Prolonged Release of IL‐10 From Enzyme‐Mediated Poly‐l‐(Tyrosine‐co‐Phenylalanine) Nanocrystals Enhances Stability and Modulates Inflammatory Responses

ABSTRACT

Protease‐catalyzed synthesis of a peptide composed of l‐tyrosine and l‐phenylalanine (2.2:1 M ratio) was achieved in a natural deep eutectic solvent (NADES) medium and subsequently employed to encapsulate the anti‐inflammatory cytokine interleukin‐10 (IL‐10). IL‐10 was loaded into enzyme‐mediated peptide nanocrystals at doses of 200 and 400 ng, achieving stable nanocomposite formulations. Release studies were conducted in phosphate‐buffered saline (PBS, pH 7.4) at 37°C under gentle agitation, revealing a sustained release profile extending up to 30–31 days, with an initial release of approximately 16% within the first 5 days and near‐complete release between Days 10 and 25, depending on loading. Encapsulation effectively protected IL‐10 from rapid degradation observed for the free cytokine under identical conditions, resulting in markedly enhanced stability. The nanocrystals were further integrated into porcine gelatin–hyaluronic acid (Ge:HA) and microbial poly(hydroxybutyrate‐co‐valerate) (PHBV) matrices, where IL‐10 release was further modulated, reaching up to ~80% release from Ge:HA and ~100% from PHBV‐based systems over 31 days. Cytotoxicity assays using primary human dermal fibroblasts confirmed excellent biocompatibility of all formulations. Moreover, studies in PMA‐activated THP‐1 macrophage‐like cells demonstrated reduced intracellular reactive oxygen species (ROS) and suppression of the pro‐inflammatory cytokine IL‐6, highlighting the combined protective and immunomodulatory effects of IL‐10 encapsulation. The presence of tyrosine residues within the nanocarrier further suggests intrinsic antioxidant contributions. Overall, these results support enzyme‐mediated peptide nanocrystals as an effective platform for the stabilization and prolonged release of IL‐10, with strong potential for treating inflammation‐related skin conditions.

Schematic representation of enzyme‐mediated peptide nanocrystal formation and IL‐10 encapsulation, providing stabilized and sustained cytokine delivery that modulates macrophage phenotype and reduces inflammatory responses.

1. Introduction

Inflammation is a highly complex and tightly regulated biological process essential for the maintenance of tissue homeostasis and host defense against harmful stimuli. While acute inflammation is a protective response, chronic inflammation is associated with the pathogenesis of a wide array of debilitating diseases, including many types of arthritis, psoriasis, or several neurodegenerative disorders, among others [1]. The action of endogenous anti‐inflammatory mediators including anti‐inflammatory drugs or biological approaches palliates the symptoms, although in chronic diseases it ends up affecting organs, especially the kidney and stomach, but they also damage the liver, heart, and the cardiovascular system [2].

In this regard, the use of natural anti‐inflammatory protein signalers is pointed out as a low toxic alternative, and among them, the interleukin 10 (IL‐10) plays a central role in the inflammation processes [3]. IL‐10 is an immunoregulatory cytokine produced by a variety of immune and nonimmune cells, including Treg, monocytes, macrophages, and epithelial cells. It exerts potent anti‐inflammatory effects by inhibiting the production of pro‐inflammatory cytokines (such as TNF‐α, IL‐6, and IL‐1β), suppressing the antigen‐presenting capacity of dendritic cells and macrophages, and promoting the differentiation of regulatory immune cell subsets [4]. Additionally, tyrosine‐based materials have been reported to stimulate IL‐10 production by immune system cells [5]. Due to these properties, IL‐10 has attracted significant interest as a therapeutic agent for controlling chronic and excessive inflammation. However, despite its strong biological rationale, the clinical translation of IL‐10‐based therapies has faced multiple challenges, including short in vivo half‐life, rapid degradation, limited tissue penetration, and the requirement for high systemic doses, which often lead to off‐target effects and reduced therapeutic efficacy, besides the associated treatment costs [6]. In response to these limitations, polymer‐based drug delivery systems have been explored to optimize the pharmacokinetics and therapeutic action of IL‐10 [7, 8]. Among these, peptide‐based delivery systems have emerged as promising biomaterials [9]. Peptides offer several advantages including biocompatibility, biodegradability, and amenable to chemical modification, allowing for the design of highly specific and functionalized molecules. Furthermore, some synthetic peptides possess intrinsic anti‐inflammatory or immunomodulatory properties, adding another therapeutic layer to their use [10]. Examples of this are tyrosine‐based peptides, owing to the phenol groups [11, 12, 13]. These peptides can modulate immune responses by interacting with cell surface receptors, inhibiting pro‐inflammatory signaling pathways, or mimicking regulatory proteins. Beyond their role as therapeutic agents, peptides are increasingly recognized as delivery vehicles (carriers) for therapeutic agents [14]. When used as carriers for IL‐10, peptides can be engineered to improve cytokine stability, facilitate targeted delivery to inflamed tissue, and enable controlled or stimuli‐responsive release. Strategies for peptide‐based IL‐10 delivery include the use of peptide hydrogels, nanocarriers, cell‐penetrating peptides (CPPs), and peptide‐conjugated nanoparticles, each designed to address specific barriers in drug delivery, such as enzymatic degradation, limited cellular uptake, or poor bioavailability [9, 15]. The combination of IL‐10's anti‐inflammatory activity with the targeting and delivery capabilities of peptides offers a synergistic therapeutic strategy that could significantly enhance treatment outcomes for many inflammatory conditions.

In this regard, the protease‐catalyzed peptide synthesis (PCPS) provides several advantages over traditional chemical routes, specifically in terms of selectivity, environmental impact, and reaction conditions [16, 17]. Unlike conventional approaches that often require harsh reagents, protecting groups, and high temperatures, enzymatic synthesis operates under mild conditions, reducing the risk of toxicity in products, racemization, and side reactions. Hydrolase‐type enzymes such as proteases exhibit high chemo‐, regio‐, and stereoselectivity, enabling the efficient formation of peptide bonds with minimal byproduct formation [18]. These features make PCPS attractive, and we would like to explore them for biomedical applications; however, despite these advantages, previous reports often use aqueous media where the reversible proteolytic activities render low molecular weight oligopeptides with four or fewer repeat units [16]. In this regard, we have recently reported enzyme‐mediated routes to peptides using low as possible in the media, thereby limiting the reversible hydrolysis to achieve peptide chains by PCPS in low toxic routes using natural deep eutectic solvent (NADES) media and subtilisin Carlsberg protease biocatalyst [19].

The present work demonstrates the enhanced stability of IL‐10 when loaded into nanocrystals (NC) of enzymatically synthesized poly‐r‐(l‐tyrosine‐co‐l‐phenylalanine) (ePYF) by our chemoenzymatic methodology using NADES medium. This enzyme‐mediated peptide‐based NC‐IL‐10 presents no cytotoxicity using primary human fibroblasts, and the study of IL‐10 release, including those from gelatin and microbial poly(hydroxybutyrate‐valerate) (PHBV) as dressings for their potential applicability, is addressed. PHBV is a microbial‐derived biodegradable polyester widely explored for biomedical applications due to its excellent biocompatibility, low inflammatory response, and controllable degradation behavior [20]. The presence of hydroxyvalerate units reduces crystallinity and improves flexibility and processability compared to the other more studied microbial polyester poly(3‐hydroxybutyrate) (PHB) [21], enabling the fabrication of mechanically stable films suitable for wound dressings. Importantly, PHBV undergoes surface erosion into non‐toxic degradation products, allowing modulation of water diffusion and sustained release of therapeutic agents [22] These properties make PHBV an appropriate supporting matrix for peptide‐based nanocrystals, where it can enhance local retention and prolonged delivery of IL‐10 in cutaneous inflammation and wound healing applications. The use of activated THP‐1 into macrophages by PMA is useful to validate our proposed prolonged‐delivery system by measuring intracellular ROS and the pro‐inflammatory cytokine IL‐16, and the results are discussed.

2. Materials and Methods

2.1. Materials

l‐Tyrosine and l‐Phenylalanine ethyl esters (≥ 99% purity) were supplied by Merck (México), desalting and purification as follows: a potassium carbonate solution (5.38 g in 10 mL water), to which 6 g of l‐PheOEt·HCl or l‐TyrOEt·HCl dissolved in 22 mL water was added. The mixture was stirred for 1 h at room temperature, followed by four washes with ethyl acetate (20 mL each). Then, the organic phase solvent was removed by rotary evaporation at 45°C, 80 rpm, and 20 mmHg, thereby obtaining the desalted monomers. Serine protease S8 endoprotease (EC 3.4.21.62), derived from Bacillus licheniformis (approximately 27 kDa, 110 U), was a kind gift from ENMEX S.A. de C.V (Carry Group Mexico). Glycerol (Gly) and choline chloride (ChCl) were supplied by Merck (Mexico). For preparation of the eutectic mixture of at a molar ratio of 1:2 (Gly:ChCl), supplemented with 30 vol% sodium phosphate buffer (50 mM, pH 7.5), the amount of solid ChCl and liquid Gly were heated to 80°C under stirring on a magnetic stirrer/heating plate (IKA, Germany) until a homogeneous solution formed. Then, the appropriate amount of buffer was added, and stirring continued for an additional 5 min. Recombinant human IL‐10 (18.6 kDa, 161 amino acids, ≥ 98% purity, Preprotech) was received in a total amount of 10 μg and aliquoted into 25 tubes of 40 μL each, using 5 mM sodium phosphate buffer, pH 7.2, to achieve a final concentration of 1.0 mg mL⁻¹. Roswell Park Memorial Institute medium (RPMI), and Dulbecco's Modified Eagle Medium (DMEM) were supplied by Gibco (USA) with l‐glutamine, fetal bovine serum (FBS), and penicillin/streptomycin were supplied by Gibco (USA). THP‐1 cell line was supplied by ATCC TIB‐202. Sigma‐Aldrich supplied 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and hydrochloric acid (HCl). Phosphate‐buffered saline (PBS) was supplied by Gibco (USA). EDC (N‐ethyl‐N′‐(3‐dimethylaminopropyl) carbodiimide, 0.096 g, commercial grade) and NHS (N‐hydroxysuccinimide, 0.012 g, 98% purity), both supplied by Sigma‐Aldrich. Trypsin–EDTA (1X, 25200‐056) was obtained from Gibco, Life Technologies Corporation, USA. Trypan blue dye (T6146‐5g) were provided by Sigma, USA. Hyaluronic acid (HA) was Durolane, Gelatin (250 Bloom, Powder, NF; Spectrum Chemical MGF Corp.). All reagents were used as received unless otherwise stated.

2.2. Enzymatic Synthesis of ePYF

For the synthesis of ePYF, a 1:1 M ratio of both substrates (3:3 mmol) was placed into flask (purified l‐PheOE 0.584 g and l‐TyrOEt 0.632 g) and dissolved with 20 mL of the previously prepared NADES. The reaction was initiated by adding 30 mg of the enzyme (Uo = 7.02 U mg⁻¹ min⁻¹) and stirring (250 rpm) for 3 h at 45°C. After reaction time, the precipitated product was filtered through a Whatman (0.45 μm), and the recovered solids were reconstituted in Milli‐Q water (using the minimal volume required to obtain a paste). The paste was combined with cold ethanol (5°C) in excess (×5 volumes) and stirred for 5 min and lyophilized for 24 h or until dry to attain a white coarse powder in 54.67% ± 6.9% yield. The lyophilized copolymer was stored at −80°C. As a negative control, the same protocol was identically carried out without enzyme. Formation of crystals took place by dissolving the ePYF in a water/isopropanol mixture and let evaporate at 4°C for 24 h.

2.3. Structural and Morphological Characterization of ePYF Nanocrystals

¹H NMR spectra were recorded on a Varian Unity Innova spectrometer (USA) at 400 MHz using deuterated water. Infrared (IR) spectra were acquired on an ATR‐FTIR spectrometer (Perkin Elmer Spectrum 100) over the range 250–4000 cm⁻¹. For optical microscopy, after crystallization of ePYF NC, 20 μL of each sample were deposited on a glass slide and allowed to dry in a cold room at 4°C to enable slow solvent evaporation. The nanocrystals formed on the slides were observed using a Zeiss Axioskop 40 Pol polarized‐light microscope (Carl Zeiss, Göttingen, Germany) with a 40× objective. For scanning electron microscopy (SEM), the nanocrystals were formed on coverslips and observed using a JEOL JSM‐5900‐LV scanning electron microscope (Japan). Transmission electron microscopy (TEM) analysis was performed using a Technai G² 20 LaB₆ transmission electron microscope (Thermo Fisher Scientific, FEI) at an accelerating voltage of 120 kV. Specimens were blotted onto precleaned (17r plasma Diener Electronics, 1 min) carbon coated TEM grids (Quantifoil, Germany). Images were captured using an OSIS Mega View camera (Olympus Imaging Systems) or a 4 k × 4 k Eagle CCD camera. Images processing, including contrast and background adjustment, was carried out using ImageJ software. X‐ray diffraction (XRD) spectra were recorded in a Powderdiffraktometer System Stadi P der Firma Stoe Darmstadt (Germany) equipped with a Dectris Mythen 1 K detector, a Ge‐ Monocromator Long fine focus Cu‐Röhre, and a 40 kV, 40 mA generator (PSD step: 2.1, 20 s/step).

2.4. DPPH Radical Inhibition Assay

The inhibition effect of peptide on DPPH radical was measured modifying the method of Hsu et al. (2008) [23]. A peptide sample (15 mg) was dissolved in deionized water (DW) (25 mL) and then aliquots were diluted in DW (80%, 60%, and 40%) successively. Subsequently, 1 mL of the samples (PS) were mixed by agitation with 1 mL of a DPPH‐methanol solution (80 μM) and maintained in dark conditions during 30 min at room temperature. After this time, absorbance was measured in a Genesys 10S UV–vis spectrophotometer (Thermo Fisher Scientific, USA) at 517 nm. Additionally, samples and DPPH blanks were measured by mixing 1 mL of each sample with 1 mL of methanol (PBs), as well as 1 mL of DW with 1 mL of DPPH methanol solution (DB), respectively. The inhibition (I) percentage was calculated according to Equation (1):

$$I\left(\%\right)=100\left(1-\frac{\left(\mathrm{PS}-\mathrm{PB}\right)}{\mathrm{DB}}\right)$$ (1)

The results of the inhibition percentage were plotted against peptide concentration (PC) and fitted to estimate the IC₅₀ value (concentration at which 50% of DPPH solution is scavenged).

2.5. IL‐10 Loading Into Nanocrystals NC:IL‐10

For NC:IL‐10 preparation, a stock solution of IL‐10 (1 mg mL⁻¹) of the prepared IL10 solution was diluted to obtain aliquots of 10 μg μL⁻¹. Loading was achieved by mixing NC and IL‐10 solutions at mass ratios of 1:1 and 1:0.5, resulting in final IL‐10 concentrations of 400g and 200 ng, respectively, in PBS, pH 7.4. To each formulation, 1% (v/v) isopropanol (≥ 99.5% purity) was added as a cosolvent. Finally, the formulations were deposited on coverslips and allowed to dry at 4°C to induce self‐assembly.

2.6. Measurement of the IL‐10 Release From ePYF Nanocrystals

Treatments were immersed in 500 μL of release medium (PBS pH 7.4). For IL‐10 containing formulations, 20 ng was added at 1:50 mass ratio relative to the nanocrystal. Sacrifice of samples was done by preparing sufficient replicates for each of the time points: 0, 24, and 72 h, and 5, 10, 15, 20, 25, and 30 days. Release conditions were 37°C with stirring at 50 rpm. At each time point, supernatants were collected for centrifugation at 13,680g for 5 min and stored at −80°C until analysis. IL‐10 release was quantified via enzyme‐linked immunosorbent assay (ELISA) using the Human IL‐10. Development kit (ELISA ABTS, Preprotech). Optical density was analyzed at 415 nm using a microplate photometer (Thermo Fisher Scientific Multiskan FC). IL‐10 concentrations were calculated using standard curves for IL‐10 of more than five points and with R ² > 0.98, and results were expressed in pg mL⁻¹.

2.7. Fabrication of Gelatin‐Hyaluronic Acid Films (Ge:HA)

Gelatin was weighed (0.6 g) and dissolved in 12 mL Milli‐Q water at 50°C for 30 min under stirring. Afterwards, the solution was cooled to 25°C, and 3 mL HA (20 mg mL⁻¹ in stabilized solution with NaCl, pH 7.4; Bioventus) was added. The mixture was stirred at 100 rpm for 5 min at room temperature, avoiding bubble formation. To form films, 5 mL of the Ge:HA gel was deposited into the lid of 60 × 15 mm Petri dishes (three replicates). The base of the dish was placed on the lid to exert pressure and flatten the gel, thereby obtaining a film shape. The samples were then stepwise frozen: first at −20°C for 2 h, followed by −80°C for 12 h. Finally, the gels were lyophilized. The lyophilized Ge:HA films were crosslinked by EDC/NHS bioconjugation. A solution of EDC and NHS was dissolved in 10 mL PBS. The films were demolded and immersed in the EDC:NHS solution (50:10 mmol, respectively) at 4°C for 24 h. After this period, the films were washed three times with DW to remove excess reagents and air‐dried at room temperature.

2.8. Production of Poly(3‐Hydroxybutyrate‐co‐3‐Hydroxyvalerate) (PHBV) Film

PHBV was synthesized by microbiological fermentation using Cupriavidus necator and a mixed carbon source of fructose and sodium propionate. The bacterial strain was first activated in a nutrient medium containing 3 g L⁻¹ beef extract and 5 g L⁻¹ peptone. After 24 h of incubation, the cells were transferred into 150 mL of seed culture medium composed of KH₂PO₄ (1.75 g L⁻¹), MgSO₄·7H₂O (1.2 g L⁻¹), citric acid (1.7 g L⁻¹), and NH₄Cl (2 g L⁻¹). Fructose (15 g L⁻¹) and sodium propionate (4 g L⁻¹) served as carbon sources. The pH of the culture was adjusted to 6.8 with NaOH. Mineral salts, carbon sources, and other components were sterilized separately prior to mixing. Fermentation was carried out at 30°C with shaking at 250 rpm for 72 h. At the end of the fermentation period, the culture was centrifuged at 9000 rpm for 10 min to collect the biomass. The supernatant was discarded, and the cell pellet was dried at 70°C for 48 h. The dried biomass was finely ground using a mortar and pestle, and PHBV was extracted by adding chloroform at a 1:10 (w/v) ratio, followed by stirring at 60°C for 1 h. The hydrophilic fraction was removed by washing the extract with five volumes of water in a separation funnel. The organic phase, containing PHBV, was dried overnight to recover the polymer, which was then weighed to determine yield. The resulting PHBV exhibited a translucent and flexible appearance with a lower density compared to the PHB homopolymer. The molar fraction of 3‐hydroxyvalerate was 0.4, as determined by ¹H NMR analysis.

2.9. Prolonged IL‐10 Release From ePYF Nanocrystals

IL‐10 was added in a ratio of 1:1 at 200 ng and 1:2 at 400 ng relative to the nanocrystal. Each treatment was immersed in 2 mL of release medium, constituted by PBS buffer at pH 7.4. IL‐10 release was conducted at 37°C with constant agitation at 50 rpm over a period of 31 days. Daily, 0.5 mL of the release medium was withdrawn and replaced with fresh PBS of equal volume in the same tube (Falcon). Collected samples were stored at −80°C until analysis. Released IL‐10 was quantified by ELISA, using the human IL‐10 standard development kit (ELISA ABTS, PeproTech). The assays were conducted by triplicate. The analysis of the drug release mechanisms was performed using the Korsmeyer–Peppas model, which describes the release of drugs from polymeric systems through Equation (2):

$$\frac{\mathit{Mt}}{M\infty }=K·\mathit{tnM}\infty \mathit{Mt}=K·\mathit{tn}$$ (2)

where Mt/M∞ represents the fraction of drug released at time t; K is the release rate constant incorporating the structural characteristics of the system; n is the release exponent, which indicates the predominant transport mechanism.

To determine the n value, the experimental release data were limited to Mt/M∞ = 0.6 (corresponding to a maximum of 60% of the total drug content), since beyond this point the assumptions of the model are no longer valid. The equation was linearized by applying a logarithmic transformation and subsequently fitted to a linear regression model. The value of the release exponent n was directly obtained from the slope of the resulting regression line. Accurate data were fit to a coefficient of determination (r ²) above 0.9. Finally, the value of n was interpreted according to the geometry of the system to elucidate the drug release mechanism.

2.10. Culture of Human Fibroblast and THP‐1 Cells

Dermal fibroblast (Fb) cells were obtained from primary skin cultures and isolated from skin biopsies from esthetic surgeries undergoing elective abdominoplasty with a previously informed consent signature and the process was approved by the Institutional Committee on Human Research of the Instituto Nacional de Rehabilitación Luis Guillermo Ibarra Ibarra (Mexico) number INR 107/24. Dermal tissue was digested with 30 mg mL⁻¹ type I collagenase for 2 h. The digested material was filtered through a 70 μm nylon cell strainer, and the filtrate was inactivated using DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS), 50 U mL⁻¹ penicillin, and 50 μg mL⁻¹ streptomycin. The resulting primary fibroblast (Fb) cell line was cultured at 37°C in a humidified incubator containing 5% CO₂ and 95% relative humidity (Heracell 150i, Thermo Fisher Scientific) until reaching approximately 80% confluency in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The human monocytic cell line THP‐1 (ATCC TIB‐202) was maintained in RPMI medium containing L‐glutamine, supplemented with 10% FBS and 1% penicillin/streptomycin, under identical incubation conditions (37°C, 5% CO₂, 95% relative humidity). For macrophage activation, 100 ng mL⁻¹ phorbol 12‐myristate 13‐acetate (PMA) was added to every 10⁶ cells in RPMI medium without FBS and incubated for 3 h [24]. Following incubation, cells were centrifuged, resuspended in RPMI containing 10% FBS and 1% penicillin/streptomycin, and cultured vertically for 24 h. Activation was verified by the presence of an adherent monolayer at the base of T75 flasks.

2.11. Human Fibroblast Viability by MTT and CalceinAM/EthD‐1 Assay

Prior to the assays, nanocomposite (NC) and NC:IL‐10 samples were sterilized under UV light (254 nm) for 10 min using a Hoefer UVC500 Ultraviolet Crosslinker. For each treatment, 50,000 cells were seeded per well in 24‐well plates and incubated for 1 h at 37°C in a humidified atmosphere containing 5% CO₂. Treatments were then added, and the final volume in each well was adjusted to 1 mL with DMEM. Plates were incubated for 24 h under the same conditions. After incubation, the culture medium was removed, wells were washed twice with PBS, and replaced with fresh DMEM. A volume of MTT solution (5 mg mL⁻¹ in DMEM) equivalent to 10% of the culture volume was added to each well, followed by incubation for 3 h at 37°C with 5% CO₂. The medium was then removed, and the resulting formazan crystals were dissolved in an isopropanol (ISO):DMSO (1:1, v/v) solution using the same volume as the culture medium. Aliquots of 100 μL from each well were transferred to a 96‐well plate, and absorbance was recorded at 550 nm using a microplate reader. For the calcein‐AM/EthD‐1 assay, cells were treated for 24 h under identical conditions. After removing the medium and washing twice with PBS, fresh medium was added, followed by the addition of calcein‐AM (2 μM) and EthD‐1 (4 μM). The plates were incubated for 45 min at 37°C with 5% CO₂, and fluorescence images were captured using an Axiovert 25 inverted microscope.

2.12. Intracellular ROS and IL‐6 Measurements in Activated THP‐1 Cells

THP‐1 cells activated with phorbol 12‐myristate 13‐acetate (PMA) were cultured with nanocomposites (NC) containing ePYF, IL‐10 (200 ng mL⁻¹ in PBS), or NC:IL‐10 for 24, 48, and 72 h. PMA‐activated cells without treatment served as inflammation controls. In each well, 1 × 10⁶ cells mL⁻¹ were seeded. At each time point, both supernatants and cell pellets were collected separately. Supernatants were stored at −20°C until analysis of IL‐6 levels. Reactive oxygen species (ROS) generation was assessed using the CellROX Green assay (Invitrogen, Thermo Fisher Scientific). For each treatment, 1 μL of CellROX Green reagent and 9 μL of DMSO were added to cells, and RPMI medium supplemented with 1% FBS was added to a final volume of 100 μL. Samples were incubated for 1 h at 37°C in a 5% CO₂ humidified atmosphere. After incubation, cell counts were performed using a Tali Image‐Based Cytometer (Invitrogen, Thermo Fisher Scientific). Quantification of IL‐6 was carried out using a Human IL‐6 ELISA Kit (PeproTech) according to the manufacturer's protocol. All ELISA results were interpolated from calibration curves containing more than five standard points with R ² > 0.98.

2.13. Statistical Analysis

All experiments were performed in triplicate and repeated in at least three independent assays. Data were analyzed using GraphPad Prism version 9 (GraphPad Software, San Diego, CA, USA). Statistical significance was evaluated using one‐way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Differences were considered statistically significant at p < 0.05.

3. Results and Discussion

3.1. ePYF Characterization

The ¹H NMR spectrum of ePYF is shown in Figure 1, with the assigned signals for ePYF showing a random incorporation of repeat units as the low‐field multiple, broadened, and overlapping signals with relative intensities reflecting the expected random distribution of sequences. All chemical shifts in the proposed structure are assigned, and the molar ratio of residues in the peptide sample renders a 2.2:1 M ratio of Tyr:Phe in the peptide. On the other hand, signals corresponding to residual choline are present. The integration of the corresponding signals indicates a molar ratio of Tyr:Ch = 5.3:1. It is worth noting that choline is non‐toxic; therefore, further purification of the peptide is not pursued. Choline is an essential nutrient that performs key functions in the body, including cellular metabolism, membrane synthesis, neuronal signaling, and lipid transport. Although small amounts can be synthesized by the liver, endogenous production is insufficient, so it must be obtained through the diet. Choline is also known to have biomedical applications, such as in hydrogel formation [25]. For this reason, there is no further purification of the product performed, which also helps to keep costs of potential therapeutics down. Additionally, the other component of the NADES, glycerol, is not detected and is therefore assumed to have been removed during the purification process.

FIGURE 1.

¹H NMR spectrum for ePYF.

The FTIR spectra of ePYF and ePYF nanocrystals are shown in Supporting Information Data 1. Optical microscopy images of the NCs are provided in Supporting Information Data 2. The analysis of SEM and TEM micrographs (Figure 2) revealed that the NCs have an average diameter of 488 ± 210 nm, indicating a relatively narrow size distribution suitable for biomedical applications [26]. The use of nanomaterials enables a reduction in the amount of material required for therapeutic applications [27]. In this context, ePYF nanocrystals were found to be noncytotoxic to dermal fibroblasts and to sustain detectable IL‐10 release for up to 32 days. This represents a significant improvement over free IL‐10, which rapidly loses stability and becomes undetectable within 1 day under the same release assay conditions. Given that the biological activity of IL‐10 is mediated through its interaction with specific cell‐surface receptors, the nanomaterial was initially expected to function solely as a controlled‐release carrier. Nevertheless, the observed outcomes suggest that additional factors may be involved, and further studies will be necessary to elucidate the underlying mechanisms of action.

FIGURE 2.

Representative SEM micrographs for (A) SEM and (B) TEM micrograph of produced NC‐ePYF.

3.2. IL‐10 Release From Peptide NCs

As shown in Figure 3, the release profile of the free interleukin clearly indicates degradation, which is expected due to the low stability in physiological medium for this protein, as reported elsewhere [28], thereby the use of IL‐10 individually as an effective therapeutic agent against inflammation is challenging [6]. Conversely, the same amount of IL‐10 when encapsulated within the nanocarriers (NCs) exhibits a sustained release, reaching completion by day 25. Noteworthy, defining a single “therapeutic” concentration of IL‐10 is challenging, as its immunomodulatory activity depends strongly on the biological context, including the local microenvironment, target tissue, cell type, and receptor expression. Nevertheless, several studies have established biologically relevant dose ranges. For instance, IL‐10 at 250 ng/mL has been shown to induce macrophage polarization toward the anti‐inflammatory M2 phenotype in vitro. Consistently, in a murine model of myocardial infarction, administration of 250 ng IL‐10 improved contractile function and reduced left ventricular dilatation over a 6‐week period compared with saline controls [13]. Lower concentrations have also demonstrated efficacy in inflammatory models. In an in vitro osteoarthritis model using chondrocytes and synoviocytes, IL‐10 at 10–20 ng/mL significantly reduced IL‐1β and TNF‐α production [29]. Moreover, in an ex vivo human study, subcutaneous or intravenous administration of IL‐10 at 25 μg/kg in healthy volunteers suppressed TNF‐α and IL‐1β production in LPS‐stimulated peripheral blood cells for up to 96 h [30]. Collectively, these findings indicate that the IL‐10 doses evaluated in the present study fall within ranges previously reported to elicit meaningful biological responses in vitro and ex vivo. Nonetheless, further validation in appropriate animal models will be required to fully assess the in vivo immunomodulatory effects of the IL‐10‐loaded materials.

FIGURE 3.

IL‐10 release alone (blue circles) and from loaded NC (red squares).

These results demonstrate the protective effect of enzymatically synthesized ePYF on IL‐10, supporting its potential for preservation and NC:IL‐10 as a sustained‐release system. This effect is consistent with recent studies showing that peptide nanocarriers can enhance cytokine stability and release profiles, making them suitable for therapeutic applications [9, 31]. The release in the initial 5 days with NC:IL‐10 is 16%. Then it increases onwards, having the highest rate of nearly 100% between Days 10 and 20. The simulation of physiological media using collagenase or trypsin can help to better estimate the lifetime of IL‐10 under more realistic conditions. In addition, the use of an animal model could provide further information about the therapeutic effects of these IL‐10‐loaded nanomaterials in the treatment of inflammation and allow a more comprehensive evaluation of their biological impact. We expect to perform these studies in a future publication, since experiments involving animal models require longer timelines and additional regulatory procedures. According to previous release models, this phenomenon can be explained by the penetration of water into the matrix of the peptide NC, which leads to crystal hydration and subsequent disorganization [32]. This process generates amorphous regions that facilitate drug diffusion. This process generates the increase of the amorphous regions that facilitate drug diffusion. The X‐ray diffraction spectrum for the peptide is shown in Figure 4 where there is a high crystallinity in the original encapsulating material (see supplementary data file 3 for enlarged XRD diffraction spectrum).

FIGURE 4.

XRD transmission spectrum for ePYF loaded with IL‐10 measured at step 0.05 and 40 s.

Similarly, the NCs used in this study appear to exert a protective effect by minimizing direct contact between IL‐10 and water molecules, thereby reducing hydrolytic degradation and aggregation that often encompass the explanations for the in vitro IL‐10 degradation. In aqueous solutions, ions can interact with exposed hydrophobic and hydrophilic domains of this protein, inducing conformational changes that promote such denaturation and aggregation [33]. We suggest that when IL‐10 is released, a fraction may still retain residual interactions with NC fragments or localized microenvironments (e.g., hydrophobic microdomains, phenylalanine‐tyrosine‐rich regions). These interactions could help stabilize IL‐10 activity, thus allowing detection by the antibodies in the ELISA. Furthermore, regarding the description of its release kinetics, the NC releases IL‐10 progressively as the material swells upon water absorption, thus enabling a diffusion‐controlled release process.

3.3. IL‐10 Delivery Using Biocompatible Ge:HA and PHBV Films

In addition, the release of IL‐10 is studied from potential wound dressings as an approach toward real cutaneous application. For this purpose, porcine gelatin mixed with HA (Ge:HA) and microbiologically produced PHBV were employed. The latter is a biodegradable and biocompatible microbial‐derived biopolymer that has gained popularity in the field of biomedicine due to enhanced therapeutic efficacy and reduced side effects [22].

Supporting Information Data 4 include a representative image of the gelatin film used and Supporting Information Data 5 shows the 1H NMR and FTIR spectra for both films. SEM micrographs of Ge:HA and PHBV are shown in Figure 5. To assess this approach, IL‐10 is loaded in the ePYF system at 200 and 400 ng and placed onto the films. All experiments for both interleukin loads display a significant reduction in the release rate when IL‐10 is encapsulated in nanocarriers, as shown in Figure 6.

FIGURE 5.

SEM microscopy images. (A) GeHA films and (B) PHBV.

FIGURE 6.

IL10 release with (A) 200 ng and (B) 400 ng loadings in the systems.

It is noteworthy that free IL‐10 placed over the films also exhibits some stabilization at both concentrations, although the release is consistently faster compared to NC:IL‐10 alone. Nevertheless, for the 200 ng IL‐10 load, the rate reaches 100% in the PHBV NC‐IL‐10 films by Day 31, while under the same conditions, Ge:HA films achieve approximately 80% release. For the 400 ng load, there is again a significantly more sustained release in the samples based on the porcine gelatin–hyaluronic acid mixture. Our discussion of the kinetics is based on the Korsmeyer–Peppas model for release systems, in which the exact mechanism, whether diffusion, erosion, or a combination of both, is not precisely known, and where the geometry of the system has a significant influence [34]. This model, applicable to hydrophilic polymers, allows the identification of the release mechanism through the diffusional exponent (n), whose interpretation depends on the system's geometry (slab, cylinder, or sphere) and is only applicable below 60% release of the initial loading. In this model, n ≤ 0.45 (all with r ² > 0.9) is consistent with a Fick‐like pattern where the drug is released primarily via diffusion through pores or a polymeric network, and hence the release slows over time. The obtained n values shown in Table 1 for our NCs with cylindrical geometry are interpreted to elucidate the predominant drug release mechanism.

TABLE 1.

IL‐10 release up to 60% with n values (r ² > 0.9) in the different systems.

Release system	IL‐10 loads	n
NC:IL‐10	200	0.45
NC:IL‐10	400	0.56
Ge:AH:IL‐10	200	0.48
Ge:AH:NC:IL‐10	200	0.58
Ge:AH:IL‐10	400	0.696
Ge:AH:NC:IL‐10	400	0.49
PHBV:IL‐10	200	0.46
PHBV:NC:IL‐10	200	0.25
PHBV:IL‐10	400	0.51
PHBV:NC:IL‐10	400	0.37

In our results, n = 0.45 for the NC:IL‐10 system with 200 ng load indicated a release primarily governed by a Fick‐like diffusion mechanism. This indicates that the NCs are stable in an aqueous medium, undergoing slow hydration, which allows IL‐10 to diffuse without significant erosion and thus enables a sustained release over the 31 days of this study. For NC:IL‐10 with 400 ng load, however, the n value reaches 0.54 which implies a combined mechanism in which relaxation and/or erosion of the NC of ePYF contributes to IL‐10 release.

This behavior highlights the influence of the loading amount in the release, providing an additional parameter to control therapeutic delivery. Similarly, for the Ge:AH:NC:IL‐10 system, the obtained n = 0.58 (200 ng) and n = 0.48 (400 ng) classify as anomalous or by a no‐Fick mechanism. While the Ge:AH film might act as a substantial barrier that limits IL‐10 release, the incorporation of NCs exerts an influence on the release process. Although the behavior falls within the non‐Fickian release range, the value of 𝑛 being near the lower boundary suggests a greater contribution from diffusion and polymer relaxation phenomena [35]. Nevertheless, in both systems, a 31‐day release profile represents a desirable outcome for prolonged and controlled drug delivery. It is worth noting that NC:IL‐10 presents a Fickian release mechanism and, when onto the gelatin, displays non‐Fickian behavior, implying a dual mechanism. This type of release has been reported in other controlled systems for encapsulated drugs in the treatment of inflammatory processes, thus establishing our system as a potential low‐toxic strategy for medical applications [36]. For the release from PHBV films we observe different results, the PHBV:IL‐10 (200 ng) value indicates that the release of the IL‐10 from the PHBV matrix occurs mainly through simple Fick‐like diffusion, whereas PHBV:NC:IL‐10 (200 ng) indicates very slow and restricted Fickian diffusion, and this is a similar behavior for PHBV:NC:IL‐10 (400 ng). Future in vivo investigations are needed to confirm the practical effectiveness of these dressings in treating wounds or other cutaneous illnesses. Nevertheless, the results indicate that IL‐10 release from the nanocarriers is slower than for free IL‐10 at the same concentrations, even in the presence of film dressings, confirming the potential advantage of using these peptide‐based nanocarriers in biomedical applications.

3.4. Cell Viability and Cytotoxicity in Primary Human Fibroblast Cultures

The MTT assay results (Figure 7E) indicate that all treatments maintained mitochondrial activity, with no significant differences observed for treatments compared to the PBS control. These findings are consistent with previous studies reporting the absence of IL‐10 toxicity [37] and with others showing that poly(l‐Phenylalanine) nanomaterials are biocompatible in synoviocyte cultures [38]. Collectively, these results prove that the NC of enzymatically synthesized ePYF here are inherently biocompatible and do not compromise cellular metabolism, reinforcing their potential for sustained‐release therapeutic applications.

FIGURE 7.

CalceinAM/EthD‐1 assay for fibroblasts treated with NC of ePYF and IL‐10 (A); control (cells with PBS) (B); NC of ePYF; (C) IL‐10 treatment; and NC:IL‐10 (D). Scale bars correspond to 100 μm. MTT assay for NC of ePYF, and IL‐10 loaded NC ePYF, PBS and free IL‐10 measurements as controls (E).

Similarly, the CalceinAM/EthD‐1 cell viability assay (Figure 7) showed that treatments with NC, IL‐10, and NC:IL‐10 elicited cellular responses comparable to the PBS control. No significant membrane damage or loss of viability was observed under any of the tested conditions, confirming the MTT assay results. Fibroblasts displayed elongated morphology, good adherence, and homogeneous confluence. Furthermore, the absence of red‐stained cells by EthD‐1 (Figure 7A–D) confirms the lack of cytotoxicity in our samples. The consistency between these assays strengthens the conclusion that the studied nanocarrier systems are safe for human fibroblast cultures and suitable for further biomedical development.

3.5. Intracellular ROS and IL‐6 Measurements in Activated THP‐1 Macrophages

Figure 8 shows the intracellular ROS in activated THP‐1 cells exposed to treatments for 24 (a), 48 (b), and 72 h (c), with two independent replicates for each experimental condition. THP‐1 cells are a human monocytic leukemia cell line that, upon PMA stimulation, undergo morphological and functional changes to acquire macrophage‐like characteristics, making them an effective in vitro model for studying both the acute and chronic phases of inflammation [39]. This model is reliable for testing potential anti‐inflammatory therapies in a controlled and reproducible manner [40, 41]. In the analysis at 24 h (Figure 8a), statistically significant differences are observed for IL‐10 and NC:IL‐10 treatments compared to the positive control (PMA). At 48 h, the same trend toward reduced ROS levels is observed although not statistically different. This sustained reduction of ROS suggests that the NC:IL‐10 system may provide not only anti‐inflammatory but also cytoprotective benefits, particularly in diseases where oxidative stress contributes to tissue damage in skin diseases such as premature aging, psoriasis, acne and atopic dermatitis [42, 43]. This finding also suggests a possible antioxidant effect associated with NC. However, despite several reports on the antioxidant effect of tyrosine‐derived polymers [11, 12] further studies will be required to assess the stability and reproducibility of this response for ePYF.

FIGURE 8.

Percentage of ROS (a) 24 h, (b) 48 h, and (c) 72 h for the treatments: control + PMA, NC, IL‐10, NC:IL‐10, and control—nonactivated THP cells.

Additionally, DPPH assay to assess the inhibition of free‐radicals by the peptide material is carried out, therein with a linear regression of I = 44.75PC—1.9518 (R ² = 0.95) displays IC₅₀ = 1.2 mg/mL, which demonstrates the potential antioxidant characteristics for the enzymatically synthesized peptide (see supplementary data file 6 for the graphical representation of the linear regression of DPPH radical inhibition).

Similar studies by Li and coworkers [44] in preactivated macrophages exposed to free and NC‐loaded IL‐10 in conjugated liposomes display reductions in ROS levels. In these studies, LPS‐activated macrophages show high fluorescence, indicating elevated ROS concentrations. In contrast, macrophages treated with IL‐10 or IL‐10‐liposomes show approximately 60% reduction in ROS, indicating that both systems effectively reduce ROS levels. In our experiments there are no statistically significant differences detected at 48 and 72 h (Figure 7B,C) with IL‐10 and NC:IL‐10 compared to the control, despite a clear trend toward decreased ROS levels after 1 day.

On the other hand, IL‐6 is a central regulator of the inflammatory response and one of the first cytokines to increase following the infection, trauma, or surgery. Its levels rise rapidly within a few hours and correlate directly with the severity of the inflammatory process [45]. According to the assays performed to measure IL‐6 in THP‐1 cells (Figure 9), a progressive increase in IL‐6 is observed for the control sample at 24 h, the value exceeds 50 pg mL⁻¹ and reaches 300 pg mL⁻¹ at 72 h, indicating a sustained intensification of the inflammatory response. In contrast, the treatment groups show a decrease in IL‐6 production. Interestingly, the NC alone reduces levels at the first 24 h, which agrees with Pareek et al. [46] where the use of N‐acryloyl‐l‐phenylalanine methyl ester for nanoparticle generation displays, in an in silico analysis, a more negative binding energy with IL‐6 (−6.17 kcal mol⁻¹) compared to ibuprofen (−5.90 kcal mol⁻¹), suggesting greater anti‐inflammatory potential via IL‐6 modulation.

FIGURE 9.

Graphical representation of IL‐6 measurements for the control + PMA, NC, IL‐10, NC:IL‐10, and control–nonactivated THP cells at 24 (A), 48 (B), and 72 h (C).

In the same study, the semiquantitative RT‐PCR relative expression of IL‐6 in RAW 264.7 macrophages treated with phenylalanine nanoparticles shows significant reduction in IL‐6 to 0.139 at a dose of 50 μg mL⁻¹ compared to the normalized control (1.0). These observations indicate that phenylalanine, as well as tyrosine in our case, might exert a sustained anti‐inflammatory effect by reducing IL‐6 levels. No significant difference in IL‐6 reduction was observed between free IL‐10 and NC:IL‐10 at 24 h, although both showed marked decreases at 48 and 72 h. However, this sustained anti‐inflammatory effect at 24 h was evident for the pure peptide NC, likely due to the phenolic groups as reported elsewhere for intrinsic antioxidant activity of tyrosine‐containing peptides [11]. It is hypothesized that the nanocrystals entrap IL‐10 within their structure, thereby protecting the cytokine from biodegradation while enabling a slower and more sustained release. Because IL‐10 exerts its biological effects through binding to specific cell‐surface receptors and subsequent activation of intracellular signaling pathways [47], the primary function of the nanocrystals is presumed to be the regulation of IL‐10 extracellular availability. At present, there is no evidence to suggest that these nanocrystals are internalized by cells or that they directly trigger intracellular signaling pathways leading to reduced production of pro‐inflammatory cytokines. Further experimental studies will therefore be required to validate this hypothesis and to clarify the underlying mechanisms of action.

4. Conclusions

We have developed a nanocarrier system based on enzymatically synthesized poly‐l‐(tyrosine‐co‐phenylalanine) capable of encapsulating IL‐10 with high stability and sustained release over extended periods. The low cytotoxicity has been demonstrated in primary cultures of human skin fibroblasts. The nanocarriers effectively modulated inflammatory responses in THP‐1‐derived macrophage‐like cells, as evidenced by reduced IL‐6 secretion and intracellular ROS levels. These findings highlight the potential of this delivery platform for the treatment of inflammation‐associated skin conditions and provide a foundation for further studies toward translational applications in inflammatory diseases and wound healing.

Author Contributions

F.L.‐T., C.G.H.‐V., Y.C.‐E., and F.G.‐L. performed experimental work and data analysis. C.M. contributed to the methodology and validation. C.G.‐S. and U.S.S. provided supervision, conceptual guidance, and access to research facilities. S.S. and S.H. conducted microscopy analyses and contributed to data interpretation. Y.Z.‐C. assisted with biological assays and data evaluation. R.S.‐S. and M.G. conceived and designed the study, coordinated the project, secured funding, and wrote the manuscript. All authors discussed the results, reviewed, and approved the final version of the manuscript.

Funding

This work was supported by Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (PAPIIT IN200126), Secretaría de ciencia, humanidades, tecnología e innovación, Deutsche Forschungsgemeinschaft, PolyTarget project number (316213987; projects B02 and Z01), and European funds for regional development, EFRE Project number (INST 275/257‐1).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting Information: Data File 1. FTIR for ePYF and nanocrystals of ePYF.

Supporting Information: Data File 2 Optical microscopy image of the ePYF nanocrystals.

Supporting Information: Data File 3 Enlarged X‐ray diffraction spectrum form 0–100 2Theta.

Supporting Information: Data File 4 GE:HA film.

Supporting Information: Data File 5 ¹H NMR and FTIR spectra for Ge:HA film.

Supporting Information: Data File 6 Linear regression for DPPH inhibition percentages of aqueous solutions of peptides.

Data Availability Statement

All data generated or analyzed during this study are included in this published article and its Supporting Information files.