Aim: Prepare liquid and solid media for bacterial culture.

Materials:

• • 10 g of tryptone
• • 5 g yeast extract
• • 15 g NaCl
• • 15 g agar *if LB agar
• • Distilled water q.s.p to 1 L

Procedure

1. 1. Mix all the ingredients without specific order of addition (besides adding distilled water last).
   *There is no need to use volumetric flasks (which should NOT be autoclaved), or to use a precision balance to weigh the ingredients.

2. 2. Add water up to 1 L.

3. 3. The medium should be transferred to a glass bottle before autoclaving.
   *Antibiotics are added when pouring plates.

Aim: Conserve bacterias at -80°C. Glycerol is an osmoprotectant agent. An overnight preculture has to be prepared to obtain a maximum of cells to conserve.

Materials:

• • Sterile glycerol (final concentration 15%)
• • 1.5 mL Eppendorf tubes

Procedure

1. 1. Manipulate under PSM.

2. 2. In an Eppendorf tube add 828 µL of preculture and 172 µL of glycerol 87%. *Be careful the glycerol is viscous, so pipet slowly.

3. 3. Immediately stock at -80°C.

Depending on to the goal of our experiments, either a commercial gel either a SDS-page gel was used.

Materials:

• • TGS running Buffer (Tris 25 mM. Glycine 200 mM. SDS 0.1%)
• • 40% acrylamid (euromedex EU0062-B)
• • 1 M Tris-Hcl pH 8.8
• • 1 M Tris-Hcl pH 6.8
• • 10% SDS (Sodium Dodecyl Sulfate)
• • 10% Ammonium persulfate (APS)
• • TEMED (tetramethylethylenediamine)
• • Electrophoresis components
• • 4X Nude PAGE LDS Sample Buffer Thermo Fisher Scientific
• • Bio-rad T100 Thermal Cycler
• • SDS-page ladder: PageRuler™ Prestained Protein Ladder, Thermo Scientific
• • SDS-page kit: SE250 Mighty Small II Mini Vertical Protein Electrophoresis Unit
• • Commercial kit ladder: Precision Plus Protein™ Unstained Protein Standards, Bio-Rad
• • Commercial kit: Bio-Rad MINI-protean Tetra-Cell, 2-Gel system
• • Generator: Biorad PowerPac™ HC

Procedure:

I. SDS-page Gel preparation

• Stacking gel: prepare 4 mL of 5% polyacrylamide

Composant	Volume
ddH20	2.87 ml
40% acrylamide mix	0.5 ml
1.0 M Tris pH 6.8	0.5 ml
10% SDS	0.04 ml
10% ammonium persulfate (freshly prepared)	0.04 ml
TEMED	0.004 ml

• Separation gel: prepare 10 ml of 10% polyacrylamide

Composant	Volume
ddH20	4.9 ml
40% acrylamide mix	2.475 ml
1.0 M Tris pH 8.8	2.5 ml
10% SDS	0.1 ml
10% ammonium persulfate (freshly prepared)	0.1 ml
TEMED	0.004 ml

II. SDS-PAGE revelation

• 1. 3 µL of 4X Nude page LDS Sample Buffer were added into 10 µL of each of the saved samples.
• 2. 4 µL of protein ladder was added:
  • • SDS-page gel: PageRuler™ Prestained Protein Ladder, Thermo Scientific for SDS
  • • Commercial gel: Precision Plus Protein™ Unstained Protein Standards, Bio-Rad
• 3. Samples were heated at 95°C for 5 minutes, and then loaded into the wells of the SDS-PAGE gel/strain free commercial gel.
• 4. TGS running buffer was finally added before the run (generator: 200 V).
• 5. Revelation:
  • • Coomassie Blue for SDS-page gel: all proteins are revealed.
  • • Stain free for commercial gel: all proteins with tryptophan are revealed.
  • • GreenLys: all proteins who have incorporated GreenLys during their synthesis are revealed by a reading at 488 nm.

I. Protein expression

Materials:

• • LB medium
• • Ampicilline 100 mg/mL
• • E.coli BL21 (DE3)
• • IPTG 1 M
• • Resuspension buffer (20 mM NaH2PO4 H2O, 500 mM NaCl + 1mM PMSF + 10% (v/v) glycerol + 20 mM imidazole - pH 7.4)
• • VWR Water Bath
• • V-1200 spectrophotometer UVisco
• • Centrifuge SIGMA 3-18KS
• • INFORS HT Multitron Standard (AG CH-4103 Bottmingen)

Procedure:

1. 1. E.coli BL21 (DE3) was transformed with the plasmid containing DhdR gene. See the transformation protocol above.

2. 2. In a 250 mL flask, 50 mL of LB medium were poured and supplemented with 25 µL of Ampicillin (final concentration 50 µg/mL). Culture media was inoculated with 500 µL of an overnight culture.

3. 3. Cells were grown at 37°C 120 rpm until OD600 reached 0.5-0.6.

4. 4. Culture media was cooled down at 16°C and protein expression was induced by addition of 500 µL of IPTG (final concentration 1 mM) at 16°C overnight.

5. 5. Culture was stopped by centrifugation (4.000 g for 20 minutes) at 4°C.

6. 6. The pellet was resuspended in 10 mL of resuspension buffer and then stored at -20°C, protected from sunlight.

II. Protein purification, dialysis and revelation

Materials:

• • VWR Water Bath
• • Refrigerated Microfuge SIGMA 1-14K
• • Bio-rad T100 Thermal Cycler
• • Fisherbrand™ Model 505 Sonic Dismembrator
• • [Clontech] TALON® Metal Affinity Resin
• • Purification column
• • Dialysis tube
• • Pliers
• • Equilibration buffer: 20mM NaH2PO4 H2O, 500 mM NaCl + 20 mM imidazole (pH to 7.4)
• • Elution buffers: 20mM NaH2PO4 H2O, 500 mM NaCl + (20mM, 50 mM, 100 mM, 250 mM and 500 mM imidazole, depending on the fraction) (pH to 7.4)
• • Dialysis buffer: 50mM NaH2PO4 H2O and 150 mM NaCl (pH to 7.2)
• • 4 X Nude page LDS Sample Buffer Thermo Fisher Scientific
• • Gel and electrophoresis components

Procedure:

1. 1. Resuspended cells were lysed by sonication during 1 minute at 25% power.

2. 2. Cell free extract (CFE) was recovered by centrifugation at 13.300 g for 20 min at 4°C and stored on ice.

3. 3. A 20mL purification column was filled by decantation with 2 ml of 50 % TALON® Metal Affinity Resin and extensively washed with 5 CV of MilliQ water.

4. 4. 10 CV of equilibration buffer was added.

5. 5. The CFE was poured by decantation, mixed gently to the resin and placed at 4°C for 10 minutes.

6. 6. Unbounding fraction was eluted using 5 CV of equilibration buffer 3 times.

7. 7. Protein of interest was eluted using 3 CV of elution buffer supplemented with increasing concentration of imidazole.

8. 8. Presence of pure protein in different fractions was assessed by SDS-PAGE. Cf SDS-PAGE Gel migration protocol with 13% acrylamide.

9. 9. Pure fractions were pooled and extensively dialysed in the dialysis buffer. Dialysed proteins have been used in the PURE system.

10. 10. Cobalt resin was regenerated using 2 CV of 500 mM imidazole ; 5 CV of MilliQ water ; then with 1 CV of MES (20 mM, pH =5) ; 5 CV of MilliQ water and finally stored with 1 CV of 20% ethanol.

I. Protein expression

Materials:

• • E. coli BL21 cells VHH anti-HER2
• • LB medium
• • Kanamycin 50 mg/mL
• • IPTG 1 M
• • INFORS HT (AG CH-4103 Bottmingen)
• • Spectrophotometer Jenway 7205

Procedure:

1. 1. Inoculation of the E. coli BL21 cells VHH anti-HER2 from glycerol stock. Cf Inoculation Protocol.

2. 2. In a 250 mL flask, 50 mL of LB medium were poured and supplemented with Kanamycin at a final concentration of 50 µg/mL. Culture media was inoculated with 500 µL of an overnight pre-culture and Cells were grown at 37°C 150 rpm until OD600 reached 0.5-0.6.

3. 3. AtOD600≈ 0,5-0,6, the erlenmeyer was retrieved and cooled down iced water tank shaking continuously. When the temperature of 20°C is reached, the culture is incubated at 18°C, 150 rpm until OD600≈ 0,8-1, and the protein expression is induced by the addition of IPTG at a final concentration of 1 mM.

4. 4. The culture was incubated at 18°C, 150 rpm for 20 hours.

II. Protein extraction

Materials:

• • TSE Buffer: 200 mM Tris-HCl, pH 8, 500 mM sucrose, 1 mM EDTA
• • Binding buffer: 50 mM sodium phosphate, 300 mM NaCl, pH 7.4
• • Slide-A-Lyzer™ MINI dialysis devices (3.5 k molecule retention threshold)
• • Centrifuge SIGMA 3-18KS

Procedure:

1. 1. The culture was harvested at 8.000 xg, 4°C, for 20 minutes and the supernatant pulled off.

2. 2. The cell pellet was resuspended in 3 mL of buffer TSE buffer per gram of cells.

3. 3. The resuspended cells were incubated at room temperature for 10 minutes.

4. 4. The cells were cold-shocked by adding 3 mL of ice-cold sterile MQ water per gram of cells.

5. 5. The suspension was incubated on ice for 10 minutes.

6. 6. The cells free extract (FCE) was recovered by spinning down at 8.000 xg, 4°C, for 20 minutes.

7. 7. CFE was buffer exchanged by extensive Dialysis on binding buffer, following the fabricant protocol.

III. Protein purification

Materials:

• • Dialysed CFE
• • [Clontech] TALON® Metal Affinity Resin
• • Purification column
• • Binding buffer: 50 mM sodium phosphate, 300 mM NaCl, pH 7,4; 4°C
• • Washing buffer: 50 mM sodium phosphate, 300 mM NaCl, 5 mM imidazole, pH 7,4; 4°C
• • Elution buffers: 50mM sodium phosphate, 300 mM NaCl + (10mM, 20mM, 200mM, 250 mM imidazole), pH 7,4; 4°C
• • Recuperation buffer (PBS): 50 mM sodium phosphate; 4°C
• • Slide-A-Lyzer™ MINI dialysis devices (3.5 k molecule retention threshold)
• • 4X Nude page LDS Sample Buffer Thermo Fisher Scientific
• • Gel and electrophoresis components

Procedure:

1. 1. A 20 mL purification column was filled by decantation with 2 mL of 50% TALON® Metal Affinity Resin extensively washed with 5 CV of MilliQ water.

2. 2. The resin was equilibrated with 10 CV of binding buffer.

3. 3. CFE was poured by decantation.

4. 4. Unbounding fraction was eluted using 5 CV of washing buffer three times.

5. 5. Protein of interest was eluted using 3 CV of elution buffer supplemented with increasing concentration of imidazole.

6. 6. Presence of pure protein in the different elution fractions was assessed by SDS-PAGE at 15% acrylamide.

7. 7. Pure fractions were pooled and extensively dialysed on a recuperation buffer (PBS).

8. 8. Fractions were conserved at -80°C after a nitrogen bath.

9. 9. Cobalt resin was regenerated using 2 CV of 500 mM imidazole ; 5 CV of MilliQ water ; then with 1 CV of MES (20 mM, pH =5) ; 5 CV of MilliQ water and finally stored with 1 CV of 20% ethanol.

I. In vitro protein synthesis with PUREfrex2.0

Materials:

• • Solution I (amino acids, NTPs, tRNAs and substrates for enzymes etc...) (GeneFrontier PF201-0.25-EX). Storage at -80°C.
• • Solution II (Proteins in 30% glycerol buffer) (GeneFrontier PF201-0.25-EX). Storage at -80°C.
• • Solution III (Ribosome) (GeneFrontier PF201-0.25-EX). Storage at -80°C.
• • FluoroTect GreenLys solution (Promega L5001)
• • SP6 RNA Polymerase with its dilution buffer (Sigma-Aldrich 10810274001)
• • RNase A (Sigma R6148-25 mL)
• • DHFR DNA (GeneFrontier PF201-0.25-EX): 20 ng/µL.
• • SUPERase (Invitrogen AM2694 Thermo fisher scientific): 20 U/µL.
• • PCR machine (Bio-rad T100 Thermal Cycler)

Procedure:

• 1. The following reaction mixture was prepared in Eppendorf tubes to synthesize proteins in 20 μL:

  	Sample of interest	Positive control	Negative control
  Nuclease-free water	7-X µL	6 µL	6.5 µL
  Solution I *	10 µL	10 µL	10 µL
  Solution II *	1 µL	1 µL	1 µL
  Solution III *	2 µL	2 µL	2 µL
  GreenLys (only for co-translational labeling)	0.5 µL	0.5 µL	0.5 µL
  DNA (1-3 ng/µL per 1 kb)	X µL (0.5 to 3 ng/μL per 1 kbp)	0.5 µL (present in the kit)	/
  Total	20 µL

  * Solutions I, II and III should be divided into aliquots to avoid refreeze and thaw as much as possible.
  Note: The experiments were conducted with 5 nM of DNA. When adding other components (e.g., 2-HG, DhdR, Tegafur), the volume of water was adjusted accordingly, or the same volume of the vehicle solution was added as a control.
• 2. When needed, 1 µL of SP6 RNA polymerase was added and the volume of water was adjusted accordingly.
• 3. Proteins of interest were synthesized by incubating the tubes for 4 to 6 hours at 37°C (check the optimal temperature for your protein).
• 4. The pre-run solution was incubated for 15 minutes at 37ºC with 0.5 µL of RNase A at 20 mg/ml to digest unreacted FluoroTect GreenLys.
• 5. 5 µL of reaction mix was transferred into an Eppendorf tube.
• 6. An equal volume of water and Sample Buffer was added to the reaction mixture, which was then heated for 3 minutes at 95°C for protein denaturation.
• 7. 15 μL of each sample was run on an SDS-PAGE gel for protein analysis. (cf Protein electrophoresis).
• 8. A Coomassie Blue stain was used to visualize the total protein content. In addition, the gel was imaged with a fluorescence gel imager at 488 nm excitation wavelength to visualize GreenLys-labeled proteins.

II. In vitro protein synthesis with PUREfrex2.1

Materials:

• • Solution I (amino acids, NTPs, tRNAs and substrates for enzymes etc...) (GeneFrontier PF213-0.25-EX). Storage at -80°C.
• • Solution II (Proteins in 30% glycerol buffer) (GeneFrontier PF213-0.25-EX). Storage at -80°C.
• • Solution III (Ribosome) (GeneFrontier PF213-0.25-EX). Storage at -80°C.
• • FluoroTect GreenLys solution (Promega L5001)
• • RNase A (Sigma R6148-25 mL)
• • Cysteine (GeneFrontier PF213-0.25-EX).
• • GSH (GeneFrontier PF213-0.25-EX).
• • GSSG (GeneFrontier PF213-0.25-EX).
• • DsbC with its dilution buffer: DsbC must be diluted from 320 µM to 80 µM (GeneFrontier PF213-0.25-EX).
• • DTT (GeneFrontier PF213-0.25-EX).
• • DHFR DNA (GeneFrontier PF201-0.25-EX): 20 ng/µL
• • SUPERase (Invitrogen AM2694 Thermo fisher scientific): 20U/µL
• • PCR machine (Bio-rad T100 Thermal Cycler)
• • Heat bath

Procedure:

• 1. The reaction mixture was assembled in an Eppendorf tube as follows:

  	Sample of interest	Positive control	Negative control
  Nuclease-free water	20-X	3.25 µL	3.75 µL
  Solution I	8 µL	8 µL	8 µL
  Solution II	1 µL	1 µL	1 µL
  Solution III	2 µL	2 µL	2 µL
  Cysteine	1 µL	1 µL	1 µL
  GSH	1 µL	1 µL	1 µL
  GSSG	1 µL	1 µL	1 µL
  DTT	1 µL	1 µL	1 µL
  DsbC (diluted)	1 µL	1 µL	1 µL
  GreenLys (only for co-translational labeling)	0.5 µL	0.5 µL	0.5 µL
  SUPERase	0.75 µL	0.75 µL	0.75 µL
  DNA (1-3 ng/µL per 1 kb)	X	0.5 µL (present in the kit)	/
  Total	20 µL

  * Solutions I, II, and III should be divided into aliquots to avoid refreeze and thaw as much as possible.
  Note: The experiments were conducted with 5 nM of DNA. When adding other components (e.g., 2-HG, DhdR, Tegafur), the volume of water was adjusted accordingly, or the same volume of the vehicle solution was added as a control.
• 2. When needed, 1 µL of SP6 RNA polymerase was added and the volume of water was adjusted accordingly.
• 3. Proteins of interest were synthesized by incubating the tubes for 4 to 6 hours at 37°C (check the optimal temperature for your protein).
• 4. The pre-run solution was incubated for 15 minutes at 37ºC with 0.5 µL of RNase A at 20 mg/ml to digest unreacted FluoroTect GreenLys.
• 5. 5 µL of reaction mix was transferred into an Eppendorf tube.
• 6. An equal volume of water and Sample Buffer was added to the reaction mixture, which was then heated for 3 minutes at 95°C.
• 7. 15 μL of each sample was run on an SDS-PAGE gel for protein analysis. cf protein electrophoresis protocol.
• 8. A Coomassie Blue stain was used to visualize the total protein content. In addition, the gel was imaged with a fluorescence gel imager at 488 nm excitation wavelength to visualize GreenLys-labeled proteins.

III. In vitro proteins synthesis with PUREfrex2.1 custom kit

Materials:

• • Solution I (amino acids, NTPs, tRNAs and substrates for enzymes etc...) (GeneFrontier PFC-Z117-1-EX). Storage at -80°C.
• • Solution II (Proteins in 30% glycerol buffer) (GeneFrontier PFC-Z117-1-EX). Storage at -80°C.
• • Solution III (Ribosome) (GeneFrontier PFC-Z117-1-EX). Storage at -80°C.
• • T7 RNAP with its dilution buffer; T7 RNAP must be diluted to 20μL/mL of buffer (Gene Frontier PFC-Z117-1-EX). Storage at -80°C.
• • SP6 RNA Polymerase with its dilution buffer (Sigma-Aldrich 10810274001)
• • FluoroTect GreenLys solution (Promega L5001)
• • RNase A (Sigma R6148-25 mL)
• • Cysteine (GeneFrontier PFC-Z117-1-EX).
• • GSH (GeneFrontier PFC-Z117-1-EX).
• • GSSG (GeneFrontier PF005-0.5-EX).
• • DsbC with its dilution buffer: DsbC must be diluted from 320 µM to 80 µM (GeneFrontier PF005-0.5-EX).
• • DTT (GeneFrontier PFC-Z117-1-EX).
• • DHFR DNA (GeneFrontier PFC-Z117-1-EX): 20 ng/µL
• • SUPERase (Invitrogen AM2694 Thermo fisher scientific): 20 U/µL
• • GroE mix with its dilution buffer : 2-fold dilution necessary (GeneFrontier PF004-0.5-EX)
• • PCR machine (Bio-rad T100 Thermal Cycler)
• • Heat bath.

Procedure:

• 1. The reaction mixture was assembled in an Eppendorf tube as described above in II.In vitro proteins synthesis with PUREfrex2.1.
• *Note: The experiments were conducted with 10 nM of DNA.
• 2. When needed, 1 µL of SP6 RNAP was added to the reaction mix.
• 3. When needed, 1 µL of T7 RNAP was added to the reaction mix.
• 4. When needed, 1 µL of GroE mix was added to the reaction mix.
• Note: If more components were added, the volume of water was adjusted accordingly, or the same volume of the vehicle solution was added as a control.
• 5. Proteins of interest were synthesized by incubating the tubes for 4 to 6 hours at 37°C (check the optimal temperature for your protein).
• 6. The pre-run solution was incubated for 15 minutes at 37ºC with 0.5 µL of RNase A at 20 mg/ml to digest unreacted FluoroTect GreenLys.
• 7. 5 µL of reaction mix was transferred into an Eppendorf tube.
• 8. An equal volume of water and Sample Buffer was added to the reaction mixture, which was then heated for 3 minutes at 95°C.
• 9. 15 μL of each sample was run on an SDS-PAGE gel for protein analysis. cf protein electrophoresis.
• 10. A Coomassie Blue stain was used to visualize the total protein content. In addition, the gel was imaged with a fluorescence gel imager at 488 nm excitation wavelength to visualize GreenLys-labeled proteins.

Materials:

• Spectrophotometer (Thermo Scientific Varioskan lux)
• PURE system samples
• Black 384-well microplate (Corning Product Number 3658)

Procedure:

• 1. 15 µL of each sample was added in a well of the microplate.
• 2. The excitation wavelength was set to 488 nm. Emission signal was measured at a wavelength of 510 nm.

Materials:

• • DOPC (Avanti Polar Lipids 850375C-25MG)
• • DOPG (Avanti Polar Lipids 840475C-25MG)
• • PEG-Biotin (Avanti Polar Lipids 880129P-10MG)
• • PE-TopFluor594(Avanti Polar Lipids 810387C-250UG)
• • DSPE-PEG Folate (Avanti Polar Lipids 880123X-500UG)
• • DGS-NTA (Ni) (Avanti Polar Lipids 790404C-5MG)
• • Chloroform
• • Glass beads, acid-washed 212-300µm, Sigma-Aldrich
• • PUREfrex solutions (20 µL)
• • DNase I (New England Biolabs M0303S): 2000 U/mL
• • Rotavapor (HiVap Heidolph)
• • Dessicator
• • Hamilton syringes
• • Zeiss Axio Observer with a camera ANDOR (Zyla sCMOS) and a HXP 120V lamp with appropriate filter cubes for our fluorescent dyes.
• • Celldiscoverer 7 Zeiss: 20x air objective with extra 2x magnification, appropriate wavelengths and filters for Topfluor594 and GFP. Measurements were performed at the photonic platform LITC at CBI (Centre de Biologie Intégrative, Toulouse).
• • Nikon Eclipse Ti: measurements were performed at the photonic platform LITC at CBI (Centre de Biologie Intégrative, Toulouse).

Procedure:

Preparation of lipid-coated beads:

• 1. For liposomes decorated with anti-HER2 nanobodies (anti-HER2 nb) and folate: a mixture of DOPC (77% molar), DOPG (19.5% molar), PEG-biotin (0.5% molar), Topfluor594 (0.1% molar), DSPE-PEG Folate (0.1% molar) and DGS-NTA(Ni) (2% molar) lipids, all dissolved in chloroform, was prepared in a round-bottom flask to obtain a total mass of 5 mg.
  For “standard” liposomes: a mixture of DOPC (80% molar), DOPG (20% molar), PEG-biotin (0.5% molar) and Topfluor (0.1% molar), all dissolved in chloroform, was prepared in a round-bottom flask to obtain 5 mg/mL of lipids.
  *Note: The Hamilton syringes were washed several times with chloroform between each sample.
• 2. 2.4 g of glass beads was poured into the lipid mixture.
• 3. The solution was subjected to 3-4 hours of rotary evaporation at room temperature at 300 mbar, followed by 2 hours of rotary evaporation at 20 mbar.
• 4. The solution was stored under argon at -20°C.

Liposome formation: Swelling of the lipid film by gentle rehydration with the PURE system solution:

• 1. A 20 μL PUREfrex2.0/2.1 reaction solution was assembled. cf PURE system protocol.
• 2. To the well-mixed reaction, 10 mg of lipid-coated beads freshly pre-dessicated for at least 30 minutes were added.
• 3. The 1.5 mL Eppendorf tube containing the bead-PUREfrex mixture was incubated at 4°C for about 1 hour. The tube was gently rolled in the hand a few times during incubation for uniform liposome swelling.
• 4. The mixtures were then subjected to four freeze/thaw cycles (5 seconds in liquid nitrogen followed by 6 minutes on ice).
• 5. From this step onward, the liposome suspension was handled gently and only with cut pipette tips to prevent liposome breakage.
• 6. Finally, 10 μL of supernatant liposome suspension was transferred to a PCR tube, where it was mixed with 0.5 units of DNase I (NEB) to avoid expression outside liposomes.
• 7. The reaction solution was incubated at 37°C in a thermocycler for 4 hours.
• 8. Extra step for anti-HER2 nb liposomes: 10 µL of liposomes were mixed with 50 µL of anti-HER2 nb (stock at 17 µM) and incubated for 30 minutes at 37°C in an Eppendorf tube.

The protocol was modified from Step 7 when anti-HER2 nb-coated liposomes were injected on top of cancer cells:

• 7. 10 µL of liposomes were mixed with 50 µL of anti-HER2 nb (stock at 17 µM) for 30 minutes, at 37°C in an Eppendorf tube.
• 8. The medium of the 9-cm² petri dish containing Caco-2 cells at the optimal density was removed.
• 9. 1 mL of culture medium (Dulbecco's Modified Eagle Medium (SIGMA: D0819-500ML) supplemented with Foetal Bovine Serum (SIGMA: F7524-500ML), MEM Non-Essential Amino Acid solution (SIGMA: M7145-100mL), and Penicillin Streptomycin (GIBCO: 15140-122)) was added to the solution containing liposomes and nanobodies.
• 10. The growth medium was added on Caco-2 cells and incubated for 4-6 hours at 37°C.

Imaging PURE system-containing liposomes and cultured cancer cells by optical microscopy

• Celldiscoverer 7 Zeiss: Samples were mounted on an environmental chamber set to 37°C with CO2. A 20x air objective with an extra 2x magnification, appropriate excitation wavelengths and filters for Topfluor594 and GFP were used. Measurements were performed at the photonic platform LITC at CBI (Centre de Biologie Intégrative, Toulouse).

Imaging PURE system-containing liposomes by optical microscopy

• 1. Nikon Eclipse Ti: Measurements were performed at the photonic platform LITC at CBI (Centre de Biologie Intégrative, Toulouse).
• 2. Axio Observer Zeiss equipped with an 100x oil-immersion objective and appropriate wavelengths and filter cubes for Topfluor594 and GFP were used. A camera ANDOR (Zyla sCMOS) was used.

Materials:

• • DOPC (Avanti Polar Lipids 850375C-25MG)
• • DOPG (Avanti Polar Lipids 840475C-25MG)
• • DGS (Avanti Polar Lipids 790404C-5MG)
• • PEG-Biotin (Avanti Polar Lipids 880129P-10MG)
• • Topfluor594 (Avanti Polar Lipids 810387C-250UG)
• • PBS (Phosphate buffered saline)
• • Chloroform
• • Büchi Rotavapor® RE 121
• • Dessicator
• • Liposofast Basic Avestin Extruder with polycarbonate membranes 400-nm pore size (Avestin)
• • Axio Observer Zeiss, 40x oil-immersion objective with a camera ANDOR (Zyla sCMOS) and a HXP 120V lamp with appropriate filter cubes for our fluorescent dyes
• • Celldiscoverer 7 Zeiss: Samples were mounted on an environmental chamber set to 37°C with CO2. A 20x air objective with an extra 2x magnification, appropriate excitation wavelength and filter for Topfluor594 were used. Measurements were performed at the photonic platform LITC at CBI (Centre de Biologie Intégrative, Toulouse).

Procedure:

Lipid film formation

• 1. A mixture of DOPC (80% molar), DOPG (20% molar), DGS (2% molar), PEG-biotin (0.5% molar) and Topfluor594 (0.1% molar) lipids, all dissolved in chloroform, was prepared in a round-bottom flask to obtain a total mass of 2 mg.
• 2. The solution was subjected to 2 hours of rotary evaporation at room temperature (atmospheric pressure) and a speed of 200 rpm/minutes.
• 3. The round-bottom flask was placed in a dessicator for 1-2 hours and stored at -20°C.

Liposome formation

• 1. Lipids in the round-bottom flask were resuspended into 1 mL of PBS to obtain a final concentration of 2 mg/mL lipids.
• 2. With the extruder, the suspension of multilamellar liposomes was forced through polycarbonate filters with 400-nm pore size. The process was repeated four to seven times to obtain homogeneous sizes of liposomes.
• 3. Liposomes were then stored at -20°C.

Functionalization of anti-HER2 nb liposomes

• 1. 50 µL of the liposome solution (stock at 2 mg/mL) was mixed with 50 µL of anti-HER2 nb (stock at 17 µM) for 30 minutes at 37°C.

Procedure:

• 1. The medium of the 9-cm² petri dish containing Caco-2 cells at the optimal density was removed and replaced with 1 mL of sterile PBS.
• 2. The anti-HER2-nanobodies-functionnalized liposomes were added to 900 µL of sterile PBS. cf In-liposome gfp expression and Preparation of 400-nm fluorescent liposomes decorated with anti-HER2 nb.
• 3. This solution was incubated with Caco-2 cells for an additional 10-30 minutes at 37°C.
• 4. Optical microscopy:
  • Celldiscoverer 7 Zeiss: Samples were mounted on an environmental chamber set to 37°C with CO2. A 20x air objective with an extra 2x magnification, appropriate excitation wavelength and filter for Topfluor594 were used. Measurements were performed at the photonic platform LITC at CBI (Centre de Biologie Intégrative, Toulouse).
  • Axio Observer Zeiss equipped with an 40x oil-immersion objective and appropriate wavelengths and filter cubes for Topfluor594 were used. Cells were imaged with the brightfield mode. A camera ANDOR (Zyla sCMOS) was used.