug Delivery to Cerebral Ischemia Area using Neutrophil-Mediated Nanoparticles. Theranostics 7, 3260−3275. (19) Tang, L., Zheng, Y., Melo, M. B., Mabardi, L., Castano, A. P., Xie, Y. Q., Li, N., Kudchodkar, S. B., Wong, H. C., Jeng, E. K., Maus, M. V., et al. (2018) Enhancing T cell therapy through TCR-signalingresponsive nanoparticle drug delivery. Nat. Biotechnol. 36, 707−716. (20) Petersburg, J. R., Shen, J., Csizmar, C. M., Murphy, K. A., Spanier, J., Gabrielse, K., Griffith, T. S., Fife, B., and Wagner, C. R. (2018) Eradication of Established Tumors by Chemically SelfAssembled Nanoring Labeled T Cells. ACS Nano 12, 6563−6576. (21) Schmid, D., Park, C. G., Hartl, C. A., Subedi, N., Cartwright, A. N., Puerto, R. B., Zheng, Y., Maiarana, J., Freeman, G. J., Wucherpfennig, K. W., et al. (2017) T cell-targeting nanoparticles focus delivery of immunotherapy to improve antitumor immunity. Nat. Commun. 8, 1747. (22) Pishesha, N., Bilate, A. M., Wibowo, M. C., Huang, N. J., Li, Z., Dhesycka, R., Bousbaine, D., Li, H., Patterson, H. C., Dougan, et al. (2017) Engineered erythrocytes covalently linked to antigenic
using DAPI/antifade mounting media (Vector Laboratories). Slides were imaged with a Nikon Eslipse AR1HD confocal microscope. Quantification of Number of IgG per Cell. To determine the number of IgG molecules, the cells were bath sonicated and loaded in 2 μL triplicates on a 0.22 mm nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% milk and incubated with secondary IRDye 680RD goat antihuman IgG. No labeling or blocking was done for experiments where cells were painted with prelabeled IRDye680−IgG. The standard curve of the antibody was prepared with control cell lysates and dotted alongside the samples. The membrane was scanned at 700 nm using Li-COR Odyssey (Li-COR Biosciences, Lincoln, NE). Control experiments showed no detectable signal from the DOCy7 anchor in the 700 nm channel (additional Supporting Information). The integrated density of a 16-bit TIFF image of dots was measured with ImageJ and plotted as a function of IgG concentration. The number of IgG/RBC was calculated using a standard curve for the respective IgG control.
■
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.9b00202. A. Supplemental Methods: 1) synthesis and characterization; 2) metabolomics and B. Supplemental figures: 1) DOCy7 excitation and emission spectra; 2) HPLC spectrum of IgG−TCO; 3) flow cytometry controls; 4) flow cytometry of mouse RBCs; 5) metabolomics of painted RBCs; 6) flow cytometry controls (PDF) IgG calculations (XLSX)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Angelo D’Alessandro: 0000-0002-2258-6490 Dmitri Simberg: 0000-0002-5288-6275 Notes
The authors declare no competing financial interest.
■ ■
ACKNOWLEDGMENTS The study was supported by the NIH grants EB022040 and CA194058 to D.S. REFERENCES
(1) Buzhor, E., Leshansky, L., Blumenthal, J., Barash, H., Warshawsky, D., Mazor, Y., and Shtrichman, R. (2014) Cell-based therapy approaches: the hope for incurable diseases. Regener. Med. 9, 649−72. (2) Hu, X., Leak, R. K., Thomson, A. W., Yu, F., Xia, Y., Wechsler, L. R., and Chen, J. (2018) Promises and limitations of immune cellbased therapies in neurological disorders. Nat. Rev. Neurol. 14, 559− 568. (3) Tang, J., Hubbard-Lucey, V. M., Pearce, L., O’Donnell-Tormey, J., and Shalabi, A. (2018) The global landscape of cancer cell therapy. Nat. Rev. Drug Discovery 17, 465−466. (4) Barker, R. A., Drouin-Ouellet, J., and Parmar, M. (2015) Cellbased therapies for Parkinson disease-past insights and future potential. Nat. Rev. Neurol. 11, 492−503. H
DOI: 10.1021/acs.bioconjchem.9b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX
Article
Bioconjugate Chemistry peptides can protect against autoimmune disease. Proc. Natl. Acad. Sci. U. S. A. 114, 3157−3162. (23) Hu, Q. Y., Sun, W. J., Wang, J. Q., Ruan, H. T., Zhang, X. D., Ye, Y. Q., Shen, S., Wang, C., Lu, W. Y., Cheng, K., et al. (2018) Conjugation of haematopoietic stem cells and platelets decorated with anti-PD-1 antibodies augments anti-leukaemia efficacy. Nature Biomedical Engineering 2, 831−840. (24) Shi, J., Kundrat, L., Pishesha, N., Bilate, A., Theile, C., Maruyama, T., Dougan, S. K., Ploegh, H. L., and Lodish, H. F. (2014) Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes. Proc. Natl. Acad. Sci. U. S. A. 111, 10131−6. (25) Shi, G., Mukthavaram, R., Kesari, S., and Simberg, D. (2014) Distearoyl anchor-painted erythrocytes with prolonged ligand retention and circulation properties in vivo. Adv. Healthcare Mater. 3, 142−8. (26) Teramura, Y., Kaneda, Y., and Iwata, H. (2007) Isletencapsulation in ultra-thin layer-by-layer membranes of poly(vinyl alcohol) anchored to poly(ethylene glycol)-lipids in the cell membrane. Biomaterials 28, 4818−4825. (27) Mukthavaram, R., Shi, G., Kesari, S., and Simberg, D. (2014) Targeting and depletion of circulating leukocytes and cancer cells by lipophilic antibody-modified erythrocytes. J. Controlled Release 183, 146−53. (28) Ferrell, J. E., Lee, K. J., and Huestis, W. H. (1985) Lipid Transfer between Phosphatidylcholine Vesicles and Human-Erythrocytes - Exponential Decrease in Rate with Increasing Acyl ChainLength. Biochemistry 24, 2857−2864. (29) Progatzky, F., Dallman, M. J., and Lo Celso, C. (2013) From seeing to believing: labelling strategies for in vivo cell-tracking experiments. Interface Focus 3, 20130001. (30) Zagorodnyuk, V. P., Kyloh, M., Nicholas, S., Peiris, H., Brookes, S. J., Chen, B. N., and Spencer, N. J. (2011) Loss of visceral pain following colorectal distension in an endothelin-3 deficient mouse model of Hirschsprung’s disease. J. Physiol. 589, 1691−706. (31) Gullapalli, R. R., Demirel, M. C., and Butler, P. J. (2008) Molecular dynamics simulations of DiI-C18(3) in a DPPC lipid bilayer. Phys. Chem. Chem. Phys. 10, 3548−60. (32) Smith, W. J., Tran, H., Griffin, J. I., Jones, J., Vu, V. P., Nilewski, L., Gianneschi, N., and Simberg, D. (2018) Lipophilic indocarbocyanine conjugates for efficient incorporation of enzymes, antibodies and small molecules into biological membranes. Biomaterials 161, 57−68. (33) McKay, C. S., and Finn, M. G. (2014) Click chemistry in complex mixtures: bioorthogonal bioconjugation. Chem. Biol. 21, 1075−101. (34) Abraham, R. T., and Weiss, A. (2004) Jurkat T cells and development of the T-cell receptor signalling paradigm. Nat. Rev. Immunol. 4, 301−8. (35) Smith, M. R. (2003) Rituximab (monoclonal anti-CD20 antibody): mechanisms of action and resistance. Oncogene 22, 7359− 68. (36) Larrar, S., Guitton, C., Willems, M., and Bader-Meunier, B. (2006) Severe hematological side effects following Rituximab therapy in children. Haematologica 91, ECR36.
I
DOI: 10.1021/acs.bioconjchem.9b00202 Bioconjugate Chem. XXXX, XXX, XXX−XXX
