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\DOI{10.5802/crchim.251}
\datereceived{2023-01-27}
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\dateposted{2023-10-18}
\begin{document}

\begin{noXML}

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{Chemical Biology}
%\def\cdr@specialtitle@french{Ch\'emobiologie}
%\makeatother

\title{1.4~nm gold nanoparticle-antibody conjugates for in situ gold
immunolabelling after transduction into living human cells}

\alttitle{Anticorps conjugués à des particules d’or de 1,4 nm et leur transduction dans des cellules vivantes pour de l’immunomarquage à l’or in situ : conjugués anticorps-particule d’or}

\author{\firstname{Nadja} \lastname{Groysbeck}\CDRorcid{0000-0002-8672-555X}}
\address{Universit\'{e} de Strasbourg - CNRS, UMR7242 Biotechnologie et
Signalisation Cellulaire, 300 Bd S\'{e}bastien Brant, CS 10413, 67412
Illkirch, France}
\email[N. Groysbeck]{nadja.groysbeck@gmail.com}

\author{\firstname{Anne Marie} \lastname{Haeberl\'{e}}}
\address{Centre National de la Recherche Scientifique, Universit\'{e}
de Strasbourg, Institut des Neurosciences Cellulaires et
Int\'{e}gratives, F-67000 Strasbourg, France}
\email[A. M. Haeberl\'{e}]{haeberle@inci-cnrs.unistra.fr}
                             
\author{\firstname{St\'{e}phane} \lastname{Ory}\CDRorcid{0000-0003-4359-1157}}
\addressSameAs{2}{Centre National de la Recherche Scientifique, Universit\'{e}
de Strasbourg, Institut des Neurosciences Cellulaires et
Int\'{e}gratives, F-67000 Strasbourg, France}
\email[S. Ory]{ory@inci-cnrs.unistra.fr}

\author{\firstname{Victor} \lastname{Hanss}\CDRorcid{0000-0001-5603-8321}}
\address{Centre for Integrative Biology (CBI), Department of Integrated
Structural Biology, Institut de G\'{e}n\'{e}tique et de Biologie
Mol\'{e}culaire et Cellulaire (IGBMC), 1 rue Laurent Fries, BP10142,
F-67404 Illkirch Cedex, France}
\email[V. Hanss]{hanssv@igbmc.fr}

\author{\firstname{Mikhael} \lastname{Eltsov}\CDRorcid{0000-0003-2298-8964}}
\addressSameAs{3}{Centre for Integrative Biology (CBI), Department of Integrated
Structural Biology, Institut de G\'{e}n\'{e}tique et de Biologie
Mol\'{e}culaire et Cellulaire (IGBMC), 1 rue Laurent Fries, BP10142,
F-67404 Illkirch Cedex, France}
\email[M. Eltsov]{eltsovm@igbmc.fr}

\author{\firstname{Patrick} \lastname{Schultz}\CDRorcid{0000-0002-7310-6186}}
\addressSameAs{3}{Centre for Integrative Biology (CBI), Department of Integrated
Structural Biology, Institut de G\'{e}n\'{e}tique et de Biologie
Mol\'{e}culaire et Cellulaire (IGBMC), 1 rue Laurent Fries, BP10142,
F-67404 Illkirch Cedex, France}
\email[P. Schultz]{pat@igbmc.fr}

\author{\firstname{Guy} \lastname{Zuber}\CDRorcid{0000-0001-8032-2410}\IsCorresp}
\addressSameAs{1}{Universit\'{e} de Strasbourg - CNRS, UMR7242
Biotechnologie et Signalisation Cellulaire, 300 Bd S\'{e}bastien Brant,
CS 10413, 67412 Illkirch, France}
\email[G. Zuber]{zuber@unistra.fr}

\shortrunauthors

\keywords{\kwd{Gold nanoparticle}
\kwd{Antibody}
\kwd{Conjugate}
\kwd{Intracellular delivery}
\kwd{Gold immunolabelling}}

\altkeywords{\kwd{Particules d'or nanom\'etrique}
\kwd{Bioconjugaison}
\kwd{Anticorps}
\kwd{Transduction}
\kwd{Immunomarquage \`a l'or}}

\thanks{This research was supported by the ITI Innovec (IdEx
(ANR-10-IDEX-0002), SFRI (ANR-20-SFRI-0012)), the French Infrastructure
for Integrated Structural Biology (FRISBI ANR-10-INBS-05), and the ITMO
Cancer (ColorME, 22P096-00). NG received a PhD fellowship from the IdEX
Unistra (Universit\'{e} de Strasbourg and Investissements d'Avenir). VH
received a PhD fellowship from the CNRS}

\begin{abstract}
Despite advances in Electron Microscopy (EM) that enable to image
protein assemblies within vitreous sections of cells at nearly atomic
resolution, labelling is still necessary to locate small proteins or
rare complexes. Gold immunolabelling has been used for decades to
localise specific proteins within cellular sections. {However, current
gold particle-antibody conjugates are not built with enough chemical
precision to match the current resolution offered by} cryo-EM
{methodology.} {Furthermore, as a close to native specimen state can
only be achieved by strict preservation of {a} frozen hydrated state,
it is required} {to deliver gold labelling agents into living cells
prior to their vitrification.} Several 1.4~nm gold
nanoparticle-antibody conjugates were synthesised. Their abilities to
bind to and label their corresponding epitopes within living cells
after cytosolic delivery {by electroporation} are documented here.
\end{abstract}

\begin{altabstract}
Les progr\`es de la microscopie \'electronique (ME) en conditions
cryog\'eniques permettent d'imager les assemblages de prot\'eines \`a une
r\'esolution quasi atomique. L'immunomarquage \`a l'or est utilis\'e depuis
des d\'ecennies pour localiser des prot\'eines sp\'ecifiques dans des
sections cellulaires, mais les sondes actuelles \`a base d'or et
d'anticorps ne sont plus adapt\'ees aux exigences de la cryo-EM. Le
maintien de l'\'etat hydrat\'e congel\'e de l'\'echantillon et la stricte
exclusion de la r\'eticulation chimique ou des r\'eactifs de contraste
exigent que les agents de marquage \`a l'or soient d\'elivr\'es dans les
cellules vivantes avant leur vitrification. Plusieurs conjugu\'es
nanoparticules d'or-anticorps de 1,4 nm ont \'et\'e synth\'etis\'es. Leur
capacit\'e \`a se lier \`a leurs \'epitopes correspondants et \`a les marquer
dans des cellules vivantes apr\`es livraison cytosolique est document\'ee
ici.
\end{altabstract}

\maketitle

\clearpage

\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}


Understanding how multiple components of a human cell interact together
to sustain the basic mechanisms of life remains mostly unknown but
makes fast progress through the combination of multiple experimental
approaches. Encyclopedia containing the human genome~\cite{1} and
tissue-based maps of the human proteome~\cite{2} enable us to define
gene expression profiles and subsequently the protein levels that
compose each human cell type. Advanced quantitative methods
(quantitative Polymerase Chain Reaction, mass spectrometry) estimate
the abundance of mRNA and proteins per cell~\cite{3}. Progresses in
microscopy allow imaging protein--protein interactions at resolution
approaching (macro)molecule level for super resolution fluorescent
microscopy and nanometric resolution for cryo-Electron Microscopy
(EM)~\cite{4}. At nanometric dimensions, specimen preservation is
crucial and dehydration or chemical cross-linking should be avoided to
prevent structural reorganisation of cellular content. In this
respect, eukaryotic cells, including mammalian ones, can be vitrified
either by plunge freezing or by high-pressure freezing, to preserve a
close-to-native frozen hydrated state, and sectioned below
${-}$140~\textdegree C for cryo-EM imaging~\cite{5}.
Abundant proteins forming filaments or large assemblies such as
ribosomes or nuclear pores are easily recognised in cryo-electron
micrographs due to their characteristic shape~\cite{4,6}. However, less
abundant biomolecular complexes or small proteins cannot be recognised
unambiguously in electron tomograms and their identification relies on
labelling tools. Electron-dense gold nanoparticles (AuNPs) conjugated
to antibodies are popular tags for EM immunolabelling since their high
electron scattering, round shape, and specific dimension provide
sufficient contrast to identify the  probe~\cite{7}. Colloidal AuNPs
with size larger than 5~nm in diameter are readily detected even when
the cellular sample is contrasted. They offer a large surface area onto
which many proteins  adsorb~\cite{8}. This ability has been exploited
to make immunocolloids of popular usage~\cite{7}. However, size
restricts penetration of those colloids in gel-like biological 
samples~\cite{9}. It has led to the development of gold nanoparticles
with diameters below 2~nm and to modification of the gold-to-antibody
conjugation because proteins do not adsorb tightly enough on the
surface of gold particles below  2~nm~\cite{10}. Phosphine-coated
1.4~nm Nanogold$^{\text{\textregistered}}$~\cite{11} and 0.8~nm ultrasmall
AuNPs~\cite{12} have increased labelling efficiency but to the cost of
an extra step consisting in increasing the size of the gold particles
by silver deposition to reveal their positions in the cell sections.
Due to the impermeability of the plasma membrane, labelling of specific
intracellular targets is generally performed post-embedding on cell
sections at temperatures  above 0~\textdegree C~\cite{13}. This protocol
is not consistent with high-resolution cryo-EM because vitrified
cellular samples cannot be warmed up after sectioning. Therefore, the
gold nanoparticles-antibody conjugates need to be delivered into living
cells, prior to the vitrification step, using weakly perturbing
transduction  methods~\cite{14}. Ultra-small 0.8~nm AuNPs conjugated to
antibodies directed against RNA polymerase II were shown to be
delivered into living HeLa cells using a cationic lipid formulation, and
the gold labels were detected within the nucleus by Scanning Electron
Microscopy (SEM) after resin embedding and silver 
enhancement~\cite{15}. To develop gold particles of sizes directly
visible in vitrified cell sections by cryo-EM, mercaptobenzoic acid
(MBA)-coated AuNPs of defined sizes around  2~nm~\cite{16,17} appeared
extremely promising~\cite{18,19}. We previously attempted to
substitute MBA coating on AuNPs {by} thionitrobenzoate  (TNB)~\cite{20}
to increase ligand exchange kinetics~\cite{21}. During the
synthesis of 1.4~nm AuNP with sodium borohydride, a fraction of 
nitro groups underwent reduction to amines, yielding AuNP with a mixed
coordination layer consisting of TABs and TNBs
(TAB-, TNB-AuNP) named AuZ. This mixed coverage was
seen advantageous, in terms of ligand exchange with incoming thiols on
the one hand, and colloidal stability on the other hand. Indeed, 
ligand exchange onto the AuNPs is rarely quantitative and
preferentially occurs with the negatively charged TNBs, leaving
zwitterionic TABs on the surface as putative protecting groups against
strong associations to proteins~\cite{22}.\looseness=1

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1}Illustration of gold immunolabelling in living
cells. The strategy relies on the synthesis of specific AuNP-mAb
conjugates and delivery procedure for the probes to diffuse, 
even into the cell nucleus, and bind to their specific targets.
\vspace*{-5pt}}
\end{figure*}


In this study, we linked several commercial monoclonal antibodies
(mAbs) to 1.4~nm AuZ gold nanoparticle 
to produce AuNP-mAb conjugates. First,
we verified that these conjugates specifically bind to their
intracellular targets using fixed and permeabilised cell lines in a
classical gold immunolabelling procedure. We confirmed that
shielding the gold surface of the AuNP-mAb conjugate with 2~kDa
polyethyleneglycol drastically reduced unspecific adsorption of
nanoparticles to extracellular constituents but also to intracellular
ones~\cite{23,24}. The AuNP-mAb conjugates were then delivered
inside living cells by  electroporation~\cite{25,26} and the cells
containing cytosolic AuNP-mAb conjugates were cultivated before
monitoring the cell distribution of the probes (Figure~\ref{fig1}). Our data
demonstrate that the 1.4~nm AuNP-mAbs enter the cytoplasm where
they circulate and associate to their targets. Accumulation of the
probes in living cells was mAb-dependent. The accumulation patterns of
the AuNP-mAb conjugates anti-GFP, anti-RPB1 (7G5, anti-RNA polymerase
II) and anti-nucleoporins were analogous to the classical immunogold
labelling ones indicating that the probes 
{{bind}} {{in cellulo}}
to their targets without perturbing their function.
Cytoplasmic-delivered anti-$\upalpha$-tubulin and
anti-$\upbeta$-tubulin conjugates in contrast promoted
intracytoplasmic aggregates, indicating that real-time binding of these
conjugates perturbs microtubule dynamics.

\section{Results and discussion}

\subsection{Synthesis of gold nanoparticle-antibody conjugates}

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Synthesis and characterisation of the
AuNP-antibody conjugate. (A) Disulfide bonds of the mouse monoclonal
antibody targeting RPB1 of RNA polII (clone 7G5) are reduced at the
hinge area with TCEP. The sulfhydryl groups at the hinge can then react
with AuZ by thiolate exchange. After clearing the conjugate from
excess AuZ, the remaining reacting thiolates on the AuNP surface are
exchanged with thiolated 2~kDa polyethyleneglycol (PEG). (B)~Non-reducing
SDS-PAGE analysis of the reaction intermediates. Illustration of the
antibody derived from protein data bank entry id  1IGY \cite{46}. 
(C)~Gallery of two-dimensional class averages from negatively stained
7G5AuPEG particles. (D) Representative class averages representing 2-D
views of unlabelled (top) or gold-conjugated (bottom) IgG molecules.
The bar represents 15~nm in C and D.\vspace*{-2pt}}
\end{figure*}

The 1.4~nm AuZ used in this study has been previously described and
comprehensively characterised~\cite{20}. To conjugate the mouse
monoclonal antibodies to AuZ, we took advantage of the disulfide
{bonds} present in the hinge region. These disulfide bonds are easily
reduced to sulfhydryl groups using Tris(2-carboxyethyl)phosphine 
(TCEP)~\cite{27}, thus creating a conjugation site for the 150~kDa mAb onto 
{TAB-, TNB-AuNP}
at about 7~nm of the recognised antigen
paratope  (Figure~\ref{fig2})~\cite{28}. The {various} reaction intermediates
can be monitored using a non-reducing SDS-PAGE experiment as
illustrated by the conjugation of AuZ to the 7G5 mAb (Figure~\ref{fig2}B). The
7G5 mAb targets the 52 times-repeated YSPTSPS sequence at the
C-terminal domain of the largest subunit (RPB1) of mammalian RNA
polymerase II (Pol~II). TCEP at a concentration of 1~mM fully cleaved
the disulfide {bond} holding the heavy chains together as seen in the
SDS-PAGE analysis (lane 7G5-SH). The mAb with an initial apparent
molecular weight (MW) of 150~kDa was completely transformed into a 
75~kDa protein corresponding to a single copy of the heavy and light
chains. The reduced mAb was then reacted with a 1.5 molar excess of AuZ
to yield a new component with an apparent MW slightly above 150~kDa
(lane 7G5Au). Excess AuZ and the first released TNBs were removed
by size exclusion chromatography. The purified 7G5Au was then
reacted with an excess of thiolated 2~kDa polyethyleneglycol (PEG) to
chemically neutralise the remaining reacting TNBs present on the gold
particle  surface~\cite{29} and to shield the gold surface from
unspecific binding~\cite{8,23}. A material with a highly retarded
electrophoretic mobility was observed (lane 7G5AuPEG), indicative of
the shielding ability of PEG. 

To assess the molecular organisation of the probe, the purified
antibody-gold particle was visualised by transmission {EM}
after negative staining with 2\% uranyl acetate. Single
particle analysis was performed to observe the position of the AuNP
relative to the antibody, to determine the proportion of Au-bound IgG
molecules and to assess the integrity of the AuNP-bound antibody. The
negatively stained protein moieties create a stain exclusion volume
which diffuses less electrons than the surrounding medium. This
property was used to select 80431 molecular images from 1125
micrographs that were aligned and clustered into 100 classes using 
Xmipp~\cite{30} (Figure~\ref{fig2}C). After rejection of false positives, the
remaining 36671 images were clustered into 100 classes. About 88\% of
the uranyl acetate-excluding materials were found associated with an
electron-dense particle whose size is consistent with the input AuNP.
The other images were found in classes representing characteristic
views of non-conjugated IgG molecules such as 15~nm long, Y-shaped
molecules (Figure~\ref{fig2}D, top). The classes showing a dark electron-dense
gold nanoparticle were heterogeneous and showed several extensions
about 5~nm in size which could each correspond to Fab or Fc fragments.
The 3-branched classes resemble the Y-shaped IgG molecule while the
other classes are likely to represent two IgG molecules bound to the
same AuNP particle with different rotational registers (Figure~\ref{fig2}D,
bottom). Strikingly, the electron-dense gold particle was always
located at the centre of the multibranched structures, indicating that
the AuNP associates with the hinge region of the IgG which exposes
several thiolates following mild reduction  step~\cite{31}. When the
AuNP is bound to a single IgG molecule, the latter adopts a classical Y
shape, suggesting that conjugation does not affect the molecular
organisation of the antibody. 

Binding of AuNP-mAb conjugates 7G5Au and 7G5AuPEG to RPB1 was first
assayed using a classical immunocytochemistry procedure. HeLa cells
cultivated on glass coverslips were fixed with 4\% EM-grade
paraformaldehyde for 20~min and their plasma membrane was permeabilised
with 0.1\% Triton X-100. The different 7G5 mAb conjugates were
incubated with the permeabilised cells, excess probe was washed away
and bound conjugates were detected with a secondary fluorescent
goat anti-mouse antibody (Figure~\ref{fig3}A). The 7G5 mAbs were found
predominantly in the cell nucleus with reduced staining in the
nucleolus, as expected for the nuclear RNA pol II enzyme. Enrichment of
fluorescence was observed within foci in accordance with the hypothesis
of transcription occurring mainly in factories or condensates containing
several RNA pol II  molecules~\cite{32}. A similar pattern was
observed with the AuNP-mAb 7G5AuPEG component (Figure~\ref{fig3}B). The
intensity of the green fluorescence was nonetheless diminished as
compared to the 7G5 mAbs  signal. This feature might originate from the
steric hindrance of  PEGs~\cite{23} as well as from the
fluorescence-quenching properties of AuNPs~\cite{33} leading to reduced
association of the secondary Abs and dimming of the fluorescent signal,
respectively. To map the distribution of gold nanoparticles, a
similar immunolabelling experiment was performed using 7G5Au 
(Figure~\ref{fig3}C) and 
7G5AuPEG (Figure~\ref{fig3}D), and a silver-enhancement procedure to
reveal the AuNPs only (Supporting information, Figure~S1 shows that the
control HeLa cells remain unstained after incubation with the
silver-enhancement solution). The 7G5Au probe showed a nuclear
enrichment, but significant amounts of silver also stained the
cytoplasm. Passivation of AuNPs with PEG 2~kDa diminished unspecific
association to the cytoskeleton and provided a sharp nuclear gold
immunolabelling in which dark spots are revealed. These dense spots
might correspond to clustering of several RNA pol II molecules. The
stealth property of PEGs surrounding AuZ was highly efficient since
passivation of 7G5Au with the gold particle-stabilising CALNNG 
peptide~\cite{34} produced strong unspecific labelling of the
cytoskeleton (Supporting information, Figure~S2).

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Immunofluorescence and gold immunolabelling
analysis of the {indicated} monoclonal antibody anti-RPB1 (clone 7G5)
{derivatives} on fixed and permeabilised HeLa cells. {Images A and B:}
the mouse antibody was fluorescently detected with an AlexaFluo488-IgG
anti-mouse conjugate. Images C, D and E: the gold particles were
amplified with silver {for detection} by bright-field imaging.
Conditions: fixation with 4\% (v/v) PFA in 0.1 M phosphate buffer pH
7.6 for 20~min, permeabilisation with 0.1\% (v/v) Triton
X-100$^{\text{\textregistered}}$, incubations with primary and secondary
antibodies at 13~nM.}
{\vspace*{4pt}}
\end{figure*}

\begin{table*}
\caption{\label{tab1}List of antibodies used for conjugation}
\begin{tabular}{ccc}
\thead
{Name} &  {Target} &  {Source} \\
\endthead
{7G5} & RPB1 of RNA polII [TSPSYSP]$_{3}$ at C ter & IGBMC \\
{7C2} & RPB1 of RNA polII [YSPTSPS{]}$_{3}$ at C ter &  IGBMC \\
{Mab414} &  Nucleoporins (Nuclear pores) &  Biolegend \\
{6F8-D9} & Histone H4 trimethyl Lysine20 (H4K20me3) &  Biolegend \\
{B-5-1-2} &  ${\upalpha}$-tubulin (C-t)  & ThermoFisher \\
{TU27} &  ${\upbeta}$-tubulin (C-t)  & Biolegend \\
{2A3} &  Green fluorescent protein (GFP) & IGBMC
\botline
\end{tabular}
\end{table*}


Commercial monoclonal antibodies (listed in Table~\ref{tab1}) targeting RPB1
of RNA polII (7C2)~\cite{35}, {nucleoporins (Mab414), the trimethyl
lysine 20 of histone H4 (6F8-D9),} ${\upalpha}${-tubulin (B-5-1-2),}
${\upbeta}${-tubulin (TU27) and the green fluorescent protein
(GFP)(2A3) were first checked for their immunofluorescence specificity
(Supporting information, Figure~S3). They were then conjugated to AuZ
and passivised with PEG using the same protocol as for 7G5 (Supporting
information, Figure S4). Gold immunolabelling experiments on adherent
cell lines led to images with precise localisation of targets using
bright-field microscopy. The anti-nucleoporins conjugated to pegylated
AuNPs (MAb414AuPEG) labelled the Nuclear Pore Complexes embedded within
the nuclear envelope (Figure~\ref{fig4}A). The post-translational
modification of histone H4 at lysine 20 {was}
also detected in the
nucleus (Figure~\ref{fig4}B). Microtubules were also clearly revealed in the
cytoplasm using the anti-}${\upalpha}${-tubulin B-5-1-2AuPEG 
(Figure~\ref{fig4}C) and 
the anti-}${\upbeta}$-tubulin TU27AuPEG (Figure~\ref{fig4}D). The
monoclonal anti-RPB1 7C2AuPEG was not as \mbox{effective} as 7G5AuPEG in terms
of labelling sharpness (Figure~\ref{fig5}A). Finally, the anti-GFP 
2A3AuPEG~\cite{12,36} {probe labelled extremely well the GFP-labelled
histone H2B within the nucleus of stably transformed HeLa cells that
express the GFP-H2B fusion (Figure~\ref{fig5}C) but did not bind to wild-type
HeLa devoid of GFP (Figure~\ref{fig5}D). The importance of passivation by
grafting PEG onto the gold surface to limit unspecific binding to
cytoplasmic components was also confirmed for that antibody (Supporting
information Figure~S5).} 

\begin{figure*}
{\vspace*{2pt}}
\includegraphics{fig04}
{\vspace*{2pt}}
\caption{\label{fig4}Gold immunolabelling analysis of the indicated
monoclonal antibodies (mAbs) conjugated to 1.4~nm AuNP passivised with
2~kDa PEG. H2B-GFP HeLa cell line stably expresses the histone H2B fused
to GFP. The gold particles were enhanced by silver for bright-field
imaging.}
{\vspace*{2pt}}
\end{figure*}

\begin{figure*}
{\vspace*{3pt}}
\includegraphics{fig05}
{\vspace*{3pt}}
\caption{\label{fig5}Gold immunolabelling analysis of the indicated
monoclonal antibodies (mAbs) conjugated to 1.4~nm AuNP passivised with
2~kDa PEG. H2B-GFP HeLa cell line stably expresses the histone H2B fused
to GFP. The gold particles were enhanced by silver for bright-field
imaging.}
{\vspace*{2pt}}
\end{figure*}

Altogether, those first experiments demonstrated that 1.4~nm AuZ can
be conjugated to several mAbs at the hinge region to produce effective
gold labelling probes for classical immunocytochemistry procedures.
Shielding the 1.4~nm gold nanoparticle with 2~kDa PEG was beneficial
and considerably limited unspecific binding to cell components.

\subsection{Fate of AuNP-mAb conjugates following cytosolic delivery
into living HeLa cells} 

\begin{figure*}
{\vspace*{-2pt}}
\includegraphics{fig06}
{\vspace*{-2pt}}
\caption{\label{fig6}\fontsize{9.9}{11.8}\selectfont The AuNP-mAb conjugates 
($0.2~\upmu\mathrm{g}{\cdot}\upmu$L$^{-1}$) were delivered in HeLa
cells by transient permeabilisation of the cell membrane using 3
electric pulses at $517~\mathrm{V}{\cdot}\mathrm{cm}^{-1}$. 
Distribution of the mAbs and conjugates inside living cells was
evaluated after culture during 20~h unless stated. Fixation: 4\% PFA,
20~min. Permeabilisation: 0.1\% Triton X-100. AuNPs were 
silver-enhanced. (A)~Scheme of the procedure. (B)~Images of the targeted
H2B-GFP in HeLa cells (left) and of the delivered anti-GFP 2A3
(right). The mAb was detected using AlexaFluor568 goat anti-mouse (in
red). (C,~D)~Detection of AuNPs by silver enhancement in cells
incubated with 2A3AuPEG without application of electric pulses (left
images) and with application of electric pulses. (E--H) 
Delivered mAbs were detected in cells using the antibody or the
gold domain.}
\end{figure*}

\begin{figure}
\includegraphics{fig07}
\caption{\label{fig7}Electron micrograph of a HeLa cell section
containing 7G5AuPEGs in the nucleus. Cells were incubated with
7G5AuPEG (0.2~$\upmu\mathrm{g}{\cdot}\upmu$L$^{-1}$) and the plasma
membrane was transiently permeabilised with 3 electric pulses at 
517~V${\cdot}$cm$^{-1}$. Cells were cultivated for 20~h, fixed with 2\%
glutaraldehyde and processed for classical TEM observation. Gold
particles were enhanced with silver.}
\end{figure}



The ability of the AuNP-mAb conjugates to diffuse inside living cells,
bind to their targets and label them was assayed (Figure~\ref{fig6}).
To introduce the probes into the cytoplasm of living cells, transient
holes in the plasma membrane of freshly trypsinised cells were created
using an electroporation device and \mbox{optimised} pulses~\cite{26,37}.
{After the electric pulses and re-closure of the plasma membrane,
HeLa cells containing cytoplasmic mAbs and AuNP-mAb conjugates were
generally cultivated for 20~h to let them adhere onto 
{glass coverslips.}
Cells were then processed for detection of antibodies and of AuNPs
(Figure~\ref{fig6}A). After delivery into the cytoplasm of HeLa cells
expressing H2B-GFP, the unconjugated anti-GFP 2A3 accumulated into the
cell nuclei and distributed similarly to the GFP, suggesting an
effective binding of the antibody to its target (Figure~\ref{fig6}B).
Entry of 150~kDa Abs into the cell nucleus {was}
unexpected since nuclear
pore complexes usually prevent nuclear entry of proteins with a
molecular weight above 60~kDa unless they are equipped with Nuclear
Localisation Signals (NLS). The anti-GFP antibody is likely transported
into the nucleus after binding to the NLS-containing GFP-H2B protein
when it is synthetised in the cytoplasm by a piggyback 
mechanism~\cite{38}. {When delivered into the cytoplasm of wild-type
HeLa cells devoid of GFP-H2B, the \mbox{anti-GFP} 2A3AuPEG probe diffused in the
cytoplasm but was excluded from the nucleus (Figure~\ref{fig6}C). In
contrast, the anti-GFP 2A3AuPEG delivered into the cytoplasm of HeLa
cells expressing H2B-GFP accumulated} {in the nuclei}} {in a fashion
resembling 2A3 (Figure~\ref{fig6}D), indicating that AuNP conjugation
does not impair nuclear import of this specific mAb anti-GFP. The
observed nuclear accumulation patterns relied on the presence of
conjugates in the cytoplasm of living cells since \mbox{electric} pulses for
electroporation were required (left-hand panels in Figure~\ref{fig6}C 
and~D).} 
{{In cellulo}}
{labelling with anti-nucleoporins
Mab414 and Mab414AuPEG yielded images (Figure~\ref{fig6}E) which were
highly reminiscent of the classical immunocytochemistry images
(Figure~\ref{fig4}A). Cytoplasmic-delivered
anti-}${\upalpha}${-tubulin TU27AuPEG and anti-}${\upbeta}$-tubulin
B-5-1-2AuPEG did not reveal an extensive microtubule network but rather
accumulated as aggregates. This type of labelling and accumulation
might be related to the dynamic nature of the microtubule network and
to the electroporation procedure that involves a transient suspension
of the cells using a trypsin treatment. Reshuffling of the microtubule
network might take place during re-adhesion of the cells and after the
electric pulses which are also known disruptors of 
microtubules~\cite{39,40}. {The cytoplasm-delivered 7G5AuPEG and
7C2AuPEG also accumulated in the nucleus (Figure~\ref{fig6}H) similarly
to the unconjugated  mAbs~\cite{37}. {Similarly to the classical gold
immunolabelling protocol, passivation of the AuNPs with PEG was also
extremely useful for nuclear accumulation. Indeed, capping the AuNP of
7G5Au with CALNNG} {peptide}} {did not lead to nuclear accumulation of
7G5AuC 24~h following electroporation (Figure~S6). Finally, the
nuclear accumulation appears not 
{as with the anti-GFP conjugates.}
This property is likely related to the production of RNA
pol II in the cytoplasm which is lower than that of H2B-GFP, limiting}
{\textit{de facto}} the entry of Ab-bound RNA pol  II~\cite{36,37}.
The 7G5AuPEG labelling was further investigated by transmission
electron microscopy (TEM) (Figure~\ref{fig7}). Twenty hours after
electroporation, cells were fixed, the gold signal was amplified by
silver enhancement for better visualisation and thin sections of
resin-embedded cells were observed by TEM. An image of a cell section
displaying the nucleus and cytoplasm was recorded. The enhanced AuNPs
were almost exclusively found in the cell nucleus, confirming that the
RNA polII-driven accumulation of the corresponding AuNP-mAb occurred in\break
this cell.

\section{Conclusion}

We hereby provide a synthesis protocol for preparing mAbs conjugated to
1.4~nm AuZ gold nanoparticles by taking advantage of the disulfide {bonds}
which connect the hinge region of the antibody heavy chains. The
reaction process can be easily monitored by non-reducing SDS-PAGE
analysis which reveals the key reaction intermediates. Passivation of
the gold surface with 2~kDa polyethyleneglycol provided probes with
excellent gold immunolabelling properties such as solubility, absence
of in vivo aggregation, good diffusion properties, and dramatically
diminished the unspecific binding to cellular {components.} Moreover,
these conjugates can be delivered into the cytoplasm of living cells by
electroporation, where they diffuse and bind to their targets. The
piggyback mechanism allowing nuclear entry of AuNP-mAb only upon
binding to NLS-containing targets can be extremely useful as filter
since unbound AuNP-mAbs remain in the cytoplasm. Our first proof of
concept data demonstrated that AuNP-mAbs can be delivered into living
cells, bind to their specific targets and accumulate at locations where
their targets go. In principle, the anti-GFP antibody has the potential
to become a rather generic gold immunolabelling probe. Standard
immunolabelling procedure and silver enhancement will enable to
monitor the labelling dynamic and efficiency, to adjust the amounts of
the electroporated probes and the incubation time (and maybe other
parameters) {to ensure that the most specific} labelling {is observed
later in cryo-EM. {Specific} monoclonal antibodies bind to a specific
protein domain and then interfere with the functioning of their
targets. After labelling with AuNPs and delivery inside living cells,
these interfering probes might help decipher the function of
protein/protein interactions and dynamics of exposure to Ab binding.
Although the electroporation procedure is convenient and has
demonstrated efficiency, the electric pulses are not neutral to cell
physiology. Cells mostly recover but the cell cytoskeleton is
perturbed. Alternatively, differentially disruptive liposomal
formulations have been used as delivery solutions and can be employed
as well.}

\section{Experimental section}

\subsection{Materials} 
The 2~kDa alpha-methoxy-omega-mercapto poly\ubreak(ethylene glycol) (PEG-SH)
was ordered from Iris Biotech. EM-grade
paraformaldehyde (20\% w/v solution) and glutaraldehyde (25\% w/v
solution) were purchased from Electron \mbox{Microscopy} Sciences. Other
reagents were obtained from Sigma Aldrich, Carl Roth, VWR Chemicals,
Euromedex or Honeywell. They were used without further purification
unless stated otherwise. All solutions and buffers were made with water
purified with a Millipore Q-POD apparatus. Precision Plus Protein
Standard Dual Xtra (BioRad) was used as protein ladder for SDS-PAGE
analysis. Monoclonal antibodies were purchased from commercial sources
and were devoid of bovine serum albumin or gelatine. They were cleared
from sodium azide or any other preservatives by size exclusion
chromatography (Sephadex G-25) and eventually reconcentrated by
ultracentrifugation.

\subsection{Gold nanoparticle synthesis}
The 1.4~nm AuZ was synthesised and characterised in a previous 
publication~\cite{18,19,20}. {The concentration of gold
nanoparticles was determined by UV--vis spectrophotometry using the
absorbance at 520~nm and an extinction  coefficient}
${\upvarepsilon}_{520}$  of $2.7\times  10^{5}~\mathrm{mol}^{-1} 
{\cdot}\mathrm{L}{\cdot} \mathrm{cm}^{-1}$.

\subsection{Reduction of monoclonal antibody}
Before performing any
conjugation, reduction of the monoclonal antibody at the hinge area was
\unskip\break
monitored by SDS-PAGE using a non-reducing loading buffer. The antibody
(2~$\upmu\mathrm{g}{\cdot}\upmu$L$^{-1}$ or 13.34~$\upmu$M,
2~$\upmu$L) was titrated with TCEP (final concentrations: 0.1, 0.25,
0.5, 1, and 2~mM). After 16~h incubation at  20~\textdegree C, 
{the reaction mixtures were mixed with}
30\% (w/v) glycerol containing 0.1\% (w/v)
SDS and 0.05\% (w/v) bromophenol blue (0.4 ~$\upmu$L) and analysed by
{SDS-PAGE.} The minimal TCEP concentration producing full
disappearance of the band at about 150~kDa was selected for
medium-scale synthesis.

\subsection{Synthesis of 7G5AuPEG} 
The monoclonal antibody in PBS
containing 1~mM EDTA (2~$\upmu\mathrm{g}{\cdot}\upmu$L$^{-1}$ or 
13.34~$\upmu$M, 100~$\upmu$L) was reduced by addition of
0.1 M TCEP, pH 7.0 (1~$\upmu$L) to a final concentration of 
1~$\upmu$M. After a 12 h incubation at 20~\textdegree C, an
aliquot was withdrawn for analysis and the mixture was then quickly
added and mixed with Auz (25~$\upmu$L of 80 ~$\upmu$M
solution). After overnight incubation at 20~\textdegree C, the crude
mixture was purified by size exclusion chromatography (Biorad P100, 
2~mL of resin, elution with PBS). Thiolated polyethyleneglycol 2000 
(2.7~$\upmu$L of a 20~mM aqueous concentration) was then added. The
mixture was incubated at room temperature for 16 h and the crude
mixture was purified by ultracentrifugation (7 cycles of 0.5 mL sterile
PBS) using a 0.5~mL Microcon$^{\text{\textregistered}}$ centrifugal filter device
with a 100~kDa cutoff (Merck, Molsheim). At the end of the process, the
gold nanoparticle-antibody conjugate was recovered in 
60~$\upmu$L PBS and analysed by SDS-PAGE to estimate the
concentration and conjugation efficiency. The other gold
nanoparticles-antibody conjugates were similarly\break prepared. 

\subsection{Electroporation protocol}
Transient permeabilisation of
the plasma membrane of cells was conducted as previously described
using the Neon$^{\text{\textregistered}}$ transfection system and three 10~ms
pulses at 1550~V~\cite{20,26}. Typically, HeLa cells 
($2\times 10^{5}$ cells in 10~$\upmu$L of the Neon Buffer
R) were mixed with each antibody (2.5~$\upmu$L of 
$2~\upmu\mathrm{g}{\cdot}\upmu$L$^{-1}$ solution in
PBS). After the electric pulses, cells were diluted in prewarmed
antibiotic-free cell culture medium (1~mL). The cells were
recovered by gentle centrifugation, suspended into the warmed 
antibiotic-free  complete cell culture medium and plated in
24-wells plates for cell adherence and growth onto glass coverslips
and left in a cell culture incubator, usually for 18~h.


\subsection{Cell culture} 
Cells were cultured in a 37~\textdegree C
humidified incubator supplied with 5\% CO$_{2}$. HeLa,
CaSki, and stably transformed H2B-GFP HeLa cells were maintained in
Dulbecco's modified Eagle medium containing 2~mM L-glutamine, 10~mM
HEPES buffer, pH 7.0, 10\% heat-inactivated foetal bovine serum (FBS)
and 50~${\upmu}$g/mL gentamycin. For immunocytochemistry,
immunofluorescence and pre-embedding immuno-EM experiments, cells
(25,000 cells/well) were seeded into 24-well plates in 0.5~mL cell
culture medium in which 13 mm diameter glass coverslips were deposited.
The cells were left to adhere overnight by culture in a 37~\textdegree
C humidified incubator supplied with 5\% CO$_{2}$.

\subsection{Immunocytochemistry for bright-field and fluorescence imaging}
Coverslip-adhered cells were fixed with 4\% PFA in 100~mM Sorenson's
buffer, pH 7.6 for 20~min at 20~\textdegree C. The coverslips were then
washed with PBS ($3\times 0.5$~mL, 5~min) containing 50~mM glycine (0.5~mL,
20~min) and cell plasma membranes were permeabilised with 0.1\%
Triton X-100 in PBS (0.5~mL, 5~min). Coverslips were soaked in PBS
containing 10\% (w/v) BSA for 1~h, washed with 0.2\% acetylated BSA
(BSA-c) in PBS ($2\times 0.5$~mL, 5~min) and then incubated with 6 to 10 nM
of the conjugates in 0.2\% BSA-c containing 10\% FCS, 0.5~mL for 1 h.
Next, cells were washed with 0.2\% BSA-c ($2\times 0.5$~mL, 5~min) and
with 80~mM citrate buffer pH 6.7 containing 2\% sucrose ($3\times 0.5$~mL, 
5~min). Gold was revealed by silver enhancement according to a
modified Danscher  protocol~\cite{41} using silver acetate and propyl
gallate~\cite{42}. For fluorescence imaging, the primary antibodies
were labelled using secondary fluorescent anti-mouse conjugates
(AlexaFluor568 goat anti-mouse, Invitrogen A11031 and AlexaFluor488
goat anti-mouse, Invitrogen A11029). Fluorescence observations were
carried out using a Leica DM5500B microscope equipped with an HCX PL
Apo $63\times 1.40$ oil PH3CS objective and a Leica DFC350FX camera.

\subsection{Preparation of cells containing gold nanoparticle-antibody
conjugates for bright-field microscopy imaging}
After the indicated time periods following application of the 
electric pulses, the adherent cells cultivated on
{glass coverslips} were washed with PBS 
($2\times 1$~mL). The cells were fixed with 4.0\% paraformaldehyde (PFA) (1~mL)
for 20~min or 2\% glutaraldehyde in 100~mM Sorenson's buffer, pH 7.6
for 1~h. After removal of the fixative solution and 3 PBS washes (1~mL),
the plasma membrane was permeabilised using 0.1\% (w/v) saponin or
Triton X-100 (1~mL, 15~min) in 0.1~M Sorenson's buffer pH 7.6. The
phosphate-buffered solution was replaced by a 0.1~M citrate solution,
pH 6.7 containing 2\% sucrose (5 washes, 1~mL). Development of 
AuNPs~\cite{41} was then done in a dark room for 30~min using freshly
prepared 6~mM silver acetate solution in 0.16~M sodium citrate, pH 6.7
containing 20\% gum arabic and 2~mM propyl  gallate~\cite{42}. The
silver-promoted enhancement of AuNPs was stopped with 0.16~M sodium
citrate solution, pH 6.7 (1~mL, several washes). The 
{glass coverslips}
were finally mounted onto $3\times 1$ inch microscope slides (knittelglass.com)
using Fluoromount-G (Southern Biotech, Ref. 0100-01, batch
I2819-WC79B). Observations were carried out using a Leica DM5500B
microscope equipped with an HCX PL Apo $63\times 1.40$ oil PH3CS objective
and a Leica DFC350FX camera.

\subsection{Cellular specimen preparation for {EM}}
20~h after the electroporation treatment, the adherent cells were washed
with warmed  (37~\textdegree C) Sorenson's buffer ($3 \times 2$~mL). The
cells were then fixed with 2.0\% glutaraldehyde (1~mL) for 24~h. They
were then washed with TEM storage solution ($5\times$, 1~mL).
Enhancement of the gold particles with silver was performed using the
SE-EM kit (Aurion, the Netherlands) according to the manufacturer's
protocol. The cells were then contrasted with 0.5\% osmium for 20~min
followed by 2\% uranyl acetate both diluted in water. The specimens
were dehydrated in a solution containing increasing concentrations of
ethanol and flat-embedded in Epon (Ladd Research Industries, Williston,
Vermont, USA). The resin-embedded specimens were sectioned into 100~nm
thick slices which were deposited on an electron microscope grid.

\subsection{{TEM} and image analysis}

Cellular specimens were imaged on a Hitachi H7500 transmission electron
microscope (Hitachi High Technologies Corporation) equipped with an AMT
Hamatsu digital camera (Hamatsu Photonics). For EM
imaging of 7G5AuPEG we used nickel EM grids covered
with a thick holey carbon film (Quantifoil R2/2). The grids were topped
with a thin continuous carbon layer and rendered hydrophilic by a 90s
treatment in a Fischione 1070 plasma cleaner operating at 30\% power
with a gas mixture of 80\% argon:20\% oxygen. The 7G5AuPEG solution was
diluted to a final concentration of 50~$\upmu$g/mL in Buffer A
(Tris 10~mM, pH 7.4, NaCl 50mM) and crosslinked for 30~s at a final
glutaraldehyde concentration of 0.3\%. Three~$\upmu$L of this
solution were deposited on the glow-discharged EM grid and the
particles were allowed to adsorb for 1~min. The grid was washed once on
a drop of buffer A and negatively stained with a 2\% uranyl acetate
solution. The sample was then imaged in a Tecnai F20 (FEI) operating at
200~kV and equipped with a 2K CCD camera (Gatan). Micrograph montages
were recorded at an image magnification of 50~kX with a 2.01~\AA{}~pixel 
size at ${-}2~\upmu$m defocus using the SerialEM 
software~\cite{43}. For single particle analysis, micrographs were
imported within the Scipion framework for image processing 
software~\cite{44} and Fourier band-passed filtered using the xmipp3
protocol~\cite{30}. {Reference-free}
picking was performed using the
eman2 protocol to generate a dataset of 80431  particles~\cite{45}. The
particle set was aligned, masked, 2-D classified, cleaned to obtain a
total of 36671 molecular images and classified again into 100 classes
using xmipp3 protocols.

\section*{Declaration of interests}
The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.

\CDRGrant[ANR]{ANR-10-IDEX-0002}
\CDRGrant[ANR]{ANR-20-SFRI-0012}
\CDRGrant[ANR]{ANR-10-INBS-05}
\CDRGrant[ColorME]{22P096-00}

\back{}

\section*{Supplementary data}

Supporting information for this article is available on the journal's
website under \printDOI\ or from the author.  

\CDRsupplementaryTwotypes{supplementary-material}{\cdrattach{crchim-251-suppl.pdf}}

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