# Morphology-Dependent Bioadhesion and Bioelimination of Hyaluronan Particles Administered in the Bladder

Raul Diaz-Salmeron, Balthazar Toussaint, Catherine Cailleau, Gilles Ponchel, Kawthar Bouchemal

► To cite this version:

Raul Diaz-Salmeron, Balthazar Toussaint, Catherine Cailleau, Gilles Ponchel, Kawthar Bouchemal. Morphology-Dependent Bioadhesion and Bioelimination of Hyaluronan Particles Administered in the Bladder. Advanced NanoBiomed Research, In press, 2 (5), pp.2100138. 10.1002/anbr.202100138. hal-03562452

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**Title** Morphology-Dependent Bioadhesion and Bioelimination of Hyaluronan Particles
Administered in the Bladder

Raul Diaz-Salmeron, Balthazar Toussaint, Catherine Cailleau, Gilles Ponchel, Kawthar Bouchemal*

R. D-S. Author 1, B. T. Author 2, C. C. Author 3, G. P. Author 4, K. B. Author 5

Université Paris-Saclay, Institut Galien Paris Saclay, CNRS UMR 8612, 92296, Châtenay-
Malabry France

E-mail: kawthar.bouchemal@universite-paris-saclay.fr

**Keywords:** hyaluronan, nanoparticles, nanoplatelets, bioadhesion, bioelimination, bladder, glycosaminoglycans

**Abstract**

Intravesical instillation allows for direct exposure of the urothelium to a drug. However, the therapeutic efficacy is often limited by rapid elimination of the drug from the bladder. In this investigation, we hypothesize that the morphology of nanoparticulate drug delivery systems could significantly impact bioadhesion and bioelimination from the bladder. Bioadhesion kinetics evaluated *ex vivo* on rat bladder mucosa demonstrate that particles with a flattened morphology, denoted as nanoplatelets, are rapidly attached to the mucosa at a lower concentration than nanospheres. The two particles have comparable surface potentials and equivalent volumes and are composed of hyaluronan, a nonsulfated polysaccharide that plays a significant role in restoring the bladder glycosaminoglycan layer. The bioaccumulation and bioelimination studied *in vivo* reveal that the nanoplatelets are eliminated from the rat bladders less rapidly than nanospheres. This investigation suggests that the bioadhesion of HA could be improved by optimizing particle morphology, opening new opportunities for the treatment of local urothelial diseases, such as interstitial cystitis/painful bladder syndrome, by restoring the loss of the glycosaminoglycan layer.

**1. Introduction**

Interstitial cystitis/painful bladder syndrome (IC/PBS) is a chronic and disabling disease responsible for pelvic pain and secondary infections.[1] This disease is generally undiagnosed, and patients often have symptoms for years before a firm diagnosis is made.[2] Although the
etiology is unknown, theories have associated IC/PBS with inflammatory responses, autoimmune disorders, microbial infections, and bladder wall defects characterized by a loss of the glycosaminoglycan (GAG) layer on the urothelial cell surface. Due to GAG bladder defects, the permeability of the urothelium to toxins found in the urine becomes excessive, resulting from diffuse transmural inflammation.[3] Among the different GAGs present on the cell surface, hyaluronan (HA) is a nonsulfated polysaccharide that plays a significant role in restoring the bladder GAG layer. The instillation of HA solution (also known as Cystistat®) directly in the bladder through a urethral catheter is often used as a therapeutic strategy for IC/PBS to promote restoring the defective GAG layer at the urothelium surface. Although Cystistat® was shown to have fewer side effects than other therapeutic strategies,[4] it has a long-term beneficial effect in only two-thirds of patients.[5] The reason for low efficacy could be due to several factors, including the dilution of the drug in the urine and drug elimination by spontaneous voiding. Indeed, the drug as a solution is held inside the bladder no more than two hours after intravesical administration. Therefore, the drug concentrations in the urothelium are expected to be low and transient. The drug concentration and the period of exposure of the drug to the urothelium are the two main parameters that showed importance for the efficacy of the drugs administered inside the bladder. In this context, efforts have been devoted to increasing the residence time of the active drug and maintaining an effective drug concentration close to the urothelium.[6] Different strategies have been envisioned by increasing either the viscosity or the bioadhesion of the drug delivery systems.[7] Thermosensitive hydrogels alone or containing adhesive polymers such as chitosan (CS), polycarbophil,[8] and hydroxypropylcellulose[9] have been used as a drug reservoir to extend the residence time in the bladder. However, high viscosity could increase the risk of urinary obstruction. Another strategy to increase the drug concentration and the residence time on the mucosa consists of encapsulating a drug inside bioadhesive particles that have a spherical morphology.[10] Bioadhesion was shown to improve by manipulating the surface properties or the size of the particles.[11] For the treatment of IC/BPS, as the active drug is HA itself,[12] designing carriers that can increase the polysaccharide concentrations close to the cell wall for restoring the GAG layer represents an unmet challenge for the scientific community. Indeed, HA shows low bioadhesive behaviors.[13] In particular, HA had a low adhesion time on the mucosa and a low value of the work of adhesion.[14] In this context, the strategy envisioned in this study was to consider particle morphology as a relevant parameter to improve bioadhesion. Particle morphology could affect the bioadhesion mechanism,[15] including the diffusion kinetics of the particle in the lumen (transport phase) and the ability of the particles to be immobilized by the glycome at the
urothelium surface (attachment phase).[16] Herein, the bioadhesive behaviors of nonspherical HA particles, denoted as nanoplatelets (NPs), were investigated. NPs have flat surfaces and a high aspect ratio (AR), as characterized by atomic force microscopy (AFM) under liquid conditions (AR up to 20).[17] The NPs were formed by supramolecular and hierarchical self-assembly in water of HA hydrophobically modified with palmitic acid (HA-PA) and α-cyclodextrin.[18] In this investigation, the bioadhesion behaviors of NPs were evaluated *ex vivo* and *in vivo* in rats after intravesical instillation. The results were compared to nanospheres (NSs) with a comparable surface composition and surface potential.

## 2. Results and discussion

### 2.1. Physicochemical characterizations

Two types of particles were investigated in this study, HA-NSs, and HA-NPs, the latter of which have a typical flattened morphology, hexagonal shape, and sharp edges.[17, 18c, 19] Physicochemical characterization of the HA-NPs and HA-NSs was previously performed in our research group by transmission electron microscopy (TEM), cryo-TEM, scanning electron microscopy (SEM), AFM imaging, and analysis of the surface potentials.[17, 18c, 19] HA-NPs dimensions determined by AFM under liquid conditions were ~100 – 137 nm in length and ~7 – 11 nm in height.[17] HA-NSs mean diameter evaluated from TEM and cryo-TEM was ~160 nm, while the height ranged from 130 to 150 nm, characterized by AFM.[20] The mean hydrodynamic diameter of CS spherical particles (CS-NSs), used as a positive control of bioadhesion,[21] was ~187 nm.[19] The diameter was characterized by quasi-elastic light scattering and AFM.[19] Herein, TEM observations used as a rapid characterization method confirm that the fluorescent labeling of the particles did not alter their morphology and size (Figure 1). The surface potentials characterizations revealed that after fluorescent labeling of HA-NPs and HA-NSs, the zeta potentials remained negative, and on the same order of magnitude (Table 1). Concerning CS-NSs, the labeling with rhodamine B decreased the zeta potential from 50.8 ± 2.5 mV to 32.2 ± 1.2 mV. Although the decrease in the zeta potential, previous investigations showed that the CS-NSs kept their bioadhesive behaviors after rhodamine B labeling.[21]

In this investigation, suspension of HA-PA in water, which serves for the preparation of the HA-NPs by assembly with α-cyclodextrin, was used as a control of bioadhesion. We hypothesized that the amphiphilic nature of the compound could improve the interactions with the urothelium. It was previously demonstrated that HA hydrophobically modified with alkyl chains forms macromolecular micelles in water above the critical micelle concentration
(37.3 µg mL⁻¹ for a degree of substitution of 12.7%).[22] TEM observations of HA-PA suspension in water revealed the existence of ~200 nm round-shaped particles in which the hydrophobic alkyl chains could be inside the micelles surrounded by the hydrophilic HA backbone.[18c]

<figure><img src="image_2.png" /><figcaption>**Figure 1:** TEM images of non-labeled particles (a, d, g), Cy5.5-labeled particles (b, e, h), and rhodamine B-labeled particles (c, f). The formulations were: HA-NPs (a, b, c), HA-NSs (d, e, f), or CS-NSs (g, h). Scale bars: 500 nm for (a, b, c, f, and h), 200 nm for (e), and 100 nm for (d and g).</figcaption></figure>

<table><caption>**Table 1:** Zeta potentials (ζ) of NPs and NSs composed of hyaluronan (HA) or chitosan (CS). The particles were labeled with Cyanine5.5* (Cy5.5) or rhodamine B**.</caption><thead><tr><th>Formulation</th><th>ζ (mV) of non-labeled particles</th><th>ζ (mV) of fluorescently labeled particles</th></tr></thead><tbody><tr><td>HA-NP</td><td>-29.1 ± 0.3</td><td>-31.3 ± 1.1*</td></tr><tr><td>HA-NS</td><td>-37.6 ± 1.8</td><td>-39.9 ± 0.9*<br>-34.2 ± 1.1**</td></tr><tr><td>CS-NS</td><td>50.8 ± 2.5</td><td>32.2 ± 1.2**</td></tr></tbody></table>

## 2.2. Ex vivo bioadhesion studies

### 2.2.1. Bioadhesion kinetics

Bioadhesion kinetics were studied to evaluate the impact of the particle morphology on the diffusion phase since this phase contributes to determining the time necessary to reach
adhesion equilibrium. The kinetics of bioadhesion of HA-NPs and HA-NSs were investigated on freshly excised bladder mucosa of healthy rats. The bioadhesive behaviors of the NSs and the NPs were analyzed within the framework of adsorption theories.[16a, 16b] For this purpose, the weights of the particles remaining attached to the mucosal surface after three cycles of washing were determined after different contact times of the suspensions with the urothelium. For this kinetic study, desorption was neglected.[16b] It was considered that the particles resisting the three washing cycles were irreversibly adsorbed. The bioadhesion kinetics were determined by keeping the other parameters influencing the bioadhesion constant (e.g., the surface of the mucosa, the temperature of the adhesion experiment, and particle concentration). The bioadhesion kinetics in Figure 2.a and 2.b showed that the HA-NPs adhered more quickly to the bladder mucosa than HA-NSs. The NSs rapidly reached adhesion saturation after 30 min. For the NPs, 60 min was necessary to reach saturation (Figure 2.a).

The initial phase of the adsorption kinetics up to 30 min of contact (Figure 2.b) was theoretically approached according to the diffusion model proposed by Durrer and coworkers.[16a, 16b] The rate constant ($k$) was calculated from the first phase of the adsorption kinetics following Equation 1. The results in Figures 2.c and 2.d show that the constant $k$ was twofold higher for the HA-NPs than for the NSs ($k_{NPs} = 12 \times 10^{-5} \text{min}^{-1}$ and $k_{NSs} = 5.73 \times 10^{-5} \text{min}^{-1}$). The constant $k$ depicts the diffusion of the particles in the suspension medium, suggesting that the NPs diffused faster than the NSs to the urothelium.

The number of NS or NP layers attached to the rat bladder mucosa was calculated considering their dimensions and the theoretical number of NSs or NPs enabling them to cover the entire mucosal surface, which was equal to $0.87 \text{ cm}^2$ (Equation S1-S9 in the Supporting Information). This method of expressing the results suggested that the particles behave very differently and accumulate as multiple layers at the urothelial surface. The calculated number of particle layers was 26 times higher for HA-NPs than for HA-NSs (315 layers of HA-NPs vs. 12 layers of HA-NSs). The number of adhered NP layers was 11 times higher than that of CS-NSs (28 layers) used as a control bioadhesive particles (Table 2).

These results could be explained by the ability to form multiple layers of particles at the urothelial surface. After the formation of the first layers involving NP-cell surface interactions, additional layers of NPs could be progressively deposited over time until saturation. Theoretical studies can be found in the literature showing that oblate and disc particles exhibited significantly higher adhesion probabilities than spherical particles.[23] The adhesion probability of rod-shaped particles with an AR of 5 was estimated to be approximately 20
times higher than that of spherical particles. Similarly, the oblate-shaped particles exhibited an almost ten times higher adhesion probability than the spherical particles.[23]

<figure><img src="image_3.png" /><figcaption>**Figure 2.** (a) Bioadhesion kinetics of HA-NPs (♦) and HA-NSs (○). The contact time of NPs or NSs on the mucosa varied from 2.5 min to 120 min for (a) and from 2.5 min to 30 min for (b). Dash points in panels (c) and (d) represent theoretical plots for the experimental data given in (a) for HA-NPs (▲) and for HA-NSs (■) following Equation 1. The rate constants were $k_{NPs} = 12 \times 10^{-5} \text{min}^{-1}$ for HA-NPs and $k_{NSs} = 5.73 \times 10^{-5} \text{min}^{-1}$ for HA-NSs. The surface of bioadhesion was 0.87 cm$^2$, and the bulk concentration of HA in NP and NS suspensions was maintained constant at 10 mg mL$^{-1}$. The time necessary to reach near-equilibrium bioadhesion was estimated at 120 min from the plateau observed in (a). The temperature was maintained at 25°C. *n* = 6 rats for each group.</figcaption></figure>

<table><caption>**Table 2.** The number of attached particles (HA-NPs, HA-NSs, and CS-NSs) labeled with rhodamine B and the theoretical number of particle layers formed on 0.87 cm$^2$ of the bladder mucosa.</caption><thead><tr><th>Formulation</th><th>Adhesion contact time (min)</th><th>Number of attached particles (× 10<sup>10</sup>)</th><th>Number of particle layers</th></tr></thead><tbody><tr><td rowspan="7">HA-NPs</td><td>2.5</td><td>3.73</td><td>59</td></tr><tr><td>5</td><td>1.75</td><td>28</td></tr><tr><td>10</td><td>3.51</td><td>56</td></tr><tr><td>30</td><td>9.88</td><td>156</td></tr><tr><td>45</td><td>17.10</td><td>271</td></tr><tr><td>60</td><td>19.92</td><td>314</td></tr><tr><td>120</td><td>20.00</td><td>315</td></tr><tr><td rowspan="7">HA-NS</td><td>2.5</td><td>1.54</td><td>3</td></tr><tr><td>5</td><td>1.28</td><td>3</td></tr><tr><td>10</td><td>3.62</td><td>4</td></tr><tr><td>30</td><td>4.02</td><td>10</td></tr><tr><td>45</td><td>4.03</td><td>10</td></tr><tr><td>60</td><td>4.27</td><td>11</td></tr><tr><td>120</td><td>5.04</td><td>12</td></tr><tr><td>CS-NS</td><td>120</td><td>11.03</td><td>28</td></tr></tbody></table>
### 2.2.2. Effect of temperature on bioadhesion

In this set of experiments, the near-equilibrium time determined in previous kinetic studies was maintained at 120 min, and the bioadhesion of HA-NPs was studied at 25°C and 37°C. The adhesive behaviors of HA-NPs were compared to other HA formulations (HA, HA-PA, and HA-NSs). The concentration of the polysaccharides in each formulation was kept constant at 10 mg mL⁻¹. According to the results in Figure 3.a (at 25°C) and 3.a' (at 37°C), the HA solution exhibited a low bioadhesion (0.41 g m⁻²), in agreement with previous findings.[13] ¹⁴] The amphiphilic HA-PA did not show any significant improvement in bioadhesion, compared to HA solution, regardless of the temperature of the experiment (Figure 3.b at 25°C and 3.b' at 37°C). In contrast, the formulation of HA-PA as HA-NPs by self-assembly with α-cyclodextrin resulted in the adhesion of a high amount of HA at the urothelium surface at both temperatures. The attached HA was 4.93 g m⁻² at 25°C (Figure 3.d) and 6.21 g m⁻² at 37°C (Figure 3.d'). Interestingly, at 37°C, the bioadhesion of HA-NPs was 2.38-times higher than that of the bioadhesive control CS-NSs (Figure 3.c'). Concerning HA-NSs, the adhesion at 25°C was as low as HA solution and HA-PA suspension (Figure 3.c). Increasing the temperature from 25°C to 37°C induced an increase in the bioadhesion of HA-NSs from 0.34 g m⁻² to 1.23 g m⁻². However, the attached HA remained five-fold lower for HA-NSs than that of HA-NPs at 37°C (Figure 3.c'). Therefore, it could be concluded that the HA-NPs exhibited the highest bioadhesion compared to the other formulations. The adhesion of particles studied in this investigation on the urothelium involves several types of noncovalent bonding, such as hydrogen, hydrophobic, and electrostatic interactions. When the temperature increased, the hydrogen bond stability decreased, but the stability of the hydrophobic interactions remained essentially constant.[24] A temperature increase results from different events, including the partial breaking of hydrogen bonds, an increase in hydrophobic interactions, and a decrease in the viscosity of the solution in which the particles are suspended. Furthermore, increasing the temperature of the experiment results in an increase in the particle diffusivity, as previously reported for HA-NPs and HA-NSs.[20] However, at 37°C, HA-NPs moved faster in water than HA-NSs and the surface explored by the HA-NPs was 33 times higher than that explored by HA-NSs, explaining the high bioadhesion of the NPs.[20] Another explanation of the increase in bioadhesion with increasing temperature from 25°C to 37°C could be the partial dehydration of the thin glycome layer on the urothelial cells. Previous investigations highlighted the role of water movement in bioadhesion since the adhesive and cohesive nature of the mucus gel increased when the water content decreased.[25]
<figure><img src="image_4.png" /><figcaption>**Figure 3.** Bioadhesion of HA formulations (HA (a, a'), HA-PA (b, b'), HA-NSs (c, c'), and HA-NPs (d, d')) was determined after 120 min of contact with the rat bladder mucosa. CS-NSs in (e, e') were used as a positive control for bioadhesion. The initial concentration of the polysaccharides (HA or CS) for all the formulations was kept constant at 10 mg mL⁻¹. The temperature of the experiment was varied as 25°C and 37°C. Two asterisks and ns (no significant differences) denote *p*-values < 0.01 and > 0.05, respectively, after one-way ANOVA followed by Tukey's multiple comparison post-test comparing the mean between different groups. *n* = 6 rats for each group.</figcaption></figure>

**2.2. In vivo evaluation of the residence time of HA formulations after intravesical administration to rats**

In this set of experiments, the residence time of HA-NSs and HA-NPs was evaluated in living healthy rats after intravesical administration. An HA-PA suspension and a solution of Cy5.5 were used as controls. The fluorescence of Cy5.5 in the bladders was monitored up to 7 days from the abdomen side of anesthetized rats using a real-time *in vivo* imager. Representative images of rat abdomen fluorescence of HA-NPs, HA-NSs, and HA-PA suspension at 24 h, 48 h, and 72 h are given in Figure 4. The fluorescence observed from the *in vivo* images (as presented in Figure 4) was then quantified by calculating the average radiant efficiency using the same threshold and the maximum and the minimum color scales for each image. Typical biphasic curves were obtained when the average radiant efficiency was plotted vs. time (Figure 5). Regardless of the formulation, the fluorescence signal first increased, followed by a second phase where the signal decreased. The increase in the fluorescence signal corresponds to the diffusion of the formulations in the bladder and their attachment to the urothelium surface. The decrease in the fluorescence signal corresponds to the bioelimination of the formulations by the natural voiding of the bladder. For NSs, the fluorescence increased until one-hour postadministration. Then, the signal continuously decreased until 3 h. The fluorescence was near zero after this period (Figure 5). Those results agreed with *ex vivo* experiments revealing that HA-NSs had low bioadhesion to the urothelium (Figure 3.c and 3').
For the HA-PA suspension, the fluorescence signal was higher than that for HA-NSs. Contrarily to HA-NSs, the fluorescence signal of HA-PA was still present after 72 h postadministration (Figure 4.b). The higher signal of HA-PA observed *in vivo* could be due to its higher flexibility and amphiphilicity compared to HA-NSs, which could be favorable for their diffusion in the urine and interaction with the urothelium surface. However, the high signal of HA-PA *in vivo* was unexpected regarding the *ex vivo* results, which showed that a small amount of HA-PA remained attached to the urothelium. The discrepancies in the *ex vivo* and *in vivo* results could be explained by the *ex vivo* experimental conditions of bioadhesion, which do not mimic what happens *in vivo*. We hypothesize is that HA-PA suspension could attach to the urothelium, but the three cycles of washing performed for *ex vivo* experiments may be too drastic compared to what happens *in vivo*, resulting from HA-PA removal.

Concerning HA-NPs, *ex vivo* bioadhesion experiments agreed well with *in vivo* data. Those results could be explained by the fast diffusion of HA-NPs through aqueous media, [20] and firm attachment to the urothelial cells through receptor-mediated mechanisms.[17] Several studies revealed that micro- and nanoparticles that have a high AR were taken up by cells faster and in large amounts than spheres. For a review article on shape-dependent endocytosis, see reference.[26] Although the mechanism of fast and higher cell uptake of particles with high AR is not well elucidated, it is believed that the high particle surface area could lead to strong interaction with the cell membrane.[27] The flat surface of the particles could result from strong multivalent interactions characterized by simultaneous binding of multiple ligands to multiple receptors.[28] Both experimental and theoretical works unambiguously indicate that ligand/receptor interaction strength and specificity could be improved by multivalency.[29] Besides the high AR of HA-NPs, the sharpness of the edges could be favorable for their internalization by cells. Previous works revealed that particles with sharp edges or irregular surfaces were faster internalized by cells than spheres or discs with a smooth surface.[30] Altogether, it could be concluded that due to their specific physicochemical properties, including the morphology, and biophysical behaviors in the bladder, the HA-NPs were able to counteract micturition and dilution after *in vivo* administration.
<figure><img src="image_5.png" /><figcaption>**Figure 4.** IVIS Lumina scans of rat abdomens after intravesical administration of Cy5.5-labeled HA formulations (HA-NSs (a), HA-PA (b), and HA-NPs (c)). Views were obtained at 24 h, 48 h, and 72 h after intravesical instillation. The concentration of the HA in the formulations was maintained at 10 mg mL⁻¹.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 5.** (a) Average radiant efficiency (RE) of rat bladder abdomens after the administration of Cy5.5-labeled HA formulations: HA-NPs (♦), HA-PA (▲), or HA-NSs (●). A solution of free Cy5.5 (■) was used as a negative control. Quantitative analysis of the bladders was performed with the same threshold and the same region of interest (ROI) from 1 h to 168 h postadministration. (b) A magnification of panel (a) by focusing on 1 to 24 h postadministration. The four columns in (c) represent the average RE of the rat's abdominal regions after 24, 48, and 72 h postadministration of the formulations: HA-NPs (black), HA-PA (dark grey), or HA-NSs (light grey) or a solution of free Cy5.5 (white). The two columns in (d) represent a magnification of the average RE of HA-NSs (light</figcaption></figure>
grey) or a solution of free Cy5.5 (white). One, two, three, and four asterisks denote *p*-values < 0.05, < 0.01, < 0.001, and < 0.0001, respectively, after the two-way Kruskal-Wallis test followed by Tukey's multiple comparison post-test. *n* = 6 rats for each group.

**3. Conclusion**

In summary, in this investigation, we quantitatively studied the effect of particle morphology on bioadhesion and bioelimination from rat bladder after intravesical administration. Herein, we demonstrated that a flat particle morphology was favorable for both the diffusion phase and the attachment phase of bioadhesion. Furthermore, the improvement in the bioadhesive behaviors of HA particles by optimizing their morphology resulted in lower bioelimination from the mucosal epithelium of live rats. However, we also highlighted discrepancies between *ex vivo* and *in vivo* results for HA hydrophobically modified with palmitic acid, which could be due to the three cycles of washing performed *ex vivo*. Those conditions could not mimic *in vivo* situations leading to material removal over washing cycles. Future investigations should be performed *ex vivo* by quantifying the polysaccharide remaining attached to the urothelium by varying the number of washing cycles. This optimization will lead to identifying the experimental conditions for a better correlation of *ex vivo* bioadhesion experiments with *in vivo* data. Finally, future investigations will be focused on the evaluation of the ability of HA formulations to cure bladder diseases such as IC/PBS.

**4. Experimental section**

**4.1. Materials**

HA (~51 × 10³ g·mol⁻¹ according to the manufacturer) was purchased from LifeCore Biomedical (Minnesota, USA). Cyanine 5.5-NH₂ (Cy5.5-NH₂, λ<sub>ex</sub> 679 nm, λ<sub>em</sub> 705 nm) was purchased from Lumiprobe (Montluçon, France). Cy5.5-labeled-*O*-palmitoyl hyaluronan (HA-PA) was synthesized and characterized as published elsewhere.[18c] CS (~20 × 10³ g·mol⁻¹ according to the manufacturer) was purchased from Amicogen (Seoul, Korea). Isobutyl cyanoacrylate monomer was obtained from WPI (Friedberg, Germany). PolyFluor® 570 (methacryloxyethyl thiocarbamoyl rhodamine B) was purchased from Biovalley Polyscience Inc® (Warrington, USA). Acetonitrile was obtained from Carlo Erba® (Val de Reuil, France). NaCl, α-cyclodextrin, phosphate buffer saline (PBS, pH~7.0~ 7.3), NaOH (1 N), sodium dodecyl sulfate, and nitric acid were purchased from Sigma Aldrich (St. Quentin Fallavier, France). A dialysis membrane with a molecular weight cutoff ~100,000 g·mol⁻¹ was purchased from Spectra/Por (Biovalley, Marne la Vallée, France). Milli-Q® water was used for all the experiments (resistivity 18.2 MΩ·cm at 21°C, Millipore purification system, Millex, SLAP 0225, Millipore, France).
## 4.2. Methods

### 4.2.1. Preparation and characterization of NSs, NPs, and controls

**NSs preparation:** NSs were prepared by emulsion polymerization by adapting previous works on polysaccharide NSs.[19, 31] Non-labeled poly(isobutyl cyanoacrylate) NSs coated with HA (denoted HA-NS) or CS (CS-NS) were prepared by dissolving of HA or CS (0.069 g) in dilute nitric acid (5 mL) at a concentration of 0.2 mol L⁻¹ under magnetic stirring in a glass tube at 40°C for 30 min. After HA dissolution, isobutyl cyanoacrylate monomer (0.250 mL) was added under vigorous magnetic stirring. The reaction was allowed to continue at 40°C under stirring for 24 h. The sample was cooled to room temperature. Then, NSs were purified first by filtration through a 1 µm filter to remove polymer aggregates and then by dialysis twice for 30 min, twice for 60 min, and once overnight against water (1 L). Finally, the pH of each NS suspension was adjusted to 6.5 with NaOH (1 N). HA NSs labeled with Cy5.5 were prepared according to the same method. In this case, native HA was previously coupled to Cy5.5 fluorescent dye,[18c] and polymerized with IBCA under vigorous magnetic stirring. HA or CS NSs labeled with rhodamine were prepared similarly, except that 5 min after the addition of isobutyl cyanoacrylate, a solution of PolyFluor®570 (1 mL) (a fluorescent competitive monomer at a 2 mg mL⁻¹ concentration in acetonitrile) was added. The reaction was allowed to continue at 40°C under stirring for 24 h. Then, the NSs were purified by filtration followed by dialysis according to the process described above for non-labeled NSs.

**NPs preparation:** HA NPs labeled with Cy5.5 were prepared by adding α-cyclodextrin solution (500 mg) in water to a small vial containing Cy5.5-labeled-*O*-palmitoyl hyaluronan (10 mg). α-cyclodextrin solution was prepared in water at a 100 mg mL⁻¹ concentration. The weight was then brought up to 1 g with the addition of water and mixed at room temperature under magnetic stirring for 72 h, as previously optimized.[18c]

**Preparation of controls:** Hydrophobically modified HA suspensions in water (HA-PA) were prepared according to the same protocol used for NPs, without adding α-cyclodextrin. Solutions containing native HA were freshly prepared by mixing HA (10 mg) in water (1 mL).

Noteworthy, the stability of NPs and NSs over storage at variable temperatures and in biological media was already demonstrated.[32]

### 4.2.2. Physicochemical characterizations

The zeta potentials of NSs and NPs were calculated at 25°C by measuring the electrophoretic mobility using a Zetasizer (Nanoseries Nano-ZS, Malvern Instruments, France).[18c, 33] The scattered angle was 173°. The suspensions were diluted to (1:33 v/v) in NaCl (1 mM).
Measurements were performed on three independent formulations. The morphology of NSs and NPs labeled or not with fluorescent dyes was characterized by TEM according to previous works.[18c, 33] The images were obtained at 80 kV transmission using a transmission electron microscope (JEOL 1400) coupled to TEM Domain Center software. The suspensions were diluted in water (1/30 and 1/100 for NPs and NSs, respectively). The dilutions were optimized to allow the observation of particles with good contrast. Then, dilute suspensions (5 µL) were placed on a carbon grid. After 3 min of drying at room temperature, NPs were directly visualized under the microscope, while NSs were stained with a phosphotungstic acid solution before observation.

### 4.2.3. *Bioadhesion ex vivo assays*

**Preparation of the bladder mucosa:** Fresh bladders were removed from euthanized female Wistar rats (250-300 g, Janvier Labs, Saint Berthevin, France) as described in the Supporting Information. Then, the bladders were excised and opened lengthwise and longitudinally. The bladders were then spread on an aluminum plate. A tight rubber seal with a diameter of 0.87 cm² was positioned on the mucosa. Then, a second aluminum plate with a cylindrical slit in the center, able to accommodate 700 µL, was coupled to the tight seal and the first plate (Figure S1 in the Supporting Information).

**Bioadhesion kinetics:** Suspensions of fluorescently labeled HA-NPs (labeled with Cy5.5) and spherical particles (labeled with rhodamine B) (600 µL at a 10 mg mL⁻¹ concentration) were put in contact with a 0.87 cm² delimited bladder mucosa at different time intervals (2.5, 5, 10, 30, 45, 60 and 120 min) to determine the time necessary to reach an equilibrium adhesion plateau. The temperature was maintained at 37°C.

**Investigation of the impact of particle morphology on Bioadhesion:** Once the attachment equilibrium was determined, the impact of particle morphology on the bioadhesion was determined after two hours of incubation. The tested formulations were HA-NPs and HA-NSs. The temperature was varied from 25°C to 37°C. The bioadhesion results were compared to different controls: a native HA solution, hydrophobically modified HA suspension (HA-PA), and CS-NSs suspension (used as a positive control of bioadhesion). The HA concentration was maintained at 10 mg mL⁻¹ regardless of the formulation.

**Quantification of attached materials:** At the end of bioadhesion experiments, the excess fluorescently labeled formulations were aspirated using a pipette and rinsed three times with a PBS solution (600 µL) for 1 min each rinsing cycle to eliminate unattached materials (HA-NPs, HA-PA, HA, HA-NSs or CS-NSs). Subsequently, the bladder tissue, including the
attached materials, was harvested, and solubilized in 2.5 mL of a solution of NaOH (1 wt%) and sodium dodecyl sulfate (2 wt%). Samples were ultrasonicated for two hours and left overnight at room temperature under magnetic stirring. The attached materials were quantified from the absorbance signal and UV spectra of extracted samples containing the polymers (Lambda 25 UV/VIS Spectrometer, PerkinElmer, Waltham, MA, USA) (Supporting Information, Figures S2 to S7). The assay of HA associated with urothelial tissues was based on the determination of the concentrations of the fluorescent probes (rhodamine and Cy5.5). Since the medium was complex, the second derivatives of the UV spectra were used to isolate the spectral variations related to these molecules from the signal originating from the media. The spectra were analyzed by the Savitzky-Golay algorithm, and convolution coefficients were those needed to perform a quadratic/cubic polynomial adjustment with a window size of 15 values. The wavelengths ranged from 400 nm to 700 nm for rhodamine B and from 500 nm to 800 nm for Cy5.5. The amplitude between the minima and the maxima observed on second-derivative spectra was used as a signal proportional to the concentration. For rhodamine, the minimum signal was observed at 545 nm, and the maximum was observed at 566 nm. For Cy5.5, the minimum and the maximum signals were observed at 679 nm and 705 nm, respectively. The experimental data were compared to the corresponding calibration curves generated under the same experimental conditions (Supporting Information Figures S3, S5, and S7). The results were expressed as the amount of the attached polymer per apparent surface of the bladder mucosa (g.m⁻²). For mechanistic analysis, the number of attached NSs and NPs was calculated and expressed as the number of theoretical layers covering the exposed surface of the mucosa. The rate constants k characterizing the adsorption kinetics were estimated according to Durrer et al.,[¹⁶ᵃ, ¹⁶ᵇ] from the plot of ln[Cₜ/(Cₜ − Cₜ)] vs. time t following Equation 1:

$$ \ln[C_t/(C_t - C_f)] = \ln[C_0/(C_0 - C_f)] + [C_f/(C_0 - C_f)] k t \quad (1) $$

where C₀, Cₜ, and Cₜ are the particle concentrations (mg mL⁻¹) at time 0, at time t, and at equilibrium, respectively. The concentrations Cₜ and Cₜ were calculated as the difference between the initial material concentration in the suspension medium (10 mg mL⁻¹) and the adsorbed material amount at time t and at equilibrium, respectively.

### 4.2.4. In vivo evaluation of the residence time of the formulations in rat bladders

**Animals:** Adult female Wistar rats (~250 – 300 g, Janvier Labs, Saint Berthevin, France) were used to evaluate the residence time of the formulations in the bladders. The rats were
housed in a standard controlled environment at 22°C, 60% relative humidity, a 12-h light/12-h dark cycle with lights on at 8 am, and access to food and water *ad libitum*. This study was conducted following the ethical standards approved by the Animal Ethics Committee of the University of Paris-Saclay following EU Directive 2010/63/EU for animal experiments (agreement N°15378-2018060610295653).

**Investigation of the residence time of the formulations in rat bladders:** The residence time of the formulations in rat bladders was determined *in vivo* after intravesical instillation of fluorescently labeled formulations (500 µL, 10 mg mL⁻¹ containing 0.15% of Cy5.5) or PBS (used as a negative control) in healthy rats (n = 6 animals per group). The residence time of each formulation was recorded at 0, 1, 2, 3, 4, 5.5, and 7 h, and from 1 to 7 days with an IVIS Lumina LT Series III System (Caliper Life Sciences) using a 660 nm excitation laser and a 710 nm emission filter. During imaging experiments, the animals were kept under anesthesia with 3% isoflurane in oxygen flow (1 L min⁻¹) on the imaging stage. The abdomen regions of rats were shaved before imaging. The images and the measurements of the fluorescent signals were acquired and analyzed with Living Imaging software. A manual threshold was selected for each image to measure the averaged radiant efficiency. Fluorescent background signals using a 580 nm excitation laser and 710 nm emission filter were subtracted from the fluorescent signal using the same selected threshold in both images.

### 4.2.5. Statistical analyses

Statistical analyses were performed using GraphPad Prism Version 7.0 software. The normality of all experimental values was examined with a Shapiro-Wilk test. The weights of the attached polysaccharides per m² of the urothelial surface were statistically assessed with a one-way analysis of variance test (ANOVA) followed by a Tukey's multiple comparison post-test comparing the mean between different groups. Bioadhesion in vivo values were statistically examined with a two-way Kruskal-Wallis test followed by a Tukey's multiple comparison post-test. The level of significance for all statistical analyses was set at *p*-value < 0.05

## Supporting Information

Supporting Information is available from the Wiley Online Library or from the author.

## Acknowledgements
The present work has benefited from the Imagerie- Gif core facility supported by l'Agence Nationale de la Recherche (ANR-11-EQPX-0029/Morphoscope, ANR-10-INBS-04/FranceBioImaging; ANR-11-INDEX-0003-02/Saclay Plant Sciences). KB received funding from the Institut Universitaire de France and from the Agence Nationale de la Recherche, ANR-17-CE09-0038-1 (ANR JCJC Acronym Shape), ANR-21-CE09-0015-01 (ANR PRC Acronym CoroNa). Raul Diaz-Salmeron received a grant from the French Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation (MESRI).

## Conflict of Interest

The authors have no conflicts of interest.

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The table of contents entry

Intravesical instillation is limited by rapid drug elimination. Herein, we demonstrate that the flat morphology of hyaluronan particles significantly improves bioadhesion and decreases bioelimination from the bladder. Bioadhesion kinetics are evaluated *ex vivo* on rat bladder mucosa, while particle bioelimination was studied *in vivo* using a real-time imager system.
This investigation could open opportunities for efficient drug delivery

Raul Diaz-Salmeron, Balthazar Toussaint, Catherine Cailleau, Gilles Ponchel, Kawthar Bouchemal*

R. D-S. Author 1, B. T. Author 2, C. C. Author 3, G. P. Author 4, K. B. Author 5

Université Paris-Saclay, Institut Galien Paris Saclay, CNRS UMR 8612, 92296, Châtenay-
Malabry France

E-mail: kawthar.bouchemal@universite-paris-saclay.fr

**Title** Morphology-Dependent Bioadhesion and Bioelimination of Hyaluronan Particles
Administered in the Bladder

ToC figure

<figure><img src="image_7.png" /></figure>
# Supporting Information

**Title:** Morphology-Dependent Bioadhesion and Bioelimination of Hyaluronan Particles
Administered in the Bladder

Raul Diaz-Salmeron, Balthazar Toussaint, Catherine Cailleau, Gilles Ponchel, Kawthar Bouchemal*

Université Paris-Saclay, Institut Galien Paris Saclay, CNRS UMR 8612, 92296, Châtenay-
Malabry France

E-mail: kawthar.bouchemal@universite-paris-saclay.fr

## 1. Detailed protocol for particle Bioadhesion

a) After rat euthanasia, bladders were removed.

b) Each bladder was placed on a sterile compress moistened with phosphate buffer saline (PBS, pH 7.4, Sigma-Aldrich, Saint-Quentin Fallavier, France).

c) The bladders were longitudinally opened using surgical pliers and small scissors.

d) Once the bladders were open, they were spread out by keeping the mucous side at the top.

e) The mucous membrane was rinsed with PBS to eliminate the residual urine.

f) After bladder spreading (mucous side up), it was deposited on the lower Bioadhesive plate.

g) A 10.5-mm-diameter seal was placed on the mucosa to avoid fluid loss and leakage (Figure S2.a).

h) The upper metallic plate with an 8-mm-diameter cylindrical slit was placed on the lower metallic plate containing the mucosa and the seal (Figure S1.b).

i) The system was tightened with the fixer clamps (Figure S1.b).

j) Each tested preparation (650 µL) was then added.

k) Bioadhesion assays were then performed as described in the main manuscript.
<figure><img src="image_8.png" /><figcaption>**Figure S1.** (a) Picture of the lower side of the metallic plate with a rubber (10.5 mm in diameter). (b) picture of the Bioadhesion system with the seals.</figcaption></figure>

The dimensions of the bioadhesion system are as follows:

- The thickness of each plate rubber: 7 mm

- The width of the plate rubbers: 3.9 cm

- The length of the plate rubbers: 8 cm

- The diameter of the seal $d_{Seal}$ = 10.5 mm = 1.05 cm

- The surface of adhesion: $S_{Adhesion}$ = 0.87 cm²

## 2. Calibration curves

### 2.1. Calibration curve of hyaluronan poly(isobutyl cyanoacrylate) nanospheres labeled with rhodamine B

**Step 1:** Preparation of samples containing NS at different concentrations:

a) First, a sample of 10 mL of hyaluronan nanospheres (HA-NS) labeled with rhodamine B or Cynaine5.5 (Cy5.5) was prepared as described in the main text. The final concentration of nanospheres (NSs) was $[C]_{0NS}$ = 20 mg·mL⁻¹ as determined by gravimetric analysis.

b) The stock sample was first diluted to 10 mg mL⁻¹ with water, then diluted again with water to reach the following concentrations: 0.75, 0.50, 0.25, 0.10, 0.05, and 0.01 mg mL⁻¹.

**Step 2:** NSs were then degraded to release the fluorescent dye contained in the particle core as described below:

a) The samples were ultrasonicated for 2 h and magnetically stirred overnight.

b) Samples were diluted at 1/4 with a solution of NaOH and sodium dodecyl sulfate (SDS) (250 µL NS sample mixed to 750 µL of NaOH/SDS solution).

c) Samples were then diluted at 1/4 by adding the extraction solvents (250 µL NS sample mixed to 250 µL of ethanol and 500 µL of acetonitrile). Briefly, a suspension composed of bladder tissue digested in 1% NaOH/ 2% SDS (250 µL) was added to small vials. Then, protein precipitation was performed with the addition of 250 µL of ethanol followed by 30 s of vortex mixing and 500 µL of acetonitrile followed by 30 s of vortex mixing.[1] After being left 5 min at room temperature to optimize and allow total protein precipitation, samples were centrifuged at 2200 g for 10 min at room
temperature, and 800 µL of the supernatant were collected into 1.5 mL-polypropylene tubes.

d) The concentrations of rhodamine B and Cy5.5 were determined by a UV/VIS spectrophotometer (Perkin Elmer, Waltham, MA, USA) at 552 nm and 680 nm, respectively.

e) Data were analyzed by transforming the absorbance spectra into second derivatives.

Typical spectra were reported in Figure S2 and the calibration curve in Figure S3.

<figure><img src="image_9.png" /><figcaption>**Figure S2.** Second-order derivative spectra of NS suspensions composed of poly(isobutyl cyanoacrylate) core coated with HA and labeled with rhodamine B (HA-NS). NS concentration ranged from 0.0025 to 0.1875 mg mL⁻¹.</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>**Figure S3.** Calibration curve of NS suspensions composed of HA-NS labeled with rhodamine B.</figcaption></figure>
## 2.2. Calibration curve of chitosan poly(isobutyl cyanoacrylate) NSs labeled with rhodamine B

The calibration curve was obtained following the same steps (from step (a) to step (f)) as described in the previous section 2.1.

<figure><img src="image_11.png" /><figcaption>**Figure S4.** Second-order derivative spectra of NS suspensions composed of NS core coated with CS and labeled with rhodamine B (CS-NS). NS concentration ranged from 0.0025 to 0.1875 mg mL⁻¹.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Figure S5.** Calibration curve of NS suspensions composed of poly(isobutyl cyanoacrylate) core coated with CS and labeled with rhodamine B.</figcaption></figure>

## 2.3. Calibration curve of HA nanoplatelets and HA-NSs labeled with Cy5.5

**Step 1:** Preparation of samples containing HA particles (HA-NPs or HA-NSs) at different concentrations:

a) First, 3 mL of fluorescent particles were prepared as described in the main text.

b) Suspensions of particles labeled with Cy5.5 were diluted with water to reach the following concentrations expressed in HA: 0.01, 0.05, 0.10, 0.25, 0.50, and 0.75 mg mL⁻¹.
**Step 2:** The particles were then degraded to release the fluorescent dye:

a) The samples were ultrasonicated for 2 h and magnetically stirred overnight.

b) Samples were diluted at 1/4 by adding the extraction solvents (250 µL of particle sample mixed with 250 µL of ethanol and 500 µL of acetonitrile). Briefly, a suspension composed of bladder tissue digested in a mixture of NaOH/SDS (1/2%) (250 µL) was added to small vials. Then, protein precipitation was performed with the addition of 250 µL of ethanol followed by 30 s of vortex mixing and 500 µL of acetonitrile followed by 30 s of vortex mixing.[2] After being left 5 min at room temperature to optimize and allow total protein precipitation, samples were centrifuged at 2200 g for 10 min at room temperature, and 800 µL of the supernatant were collected into 1.5 mL-polypropylene tubes.

c) The concentrations of Cy5.5 were determined by a UV/VIS spectrophotometer at 552 nm and 680 nm, respectively.

d) Data were analyzed by transforming the absorbance spectra into second derivatives.

Typical spectra were reported in Figure S6 and the calibration curve in Figure S7.

<figure><img src="image_13.png" /><figcaption>**Figure S6.** Second-order derivative spectra of HA-NP suspensions labeled with Cy5.5 at different concentrations.</figcaption></figure>

<figure><img src="image_14.png" /></figure>
**Figure S7.** Calibration curve of HA-NP suspensions labeled with Cy5.5.

**3. Data analysis and details of calculations**

From bioadhesion experiments, the mass of attached N particles per m² of bladder mucosa ($m_T$, g.m⁻²) was calculated from the calibration curves.

**3.1. NSs**

The total number of attached NSs ($N_{NS}$) was calculated following the Equation S1-S3, where $ρ$ is the bulk density of preparations ($ρ_{NS}$ =1.2 g.cm⁻³ determined by a pycnometer device at 20°C), $V_{(N_{NS})}$ is the total volume occupied by N NSs and $r_{h_{NS}}$, and $d_{h_{NS}}$ are the mean hydrodynamic radius and diameter of NSs.

$$N_{NS} = \frac{m_{T(N_{NS})}}{\rho_{NS} V_{(N_{NS})}} \qquad (S1)$$

$$N_{NS} = \frac{m_{T(N_{NS})}}{\rho_{NS} \left(\frac{4}{3}\right) \pi r_{h_{NS}}^3} \qquad (S2)$$

$$N_{NS} = \frac{m_{T(N_{NS})}}{\rho_{NS} \left(\frac{4}{3}\right) \pi \left(\frac{d_{h_{NS}}}{2}\right)^3} \qquad (S3)$$

It is possible to calculate the number of layers that theoretically cover the 0.87 cm² of the bladder surface:

$$N_{NS/Layer} = \frac{S_{mucosa}}{S_{NS/Layer}} = \frac{0.87}{\pi \left(\frac{d_{h_{NS}}}{2}\right)^2} \qquad (S4)$$

$$N_{Layers} = Y = \frac{N_{NS}}{N_{NS/Layer}} \qquad (S5)$$

**3.2. NPs**

The same calculations were used for HA-NPs suspensions except that the surface of the hexagons was calculated from the equation: $S_{NP} = \frac{3\sqrt{3}}{2} a_{NP}^2$, where $α_{NP}$ is the hexagon side determined from our previous work.[3]

$$N_{NP} = \frac{m_{T(NP)}}{\rho_{NP} V_{(N_{NP})}} \qquad (S1')$$

$$N_{NP} = \frac{m_{T(N_{NP})}}{\rho_{NP} S_{w_{NP}}} \qquad (S6)$$

$$N_{NP} = \frac{m_{T(N_{NS})}}{\rho_{NS} \frac{3\sqrt{3}}{2} a_{NP}^2 w_{NP}} \qquad (S7)$$

$w_{NP}$ corresponds to the width of the NPs determined from our previous work.[3]

It is also possible to calculate the number of layers that theoretically cover the 0.87 cm² of the bladder surface:

$$N_{NP/Layer} = \frac{S_{mucosa}}{S_{NP/Layer}} = \frac{0.87}{\frac{3\sqrt{3}}{2} a_{NP}^2} \qquad (S8)$$
$$N_{Layers} = Y = \frac{N_{NP}}{N_{NP/Layer}} \qquad (S9)$$

References

[1] aJ. Abian, M. Carrascal, in *Journal of chromatography library, Vol. 68*, Elsevier, **2003**, pp. 39-73; bJ.-H. Chen, S. Singer, in *The Handbook of Metabonomics and Metabolomics*, Elsevier, **2007**, pp. 149-169.

[2] B. Toussaint, F. Lanternier, C. Woloch, D. Fournier, M. Launay, E. Billaud, E. Dannaoui, O. Lortholary, V. Jullien, *Journal of Chromatography B* **2017**, 1046, 26-33.

[3] R. Diaz-Salmeron, G. Ponchel, K. Bouchemal, *European Journal of Pharmaceutical Sciences* **2019**, 133, 251-263.