Exploring Protein Corona Formation on
Fucoidan-Coated Poly(isobutyl cyanoacrylate)
Nanoparticles: Implications for Drug Delivery

Iago Dillion Lima Cavalcanti, José Cleberson Santos Soares, Sinara Mônica

Vitalino De Almeida, Otília Pessoa, Evandro Silva, Marcelo Sobral Da Silva,

Francisco Humberto Xavier Junior, Eloisa Berbel Manaia, Gilles Ponchel,

Nereide Stela Santos Magalhães, et al.

► To cite this version:

Iago Dillion Lima Cavalcanti, José Cleberson Santos Soares, Sinara Mônica Vitalino De Almeida, Otília Pessoa, Evandro Silva, et al.. Exploring Protein Corona Formation on Fucoidan-Coated Poly(isobutyl cyanoacrylate) Nanoparticles: Implications for Drug Delivery. ACS Applied Nano Materials, 2025, 8 (19), pp.9878-9892. 10.1021/acsanm.5c00999. hal-05462873

HAL Id: hal-05462873

https://cnrs.hal.science/hal-05462873v1

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# Exploring Protein Corona Formation on Fucoidan-Coated Poly(isobutyl cyanoacrylate) Nanoparticles: Implications for Drug Delivery

Iago Dillion Lima Cavalcanti, José Cleberson Santos Soares, Sinara Mônica Vitalino de Almeida, Otília Pessoa, Evandro Silva, Marcelo Sobral da Silva, Francisco Humberto Xavier Junior, Eloisa Berbel Manaia, Gilles Ponchel, Nereide Stela Santos Magalhães,* and Mariane Cajubá de Britto Lira Nogueira*

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Cite This: ACS Appl. Nano Mater. 2025, 8, 9878–9892

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Supporting Information

**ABSTRACT:** Serum proteins play an essential role in nanosystems' stealth properties governing their distribution and targeting to specific sites. Opsonin proteins can induce nanoparticle degradation, elimination, and reduced therapeutic efficiency. The objective is to evaluate how PIBCA nanoparticles coated with fucoidan (NP-Fuc) interact with serum proteins, clarifying their behavior within the body and understanding whether this can impact the *in vivo* application of these nanoparticles. Static and dynamic interaction studies were carried out with bovine serum albumin (BSA), fetal bovine serum (FBS), and human serum using nanosight, absorption, and fluorescence spectroscopy. Scanning electron microscopy, infrared spectroscopy, and hydrogen nuclear magnetic resonance studies were conducted. Static and dynamic studies showed interaction of NP-Fuc with BSA, evidenced by the increase in nanoparticle size (from 349.5 ± 4.34 nm to 2 µm). Additionally, nanosight analysis also showed the interaction between BSA and NP-Fuc according to the refringence and the hydrodynamic radius (cloud effect) around the nanoparticles. The adsorption technique showed the interaction due to changes in BSA's absorption and fluorescence profiles. Infrared and ¹H NMR spectra showed the presence of BSA in the NP-Fuc-BSA nanoparticles, indicating the protein binding to nanoparticles. Considering the results, NP-Fuc interacts with BSA to form an NP-Fuc-BSA complex. Further studies are important to optimize the size of NP-Fuc-BSA as a promising nanocarrier for drug release.

**KEYWORDS:** fucoidan, core-corona nanoparticles, serum proteins, polimeric nanoparticles, albumin

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## 1. INTRODUCTION

Polymeric nanoparticles have been increasingly used to develop nanomedicines due to their low toxicity, controlled release, and drug encapsulation properties, besides the usefulness for the development of nanovaccines.¹⁻³ Given the high importance of nanoparticles in the pharmaceutical scenario and their use in diagnosis and therapy, evaluating nanoparticles' behavior in the body is crucial.⁴⁻⁶

There is a wide range of polymers that can be used in the development of nanoparticles, which can be polymers of natural origin (chitosan, albumin, sodium alginate, and gelatin) or synthetic polymers (Polylactides (PLA), poly(lactic coglycolic) (PLGA), polyglycols (PGA), poly(vinyl alcohol) (PVA), polyacrylamide (PAM), and polyethylene glycol (PEG)).⁷ Isobutyl cyanoacrylate (IBCA) is a monomer derived from alkyl cyanoacrylate that has been used in the preparation of nanoparticles with great potential as a drug delivery system.⁸⁻¹⁰ Our research group has been working with

IBCA nanoparticles, which is a biodegradable synthetic monomer with great potential as a controlled drug delivery system.⁸,⁹,¹¹,¹² In a recent study, Lira Nogueira et al.¹⁰ evaluated the endocytosis pathways of nanoparticles coated with fucoidan (NP-Fuc), seeking to identify the endocytic pathway of nanoparticles in macrophages cells line (J774A.1 cells), showing that nanoparticles with a charge close to neutrality appear to be less captured when compared to those with a negative charge, emphasizing the importance of

Received: February 24, 2025

Revised: April 22, 2025

Accepted: April 24, 2025

Published: May 6, 2025

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<figure><img src="image_10.png" /><figcaption>**Figure 1.** Synthesis scheme for fucoidan-coated nanoparticles (NP-Fuc).</figcaption></figure>

evaluating the *in vivo* interactions of these nanoparticles with proteins and how the charge can influence this process.

Fucoidan is a sulfated polysaccharide that provides a negative charge to nanoparticles, which can confer stealth properties and thereby reduce the level of binding of opsonins. In addition, fucoidan has biological activities such as antioxidant, anti-inflammatory, and antitumor.<sup>13-15</sup> Due to its wide range of therapeutic properties and the versatility of its structure, fucoidan has attracted significant attention as a promising polysaccharide for future therapeutic applications. Its ability to be incorporated into nanosystems—particularly as a targeting molecule—has further reinforced its potential in the development of advanced drug delivery strategies.<sup>8,10,16,17</sup> Thus, NP-Fuc can be used as a therapeutic molecule and as a nanocarrier encapsulating drugs that can enhance its pharmacological activity.<sup>8,9,18-20</sup>

In the body, nanoparticles can interact with several biomolecules, including lipids, proteins, and nucleic acids, among others. The interaction of nanoparticles with some serum proteins could be undesirable and desired. It is undesirable when a nanocarrier interacts with some opsonins, which can accelerate the nanosystem's degradation, reducing its half-life time.<sup>1,21,22</sup> On the other hand, interactions between nanosystems and some blood proteins may be desired to facilitate the distribution of these nanocarriers in the human body.<sup>23-25</sup> Serum proteins such as albumin, being natural carrier proteins in the body, can act in the distribution of molecules in the body, allowing for drug transport. Thus, they can be allied in the distribution of nanoparticles to target sites, potentially forming a core-corona.<sup>25-28</sup>

As recently demonstrated by Ribeiro et al.,<sup>29</sup> the binding strategy of albumin and other substances to form more efficient therapeutic products has been widely used, especially for anticancer medicines such as Paclitaxel (Abraxane), Docetaxel, Doxorubicin, and 5-Fluorouracil. The resulting drug complexes presented lower toxicity and greater efficiency, since albumin can drive and accumulate at tumor sites. For this reason, human serum albumin (HSA) and bovine serum albumin (BSA) can be applied both for obtaining conjugated-prodrugs or nanoparticles<sup>29</sup> as well as for *in vitro* evaluation of interaction profiles, which can help the understanding of *in vivo* biological activity of some new drugs.<sup>30,31</sup>

Therefore, in order to understand whether nanosystems have *in vivo* stability and can be safely applied intravenously in the future without jeopardizing their controlled release, it is important to know their interactions with serum proteins.<sup>32-34</sup> In this context, the interactions of NP-Fuc with serum proteins and BSA were evaluated, seeking to identify whether NP-Fuc can be used as long-circulating stealth nanosystems in the body.

## 2. MATERIALS AND METHODS

**2.1. Materials.** Fucoidan was obtained from the brown algae *Fucus vesiculosus* extracted by researchers from our research group.<sup>35</sup> Oncocalyxone A (oncoA) was isolated from the heartwood of the *Cordia oncocalyx* plant and was previously extracted and supplied by researcher Prof. Otilia Pessoa.<sup>36</sup> IBCA was obtained from Henkel Biomedical (Dublin, Ireland); hydrochloric acid (HCL), Bovine fetal serum (FBS), and BSA were purchased from Sigma-Aldrich. Human serum was extracted from the whole blood of a healthy human, following ethical guidelines after approval by the Human Research Ethics Committee of UFPE (Brazil), with protocol no. 3.794.871 and CAEE 25179719.1.0000.5208. It is hereby declared that the Ethics Committee of UFPE follows the Code of Ethics of the World Medical Association (Declaration of Helsinki).

**2.2. Methods.** **2.2.1. Development, Purification, and Characterization of Nanoparticles.** The nanoparticles were obtained by the anionic emulsion polymerization (AEP) technique, as described by Cavalcanti et al.<sup>8</sup> For NP-Fuc, fucoidan was dissolved in ultrapure water (Milli Q), and the pH was adjusted to 2.5 using HCl (1 M). After this step, the solution was stirred (1.200 rpm), and then 50 µL of IBCA was added. Finally, the nanoparticles were kept for 3 h until the polymerization process was completed. Figure 1 illustrates the nanoparticle synthesis methodology. For oncoA-loaded nanoparticles (oncoA-NP-Fuc), a solution of oncoA (5 mg/mL) in acetonitrile:water (50:50 v/v) was added after 5 min of the addition of the IBCA.

The obtained nanoparticles were purified using a dialysis membrane (Spectra-por membrane 100 000 g/mol molecular weight cutoff (MWCO), Biovalley, Marne la Vallée, France) in 1 L of distilled water, changed after 30 min three times, and kept under stirring (300 rpm) overnight.

The obtained nanoparticles were characterized in terms of size, polydispersity index (PDI), and ζ potential in the Zetasizer ZS90 (Malvern Instrument) to confirm the development of the nanoparticles.<sup>8</sup>

**2.2.2. Interaction of Serum Proteins with NP-Fuc by the Static and Dynamic Incubation Method.** The adsorption of proteins on the surface of nanoparticles was evaluated from static or dynamic
incubation of nanoparticles using protein from FBS, BSA, and human serum, with subsequent surface charge and particle size analysis.37 To obtain human serum, 10 mL of whole blood was collected from a healthy human and centrifuged at 3000 rpm for 15 min after coagulation. After that, the serum was collected and used in the experiment. The unused serum was kept refrigerated at -20 °C. The experiments followed ethical guidelines after approval by the UFPE Human Ethics Committee, with protocol no. 3.794.871 and CAAE 25179719.1.0000.5208.

For static incubation, 300 µL of nanoparticle dispersion (NP-Fuc) was diluted with 300 µL of FBS, BSA (60 mg/mL in 10 mM Tris-HCl buffer, pH 7.4, to obtain the protonation state of polar amino acids under physiological conditions), or human serum. The mixture was incubated for 0, 30, 60, and 120 min at 37 °C. An aliquot (50 µL) was withdrawn after each defined time and diluted in 950 µL of ultrapure water (1:20 v/v) for ζ potential and particle size analysis. The dynamic incubation study follows the static incubation method, differing only in that the samples were incubated at 37 °C and kept under stirring (100 rpm).37

### 2.2.2.1. BSA Interaction with oncoA-NP-Fuc after Dynamic Incubation in the Nanosight.
Because of the results of a significant change in the size of the nanoparticles after incubation with BSA in the previous studies of static and dynamic incubations, only BSA was selected to continue the interaction study. In order to complement the results obtained with the zetasizer, another technique was used to assess the size and concentration of nanoparticles after interaction with BSA using the NanoSight NS300. For this study, oncoA-NP-Fuc was also included due to the intrinsic fluorescence of oncoA.38 The oncoA was encapsulated in the nanoparticles, thus not interfering with the surface of these nanocarriers, but promoting an increase in the intensity of the light captured by the nanosight, and because the device's fluorescence modulus allows a cleaner signal to be obtained, thus making it easier to interpret the results.39

For the analysis, the nanoparticles (NP-Fuc and oncoA-NP-Fuc) in a volume of 200 µL were incubated with 200 µL of BSA (60 mg/mL in 10 mM Tris-HCl buffer pH 7.4 to obtain the protonation state of polar amino acids under physiological conditions) and kept under agitation at 100 rpm at 37 °C. At 0, 10, 30, and 120 min, 10 µL of aliquots were taken and diluted in 990 µL of ultrapure water for analysis in the NanoSight NS300.

### 2.2.2.2. Nanoparticle Tracking Analysis (NTA).
NTA measurements were performed with a NanoSight NS300 system (Malvern Technologies, Malvern, UK) configured with a 488 nm laser and a high-sensitivity scientific CMOS camera. The samples were injected into the sample chamber with 1 mL of sterile syringes, until the liquid reached the tip of the nozzle. Nanosight 3.2 software was used for all analyses using standard settings. The camera level for each sample was manually adjusted to achieve optimal visualization of particles (sample concentration ranging from $1 \times 10^8$ to $1 \times 10^9$ particles/mL diluted with ultrapurified water). The detection threshold was set for maximum sensitivity with a minimum of background noise. All counts were performed at 25 °C in replicates of 3 for each sample, collecting a video of 30–40 s. To minimize variability, all camera and detection threshold settings were kept the same for each mode when multiple experiments were performed on a single sample source. The validity of the reported particle size was periodically assessed for the NS300 unit using 100 and 200 nm polystyrene beads (Malvern Technologies, Malvern, UK).

### 2.2.3. Interaction of BSA on the Surface of NP-Fuc.
The interaction of BSA on the surface of NP-Fuc was evaluated by the contact method, adapted from the study by Li et al.33 and Golafshan et al.34 Different concentrations of NP-Fuc ($1.73 \times 10^{11}$, $3.46 \times 10^{11}$, $6.92 \times 10^{11}$, $1.04 \times 10^{12}$, and $1.38 \times 10^{12}$ particles/mL) were incubated with a BSA solution (1 mg/mL) in 10 mM Tris-HCl buffer (pH 7.4) under agitation (100 rpm) for 30 min at 37 °C. After incubation, the solutions were analyzed in a UV-vis spectrophotometer (Shimadzu UV-1800) using the albumin wavelength of absorbance (280 nm) and in fluorescence spectrum (Jasco spectrofluorometer FP-6300) in the wavelength range of 300–400 nm with excitation of 280 nm. The excitation and emission slits were

2.5 nm. With the data achieved in the absorption spectrum, the intrinsic binding constant (Kb) was obtained using eq 1.40

$$ \frac{[\text{NP} - \text{Fuc}]}{(\varepsilon_a - \varepsilon_f)} = \frac{[\text{NP} - \text{Fuc}]}{(\varepsilon_b - \varepsilon_f)} + \frac{1}{\text{Kb}(\varepsilon_b - \varepsilon_f)} \quad (1) $$

In the equation, the values of $ε_a$, $ε_b$, and $ε_f$ represent the apparent, limited, and free extinction coefficients, respectively. The graph [NP-Fuc]/($ε_a - ε_f$) versus [NP-Fuc] was used to obtain Kb values from the ratio between the slope and the intercept. Data were tabulated using the SigmaPlot 10.0 software.

For the fluorescence data, the different intensities obtained after exposure of the nanoparticles with BSA were analyzed for the emission suppression behavior using the Stern–Volmer (Ksv) eq 2.41

$$ F_0/F = 1 + k_q \tau_0 [Q] = 1 + K_{sv}[Q] \quad (2) $$

In the equation, $F_0$ and F represent the steady-state fluorescence intensities of BSA in the absence and presence of nanoparticles, respectively, and [Q] is the concentration of BSA, $k_q$ is the bimolecular extinction rate constant, and $τ_0$ is the average lifetime of nanoparticles in the absence of BSA. The linear regression constant was obtained by graph relative emission intensity ($F_0/F$) versus [Q]. The software used for data tabulation was SigmaPlot 10.0.

### 2.2.4. Morphological Analysis.
The morphological analysis was performed by scanning electron microscopy (SEM). Initially, 300 µL of BSA solution (5 mg/mL) was added to a microtome containing 300 µL of NP-Fuc and kept under stirring for 1 h (100 rpm at 37 °C). After incubation, the mixture was diluted (1:10 (v/v)), and the sample was spread on stubs. The material was dried in an oven at 37 °C for 24 h and then metalized in gold and palladium (FINE COAT, ION SPUTTER JFC-1100) and analyzed in a SEM (EVO, LS15). For the analysis of energy dispersive spectroscopy (EDS), an SEM coupled to the EDS (OXFORD BRAND DETECTOR X-act MODEL) (15kv throttle voltage and 15 mm working distance) was used.

### 2.2.5. Spectroscopic Analysis.
Fourier transform infrared (FTIR) spectra of samples Fuc, Fuc-BSA (interaction between fucoidan and BSA), NP-Fuc, NP-Fuc-BSA, BSA, and physical mixture (BSA and NP-Fuc) were obtained with a resolution of $4 \text{ cm}^{-1}$ in the region 4000 to $500 \text{ cm}^{-1}$ using an FT-IR/FT-NIR (PerkinElmer, Spectrum 400). Data were processed, and graphs were plotted in Origin 8 software (OriginLab Northampton).

### 2.2.6. Thermogravimetric Analysis.
The thermogravimetric curves (TG) were obtained using the equipment DTG-60H (Shimadzu) in a nitrogen atmosphere with a flow of 50 mL/min, while differential scanning calorimetry (DSC) was obtained using equipment DSC-60 (Shimadzu). Thermograms of BSA, Fuc, NP-Fuc, Fuc-BSA, NP-Fuc-BSA, and physical mixture (BSA and NP-Fuc) were obtained using 5 mg of lyophilizate, sealed in aluminum crucibles, and analyzed from a temperature of 25 to 500 °C using a heating rate of 10 °C/min.

### 2.2.7. ¹H NMR Analysis.
Hydrogen nuclear magnetic resonance (¹H NMR) was used to investigate the interaction between BSA and NP-Fuc. For this, NP-Fuc was initially prepared using deuterium oxide ($D_2O$) as a vehicle instead of ultrapure water, following the methodology described in the previous section (item 2.1.1). Then, it was incubated at 37 °C and shaken at 100 rpm, with BSA at different concentrations (0.14, 0.73, 1.82, 3.64, and 20 mg/mL in $D_2O$). The variation in the concentration of BSA was carried out to identify the changes in the peaks of the ¹H NMR spectra and to identify the binding mechanisms between BSA and the nanoparticles. $D_2O$ with 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TMS) was used as the standard. Varian 500 MHz heated to 37 °C was used for the analysis. For the acquisition of the ¹H NMR spectra, the pulse sequence (zg) was used, the waiting time to start the acquisition (D1) was 1.00 s (s), and the pulse duration (P1) for hydrogen was 8, 00 µs. The spectral width was 10,000 Hz, the acquisition time (AQ) was 1.6384 s, and 16 FIDs were accumulated (NS). Changes in the chemical shifts of protons have been documented.
### 3. RESULTS AND DISCUSSION

**3.1. Development and Characterization of Nano-particles.** The nanoparticles obtained had a size around 357 ± 3.26 nm with a ζ potential of -45.83 ± 0.22 mV, which agrees with the results obtained in the studies by Lira et al.,⁹ Cavalcanti et al.,⁸ and Cavalcanti et al.³⁸ Table 1 presents the

<table><caption>Table 1. Characterization of Nanoparticles</caption><thead><tr><td>formulation</td><td>diameter (nm)</td><td>PDI</td><td>ζ potential (mV)</td></tr></thead><tbody><tr><td>NP-Fuc</td><td>357 ± 3.26</td><td>0.06 ± 0.00</td><td>-45.83 ± 0.22</td></tr><tr><td>oncoA-NP-Fuc</td><td>305 ± 0.91</td><td>0.13 ± 0.00</td><td>-39.80 ± 1.40</td></tr></tbody></table>

results obtained in the zetasizer regarding the size, PDI, and ζ potential of the nanoparticles. The negative charge of the nanoparticles is related to the presence of the sulfate groups of fucoidan.⁸,⁹

The reduction in nanoparticle size after encapsulation of oncoA may be related to the decrease in fucoidan on the surface of the nanoparticles, since there is also a reduction in the surface charge of the nanoparticles. These results were also observed by Cavalcanti et al.⁸

**3.2. Interaction of Serum Proteins with NP-Fuc by the Static and Dynamic Incubation Method.** Serum proteins

have been employed for evaluation of NP features related to biodistribution, biocompatibility, and therapeutic efficacy, since after administration, the system containing the drug needs to be distributed through the blood to find the specific site of action.²⁵ Here, the interaction of NP-Fuc with proteins was carried out using static and dynamic methods with human serum, FBS, and BSA. BSA was kept at pH 7.4 in 10 mM Tris-HCl buffer to obtain the polar amino acid protonation state of albumin under physiological conditions.

Evaluating the interaction between the static and dynamic methods allows us to see whether the agitation of the system favors the interaction of the nanoparticle with the proteins. In view of the results shown in Figure 2, it can be seen that there was no significant difference in the interaction between the static and dynamic methods. Although there was no change in the size of NP-Fuc after contact with human serum and FBS, it can observe a change in the ζ potential, with an increase in surface charge from -45.60 ± 0.30 to -21.58 ± 0.77 mV. This change may indicate the interaction of serum proteins with NP-Fuc. Considering that albumin is the most abundant serum protein, comprising about half of the total protein mass, the changes observed in nanoparticles charge can be due to the interaction between this protein and NP-Fuc.⁴²,⁴³

<figure><img src="image_11.png" /><figcaption>Figure 2. Size and ζ potential in static (■-) and dynamic (-●-) interaction studies of NP-Fuc with BSA, human serum, and FBS.</figcaption></figure>
<figure><img src="image_12.png" /><figcaption>**Figure 3.** Concentration and distribution of nanoparticles and insert containing video image of oncoA-NP-Fuc without BSA (A), oncoA-NP-Fuc + BSA at time zero (B), after incubation for 10 min (C), 30 min (D), and 120 min (E).</figcaption></figure>

The absence of an increase in nanoparticle size following interaction with human serum and fetal bovine serum may be attributed to molecular competition within the serum as well as the inhibition of aggregate formation between albumin and the nanoparticles. We propose that when free albumin interacts with nanoparticles in isolation, it can form multiple layers around them, leading to an observable increase in the size. In contrast, this multilayer formation likely does not occur in serum due to the complex environment containing a variety of biomolecules. These competing interactions may limit protein adsorption to a single tightly bound layer (hard corona), which does not significantly alter the nanoparticle size. Nevertheless, this binding is sufficient to induce measurable changes in surface charge, confirming the interaction between the nanoparticles and serum components.

Albumin binds to several molecules and plays a fundamental role in the transport and distribution of these molecules, helping them to reach their target sites.⁴²,⁴⁴ For better evaluation of the interaction between NP-Fuc and albumin, BSA was used due to its homology with HSA and its value for

this type of *in vitro* analysis.²⁹ When BSA was used alone, a clear modification was observed in NP-Fuc size (Figure 2 BSA), the addition of BSA seems to induce the formation of aggregates, with an aggregate size of around 2 µm. Despite the formation of aggregates after incubation with BSA, it was observed that in the serum samples there was no change in the size of the nanoparticles, showing that for intravenous administration the nanoparticles would maintain their size, even with the binding of plasma proteins.

The identification that molecules present in human serum or FBS interact with the nanoparticles is evidenced by the modification of the surface charge, which indicates that possible serum proteins can bind to the surface of the nanoparticles, forming the core-corona, thus modifying the charge of these nanosystems. These results corroborate with the study by Coty et al.⁴⁵ which showed that PIBCA nanoparticles coated with sulfated dextran can have affinities with different whey proteins. The different charge results of the nanoparticles after exposure with BSA (around -10 mV) and with human serum and FBS (around -20 mV) may be related
<figure><img src="image_13.png" /><figcaption>Chart 2. Videos of the Nanoparticles Obtained from Nanosight for the Samples of oncoA-NP-Fuc without BSA (A), oncoA-NP-Fuc + BSA at time zero (B), after incubation for 10 min (C), 30 min (D), and 120 min (E)</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Figure 4.** Absorbance (A) and fluorescence (B) profiles of BSA in the presence and absence of different NP-Fuc concentrations.</figcaption></figure>

to the interaction of the nanoparticles not only with albumin but also with other proteins present in serum.⁴⁵

According to the literature, fucoidan is a sulfated polysaccharide that interacts with BSA forming interpolymer complexes, so the presence of fucoidan on the surface of nanoparticles may be responsible for the interaction with BSA.⁴⁶,⁴⁷ It can be suggested that the interaction of NP-Fuc with BSA could increase the half-life of the NP in the body, due to the formation of the core-corona that can mask the presence of the nanoparticles.¹⁰,²⁸,⁴⁸ The presence of the corona protein on the surface of the nanoparticles may confer stealth properties, preventing the activation of the complement system and thus preventing the phagocytosis of the nanoparticles and allowing them to remain in the body for longer, thus favoring the maintenance of the activities of the nanocarriers such as targeting and controlled release of the encapsulated drug.⁴⁹,⁵⁰

According to Hyun et al.,⁵¹ the presence of albumin on the surface of nanoparticles can act by inhibiting the recognition and clearance of nanoparticles by the phagocytic system, serving as a disopsonin. Complementary assays were then performed to analyze the complex formed by NP-Fuc and BSA.

### 3.2.1. BSA Interaction with oncoA-NP-Fuc after Dynamic Incubation in the Nanosight.
Nanosight is a device that allows tracking analysis of nanoparticles regarding their size (range 10 to 1000 nm) in solution. Nanosight allows for individual and simultaneous analysis, allowing the analysis of size distribution and quantification of nanoparticles, with a

fluorescence module that provides the differentiation of labeled nanoparticles.⁵²

In this study, because NP-Fuc nanoparticles were not labeled with a fluorescent marker, no clear data were collected on the study of the interaction of BSA with the nanoparticles due to the low intensity of captured light (see Figure S1 and Chart 1A–1E). Therefore, oncoA-NP-Fuc nanoparticles were chosen, which are labeled with oncoA, which exhibits intrinsic fluorescence (wavelength emission = 430 nm).³⁸

As expected, Figure S2 shows that the analyses with oncoA-NP-Fuc showed a higher intensity of captured light than NP-Fuc due to the fluorescence of oncoA. The concentration graphs (Figure 3) show that the nanoparticles presented a size in the oncoA-NP-Fuc group without BSA around 247 ± 49.2 nm (Figure 3A) (there is a small peak at 444 nm, which is in line with the variation shown in the PDI, in the studies on the zetasizer, Table 1) and this size increased slightly until the time of 120 min for 285 ± 74.2 nm (Figure 3E); however, the concentration of nanoparticles decreased from time zero (Figure 3B) which had a concentration of 3.62e+09 to 2.06e+09 particles/mL in 30 min (Figure 3D).

Although the concentration graphs show little variation in size, it can be seen that as the exposure time of the nanoparticles with BSA increases, the concentration of the nanoparticles decreases. It can be seen that this reduction is probably related to the fact that Nanosight detects particles around 10 nm to 1 µm⁵² and according to the static and dynamic studies, the nanoparticles after interacting with BSA
<figure><img src="image_15.png" /><figcaption>Figure 5. Fucoidan-coated nanoparticles (A) and fucoidan-coated nanoparticles after BSA adsorption (B).</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>Figure 6. EDS analysis coupled to SEM of NP-Fuc. (A) Merge from maps of chemical elements present in nanoparticles; (B) presence of oxygen; (C) presence of Carbon; (D) spectrum of the sum of maps; (E) apparent concentration of chemical elements. Legends: O = Oxygen; C = Carbon.</figcaption></figure>

have a size above 1 µm (Figure 2), and are therefore outside the detection of the equipment. These results then corroborate the incubation study since the reduction in concentration may mean that the equipment is detecting fewer nanoparticles because there is an increase in aggregates.

Another fact that could not be neglected regarding the possible interaction between BSA and oncoA-NP-Fuc is the refringence observed around the nanoparticles already at time zero (Chart 2B) forming a cloud and indicating that something has bound the nanoparticles and increased their size. Comparing the interaction videos (Chart 2B–E2) with the video of nanoparticles without BSA (Chart 2A), there is no intense refringence as in the other groups. In view of this, the data from nanosight corroborate the data presented in the zetasizer, in which the binding of BSA seems to occur immediately since at time zero it can already observe the refringence in the nanoparticles.

**3.3. Adsorption of BSA on the Surface of NP-Fuc.** BSA is obtained from bovine serum and has been employed in several studies to evaluate drug–protein interactions besides the employment as a nanocarrier.⁴⁴,⁵³ Most of the studies are performed by absorption and fluorescence spectroscopies because the UV–vis absorption at 280 nm is associated with its aromatic amino acids (Trp, Tyr, and Phe) and highly

sensible fluorescence changes after interaction with other molecules.²⁹ The results obtained from the adsorption study show the interaction of BSA with the nanoparticles by both absorbance and fluorescence analyses (Figure 4). It can be seen in Figure 4A that the increase in the concentration of NP-Fuc increased the absorbance of BSA with an $R^2$ value of 0.9951. The increase in absorbance may be related to the adsorption of BSA on the surface of NP-Fuc forming a complex in the ground state of NP-Fuc-BSA, in which the newly formed complex also presents absorption at 280 nm, thus justifying the increase in the absorption, like observed by Kathiravan et al.⁵⁴ According to the value of Kb, the value calculated was $5.82 \times 10^4 M^{-1}$, which shows the binding affinity between BSA and the NP-Fuc.

Fluorescence studies are useful to explore BSA interaction due to the possibility of identifying differences in protein emission properties after the interaction occurs.³¹ Figure 4B shows the fluorescence profile of BSA in the absence and presence of NP-Fuc. A reduction in BSA fluorescence was observed depending on the NP-Fuc concentration with an $R^2$ value of 0.9853. The interaction of NP-Fuc with BSA changes the hydrophobic environment where the amino acid tryptophan is present, thereby inducing fluorescence suppression.³¹,⁵⁵ The Ksv calculation showed a value of $10.41 \times 10^4$
<figure><img src="image_17.png" /><figcaption>**Figure 7.** EDS analysis coupled to SEM (A) Merge from maps of chemical elements present in nanoparticles; (B) presence of oxygen; (C) presence of carbon; (D) presence of nitrogen; (E) spectrum of the sum of maps; (F) apparent concentration of chemical elements. Caption: O = Oxygen; C = Carbon; N = Nitrogen.</figcaption></figure>

<table><caption>**Table 2.** Wavenumber of the Absorption Bands in the Infrared Spectra of the Samples</caption><thead><tr><th>samples</th><th>O-H (cm<sup>-1</sup>)</th><th>C-H (cm<sup>-1</sup>)</th><th>C=O (cm<sup>-1</sup>)</th><th>amide I (cm<sup>-1</sup>)</th><th>amide II (cm<sup>-1</sup>)</th><th>S=O (cm<sup>-1</sup>)</th><th>C-O (cm<sup>-1</sup>)</th><th>S=O (cm<sup>-1</sup>)</th></tr></thead><tbody><tr><td>NP-Fuc-BSA</td><td>3532</td><td>3100</td><td>1773</td><td>1601</td><td>1504</td><td>1371</td><td>1157</td><td>949</td></tr><tr><td>NP-Fuc</td><td>3531</td><td>3111</td><td>1771</td><td></td><td></td><td>1372</td><td>1153</td><td>949</td></tr><tr><td>Fuc-BSA</td><td>3414</td><td>3082</td><td>1773</td><td>1668</td><td>1524</td><td>1366</td><td>1169</td><td>940</td></tr><tr><td>BSA</td><td>3407</td><td>3086</td><td>1774</td><td>1664</td><td>1524</td><td></td><td></td><td></td></tr><tr><td>Fuc</td><td>3376</td><td>2969</td><td>1678</td><td></td><td></td><td>1341</td><td>1126</td><td>928</td></tr><tr><td>physical mixture</td><td>3425</td><td>3094</td><td>1778</td><td>1657</td><td>1517</td><td>1376</td><td>1163</td><td>959</td></tr></tbody></table>

M⁻¹, thus evidencing the interaction between BSA and static-type NP-Fuc, as the fluorescence suppression seems to be related to the formation of the NP-Fuc-BSA complex. The quenching of BSA fluorescence was also observed in the study by Kathiravan et al.,⁵⁴ corroborating the results obtained in this study.

### 3.4. Morphological Analysis of NP-Fuc Bound to BSA.

The study of the morphology of the NP-Fuc nanoparticles (Figure 5A) using the SEM technique showed nanospheres with a size around 282.65 ± 3.75 nm, with a homogeneous size distribution, corroborating the results presented by Cavalcanti et al.⁸ and Lira et al.⁹ On the other hand, NP-Fuc showed a larger size after interaction with BSA (Figure 5B), around 2.4 ± 0.5 µm, which indicates the BSA adsorption on the surface of these nanoparticles, corroborating with the results obtained in the static and dynamic incubation study (Section 3.2) whose nanoparticles had similar sizes.

According to the study by Sripriyalakshmi et al.,⁵³ BSA nanoparticles tend to have a spherical shape and uniform distribution. Sripriyalakshmi et al.⁵³ had obtained SEM images very similar to those presented in our study, which may indicate the presence of BSA in NP-Fuc. The adsorption of BSA on NP-Fuc can improve the biodistribution of these nanoparticles in the organism; thus, the possibility of developing NP-Fuc coated with BSA as an effective nano-system in the delivery of drugs is of great interest.

### 3.5. EDS Analysis.
The analysis of EDS coupled to SEM in Figure 6 showed that oxygen and carbon are present in NP-Fuc composition, in line with the study by Cavalcanti et al.⁸ Even though fucoidan is composed of sulfate groups,⁸ it was not possible to detect the sulfur element in NP-Fuc, probably due

to the low concentration of sulfur in the nanoparticles formulation.

The analysis of EDS with NP-Fuc-BSA (Figure 7) showed in its composition the presence of oxygen, carbon, and nitrogen elements. Albumin is formed by amino acids whose main elements include carbon, hydrogen, oxygen, and nitrogen,⁵⁶,⁵⁷ which is in line with the data obtained. Therefore, the presence of nitrogen in the nanoparticles is related to the adsorption of BSA due to the amide groups present in the protein, being another indication of the adsorption of BSA in nanoparticles.⁵⁴,⁵⁸,⁵⁹

### 3.6. Spectroscopic Analysis.
Finally, infrared spectroscopy was performed to confirm the interaction between NP-Fuc and BSA, and the results can be seen in Table 2 and Figure 8. NP-Fuc sample has an absorption band at 1372 cm⁻¹ and another at 1470 cm⁻¹ (S=O) referring to the sulfate groups present in the fucoidan and the absorption band at 1153 cm⁻¹ seems to refer to the CO bond, in addition to the absorption bands at 3531 cm⁻¹ (referring to the OH bond) and 3111 cm⁻¹ (referring to the CH sp3 elongation), corroborating the results obtained by Cavalcanti et al.⁸

Regarding the BSA profile, the absorption bands at 3407 and 3086 cm⁻¹ are attributed to -OH stretching vibration and CH sp3 elongation, respectively. It is possible to observe an absorption band at 1664 and 1524 cm⁻¹ referring to amide I (C=O stretching vibrations) and amide II (the coupling of bending vibrate of N-H and stretching vibrate of C-N) respectively.⁵⁴,⁵⁸⁻⁶⁰

The profile of the nanoparticles after contact with BSA (NP-Fuc-BSA) proves the interaction since the amide I absorption band of BSA is smaller and the amide II band is absent, which leads us to suppose that probably the interaction of BSA with
<figure><img src="image_18.png" /><figcaption>**Figure 8.** Infrared spectra of the physical mixture (black), BSA (purple), Fuc (orange), Fuc-BSA (green), NP-Fuc (blue), and NP-Fuc-BSA (red).</figcaption></figure>

NP-Fuc occurs between sulfate groups of fucoidan and amide groups of BSA. And apparently, it seems that the conformation of fucoidan on the surface of NP-Fuc influences this binding, since in the spectrum of Fuc-BSA and the physical mixture it can observe the characteristic absorption bands of BSA.

**3.7. Thermogravimetric Analysis.** Thermogravimetric curves allow evaluation of the degradation of samples with increasing temperature. According to Figure 9A, NP-Fuc starts decomposing at a temperature of around 200 °C with complete degradation at around 275 °C, and these results are like those presented by Cavalcanti et al.⁸ The interaction of BSA with both Fuc and NP-Fuc improves its decomposition profile, to which it was observed that NP-Fuc-BSA starts its decomposition at around 250 °C with complete degradation at around 375 °C. When it was compared these NP-Fuc-BSA results with the physical mixture, it was observed an overlap in the decomposition profile of NP-Fuc and BSA. These results suggest that there is an interaction between BSA and NP-Fuc after incubation and that this interaction seems to confer greater thermal resistance to the nanoparticles.⁶¹,⁶²

As for the differential scanning calorimetry results (Figure 9B), they are similar to those found in the literature. It can observe two endothermic events characteristics of BSA (around 50 and 220 °C) corroborating with the study by Kim et al.,⁶⁶ while the nanoparticles showed an endothermic peak around 250 °C, in line with the study by Cavalcanti et al.⁸ It can be observed that the Fuc-BSA interaction presented an event similar to that of the NP-Fuc and that the NP-Fuc-BSA formulation showed only one endothermic event around 200 °C with a characteristic similar to the second endothermic peak of BSA, evidencing the presence of BSA in the nanoparticles, different from the physical mixture that shows the overlap of events characteristic of NP-Fuc and BSA.

The enhanced thermal resistance of the nanoparticles resulting from BSA binding may be attributed to the strong interaction between albumin and fucoidan on the nanoparticle surface. This interaction likely contributes to the stability of the nanoparticles, during both circulation in the body and storage, particularly in formulations containing BSA-coated NP-Fuc. These findings are promising and pave the way for future studies on NP-Fuc-BSA.

**3.8. ¹H NMR Analysis.** According to the spectra obtained from the ¹H NMR, it can see in Figure 10 that there is a signal in the BSA spectrum around 6.5 to 7.5 ppm that is related to aromatic protons and amides and around 0 to 4.5 ppm referring to aliphatic protons, results like the study of Ferrari et al.⁶³ The presence of well-defined signals in the region of ~2.3–3.0 in the BSA spectrum may be related to the presence of amine groups.⁶⁴ On the other hand, the Fuc and NP-Fuc spectra are similar, showing the presence of peaks that characterize fucoidan, such as in the region from 3.4 to 4.5, which refers to sulfate groups, as well as in the region 1.0 to 1.8, referring to methyl groups.⁶⁵,⁶⁶

It can be seen in Figure 10 that some BSA peaks were shifted after incubation with Fuc (purple line) and NP-Fuc (yellow line), becoming more evident in Figure 10B that there was a shift in the signals in the region of 2.3 to 3.0 ppm that is related to the amine groups of BSA, indicating an interaction of fucoidan with BSA probably in these groups. It was also observed that in the region of 3.4 to 4.5 ppm of Fuc and NP-

<figure><img src="image_19.png" /><figcaption>**Figure 9.** Thermogravimetric curves (A) and differential scanning calorimetry (B) of BSA (purple), Fuc (pink), NP-Fuc (blue), Fuc-BSA (green), NP-Fuc-BSA (red), and a physical mixture (black).</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>**Figure 10.** ¹H NMR spectra of BSA (blue), Fuc (red), NP-Fuc (green), Fuc-BSA (purple), and NP-Fuc-BSA (yellow). (A) full spectra; (B) region from 0 to 5 ppm; (C) region from 5 to 9 ppm.</figcaption></figure>

Fuc there was a reduction in the intensity of these peaks that are related to the sulfate groups of fucoidan.

In Figure 10C, it can be seen that there is a reduction in the BSA signal between 6.5 and 7.5 ppm referring to the amide groups. These results show that this probable link between BSA and NP-Fuc may be related to the presence of sulfate groups of fucoidan with amine and amide groups of BSA, corroborating the results obtained in the infrared analysis.

In Figure 11, it can be seen the appearance of the signal at ~2.3–2.5 ppm of BSA (light green line) at a concentration of 20 mg/mL (orange line) with a shift to 2.4–2.6 ppm. It is also possible to observe that with increasing BSA concentration,

there is an increase in the signal at ~4.8–5.0 ppm and the appearance of a signal at ~4.6–4.8 ppm. According to Rashed et al.,⁶⁷ these signals appear to be related to ring protons of D-glucose and D-galactose of fucoidan.

The interaction of BSA through the amide groups with the sulfate groups of fucoidan present on the surface of the nanoparticles can help in the distribution of the nanoparticles in the body since albumin is the main transport protein. This can directly impact the distribution and direction of the action of drugs encapsulated in these nanoparticles and can help in their bioavailability and biodistribution process. Despite consistent results, studies must be carried out to better
<figure><img src="image_21.png" /><figcaption>**Figure 11.** Incubation of NP-Fuc (blue) at concentrations of 0.14 (red), 0.73 (dark green), 1.82 (purple), 3.64 (yellow), and 20 mg/mL (orange) BSA (light green).</figcaption></figure>

understand these mechanisms, and this intensity of interaction can also impair the controlled release of the drug due to the strong interaction of albumin and fucoidan, thus preventing its decomposition for controlled release of the drug. According to previous studies,⁸⁻¹⁰ NP-Fuc does not present cytotoxicity against macrophages, nor does it induce hemolysis in red blood cells, results that reinforce the use of these nanoparticles in future *in vivo* studies.

## 4. CONCLUSIONS

The interactions between nanoparticles with serum proteins can promote their rapid degradation due to interaction with opsonins or promote greater circulation in the bloodstream when these interact with albumin, which is considered to be the most abundant carrier protein in the human body. Therefore, here, it was evaluated the *in vitro* interaction of fucoidan-coated nanoparticles (NP-Fuc) with BSA. It was observed that the NP-Fuc interacted with BSA. After the static and dynamic interaction studies, there was a change in the size of the nanoparticles exposed only to BSA and although the increase in size indicated aggregation of the nanoparticles with BSA, the results with FBS and human serum did not interfere with the size of the nanoparticles, which was positive, as it indicates that in the body, the nanoparticles will not induce the formation of aggregates and pose health risks. However, the change in the surface charge of the nanoparticles still emphasizes that plasma proteins interact with the nano-particles. The NTA technique allowed us to complement the incubation studies, showing that the surface of the nano-particles is modified, increasing their size due to the increase in

refringence with the formation of a cloud around the nanoparticles, thus indicating the presence of BSA on the surface of the nanoparticles. This interaction was also confirmed by the modification in BSA's absorption and fluorescence profile after contact with NP-Fuc. The SEM results show that after BSA adsorption, the nanoparticles increase in size with irregular morphology.

Infrared spectra demonstrate the presence of BSA in NP-Fuc, thus, indicating the interaction between the two molecules. The reduction in the intensity of the absorption bands of the amide groups is suggestive of the interaction of NP-Fuc in this region of the BSA. These results complement the results obtained in the ¹H NMR, suggesting that the binding mechanism of NP-Fuc with BSA occurs through the sulfate groups of fucoidan with the amide and amine groups of BSA. Furthermore, the results of the thermal analysis demonstrate that the interaction between BSA and NP-Fuc seems to confer greater thermal resistance to the nanoparticles.

These results encourage the use of these nanocarriers as promising polysaccharide-coated polymeric NPs due to their versatility, which, on the one hand, can be used as a targeting nanosystem without causing risk in the form of aggregates with plasma proteins, with rapid distribution in the body due to the probable adsorption of albumin, and, on the other hand, increase the half-life of circulating NPs in the body. In addition, the discovery that albumin interacts with NP-Fuc is important for developing nanoparticles containing albumin adsorbed on the surface. Further studies are needed to identify the best albumin/nanoparticle ratio to obtain an ideal size for intravenous administration (with respect to the range of less than 400 nm).
Despite the promising results, *in vivo* studies are needed to confirm the safety of using NP-Fuc in intravenous administration, confirming that the interaction of NP-Fuc with blood components will not interfere with the behavior of the nanoparticles in *in vivo* models.

<figure><img src="image_22.png" /></figure>

## ASSOCIATED CONTENT

<figure><img src="image_23.png" /></figure>

**Supporting Information**

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsanm.5c00999.

Intensity of light captured in the nanosight according to the size of the nanoparticles in the groups NP-Fuc (Figure S1); intensity of light captured in the nanosight according to the size of the nanoparticles in the groups oncoA-NP-Fuc (Figure S2) (PDF)

<figure><img src="image_24.png" /></figure>

## AUTHOR INFORMATION

### Corresponding Authors

Nereide Stela Santos Magalhães — Instituto Keizo-Asami (iLIKA), Universidade Federal de Pernambuco (UFPE), 50670-901 Recife, Brazil; Phone: + 55 81 21268587; Email: nereide.magalhaes@ufpe.br

Mariane Cajubá de Britto Lira Nogueira — Instituto Keizo-Asami (iLIKA), Universidade Federal de Pernambuco (UFPE), 50670-901 Recife, Brazil; Laboratório de Nanotecnologia, Biotecnologia e Cultura de Células, Centro Acadêmico de Vitória, Universidade Federal de Pernambuco (CAV/UFPE), 55608-680 Recife, Brazil; ● orcid.org/0000-0002-7556-4746; Email: mariane.lira@ufpe.br

### Authors

Iago Dillion Lima Cavalcanti — Instituto Keizo-Asami (iLIKA), Universidade Federal de Pernambuco (UFPE), 50670-901 Recife, Brazil; Laboratório de Nanotecnologia, Biotecnologia e Cultura de Células, Centro Acadêmico de Vitória, Universidade Federal de Pernambuco (CAV/UFPE), 55608-680 Recife, Brazil; ● orcid.org/0000-0001-6625-8395

José Cleberson Santos Soares — Laboratório de Química e Inovação Terapêutica (LQIT), Universidade Federal de Pernambuco (UFPE), 50670-901 Recife, Brazil

Sinara Mônica Vitalino de Almeida — Universidade de Pernambuco (UPE), 55294-902 Garanhuns, Brazil; ● orcid.org/0000-0002-2605-4192

Otilia Pessoa — Laboratório de Análise Fitoquímica de Plantas Medicinais II, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60451-970 Fortaleza, Brazil

Evandro Silva — Programa de Pós-Graduação em Produtos Naturais e Sintéticos Bioativos (PPGPNSB), Universidade Federal da Paraíba (UFPB), Campus Universitário I, Castelo Branco III. Cidade Universitária, CEP: 58051-900 João Pessoa, Paraíba, Brasil; Instituto de Pesquisa em Fármacos e Medicamentos (IPeFarm/UFPB), Campus Universitário I, Castelo Branco III. Cidade Universitária, CEP: 58051-900 João Pessoa, Paraíba, Brasil

Marcelo Sobral da Silva — Programa de Pós-Graduação em Produtos Naturais e Sintéticos Bioativos (PPGPNSB), Universidade Federal da Paraíba (UFPB), Campus Universitário I, Castelo Branco III. Cidade Universitária, CEP: 58051-900 João Pessoa, Paraíba, Brasil; Instituto de Pesquisa em Fármacos e Medicamentos (IPeFarm/UFPB),

Campus Universitário I, Castelo Branco III. Cidade Universitária, CEP: 58051-900 João Pessoa, Paraíba, Brasil

**Francisco Humberto Xavier Junior** — Programa de Pós-Graduação em Produtos Naturais e Sintéticos Bioativos (PPGPNSB), Universidade Federal da Paraíba (UFPB), Campus Universitário I, Castelo Branco III. Cidade Universitária, CEP: 58051-900 João Pessoa, Paraíba, Brasil; Departamento de Ciências Farmacêuticas, Laboratório de Biotecnologia Farmacêutica (BioTecFarm/UFPB), Campus Universitário I, Castelo Branco III. Cidade Universitária, CEP: 58051-900 João Pessoa, Paraíba, Brasil

Eloisa Berbel Manaia — Faculté de Pharmacie Institut Galien Paris-Saclay - UMR-CNRS 8612, Université Paris, 91400 Saclay, France; ● orcid.org/0000-0001-6352-1602

Gilles Ponchel — Faculté de Pharmacie Institut Galien Paris-Saclay - UMR-CNRS 8612, Université Paris, 91400 Saclay, France

Complete contact information is available at: https://pubs.acs.org/10.1021/acsanm.5c00999

### Funding

The Article Processing Charge for the publication of this research was funded by the Coordenacao de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil (ROR identifier: 00x0ma614).

### Funding

I.D.L.C. is supported by the Brazilian agencies Foundation for Research Support of the State of Pernambuco (FACEPE) Postdoctoral scholarship (APQ-0490-4.03/24 and DCR-0009-4.03/24) and Brazilian Council for Scientific and Technological Development (CNPq) (303224/2024-0), and M.C.d. B.L.N. is supported by the PropesquilUFPE (23076.067716/2023-79 and 23076.051304/2023-10) and FACEPE APQ-0791-4.03/22. The Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the Programa Institucional de Internalização (Print) (#2761/2018).

### Notes

The authors declare no competing financial interest.

<figure><img src="image_25.png" /></figure>

## ACKNOWLEDGMENTS

The authors thank the Laboratory of Microscopy of the National Institute of Technology in Union and Coating of Materials (INTM) for the collaboration with the analyses in the SEM, the Laboratory of Polymers and Nanostructures of UFPE for the collaboration with the FTIR studies, the Laboratory of Coordination Compounds and Surface Chemistry at UFPB for thermogravimetric analysis and to the Multiuser Laboratory of Characterization and Analysis at UFPB for the ¹H NMR analyses. The authors are grateful to National Council for Scientific and Technological Development (CNPq) for the financial support, with a research grant to NSSM (#307968/2019-7) and IDLC for the Postdoctoral scholarship (#303224/2024-0) and FACEPE (APQ-0490-4.03/24 and DCR-0009-4.03/24). We are also grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the Programa Institucional de Internalização (Print) (#2761/2018) for Financial Support. M.C.d.B.L.N. thanks Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco - FACEPE (APQ-0791-4.03/22), Propesqi UFPE Qualis A (23076.067716/2023-79), and Auxílio Pesquisador (23076.051304/2023-10).
## ABBREVIATIONS

BSA - bovine serum albumin

FBS - fetal bovine serum

HSA - human serum albumin

IBCA - isobutyl cyanoacrylate

PIBCA - poly(isobutyl cyanoacrylate)

NP-Fuc - fucoidan-coated PIBCA nanoparticles

NP-Fuc-BSA - fucoidan-coated PIBCA nanoparticles with adsorbed BSA

oncoA-NP-Fuc - oncoA-loaded nanoparticles

SEM - scanning electron microscopy

FTIR - Fourier transform infrared

Ksv - Stern-Volmer equation

EDS - energy dispersive spectroscopy

¹H NMR - hydrogen nuclear magnetic resonance

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