# Size and phase preservation of amorphous calcium carbonate nanoparticles in aqueous media using different types of lignin for contrast-enhanced ultrasound imaging

Lauriane Chuzeville, Abderrahmane Aissani, Ahilan Manisekaran, Yves Fleming, Patrick Grysan, Servane Contal, Aline Chary, David Duday, Olivier Couture, Resmi Anand, et al.

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Lauriane Chuzeville, Abderrahmane Aissani, Ahilan Manisekaran, Yves Fleming, Patrick Grysan, et al.. Size and phase preservation of amorphous calcium carbonate nanoparticles in aqueous media using different types of lignin for contrast-enhanced ultrasound imaging. Journal of Colloid and Interface Science, 2024, 658, pp.584-596. 10.1016/j.jcis.2023.12.106. hal-04755761

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# Size and phase preservation of amorphous calcium carbonate nanoparticles in aqueous media using different types of lignin for contrast-enhanced ultrasound imaging

<figure><img src="image_3.png" /></figure>

Lauriane Chuzeville<sup>a, b</sup>, Abderrahmane Aissani<sup>c</sup>, Ahilan Manisekaran<sup>a, b</sup>, Yves Fleming<sup>a</sup>, Patrick Grysan<sup>a</sup>, Servane Contal<sup>d</sup>, Aline Chary<sup>d</sup>, David Duday<sup>a</sup>, Olivier Couture<sup>c</sup>, Resmi Anand<sup>a</sup>, Jean-Sébastien Thomann<sup>a, *</sup>

## GRAPHICAL ABSTRACT

<figure><img src="image_4.png" /></figure>

## ARTICLE INFO

**Keywords:**

Calcium carbonate nanoparticles

Lignin

Core-shell particles

Contrast-enhanced ultrasound imaging

## ABSTRACT

**Hypothesis:** Calcium carbonate (CaCO₃) nanoparticles could have great potential for contrast-enhanced ultrasound imaging (CEUS) due to their gas-generating properties and sensitivity to physiological conditions. However, the use of nano CaCO₃ for biomedical applications requires the assistance of stabilizers to control the size and avoid the fast dissolution/recrystallization of the particles when exposed to aqueous conditions.

**Experiments:** Herein, we report the stabilization of nano CaCO₃ using lignin, and synthesized core-shell amorphous CaCO₃-lignin nanoparticles (LigCC NPs) with a diameter below 100 nm. We have then investigated the echogenicity of the LigCC NPs by monitoring the consequent generation of contrast in vitro for 90 min in linear and non-linear B-mode imaging.

<footnote><sup>a</sup> Materials Research and Technology (MRT), Luxembourg Institute of Science and Technology, 5 Avenue des Hauts Fourneaux, Esch/Alzette L-4362, Luxembourg</footnote>

<footnote><sup>b</sup> University of Luxembourg, Department of Physics & Materials Science, 162a Avenue de la Faiencerie, 1511 Luxembourg city, Luxembourg</footnote>

<footnote><sup>c</sup> Sorbonne Université, CNRS, INSERM, Laboratoire d'Imagerie Biomédicale, 15 rue de l'école de médecine, 75006 Paris, France</footnote>

<footnote><sup>d</sup> Environmental Research and Innovation (ERIN) Department, Luxembourg Institute of Science and Technology, 5 Avenue des Hauts Fourneaux, Esch/Alzette L-4362, Luxembourg</footnote>

<footnote>* Corresponding author at: Materials Research and Technology (MRT), Luxembourg Institute of Science and Technology, 5 Avenue des Hauts Fourneaux, Esch/Alzette L-4362, Luxembourg.</footnote>

<footnote>E-mail address: jean-sebastien.thomann@list.lu (J.-S. Thomann).</footnote>
**Findings:** This research explores how lignin type and structure affect stabilization efficiency, lignin struc-
turation around CaCO₃ cores, and particle echogenicity. Interestingly, by employing lignin as the stabilizer, it
becomes possible to maintain the echogenic properties of CaCO₃, whereas the use of lipid coatings prevents the
production of signal generation in ultrasound imaging. This work opens new avenue for CEUS imaging of the
vascular and extravascular space using CaCO₃, as it highlights the potential to generate contrast for extended
durations at physiological pH by utilizing the amorphous phase of CaCO₃.

**1. Introduction**

Ultrasound imaging is currently the second most prescribed imaging
diagnosis modality, as a real-time, non-invasive and non-ionizing pro-
cedure [1]. However, image quality remains limited due to the poor
contrast difference between soft tissues, affecting the discrimination
between tumoral and healthy organs, for instance [2]. The development
of contrast-enhanced ultrasound imaging (CEUS) has broadened the
potential of the technique by improving the sensitivity, resolution, and
image quality [2-5]. Gas-filled microbubbles stabilized by a thin lipid
shell, with an average diameter generally between 1 and 10 µm [2,6-8],
are currently used as ultrasound contrast agents (UCAs). However, due
to their large size, these UCAs remain blood-pool agents confined in the
vascular system and are not able to accumulate into tumor or inflam-
mation sites through tissue extravasation [7,9,10]. Moreover, the cur-
rent UCAs suffer from a short half-life in the blood, nearly 3–5 min of
tumor imaging, which significantly impacts their circulation time and
the duration of contrast enhancement [9,11–13].

The development of solid nanoparticles seems a promising solution to overcome the drawbacks of current UCAs, owing to their slow biodegradability and controlled nanosize [7,14–16]. However, solid particles generate a poor contrast in ultrasound imaging due to their reduced impedance mismatch with the surrounding tissue and relative incompressibility [6].

An interesting approach is to use hybrid polymer-calcium carbonate nanoparticles to generate gaseous inclusion within the tumor after the extravasation of the particles [9,10,17–20]. The degradation of calcium carbonate (CaCO₃) continuously generates CO₂ nanobubbles coalescing into CO₂ microbubbles, known for their echogenic properties in ultrasound imaging [9,10,18,21]. Furthermore, CaCO₃ nanoparticles have been demonstrated as tumor growth inhibitors due to their ability to modulate the pH of tumor microenvironments locally [22].

One of the main disadvantages of CaCO₃ nanoparticles for biomed-
ical applications is their high instability and tendency to self-aggregate
in aqueous media [23,24]. Typically, an optimal nanoparticle size of 10
to 300 nm is necessary to enhance the permeability and retention (EPR)
effect for particle accumulation at tumor and inflammation sites
[25,26]. The stabilization of CaCO₃ nanoparticles for CEUS is generally
performed by processes inspired by biomineralization, using charged or
zwitterionic polymers to control the size of CaCO₃ particles
[11,19,27–30]. The resulting particles are hybrid polymer-CaCO₃ ma-
terials presenting a composite structure in which CaCO₃ is embedded in a
polymeric matrix [9,27,29,31]. The disadvantage of these composite
structures is the limited amount of CaCO₃ material in the particle due to
the presence of the polymer matrix, leading to the need for high particle
concentration to generate contrast (4—25 mg·mL⁻¹) [17,19,20].
Furthermore, there is currently no evaluation of the stabilizer influence
on particle echogenicity.

In this work, we report on the development of lignin-CaCO₃ core–
shell nanoparticles (NPs) using a core of amorphous CaCO₃. By using
CaCO₃-lignin core–shell structures, we aim to optimize the quantity of
CaCO₃ in a single particle and generate *in vitro* contrast enhancement at
lower concentrations. Among its various forms, amorphous calcium
carbonate (ACC) possesses the ability to release CO₂ in physiological and
tumor conditions and can be synthesized at the nanoscale with sizes of
less than 150 nm [32–35]. However, ACC is the most sensitive form of
CaCO₃ to water and requires a stabilization step to prevent its rapid

dissolution and crystallization when it comes into contact with biolog-
ical fluids. Lignin is an abundantly low-cost natural polyelectrolyte that
has recently been investigated by the scientific community for high-
value applications, including biomedical applications, due to its
unique chemical, mechanical, and biological properties [36–38].
Recently, lignin and lignin monomers have been reported for controlling
the size and phase of CaCO₃ microparticles [34,35,39]. However, the
synthesis of CaCO₃ NPs with sizes < 1 µm and the stabilization of the
amorphous form of CaCO₃ using lignin have not yet been described in
the literature.

We previously reported on the stabilization of ACC NPs in water by a
2-minute solvent-shifting process, utilizing the natural electrostatic af-
finity of negatively charged phospholipids for calcium ions [40]. Simi-
larly, lignin possesses an amphipathic structure rich in hydroxyl (–OH)
and carboxyl groups (–COOH) [41], known for their affinity with cal-
cium ions [42]. Moreover, the widespread availability and cost-
effectiveness of lignin make it a more suitable candidate for this appli-
cation. Consequently, we applied a similar solvent-shifting process for
the stabilization of CaCO₃ NPs in water using lignin.

One reason for the slow emergence of high value applications using
lignin arises from the complexity and variability of lignin chemical
structure [43–45]. Therefore, we selected four different lignins to
investigate the influence of the lignin source, chemical composition, and
physicochemical properties on the stabilization efficiency of ACC NPs in
water. A kraft lignin (KL), two soda lignins (SL24 and SL60), and an
organosolv lignin (OL) were used in this study. KL was extracted using
the kraft sulfur process whereas, soda lignins and OL were extracted by
sulfur-free processes [48,49]. The source and the extraction processes
are known to significantly influence the structure and properties of
lignins [46,47,50–52]. The chemical structure and average molecular
weights of lignins are characterized by phosphorus-31 nuclear magnetic
resonance (³¹P NMR) spectroscopy and gel permeation chromatography
(GPC), respectively.

The echogenic properties of the resulting LigCC NPs are investigated
and benchmarked against CaCO₃ nanoparticles stabilized by an egg-
sourced phospholipid [40]. The influence of the material used for the
external coating of ACC NPs on the echogenic properties of the final
particles is discussed. The cytotoxicity of the different particles is eval-
uated on several cell models as a preliminary validation for the
compatibility of the particles with intravenous injection.

**2. Materials and methods**

**2.1. Materials**

Calcium chloride hexahydrate (CaCl₂·6H₂O, grade: BioUltra ≥ 99.0 %), kraft lignin (alkali), chromium (III) acetylacetonate, pyridine (99.8 % anhydrous), poly-L-lysine 0.1 % solution (molecular weight 70,000–150,000) and ammonium hydrogen carbonate (NH₄HCO₃, BioUltra, ≥99.5%) were purchased from Sigma Aldrich (Overijse, and Diegem, Belgium). 2-Chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholine (95 %) and endo-N-hydroxy-5-norbornene-2,3-dicarboximide (97 %) were purchased from Sigma Aldrich (Gillingham, United Kingdom). Chloroform-d (99.80 %) was purchased from Deutero Gmbh (Kastellaun, Germany). The soda Protobind® lignins 2400 and 6000 were purchased from Tanavis AG (Rüschlikon, Switzerland). Organosolv lignin was purchased from Chemical Point UG (Oberhaching, Germany).
Absolute ethanol (analytical reagent grade) was purchased from VWR International (Fontenay-sous-Bois, France). All solvents and chemicals were used without further purification.

## 2.2. Preparation of the LigCC NPs

LigCC NPs were prepared by using a two-step procedure. First, ACC NPs were synthesized using a previously reported method [40]. Briefly, a $CaCl_2 \cdot 6H_2O$ solution of $4.4 \text{ mg.mL}^{-1}$ in ethanol and solid $NH_4HCO_3$ (4 vials) were placed in a vacuum chamber at 500 mbar pressure for 113 h. The particles were recovered by centrifugation at 30,000 g for 10 min. The particles were further dispersed in absolute ethanol using an ultrasonic bath for 5 min. The resulting solution was stored at 4 °C. The mass concentration of the ACC NPs colloidal suspension after synthesis was determined by drying 20 mL of ACC NPs suspension at room temperature under vacuum.

The stock solutions of different types of lignin-ethanol soluble fractions were prepared by mixing 1 g of lignin in 1 L of ethanol using an ultrasonic bath for 20 min. The solutions were left to rest for 12 h for the insoluble part of lignin to sediment. The supernatants of the different lignin solutions were collected. The mass concentration of each supernatant was determined using the procedure described above for the mass concentration of the ACC NPs suspension. Afterwards, all solutions were adjusted to 0.5 mg/mL by diluting with the proper volume of ethanol.

In the second step, ACC NPs and lignin at a mass ratio ACC NPs/Lignin of 0.75 in absolute ethanol was mixed using an ultrasonic bath for 5–20 min. Afterwards, the ethanol solution is added by a one pot addition in water under magnetic or mechanical stirring. The addition can be performed using a pipette followed by several aspiration cycles and 10 s high-rpm vortex mixing, or by simply pouring the solution in water under magnetic stirring (>500 rpm).

## 2.3. Hydrodynamic size and surface morphology of the LigCC NPs

ACC NPs and LigCC NPs hydrodynamic size distribution and zeta potential were characterized by dynamic light scattering (DLS) and micro-electrophoretic light scattering respectively, in absolute ethanol [53] for ACC NPs and in water at pH 7 for LigCC NPs, using a Zetasizer (Nano-ZS90, Malvern Instruments Ltd., UK) The nanoparticles suspensions were neither diluted nor filtered prior to the analysis. A minimum of three measurements are performed per sample. The refractive indexes were 1.681 for ACC NPs and 1.59 for LigCC NPs, with an absorbance of 0.

The size and morphology of ACC NPs and LigCC NPs were characterized by scanning electron microscopy (SEM). A droplet of nano-particles suspension was deposited on to copper tape or silicon wafer, and air-dried. ACC NPs were imaged immediately after drying to avoid crystallisation due to air moisture contamination. SEM images were performed with a Helios Nanolab 650 Scanning Electron Microscope (FEI, Eindhoven, Netherlands), operating at a voltage of 2–10 kV and current of 25–100 pA.

The core-shell structure of the particles was imaged by high-resolution scanning transmission electron microscopy (STEM). The images were performed with a Helios Nanolab 650 microscope (FEI, Eindhoven, Netherlands), at an acceleration voltage of 10 kV for the sample organosolv, and 30 kV for the other samples, and a current of 50 µA. All STEM images were performed in bright field mode.

## 2.4. X-ray diffraction analysis (XRD)

The measurements were performed on a Panalytical X'Pert Pro XRD equipped with a Cu anode in reflection geometry. In order to ensure that the same surface is analysed a programmable divergence slit as well as a programmable anti scatter slit were used. During the measurement, the samples were spun at 4 s per round.

**Sample preparation:** The samples to be analyzed were deposited

onto a zero-background holder. After covering the latter with a thin layer of Kapton® film, the sample holder was put into the spinner. The diffraction pattern was obtained following the subtraction of the nano-particle holding solution and the corresponding normalized solution on its own.

## 2.5. Nuclear magnetic resonance (NMR) spectroscopy

The ethanol soluble fraction of the different types of lignin was extracted as follows: 1 g of lignin was dissolved in 1 L of ethanol using an ultrasonic bath for 20 min. The solutions were allowed to rest for 12 h to allow the insoluble part of lignin to sediment. The supernatants of the different lignin solutions were collected and evaporated by a rotary evaporator (Heidolph, Germany). The dried ethanol soluble fractions of the lignin were then extracted from the balloon using water. After filtration through filter paper, the ethanol soluble fractions of different types of lignin were dried in an oven at 50 °C overnight. The resulting power was characterized by NMR.

The $^{31}$P nuclear magnetic resonance (NMR) spectroscopy analysis was performed using an AVANCE III HD spectrometer (Bruker, Fällanden, Switzerland) equipped with a 5 mm BBO-probe, operating at a proton frequency of 600 MHz. The protocol was adapted following the work reported by A. Adjaoud et al. [51]. 30 mg of lignin were dissolved in 700 µL of a solvent mixture containing deuterated chloroform (CDCl₃) and anhydrous pyridine in a 1/1.6 (v/v) ratio. This solvent mixture served also for the preparation of the relaxation agent (RA) tris(acetylacetonato)chromium(III) (14 µmol mL⁻¹) and the internal standard (IS) endo-N-hydroxy-5-norbornene-2,3-dicarboximide (108 µmol mL⁻¹) solutions. The derivatization reaction performed prior to the $^{31}$P NMR analysis, namely the phosphitylation, was performed by adding to the mixture of lignin 100 µL of the RA stock solution, 100 µL of the IS stock solution, and 100 µL of the phosphitylation reagent 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP). All $^{31}$P NMR spectra were calibrated on the water-derivatized product at 132.2 ppm. The baseline corrections were performed automatically. The phase corrections were performed manually. The chemical shifts used for the quantification were determined by the adapting the works of Adjaoud et al. [51], Pu et al. [54], Izaguirre et al. [55] and Balakshin et al. [56] to our spectra.

## 2.6. Gel permeation chromatography (GPC)

Gel permeation chromatography (GPC) analyses were performed on a 1200 Infinity gel permeation chromatograph (Agilent technologies, Craven Arms, United Kingdom) equipped with an integrated IR detector, a PL PolarGel-M column and a PL PolarGel-M guard column (Agilent technologies, Craven Arms, United Kingdom). Lignin samples were dissolved at a mass concentration of 3 $\text{mg.mL}^{-1}$ in a 0.1 M solution of Li($\text{CF}_3\text{SO}_2)_2\text{N}$ in anhydrous N, N-dimethyl formamide (DMF, 99.8 %). The resulting lignin solutions were filtered on PTFE 0.2 mm filters. 200 µL of filtered solution were injected into the PolarGel-M column (7.5 x 300 mm). The eluent was a 0.1 M solution of lithium bis(trifluoromethanesulfonyl)amide ($\text{Li(CF}_3\text{SO}_2)_2\text{N}$) in DMF at a flow rate of 1 mL.min⁻¹. The column was maintained at 50 °C during the analysis. Poly(methylmethacrylate) standards (EasiVial PMMA, Agilent Technologies, Mp = 550 – 1568000 g.mol⁻¹) were used to perform calibration. The number average molecular weight (Mn) and the weight average molecular weight (Mw) were obtained using the resulting calibration curve. The dispersity indexes (D'M) were calculated using the ratio Mw / Mn.

## 2.7. Thermogravimetric analysis

The approximate composition of lignin and calcium carbonate in LigCC NPs were determined by thermogravimetric analysis (TGA 2, Mettler Toledo GmbH). In brief, a suspension of LigCC NPs was lyophilized immediately after lignin coating to obtain dry powder.
Subsequently, 10 mg of the dried LigCC NP powder was placed in the pan and subjected to heating from 25 °C to 900 °C at a rate of 10 °C/min under an oxygen atmosphere.

## 2.8. Atomic force microscopy (AFM)

LigCC NPs suspension were deposited on poly-L-lysine coated silicon wafer and air-dried before analysis. The modification with poly(L-lysine) was used for the particle adsorption on the silicon wafer. Poly(L-lysine) served as anchoring polymer for the particles. The protocol was inspired from the work of Zou et al. [57]. A silicon wafer was cleaned by UV/ozone exposure for 20 min in an UVO Cleaner® (Jelight Company, Inc, Irvine, USA) and further immersed into a 0.1 % poly(L-lysine) solution for 90 min. After rinsing with deionized water and drying under nitrogen atmosphere, the wafer was cut to the adequate size. A drop of LigCC suspension was finally deposited on the surface and air-dried. The topographic and mechanical properties of the samples were investigated by the mean of an MFP3D Infinity AFM (Asylum Research, Santa Barbara, CA) working in bimodal Amplitude Modulation-Frequency Modulation (AM-FM) configuration. Topography is obtained by maintaining the amplitude of the first eigenmode (c. 265 KHz) of the cantilever constant. The Lost Tangent (LT) is derivated for the phase signal of the first resonance. The second eigenmode (c. 1.52 MHz) is used to detect frequency shift at the resonance via a Phase Lock Loop, poly(L-lysine), set at 90°. These frequency variations are proportional to the elastic modulus (EM) of the sample. The AFM tip (AC160TS, Olympus, Japan) cantilever stiffness and inverse optical sensitivity are calibrated prior to the measurements by a Sadler non-contact method. Prior to the calibration, the tip is scanned in contact mode over a titanium dioxide (TiO₂) surface to round up its apex, the increased curvature radius, despite the degraded lateral resolution is insuring more stable operation in EM characterization.

A Hertz punch model is chosen to reflect the hertzian interactions between the tip and the surface. The only free parameter to be determined is the AFM tip radius. This value is obtained by analysing a reference sample of a blend mix of polystyrene and low-density polyethylene. The value of the elastic modulus of the polystyrene phase is 2.7 GPa and the tip radius is then calculated. The reference sample is then replaced by the samples to be quantitatively measured. Between each serial of samples, the reference is checked again to adjust for any deviations of the system. Measurements are done under ambient conditions. Data are treated under MountainSPIP (DIGISURF, France). From the topography analysis the nanoparticles are individually segmented in two steps. First, particles are isolated from the background by applying a height threshold, then a watershed segmentation is applied to obtain individuals particles detection. The obtained mask is then applied to the EM and LT images to obtain the maximum height (assumed to be the diameter) and the corresponding average EM and LT values of the particle.

## 2.9. In vitro cell viability

### Cell culture

EAhy926 cells were cultivated in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Erembodegem, Belgium) plus 10 % Fetal Bovine Serum (FBS, Sigma, Overijse, Belgium), 1 % of penicillin-streptomycin (Sigma, Overijse, Belgium) and 25 mM of HEPES (Sigma, Overijse, Belgium) (further referred as EA.hy926 medium). Cells were passaged twice a week using TryPLE (Gibco, Erembodegem, Belgium) and medium was changed alternate days. THP-1 cells were cultivated in RPMI (Roswell Park Memorial Institute 1640, Gibco, Erembodegem, Belgium) basal medium plus 10 % FBS (Fetal Bovine Serum, Sigma, Overijse, Belgium), 1 % of penicillin-streptomycin and 50 µM of β-mercaptoethanol (Biorad, Temse, Belgium). Cells were passaged twice a week.

### Exposure conditions

Initial stock solutions of the different particles were prepared in

sterile water at a concentration of 0.4 mg.mL⁻¹ NPs. EAhy926 cells were seeded in 96-well plates (0.27 x 106 cells.mL⁻¹, 200 µL per well). After 24 h, the cells were exposed to several concentrations of NPs: 0, 0.31, 0.63, 1.25, 2.5, 5, 10, 30 and 40 µg.mL⁻¹ suspended in EA.hy926 medium with 10 % of sterile water with the NPs. THP-1 cells were seeded in 96-well plates (1 x 10⁶ cells.mL⁻¹, 200 µL per well) and directly exposed to several concentrations of NPs: 0, 0.31, 0.63, 1.25, 2.5, 5, 10, 30 and 40 µg.mL⁻¹ suspended in THP-1 medium with 10 % of sterile water with the NPs.

After 24 h of exposure to the NPs, cells were incubated in the dark at 37 °C with 400 µM of resazurin (Sigma, Overijse, Belgium) for 1 h. Fluorescence intensity was measured using a fluorescence microplate reader (Spark 20 M, Tecan, Mechelen, Belgium) with excitation at 530 nm and emission collected at 590 nm. Relative cell viability was calculated using the following equation.

$$Cellviability = \frac{(Fluorescenceintensityofthesample - Blank)}{(Fluorescenceintensityofthevehiclecontrol - Blank)} \times 100$$

## 2.10. In vitro ultrasound imaging

The particle echogenic properties were evaluated using a Verasonics 256 channel system equipped with the high-performance linear array transducer from General Electrics GEL3.12. The L3-12 probe is a broad-spectrum 2–11 MHz linear probe equipped with an array of 256 transducers. The right and left side of the probe can work separately on two different samples (SI. Fig. S1). The software MATLAB was used for programming the sequences and treat the data. The acquisitions were performed at a centered frequency of 5 MHz and a MI of 0.21, representative parameters of the clinical ultrasound imaging conditions. The ultrasound acquisition was performed using a standard 3-pulses amplitude modulation sequence with three angles (-3° 0° 3°).

# 3. Results and discussion

## 3.1. Lignin stabilization of ACC NPs in water

In this study, ACC NPs with a mean diameter of 71 ± 13 nm were prepared using ammonia diffusion method in absolute ethanol. This method, previously optimized in a previous work (Fig. 1a), allowed for the facile production of homogeneous ACC nanospheres without the need for additional additives. The ACC NPs exhibited a positive surface charge in ethanol, as indicated by a zeta potential value of + 26.0 mV [40], consistent with our previous findings.

To stabilize ACC NPs in aqueous media, lignin was employed using a solvent-shifting process. The ACC NPs and various lignin's were mixed in ethanol for 2 min before being injected into deionized water [40]. Only the lignin ethanol soluble fraction (ESF) was used for the process, as lignin is partially soluble in ethanol.

The resulting nanoparticles were referred to as lignin-coated ACC (LigCC) NPs, were dispersed in water for further studies. Various characterization techniques were employed, including XRD, DLS, SEM, and STEM (Fig. 1c-n). The XRD spectra were representative of the CaCO₃ amorphous phase in ethanol, with two broad peaks at around 25° and 40° (Fig. 1b) [40,58]. All lignins successfully preserved the amorphous phase of CaCO₃ after dispersion in water, despite their differences in source, structure, and properties. The preservation of the amorphous phase of CaCO₃ is significantly important as it is known to possess unique properties compared to its crystalline counterparts.

KL and SL24 LigCC NPs exhibited homogeneous nature in terms of size and morphology, with an average hydrodynamic diameter of 77 ± 20 nm and 68 ± 10 nm, respectively. On the other hand, SL60 and OL LigCC NPS exhibited a heterogeneous nature in hydrodynamic size, with three distinct populations identifiable through DLS analysis. The size heterogeneity as further confirmed by SEM and STEM imaging. Interestingly, SEM and STEM images showed the presence of aggregates
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Stabilization of ACC NPs in water by different lignins. (a) SEM image of ACC NPs in ethanol. (b) XRD spectrum of LigCC NPs in water. SEM images of (c) KL LigCC NPs (d) SL24 LigCC NPs (e) SL60 LigCC NPs and (f) OL LigCC NPs. Particle size diameter of (g) KL LigCC NPs (h) SL24 LigCC NPs (i) SL60 LigCC NPs (j) OL LigCC NPs measured using DLS. STEM images of (k) KL LigCC NPs (l) SL24 LigCC NPs (m) SL60 LigCC NPs and (n) OL LigCC NPs.</figcaption></figure>

composed of several ACC cores stabilized by a single lignin shell. In addition to this, SL60 and OL LigCC NPs also contained a population of non-aggregated particles with an average diameter of 79 ± 10 nm and 75 ± 10 nm, respectively.

For SL60 LigCC NPs, the dominant population size was centered on 100 nm. The formation of aggregates seemed limited compared to the stabilization of single particles (Fig. 1-i). For OL LigCC NPs, the dominant population size was centered on 330 nm, suggesting a majority of multi-core aggregates in solution rather than single-core particles (Fig. 1-j).

The core-shell structure of the particles was characterized by STEM imaging. The shells formed by SL24 and KL ESF appeared homogeneous in thickness with values ranging from 14 to 20 nm for both samples. Shells formed by SL60 and OL ESF were heterogeneous in thickness, with regions of high thickness up to ~ 20–30 nm and low thickness regions where the shell thickness could go down to ~ 3–5 nm.

The preservation of the ACC core in deionized water was confirmed by STEM imaging. Additionally, it is known that the dissolution of CaCO₃ in deionized water can increase the pH of the solution [22]. When monitoring the pH four weeks after the synthesis of LigCC NPs, no pH variations or alkalization of the solutions were observed. This observation confirms the stability of the LigCC particles and the preservation of the amorphous CaCO₃ core in deionized water.

The zeta potential of the particles was measured to confirm the structuration of lignin around the ACC cores and assess particle stability. Zeta potential measurements for the four LigCC NP samples exhibited similarities, with values ranging from -14.8 ± 1.2 mV to -22.7 ± 0.7 mV (Table 1). It is worth noting that the zeta potential of bare ACC NPs

<table><caption>**Table 1**
LigCC NPs zeta potential in deionized water at pH 7.</caption><thead><tr><th>Lignin (shell)</th><th>KL</th><th>SL24</th><th>SL60</th><th>OL</th></tr></thead><tbody><tr><td>Zeta potential(mV)</td><td>-15.6 ± 0.5</td><td>-14.8 ± 1.2</td><td>-18.7 ± 0.5</td><td>-22.7 ± 0.7</td></tr></tbody></table>

dispersed in ethanol was positive, with values of + 26.0 mV. The shift from positive to negative zeta potential values provides evidence for the formation of a lignin layer around the ACC cores [59].

Zeta potential can be a marker of stability for dispersions containing particles that are not sterically stabilized [60,61]. SL60 and OL LigCC NPs were the particles with the highest values of zeta potential, and as a consequence, the strongest electrostatic repulsion between the particles. Therefore, the aggregates evidenced by DLS and electron microscopy for SL60 and OL LigCC NPs were probably formed during the ethanol injection process, and not by particle aggregation in solution.

## 3.2. Lignin structural and chemical characterization

The effectiveness of lignin structuration around ACC NPs in water seemed to vary depending on the type of lignin used as a stabilizer. To understand the observed differences in particle stability and lignin structuration around the ACC cores, the different lignin ESF were characterized by gel permeation chromatography (GPC) and phosphorous NMR ($^{31}$P NMR).

The average molecular weights and dispersity index $D$-stroke (DM) of lignin ESF are presented in Fig. 2. The weight average molecular weight
<figure><img src="image_6.png" /></figure>

<table><caption>**Fig. 2.** GPC characterization of the different lignin ESF. **Top:** Chromatograms normalized to the unit. **Bottom:** Table summarizing the Mn (g.mol⁻¹), Mw (g.mol⁻¹) and DM values.</caption><thead><tr><td>Lignin ESF</td><td>M<sub>n</sub> (g.mol<sup>-1</sup>)</td><td>M<sub>w</sub> (g.mol<sup>-1</sup>)</td><td>D<sub>M</sub></td></tr></thead><tbody><tr><td>KL</td><td>460</td><td>1368</td><td>2.9</td></tr><tr><td>SL24</td><td>594</td><td>1827</td><td>3.1</td></tr><tr><td>SL60</td><td>731</td><td>2003</td><td>2.7</td></tr><tr><td>OL</td><td>510</td><td>4100</td><td>8</td></tr></tbody></table>

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Schematic representation of the possible impact of lignin Mw and conformation in ethanol on the formation of aggregates during the ethanol-injection process.</figcaption></figure>
Mw of OL and SL60 ESF was higher (>2000 g.mol-1) than the Mw of SL24 and KL ESF (<2000 g.mol-1). This may arise from the higher solubility of SL60 and OL in ethanol (77 % and 80 %, respectively) compared to KL and SL24 (54 % and 59 %, respectively) (Table S1, SI†).

In ethanol, lignin adopts a more extended and unfolded configuration, in opposition to the crumbled globular-like conformation adopted in water (Fig. 3) [62]. Therefore, in ethanol, longer lignin chains are more likely to interact with multiple ACC NPs and form aggregates after injection of the ethanol mix in water. This could explain the structuration of SL60 and OL lignin around several ACC cores. The Mw of OL ESF was at least 50 % higher than the other lignin ESF. However, the difference between SL24 ESF and SL60 ESF is inferior to 200 g.mol⁻¹ and probably not sufficient to explain the differences observed in term of ACC NPs stabilization. This conclusion suggests the influence of other factors on the structuration of lignin around ACC NPs.

At equivalent mass concentration, an ethanol solution of OL ESF contains significantly less individual polymer chains than the other lignin ESF solutions (Fig. 3). The structuration of OL ESF around individual particles of ACC NPs may not be favorable in terms of lignin molecular concentration. This hypothesis seems in accordance with the DLS characterization of OL LigCC NPs, which shows a majority of aggregates in solution compared to single particles (Fig. 1-j). The OL ESF dispersity index is higher compared to other lignins, which may contribute to the formation of different LigCC aggregate/particle sizes after injection in water.

The lignin content of -OH and -COOH functional groups is an important characteristic of the lignin structure. The amphipathic and polyelectrolyte nature of lignin is conferred by the combination of hydrophilic groups (-OH, -COOH) and hydrophobic regions (aromatic moieties) within its chemical structure [41]. Variations in the hydrophilic group content among different lignins can alter the ratio of hydrophilic to hydrophobic regions, which could affect the efficiency of the protection against water conferred by the lignin shell.

Moreover, hydroxyl and carboxyl groups are organic functional groups with an affinity for calcium ions [63,64]. Variation in the content

of -OH and -COOH groups from one lignin to another can influence lignins affinity for calcium ions and the surface of ACC NPs [63,64].

Fig. 4 displays the ³¹P NMR quantification of the -OH and -COOH functional groups of the different lignins. The chemical shifts used for the quantification were determined using the works of Pu et al. [54], Izaguirre et al. [55] and Balakshin et al. [56] (Fig. 4a, Table S2 SI†). We used the internal standard endo-N-hydroxy-5-norbornene-2,3-dicarboximide with a chemical shift of ~ 152 ppm, outside of the chemical shift range of lignin-derivatized hydroxyl groups [65].

A higher -OH content in lignin indicates a higher hydrophilicity and a higher affinity for calcium ions and ACC NPs [6364]. The quantification by ³¹P NMR shows that the SL24 and SL60 ESF possess a higher content of -OH groups than the KL and OL ESF. OL ESF possesses the lowest total -OH group content. The variations in the lignin ESF hydrophilicity and total -OH group content do does not seem to be correlated to the differences observed in the lignin structuration around ACC NPs.

The -COOH content of OL ESF is significantly higher than the ESF of other lignins (Fig. 4). Lignin -COOH groups possess pKa values of around 5.5 [6667]. LigCC particles were formed in deionized water at a neutral pH of 6.5–7. At these pH values, the majority of the -COOH groups were deprotonated under the form -COO-, with a net negative charge. As a consequence, a higher content of -COOH groups leads to a higher affinity of the lignin ESF for Ca²⁺ and for the positively charged surface of ACC NPs. However, the variations of -COOH content per g of lignin ESF do not seem to be correlated either to the lignin structuration around ACC NPs or to the formation of LigCC NPs aggregates.

The highest ratio of aliphatic:phenolic hydroxyl groups was observed for the SL24 ESF, followed by OL ESF, SL60 ESF, and KL ESF. The ratio of aliphatic:phenolic -OH could influence the lignin structuration around the ACC cores as the reactivity and accessibility of these two hydroxyl groups are different. However, variations in the aliphatic:phenolic -OH ratio do not seem to be correlated to the differences observed in the lignin structuration around single or multiple ACC cores.

TGA studies on LigCC NPs (kraft) revealed their degradation

<figure><img src="image_8.png" /><figcaption>Fig. 4. Quantitative ³¹P NMR characterization of the different lignins. The quantification was performed on the ethanol soluble fraction of the lignins. (a) ³¹P NMR spectra of the ESF of each lignin with the integration intervals used for the -OH group quantification. (b) -OH content in mmol.g⁻¹. (c) Aliphatic/phenolic -OH ratios.</figcaption></figure>
behavior, providing insights into the composition of organic (lignin) and inorganic content (calcium) (Fig. S9 SI†). The observed weight loss of LCC NPs up to 150 °C could be attributed to the evaporation of volatile solvents, such as water and ethanol. This is likely because the freeze-drying process couldn't eliminate ethanol, given its very low freezing temperature (–114 °C). The first transition from 150 °C to 450 °C is considered the first stage of degradation, attributed to the thermal degradation of lignin. The transition, occurring from 450 °C to 615 °C, is likely due to the decarbonation of CaCO₃. An approximate weight percentage of CaO is calculated to be 22 %. This low percentage of CaCO₃ indicates a denser coating of lignin and a porous structure of CaCO₃.

### 3.3. Evaluation of the particle viscoelastic properties

The viscoelastic properties of the LigCC NPs lignin shells were evaluated using atomic force microscopy (AFM), in rapid bimodal amplitude modulation-frequency modulation (AM-FM) (Fig. 5, Fig. S5 SI†). The LigCC particles were immobilized on poly(l-lysin) functionalized silicon wafer, as they are negatively charged, and dried. All LigCC shells present a Young's modulus (EM) value superior to 1 GPa with lost tangent values inferior to 0.5. These values suggest a limited flexibility and deformability of the lignin shells. The shell of OL LigCC NPs appears stiffer than other lignin shells, with average EM values above 1.5 GPa.

The particle heights of SL24, OL and SL60 LigCC NPs were smaller than that of KL LigCC NPs, suggesting a possible dissolution or degradation of the ACC core during the thermal drying process following the deposition of the particles on the poly(l-lysine) silicon wafer (Fig. 5). For OL and SL60 LigCC NPs, a degradation of the ACC core could arise from the shell thickness variations observed for these particles. Regions with a thin lignin shell between ~ 3 and 5 nm may be more fragile or more permeable to water. SL24 ESF was the most hydrophilic lignin fraction. The SL24 shell might provide a less efficient protection against water

<figure><img src="image_9.png" /></figure>

<table><caption>Fig. 5. Quantitative nanomechanical mapping of the LigCC NPs performed using AM-FM. (a) An illustrated example of a 2x2 micron zone is presented for KL LigCC NPs for each analyzed parameter. Scale bars: 500 nm. (b) Height and lost tangent values for all particles. (c) Particle EM values.</caption><thead><tr><td>Lignin type</td><td>Height (nm)</td><td>tanδ</td></tr></thead><tbody><tr><td>KL</td><td>55.7 ± 15</td><td>0.25 ± 0.09</td></tr><tr><td>SL24</td><td>36.5 ± 10</td><td>0.24 ± 0.08</td></tr><tr><td>SL60</td><td>31.1 ± 8</td><td>0.19 ± 0.09</td></tr><tr><td>OL</td><td>28.7 ± 8</td><td>0.24 ± 0.05</td></tr></tbody></table>

than other lignins.

### 3.4. *In vitro* evaluation of the particle echogenic properties

The *in vitro* echogenic performances of the particles were evaluated in linear and nonlinear B-mode imaging, in order to evidence the gas generation properties of the particles. The imaging was performed after dilution of the particles in a buffer of pH 7.4 or in a buffer of pH 5. The pH 7.4 buffer represents the pH conditions found in the vascularization, and the pH 5 buffer represents the acidic conditions found in the lysozyme, after particle extravasation and internalization in tumor cells, for instance. This information is essential for assessing the potential clinical and diagnostic applications of these nanoparticles and their behaviours within the diverse pH environments found in biological systems for instance in diseases such as cancer or inflammation, pH of the local tissue can deviate from the normal pH. By characterizing the behaviour of our nanoparticles in such conditions, we can gain insights into their potential as contrast agents for detecting or monitoring these pathological states as well.

The echogenic properties of the particles were monitored for 90 min at low particle concentration (0.02 mg.mL⁻¹) (Figs. 6 & 7). The echogenic properties of the particles were evaluated at 5 MHz with an amplitude representative of clinical diagnostic ultrasound imaging. As a control, the contrast of water and buffers were measured.

The influence of the material used for the stabilization of ACC NPs on the echogenic properties of the final particles is studied by comparing the echogenicity of LigCC NPs to the echogenicity of lipid-coated ACC NPs (LCC NPs). The LCC NPs were synthesized using the ACC cores used for the synthesis of LigCC NPs. The average dry size of the LCC NPs is ~ 70 nm, similar to the size of single core LigCC NPs.

#### 3.4.1. Linear B-mode imaging

The results obtained in linear B-mode imaging are presented in Fig. 6. The image intensity of a 5 mm-diameter disk in the center of each vial are displayed (Fig. 6a-b) and averaged for quantification (Fig. 6c-d). This region constitutes the region of interest (ROI).

LigCC particles appear to be echogenic materials at pH 7.4 and pH 5. The signal intensity in ROI increases rapidly after the injection of the LigCC particles, with the strongest backscattered intensities measured between 30 s and 3 min.

Variations in signal intensity and the signal attenuation profile are observed between the different particles, according to the lignin used for the shell. The best performances are obtained using KL LigCC NPs, at pH 7.4 and pH 5. While a contrast-enhancement remains visible after 90 min using KL LigCC NPs at both pH, the signal of SL24 and SL60 LigCC NPs was significantly reduced after 10 min.

The signal reflected from SL24 LigCC NPs drops below the signal of the other particles after 20 min for both pH conditions. This observation suggests a faster degradation of the ACC core using SL24, which could be the consequence of the higher hydrophilicity of SL24 ESF compared to other lignins [10].

Surprisingly, no contrast is observed in the ROI using LCC NPs for the whole duration of the monitoring. The use of negatively charged lipids as stabilizers seems to prevent the generation of contrast in ultrasound imaging at 5 MHz. In a previous study, the stabilization of ACC using negatively charged phospholipids was demonstrated to lead to the infiltration of lipids into the porosity of the particles. This specific structuration of the lipids may prevent the degradation of the ACC core and the consequent generation of echogenic CO₂ microbubbles. Additional studies would be required to determine whether the absence of contrast is due to the stabilization of CO₂ nanobubbles by the lipids, preventing the bubble coalescence, or to the absence of CO₂ release.

In light of these results, the choice of external coating for ACC NPs appears determinant to preserve the echogenic properties of the ACC core, and to optimize the signal intensity and attenuation in time.

To the best of our knowledge, there is no report on the echogenic
<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Linear backscattering intensity of the LigCC NPs and EPG-LCC NPS at pH 5 and pH 7.4. B-mode images of the different particles at 0.5, 3, 5, 10, 20, 30, 60, 90 min after exposition to (a) pH 7.4 or (b) pH 5. The image intensity of a 5 mm-diameter disk in the center of each vial are displayed. The surrounding vial and agar containers are masked. The images are displayed on a fixed 10 to 50 dB dynamic range with pure water as 0 dB reference. Relative intensities of the different particles in time after exposition to (c) pH 7.4 or (d) to pH 5. The intensities are representative of the signal in the ROI.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 7.** Nonlinear imaging with amplitude modulation of the different LigCC particles and EPG-LCC NPS at pH 5 and pH 7.4. B-mode images of the nonlinear signal backscattered from the different particles at 0.5, 3, 5, 10, 20, 30, 60, 90 min after exposition to (a) pH 7.4 or (b) pH 5 conditions. The center of each vial is displayed with a 0 to 40 dB dynamic range with respect to water. Nonlinear relative intensities of the different particles in time after exposition to (c) pH 7.4 or (d) to pH 5.</figcaption></figure>

properties of the amorphous form of CaCO₃. Studies on ACC NPs for drug delivery purposes reported that ACC particles were sensitive to aqueous conditions at physiological pH (pH 7.4). Our work suggests that ACC NPs coated with lignin remain sensitive to aqueous conditions at pH 7.4 and are echogenic materials suitable for pH conditions ranging from 7.4 to 5.

Previous studies evaluating the potential of hybrid polymer-CaCO₃ as UCAs reported a contrast enhancement triggered by the medium acidity, due to the selective degradation of CaCO₃ under mildly acidic conditions [10,17-20]. Our work suggests that the use of the amorphous

form of CaCO₃ allows the generation of contrast in ultrasound imaging in a larger pH range. These performances could be interesting for the contrast enhancement of the vascular and extravascular space.

LigCC NPs were stored and stable in deionized water, and were demonstrated to be stable in aqueous conditions at a neutral pH, with values ranging between 6 and 7. Therefore, the results suggest that the generation of contrast from the LigCC NPs at pH 7.4 is triggered by the ultrasound procedure.

Min et al. recently determined that the rapid dissolution of CaCO₃ at an acidic pH was linked to a stronger signal attenuation in ultrasound
imaging for CaCO₃ particles exposed to acidic conditions [10]. For LigCC NPs, a shorter time of contrast enhancement was noticed at pH 5 for SL60, SL24, and OL LigCC NPs, compared to the results obtained at pH 7.4. However, for KL LigCC NPs, the contrast enhancement could be observed for up to 90 min at pH 5, and the signal attenuation profile between the two pH conditions was similar.

The preservation of long contrastenhancement time at an acidic pH, usually hampered by the fast dissolution of CaCO₃, could be of great interest for the imaging of tumoral or inflammatory spaces.

### 3.4.2. Non-linear B-mode imaging

The generation of gas microbubbles was confirmed using nonlinear B-mode imaging with amplitude modulation. Indeed, the signal observed using linear ultrasound imaging included the signal resulting from the difference in acoustic impedance between different structures. The signal in linear imaging is not sufficient to confirm that the echogenicity of LigCC NPs arises from the generation of CO₂ microbubbles.

Nonlinear imaging specifically characterizes the nonlinear response of a structure or sample. The type of nonlinear imaging used in this study is generally sensitive to the microbubble contrast agents used in clinical practice.

A signal was recorded in nonlinear imaging for all LigCC NPs samples, but no contrast was observed using LCC NPs. The tendencies observed in nonlinear B-mode imaging in terms of signal intensity variation between the samples and of signal attenuation were similar to the results obtained with linear imaging B-mode imaging (Fig. 7).

The nonlinear imaging of LigCC NPs evidences the presence of nonlinear scatterers for all samples. The presence of a nonlinear signal could arise from the deformability of the LigCC particles, however, the evaluation of the LigCC NPs viscoelastic properties suggested stiff particles with low deformability potential. Therefore, the presence of a nonlinear signal backscattered from LigCC NPs samples is preferentially associated with the generation of CO₂ microbubbles.

To corroborate the conclusions regarding the generation of gas microbubbles, the signal variation over 600 ms of imaging was evaluated at each time interval for all particles (Fig. S6, SI†). The evaluation of the signal variation is used to reveal zones containing moving or dynamic echogenic objects, such as microbubbles. Significant signal variations can be recorded from disappearing or moving echogenic particles or bubbles.

The measurement of the signal variation produced by LigCC NPs at each time interval confirms the generation of CO₂ microbubbles. Significant variations were observable for each LigCC sample. The signal variations observed for EPG-LCC NPs were negligible, which suggests that the absence of a signal from the LCC NPs is due to the absence of the generation of CO₂ microbubbles.

For KL LigCC NPs, sparse zones of signal variation remain visible 90 min after injection of the particles in the different buffers, which corroborates the presence of CO₂ microbubbles after 90 min of ultrasound imaging.

For several particles, a second event of signal increase was observed between 20 min and 1 h (Fig. 6 & Fig. 7). This phenomenon could arise from a destabilization of the lignin shell after several acoustic stimulation, leading to an acceleration of the dissolution of the ACC core. It could also arise from a second event of nucleation and coalescence of CO₂ microbubbles due to cavitation phenomenon. Further investigations would be required to determine the origin of the second event of signal increase.

In clinical ultrasound imaging, the contrast enhancement induced by the injection of current UCAs such as SonoVue lasts only a few minutes due to the continual elimination of the microbubbles by the lungs and the liver. Although these effects are also expected to clear the CO₂ microbubbles generated by LigCC NPs, the progressive dissolution of the ACC core in contact with aqueous media may ensure a continuous production of CO₂ microbubbles and preserve the contrast for longer durations. This hypothesis was verified in vitro. The signal profile

generated by KL LigCC NPs was compared to the signal profile generated by SonoVue, the UCA currently clinically approved and used in Europe (Fig. S7 & Fig. S8, SI†).

The attenuation of the signal generated by SonoVue is stronger than the signal attenuation observed for KL LigCC NPs over time. Furthermore, although the signal of SonoVue completely disappears after 30 min, the KL LigCC NPs signal can still be observed after 90 min of observation. *In vitro*, microbubbles disappear through the action of buoyancy and dissolution. Other tests should be performed *in vivo* to confirm these observations regarding the differences in signal attenuation and contrast time.

### 3.5. Cytotoxicity evaluation

Prior to cytotoxicity evaluation, we have analyzed the stability of our particles in FBS. The Kraft LigCC nanoparticles demonstrate a degree of stability in Fetal Bovine Serum (FBS). Zeta potential measurements reveal that the nanoparticles in FBS have a slightly less negative zeta potential (-13.7 ± 0.5 mV) compared to those in water (-15.6 ± 0.5 mV). Despite this, the zeta potential values for both conditions are within a range considered indicative of colloidal stability. The toxicity of the particles was evaluated on two different human cell lines relevant for intravenous injection: a model for monocytes which *in vivo* circulate in the blood and continually migrate to tissues (THP-1) and a model for endothelial cells, forming the interior surface of the blood vessels (EA. hy926). The viability of THP-1 and EA.hy926 cells was assessed after 24 h incubation with different concentrations of particles, and relatively compared to a solution containing no particles as a negative control (Fig. 8).

The toxicity was evaluated according to the sample mass concentration, theoretically estimated from the initial ACC NPs mass and initial lignin mass in solution. EPG LCC NPs were included to benchmark the toxicity of lignin-coated and lipid-coated CaCO₃. The toxicity was evaluated for a concentration range from 0.31 µg.mL⁻¹ to 40 µg.mL⁻¹ for all particles.

For all particles, the cell viability is above 80 % for the majority of the concentrations tested, for both cell lines. Although for some isolated concentrations the cell viability drops under 80 %, no dose-response effect is observed for all samples. Furthermore, the cell viability remains above 85 % for all particles at 40 µg.mL⁻¹, the highest concentration evaluated in this study. These results are promising regarding the non-toxicity and biocompatibility of the lignin-coated and lipid-coated CaCO₃, and for their compatibility with intravenous injections.

## 4. Conclusions

The present study highlights the significant role that lignin, as a stabilizer, plays in preserving the properties of calcium carbonate nanoparticles (CaCO₃ NPs) in aqueous media. The core-shell structure of LigCC NPs, comprising an amorphous calcium carbonate (ACC) core and a lignin shell, acts as a robust protecting barrier, preventing crystallization and dissolution of ACC. The choice of lignin type influences the structuration around ACC NPs, with higher molecular weight lignins favoring the encapsulation of multiple ACC cores. Echogenic properties demonstrated by LigCC NPs, even at low concentrations, make them a promising candidate for clinical ultrasound imaging. However, the study underscores the critical impact of the stabilizer material on the echogenic response, emphasizing the need for further investigations into optimizing signal intensity, pH responsiveness, and signal stability over time. These findings have the potential to advance the field of contrast-enhanced ultrasound imaging by providing valuable insights into nanoparticle stabilization mechanisms.

### Funding

MASSENA PRIDE/15/10935404 project, funded by the Fonds National de la Recherche of Luxembourg (FNR).
<figure><img src="image_12.png" /><figcaption>**Fig. 8.** Viability of EA.hy926 (endothelial) and THP-1 (immune) cell lines exposed for 24 h to the different LigCC and LCC NPs and relatively compared to the control (0 µg.mL⁻¹). Data represents the mean of four independent replicates ± SD. * indicates a significant difference as compared to the control (One-way ANOVA followed by a Dunnett's multiple comparison post-hoc test).</figcaption></figure>

### CRediT authorship contribution statement

**Lauriane Chuzeville:** Methodology, Validation, Investigation, Writing – original draft. **Abderrahmane Aissani:** Methodology, Validation, Investigation. **Ahilan Manisekaran:** Methodology, Investigation. **Yves Fleming:** Investigation, Methodology. **Patrick Grysan:** Investigation, Methodology, Formal analysis. **Servane Contal:** Investigation, Methodology. **Aline Chary:** Formal analysis, Methodology, Validation. **David Duday:** Writing – review & editing. **Olivier Couture:** Conceptualization, Validation, Formal analysis, Writing – review & editing, Supervision. **Resmi Anand:** Methodology, Investigation, Formal analysis, Writing – original draft, Validation. **Jean-Sébastien Thomann:** Conceptualization, Validation, Formal analysis, Writing – review & editing, Supervision, Funding acquisition.

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J-S. Thomann has patent # LU102358B1 ; WO2022144136A1 pending to Luxembourg Institute of Science and Technology (LIST). L. Chuzeville has patent # LU102358B1;WO2022144136A1 pending to Luxembourg Institute of Science and Technology (LIST).

### Data availability

Data will be made available on request.

### Acknowledgement

The authors acknowledge the Fonds National de la Recherche (FNR) of Luxembourg for supporting this work through the MASSENA PRIDE/15/10935404 project. The authors acknowledge Dr. Lori Bridal, Dr. Pascal Dargent, Dr. Jerome Gateau and Antoine Coudert from the Laboratoire d'Imagerie Biomédicale (LIB) in Paris for their valuable scientific insights into the ultrasound imaging results and their assistance in the characterization of the echogenic properties of the particles. The authors acknowledge Dr. Reiner Dieden and Coraline Sirot from the Luxembourg Institute of Science and Technology (LIST) for their assistance in the lignin characterization by ³¹P NMR. The authors acknowledge Dr. Alexander Shaplov from LIST for his assistance in the lignin

characterization by GPC. Lastly, the authors acknowledge Dr. Arno Gutleb from LIST for his assistance in the interpretation of the results of the cytotoxicity assessment.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jcis.2023.12.106.

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