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\DOI{10.5802/crchim.383}
\datereceived{2024-08-09}
\daterevised{2025-01-15}
\dateaccepted{2025-02-05}
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\begin{noXML}

\CDRsetmeta{articletype}{research-article}

\title{Synthesis of long-wavelength-absorbing 
photosensitizer/nanoparticle conjugates and their {in vitro} PDT
evaluation on colorectal cancer cell lines}

\alttitle{Synth\`{e}se de conjugu\'{e}s
photosensibilisateur/nanoparticules absorbant \`{a} de grandes
longueurs d'onde et leur \'{e}valuation PDT \textit{in vitro}  sur des
lign\'{e}es cellulaires de cancer colorectal}

\author{\firstname{Luce Janice} \lastname{Ndzimbou}\CDRorcid{0009-0009-5626-3305}}
\address{Univ. Limoges, LABCiS, UR 22722, F-87000 Limoges, France}

\author{\firstname{Rayan} \lastname{Chkair}\CDRorcid{0009-0000-1530-6676}}
\addressSameAs{1}{Univ. Limoges, LABCiS, UR 22722, F-87000 Limoges, France}

\author{\firstname{Gautier Mark Arthur} \lastname{Ndong Ntoutoume}}
\addressSameAs{1}{Univ. Limoges, LABCiS, UR 22722, F-87000 Limoges, France}

\author{\firstname{Mona} \lastname{Diab-Assaf}}
\address{Doctoral School of Sciences and Technology, Lebanese
University, Hadath El Jebbeh, Beyrouth 21219, Lebanon}

\author{\firstname{Guillaume} \lastname{Chemin}\CDRorcid{0000-0003-0004-6618}}
\addressSameAs{1}{Univ. Limoges, LABCiS, UR 22722, F-87000 Limoges, France}

\author{\firstname{Bertrand} \lastname{Liagre}\CDRorcid{0000-0003-4148-2598}}
\addressSameAs{1}{Univ. Limoges, LABCiS, UR 22722, F-87000 Limoges, France}

\author{\firstname{Fr\'{e}d\'{e}rique} \lastname{Br\'{e}gier}\CDRorcid{0000-0002-9880-7616}}
\addressSameAs{1}{Univ. Limoges, LABCiS, UR 22722, F-87000 Limoges, France}

\author{\firstname{Vincent} \lastname{Sol}\CDRorcid{0000-0003-0175-0156}\IsCorresp}
\addressSameAs{1}{Univ. Limoges, LABCiS, UR 22722, F-87000 Limoges, France}
\email[V. Sol]{vincent.sol@unilim.fr}

\keywords{\kwd{Purpurinimide}
\kwd{Photosensitizers}
\kwd{Cellulose nanocrystals}
\kwd{Photodynamic therapy (PDT)}
\kwd{Colorectal cancer}}

\altkeywords{\kwd{Purpurinimide}
\kwd{Photosensibilisateurs}
\kwd{Nanocristaux de cellulose}
\kwd{Th\'{e}rapie photodynamique (PDT)}
\kwd{Cancer colorectal}}

\begin{abstract}
Starting from purpurin-18, a stable purpurin-18 imide derivative with
intensive absorption in the near infrared has been synthesized. The
opening of the exocycle anhydride by a primary alkylamine bearing an
adamantyl group, allows after cyclization the formation of an imide
function. This reaction led to the purpurinimide adamantane derivative
(PIA) which enables the inclusion of a photosensitizer in the cavity of
cationic cyclodextrin coupled to cellulose nanocrystals
(CNCs/\tralicstex{β-CD\textsuperscript{+}}{ ${\upbeta}$-CD$^{+}$}). Preliminary  {in vitro} results
showed that \tralicstex{CNCs/β}{$\mathrm{CNCs}{/}{\upbeta}$}-CD\tralicstex{\textsuperscript{+}}{$^{+}$} complexes improved
internalization of photosensitizers into HCT116 and HT-29 colorectal
cancer cell lines. Moreover, MTT (3-{[}4,5-dimethylthiazol-2-yl{]}-2,5
diphenyl tetrazolium bromide) assay of CNCs/\tralicstex{β-CD\textsuperscript{+}/PIA}{${\upbeta}$-CD$^{+}/\mathrm{PIA}$}
complexes showed significant dose-dependent phototoxicity after
illumination with red light, with IC\textsubscript{50} values of 2.5 and 8.7 nM,
for HT 116 and HT-29, respectively, and very low dark toxicity. The
current results suggest that these nanoparticle/photosensitizer
complexes are promising and useful tools for photodynamic therapy
(PDT).
\end{abstract}

\begin{altabstract}
\`A partir de la purpurine-18, un d\'eriv\'e stable de la
purpurine-18 imide avec une absorption intensive dans le proche
infrarouge a \'et\'e synth\'etis\'e. L'ouverture de l'anhydride
exocyclique par une alkylamine primaire portant un groupement
adamantyle, permet apr\`{e}s cyclisation intramol\'eculaire de
cr\'eer une fonction imide. Cette r\'eaction a conduit au
d\'eriv\'e PurpurinImide Adamantane (PIA) qui permet l'inclusion
d'un photosensibilisateur dans la cavit\'e de la cyclodextrine
cationique coupl\'ee aux nanocristaux de cellulose
(CNCs/\tralicstex{β-CD\textsuperscript{+}}{ ${\upbeta}$-CD$^{+}$}). Des r\'esultats pr\'eliminaires
\textit{in vitro} ont montr\'e que les complexes
CNCs/\tralicstex{β-CD\textsuperscript{+}}{ ${\upbeta}$-CD$^{+}$} am\'elioraient l'internalisation des
photosensibilisateurs dans les lign\'ees cellulaires de cancer
colorectal HCT116 et HT-29. En outre, le test MTT
(3-[4,5-dimethylthiazol-2-yl{]}-2,5 diphenyl tetrazolium bromide)
des complexes CNCs/\tralicstex{β-CD\textsuperscript{+}}{ ${\upbeta}$-CD$^{+}$}/PIA a montr\'e une
phototoxicit\'e dose-d\'ependante significative apr\`{e}s
illumination \`{a} la lumi\`{e}re rouge, avec des IC$_{50}$ de 2,5 et
8,7 nM, pour HT 116 et HT-29 respectivement, et une tr\`{e}s faible
toxicit\'e \`{a} l'obscurit\'e. Les r\'esultats actuels
sugg\`{e}rent que ces complexes nanoparticules/photosensibilisateur
sont des outils prometteurs et utiles pour la th\'erapie
photodynamique (PDT).
\end{altabstract}

\shortrunauthors

\thanks{Campus France, Agence Nationale des Bourses du Gabon, Ligue
Nationale contre le Cancer (CD 87 and CD 23), Conseil R\'egional de
Nouvelle Aquitaine}

\maketitle

\twocolumngrid

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\let\rmmu\upmu

\section{Introduction}

Colorectal cancer is the second most deadly cancer, accounting for
9.4\% of the nearly 10 million worldwide cancer deaths in 2020~\cite{1},
and it remains a major public health problem. Surgery is generally the
main option for treating colorectal cancer, often preceded and/or
followed by chemotherapy or radiotherapy. These traditional therapies
are often accompanied by myelosuppression, multidrug \mbox{resistance}, and
numerous side effects linked to damages to healthy cells. Today
photodynamic therapy (PDT) is a promising alternative treatment due to
a series of advantages such as fewer side effects and toxicity, and PDT
does not induce intrinsic or acquired resistance mechanisms~\cite{2}.
This therapeutical approach requires the presence of molecular oxygen,
the use of a photosensitizer (PS), and irradiation with light of
appropriate wavelength. Upon irradiation, the PS is excited to a
higher-energy state from which, by energy or electron transfer to
ground state/air oxygen ($^{3}\mathrm{O}_{2}$), it generates singlet
oxygen ($^{1}\mathrm{O}_{2}$) and other reactive oxygen species (ROS)
which induce multiple damage to the tumor cell~\cite{3,4}. Ideally, PSs
should be characterized by a high $^{1}\mathrm{O}_{2}$ quantum yield
and would not exhibit cytotoxicity in the dark; in addition, they must
be able to absorb light in the near-infrared (NIR) spectral region
since {NIR light within the phototherapeutic window
(${\lambda}=650$--850~nm) can penetrate rather deeply into tissues}.
Another key point to avoid unwanted side effects is the preferential
accumulation of PS in tumors~\cite{5,6}. Thus, one approach to target
solid tumors is to exploit the enhanced permeability and retention
(EPR) effect~\cite{7}. Over the past decade a large number of
nanoparticle-based PS delivery systems have been developed~\cite{8,9}.
These nanocarriers include silica-\cite{10}, gold-\cite{11}, and
natural polymer-based nanoparticles~\cite{12}. Polysaccharides have
been widely explored for the design and development of nanocarriers due
to their numerous advantages, including high water solubility, good
biocompatibility, low toxicity, biodegradability and abundance of
sources. We have recently reported the synthesis of nanoparticles
consisting of covalently bonded porphyrins\cite{13} or pheophorbide
\textit{a} to modified xylan~\cite{14}. These nanoparticles exhibited a
dose-dependent phototoxicity only when irradiated with red light but
their aggregation tendency, leading to a reduction in photodynamic
efficiency, is a major drawback. To overcome this issue, nanoconjugates
consisting of negatively charged cellulose nanocrystals (CNCs) and
cationic ${\upbeta}$-cyclodextrin were developed to encapsulate
protoporphyrin IX derivatives bearing an adamantane group~\cite{15}.
The  {in vitro} evaluation of this nanoparticle-based PS against
HT-29 colorectal cancer cells showed a clear increase in efficacy
compared with the free PS. This nanoplatform appears to be a very
promising tool for the vectorization of PSs. Purpunin-18 (Pp-18)
possesses a strong absorption band in the red region (702~nm in
CHCl$_{3}$), making it a potential PS for PDT, as red light penetrates
deeply into animal tissues~\cite{16}. Unfortunately, direct use of
Pp-18 as PS is limited, due to the presence of a cyclic anhydride,
which readily opens after alkaline treatment or in biological media to
form chlorin p6 with a hypsochromic shift of the Q absorption band
around 662--665~nm~\cite{17,18,19}. In addition, the high
hydrophobicity of Pp-18 leads to its aggregation at physiological pH
and reduces its bioavaibility~\cite{20,21}. To overcome these issues,
it has been shown that it is possible to generate more \mbox{stable} PSs such
as purpurinimides with a bathochromic red-shift and excellent 
{in vitro} and {in vivo} PDT efficacy~\cite{22,23}. CNCs
were obtained by sulfuric acid hydrolysis of microcrystalline
cellulose, which removed amorphous regions and introduced negatively
charged sulfate groups that cause electrostatic repulsion forces
between suspended particles~\cite{24,25}.  These negative charges
promote the addition, via electrostatic coupling, of positively charged
${\upbeta}$-CycloDextrin (${\upbeta}$-CD$^{+}$) previously
functionalized with glycidyltrimethyl ammonium chloride
(GTAC)~\cite{26}.

In this work, to obtain a more stable purpurinimide PS than Pp-18 with
red-shift light absorption, we transformed the Pp-18 anhydride ring
into a cyclic imide to which an adamantyl group is attached. Indeed the
adamantyl moiety, which is a spherical entity with a diameter of 
6.5~\AA, perfectly fits into the cavity diameter of ${\upbeta}$-CD
(6.0--7.0~\AA)~\cite{27}. Moreover, ${\upbeta}$-CD/adamantane
complexes have found several important applications in supramolecular
chemistry and biomedical applications due to their high 
stability~\cite{28}. In a second step, a complex between the newly
synthesized PS and cellulose--cyclodextrin nanoparticles was formed.
Finally, using PDT, the antiproliferative activity of this conjugate
against HCT116 and HT-29 colorectal cancer cell lines was
tested~\cite{27}.

\section{Results and discussion}\label{sec2}

\subsection{Synthesis and characterization of the purpurinimide
adamantane derivative (PIA)}\label{sec21}

 The hemisynthesis of Pp-18 from chlorophyll \textit{a} was first
performed following a previously reported  procedure~\cite{29}.
Pp-18 was reacted with ${N}$-(2-amino\-ethyl)adamantyl-1-carboxamide
hydrochloride in the presence of diisopropylamine (DIPEA) to produce a
mixture of two chlorin-\textit{p6}  derivatives (not isolated) via the
opening of the Pp-18 anhydride exocycle. After solvent evaporation and
treatment of the crude reaction mixture with acetic anhydride, an
intramolecular cyclization of the two chlorin p6 derivatives took
place. After purification by preparative thin layer chromatography, the
purpurinimide adamantane derivative (PIA) was obtained in 32\% yield
(Scheme~\ref{sch1}). The chemical structure of PIA was established by 
IR, $^{1}$H, $^{13}$C NMR and HRMS \mbox{(Figures~S5--S10).}

\begin{scheme*}
\includegraphics{sc01}
{\vspace*{5pt}}
\caption{\label{sch1}Synthesis of the purpurinimide adamantane
derivative (PIA).}
{\vspace*{-3pt}}
\end{scheme*}

UV--Visible monitoring of the reaction showed that conversion of the
anhydride of purpurin-18 to the cyclic imide of PIA was accompanied by
a shift in the absorption maximum from 702 to 712~nm in CHCl$_{3}$
(Figure~\ref{fig1}). 

\begin{figure}
\includegraphics{fig01}
\caption{\label{fig1}UV--Visible absorption spectra of Pp-18 and PIA in
CHCl$_{3}$.}
\end{figure}

\subsection{Synthesis and characterization of cellulose \break
nanocrystals/cationic ${\upbeta}$-cyclodextrin/purp\-urinimide
adamantane derivative (CNCs/${\upbeta}$-CD$^{+}$/PIA)
complexes}\label{sec22}

\begin{scheme*}
{\vspace*{3pt}}
\includegraphics{sc02}
{\vspace*{9pt}}
\caption{\label{sch2}Synthesis of CNCs/{${\upbeta}$}-CD$^{+}$/PIA
complexes: (a)~synthesis of CNCs, (b)~synthesis of ${\upbeta}$-CD$^{+}$,
(c)~inclusion of PIA into CNCs/${\upbeta}$-CD$^{+}$ complexes.}
{\vspace*{2pt}}
\end{scheme*}

\begin{table*}[t!]%tab1
\caption{\label{tab1}Size distribution, PDI and zeta potential
(${\zeta}$) of CNCs, CNCs/$\upbeta$-CD$^{+}$ and
CNCs/${\upbeta}$-CD$^{+}$/PIA}
\begin{tabular}{cccc}
\thead
 & CNCs & CNCs/$\upbeta$-CD$^+$ & CNCs/$\upbeta$-CD$^+$/PIA  \\
\endthead
Size (nm) & 165.5 ${\pm}$ 2.1 & 438.97 ${\pm}$ 13 & - \\
PDI & 0.296 & 0.196 & - \\
${\zeta}$ (mV) & ${-}$51.8 ${\pm}$ 1.5 & 29.77 ${\pm}$ 2.67 & 19.97 ${\pm}$ 0.15
\botline
\end{tabular}
{\vspace*{1pt}}
\end{table*}


After sulfuric acid hydrolysis of microcrystalline cellulose, cellulose
nanocrystals (CNCs) were obtained in 29\% yield according to previously
described methods (Scheme~\ref{sch2}a)~\cite{24,25}. The high zeta
potential value of ${-}$51.8  ${\pm}$ 1.5 mV exhibited by CNCs 
(at~1.52/1000~w/w, Figure~S1a) confirmed the presence of negatively charged
sulfate groups on the surface of CNCs~\cite{24,30}. This absolute
value, higher than 30 mV, indicated a high degree of stability of the
CNCs in aqueous suspension~\cite{31,32}.  FTIR spectra of CNCs showed
peaks at 1205~cm$^{-1}$ and 817~cm$^{-1}$ that were assigned to sulfate
esters groups on the CNCs surface, resulting from hydrolysis with
sulfuric acid~\cite{33,34}  (Figure~S10). In parallel, positive charges
were introduced on ${\upbeta}$-cyclodextrin by functionalization with a
large excess of glycidyltrimethylammonium chloride (GTAC) in alkaline
aqueous medium (Scheme~\ref{sch2}b) according to Chisholm and
Wenzel~\cite{35}. Characterization of the final product is in agreement
with the literature data (Figures~S2 and S3)~\cite{26,35}.  Finally,
mixing ${\upbeta}$-CD$^{+}$  with CNCs resulted in the formation of
CNCs/${\upbeta}$-CD$^{+}$ complexes via electrostatic interactions
(Scheme~\ref{sch2})~\cite{26}.  These complexes were then separated
from free ${\upbeta}$-CD$^{+}$ by centrifugation for 10~min at 
13,000~rpm. To see how the characteristics have evolved after coupling, zeta
potential measurements were conducted on CNCs/${\upbeta}$-CD$^{+}$
without dilution to determine their overall surface charge; their
hydrodynamic size was measured on a 21-fold dilution (Table~\ref{tab1},
Figure~S1b). A significant increase in zeta potential 
from ${-}51.8\pm 1.5$~mV for CNCs to 29.77 ${\pm}$ 2.67 mV for
CNCs/${\upbeta}$-CD$^{+}$ was observed (Table~\ref{tab1}). The latter value,
close to 30 mV, indicated that the dispersion was stable~\cite{32}.
Analyzed by dynamic light scattering (DLS), this suspension showed an
apparent hydrodynamic particle size of 438.97 ${\pm}$ 13~nm compared to
165.5 ${\pm}$ 2.1~nm for CNCs (Figure~S1, Table~\ref{tab1}). The FTIR spectrum
of CNCs/${\upbeta}$-CD$^{+}$ differs from that of CNCs, with the
appearance of a new peak at 1478~cm$^{-1}$ attributed to the
--N$^{+}$--CH$_{3}$ group of ${\upbeta}$-CD$^{+}$, indicating the
presence of ${\upbeta}$-CD$^{+}$ on the CNCs surface (FTIR spectrum of
CNCs/${\upbeta}$-CD$^{+}$/PIA, Figure~S10). These data are a good
indication that positively charged ${\upbeta}$-CD$^{+}$ was
successfully complexed with CNCs. PIA was loaded into the cavity of the
complexed ${\upbeta}$-CD$^{+}$  (Scheme~\ref{sch2}c) thanks to the
hydrophobic nature of the adamantane group. Briefly, a solution of PIA
in acetone was mixed with aqueous CNCs/${\upbeta}$-CD$^{+}$  under
ultrasound and magnetic stirring. At the end of the reaction, complexes
were purified by centrifugation for 10~min at 10,000~rpm. A decrease in
zeta potential from 29.77 ${\pm}$ 2.67 mV for CNCs/${\upbeta}$-CD$^{+}$
to $19.97 \pm 0.15$ for CNCs/${\upbeta}$-CD$^{+}$/PIA was observed
(Table~\ref{tab1}). This slight decrease could be explained by the carboxylate
group of PIA. The new value around 20 mV indicated that the dispersion
was relatively to moderately stable~\cite{32}.  The successful
inclusion of PIA in complexes was also confirmed by FT-IR spectroscopy
(Figure~S10) with peaks at 1724~cm$^{-1}$,  1679~cm$^{-1}$,
1641~cm$^{-1}$, 1600~cm$^{-1}$, due to, respectively, vibrations of
C${=}$O, C${=}$O (amide bond), N--H (amide function), C${=}$C. The
concentration of PIA in CNCs/${\upbeta}$-CD$^{+}$ complexes was
determined by UV--Vis spectroscopy around $3.65\times 10^{-4}$~mol/L.

\subsection{In vitro biological studies}\label{sec23}

Uptake of PIA and CNCs/${\upbeta}$-CD$^{+}$/PIA complexes by HCT116 and
HT-29 cells was studied by fluorescence confocal microscopy
(Figure~\ref{fig2}).

\begin{figure*}
\includegraphics{fig02}
\caption{\label{fig2}Cellular internalization of PIA and
CNCs/${\upbeta}$-CD$^{+}$/PIA in human colorectal cancer cell lines HCT116
(A) and HT-29 (B). Cells were grown for 24 h in chamber slides coated
with type I collagen and acetic acid. Cells were then treated with PS
at IC$_{50}$ values. After 24 h, the red fluorescence of PS was
assessed by confocal microscopy. ImageJ software (version 1.52p) was
used to determine colocalization. White scale bar ${=}$ 20
$\rmmu$m.}
\end{figure*}

Cellular accumulation was observed after 24 h incubation with PIA and
CNCs/${\upbeta}$-CD$^{+}$/PIA. Incubations in presence of
CNCs/${\upbeta}$-CD$^{+}$/PIA led to an increased accumulation of PSs,
regardless of the cell line. These results demonstrate the important
role played by CNCs/${\upbeta}$-CD$^{+}$ in the cellular uptake of the
PS and are in agreement with our previous  results~\cite{26}.
Photodynamic activities of PIA alone or CNCs/${\upbeta}$-CD$^{+}$/PIA
complexes were {evaluated} in HCT116 and HT-29 colorectal cancer cell
lines {using} MTT (3-{[}4,5-dimethylthiazol-2-yl{]}-2,5 diphenyl
tetrazolium bromide) assays (Figure~\ref{fig3}). The cancer cell lines
were incubated with different concentrations of
CNCs/${\upbeta}$-CD$^{+}$/PIA complexes as aqueous solution or PIA
alone in DMSO at 0.13, 1.3, 6.5, 13, 26 and 39~nM for 24 h. Cells were
then illuminated for 7~min at 650~nm (light dose 
30~J/cm$^{2}$) and then incubated in the dark for 24 and 48~h. 
Within the concentration range tested, we observed a low
cytotoxicity in the dark. Under irradiation, the cytotoxicities of PIA
alone or CNCs/${\upbeta}$-CD$^{+}$/PIA were much stronger in comparison
with the control tests in\break the dark.

\begin{figure*}
\includegraphics{fig03}
\caption{\label{fig3}Cell viability in HCT116 and HT-29 colorectal
cancer cells as an index of photocytotoxicity (IR) (light, total light
dose 30~J/cm$^{-2}$; irradiation 7~min, ${\lambda}= 650$~nm) and
dark cytotoxicity (NIR) after 24 h incubation times of PIA alone or
CNCs/${\upbeta}$-CD$^{+}$/PIA complexes at concentrations of 0.13 to 39~nM.
The percentage of cell viability was determined by MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay,
24 and 48 h after irradiation. Error bars represent the standard
deviation of three replicate experiments. ($h{\nu}=$ irradiated and
no $h{\nu} =$ non irradiated). Data are represented as mean ${\pm}$
SEM ($n = 3$).  $^{***}p < 0.001$ relative to the no $h\nu$ at 48~h.}
{\vspace*{-1pt}}
\end{figure*}

CNCs/${\upbeta}$-CD$^{+}$ complexes alone were shown to only slightly
reduce cancer cell proliferation after 48~h~\cite{26}; therefore it can
be concluded that the antiproliferative efficacy observed in these
experiments, on the two cancer cell lines, is mainly due to the
presence of PIA in the CNCs/${\upbeta}$-CD$^{+}$. In both cases,
whether PIA is alone or complexed, the IC$_{50}$ values after 48 h are
only slightly lower than those obtained after 24~h (Table~\ref{tab2}).


\begin{table*}[t!]%tab2
\caption{\label{tab2}IC$_{50}$ values of PIA and
CNCs/${\upbeta}$-CD$^{+}$/PIA obtained using MTT assay}
\fontsize{9.2}{10.5}\selectfont\tabcolsep=2pt
\begin{tabular}{ccccccccccc}
\thead
Cell Line  & \multicolumn{4}{c}{HCT116} & 
\parbox[t]{2.2cm}{\centering $P$ value (48~h relative to 24~h)}
  & \multicolumn{4}{c}{HT-29} & 
  \parbox[t]{2.2cm}{\centering $P$ value (48~h relative to 24~h)}\vspace*{2pt}\\
\endthead
\parbox{2.2cm}{\centering Incubation time after irradiation (h)}\raisebox{20pt}{}  & 
\multicolumn{2}{c}{24 h} & 
\multicolumn{2}{c}{48 h} &  & \multicolumn{2}{c}{24~h} & 
\multicolumn{2}{c}{48 h} &  \vspace*{5pt}\\
 & IC$_{50}^{a}$ & IC$_{50}^{b}$ & IC$_{50}^{a}$ & 
 IC$_{50}^{b}$ &  & IC$_{50}^{a}$ & IC$_{50}^{b}$ & 
 IC$_{50}^{a}$ & IC$_{50}^{b}$ & \vspace*{5pt}  \\
PIA  & 2.6 & - & 2 & - & 0.3118 (NS) & 8.7 & - & 3.1 & - & 0.0458 (*) \vspace*{5pt} \\
\parbox{2.2cm}{\centering CNCs/$\upbeta$-CD$^{+}$/PIA}\vspace*{10pt}  & 4.3 & 22.9 & 2.5 & 13.3 & 0.0856 (NS) & 10 & 53.2 & 8.7 & 46.2 & 0.0565 (NS) \\
\parbox{2.2cm}{\centering $P$ value (PIA relative to CNCs/$\upbeta$-CD$^{+}$/PIA)}  & 0.0306 (*) & - & 0.0876 (NS) & - & - & 0.0226 (*) & - & 0.0054 (**) & - &\vspace*{2pt}
\botline
\end{tabular}
\tabnote{$^{*}$Data are expressed as mean ${\pm}$ standard error of the
mean (SEM) of three experiments. The statistical significance of
results was evaluated using GraphPad Prism 5.0 by a two-tailed unpaired
Student's $t$-test, as $^{*}p< 0.05$ was considered statistically
significant. $^{*}p< 0.05$, $^{**}p< 0.01$ *NS: not significant.}
\tabnote{$^{a}$ nmol PIA/L. $^{b}~\rmmu$g CNCs/${\upbeta}$-CD$^{+}$/PIA/mL.}
\end{table*}

As shown in Figure~\ref{fig3}, a dose-dependent antiproliferative
effect was observed in both colon cancer cell lines but HT-29 cells
were more resistant than HCT116, as previously observed during 
{in vitro} assay using both cell lines~\cite{36,37}. After 48 h
photoirradiation, IC$_{50}$ values of 2 and 3.1~nM were determined for
HCT116 and HT-29, respectively. Encapsulation into
CNCs/${\upbeta}$-CD$^{+}$ appeared to slightly decrease the toxicity of
PIA. However, the IC$_{50}$ \mbox{values} remained in the nanomolar range,
with IC$_{50}$ values of 2.5 and 8.7~nM against HCT116 and HT-29,
respectively. The IC$_{50}$ values for the
CNCs/${\upbeta}$-CD$^{+}$/PIA complex of 13.3 and 46.2
$\rmmu$g/mL against HCT116 and HT-29, respectively, are
relatively low in comparison with a biotoxicity value of 250
$\rmmu$g/mL reported in the literature~\cite{38}.
Encapsulation of PIA in complexes proves to be a relevant choice to
solubilize PIA in aqueous media and will facilitate its selective
accumulation in the tumor tissues via enhanced permeation and retention
(EPR) effect. In comparison with previous work,
CNCs/${\upbeta}$-CD$^{+}$/PIA showed stronger photocytotoxicities than
two different protoporphyrin IX (PpIX) adamantane derivatives also
encapsulated into CNCs/${\upbeta}$-CD$^{+}$ (IC$_{50}$ values of 
1000~nM and 420~nM)~\cite{15}. PIA and its nanoformulation exhibited
excellent PDT efficacies against cancer cell lines.

\section{Conclusion}\label{sec3}

In this work, the synthesis of a new purpurin-18 imide derivative
bearing an adamantane moiety and the preparation of biocompatible
CNCs/${\upbeta}$-CD$^{+}$/PIA complexes for PDT anticancer therapy were
described. $^{1}$H NMR, HRMS, UV--Visible and FTIR \mbox{spectroscopies}
indicated that the purpurin-18 imide derivative and its inclusion
complexes were successfully obtained. The biological tests demonstrated
for the resulting complexes a very good solubility in aqueous media, an
increased uptake of hydrophobic PS by cancer cells, and excellent
photodynamic efficacy (IC$_{50}= 2.5$ and 8.7~nM against HCT116 and
HT-29 cell lines respectively, 48 h after irradiation). Based on these
results, we can suggest that PIA-loaded CNCs/${\upbeta}$-CD$^{+}$ is a
promising tool for PDT anticancer therapy. 

\section{Materials and methods}\label{sec4}

\subsection{Materials}\label{sec41}

For a list of materials, see Supplementary Information (SI).

\subsection{Methods}\label{sec42}

\subsubsection{Synthesis of
13$^1$,15$^1${-}${N}$-(2-[(1-Adamantylcar\-bonyl)amino]ethylcycloimide) 
chlorin p6 (purpurinimide adamantane derivative or PIA) 
(Figure~\myRef{fig4})}\label{sec421}

Purpurin-18 (90.8~mg, 0.1608~mmol, 1 equiv) and
${N}$-(2-aminoethyl)adamantyl-1-carboxamide hydrochloride (99.8~mg,
0.3856~mmol, 2.4 equiv) were dissolved in CH$_{2}$Cl$_{2}$ (10~mL)
under argon atmosphere. DIPEA (100 $\rmmu$L, 0.5730~mmol, 
3.6~equiv) was added and the resulting solution was stirred for 72 h at
room temperature; the color of the solution changed from purple to
green indicating the formation of the chlorin p6 derivative.
CH$_{2}$Cl$_{2}$ was evaporated and the residue was dissolved in
Ac$_{2}$O (3~mL) and stirred for 20~min; the color changed from green
to purple indicating the formation of the new imide cycle. The
resulting mixture was diluted with 5\% aqueous NaHCO$_{3}$ (90~mL) and
stirred for 4 h. The product was extracted with CH$_{2}$Cl$_{2}$,
washed with deionized H$_{2}$O, and dried over MgSO$_{4}$.
After evaporation, the purpurinimide was isolated by preparative thin
layer chromatography using CHCl$_{3}$/MeOH (95:5) as the eluent and
crystallized from CH$_{2}$Cl$_{2}$/petroleum ether/cyclohexane,
yielding 39.6~mg (32\%). 

\begin{figure}
\includegraphics{fig04}
{\vspace*{-2pt}}
\caption{\label{fig4}Structure and numbering of PIA.}
{\vspace*{-2pt}}
\end{figure}

CCM Rf (CHCl$_{3}$/MeOH 95:5) ${=}$ 0.17. FTIR/ATR, ${\nu}$/cm$^{-1}$:
3333, 2961, 2904, 2850, 1725, 1679, 1641, 1600, 1524. $^{1}$H NMR 
(500~MHz, CDCl$_{3}$): ${\delta}_{\mathrm{H}}$, ppm 9.54 (s, 1~H,
H10), 9.31 (s, 1~H, H5), 8.55 (s, 1~H, H$_2$0), 7.87 (dd, 1~H,
$J=17.0$ and 11.9~Hz, H3$^{1}$-CH), 6.45 (bs, 1~H, NH), 6.28 (d,
1~H, $J=17.7$~Hz, H3$^{2}$-CH$_{2}$), 6.17 (d, 1~H,
$J=11.4$~Hz, H3$^{2}$-CH$_{2}$), 5.69 (m, 1~H, H17), 4.84 (m, 1
H, a-CH$_{2}$), 4.60 (m, 1~H, b-CH$_{2}$), 4.48 (d, 1~H, $J=13.4$~Hz, 
a-CH$_{2}$), 4.32 (m, 1~H, H18), 3.76 (s, 3~H, 12-CH$_{3}$), 3.59
(q, 2~H, $J=7.3$~Hz, 8$^{1}$-CH$_{2}$), 3.42 (d, 1~H,
$J=13.1$~Hz, b-CH$_{2}$), 3.32 (s, 3~H, 2-CH$_{3}$), 3.14 (s, 3
H, 7-CH$_{3}$), 2.70 (m, 1~H, 17$^{1}$-CH$_{2}$), 2.15-1.92 (m, 3~H,
17$^{1}$-CH$_{2}$ and 17$^{2}$-CH$_{2}$), 1.82 (d, 3~H,
$J= 8.0$~Hz, 18-CH$_{3}$), 1.73 (m, 3~H, f-Ad), 1.66 (t, 3~H,
$J=7.5$~Hz, 8$^{2}$-CH$_{3}$), 1.53--1.38 (m, 12~H, e-Ad et
g-Ad), 1.10 and ${-}$0.10 (s, 2~H, NH). $^{13}$C NMR (125~MHz, CDCl$_{3}$):
${\delta}_{\mathrm{C}}$, ppm 179.8 (1C, Adamantane C${=}$O), 176.5
(1C, C16), 175.8 (1C, C19), 170.4 (1C, COOH), 167.7 (1C, cycloimide
C15'), 164.2 (1C, cycloimide C13'), 155.8 (1C, C6), 149.9 (1C, C9),
145.6 (1C, C8), 143.4 (1C, C1), 139.0 (1C, C12), 137.25 and 137.17 (2C,
C3, C14), 136.42 and 136.4 (1C, C7 and C4), 131.5 (2C, C2, C11), 128.6
(1C, C3$^{1}$), 123.3 (1C, C3$^{2}$), 115.1 (1, C13), 107.0 (1C, C10),
102.6 (1C, C5), 97.9 (1C, C15), 94.9 (1C, C20), 53.5 (1C, C17), 50.3
(1C, C18), 40.5 (1C, \mbox{C-Ad$_{d}$),} 38.64 (3C, CH$_{2}$-Ad),
38.56 (1C, NCH$_{2}$CH$_{2}$NH), 38.2 (1C, NCH$_{2}$CH$_{2}$NH), 36.2
(3C, CH$_{2}$-Ad), 32.2 (1C, C17$^{2}$-CH$_{2}$), 31.4 (1C,
C17$^{1}$-CH$_{2}$), 27.8 (3C, CH-Ad$_{\mathrm{f}}$), 23.7 (1C,
18-CH$_{3}$), 19.4 (1C, 8-CH$_{2}$), 17.4 (1C, 8-CH$_{3}$), 12.4 (1C,
12-CH$_{3}$), 11.9 (1C, 2-CH$_{3}$), 11.1 (1C, 7-CH$_{3}$). HRMS m/z:
[M $+$ H]$^{+}$ calc for C$_{46}$H$_{53}$N$_{6}$O$_{5}$ 769.4072; found
769.4080. UV-Visible in DMSO, $\lambda_{\max}$/nm ($\epsilon$,
10$^{-3}$~L$\cdot$mol$^{-1}{\cdot}$cm$^{-1}$): 420 (78), 512 (6.4), 
550~(14), 648 (5), 705 (28). UV-Visible in CHCl$_{3}$,
$\lambda_{\max}$/nm ($\epsilon$,
10$^{-3}$~L{$\cdot$}mol$^{-1}{\cdot}$cm$^{-1}$): 421 (120), 515 (6),
554 (23), 656 (8), 712 (44).

{\vspace*{1pt}}

\subsubsection{Production of cellulose nanocrystals (CNCs)}\label{sec422}

{\vspace*{1pt}}

Microcrystalline cellulose (MCC) was used as starting material to
obtain CNCs according to previously reported methods~\cite{24,25}.
Briefly, MCC (2~g) was mixed with H$_{2}$SO$_{4}$  (40~mL of 61 wt\%)
using a magnetic stirrer at 45~\textdegree C for 1.5 h. The suspension
was diluted with cold distilled water (150~mL, 4~\textdegree C) to stop
the reaction. Then, CNCs were pelleted by centrifugation for 10~min at
3700~rpm and the supernatant was discarded. The pellets were
resuspended in H$_{2}$O to eliminate excess acid and impurities and
then centrifuged for 10~min at 3700~rpm. The supernatant was
discarded. The pellets were resuspended in H$_{2}$O and centrifuged for
10~min at 3700~rpm. The supernatant containing the CNCs was dialyzed
against distilled H$_{2}$O using membrane tubing (6--8 KDa cutoff) for
5--6 days. Dialysis H$_{2}$O was changed two or three times each day.
After dialysis, the resulting solution was completely dispersed by
ultrasonic treatment in an ice bath using a Elmasonic S15 for 15~min to
give the desired colloidal suspension. After freeze-drying, the yield
was determined as 29\% using Equation~(\ref{eq1}). Hydrodynamic size,
polydispersity index, and zeta potential measurements were carried out
with a 0.152 wt\%\break suspension. 

FTIR/ATR ${\nu}$/cm$^{-1}$: 3337, 2900, 1650, 1425, 1369, 1333, 1317,
1281, 1205, 1161, 1111, 1033, 1057, 898, 817 and 776. 

The CNCs yield (\%) was calculated according to Equation~(\ref{eq1}):
{\vspace*{1pt}}
{\begin{equation}\label{eq1}
\mbox{CNCs yield }(\%) = \frac{M_{2}\times V_{2}}{M_{1}\times {V}_{1}}\times 100
\end{equation}}\unskip
where ${M}_{1}$ is the mass of MCC, ${M}_{2}$ the mass of the aliquot
of lyophilized CNCs, ${V}_{1}$ the total volume of collected
supernatant CNCs suspension, and ${V}_{2}$ the volume of the aliquot of
CNCs suspension to be lyophilized.

\subsubsection{Cationic $\upbeta$-cyclodextrin ($\upbeta$-CD$^+$\unskip) synthesis}\label{sec423}

Cationic ${\upbeta}$-cyclodextrin was prepared by reacting the native
${\upbeta}$-cyclodextrin (${\upbeta}$-CD) with
glycidyltrimethylammonium chloride (GTAC)~\cite{35}.  Distilled water
(20~mL) and ${\upbeta}$-CD (3.008 g, 2.65~mmol, 1 equiv) were
introduced in a round-bottomed flask (100~mL). The pH was adjusted to
12 with NaOH (2M). Then GTAC (14.5~mL, 108~mmol, 40.8 equiv) was added
to the mixture. The reaction mixture was stirred at 50~\textdegree C
for 5 days. The pH was adjusted to 6 with the addition of HCl (2M) and
stirring for 30~min at rt. The reaction mixture was dialyzed against
distilled water in a dialysis tubing (MWCO Molecular weight cutoff: 500
Da) for 27 h, distilled H$_{2}$O was replaced once. After dialysis, the
volume of the colorless liquid was brought up to 250~mL with distilled
H$_{2}$O. Aliquots of this solution were lyophilized and characterized.
Degree of substitution (DS) ${=}$ 2 was determined by integrating the
appropriate signals of the  $^{1}$H NMR spectrum.

FTIR/ATR (cm$^{-1}$): 3345, 2929, 1638, 1478, 1418, 1353, 1148, 1108
and 1024. $^{1}$H NMR (500~MHz, D$_{2}$O): ${\delta}$ (ppm) ${=}$ 5.33
(d, 1~H, $J = 5.75$~Hz), 4.48 (m, 2~H), 4.35--4.26 (m, 1~H), 4.08-3.81 (m,
6~H), 3.78--3.69 (m, 2~H), 3.64 (m, 4~H), 3.6--3.42 (m, 6~H), 3.38-3.33 (m,
2~H) and 3.27 (s, 18~H). 

\subsubsection{Preparation of CNCs/$\upbeta$-CD$^+$}\label{sec424}

The CNCs/${\upbeta}$-CD$^{+}$  complex was prepared as previously
described with slight modification~\cite{26}. ${\upbeta}$-CD$^{+}$
(40~mL, 1.42~mg/mL) was added slowly to CNCs (11~mL, 1.42~mg/mL) under
ultrasound in an ice bath for 15~min and stirred overnight at rt. The
mixture was then centrifuged at 13,000~rpm for 10~min to eliminate free
${\upbeta}$-CD$^{+}$. The precipitate was dispersed in distilled
H$_{2}$O (20~mL). The final solution was used to encapsulate PIA.

\subsubsection{Preparation of purpurinimide-loaded cellulose
nanocrystals/cationic-$\upbeta$-cyclodextrin
(CNCs/$\upbeta$-CD$^+$/PIA)}\label{sec425}

PIA (5~mg) was dissolved in acetone ($800~\rmmu\mathrm{L}$) and
introduced into aqueous CNCs/${\upbeta}$-CD$^{+}$ suspension (11~mL)
under ultrasound in an ice bath for 5~min. The solution was then
protected from light and stirred for 1.25 h in an ice bath. The complex
was centrifuged at 10 000~rpm for 10~min to eliminate free PIA
remaining in the supernatant. The pellet was redispersed in distilled
H$_{2}$O (11~mL) to form the final suspension of inclusion complex.
Aliquots (0.5~mL) of this final suspension were lyophilized and then
solubilized in DMSO to determine the loading amount of PIA in
CNCs/${\upbeta}$-CD$^{+}$/PIA, by measurement of absorbance at 420~nm.
PIA concentration in CNCs/${\upbeta}$-CD$^{+}$/PIA complex was 
$3.65 \times 10^{-4}$~mol/L. To determine the overall surface charge of
CNCs/${\upbeta}$-CD$^{+}$/PIA inclusion complex, the zeta potential of
the sample was measured without\break dilution.

FTIR/ATR (cm$^{-1}$): 3338, 2969, 2904, 2852, 1724, 1679, 1641, 1600,
1526, 1479, 1455, 1426, 1369, 1136, 1315, 1281, 1248, 1205, 1161, 1110,
1056, 1032, 900, 881 and 791.

\subsubsection{In vitro photoirradiation
Studies}\label{sec426}

The two human colorectal cancer cell lines used, HT-29 and HCT116, were
provided by the American Type Culture Collection (ATCC-LGC Standards,
Mosheim, France). Both cell lines were cultured in RPMI medium
supplemented with 10\% fetal bovine serum (FBS), L-glutamine (1\%),
penicillin (100~U/mL), and streptomycin ($100~\upmu$g/mL) (all reagents
purchased from Gibco BRL, Cergy-Pontoise, France). Cells were cultured
in a humidified atmosphere with 5\% CO$_{2}$ at  37~\textdegree C. For
all experiments, HCT116 cells were seeded at $1.2\times10^{4}$, and
HT-29 cells at $2.1 \times 10^{4}$~cells/cm$^{2}$.

The PDT protocol was performed as follows:  $4 \times10^{3}$ and
$7\times10^{3}$ cells/well for HCT116 and HT-29, respectively, were
seeded into 96-well plates. Cells were grown for 24 h before exposure
or not to 0.13, 1.3, 6.5, 13, 26 and 39~nM of PIA in DMSO or
CNCs/${\upbeta}$-CD$^{+}$/PIA (aqueous suspension). After 24 h, a red
phenol-free medium was added before irradiation or not at 650~nm 
(30~J/cm$^{2}$--7~min) delivered from the light source PDT TP-1
(Cosmedico Medizintechnik GmbH, Schwenningen, Germany). After
irradiation, the cells were incubated at 37~\textdegree C for 24 and 48
h for further analysis. 

Cancer cell viability was assessed by MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method
(from Sigma-Aldrich, Saint-Quentin-Fallavier, France). MTT (5 g/L) was
added 24 and 48~h after irradiation. After 3 h of incubation, DMSO 
(100~$\rmmu$L) was added to each well to dissolve the \mbox{formazan}
crystals, and absorbance was recorded at 550~nm using a microplate
reader (Thermoscientific MULTISKAN FC). The absorbance ${A}$ values
${\pm}$ standard deviation was the mean of three experiments. The
percent of viability was calculated using  Equation~(\ref{eq2}):
{\begin{equation}\label{eq2}
\mbox{Viability } (\%) =  \frac{A_{\mathrm{treated}}}{A_{\mathrm{control}}}\times 10 
\end{equation}}\unskip
where ${A}_{\mathrm{treated}}$ is the absorbance of the treated cells and
${A}_{\mathrm{control}}$ the absorbance of the untreated cells.

\subsubsection{Cellular internalization by confocal
microscopy}\label{sec427}

To confirm the cellular uptake, HCT116 and HT-29 cells were seeded for
24~h before treatment with PIA or CNCs/${\upbeta}$-CD$^{+}$/PIA complexes at
IC$_{50}$ concentrations in chamber slides (ibidi $\rmmu$-Slide 8 well
from Clinisciences, Martinsried, Germany) coated with a type I collagen
(3~mg/mL) and with acetic acid (20~mM) gel. Photos were taken using a
confocal microscope (laser Zeiss LSM 510 Meta---${\times}1000$).
Colocalization was assessed using the ImageJ software (version 1.52p).

\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.

\section*{Acknowledgements}

The authors acknowledge logistical and financial support from Campus
France, ``Agence Nationale des Bourses du Gabon'', ``Ligue Nationale
contre le Cancer (CD 87 and CD 23)'' and ``Conseil R\'egional de
Nouvelle Aquitaine''. They wish to thank Dr.\ Cyril Colas (ICOA-Univ.
Orl\'eans) for ESI-HRMS analyses and Dr.\ Yves Champavier from BISCEm
platform for NMR analysis. They are indebted to Dr.\ Michel Guilloton
for his help in manuscript editing.

\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-383-suppl.pdf}}

\printbibliography
\refinput{crchim20240660-reference.tex}

\end{document}
