Probing Structural Confinement in Organic
Nanoparticles by Intertwining Photochromism and
Nanomechanics

Federico Zizzi, Virginie Silvestre, Eric Gautron, Nicolas Gautier, Stéphane

Cuenot, Eléna Ishow

► To cite this version:

Federico Zizzi, Virginie Silvestre, Eric Gautron, Nicolas Gautier, Stéphane Cuenot, et al. Probing Structural Confinement in Organic Nanoparticles by Intertwining Photochromism and Nanomechanics. ACS Applied Nano Materials, 2025, 10.1021/acsanm.5c04415. http://dx.doi.org/10.1021/acsanm.5c04415

HAL Id: hal-05407190

https://hal.science/hal-05407190v1

Submitted on 9 Dec 2025

**HAL** is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Probing Structural Confinement in Organic
Nanoparticles by Intertwining Photochromism and
Nanomechanics

Federico Zizzi,†,§ Virginie Silvestre,† Eric Gautron,§ Nicolas Gautier,§ Stéphane Cuenot,*,§

Eléna Ishow*,

elena.ishow@univ-nantes.fr; stephane.cuenot@cnrs-imn.fr

†Nantes Université, CNRS, CEISAM, UMR 6230, F-44000 Nantes, France. §Nantes Université, CNRS, Institut des Matériaux de Nantes Jean Rouxel, IMN, F-44000 Nantes, France.

**KEYWORDS.** AFM measurements, soft nanoparticles, elastic modulus, photochromism, azo materials, photocrosslinking, structural confinement.

**ABSTRACT.** Functional organic nanoparticles, largely praised for optoelectronics and bioimaging applications, are usually manufactured upon mere self-assembly of molecular dyes. Undesirable dissociation due to non-covalent interactions between the molecular constituents can be prevented by further photocrosslinking. In order to study the reactivity within nanoparticles and the impact of photocrosslinking on the mechanical properties of nanoparticles, we elaborate nanoparticles and films, based on a photoactivatable network issued from reacting bis-acrylate functionalized azo photochromic units and bis(dithiolane) crosslinkers of variable flexibility.
Photochromic studies, recording azo relaxation dynamics after photoisomerization, are combined to force spectroscopy investigations, using atomic force microscopy following a nanocompression method, to give access to elastic modulus at the single nanoparticle level. We find that the average thermal relaxation of the azo units in crosslinked nanoparticles is largely enhanced by comparison to that in crosslinked films, whereas close values are obtained for both the more flexible and stiffer networks. While the elastic modulus slightly increases for thin films incorporating stiffer crosslinkers, the difference is tenfold amplified in nanoparticles. These results first demonstrate that high structural confinement in nanoparticles upon nanoprecipitation favors large reactivity and thereby large incorporation of the crosslinkers in the final three-dimensional networks. Secondly, they point out that nanoparticle elasticity is mainly ruled by the stiffness of the crosslinkers. The enhanced discrepancy in elasticity at the nanoparticle level is thus tentatively interpreted as nanoconfinement effects, impacting free volume distribution and chain mobility.

INTRODUCTION

Photocrosslinking represents a buoyant field of investigations in materials science for a broad range of applications in biomedicals,¹,² opto-electronics³,⁴ or surface coatings,⁵,⁶ as light permits remote addressing and straightforward modulation thanks to its easily tunable intensity, energy and polarization.⁷ Most studies have been devoted to 3D resin bio-printing and 2D thin film patterning where stress upon matter shrinkage needs to be highly controlled in order to yield the targeted shape and geometry.⁵,⁸,⁹ Such requirement is even more stringent for photocrosslinked organic nanoparticles, usually generated from micro¹⁰ or nanoemulsion¹¹ processes, given their reduced dimensions. Knowing about their mechanical properties, especially their elastic modulus,
has actually become a subject of active research in nanomedicine for bioimaging and drug delivery
purposes since the cellular uptake mechanisms have shown strong dependence on the mechanical
interplay between cells and nanoparticles. ¹²⁻¹⁴ While assessing rheology at the macroscopic scale
usually requires dynamical mechanical analyzer or tensile tester, such measurements at the
nanoscale resort to atomic force microscopy (AFM), considered over the years as the tool of choice
to quantify the mechanical characteristics of nanoparticles. ¹⁵ The vast majority of studies has been
performed on hard nanoparticles, made of crystalline lattices or heavily crosslinked polymer
nanoparticles, for which indentation models have been developed. ¹⁶ These models have
progressively been refined to take into account adhesion effects due to water adsorption substrate
interactions, and coulombic or van der Waals interactions, the different shapes of AFM tips and
more recently, the finite-thickness of samples, a key parameter for thin and soft samples. ¹⁷,¹⁸
However, their accurate extension to soft nanoparticles, broadly used in pharmaceutics and
medicine, and characterized by extensive deformation and deswelling in their individual state,¹⁹
still remains highly challenging due to the flexible character of the molecular or macromolecular
components of the nanoparticles. ²⁰,²¹ Moreover, careful assessment of the elasticity of soft
nanoparticles through an indentation methodology faces extended molecular rearrangements
caused by the mechanical perturbation from the tip, which cannot be taken into account by models
in contact mechanics. All these treatment limitations might have been the reason for the absence,
up to now, of comparative investigations of the mechanical properties and matter structuration
thereof, when going from photocrosslinked micrometric materials to nano-objects. Hence, having
in hand a microscopic sensor, amenable to dynamic responses depending on the surrounding
stiffness, would represent a complementary strategy to bring cross-correlated results. In this
respect, azo photochromic materials, undergoing reversible E-Z photoisomerization, have long
been known for their ability to transduce optical excitation into mechanical motions.²²⁻²⁴ Their remarkable properties as photoactuators have been exploited to generate poled materials upon molecular rearrangement for high-speed optical multiplexing,²⁵ and induce extensive mass migration in polymer and small-molecule matrices to create large surface relief gratings.²⁶,²⁷ More recently, photoactuation of azo-functionalized thin films of liquid crystalline polymers led to impressive folding,²⁸,²⁹ twisting³⁰ or crawling³¹,³² under polarized light, thus mimicking the actions of microrobots. At the molecular level, the differentiated ability of the *E* and *Z* azo isomers to physically explore spatial regions has contributed to the development of photopharmacology to modulate ion channels,³³,³⁴ thereby regulating the corresponding neuronal³⁵,³⁶ or cardiac activity.³⁷ All above-mentioned photoinduced matter displacements or reorientations actually rely on the photochromic reaction, requiring large change in geometry of the azo units when reversibly transformed between the linear elongated geometry of the *E* isomer and the more compact bent shape of the *Z* isomer. This in turn implies extensive microscopic reorganization around the azo units to accommodate the photogenerated isomers, hence larger *E*-to-*Z* photoconversion is achieved in softer matrices.³⁸,³⁹ Due to their exceptional photomechanical properties, azo units can definitely be regarded as indirect probes of the stiffness degree of their surroundings. Contrarily to the abovementioned works addressing liquid crystalline polymer or polymer azo thin films, little studies have been carried out on amorphous azo nanoparticles and their resulting mechanical properties. Such scarcity is likely to stem from the low colloidal stability and fast sedimentation of azo nanoparticles, usually generated by flash precipitation in water of symmetrical azo units, allowing for reversible UV-vis photoswitching. We recently solved this issue by employing push-pull azo units whose polar backbone generates stabilizing interactions with the surrounding water molecules.⁴⁰,⁴¹ The retained azo units were also endowed with UV-vis photoswitching ability
between two states, enriched with either 63 % of *E*-isomer or 65 % of *Z*-isomer respectively.⁴¹

However, the absence of covalent bonds between the self-assembled units affected the reversibility of the nanoparticle geometry as we observed progressive disassembling and size reduction of the nanoparticles deposited on hard substrates upon *E-Z* photoswitching.⁴¹ In order to exploit the potentiality of *E-Z* isomerization to sense the mechanical properties at the nanoscale, we have devised new photoactive crosslinked nanoparticles with linkers of variable flexibility. In the studies reported herein, we thus want to show that photochromic studies combined with AFM investigations, based on a compression approach, offer a particularly attractive and converging strategy to explore and rationalize the evolution of the elastic properties when going from soft photocrosslinked films to nanoparticles. The reported results allow us to independently probe, for the first time to our knowledge, the local compactness at the molecular level and the network elasticity within flexible functional organic nanoparticles, which should provide landmarks to better control the design of nano-architectures, aimed at delivering drugs under photo- or magneto-induced stimulus.

# EXPERIMENTAL SECTION

*Materials.* All chemical reagents and solvents were purchased from commercial sources and used as received unless otherwise specified. Reactions requiring anhydrous conditions were performed under argon. Purification by column chromatography was conducted on silica gel 60 M (40-63 µm) from Macherey-Nagel and thin layer chromatography reaction monitoring on Merck Silica gel 60 F254 analytical plates with an UV coating (254 or 365 nm).

*Manufacturing of photochromic and photocrosslinkable organic nanoparticles.* A stock solution of **azoPC** photochromes with a 1.45 mmol.L⁻¹ concentration was prepared in
spectrophotometric grade tetrahydrofuran (THF). A small aliquot (100 µL) was quickly injected
in one shot into a 18 mL vial containing Millipore® water (2.5 mL) under vigorous stirring by
means of a vortex. Preparation of the photocrosslinkable **azoPC1** and **azoPC2** followed the same
protocol. First, three separate stock solutions were prepared in THF (1 mL each): **azoPC** (3.27 mg,
2.9×10⁻³ mmol), **TPO** (1.01 mg, 2.9×10⁻³ mmol), and **DT1** (6.36 mg, 11.6×10⁻³ mmol) or **DT2**
(7.16 mg, 11.6×10⁻³ mmol). All three solutions were mixed in a volume ratio of 0.5:0.05:0.25 (in
mL) corresponding to **azoPC:TPO:DT1** (or **DT2**), and the mix was completed with THF (0.20
mL) to reach a total volume of 1 mL. In this mixture, the final concentration of azo units was 1.45
mmol.L⁻¹, the photoinitiator was present at 0.1 eq. with respect to the azo units, and the **DT1** (or
**DT2**) crosslinkers were present at 2 eq. with respect to the azo units. A small aliquot (100 µL) of
the mix was then quickly injected in a 18-mL vial containing Millipore® water (2.5 mL) under
vigorous stirring by means of a vortex.

*Manufacturing of photocrosslinked thin films.* Three distinct solutions were prepared from **azoPC** (2 wt. %, 30 mg, 0.027 mmol), **TPO** (9.3 mg, 0.027 mmol), and the dithiolane crosslinkers **DT1** (58 mg, 0.106 mmol) or **DT2** (66 mg, 0.106 mmol) in chloroform (1 mL for each). The final photocrosslinkable solution was then prepared by mixing the three previous solutions, following the volume ratio of 1:0.1:0.5 (in mL) corresponding to **azoPC:TPO:DT1** (or **DT2**), and the mix was completed with THF (0.4 mL). In this way, the molar ratio of **azoPC** to dithiolane crosslinker **DT1** or **DT2** was 1:2, while that for **azoPC:TPO** was 1:0.1. Thin films were prepared by drop-casting the solution on pre-cleaned glass slides. Photocrosslinking was realized by illuminating the deposited thin film for 5 min, with a mercury light source (Hamamatsu - Ligthning Cure LC6 or OmniCure Series 2000) filtered at 365 nm (30 mW.cm⁻²). Non-polymerized material was readily removed by washing out the samples with spectroscopic grade chloroform. The resulting film
thickness was measured to be in the micrometric range by means of a mechanical profilometer
(Dektak Veeco 8).

*Dynamic light scattering measurements (DLS)*. Nanoparticle size measurements in aqueous dispersions were performed *via* DLS, well-adapted to spherical nano-objects as those herein reported, using the VASCO Kin™ nanoparticle sizer provided by Cordouan Technologies, and equipped with a 45 mW laser diode operating at 638 nm. The laser power was manually adjusted for each sample before any measurement to get satisfying results. All measurements were collected on aqueous dispersions at 25 °C in a backscattering mode at a 170° angle, over 2 min with a 5 Hz acquisition frequency, namely every 200 ms. Acquisitions and analyses were done with the Nano Kin™ software, provided by Cordouan Technologies, Cumulant-based algorithm in intensity was privileged to infer size distributions. A logarithmic scale was adopted for clarity reason, improving visualization of all samples on the same scale, with no impact on the fitted parameters.

*Transmission electron microscopy*. Nanoparticle size measurements as dry samples were performed using a MO-Jeol 1230 microscope (80 kV) and a Thermo Fisher Scientific S/TEM Themis Z (300 kV). The samples were prepared from diluted dispersions in water, that were further deposited on holey carbon-coated copper grids (300 mesh), purchased from Agar Scientific. Size distributions to provide a mean “dry” diameter $D_{TEM}$ were assessed by fitting with a lognormal distribution the histograms issued from counting between 150 and 200 nano-objects using the free ImageJ software.

*UV-absorption spectroscopy*. UV-visible absorption spectra were recorded using a Varian Model Cary 5E spectrophotometer. Molar absorption coefficients were obtained by correlating the absorption spectra of two distinct photostationary states issued from irradiation at 365 and 550 nm
(10 mW.cm⁻² irradiance for each) with ¹H NMR spectra recorded on the same solution using a Bruker spectrometer Avance Ascend HD 700 MHz equipped with an inverse cryoprobe.

*Infrared spectroscopy.* Infrared spectroscopy measurements were performed using a FTIR Bruker Tensor 27 spectrometer working in the transmission mode. All samples investigated as thin films were issued from the deposition of chloroform solutions on 1.3 mm round NaCl windows (Eurolabo) and subsequent drying in air.

*Photochromic investigations.* Solutions of azoPC compounds were prepared at a concentration of around 2×10⁻⁵ mol.L⁻¹ in THF solution while the azo nanoparticles aqueous dispersions were diluted by five with Millipore water. They were irradiated under vigorous stirring using a Hg-Xe light source (Hamamatsu, Lightning Cure LC8), equipped with a quartz fiber and single-band bandpass filters to irradiate in the visible range (550/88 nm BrightLine®, Semrock, Optoprim-France retailer), or in the UV range (365 nm MaxLamp™ Mercury line filter, Semrock, Optoprim-France retailer). The power was adjusted using a laser power meter (Nova II, Ophir) and a photodiode (PD300/UV/NIR). Back thermal relaxation was recorded from solutions promoted to their photostationary state.

*Atomic force microscopy.* All AFM experiments were performed in ambient conditions, at room temperature (20 °C) using a NanoWizard II® atomic force microscope (JPK, Germany) equipped with inverted optical microscope and x-y closed-loop control system. Cantilevers with calibrated spherical carbon-glassy tips (Biosphere™ series purchased from Nanotools) characterized by a tip radius of 100 nm ± 10 nm were used for all experiments (i.e. imaging and mechanical measurements). Their spring constants were calibrated using the thermal noise method implemented in the JPK AFM setting, with values comprised between 0.1 and 0.4 N m⁻¹. The cantilever sensitivity was systematically measured from the slope of the force-distance curve performed on silicon substrate before mechanical measurements. AFM was firstly used for imaging in intermittent contact mode thin films and
nanoparticles, and selecting nanoparticles to be probed. Then, force mapping (typically 32×32 curves) was performed on the selected area of thin films (50 µm × 50 µm) or around the selected nanoparticle (1 µm × 1 µm). Force-distance curves were carried out at a low speed of 2 µm.s⁻¹ and the maximum applied force (few nN) was chosen to keep in the linear elastic regime and avoid damaging the soft nanoparticles. The raw force-distance curves were converted into force versus tip-sample distance (F-δ) curves to apply a contact mechanics model where a sphere (AFM tip) compressed an elastic material characterized by its elastic modulus *E*. A sphere-sphere contact geometry was used to model the AFM tip-nanoparticle system, characterized by the AFM tip radius (*R*<sub>tip</sub>) and the nanoparticle radius (*R*<sub>sample</sub>), generating an effective radius (*R*) defined as (Equation 1):

$$ \frac{1}{R} = \frac{1}{R_{tip}} + \frac{1}{R_{sample}} \qquad (\text{Eq. 1}) $$

In the case of a sphere-plane contact geometry as that obtained for thin films, *R*<sub>sample</sub> tends to infinity and the effective radius corresponds to the tip radius. As we explored soft matter with small thickness deposited on a solid substrate, either structured as thin films or nanoparticles, bottom-effect corrections were included in the model to take the finite-thickness of soft samples into account, thereby avoiding overestimation of the extracted mechanical parameters. The relation F-δ for a spherical tip into an elastic material can be described by the analytical expression (Equation 2):

$$ F = \frac{4\sqrt{R}}{3(1-\nu^2)} E \delta^{3/2} \left( 1 + 1.133 \frac{\sqrt{R\delta}}{h} + 1.497 \frac{R\delta}{h^2} + 1.469 \frac{(R\delta)^{3/2}}{h^3} + 0.755 \frac{(R\delta)^2}{h^4} \right) \qquad (\text{Eq. 2}) $$

where *ν* is the sample Poisson's ratio and *h* is the finite-thickness of the elastic sample (i.e. the thickness of thin films or the nanoparticle diameter) deposited onto a rigid substrate.<sup>17,18</sup> The second to fifth terms correspond to the bottom-effect corrections, yielding the elastic modulus of the investigated material without overestimation due to the rigid support, while the first term represents the Hertz model for a semi-infinite elastic material, independent on *h*. The resulting
apparent elastic modulus was then obtained as the only adjustable parameter by fitting all
experimental curves by equation 2. A total of 1024 measurements was performed on thin films
while at least 10 measurements were selected at the center of each probed nanoparticle from force
mapping. To give a concise view of the distribution of elastic modulus values, a boxplot
representation was chosen. For each dataset, five summary statistics can easily be provided with
the minimum and maximum values, the median value, the first and third quartiles (i.e. 25% and
75% of the data) while all data are plotted. In addition, the elastic modulus values are also
discussed in terms of mean value ± standard deviation.

*Sample preparation for AFM measurements.* Mica squared plates (1 inch, Oxford) were freshly-cleaved before depositing a small aliquot of nanoparticle aqueous dispersion (100 µL), previously diluted 10 times with Millipore water. After incubation for 1 h, the mica plate was washed and let dry at ambient temperature and pressure conditions.

RESULTS AND DISCUSSION

Design of azo materials for material’s elasticity tuning

Since photochromic thin films and nanoparticles are targeted, the azo structure needs to meet
several crucial physical and chemical imperatives. Firstly, photoisomerization has to operate in the
solid state: lateral bulky substituents will then be introduced to prevent inhibition of the reaction
upon π-π stacking of the aromatic chromophores.³⁹,⁴² Secondly, physical stability of the
nanoparticles after manufacturing is required. On the one hand, this implies colloidal stability in
water in the absence of surfactant to accommodate with the simple adopted flash precipitation
process and make further mechanical comparisons between films and nanoparticles relevant. On
the other hand, structural stability is expected to avoid progressive disintegration of the self-
assembled dyes upon successive irradiation cycles.⁴¹ To achieve both requirements, polar structures, involving strong charge transfer along the azo moiety will be selected,⁴⁰,⁴¹ while photocrosslinkable units will be introduced to covalently bind the photochromic units and form a three-dimensional (3D) network. Thirdly, probing photochromism in the solid state needs repetitive addressing, permitted by the *E-Z* reaction reversibility: photoswitching between two stable states using two distinct irradiation wavelengths is ideal, which can be achieved by introducing fluorine atoms in the *ortho*-substituted azo positions.⁴³,⁴⁴ All above-mentioned architectural criteria led to the final key structure **azoPC** where the triarylamino group imparts the azo units with two main advantages (Scheme 1, Figures S1-S22). Beyond its strong electron-donating character to ensure large dipole moment, it supports an additional absorption band in the UV range, yielding unusual *Z→E* back reaction for pseudo-stilbene azo compounds in this spectral domain.⁴¹ Moreover, it opens up the possibility to graft two photopolymerizable acrylate groups and achieve 3D-crosslinked architectures. The choice for acrylate units was motivated by their high reactivity upon radical photoinitiation,⁴⁵ especially towards dithiolane units,⁴⁶ to undergo efficient dithiolane-ene copolymerization in water, with low sensitivity to oxygen.⁴⁷ Long terminal carboxyl ester chains were introduced to provide the acrylate units with enough mobility, and enable photopolymerization even in the absence of solvent, as already demonstrated with emissive acrylate monomers and the patterning of insoluble overlapping fluorescent motifs after structured illumination.⁴⁸

**Scheme 1.** Synthesis pathway toward the photocrosslinkable azo compound **azoPC**: i) *p*-acetylboronic acid, Pd(PPh₃)₄, 2M Na₂CO₃(aq) toluene, EtOH, reflux, 12 h, 88%; ii) NaBH₄, EtOH, RT, 24 h, quant.; iii) *tert*-butyldimethylsilyl chloride (TBDMSCl), imidazole, DMF, 1 h, RT, 86
%; iv) N₂H₄, Pd/C, EtOH, reflux, 3 h, 94%; v) 4-bromo-2,6-difluoronitrosobenzene, acetic acid, rt, 48 h, 58%; vi) *p*-formylboronic acid, Pd(PPh₃)₄, 2M Na₂CO₃(aq), toluene, EtOH, reflux, 12h, 82%. vii) *tert*-butylammonium fluoride (TBAF), RT, 1 h. viii) dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridinium *p*-toluenesulfonate (DPTS), 0 °C to RT, 48 h, 45%.

<figure><img src="image_2.png" /></figure>

The adopted synthetic pathway thus provided the final product **azoPC** as a viscous orange-red product, characterized by a second-order glass transitions at temperature *T*<sub>g</sub> around 13.6°C, namely below room temperature (Figure S23). Strong hydrophobicity of all components was required to favor co-assembly in water and ulterior intermolecular crosslinking with the azo-functionalized diacrylate units. Such requirement prompted us to retain two bis(1,2-dithiolane)-terminated alkyl chains **DT1** and **DT2**, showing the same length but significant difference in flexibility, owing to the stiffer isopropylidenedicyclohexyl backbone for **DT2**. The generated networks are thus expected to exhibit distinct elasticity, since photocrosslinking the thiol **DT2** analogue with acrylate monomers conducted to polymers with higher glass transition temperature *T*<sub>g</sub> (+ 13.3 °C) and
elastic modulus (+ 55.4 MPa), when compared to those obtained for the polymers incorporated the **DT1** thiol analogue (Scheme 2).⁴⁹ Both chains **DT1** and **DT2** were readily synthesized from Steglich esterification between lipoteichoic acid and the corresponding diol.

**Scheme 2.** a) Bis(1,2-dithiolane) linkers **DT1** and **DT2** with a central chain of varying flexibility.

b) Photoinduced polymerization involving diacrylate and bis(dithiolane) monomers (PI: photoinitiator, HA: protic species). For the sake of simplicity, only half of the monomers was represented while the R₂ substituents for the dithiolane ring-opening polymerization were voluntarily not placed at a specific position given the three possible orientations of the cleaved monomers before forming the disulfide bonds.

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

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

## Thin film and nanoparticle processing – evolution upon photocrosslinking

Photocrosslinking composition in terms of **azoPC**, **DT** monomers, and 2,4-6-trimethylbenzoyldiphenylphosphine (TPO) photoinitiator, was first optimized on micrometer-thick films, using a 1:2 diacrylate:bis(dithiolane) molar ratio to favor dithiolane-ene polymerization (Scheme 2b). The optimal molar ratio was found to be 1:2:0.1 for **azoPC:DT1** (or **DT2**:TPO (dubbed **azoPC1** and
**azoPC2)** and tested on glass slides by drop-casting the corresponding solution in chloroform at a 2 wt. % concentration of azo compound. Photocrosslinking, performed at 365 nm (30 mW.cm⁻² for 5 min.) where TPO absorbs the most,⁵⁰ could be followed with naked eyes. Indeed, the self-sustainable films progressively wrinkled over time, as a consequence of shrinkage effects upon polymerization, which eventually transduced into mechanical tension and spontaneous folding until forming a beading on the edge (Figure S24). Assessing photoconversion from the decrease of the weakly-absorbing UV band of 1,2-dithiolane peaking at 330 nm⁴⁶ was precluded by the strong absorption of **azoPC** in this region. We rather focused on the typical bands of **azoPC** in infrared (IR) spectroscopy, centered at 1496 cm⁻¹ (phenyl stretching modes $\bar{v}_{C=C}$), 825 cm⁻¹ (out-of-plane modes $\delta'_{C-H}$ of the *p*-substituted aromatic rings), and 809 cm⁻¹ (wagging modes $\delta^2_{C-H}$ of the C=CH₂ bonds in the acrylate units), and showing weak overlap with the IR bands of **DT1** and **DT2** (Figure S25). While the vibrational bands related to the phenyl rings are not supposed to vary, the band of the acrylate C-H wag is expected to decrease upon photopolymerization of the acrylate units. For such a purpose, **azoPC1** or **azoPC2** thin films were drop-cast on a NaCl crystal and let dry. Infrared spectra were recorded after each irradiation time and final development with chloroform (Figures S26-S27). Since the sample position could vary from one irradiation to another and loss of material occurred after development, spectral normalization with respect to the out-of-plane $\delta'_{C-H}$ mode was required. A neat decrease in the contribution of the $\delta^2_{C-H}$ wagging mode could then be noted, confirming photocrosslinking of the acrylate units (insets of Figures S26-S27). After processing the spectra, we calculated a photoconversion ratio of the acrylate units, after development, of 60 % for **azoPC1** and 51 % **for azoPC2**.

Given the reality of the photopolymerization process, we then used identical ratios of monomers to prepare nanoparticles from the corresponding solution in tetrahydrofuran, adjusted
to a 1.5 mmol.L⁻¹ **azoPC** concentration. A small aliquot of the resulting mixture was quickly
injected into a large volume of Millipore water under vigorous stirring to yield nano-suspensions.
The same nanoflash precipitation procedure was followed to yield pristine **azoPC** nanoparticles,
devoid of DT crosslinkers, to further serve as a reference. Colloidal stability was ensured by the
largely negative surface potential ζ found by zetametry to be less than -33 mV. Given the push-
pull character of the azo compounds, present in a large amount in the generated nanoparticles,
strong structuration of water molecules around the organic nanoparticles has already been reported,
hence polarization effects could be evoked as a possible reason for the measured negative ζ value.⁵¹
Photocrosslinking was eventually carried out at 365 nm (30 mW.cm⁻²), for 5 min, to ensure
progress of the reaction. Attempts to evaluate the rate of crosslinking by infrared spectroscopy
failed because of a much too low azo concentration and large water absorption that could not be
compensated using liquid cells with larger calibrated optical paths and CaF₂ windows.
Nevertheless, transmission electron microscopy clearly revealed distinct geometries before and
after UV irradiation (Figure 1).

Strong geometry changes from a hazy shape for **azoPC1** and **azoPC2**, recalling that of pristine **azoPC** nanoparticles (Figure S28), to well-defined and contrasted nanospheres for **azoPC1**<sub>XL</sub> and **azoPC2**<sub>XL</sub>, after illumination, let us reasonably suggest extensive matter contraction, yielding denser material, as a result of crosslinking. Both **azoPC1**<sub>XL</sub> and **azoPC2**<sub>XL</sub> nanoparticles displayed by diffuse light scattering (DLS) a hydrodynamic diameter *D*<sub>Z</sub> of 141 ± 28 nm and 174 ± 39 nm respectively, with a polydispersity index (PDI) varying between 0.161 and 0.204, featuring a relatively narrow size distribution with respect to the manufacturing process (Figure S29). In the dry state, as anticipated, the mean diameter *D*<sub>TEM</sub> (associated standard deviation σ), inferred from log-normal fitting of the size distribution, was found to be equal to 37 nm (0.40) and 51 nm (0.27)
<figure><img src="image_5.png" /><figcaption>**Figure 1.** TEM images of non-crosslinked and crosslinked (photo-irradiation at 365 nm, 30 mW.cm⁻², 5 min) nanoparticles deposited on holey carbon-coated copper grids from the corresponding aqueous dispersions, and associated size distribution (histogram) and log-normal fit (red curve): for azoPC:DT1:TPO (azoPC1 and azoPC1XL) and azoPC:DT2:TPO (azoPC2 and azoPC2XL).</figcaption></figure>

for azoPC1XL and azoPC2XL nanoparticles respectively (Table S1). Interestingly, azoPC2XL nanoparticles are significantly larger than azoPC1XL ones despite similar size for both DT1 and DT2 crosslinkers. Increase in the free volume and size thereof may originate from steric crowding and restricted torsion brought by the isopropylidenedicyclohexyl middle unit, which does not necessarily rhyme with more molecular “fluidity” as discussed below in the case of nanoparticles.
Influence of spatial confinement on photochromic and back thermal E/Z isomerization

Photochromic investigations were first carried out in solution to analyze the loss of degrees of freedom in the solid state. Tetrahydrofuran was chosen as a solvent to provide both high solubility and closer polarity to that of neighboring molecules once self-assembled in films or nanoparticles. UV-vis absorption spectroscopy revealed two bands centered at 470 and 330 nm (Figure 2a – Table 1). For azoPC, the first band could be ascribed to strong charge transfer along the azo unit following a ππ* transition from previous calculations, performed on very similar azo compounds comprising tert-butyl substituents instead of the polymerizable alkoxy chains.⁴¹

The second band involved two overlapping transitions from the triphenylamine core to the biphenyl units as well as to the main axis implying the azo and carbonyl electron-withdrawing groups. The latter specificity brings the potential to favor reverse photoswitching from Z→E configuration whereas push-pull azo compounds are classically endowed with the sole E→Z photoinduced isomerization upon excitation in the visible range. Following this consideration, photoisomerization was then conducted at two wavelengths, namely 550 nm and 365 nm to reach photostationary states (PSS) enriched in Z and E isomers, respectively (Figure 2a). In THF solution, thermal Z→E back relaxation of azoPC in the dark after a 550 nm excitation revealed a monoexponential recovery of the E forms, characterized by a rate constant $k_{ZE}$ found to be equal to 0.33×10⁻⁵ s⁻¹ (namely, a time constant of 3,5 days) (Figure 2b, Table 1). It agrees with the numerous studies, reporting impeded thermal relaxation due to the sterically crowded *ortho* fluorinated substituted position.⁵²
<figure><img src="image_6.png" /><figcaption>**Figure 2.** a) Absorption spectra of azoPC in THF (2.5×10⁻⁵ mol.L⁻¹) following irradiation at 550 or 365 nm (10 mW.cm⁻² - PSS designates the photostationary state reached after each irradiation). b) Thermal back relaxation from the photostationary state reached after irradiation at 550 nm (inset: time evolution of the absorbance at 468 nm). c) Absorption spectra of the E and Z-isomers calculated from ¹H NMR spectra recorded in THF-d₈ (700 MHz).</figcaption></figure>

The slow thermal recovery of the *E* isomers thus allowed us to perform ¹H NMR spectra, representative of the state achieved right after each irradiation. We used a 700 MHz spectrometer, equipped with a cryoprobe, which is particularly adapted to detect traces in the 10⁻⁵ mol.L⁻¹ concentration range. In this case, the absorption spectra can be recorded without requiring specific dilution of the solution, while risks of modifying the *E/Z* ratio upon dilution are avoided.
<table><caption>**Table 1.** UV-vis absorption and photochromic properties (minimum conversion yield $\rho_{min}$ between both photostationary states reached at 365 and 550 nm, average rate constant $\langle k_{ZE} \rangle$ of the thermal back reaction $Z \rightarrow E$) of azoPC in THF solution and processed as pure or DT1/DT2 (+ 0.1 mol. TPO) doped films or nanoparticles before (nXL) and after photocrosslinking (XL) at 365 nm.</caption><thead><tr><th rowspan="2"></th><th rowspan="2">System</th><th colspan="2">λ<sub>max</sub>(abs) (nm)</th><th colspan="2">ρ<sub>min</sub> (%)<sup>d</sup></th><th colspan="2">&lt;k<sub>ZE</sub>&gt; (10<sup>-5</sup> s<sup>-1</sup>)<sup>e</sup></th></tr><tr><th>PSS<sub>365</sub></th><th>PSS<sub>550</sub></th><th>nXL</th><th>XL</th><th>nXL</th><th>XL</th></tr></thead><tbody><tr><td rowspan="3">THF<sup>a</sup></td><td>azoPC</td><td>469</td><td>466</td><td>33.0</td><td></td><td>0.33</td><td></td></tr><tr><td>azoPC</td><td>482</td><td>481</td><td></td><td>16.0</td><td></td><td>7.85</td></tr><tr><td>azoPC1</td><td>486</td><td>482</td><td>16.7</td><td>12.5</td><td>3.03</td><td>362</td></tr><tr><td rowspan="2">Nanoparticles<sup>b</sup></td><td>azoPC2</td><td>488</td><td>486</td><td>12.0</td><td>10.0</td><td>4.71</td><td>487</td></tr><tr><td>azoPC</td><td>458</td><td>461</td><td></td><td>11.0</td><td></td><td>1.24</td></tr><tr><td rowspan="3">Films<sup>c</sup></td><td>azoPC1</td><td>488</td><td>487</td><td>15.0</td><td>9.0</td><td>5.96</td><td>110</td></tr><tr><td>azoPC2</td><td>484</td><td>483</td><td>13.0</td><td>7.0</td><td>9.93</td><td>89</td></tr></tbody></table>

Integrating the peaks specific to each isomer provided a photoconversion yield $\rho$ of 68 and 67 % following irradiation at 550 nm et 365 nm (10 mW.cm<sup>-2</sup>), respectively (Figure S22). From these yields, we could calculate the absorption spectrum of the pure Z isomer, that presents substantial difference in the visible range compared to that of the E isomer (Figure 2c). When similar

<footnote><sup>a</sup> THF solution (2×10<sup>-5</sup> mol.L<sup>-1</sup>). <sup>b</sup> Obtained after addition into Millipore water (2.5 mL) of a 1.5×10<sup>-3</sup> mol.L<sup>-1</sup> azo solution in THF (100 µL). <sup>c</sup> From spin-casting a 2 wt. % azo solution in chloroform. Film thickness *d* was measured to be 2 µm for azoPC1<sub>XL</sub> and 1 µm for azoPC2<sub>XL</sub> respectively. <sup>d</sup> Minimum photoconversion yield $\rho_{min}$= (A<sub>0</sub>-A<sub>PSS</sub>)/A<sub>0</sub> where A<sub>0</sub> and A<sub>PSS</sub> designate the absorbances before irradiation and at the photostationary state, respectively, and considering no absorbance from the Z isomers. <sup>e</sup>Experimental fit of the recovered absorbance A<sub>E</sub> of the E species upon back thermal relaxation in the dark, as follows: A<sub>E</sub>(t) = A<sub>E</sub><sup>0</sup> + Σ<sub>i</sub> a<sub>i</sub>exp(-k<sub>ZE</sub><sup>i</sup>t). Monoexponential law for solutions and multiexponential law for films and nanoparticles. For the latter, the average rate constant $\langle k_{ZE} \rangle$ is defined as $\langle k_{ZE} \rangle = \sum_i f_i k_{ZE}^{i}$ with f<sub>i</sub> = a<sub>i</sub>/Σ<sub>j</sub> a<sub>j</sub> corresponding to the amplitude fraction where a<sub>i</sub> is the pre-exponential factor associated to the rate constant k<sub>ZE</sub><sup>i</sup>.</footnote>
photochromic reactions were performed on azoPC nanoparticles or films (Figure 3a and Figures S30-S31), noticeable reduction of the minimum photoconversion yield ρ_min (calculated as the normalized ratio of the absorbance difference ($A_{PSS(365)} - A_{PSS(550)}$)/$A_{PSS(365)}$ reached at each PSS) was observed, going from 33 % in THF solution down to 16 and 11 % in nanoparticles and thin films, respectively (Figure 3b). Conversely, the average back thermal rate constant <$k_{ZE}$>, resulting from a bi- or triexponential modelling of the $Z→E$ thermal relaxation, displayed a significant increase that was even more pronounced for nanoparticles (24 times) than for films (4 times) (Table 1, Figure S32). This is the consequence of a prevailing fast rate component, especially for the nanoparticles, that is also accompanied by a lower fraction of molecules, relaxing in the same range of order than those in solutions (Table S2). Both the ρ_min decrease and <$k_{ZE}$> increase are the two sides of the same coin, namely a consequence of steric crowding and restricted mobility in the solid state, which provokes less free volume to enable $E → Z$ isomerization and stabilize the formed Z isomers as well.<sup>41,53</sup>

When comparing the effect of the nature of the dithiolane monomer **DT1** or **DT2** on the **azoPC** photochromic response before crosslinking, both the $E→Z$ photoconversion as well as the $Z→E$ back thermal relaxation were impacted. Indeed, introducing a stiffer ligand like **DT2** reduces by around 28 % the azo photoisomerization ability in nanoparticles and 13 % in films with respect to **DT1**-containing systems (**azoPC1**). As for the back thermal rate constant, its average value <$k_{ZE}$> was found 1.5 times higher in **azoPC2** than in **azoPC1** systems. Both evolutions recall the trend observed for **azoPC** when going from solution to solid state, hence we can reasonably state on a more rigid environment brought by the bulkier **DT2** monomers.
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

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

<figure><img src="image_10.png" /></figure>

**Figure 3.** Aqueous dispersions of pristine **azoPC** nanoparticles: a) absorption spectra of PSS reached under irradiation at 365 or 550 nm (10 mW.cm⁻² each), b) Comparative change in the maximum absorbance ΔA = A<sub>PSS(365)</sub> - A<sub>PSS(550)</sub> between both PPS at 365 nm (E-mostly) and 550 nm (Z-mostly) before and after photocrosslinking. Aqueous dispersions of **azoPC1** and **azoPC2** doped with TPO: time evolution of the absorbance at the absorption maximum wavelength during back thermal relaxation after reaching PSS@550 nm: c) before photocrosslinking, d) after photocrosslinking at 365 nm (5 min, 30 mW.cm⁻²).

This assumption is reinforced by the raise of <k<sub>ZE</sub>> after photocrosslinking the films and nanoparticles, where photoisomerization conversion also declines with enhanced rigidity. Such a raise is particularly striking in **azoPC1<sub>XL</sub>** and **azoPC2<sub>XL</sub>** nanoparticles, with a two-order magnitude change from 3-4×10<sup>-5</sup> to 3-4×10<sup>-3</sup> s<sup>-1</sup> before and after crosslinking, respectively (Figure 3c). The noteworthy discrepancy between nanoparticles and films after crosslinking tends to
illustrate the high spatial confinement of monomers undergone after flash nanoprecipitation, conducive to larger cross-link density, which reduces the local free volume to adapt the photogenerated Z isomers.⁵⁴

Since all measurements involving nanoparticles have been performed on aqueous dispersions, water molecules at the interface could also play a role by affecting the azo thermal relaxation. Local polarity effects due to the surrounding water molecules could here speed up the thermal relaxation process since polar media are known to decrease the potential energy barrier between the *E* and *Z* ground states of push-pull azo compounds, the *Z* isomers becoming less stabilized upon solvation. Such water-dye interactions have indeed proved to be crucial in the case of fluorescent organic nanoparticles, causing systematic decrease in emission intensity of self-assembled polar molecules, due to radiationless deactivation promoted by vibrational coupling with the water O-H stretch oscillators.⁵⁵,⁵⁶ Additionally, water molecules could also favor high packing of the photochromic material within the nanoparticles, issued from the flash precipitation of hydrophobic monomers. All these interrogations prompted us to investigate matter cohesion and distribution within single nanoparticles by resorting to AFM that permits measurements at the nanoscale after water evaporation of the aqueous dispersions.

## Comparative mechanical properties of thin films and nanoparticles by AFM nanoscale investigations

Given the honey-like and thus structureless properties of azoPC, as pristine or DT-mixed films and nanoparticles, AFM measurements were exclusively conducted on the corresponding photocrosslinked materials. Particular attention was paid to the experimental conditions, in terms of preparation and investigation of nanoparticle samples. The latter were deposited for 30 min. on freshly cleaved and ozonized mica substrates, washed once with deionized water and carefully
dried. Soft cantilevers with calibrated spherical tips (100 nm-large radius, greater than the
nanoparticle diameter) were chosen to apprehend matter cohesion by measuring the elastic
properties following a compression method.⁵⁷ Indeed, the conventional indentation methods with
a hard sharp tip, well suited for hard nanoparticles, would be too insensitive to allow for accurate
measurements on soft nanoparticles deposited on harder substrates. The samples were first imaged
and for those made of nanoparticles, areas displaying individual nanoparticles were preferentially
selected (Figure 4a) The mechanical properties of thin films and nanoparticles were then
investigated by performing force mapping with a high spatial resolution, which is essential to
derive reliable data from nanoparticle addressing. In this way, a force map was carried out by
recording an array of 32×32 force-distance curves on an area of 1 µm² around the selected
nanoparticle (Figure 4b). Such experimental conditions gave enough data to focus the analyses on
the center of nanoparticles and get reliable values. Indeed, the contact area between the tip and the
nanoparticle is well known since it can readily be modeled by a sphere-sphere contact geometry,
contrarily to that on the nanoparticle edges, considerably varying as a function of the nanoparticle
curvature and tip location. After conversion of raw approach force-distance curves into force versus
tip-sample distance (F-δ) curves, the Hertz model was subsequently applied with only one adjustable
parameter, the elastic modulus *E*, in a sphere-sphere (for nanoparticles) or sphere-plane (for thin films)
contact geometry (Figure 4c). In addition, bottom-effect corrections were included in the used
contact mechanics model to take the finite thickness of films or nanoparticles deposited on a solid
substrate into account, thereby avoiding overestimation of the extracted elastic modulus (equation
2).¹⁷,¹⁸
<figure><img src="image_11.png" /><figcaption>**Figure 4.** Methodology used to determine the elastic modulus of films and nanoparticles, illustrated by considering a 95 nm-large azoPC2<sub>XL</sub> single nanoparticle: a) AFM imaging of an individual nanoparticle deposited on a mica substrate, b) force mapping of 32×32 curves recorded over a 1 µm × 1 µm area around the selected nanoparticle, c) approach and retract force versus tip-sample distance (F-δ) curves with Hertz model fitting, d) reconstructed elastic modulus map, e) effect of the finite thickness of the nanoparticle (bottom-effect corrections) on the determined elastic modulus.</figcaption></figure>

After analyzing each force curve, the elastic modulus map could be reconstructed (Figure 4d) and the data corresponding to the center of the nanoparticle were extracted. Interestingly, considering the finite thickness of soft nanoparticles deposited on a rigid substrate significantly
affects their elastic modulus that decreases from 78.1 ± 6.7 MPa to 49.4 ± 4.9 MPa, thereby fully justifying the need for such a correction (Figure 4e).

We first investigated the **azoPC1<sub>XL</sub>** and **azoPC2<sub>XL</sub>** photocrosslinked films at two distinct regions, namely the beading (A) and the flat surface (B) differing by their thickness (4-6 versus 1-2 µm), to apprehend any effect of the latter on the elastic modulus *E*. Both regions of each film actually displayed similar values of elastic modulus around *E* = 6.9 ± 0.7 for **azoPC1<sub>XL</sub>** and 7.7 ± 0.7 MPa and **azoPC2<sub>XL</sub>**, proving the absence of thickness effect on the sample stiffness (Figure 5a). The slightly higher *E* values measured for **azoPC2<sub>XL</sub>** may reasonably account for the stiffer character of **DT2**. Finally, the MPa range found for the elastic modulus of the current azo materials correspond to those usually obtained for thiol-ene crosslinked polysiloxanes,⁵⁸ suggesting a rather loose 3D network with regard to those involving trifunctional monomers and characterized by elastic modulus in the GPa range.⁵⁹

<figure><img src="image_12.png" /></figure>
<figure><img src="image_13.png" /><figcaption>**Figure 5.** Elastic modulus values determined on photocrosslinked films and nanoparticles by AFM following a compression approach: a) optical images of azoPC1<sub>XL</sub> and azoPC2<sub>XL</sub> films and elastic modulus values measured on the beading (A) and flat surface (B) regions, b) AFM images of individual azoPC1<sub>XL</sub> nanoparticles (height in nm) and distribution of the corresponding calculated elastic modulus values, c) AFM images of individual azoPC2<sub>XL</sub> nanoparticles (height in nm) and distribution of the corresponding calculated elastic modulus values. Scale bar: 200 nm (1 µm × 1 µm image).</figcaption></figure>

Remarkably, marked discrepancy of the elastic modulus of the nanoparticles as a function of the linker flexibility could be observed. Its determination after force mapping of four nanoparticles, gave averaged values of *E* equal to 1.1 ± 0.2 MPa for **azoPC1**<sub>XL</sub> against 52.3 ± 4.4 MPa for **azoPC2**<sub>XL</sub> comprising the stiffer **DT2** crosslinker (Figures 5b-c). These values appear particularly low, by one order of magnitude, compared to those of crosslinked polystyrene (PS),⁶⁰ poly(methyl methacrylate) (PMMA)⁶¹ or poly(D,L-lactide-co-glycolic acid) (PLGA)⁶² nanoparticles, amounting to 1-4 GPa, by using a nanocompression approach or a nanoindentation methodology,
integrating deformation corrections for tips with a radius of curvature larger than the NP diameter. They rather echo those of egg yolk phosphatidylcholine liposomes (~2 MPa)⁶³ and anionic phospholipid liposomes incorporating cholesterol units (0.9-3.6 MPa)⁶⁴, considered as soft nanoparticles, or those of lipid-coated poly(D,L-lactide-co-glycolic acid) (PLGA) nanoparticles (~60 MPa),⁶⁵ regarded as semi-soft nanoparticles as a result of shell stiffening by a harder core⁶⁶ like PLGA. Compared to microgels with elastic modulus in the kPa range, as determined by AFM spectroscopy mode,⁶⁷ **azoPC1<sub>XL</sub>** and **azoPC2<sub>XL</sub>** nanoparticles appear much stiffer. This supports the reasons for limited azo photoswitching and the lack of photoinduced elasticity modulation, contrarily to what was observed in flexible azo supramolecular hydrogels.⁶⁸ These mechanical results also notably differ from those measured in films, with a 6-fold decrease of *E* for **azoPC1<sub>XL</sub>** and a 7-fold increase of *E* for **azoPC2<sub>XL</sub>**, featuring enhanced flexibility and stiffness, respectively. Such observation is to be related to the intrinsic character of the crosslinkers, **DT1** displaying more flexibility than **DT2**. This would also mean that enough crosslinkers need to be present inside the networks to offer a significantly different response. It thus tends to prove the reality of dithiolane-ene copolymerization sustaining the crosslinked network of the nanoparticles, while disulfide bond cannot be excluded given the spatial confinement of the radicals within the nanoparticles, and the water-free nanoparticle internal structure.⁴⁶ Trying to link the microscopic descriptions of matter issued from AFM studies and photochromism prompted us to eventually formulate the following assumption regarding matter organization and dynamics in organic nanoparticles. As abovementioned, the observation for both crosslinked nanoparticles of very fast and similar back thermal rate relaxations, despite strongly distinct elastic moduli, lets us suggest a dense crosslinked network, with little space to host the Z photoisomers that are constraint to quickly revert back. The large difference in elasticity as measured by AFM would thus derive from the extensive
incorporation of **DT1** and **DT2** in the network of the nanoparticles, strongly impacting the
mechanical behavior of the ensemble (much softer for **azoPC1**<sub>XL</sub>, and much stiffer for **azoPC2**<sub>XL</sub>).
By contrast, the photocrosslinked films, endowed with a slower thermal relaxation rate but similar
elastic moduli, seem to offer a looser network, suggesting that the **DT** crosslinkers are present in
a lower amount, and impact less the mechanical properties of the whole film. We thus suspect that
large matter confinement, as usually encountered in nanoparticles upon precipitation in water,
would promote close vicinity of the monomers and amplify the incorporation of **DT1** or **DT2** in
the final polymerized network, with possible disulfide bonds in more packed surroundings like
**azoPC2**<sub>XL</sub> nanoparticles. Matter confinement could also be evoked to explain the notable
difference in elasticity between **azoPC1**<sub>XL</sub> and **azoPC2**<sub>XL</sub> nanoparticles. Previous studies have
indeed reported the evolution of the elastic modulus *E* and glass transition temperature of core-
shell polymeric nanoparticles as a function of the shell thickness, using Brillouin light-scattering
spectroscopy and modulated differential scanning calorimetry, respectively.⁶⁶ They concluded that
nanoparticles, made out of a poly(butyl methacrylate) (PMBA) shell around a polystyrene core,
underwent significant increase in their elastic modulus and glass transition temperature when
decreasing the thickness of the outer PBMA layers. Such discrepancy was then ascribed to large
reduction of the mobility of the polymer chain at the free interface upon geometry confinement.
This important finding lets us suggest to better exploit the highly confined architecture of organic
nanoparticles in water so as to create dense networks with additional capability of phototunable
stiffness or deformation,⁶⁹ ruling the efficiency of cellular endocytosis for nanoparticle drug
delivery, while permitting degradability thanks to photo-, thermo- or chemo-assisted reduction of
the incorporated disulfide bonds.⁷⁰
CONCLUSIONS

Photochromic azo nanoparticles, issued from flash precipitation in water of a photocrosslinkable blend of azo-functionalized acrylate monomers and dithiolane monomers of varying flexibility, have been manufactured to gain insight into the evolution of matter cohesion at the nanoscale after photopolymerization. Substitution by a triarylamino electron-donating group gave access to the unusual Z→E back isomerization under UV irradiation after promoting the formation of Z isomers using irradiation in the visible range. Such a behavior allowed us to study the negative impact on photoconversion upon crosslinking or rigidification, while the dynamics of the back thermal relaxation dramatically increased after crosslinking. Comparison with thin films showed neat enhancement of the relaxation kinetics in nanoparticles, by one order of magnitude, suggesting a high-density network in nanoparticles after photocrosslinking, and less free volume (porosity) to adapt the newly formed isomers. This assumption was corroborated by AFM measurements on organic nanoparticles of only a few tens of nanometers in diameter, which showed large difference in the elastic modulus as a function of the stiffness of the dithiolane crosslinkers. Such a difference was found particularly pronounced with regard to that noted with crosslinked films. It emphasized this time the higher reactivity and incorporation of 1,2-dithiolane monomers in the network upon dithiolane-ene reaction as a result of extensive matter confinement during nanoprecipitation. Combining studies of the Z→E relaxation dynamics of photochromic nanoparticles with investigations of their mechanical properties at the single nanoparticle level points out the fact that highly crosslinked networks in nanoparticles do not necessarily rhyme with high stiffness, but rather than with smaller porosity. Such a porosity-elasticity duality, obtained by varying the crosslinking density and monomer flexibility respectively, appears to be particularly marked in nanoparticles and the consequence of large matter confinement upon precipitation. Hence, better
leveraging such confinement effects in nanoparticles, especially in the formation of reversible covalent bonds, would open the path to biodegradable nano-objects without compromising their mechanical resistance, and contribute to reduce the undesirable and alarming nanomaterial accumulation in our environment.

## ASSOCIATED CONTENT

**Supporting Information.** Synthesis pathways for compounds 2 to azoPC, NMR spectra, UV-vis and IR spectra, thermal analyses, optical microscopy of thin films, TEM and DLS measurements, photochromic properties.

## AUTHOR INFORMATION

### Corresponding Author

Eléna Ishow – Nantes Université, CNRS CEISAM, UMR 6230, F-44000 Nantes, France ;
orcid.org/0000-0002-2965-1224; Email: elena.ishow@univ-nantes.fr

Stéphane Cuenot, Nantes Université, CNRS, Institut des Matériaux de Nantes Jean Rouxel, IMN, F-44000 Nantes, France ; orcid.org/0000-0003-1883-3847 ; Email: stephane.cuenot@cnrs-imn.fr.

### Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

### Funding Sources
French Research National Agency (ANR) - research collaborative project "AZOTICS" (grant n° ANR-21-CE06-0034-01).

ACKNOWLEDGMENT

The French Research National Agency (ANR) is gratefully acknowledged for its essential financial
support through the research collaborative project "AZOTICS" (grant n° ANR-21-CE06-0034-
01). The authors would like to thank very much the IMN's characterization platform PLASSMAT,
Nantes, France for the TEM measurements, as well as Michael Bailly for the thermal analyses
performed at LTEN, Nantes, France. Laurence Arzel and Julie Hémez from the AMaCC platform
at CEISAM - Nantes University are strongly acknowledged for all mass spectrometry analyses.
Camille Alvarado and Angelina d'Orlando from the BIBS platform of Nantes-Angers INRA
(France) are also gratefully acknowledged for their kind advices regarding TEM image acquisition.
The Cordouan Technologies Company (Bordeaux, France) is strongly acknowledged for its
constant and rich support regarding the analyses of nanoparticle samples.

REFERENCES

(1) Zennifer, A.; Manivannan, S.; Sethuraman, S.; Kumbar, S. G.; Sundaramurthi, D. 3D bioprinting and photocrosslinking: emerging strategies & future perspectives. *Biomater. Adv. 2022*, *134*, 112576.

(2) Lim, K. S.; Galarraga, J. H.; Cui, X.; Lindberg, G. C. J.; Burdick, J. A.; Woodfield, T. B. F. Fundamentals and Applications of Photo-Cross-Linking in Bioprinting. *Chem. Rev.* **2020**, *120*, 10662-10694.

(3) Li, Y.; Chen, R. Photolithographic Patterning of Organic Semiconductors for Organic Field-Effect Transistors. *Chem. Rev.* **2025**, *125*, 4933-4973.
(4) Zuniga, C. A.; Barlow, S.; Marder, S. R. Approaches to Solution-Processed Multilayer Organic Light-Emitting Diodes Based on Cross-Linking. *Chem. Mater.* **2011**, *23*, 658-681.

(5) Blasco, E.; Wegener, M.; Barner-Kowollik, C. Photochemically Driven Polymeric Network Formation: Synthesis and Applications. *Adv Mater* **2017**, *29*, 1604005.

(6) Chen, M.; Zhong, M.; Johnson, J. A. Light-Controlled Radical Polymerization: Mechanisms, Methods, and Applications. *Chem. Rev.* **2016**, *116*, 10167-10211.

(7) Skliutas, E.; Merkininkaite, G.; Maruo, S.; Zhang, W. X.; Chen, W. Y.; Deng, W. T.; Greer, J.; Freymann, G. V.; Malinauskas, M. Multiphoton 3D lithography. *Nat. Rev. Meth. Primers* **2025**, *5*, 15.

(8) Kim, K.; Grummon, B. C.; Thrasher, C. J.; Macfarlane, R. J. Regio-Selective Mechanical Enhancement of Polymer-Grafted Nanoparticle Composites via Light-Mediated Crosslinking. *Adv. Mater.* **2025**, *37*, 2410493.

(9) Yue, K.; Trujillo-de Santiago, G.; Alvarez, M. M.; Tamayol, A.; Annabi, N.; Khademhosseini, A. Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. *Biomaterials* **2015**, *73*, 254-271.

(10) Jasinski, F.; Lobry, E.; Tarablsi, B.; Chemtob, A.; Croutxé-Barghorn, C.; Le Nouen, D.; Criqui, A. Light-Mediated Thiol–Ene Polymerization in Miniemulsion: A Fast Route to Semicrystalline Polysulfide Nanoparticles. *ACS Macro Lett.* **2014**, *3*, 958-962.

(11) Anselmo, A. C.; Zhang, M. W.; Kumar, S.; Vogus, D. R.; Menegatti, S.; Helgeson, M. E.; Mitragotri, S. Elasticity of Nanoparticles Influences Their Blood Circulation, Phagocytosis, Endocytosis, and Targeting. *ACS Nano* **2015**, *9*, 3169-3177.

(12) Guo, P.; Liu, D.; Subramanyam, K.; Wang, B.; Yang, J.; Huang, J.; Auguste, D. T.; Moses, M. A. Nanoparticle elasticity directs tumor uptake. *Nat. Commun.* **2018**, *9*, 130.
(13) Anselmo, A. C.; Mitragotri, S. Impact of Particle Elasticity on Particle-Based Drug Delivery Systems. *Adv. Drug Delivery Rev.* **2017**, *108*, 51–67.

(14) Li, M.; Gao, Z.; Lv, H.; Sekhar, K. P. C.; Song, A.; Jiang, X.; Hao, J.; Cui, J. Multilayered Nanoarchitectonics of Poly(ethylene glycol) Nanoparticles with Tunable Stiffness Modulate Bio-Nano Interactions and Targeted Drug Delivery. *ACS Nano* **2025**, *19*, 22240-22252.

(15) Dokukin, M. E.; Sokolov, I. On the Measurements of Rigidity Modulus of Soft Materials in Nanoindentation Experiments at Small Depth. *Macromolecules* **2012**, *45*, 4277-4288.

(16) Guo, D.; Xie, G.; Luo, J. Mechanical properties of nanoparticles: basics and applications. *J. Phys. D: Appl. Phys.* **2014**, *47*, 013001.

(17) Garcia, P. D.; Garcia, R. Determination of the elastic moduli of a single cell cultured on a rigid support by force microscopy. *Biophys. J.* **2018**, *114*, 2923-2932.

(18) Cuenot, S.; Fillaudeau, A.; Briolay, T.; Fresquet, J.; Blanquart, C.; Ishow, E.; Zykwinska, A. Poroelastic and viscoelastic properties of soft materials determined from AFM force relaxation and force-distance curves. *J. Mech. Behav. Biomed. Mater.* **2025**, *163*, 106865.

(19) Scotti, A.; Schulte, M. F.; Lopez, C. G.; Crassous, J. J.; Bochenek, S.; Richtering, W. How Softness Matters in Soft Nanogels and Nanogel Assemblies. *Chem. Rev.* **2022**, *122*, 11675-11700.

(20) Garcia, R. Nanomechanical mapping of soft materials with the atomic force microscope: methods, theory and applications. *Chem. Soc. Rev.* **2020**, *49*, 5850-5884.

(21) Takechi-Haraya, Y.; Goda, Y.; Izutsu, K.; Sakai-Kato, K. Improved Atomic Force Microscopy Stiffness Measurements of Nanoscale Liposomes by Cantilever Tip Shape Evaluation. *Anal. Chem.* **2019**, *91*, 10432–10440.
(22) Ikeda, T.; Mamiya, J.-i.; Yu, Y. Photomechanics of Liquid-Crystalline Elastomers and Other Polymers. *Angew. Chem. Int. Ed.* **2007**, *46*, 506-528.

(23) Priimagi, A.; Barrett, C. J.; Shishido, A. Recent twists in photoactuation and photoalignment control. *J. Mater. Chem. C* **2014**, *35*, 7155-7162.

(24) Fang Xu, B. L. F. Photoresponsive Supramolecular Polymers: From Light-Controlled Small Molecules to Smart Materials. *Adv. Mater.* **2023**, *35*, 2204413.

(25) Brasselet, S.; Zyss, J. Multipolar molecules and multipolar fields: probing and controlling the tensorial nature of nonlinear molecular media. *J. Opt. Soc. Am. B* **1998**, *15*, 257-288.

(26) Oscurato, S. L.; Salvatore, M.; Maddalena, P.; Ambrosio, A. From nanoscopic to macroscopic photo-driven motion in azobenzene-containing materials. *Nanophotonics* **2018**, *7*, 1387-1422.

(27) Natansohn, A.; Rochon, P. Photoinduced Motions in Azo-Containing Polymers. *Chem. Rev.* **2002**, *102*, 4139-4176.

(28) Yu, Y.; Nakano, M.; Ikeda, T. Directed Bending of a Polymer Film by Light. *Nature* **2003**, *425*, 145.

(29) Wani, O. M.; Zeng, H.; Priimagi, A. A light-driven artificial flytrap. *Nat. Commun.* **2017**, *8*, 15546.

(30) Iamsaard, S.; Aßhoff, S. J.; Matt, B.; Kudernac, T.; Cornelissen, J. J. L. M.; Fletcher, S. P.; Katsonis, N. Conversion of light into macroscopic helical motion. *Nat. Chem.* **2014**, *6*, 229-235.

(31) Pilz da Cunha, M.; van Thoor, E. A. J.; Debije, M. G.; Broer, D. J.; Schenning, A. P. H. J. Unravelling the photothermal and photomechanical contributions to actuation of
azobenzene-doped liquid crystal polymers in air and water. *J. Mater. Chem. C* **2019**, *7*,
13502-13509.

(32) Ma, S.; Li, X.; Huang, S.; Hu, J.; Yu, H. A Light-Activated Polymer Composite Enables On-Demand Photocontrolled Motion: Transportation at the Liquid/Air Interface. *Angew. Chem. Int. Ed.* **2019**, *58*, 2655-2659.

(33) Loudwig, S.; Bayley, H. Photoisomerization of an individual azobenzene molecule in water: an on-off switch triggered by light at a fixed wavelength. *J. Am; Chem. Soc.* **2006**, *128*, 12404-12405.

(34) Broichhagen, J.; Frank, J. A.; Trauner, D. A Roadmap to Success in Photopharmacology. *Acc. Chem. Res.* **2015**, *48*, 1947-1960.

(35) Maleeva, G.; Nin-Hill, A.; Wirth, U.; Rustler, K.; Ranucci, M.; Opar, E.; Rovira, C.; Bregestovski, P.; Zeilhofer, H. U.; König, B.; Alfonso-Prieto, M.; Gorostiza, P. Light-Activated Agonist-Potentiator of GABAA Receptors for Reversible Neuroinhibition in Wildtype Mice. *J. Am. Chem. Soc.* **2024**, *146*, 28822-28831.

(36) Leippe, P.; Donthamsetti, P.; Ko, T.; Stanley, C.; Isacoff, E.; Trauner, D. Cell-Specific Optical Control of AMPA Glutamate Receptors with a Photoswitchable Tethered Antagonist. *Angew. Chem. Int. Ed.* **2024**, *63*, e202411181.

(37) Montnach, J.; Blömer, L. A.; Lopez, L.; Filipis, L.; Meudal, H.; Lafoux, A.; Nicolas, S.; Chu, D.; Caumes, C.; Béroud, R.; Jopling, C.; Bosmans, F.; Huchet, C.; Landon, C.; Canepari, M.; De Waard, M. In vivo spatiotemporal control of voltage-gated ion channels by using photoactivatable peptidic toxins. *Nat. Commun.* **2022**, *13*, 417.

(38) Russew, M. M.; Hecht, S. Photoswitches: From Molecules to Materials. *Adv. Mater.* **2010**, *22*, 3348-3360.
(39) Ishow, E.; Lebon, B.; He, Y. N.; Wang, X. G.; Bouteiller, L.; Galmiche, L.; Nakatani, K. Structural and Photoisomerization Cross Studies of Polar Photochromic Monomeric Glasses Forming Surface Relief Gratings. *Chem. Mater.* **2006**, *18*, 1261-1267.

(40) Snell, K. E.; Mevellec, J.-Y.; Humbert, B.; Lagugné-Labarthet, F.; E., I. Photochromic organic nanoparticles as innovative platforms for plasmonic nanoassemblies. *ACS Appl. Mater. Int.* **2015**, *7*, 1932-1942.

(41) Bequet-Ermoy, E.; Silvestre, V.; Cuenot, S.; Ishow, E. Reversible Light-Triggered Stretching of Small-Molecule Photochromic Organic Nanoparticles. *Small* **2024**, *20*, 2403912.

(42) Yasuhiko Shirota, H. U., Toshiki Ujike, Satoru Yoshikawa,; Kazuyuki Moriwaki, D. N., Hideyuki Nakano. Photochromic amorphous molecular materials and their applications. *Opt. Mater.* **2002**, *21*, 249-254.

(43) Knie, C.; Utecht, M.; Zhao, F.; Kulla, H.; Kovalenko, S.; Brouwer, A. M.; Saalfrank, P.; Hecht, S.; Bléger, D. ortho-Fluoroazobenzenes: Visible Light Switches with Very Long-Lived Z Isomers. *Chem. Eur. J.* **2014**, *20*, 16492-16501.

(44) Bléger, D.; Schwarz, J.; Brouwer, A. M.; Hecht, S. o-Fluoroazobenzenes as Readily Synthesized Photoswitches Offering Nearly Quantitative Two-Way Isomerization with Visible Light. *J. Am. Chem. Soc.* **2012**, *134*, 20597-20600.

(45) Khine, Y. Y.; Nguyen, H.; Afolabi, F.; Lin, C.-C. Fast-relaxing hydrogels with reversibly tunable mechanics for dynamic cancer cell culture. *Biomater. Adv.* **2024**, *159*, 213829.

(46) Keyser, S. P.; Fairbanks, B. D.; Bahns, T.; Bowman, C. N. Dithiolane-Ene Copolymerization: Enabling Tunable, Dynamic, Dual-Cure Networks via Real-Time UV-Vis/FTIR Kinetics and Compositional Analysis. *Macromolecules* **2025**, *58*, 8468-8478.
(47) Nelson, B. R.; Kirkpatrick, B. E.; Miksch, C. E.; Davidson, M. D.; Skillin, N. P.; Hach, G. K.; Khang, A.; Hummel, S. N.; Fairbanks, B. D.; Burdick, J. A.; Bowman, C. N.; Anseth, K. S. Photoinduced Dithiolane Crosslinking for Multiresponsive Dynamic Hydrogels. *Adv. Mater.* **2024**, *36*, 2211209.

(48) Olivier, S.; Derue, L.; Geffroy, B.; Maindron, T.; Ishow, E. Small Molecule-Based Photocrosslinkable Fluorescent Materials toward Multilayered and High-Resolution Emissive Patterning. *J. Mater. Chem. C* **2015**, *3*, 8403-8412.

(49) Sahin, M.; Ayalur-Karunakaran, S.; Manhart, J.; Wolfahrt, M.; Kern, W.; Schlögl, S. Thiol-Ene versus Binary Thiol-Acrylate Chemistry: Material Properties and Network Characteristics of Photopolymers *Adv. Eng. Mater.* **2017**, *19*, 1600620.

(50) Neumann, M. G.; Miranda, W. G.; Schmitt, C. C.; Rueggeberg, F. A.; Correa, I. C. Molar extinction coefficients and the photon absorption efficiency of dental photoinitiators and light curing units. *J. Dent.* **2005**, *33*, 525-532.

(51) Daniel, J.; Bondu, F.; Adamietz, F.; Blanchard-Desce, M.; Rodriguez, V. Interfacial Organization in Dipolar Dyes-based Organic Nanoparticles probed by Second-Harmonic Scattering. *ACS Photonics* **2015**, *2*, 1209-1216.

(52) Gao, M.; Kwaria, D.; Norikane, Y.; Yue, Y. Visible-light-switchable azobenzenes: Molecular design, supramolecular systems, and applications. *Nat. Sci.* **2023**, *3*, e220020.

(53) Nakano, H.; Takanashi, T.; Kadota, T.; Shirota, Y. Formation of Surface Relief Grating Using a Novel Azobenzene-Based Photochromic Amorphous Molecular Material. *Adv. Mater.* **2002**, *14*, 1157-1160.

(54) Hu, H.; Zhou, Y.; Xi, B.; Li, Y. C. Polymer Mechanochemistry in Confined Spaces. *Angew. Chem. Int. Ed.* **2025**, *64*, e20241735.
(55) Faucon, A.; Lenk, R.; Hemez, J.; Gautron, E.; Jacquemin, D.; Le Questel, J.-Y.; Graton, J.; Brosseau, A.; Ishow, E. Fluorescent Carboxylic and Phosphonic Acids: Comparative Photophysics from Solution to Organic Nanoparticles. *Phys. Chem. Chem. Phys.* **2013**, *15*, 12748-12756.

(56) Maillard, J.; Klehs, K.; Rumble, C.; Vauthey, E.; Heilemann, M.; Fürstenberg, A. Universal quenching of common fluorescent probes by water and alcohols. *Chem. Sci.* **2021**, *12*, 1352-1362.

(57) Hoang, S.; Olivier, S.; Cuenot, S.; Montillet, A.; Bellettre, J.; Ishow, E. Microfluidic Assisted Flash Precipitation of Photocrosslinkable Fluorescent Organic Nanoparticles for Fine Size Tuning and Enhanced Photoinduced Processes. *ChemPhysChem* **2020**, *21*, 2502-2515.

(58) Kang, J.; Lim, Y. W.; Lee, I.; Kim, S.; Kim, K. Y.; Lee, W.; Bae, B. S. Photopatternable Poly(dimethylsiloxane) (PDMS) for an Intrinsically Stretchable Organic Electrochemical Transistor. *ACS Appl. Mater. Int.* **2022**, *14*, 24840-24849.

(59) Reinelt, S.; Tabatabai, M.; Moszner, N.; Fischer, U. K.; Utterodt, A.; Ritter, H. Synthesis and Photopolymerization of Thiol-Modified Triazine-Based Monomers and Oligomers for the Use in Thiol-Ene-Based Dental Composites. *Macromol. Chem. Phys.* **2014**, *215*, 1415-1425.

(60) Guo, D.; Li, J.; Xie, G.; Wang, Y.; Luo, J. Elastic Properties of Polystyrene Nanospheres Evaluated with Atomic Force Microscopy: Size Effect and Error Analysis. *Langmuir* **2014**, *30*, 7206-7212.
(61) Armini, S.; Vakarelski, I. U.; Whelan, C. M.; Maex, K.; Higashitani, K. Nanoscale Indentation of Polymer and Composite Polymer–Silica Core–Shell Submicrometer Particles by Atomic Force Microscopy. *Langmuir* **2007**, *23*, 2007-2014.

(62) Alsharif, N.; Eshaghi, B.; Reinhard, B. M.; Brown, K. A. Physiologically Relevant Mechanics of Biodegradable Polyester Nanoparticles. *Nano Lett.* **2020**, *20*, 7536-7542.

(63) Liang, X.; Mao, G.; Ng, K. Y. S. Mechanical properties and stability measurement of cholesterol-containing liposome on mica by atomic force microscopy. *J. Coll. Int. Sci.* **2004**, *278*, 53-62.

(64) Benne, N.; Leboux, R. J. T.; Glandrup, M.; van Duijn, J.; Lozano Vigario, F.; Neustrup, M. A.; Romeijn, S.; Galli, F.; Kuiper, J.; Jiskoot, W.; Slütter, B. Atomic force microscopy measurements of anionic liposomes reveal the effect of liposomal rigidity on antigen-specific regulatory T cell responses. *J. Controlled Release* **2020**, *318*, 246-255.

(65) Eshaghi, B.; Alsharif, N.; An, X.; Akiyama, H.; Brown, K. A.; Gummuluru, S.; Reinhard, B. M. Stiffness of HIV-1 Mimicking Polymer Nanoparticles Modulates Ganglioside-Mediated Cellular Uptake and Trafficking. *Adv. Sci.* **2020**, *7*, 2000649.

(66) Kang, E.; Graczykowski, B.; Jonas, U.; Christie, D.; Gray, L. A. G.; Cangialosi, D.; Priestley, R. D.; Fytas, G. Shell Architecture Strongly Influences the Glass Transition, Surface Mobility, and Elasticity of Polymer Core-Shell Nanoparticles. *Macromolecules* **2019**, *52*, 5399-5406.

(67) Rassmann, N.; Trippmacher, S.; Specht, A.; Theis, K.; Rössler, T.; Wohlrab, S.; Kellnberger, R.; Salehi, S.; Bargel, H.; Helfricht, N.; Albrecht, K.; Scheibel, T.; Groll, J.; Gekle, S.; Papastavrou, G. Determining the Elastic Modulus of Microgel Particles by Nanoindentation. *ACS Appl. Nano Mater.* **2025**, *8*, 5383-5398.
(68) Rosales, A. M.; Rodell, C. B.; Chen, M. H.; Morrow, M. G.; Anseth, K. S.; Burdick, J. A. Reversible Control of Network Properties in Azobenzene-Containing Hyaluronic Acid-Based Hydrogels. *Bioconjugate Chem.* **2018**, *29*, 905-913.

(69) Ohnsorg, M. L.; Hushka, E. A.; Anseth, K. S. Photoresponsive Chemistries for User-Directed Hydrogel Network Modulation to Investigate Cell-Matrix Interactions. *Acc. Chem. Res.* **2025**, *58*, 47-60.

(70) Choi, C.; Self, J. L.; Okayama, Y.; Levi, A. E.; Gerst, M.; Speros, J. C.; Hawker, C. J.; de Alaniz, J. R.; Bates, C. M. Light-Mediated Synthesis and Reprocessing of Dynamic Bottlebrush Elastomers under Ambient Conditions. *J. Am. Chem. Soc.* **2021**, *143*, 9866-9871.
Nanoparticles

<figure><img src="image_14.png" /><figcaption>elastic modulus</figcaption></figure>

Films

<figure><img src="image_15.png" /></figure>