Delivering a fluo-nanobody in living cells by photoporation improves

In the course of the study it became clear that not every nanobody is suitable for immunocytochemistry, which also applies to conventional antibodies. For instance, β-catenin Nb86 (another nanobody against human β-β-catenin obtained after immunization with the same β-catenin fragment from Ser389 to Leu781), labelled through pAzF, yielded poor results in a conventional staining protocol on permeabilized and PFA fixed cells (Figure 47A, cf. Figure 46B). With methanol fixation, β-catenin Nb86 was able to visualize β-catenin (Figure 47A, bottom). It was hypothesized that this could be due to cross-linking by para-formaldehyde and that delivery of the nanobody into living cells would allow it to bind its target under native conditions, prior to cross-linking. Crossing the plasma membrane, however, remains a major hurdle for nanobodies. A recently developed transfection technique termed photoporation has been reported to accomplish this⁶⁷. This is an upcoming flexible and safe physical transfection method that can deliver a wide variety of molecules into a broad range of cells. Cells are first

incubated with photothermal nanoparticles, such as graphene quantum dots used here, which can bind to the cell membrane. After washing, pulsed laser irradiation is applied rendering the cell membrane temporarily permeabilized by photothermal effects induced locally by the graphene quantum dots. Compounds in the cell medium, including labelled nanobodies, can then diffuse into the cell cytoplasm until the cell membrane is resealed.

Figure 47: β-catenin visualization using β-catenin Nb86-AF488 and the commercial antibody (a rabbit monoclonal anti-β-catenin antibody (ab32572)). The nanobody was labelled through the pAzF method. (A) Top, the β-anti-β-catenin Nb86-AF488 was used in 4% PFA fixed HNSCC61 cells in single step immunocytochemistry and did not show a specific β-catenin signal.

Bottom, methanol fixation on HNSCC61 cells was performed and the same β-catenin Nb86-AF488 staining performed, showing heightened intensity at the cell-cell contacts. (Scale bar = 10 µM). (B) The β-cat Nb86-AF488 was photoporated into HeLa cells and then fixed. As a control, a conventional staining was performed with a commercial anti-β-catenin antibody (a rabbit monoclonal anti-β-catenin antibody (ab32572)) (in red, middle panel). (Scale bar = 50 µM)

β-catenin Nb86-AF488 was delivered into living cells by photoporation and the cells fixed thereafter. It was observed that β-catenin became clearly discernible at cell-cell contacts (Figure 47B). In a photoporation time lapse experiment, it was possible to observe β-catenin at cell-cell contacts (e.g. time frame between 5h50 and 9h35 indicated with an arrow in Video 1; Video 2 is as control showing a fascin nanobody in photoporation). It was concluded that β-catenin Nb86 bound to its epitope in living cells but failed to do so

subsequent to cell fixation. Hence, photoporation may represent a new avenue for introduction of nanobodies into living cells, allowing the study of short and medium range (24h) dynamic cellular processes and the immediate effects of protein binders on their targets.

Video 1: HeLa cells photoporated with β-catenin Nb86-AF488 (labelled through the pAzF method). An image was taken every 25 min over 12h30. The arrows highlight visualization of β-catenin at cell contact site during 2 cells passing by (e.g.

time frame between 5h50 and 9h35). (Scale bar = 50 µM)

Video 2: HeLa cells photoporated with fascin Nb2-AF488 (labelled through the pAzF method). An image was taken every 25 min over 12h30. Since fascin is an F-actin bundling protein, fascin Nb2-AF488 visualizes filopodia and microspikes. This video is to be considered as a control for the β-catenin Nb86-AF488 photoporation. (Scale bar = 50 µM)

In view of these findings, cortactin Nb2, similar to β-catenin Nb86, was photoporated and the cells then fixed. Phalloidin-AF647 was added to the medium in order to co-stain F-actin with cortactin in invadopodia of HNSCC61 cells. Comparison with Figure 45 shows that a slightly better contrast was obtained and that invadopodia were highlighted by photoporation of cortactin nanobody 2-AF488 and phalloidin-AF647, as well as by the commercial cortactin antibody (anti-cortactin antibody clone 4F11) (Figure 48).

Figure 48: Cortactin Nb2 photoporation in HNSCC61 cells. A few invadopodia are arrowed. The cells were photoporated with cortactin Nb2-AF488 and phalloidin-AF647, followed by a fixation with 4% PFA and a commercial cortactin Ab staining (a mouse monoclonal anti-cortactin antibody clone 4F11). (Scale bar = 10 µM)

6.2.4 Discussion

This work compares two possibilities for incorporation of an alkyne/azido group into a nanobody, an enzymatic method in which sortase A adds an alkyne containing peptide and another in which an azido containing amino acid is incorporated at the nanobody C-terminus. The click chemistry or CuAAC reaction led to the most promising results when incorporating the unnatural para-azido Phe (pAzF) C-terminal to a nanobody. The procedure is straightforward, reliable, relatively fast and has a higher yield when start and end are taken into account. It may become broadly applicable as it can be used for

different nanobodies and coupling to a variety of moieties e.g. different fluorophores, magnetic beads, quantum dots, gold nanoparticles. The use of fluorescently-labelled nanobodies and the new avenue of introducing them into living cells by photoporation will allow study of short and medium range (24h) dynamic cellular processes targeting a broad range of proteins. Moreover, this technique reduces linkage error and will enable the imaging of endogenous proteins at higher resolution and can be of interest for super resolution microscopy. The present study used a CuAAC reaction which has the advantage of site selectivity. Unlike the maleimide method, the presence of a reactive moiety in this case depends on the incorporation of the alkyne/azido carrying amino acid through sortase or through pAzF. As there is only one sortag or one amber stop codon present, the stoichiometry is 1:1. It is known that alkyne and azido groups are highly specific towards each other and that they remain inert to other chemical groups present in proteins⁴⁴³. Hence, labelling will be near stoichiometric as only one reactive group is present on the nanobody to react in the CuAAC reaction. The method also has the advantage that no obvious decrease in yield of the purified pAzF carrying nanobody was observed compared to the unmodified nanobody or to the nanobody with sortag (which is also produced without non-natural amino acids). Recently the CuAAC reaction was used after incorporating an alkyne/azido group into a nanobody⁴⁴⁴. An alkyne containing peptide was employed in combination with the intein-mediated protein ligation (IPL) technique. By equipping the nanobody with a Cys residue between the nanobody and intein sequences, an N-S shift leading to a thioester linkage was facilitated. By using an alkyne bearing cysteine derivative, an on-column cleavage was performed, resulting in an alkynated nanobody.

The incorporation of an alkyne/azido group can also offer several other opportunities.

Previously nanobodies have been used to couple other moieties than fluorophores. They have been coupled to quantum dots and four nanobodies were selectively conjugated onto one quantum dot²⁰⁷. This was achieved by means of sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate which crosslinks the C-terminal Cys of the nanobody to an amino group on the quantum dots^{207, 208}. Quantum dots are capable of emitting enough signal to be detected deeper in tissues than Alexa fluorophores and are therefore preferred. This can easily be performed as well using the CuAAC principle. Non-fluorescent moieties are also possible. A linear polyethylene glycol (PEG) molecule was coupled to nanobodies using maleimide chemistry in order to prolong the in vivo circulation time and to study the changes in pharmacokinetics²⁰³. As discussed above, biotin added to GFP nanobodies enabled immunoprecipitation of GFP fusion proteins¹⁸⁹. Through maleimide chemistry, EGFR nanobodies were conjugated to liposomes in order to induce receptor internalization in cancer cells resulting in an inhibition of tumor cell

proliferation^{445, 446}. As these are examples of the potential of nanobodies, these can all be obtained using the CuAAC reaction as well. The alkyne-azido functionality can be further exploited as for example in Western blotting.

Others produced a green fluorescent protein (GFP) nanobody with a C-terminal tubulin-derived recognition sequence (Tub-tag), consisting of 14 amino acids (VDSVEGEGEEEGEE)188, 189, 430

. By using a tubulin tyrosine ligase (TTL), small unnatural tyrosine derivatives were attached to the Tub-tag¹⁸⁹. The authors were able to couple a 3-formyl-L-tyrosine to a Tub-tagged anti-GFP nanobody, followed by chemical labelling with Alexa Fluor 594-hydrazide. This was further reduced to a one-step protocol using a coumarin-coupled amino acid or β-(1-azulenyl)-l-alanine¹⁸⁸. However, the fluorescent substrates allowed in this one-step process are limited.

The sortase time course experiment in the present study showed reversibility of the amide-bond formation between the nanobody and GGGY-PAG peptide, resulting in a need for higher substrate concentrations⁴³⁰. Another disadvantage of sortase is that peptides used are not readily available¹⁷⁹. In addition to TTL and sortase coupling, other methods include the bacterial biotin ligase (BirA) method which is limited to biotinylation⁴³⁰, the use of transglutaminases to couple moieties on glutamines¹⁹⁸ or the lipoic acid ligase (lplA) method, which couples moieties to lysines in a lipoic acid acceptor peptide tag⁴⁴⁷. Others have used SrtA and butelase-1, which is believed to have a much higher turnover number compared to SrtA^{448, 449}.

Nanobodies are already used as a counterpart to antibodies, resulting in improved imaging resolution. NHS chemistry was employed for AF647 labelling of anti-tubulin nanobodies¹⁶⁴ and it was observed that the AF647-nanobody resolved microtubules much better than a conventional (directly labelled) anti-tubulin antibody using single molecule localization microscopy (SMLM). Others were able to visualize the nuclear pore complex with AF647-labelled nanobodies at high resolution using stochastic optical reconstruction microscopy (STORM) microscopy¹⁷⁹. The nanobodies were labelled using both NHS and maleimide. Endogenous tubulin has been targeted with labelled nanobodies by using the DNA-PAINT (DNA point accumulation for imaging in nanoscale topography) method based on sortase coupling⁴⁵⁰. Thus nanobodies may further develop into excellent tools for future super resolution microscopy in which high precision and accuracy is desired. For this it is important to minimize the linkage error, a result not only of the size of the protein moiety (the nanobody) but also of the size of the fluorescent molecule. The smaller a fluorophore, the more accuracy and resolution can be gained. If resolution at the level of tens of nm is not required, GFP fusion nanobodies can nevertheless yield interesting data as shown for protein-protein interactions¹⁰⁸.

For entry into cells, chemical permeabilization and fixation is one method, but this kills the cells and may cause blockage of the binding site or alter protein structure²⁷². Moreover, permeabilisation can also be disadvantageous as it can cause loss of proteins or even promote their relocalisation²⁷². As shown in Figure 47, β-catenin Nb86 was ineffective in reaching its target in 4% PFA fixed cells, but this could be circumvented by first delivering the nanobody by photoporation. This illustrates the effect of fixation and the advantage of addressing living cells. Introduction of recombinant nanobodies into living cells, and target binding under native conditions, combined with live cell imaging, could solve problems of fixation and permeabilisation, although the delivery of proteins is challenging²⁷². Coupling of cell-penetrating peptides (such as cyclic arginine-rich peptides⁵⁹ or penetratin^{60, 451}) to nanobodies, transportation of nanobodies in mesoporous silica nanoparticles⁶⁵ or using an E. coli type III secretion system^{39, 66} have been employed previously to address this issue. Another option is the use of streptolysin O to create pores into the cell membrane to allow diffusion of nanobodies from the cell medium into the cytosol[50]. Photoporation is a recent technique which has major advantages compared to other protein delivery techniques. It allows targeting of many cells (>80%), with low toxicity (< 2%) and a broad range of cells, even primary cells67, 68, 70, 71, 74

. A comparative experiment was performed in which FITC-Dextran-10kDa was delivered into HeLa cells using photoporation or electroporation, as a standard transfection method⁶⁷. It was observed that cell viability was higher with photoporation.

Electroporation resulted in more (>90%) positive cells, but the amount of FITC-Dextran-10kDa was lower in each cell compared to photoporation. Another advantage of photoporation is the ability to use a recipient of choice and compatibility with optical microscopy, while electroporation only allows use of non-adherent cells in a specific electroporation recipient⁶⁷. Photoporation was also compared to the commercial protein transfection (PULSin) reported to be efficient for cytosolic delivery of proteins and peptides such as antibodies and nanobodies⁶⁸. An inhomogeneous intracellular staining pattern of histone nanobodies was observed with this method together with toxicity which was not observed when using photoporation.

In conclusion, we propose that the combination of fluorescent nanobodies and photoporation may develop into a new method for studying protein behavior and function in mammalian cells. As pharmacological inhibitors are lacking for many cytoplasmic proteins that, by virtue of their properties, do not display catalytic activity, it is expected that new biological information will be obtained through photoporation of nanobodies.