Stable and Reversible Functionalization and Super-Resolution Microscopy of Live Cell Membranes

Live cell surface functionalization has been increasingly explored in recent years because of its emerging bio- medical and therapeutic applications. Known methods to functionalize live-cell surface includes time-consuming metabolic docking of non-natural functional groups on cell surfaces that are hard to use. Here, we report an easy and direct functionalization method for cell surface and cell-cell conjugation utilizing native phosphate groups on cell membranes. We used three-dimensional single-molecule localization microscopy to confirm that the chemical conjugate binds preferentially on the cell surface. We show an application of this method for live-cell imaging and enhancement of cell-cell interaction leading to increased T cell proliferation. Our simple and reversible cell-conjugation strategy would be widely useful to explore and optimize cell-cell contact-based proliferation for any cell-based therapeutic applications.


Super-resolution imaging
Super-resolution imaging was performed using a custom-built 3D super-resolution system with biplane configuration with a 100 1.35 NA silicone oil-immersion objective lens (FV-U2B714, Olympus America Inc.) and a PIFOC objective positioner (ND72Z2LAQ, Physik Instrumente). Samples were excited with a 642 nm laser (2RU-VFL-P-2000-642-B1R, MPB Communications Inc.). Single molecule emission events were captured by a scientific complementary metal-oxide-semiconductor camera (Orca-Flash4.0v3, Hamamatsu) with an effective pixel size of 120 nm. 3D volume was formed by scanning the objective lens in the axial direction with 400 nm interval per optics section. 3D superresolution images were reconstructed from 8 optics sections with each section containing 6,000 frames imaged through 3 cycles. Single molecule 3D position estimation was performed by a in situ point spread function retrieval method as described previously by F. Xu et al 1

Synthesis
Synthesis of covalent cargo backbone.

Step 1. Synthesis of benzo-triazolide of iodoacetic acid
Dry thionyl chloride (1.1 mmol) was added to a solution of 1H-benzotriazole (3 mmol) in 10 ml dry DCM taken in a 50 ml RB at room temperature under Ar atmosphere. The reaction mixture was stirred (500 rpm) for 10 minutes. Next, solid iodoacetic acid (1.0 mmol) was dissolved in 10 ml of dry DCM in a closed cap vial and added quickly. The reaction mixture was stirred for 12 h at room temperature. The white precipitate was filtered off and the filtrate was concentrated under 4 reduced pressure. After the evaporation of the solvent, the crude residue of benzotriazolide was isolated as intermediate and used in the next step reaction. This benzotriazolide intermediate should be used immediately in the next step before it turned into blackish from pale yellow/brown color, as it results in product degradation.
Step 2. Synthesis of cargo intermediate I 30 mg HBr salt of Poly-D-Lysine (M ~150 kD) was dissolved in 5 mL MeOH followed by addition of 100 µL triethyl amine. This mixture was stirred for 30 minutes at 4 0 C. Next, the crude benzo-triazolide (0.06 mmol) obtained in the step 1 was added and the reaction mixture was stirred at 4 0 C for 24 h. After evaporation of the solvent MeOH at RT (inside hood), crude residue of cargo intermediate I was isolated as precipitate which was purified by washing with acetonitrile and used in the next step. This crude cargo intermediate I should not be dried completely (to solid state), otherwise some changes may happen in the polymer and it may be difficult to dissolve in next step. This product needs to be dried until semi-solid or colloidal state of the product.
Step 3. Synthesis of cargo intermediate II Adenosine diphosphate (ADP) free acid (5.3 mg) was dissolved in 5 mL THF in an RB followed by addition of 2.4 mg of 4-dimethyl amino pyridine (DMAP) to it. This mixture was stirred for 1 h at 4 0 C followed by addition of cargo intermediate I (30 mg) dissolved in 5 ml MeOH. The resulting reaction mixture was further stirred for 24 h at 4 0 C. The reaction mixture was acidified with ice cold 1 (N) HCl to neutralize any remaining base. Finally, the solvent was evaporated at RT (inside hood) to get the crude product of covalent cargo which was purified by washing with acetonitrile. ADP used in this step should be in free acid form and not in salt form. Free acid form will help to dissolve in organic solvent like THF. This crude cargo intermediate II should be 5 washed with acetonitrile to make sure there is no small molecule like ADP, DMAP etc. Presence of these nucleophilic small molecules will compete with the amine group of the cargo back bone against the electrophilic site of the NHS-Fluorescein in the next step -leading to decrease in degree of fluorophore conjugation on the cargo back bone. Few drops of 1 (N) HCl can be used to dissolve this cargo intermediate for next step reaction if necessary.
Step 4. Synthesis of fluorophore tag covalent cargo molecule 6.0 mg of cargo intermediate II prepared in the previous step was dissolved in 1 ml of water followed by addition of 4 mL methanol and 100 µL of triethyl amine. This mixture was stirred for 30 minutes at 4 o C followed by addition of 1.0 mg NHS-Fluorescein (dissolved in 1 ml MeOH).
The resulting reaction mixture was further stirred for 12 h at 4 0 C. Un-reacted triethyl amine was neutralized by dropwise addition of ice cold 1 (N) HCl. The orange-yellow precipitate obtained from this reaction was separated and washed with cold methanol and acetonitrile and dried at room temperature (inside hood) to obtain the final fluorophore tag cargo molecule. This final cargo molecule should be neutralized with 1 (N) HCl to avoid aerial oxidation of the product and can be dried to solid state. This product can be dissolved in acidic PBS for cell conjugation and other work.

Synthesis of non-covalent phospholipid cargo
4.0 mg of HBr salt of D-lysine chain polymer (150 kD) was dissolved in 5 mL water. 10 μL triethyl amine was added and mixture was stirred for 5 minutes at room temperature (RT) to free all amine groups. Next, 2.0 mg phospholipid was dissolved in 2 mL methanol and added to it and the reaction mixture was stirred at RT. The reaction mixture was evaporated to get the crude residue and 6 washed with acetonitrile to remove any un-reacted phospholipid and get the non-covalent phospholipid cargo.

Synthesis of covalent phospholipid cargo
6.0 mg of covalent cargo was dissolved in 5 mL water followed by addition of 2.0 mg phospholipid and the reaction mixture was stirred at room temperature. The reaction mixture was evaporated at room temperature to get the crude residue, then washed with cold acetonitrile to remove any unreacted phospholipid and get the title product.

Synthesis of non-covalent fluorophore cargo
Synthesis scheme for fluorophore conjugated cargo molecules is shown in Supplementary Figure   S5. For non-covalent fluorophore cargo molecule, 8.0 mg HBr salt of D-lysine chain polymer was dissolved in 5 mL methanol by adding 100 µL triethyl amine. The resulting reaction mixture for the non-covalent cargo reactions (Supplementary Figure S4) was further stirred for 12 h at 4 0 C.
Un-reacted triethyl amine was neutralized by dropwise addition of ice cold 1 (N) HCl. The orangeyellow precipitate obtained from this reaction was separated and washed with cold methanol and dried at room temperature in vacuum to obtain the dual-conjugated fluorophore covalent cargo.

Synthesis of covalent cargo conjugated with AF647 dye
A solution of poly-D-lysine conjugated with ADP (dissolved in MeOH) was stirred for 30 minutes at 4 o C, then 1 mg of AF647 NHS ester was added. The mixture was stirred at 4 0 C overnight. The mixture was purified with protein pierce concentrator with 10K MWCO by dissolving the product in 70% ethanol.

Synthesis of covalent cargo 1 (75 kD) and covalent cargo 3 (300 kD)
Both the covalent cargo 1 and 3 were synthesized as per the above covalent cargo synthesis procedure suing 75 kD and 300 kD poly-D-lysine backbone materials respectively.

Synthesis of butoxy carbonyl-protected non-covalent fluorophore cargo (BOC-cargo)
4.0 mg HBr salt of D-lysine chain polymer (150 kD) was dissolved in 5 mL methanol followed by adding 100 µL triethyl amine. This mixture was stirred for 30 minutes at 4 0 C followed by addition of 2.0 mg NHS-fluorescein dye. The resulting reaction mixture was further stirred for 12 h at 4 0 C.
Next, excess BOC-anhydride (5.0 mg) was added to it and the reaction mixture was stirred for another 2 h. The precipitate obtained from this reaction was separated and washed with cold acetonitrile and dried at room temperature under vacuum to get the BOC-protected non-covalent fluorophore cargo.

Synthesis of magnetic bead linked cargo
4.0 mg of each non-covalent and covalent fluorophore cargo was dissolved in 5 mL methanol by adding 100 µL triethyl amine. The reaction mixture was stirred for 30 minutes at 4 0 C to make free all the amine groups. Next, 1.0 mg of NHS-magnetic beads was added to each of the reaction mixture and stirred for 12 h at 4 0 C. Un-reacted triethyl amine was neutralized by dropwise addition of ice cold 1 (N) HCl. The brown-yellow precipitate obtained from this reaction was separated and washed with cold acetonitrile and dried at room temperature under vacuum to get the magnetic bead linked respective cargos.
General procedure for cell-surface functionalization and live cell imaging 8 Jurkat T cells (1×10 5 ) were plated in a 12 well plate and treated with 100 µg/mL concentration of the cargos in growth media. The cell and cargo mixture were shaken at 120 rpm using an orbital shaker for 30 minutes at room temperature. Next, cells were stained with Hoechst 33342 (for nucleus) in growth media and washed with sterile PBS and transferred in glass bottom dish. Cells were viewed under 60X oil object (optical zoom 3) in confocal laser microscope (Nikon AR1-MP).

Fixed cell surface conjugation and imaging
Jurkat T cells were fixed by 37 ºC pre-warmed 4% paraformaldehyde (15710, Electron Microscopy Sciences) for 15 minutes and immediately transferred in glass bottom dish (MatTek Corporation).
Cells were seeded as well as fixed by centrifugation at 1000 rpm at 10 0 C for 5 minutes. Next, fixed cells were gently rinsed with PBS to remove any fixation agent and treated with the covalent fluorophore cargo (0.1 mg/mL) for 30 minutes in PBS at RT. Cells were stained with DAPI (4′,6diamidino-2-phenylindole) and washed with PBS and again centrifuged to make sure their attachment on the glass bottom surface. Finally, confocal images were captured using 60X oil object.
Legends for 3D confocal movie S1 (Cell surface conjugation of Jurkat T cell with 150 kD cargo). Jurkat T cells were treated with the covalent cargo (0.1 mg/mL) for 30 minutes in growth media at room temperature and stained with Hoechst 33342 for nucleus. Cells were then washed with PBS and confocal laser microscopic images were captured in z-stack using 60X oil object.
Nucleus is shown in blue and cell surface is shown in green.

Legends for 3D confocal movie S2 (Cell-cell conjugation of Jurkat T cells with 150 kD cargo).
Jurkat T cells were treated with the covalent cargo (100 µg/mL) for 30 minutes in growth media at room temperature and stained with Hoechst 33342 for nucleus. Cells were then washed with PBS and confocal laser microscopic images were captured in z-stack using 60X oil object. Nucleus is shown in blue and cell surface is shown in green.

Stability of the conjugated cargo molecules on surface modified Jurkat T cells
Surface conjugated Jurkat T cells (100,000 cells/well) were grown in 12 well culture plate in growth media and images were captured after 1, 3 and 6 days of incubation using confocal laser microscope. Fluorescence intensity was measured using NIS-Elemental software.

Viability assay of the surface modified Jurkat T cells
The cell viability experiment was performed using the CellTiter Blue (CTB) reagent. Surface conjugated Jurkat T cells (1×10 5 ) were seeded in each well of 96-well plates using growth media and incubated in a humidified incubator at 37 °C and 5% CO2 atmosphere. At the end of the incubation, cell titer blue reagent (10 µl) was added directly to each well and the plates were incubated for additional 3 h at 37 °C to allow cells to convert resazurin to resorufin, and the fluorescent signal was measured at 590 nm after exciting at 560 nm using a multiplate ELISA reader (Bio-Tek Synergy HT plate reader, Bio-Tek, Winooski, VT). The percentage of live cells in a cargo-conjugated sample was calculated by considering the fluorescence intensity of the vehicle treated un-conjugated Jurkat T cell sample as 100 %.

Cargo displacement reactions with surface modified Jurkat T cells in presence of no fluorophore-tag cargo molecules
Jurkat T cells were conjugated with the non-covalent and covalent fluorophore cargos for 30 minutes. Next, these surface conjugated Jurkat T cells (100,000 cells/well) were taken in 12 well culture plate and treated with no fluorophore tag non-covalent and the dual-conjugation covalent cargo for another 30 minutes in growth media at 120 rpm shaking at room temperature. Cells were treated with Hoechst 33342 for nuclear stain and washed with PBS. Finally, cells were transferred 10 in glass bottom wells and confocal images were recorded to monitor the retention of surface conjugation.

Reversibility and reusability of the covalent cargo reagent
Jurkat T cells were taken in 24 well plate and incubated with magnetic bead linked covalent fluorophore cargo reagent for 30 minutes at 120 rpm on an orbital shaker. The excess cargo and surface conjugated cell mixture were centrifuged at 500 rpm for 2 minutes to decant the 'unconjugated cargo' from the top of the well. Next, surface conjugated Jurkat T cells were taken in 24 well plate and incubated with PBS of pH 5.5 for 30 minutes at 120 rpm on an orbital shaker.
The magnetic bead linked free cargo molecules were isolated by magnetic separation from its T cell mixture. The recovered cargo solution thus obtained was adjusted to pH 7.0 by adding 1 (N) NaOH solution and re-used with combination of unconjugated cargo to incubate live Jurkat T cells for another 30 minutes at 120 rpm. The precipitated Jurkat T cells obtained in each step were stained with Hoechst for nucleus and washed with PBS to remove any trace of cargo reagent.
Confocal images were acquired using 60X oil object.

T-cell proliferation (manufacturing): T-cell culture in presence of the cargo reagent
Jurkat T cells (1×10 5 ) were seeded in 96-well plates in growth media and incubated for 3 days in presence of 10 µg/mL concentration of these cargos in a humidified incubator at 37°C and 5 % CO2 atmosphere. At the end of the incubation, cell titer blue reagent was added directly to each well and the plates were incubated for 3 h at 37°C to allow cells to convert resazurin to resorufin, and the fluorescent signal was measured using a multiplate ELISA reader (Bio-Tek Synergy HT 11 plate reader, Bio-Tek, Winooski, VT). The percentage of live cells in a cargo reagent treated sample was calculated by considering the vehicle treated Jurkat T cell sample as 100 %.

live-cell analysis).
(2×10 5 ) cells/well were taken in 48-well plates in growth media and treated with 10 µg/mL of the cargos and incubated for 6 days at 37 °C inside the IncuCyte incubator. Both the proliferation and clustering of the Jurkat T cells were monitored by real time image and video recoded by IncuCyte S3 live cell analysis system. After 6 days of treatment, the clustered Jurkat T cells in each well were treated with PBS (pH 5.5) for 10 minutes at 120 rpm in an orbital shaker. Next, images of the HCl treated cells were recorded again by the IncuCyte S3 live cell analysis system. Finally, magnetic cell separation technique was employed to isolate cargo free pure Jurkat T cells.

IncuCyte movie S3: Jurkat T cell and vehicle treatment
IncuCyte movie S4: Jurkat T cell and cargo 2 treatment  in presence of base di-methyl amino pyridine (DMAP) to get the covalent cargo (ADP was used as ¼ th equivalent with respect to total amine groups of the polymer chain, so that 50 % of the amine groups remain as free in the covalent cargo. As the nitrogen lone pair of the amine group in the adenine is less nucleophilic due to its extended conjugation with the aromatic system, therefore in presence of base, either 3'-OH or 2'-OH of adenine forms bond with the cargo intermediate I. Smallest possible unit structure of the covalent cargo was shown in this step).       observed for the treatment of the cargo 2 as compared to that of the no cargo treated Jurkat T cells.
Magnetic cell separation was used to isolate free cells from its cargo mixture.