Ethosomal Nanocarriers for Hydrophilic Peptide Encapsulation: Formulation Optimization, Stability, and In Vitro Release Performance

TL;DR

This study develops optimized ethosomes to improve the stability and controlled release of hydrophilic collagen peptides for skin care applications.

Contribution

The work presents a systematic investigation of ethosomal nanocarriers for hydrophilic peptide stabilization in dermocosmetic formulations.

Findings

Optimized ethosomes showed sustained encapsulation efficiency of 73% after 180 days.

In vitro release from ethosomes was prolonged compared to free peptide solutions.

Cytocompatibility tests confirmed safety for skin cell applications.

Abstract

Background: Hydrolyzed collagen peptides (HCP) are widely used as bioactive ingredients in anti-aging and skin rejuvenation formulations due to their role in supporting skin hydration, elasticity, and extracellular matrix integrity. However, their high hydrophilicity limits effective incorporation into lipid-based systems, and restricts controlled release from formulations. Objective: In this study, ethosomal nanocarriers were designed as a phospholipid–ethanol-based system to promote favorable molecular interactions with hydrophilic peptides, aiming to enhance the encapsulation, stability, and controlled release of HCP for dermocosmetic applications. Methods: HCP-loaded ethosomes were prepared using phospholipid (Lipoid P75) and ethanol and optimized by varying high-pressure homogenization cycles. Physicochemical properties, including vesicle size, distribution uniformity, zeta potential, pH, and long-term stability, were monitored for up to 180 days. Vesicle morphology and peptide–lipid interactions were characterized using cryo-scanning electron microscopy and FTIR spectroscopy. Encapsulation efficiency was determined by ultrafiltration, while cytocompatibility was assessed in HaCaT keratinocyte cells. In vitro release behavior was investigated using Franz diffusion cells and compared with aqueous HCP solutions. Results: All formulations exhibited nanoscale size distribution and high colloidal stability, with negative zeta potentials ranging from −42.9 to −76.7 mV. The optimized formulation demonstrated sustained encapsulation efficiency (73% after 180 days) and preservation of peptide structure, as confirmed by FTIR, indicating effective chemical stabilization within the ethosomal matrix. Cytotoxicity studies confirmed good skin cell compatibility. In vitro release studies revealed a controlled and prolonged release profile from ethosomal carriers compared with free HCP solutions, suggesting improved topical bioavailability of collagen peptides. Conclusions: To the best of our knowledge, this work provides one of the first systematic investigations of optimized ethosomal systems for the stabilization of hydrophilic collagen peptides as anti-aging dermocosmetic ingredients. These findings demonstrate that optimized HCP-loaded ethosomes represent a promising ingredient formulation platform enabling bioactive preservation, formulation stability, and controlled topical performance for collagen-based skin rejuvenation applications.

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1. Introduction

Skin aging is a multifactorial biological process that gradually reduces hydration, elasticity, and the integrity of the extracellular matrix, causing wrinkles, loss of firmness, and weaker skin barrier function [1]. Bioactive peptides have attracted considerable interest in anti-aging dermocosmetic research due to their ability to stimulate collagen synthesis, improve skin hydration, and support the extracellular matrix [2,3,4,5,6,7].

Hydrolyzed collagen peptides (HCP), in particular, are widely used as functional ingredients in cosmetic [8,9,10], nutricosmetic [11], and biomedical applications [12,13,14,15] due to their demonstrated benefits in skin regeneration, hydration, and anti-aging. In addition, there are studies indicating that collagen has the potential to function as a natural antioxidant [16].

Despite their significant biological potential, the relatively high molecular weight of these peptides limits their skin penetration [17]. When used in cosmetic formulations, their large molecular size causes them to tend to remain on the skin surface; however, even under these conditions, they can still provide benefits as effective moisturizing agents. Compared to native collagen, hydrolyzed collagen peptides have lower molecular weights; nevertheless, they are still considered relatively large for efficient transdermal transport [18].

In addition, aqueous solutions of hydrolyzed collagen often possess an undesirable odor. Therefore, the development of effective formulation strategies to mask this unwanted odor is necessary for both transdermal and oral applications. Effective encapsulation strategies play a crucial role in overcoming challenges such as limited skin penetration and undesirable odor [19]. In this context, there is a growing interest in the development of vesicular encapsulation systems that enhance the skin absorption of collagen, provide controlled release, protect peptides from environmental degradation, and eliminate unwanted odors.

Lipid-based vesicles such as liposomes have been extensively explored to improve the stability and performance of dermocosmetic bioactives [20,21]. Liposomes are self-assembled phospholipid bilayers capable of encapsulating hydrophilic agents in their aqueous core and lipophilic agents within their lipid membranes, offering improved protection and controlled release compared with simple solutions. Indeed, several commercial cosmetic products incorporate liposomal technologies to enhance ingredient delivery and stability, and liposome platforms remain among the most recognized nanocarrier systems in skincare formulation science. However, classical liposomal systems may exhibit drawbacks, including limited encapsulation efficiency for highly hydrophilic compounds and relatively rapid release of encapsulated actives during storage or after topical application. These challenges have driven the development of modified liposomal carriers with enhanced stability and performance [22,23].

Ethosomes are modified liposomal carriers that were first introduced by Touitou and co-workers in 1997 to enhance skin delivery and improve vesicle flexibility [24]. These systems are composed of phospholipids and a relatively high ethanol content (approximately 20–50%), which acts as an effective skin penetration enhancer. Under non-occlusive conditions, ethanol does not persist on the skin surface, thereby minimizing surface-related accumulation concerns [25]. The presence of ethanol strongly influences the physicochemical characteristics of ethosomes, resulting in reduced vesicle size, enhanced stability, and increased encapsulation efficiency, particularly for lipophilic active compounds, when compared with conventional liposomes [26]. Moreover, ethosomal vesicles promote fluidization of the lipid organization within the stratum corneum, facilitating penetration into deeper skin layers and contributing to improved dermal and transdermal delivery performance [27]. These properties render ethosomes highly suitable for the controlled delivery of a wide range of active molecules, including hydrophilic, amphiphilic, and lipophilic compounds [28,29,30,31,32].

Ethosomal systems have been widely investigated in academic research for dermal and transdermal delivery of a broad range of active compounds, including small molecules, antioxidants, and natural extracts [33,34,35,36,37]. In vitro and in vivo studies have reported enhanced penetration and modified release profiles compared with conventional liposomes or simple solutions [38,39,40]. In addition, to the best of our knowledge, the application of ethosomal carriers for peptide delivery has been reported in the literature, but remains relatively limited compared with studies focusing on small molecules and antioxidants [41,42]. Some studies have also explored peptide-modified ethosomes, in which the peptides are used not as the encapsulated cargo but as surface modifiers to enhance skin penetration [43,44].

Nevertheless, despite these promising characteristics, ethosomes remain far less represented in commercially available cosmetic products than liposomes. This limited translation can be attributed to several formulation challenges reported in the literature, including potential skin irritation associated with ethanol, difficulties in maintaining long-term vesicle stability, and the need for careful control of vesicle size, encapsulation efficiency, and batch reproducibility [45]. These limitations highlight the importance of systematic formulation optimization and comprehensive physicochemical characterization, even when ethosomal carriers are generally considered safe for topical applications.

While ethosomes have been extensively investigated for the delivery of small molecules and antioxidants, their application for transporting hydrolyzed collagen peptides remains insufficiently characterized. Although liposomal systems have been successfully explored for the delivery of collagen peptides [46,47,48], studies using ethosomes are still lacking, highlighting a gap in the current literature that the present study aims to address. The hydrolyzed collagen peptides used in this study can also be considered a representative peptide load, serving as a model to investigate the peptide incorporation, vesicle integrity, and release behavior in ethosomal systems. Moreover, the effects of high-pressure homogenization cycles on the structure, encapsulation efficiency, and stability of peptide-loaded ethosomes have not yet been clearly established.

This study aims to develop and characterize ethosomal formulations containing hydrolyzed collagen peptides. During the formulation process, the effects of processing parameters, particularly the number of high-pressure homogenization cycles, are investigated. In addition, by examining vesicle stability, encapsulation efficiency, and in vitro release performance, the study seeks to provide insights that may guide controlled delivery applications.

2. Results and Discussion

2.1. Effect of High-Pressure Homogenization on Vesicle Size

2.1.1. Particle Size and Uniformity of Blank Ethosomes

Formulations were prepared using Lipoid P75 as the phospholipid source with ethanol content and high-pressure homogenization (HPH) conditions (pressure: 500–1000 bar, cycles: 1–5). Blank ethosomes were used as reference systems to evaluate the effects of processing parameters on vesicle formation in the absence of peptide loading. The compositions of the empty vesicles are summarized in Table 1.

Particle size and distribution were measured using laser diffraction, reported as volume-weighted median diameter (d_0.5_, µm) and uniformity values (Table 2, Figure 1). Non-ethanolic formulations (L1–L9) served as liposomal controls to evaluate the effects of pressure (500–1000 bar) and cycle number (1, 3, 5) on vesicle size and homogeneity independent of ethanol. At lower pressures (500–750 bar, L1–L6), vesicles exhibited variable particle sizes and elevated uniformity values, indicating incomplete vesicle disruption and reorganization during homogenization. In contrast, vesicles prepared at 1000 bar (L7–L9) exhibited consistently lower and more stable uniformity values, reflecting the formation of homogeneous vesicle populations. These findings confirm that sufficient homogenization pressure is critical for reproducible vesicle structure in phospholipid-based systems.

Formulations containing 30% ethanol (L10–L15) showed enhanced vesicle formation. The presence of ethanol reduced vesicle size and improved size uniformity by promoting phospholipid bilayer fluidization and vesicle deformability. These ethosomal formulations, particularly L13–L15, which contained 5% lecithin and 0.3% vitamin E, exhibited smaller initial particle sizes (0.089–0.092 µm on Day 1) and more uniform size distributions compared to the non-ethanolic formulations. Although particle size increased slightly over the 14-day period (to 0.33–0.35 µm), this increase is attributed to vesicle maturation and structural relaxation rather than colloidal instability, as uniformity values remained within an acceptable range.

2.1.2. Particle Size and Uniformity of Peptide-Loaded Ethosomes

Hydrolyzed collagen-loaded ethosomes were prepared with 5% Lipoid P75, 0.3% vitamin E, 30% ethanol, and 1.5% hydrolyzed collagen. The number of high-pressure homogenization cycles varied between 1 and 7 for these formulations (coded T9–T12). Corresponding blank formulations without collagen (coded T21–T24) were also prepared under the same conditions. This experimental design enabled direct assessment of peptide-induced structural changes while maintaining identical processing conditions. The composition of these formulations is shown in Table 3.

As shown in Table 4 and Figure 2, both particle size and uniformity were influenced by the number of high-pressure homogenization cycles. Increasing the number of cycles consistently decreased particle size and enhanced uniformity for both blank and peptide-loaded vesicles, indicating that repeated homogenization progressively refines vesicle structure. Peptide incorporation caused a slight increase in vesicle size compared to blank formulations, reflecting successful encapsulation of the hydrophilic collagen without disrupting overall vesicle stability.

Notably, T11 (processed with five homogenization cycles) exhibited the most consistent particle size and uniformity over the 180-day storage period, demonstrating an optimal balance between sufficient mechanical energy input and vesicle structural integrity. In contrast, increasing the number of cycles beyond five (T12) did not result in further meaningful size reduction, indicating a saturation point beyond which additional homogenization provides limited structural benefit. Based on these findings, T11 was selected as the optimized formulation for subsequent stability, encapsulation efficiency, and in vitro release studies.

The particle size profile of the hydrolyzed collagen peptide-loaded ethosomes developed in our study shows smaller vesicle sizes of 90 nm than those commonly reported for peptide-encapsulated liposomal systems, which typically range from 100 to 250 nm depending on peptide hydrophilicity and the preparation method [49,50,51]. In contrast, the optimized ethosomal formulation (T11) achieved particle sizes of approximately 90 nm, indicating the effectiveness of ethosomes.

2.2. Peptide Quantification and Analytical Method Validation

Throughout the study, quantitative determination of hydrolyzed collagen peptides was required at multiple stages, including peptide loading, encapsulation efficiency calculations, and long-term stability assessments. Therefore, a reliable and validated analytical method was essential to ensure accurate and reproducible peptide quantification across all experimental phases.

The Lowry colorimetric assay was selected and employed as the primary method for hydrolyzed collagen determination in all relevant analyses. High-performance liquid chromatography (HPLC) was not used, as hydrolyzed collagen consists of a mixture of peptides with varying molecular weights and sequences rather than a single defined peptide. The Lowry assay allows total protein/peptide content measurement and is well-suited for evaluating complex peptide mixtures in formulation studies.

Lowry Method Development and Validation

The Lowry method used to determine hydrolyzed collagen content showed high analytical performance, as evidenced by its linearity, accuracy, precision, sensitivity, and robustness. Linearity was confirmed over the concentration range of 20–100 µg/mL, showing a correlation coefficient r^2^ > 0.99. Accuracy, determined by recovery studies at three concentration levels (20, 60, and 80 µg/mL), ranged between 98 and 102%, indicating good agreement between observed and theoretical values. Intra-day and inter-day precision, expressed as relative standard deviation (RSD), were below 2%, confirming method repeatability and reproducibility. The LOD and LOQ, calculated from the standard deviation of the response and the slope of the calibration curve (LOD = 3.3σ/S; LOQ = 10σ/S), demonstrated adequate sensitivity. The method remained robust against minor variations in experimental parameters and showed high specificity, with no interference from formulation excipients. Comprehensive validation data, equations, statistical analyses, and the full calibration curve are provided in the Supplementary Information (Section S1).

2.3. Physicochemical Properties and Colloidal Stability

2.3.1. pH, Conductivity, Zeta Potential, and Density

The physicochemical properties of the ethosomal formulations were evaluated to assess their suitability for dermal application. The pH, conductivity, and density values are summarized in Table 5. The pH values of all formulations ranged between 6.3 and 6.9, which is close to the physiological pH of the skin, indicating good compatibility for topical application. Conductivity values ranged from approximately 44 to 57 µS/cm, reflecting the combined influence of ethanol content and phospholipid concentration on the ionic environment of the dispersion medium. These values confirm the presence of a continuous aqueous phase while maintaining the characteristic ethosomal structure.

The densities of the formulations were found to range between 0.92 and 0.97 g/cm^3^, with only minor variations among samples. The narrow density range suggests homogeneous formulation composition and good reproducibility of the preparation process, supporting the formation of uniformly dispersed ethosomal systems without evidence of phase separation.

In addition to these parameters, zeta potential measurements were performed to characterize the surface charge properties of the vesicles (Table 5). All formulations exhibited negative zeta potential values, which can be attributed to the anionic phosphate groups of the phospholipid headgroups. On Day 1, zeta potential values ranged from −42.9 to −76.7 mV, indicating strong electrostatic repulsion between vesicles. These values are well beyond the commonly accepted ±30 mV stability threshold [52]. Such values are generally associated with good colloidal stability in phospholipid-based nanosystems.

2.3.2. Physical Stability of Ethosomal Formulations

The physical stability of the ethosomal formulations was evaluated by monitoring changes in particle size over a period of 180 days (Table 4) as described in Section 2.1.2. The absence of pronounced increases in particle size or uniformity values indicates that vesicle aggregation or fusion did not occur during the monitored period, supporting the physical stability of the systems (Table 4).

Zeta potential measurements over 30 days further supported the physical stability of the formulations (Table 5). All ethosomal systems maintained absolute zeta potential values above 30 mV throughout the observation period, suggesting sufficient electrostatic repulsion to prevent vesicle aggregation. Blank formulations generally exhibited higher absolute zeta potential values than peptide-loaded systems, which is consistent with partial surface charge shielding following hydrolyzed collagen incorporation. After drug loading, the zeta potentials of ethosomes decreased significantly, indicating the presence of charged peptides on the surface of the nanoparticles [53]. Our zeta potential results are in good agreement with the previous study by Verma et al.; in their work, econazole nitrate-loaded ethosomes exhibited zeta potential values ranging from −48.8 to −75.1 mV [54]. In another study, Al-Ameri et al. reported values of −36.3 ± 3.11 to −51.0 ± 0.52 for ethosomes prepared with soya lecithin [55].

Among the peptide-loaded formulations, T11 displayed the most consistent zeta potential profile over time, indicating a more stable vesicular surface (Figure 3). In contrast, T12 showed slightly greater fluctuations, which may be attributed to excessive high-pressure homogenization cycles inducing bilayer stress or structural rearrangement. These findings highlight the importance of optimized processing conditions in preserving vesicle integrity and short-term physical stability.

In addition to short-term observations, long-term stability of the peptide-loaded ethosomal formulations was assessed over 180 days by monitoring particle size distribution (Table 4) and encapsulation efficiency (Table 6). No significant changes in particle size were observed throughout the storage period, and encapsulation efficiency remained above 85%, indicating sustained vesicle integrity and efficient retention of hydrolyzed collagen peptides. These results, combined with the short-term zeta potential data, confirm the overall stability of the ethosomal systems.

2.4. Encapsulation Efficiency of Ethosomes

The encapsulation efficiency (EE) of the ethosomal formulations was evaluated to determine their capacity to entrap hydrolyzed collagen peptides within the vesicular structure. Quantitative determination of hydrolyzed collagen peptides was performed using the validated Lowry method described in Section 3.2. The percentage of encapsulated and unencapsulated hydrolyzed collagen was calculated as described in Section 2.5, and the results are summarized in Table 6.

On Day 1, all peptide-loaded ethosomal formulations (T9–T12) exhibited high encapsulation efficiencies, ranging from 85.5% to 86.8%, indicating effective incorporation of hydrolyzed collagen peptides into the ethosomal vesicles. These results suggest that the combination of ethanol-containing phospholipid bilayers and high-pressure homogenization provided favorable conditions for the entrapment of hydrophilic peptides. The initial encapsulation efficiency of the peptide-loaded ethosomes (85.5–86.8%) is comparable to that typically reported for optimized peptide-loaded liposomal systems (80–90%) [49,50,51].

Following storage for 180 days, a moderate decrease in EE was observed for all formulations. The encapsulation efficiency of T11 decreased from 86.8% to 73.0%, while T12 retained 70.5% of encapsulated peptide after six months. Such reductions are commonly reported for phospholipid-based vesicular systems and may be attributed to gradual bilayer rearrangement or slow peptide diffusion during prolonged storage.

Despite this decrease, all formulations maintained encapsulation efficiencies above 70% after 180 days, demonstrating satisfactory long-term peptide retention. Among the tested formulations, T11 exhibited the highest EE at both initial and final time points, suggesting that five high-pressure homogenization cycles provide an optimal balance between vesicle formation and membrane integrity. In contrast, the slightly lower EE observed for T12 may be associated with excessive homogenization-induced bilayer stress, which can compromise long-term peptide retention.

These results indicate that high-pressure homogenization plays a critical role in achieving high initial encapsulation efficiency. Among the tested formulations, T11 demonstrated superior long-term peptide retention, maintaining slightly higher encapsulation efficiency than T12 after 180 days, which suggests a more favorable vesicle structure.

2.5. Morphological and Molecular Characterization of Peptide-Loaded Ethosomes

2.5.1. Cryo-SEM Analysis

The morphological characteristics of the ethosomal formulations were evaluated using cryo-scanning electron microscopy (cryo-SEM) to visualize vesicle architecture, surface features, and overall structural integrity. Cryo-SEM was selected to preserve the native hydrated state of the lipid vesicles and minimize structural collapse commonly associated with conventional SEM preparation.

Both hydrolyzed collagen-loaded (T11) and blank (T24) ethosomes exhibited generally spherical or near-spherical morphology, confirming successful vesicle formation (Figure 4). Minor surface irregularities and apparent particle enlargement are likely artifacts caused by ice-crystal growth during cryo-fixation, a common occurrence in phospholipid-based vesicles.

Quantitative morphometric parameters were obtained from annotated cryo-SEM images using image-based area analysis (see Figure S2). Particle diameters estimated from these images were approximately 3.07 µm for the peptide-loaded ethosomes (T11, range: 2.73–3.68 µm) and 1.47 µm for the blank formulation (T24, range: 1.02–1.90 µm). These SEM-derived diameters are larger than the median diameters measured by laser diffraction (d_0.5_: 0.090–0.097 µm, Table 4); which can be attributed to sample preparation and imaging artifacts associated with SEM analysis, including vesicle deformation, partial aggregation, and two-dimensional projection of three-dimensional structures. A similar observation was reported by Chudzińska-Skorupinska et al., where the average particle diameter determined from SEM images was approximately twice the hydrodynamic diameter measured by DLS [56].

Quantitative analysis of vesicle morphology, including mean aspect ratio and image-based size variability (ISV), is summarized in Table 7. The hydrolyzed collagen peptide-loaded formulation (T11) displayed a higher mean aspect ratio (1.58 ± 0.23) compared to the blank formulation (T24, 1.37 ± 0.22), suggesting a modest change from ideal spherical geometry following peptide incorporation. This increase in aspect ratio may be associated with interactions between the encapsulated peptide cargo and the phospholipid bilayer, potentially influencing membrane curvature without disrupting vesicle formation or structural stability. Importantly, both formulations exhibited very low ISV values (<0.05), indicating narrow morphological size distributions and a high degree of vesicle homogeneity. Collectively, these findings demonstrate that the ethosomal systems preserve structural integrity and homogeneity upon peptide loading, proving their suitability as nanocarriers for peptide encapsulation.

2.5.2. Fourier Transform Infrared (FTIR) Spectroscopy

FTIR spectroscopy was used to evaluate the structural characteristics of hydrolyzed collagen peptides and to assess its compatibility with ethosomal components. The spectrum of pure hydrolyzed collagen (Figure 5) showed characteristic peptide absorption bands at 3289 cm^−1^ (Amide A, N-H stretching), 2955 and 2888 cm^−1^ (C-H stretching), and amide bands at 1635 cm^−1^ (Amide I), 1526 cm^−1^ (Amide II), and 1241 cm^−1^ (Amide III). Additional peaks at 1442, 1396, 1328, and 1078 cm^−1^ correspond to CH bending, carboxylate vibrations, and C–O stretching typical of collagen-derived peptides. These results confirm the presence of intact peptide structures despite hydrolysis, consistent with previous reports of collagen FTIR signatures prior to encapsulation [57].

In contrast, the FTIR spectra of empty and collagen-loaded ethosomes (Figure 5) displayed nearly identical band patterns dominated by phospholipid-associated peaks, including CH_2_/CH_3_ stretching around 2979 cm^−1^ and phosphate- or ester-related vibrations in the 1407-1044 cm^−1^ range. No new peaks appeared in the peptide-loaded formulation, and no characteristic lipid peaks were lost, indicating that the presence of collagen did not chemically alter the phospholipid bilayer. This behavior aligns with literature [58,59] showing that in liposomal or ethosomal systems, the lipid matrix signals often dominate and mask the weaker absorbance of entrapped peptides or small molecules.

Minor shifts, such as the movement of the Amide A/OH region from 3289 to 3340 cm^−1^ and Amide I from 1635 to 1644 cm^−1^, suggest subtle changes in hydrogen-bonding environment, likely due to peptide–lipid interactions, but do not indicate covalent interactions or structural degradation. These subtle band shifts are typical of non-covalent loading of biomolecules into lipid bilayers as reported by others [60].

Taken together, the FTIR data demonstrate that (i) collagen peptides retain their structural features, (ii) ethosomes maintain their phospholipid integrity after loading, and (iii) encapsulation occurs through non-covalent, physicochemical interactions without chemical modification.

2.6. In Vitro and Biological Evaluations

2.6.1. Cytotoxicity Assay

The safety of the formulations was evaluated using a cell viability assay on HaCaT keratinocyte cells. The cytotoxicity of pure collagen peptides, empty ethosomes (T24), and collagen-loaded ethosomes (T11) was evaluated across a range of concentrations (0.1% to 25%, v/v) as shown in Figure 6.

Pure collagen demonstrated high cell viability (88–102%) at all concentrations, confirming its biocompatibility and minimal cytotoxic effects. In contrast, the hydrolyzed collagen peptide-loaded ethosomes (T11) exhibited significantly lower cell viability, particularly at higher concentrations (25%, and 10%) with values ranging between approximately 10% and 22%. This reduced viability suggests that the formulation or the ethosomal carriers themselves may exert some cytotoxic effects at these doses. The empty ethosomes (T24) also showed lower viability at higher concentrations (25%, and 10%), but the viability markedly increased (100%) at the lowest concentration (0.1%), indicating dose-dependent cytotoxicity primarily related to the vesicular system rather than the collagen payload. A similar pattern has been reported in the literature by Bondi et al., where Gossypin-loaded and unloaded formulations showed minimal cytotoxicity at low doses (0.5 µg/mL), whereas higher doses led to reduced cell viability, particularly after extended exposure (48 h) [37].

The low viability observed at higher concentrations for both T11 and T24 may be attributed to the ethanol content or the surfactant properties of the phospholipid vesicles, which can disrupt cell membranes at elevated doses. However, at low concentration (0.1%), both ethosomal formulations are well tolerated, highlighting their potential safety for topical applications at appropriate dosages. The assay controls performed as expected, with SLS (positive control) markedly reducing cell viability, while the negative control (DMEM) maintained high viability, confirming the reliability of the test.

These results indicate that pure collagen is non-toxic across the tested range, and the ethosomal nanocarrier system exhibits good biocompatibility at lower concentrations. This supports the feasibility of using hydrolyzed collagen peptide-loaded ethosomes safely for subsequent in vitro release behavior studies using Franz diffusion cells.

2.6.2. In Vitro Release Assessment Using Franz Cells

The in vitro release behavior of hydrolyzed collagen peptides from ethosomal formulations (T11) was evaluated using Franz diffusion cells. The primary objective of this experiment was to compare the release profiles of encapsulated peptides with those of non-encapsulated aqueous peptide solutions, rather than to investigate transdermal penetration or systemic delivery.

The Franz diffusion cells were equipped with cellulose acetate dialysis membranes (12–14 kDa) and Strat-M synthetic membranes. The two membranes differ in their structural relevance. Cellulose acetate serves as a simple barrier but does not closely resemble human skin, whereas Strat-M is a synthetic membrane designed to mimic the main structural and chemical properties of human skin although it has not been approved legally as a replacement for animal or human skin in regulations [61]. Its top layer is tightly packed and coated with a lipid mixture similar to the stratum corneum, while the porous lower layer resembles the epidermis and dermis [62]. The effective diffusion surface area of each membrane was 0.21 cm^2^.

A 1.5% aqueous solution of hydrolyzed collagen peptide was used to examine the release behavior of the free peptide mixture. This solution was compared with the release profiles of the encapsulated peptide-loaded ethosomes. The blank ethosomal formulation (T24) served as a negative control. Time-dependent release was measured, and the cumulative amounts diffused per unit area were calculated and plotted (Figure 7).

As shown in the graph (Figure 7), the free peptide mixture exhibited a faster release compared to the peptide-loaded ethosomal formulation (T11). This indicates that the encapsulated peptides are released in a controlled manner due to the vesicular structure. This difference indicates that incorporation of hydrolyzed collagen peptides into ethosomal vesicles effectively modulates their release, resulting in a more sustained release behavior attributable to the vesicular structure.

When comparing the membranes, both the free peptide solution and the peptide-loaded ethosomal formulation (T11) showed faster diffusion through the cellulose acetate membrane than through the Strat-M membrane. This observation reflects the higher barrier function of the Strat-M membrane and supports its closer resemblance to skin-like diffusion resistance.

In summary, these results indicate that encapsulating peptides within ethosomal vesicles provides sustained and controlled release, enhances the delivery potential of collagen, and that the type of membrane also affects the release rate. From a dermocosmetic perspective, such release behavior is advantageous, as prolonged availability of collagen-derived peptides at the skin surface or within the upper skin layers may support hydration.

3. Materials and Methods

3.1. Materials

Reagents for protein quantification by the Lowry method included sodium carbonate (Merck, Darmstadt, Germany), sodium hydroxide (Merck, Darmstadt, Germany), sodium–potassium tartrate (ZAG Chemistry, Istanbul, Türkiye), copper sulfate pentahydrate (Merck, Darmstadt, Germany), Folin–Ciocalteu reagent (Sigma-Aldrich, Darmstadt, Germany), and bovine serum albumin (BSA, Thermo Fisher Scientific, Waltham, MA, USA). Disodium phosphate (Sigma-Aldrich, Darmstadt, Germany), monopotassium phosphate (Sigma-Aldrich, Darmstadt, Germany), sodium chloride (Merck, Darmstadt, Germany), and potassium chloride (Merck, Darmstadt, Germany) were used for the preparation of phosphate-buffered saline (PBS). Ethanol (>99%, Merck) and soybean lecithin containing phosphatidylcholine from genetically modified plants (Lipoid P75, Lipoid GmbH, Ludwigshafen am Rhein, Germany; >70% phosphatidylcholine) were used as phospholipid sources for ethosome preparation. Hydrolyzed collagen (>98%, Collagen Ar-Ge Teknoloji A.Ş., Istanbul, Türkiye), with an average molecular weight of ≤3 kDa, and vitamin E (BASF, Ludwigshafen, Germany) were used as active ingredients. Polysorbate 20 (Emulgin SML 20, BASF, Ludwigshafen, Germany) was added at 2% to the PBS medium in Franz diffusion cell experiments. Ultrapure water (Merck, Darmstadt, Germany) was used for all analyses. The XTT Cell Proliferation Kit (Roche Diagnostics, Mannheim, Germany) and DMEM (Sigma-Aldrich, Darmstadt, Germany) were used for cell viability assays.

A high-pressure homogenizer (Microfluidizer M-110P, Microfluidics, Middleborough, MA, USA) was used for ethosome preparation and particle size reduction. Particle size and distribution were analyzed using a Mastersizer 2000 (Malvern Instruments, Malvern, UK) and an Anton Paar Litesizer 500 (Anton Paar, Graz, Austria). Fourier transform infrared spectra were collected with a Thermo Scientific Nicolet iS5 FT-IR spectrometer equipped with an iD5 ATR accessory (Thermo Fisher Scientific, Waltham, MA, USA).

Absorbance for protein quantification was measured using a UV–Vis spectrophotometer (GENESYS 10S, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a 10 mm optical path quartz cuvette. Two milliliters of each sample were placed in the cuvette, and measurements were performed at 750 nm. All measurements were repeated five times, and average values were reported. Absorbance during cytotoxicity assays was measured using an iMark Microplate Absorbance Reader (Bio-Rad, California, CA, USA). pH was measured using a WTW pH meter (Xylem/WTW, Weilheim, Germany), and density was determined using an Anton Paar DMA 38 digital density meter (Anton Paar, Graz, Austria).

Skin permeation experiments were carried out using a LOGAN FDC-6 Franz diffusion cell system (Logan Instruments, Somerset, NJ, USA). Strat-M synthetic membranes (Merck, Darmstadt, Germany) and cellulose acetate membranes with a molecular weight cut-off of 12–14 kDa (Spectrum Laboratories, Rancho Dominguez, CA, USA) were used as diffusion barriers.

3.2. Determination of Hydrolyzed Collagen Content by the Lowry Method

Hydrolyzed collagen content was quantified using the Lowry method [63]. Samples and hydrolyzed collagen standards were reacted with the Lowry reagent, followed by addition of Folin–Ciocalteu reagent, and absorbance was measured at 750 nm using a UV–Vis spectrophotometer (GENESYS 10S, Thermo Fisher Scientific, Waltham, MA USA). Collagen concentrations in the samples were calculated from the corresponding calibration curve. Detailed reagent compositions, preparation steps, and full procedural parameters are provided in the Supplementary Information.

3.2.1. Method Validation

The analytical method for the quantification of hydrolyzed collagen was validated in accordance with the ICH Q2(R2) (2023) guidelines to assess linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), range, robustness, and specificity. Comprehensive validation data, equations, statistical outputs, and calibration curve plots are presented in Supplementary Information.

3.2.2. Determination of Hydrolyzed Collagen Solubility

Solubility was determined by stirring 1 g of hydrolyzed collagen in 30 mL of phosphate-buffered saline (PBS) with pH 7.2 at 25 °C and 150 rpm for 1 h using a magnetic stirrer. Three parallel replicates were conducted. Samples were filtered through a 0.45 µm membrane filter, and 1 mL of the filtrate was taken for quantitative analysis according to the method described in Section 2.2.

3.3. Preparation of Ethosomal Formulations

Blank ethosomal formulations were prepared using the cold method under mechanical stirring [64]. Briefly, synthetic phosphatidylcholine (Lipoid P75) and vitamin E were dissolved in ethanol and stirred at 300 rpm. Deionized water was then added dropwise to the ethanolic lipid phase at 30 °C under continuous stirring to induce vesicle formation. After complete addition of the aqueous phase, the dispersions were stirred for an additional 5 min to ensure homogeneity.

Hydrolyzed collagen peptide–loaded ethosomes were prepared following the same procedure, with the peptide incorporated into the aqueous phase prior to hydration. Deionized water containing hydrolyzed collagen peptides (1.5%, w/v) was added dropwise to the ethanolic lipid phase under identical stirring and temperature conditions, allowing the formation of peptide-loaded vesicles.

Following initial preparation, both blank and peptide-loaded ethosomal dispersions were subjected to size reduction by high-pressure homogenization as described in Section Size Reduction by High-Pressure Homogenization.

Size Reduction by High-Pressure Homogenization

Following initial preparation, ethosomal dispersions were subjected to high-pressure homogenization using a microfluidizer (Microfluidizer M-110P, Microfluidics, USA) to reduce vesicle size [65]. Homogenization was performed at pressures of 500, 750, or 1000 bar, and dispersions were processed for 1, 3, 5, or 7 homogenization cycles, where each cycle corresponded to a single pass through the interaction chamber. Processing temperature was maintained at 25 °C to minimize thermal effects.

Blank ethosomes were initially prepared and processed under these varying conditions to isolate the effects of homogenization pressure and cycle number on vesicle formation and size uniformity in the absence of peptide loading. Based on these results, selected processing conditions were subsequently applied to ethanol-containing and peptide-loaded ethosomal formulations. All formulation variables other than homogenization pressure and cycle number were kept constant.

3.4. Characterization of Ethosomal Formulations

3.4.1. Measurements of pH, Conductivity, Density

The physicochemical properties and stability of ethosomal formulations were characterized by evaluating pH, conductivity, density, particle size, zeta potential, and physical appearance. The pH of the ethosomal dispersions was measured using a tabletop pH meter (pH 3110, Xylem/WTW, Weilheim, Germany). Conductivity was determined using a tabletop conductivity meter (WTW pH 3110, WTW, Germany). The density of the ethosomal formulations was measured using a tabletop density meter (DMA 38, Anton Paar, Ashland, VA, USA). All measurements were performed five times for each sample.

3.4.2. Particle Size Analysis

The particle size and size distribution of empty and hydrolyzed collagen–loaded vesicular systems were determined by laser diffraction using a Mastersizer 2000 (Malvern Instruments, USA) [66,67,68]. Measurements were performed at 25 ± 2 °C after appropriate dilution with distilled water. Particle size was reported as the volume-weighted median diameter (d_0.5_), which represents the diameter at which 50% of the particle population is smaller. Each measurement was performed in five replicates, and the reported values represent the mean particle size for each formulation.

3.4.3. Size Distribution Uniformity

The homogeneity of vesicle size distributions was evaluated using a uniformity parameter derived from the volume-based particle size distribution obtained by laser diffraction (Mastersizer 2000, Malvern Instruments, USA) [69]. This parameter reflects the width of the vesicle size distribution and provides an indication of population uniformity, with lower values corresponding to more homogeneous vesicle populations and higher values indicating broader, more heterogeneous distributions.

For context, in dynamic light scattering (DLS), vesicle homogeneity is often expressed as the polydispersity index (PDI), which ranges from 0 for highly uniform populations to 1 for highly polydisperse systems. In this study, particle size distributions were obtained by laser diffraction, and the uniformity parameter used here is conceptually similar to PDI but derived from volume-based distributions rather than DLS measurements.

In this study, the uniformity values ranged from approximately 0.1 for highly uniform nanosized vesicles to over 70 for heterogeneous or partially aggregated populations. These values allow comparison of formulation and processing effects on vesicle homogeneity. The uniformity parameter is distinct from the polydispersity index (PDI) obtained from DLS, which is not reported here. All uniformity values reported in the tables and figures represent the mean of five replicate measurements for each formulation.

3.4.4. Zeta Potential Analysis

The zeta potential (ζ) of both empty and hydrolyzed collagen–loaded ethosomal formulations was determined using a Litesizer™ 500 (Anton Paar, Germany), which utilizes dynamic light scattering principles to determine electrophoretic mobility [70]. The dispersions were diluted at a 1:20 dilution factor with deionized water and transferred into 100 Ω-type zeta cuvettes. Measurements were conducted at 25 ± 2 °C, and the mean zeta potential values represent the average of five replicate readings for each sample, reflecting the surface charge and stability of the vesicular systems.

3.4.5. Physical Stability

To optimize lipid selection and identify suitable formulations, the stability of empty ethosomes was initially evaluated over 14 days. Subsequently, selected ethosomal formulations were stored at room temperature (25 ± 2 °C) for up to 180 days. Stability was assessed by monitoring changes in particle size, size distribution uniformity, zeta potential, and physical appearance (color and homogeneity) at predetermined time points (1, 7, 30, and 180 days). Any signs of aggregation, sedimentation, or precipitation were visually inspected and recorded.

3.4.6. Vesicle Morphology Characterization by Cryo-Scanning Electron Microscopy (Cryo-SEM)

The morphology of ethosomal formulations was examined using cryo-scanning electron microscopy (cryo-SEM) [66,67]. Samples were prepared with a PolarPrep 2000 cryo-preparation system (Quorum Technologies, Sussex, UK). The dispersions were frozen in nitrogen slush at −210 °C, fractured at −180 °C, and sublimed at −90 °C for 5–25 min, as optimized experimentally, to achieve optimum surface relief. The samples were subsequently coated with platinum to enhance conductivity.

Imaging was performed using an FEI Quanta 600F FEG Scanning Electron Microscope (Issy-les-moulineux, France) at −140 °C. Image analysis was carried out using the Scandium Image Analysis System (Olympus Soft Imaging Systems, Münster, Germany). Spatial calibration of the images was obtained directly from the microscope’s calibration data to ensure accurate measurement of vesicle dimensions and surface features.

Quantitative morphometric parameters were obtained from annotated cryo-SEM micrographs using image-based area analysis. Mean aspect ratio and image-based size variability (ISV) were calculated from projected vesicle contours to assess vesicle shape and morphological size dispersion. These parameters reflect morphology-derived vesicle uniformity and are distinct from hydrodynamic size or polydispersity values obtained using laser diffraction or dynamic light scattering techniques.

3.5. Determination of Encapsulation Efficiency

Encapsulation efficiency (EE) of ethosomal vesicles was evaluated by ultracentrifugation (Allegra X-30R, Beckman Coulter, Indianapolis, IN, USA). Ethosome suspensions (0.5 mL) were diluted with distilled water and centrifuged at 16,000× g for 45 min. The concentration of free (unencapsulated) hydrolyzed collagen was quantified in the supernatant using established analytical methods. EE (%) was calculated as follows:

$$[eqn]$$

where T is the total initial amount of HCP added and C is the amount of the untrapped HCP detected in the supernatant [70].

3.6. In Vitro and Biological Evaluations

3.6.1. In Vitro Cytotoxicity Assay

The cytotoxicity of ethosomal formulations and pure hydrolyzed collagen was evaluated using the XTT Cell Proliferation Assay, following the ISO 10993-5 standard for biological evaluation of medical devices [71]. Human keratinocyte cells (HaCaT cell line) were detached from culture flasks using standard passaging protocols and counted with a hemocytometer under an inverted microscope. Cells were seeded in 96-well plates at a density of 10,000 cells per well and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO_2_ to allow for cell attachment.

After attachment, cells were treated with the test samples, consisting of ethosomal formulations and hydrolyzed collagen, which were diluted in DMEM medium at four concentrations: 25%, 10%, 1%, and 0.1%. Sodium lauryl sulfate (SLS) at 0.1% and 0.01% served as the positive control to confirm assay responsiveness, while DMEM medium alone was used as the negative control.

Following 24 h of incubation at 37 °C and 5% CO_2_, XTT reagent and its activator were added to each well to achieve a final concentration of 0.3 mg/mL, and the plates were incubated for an additional 4 h. The absorbance at 450 nm was recorded using a microplate reader (iMark, Bio-Rad, California, CA, USA). Cell viability was calculated as the ratio of absorbance in treated wells relative to negative control wells, using the formula:

$$[eqn]$$

All concentrations were tested in technical replicates (four wells per concentration) with three readings taken per well to improve reliability. The mean values ± standard deviation were calculated, and the percentage of viable cells at different sample concentrations was plotted to evaluate cytotoxicity.

3.6.2. In Vitro Release Assessment Using Franz Diffusion Cells

The in vitro release of hydrolyzed collagen entrapped in ethosomal formulations was evaluated using a semi-automatic Franz diffusion cell system (Franzcell Transdermal FDC-6, Logan, UT, USA) [60]. The receptor compartment of each cell had a volume of 11 mL and was continuously stirred with a magnetic bar. The temperature was maintained at 37 ± 1 °C using a recirculating water bath to simulate physiological conditions.

The barrier between the donor and receptor compartments consisted of either a synthetic cellulose acetate membrane or Strat-M^®^ membrane. The surface area of the membranes used was 0.21 cm^2^. Membranes were pre-soaked in distilled water for 2 h prior to use to ensure proper hydration.

For the donor phase, 1 mL of ethosomal solution containing the active ingredient (hydrolyzed collagen) was applied to the membrane surface. 1 mL aliquots of the receptor solution were withdrawn at predetermined time points (1, 2, 4, 8, 12, 24, and 48 h) and analyzed to determine the concentration of the active compound, following the methods described in Section 2.2.

The receptor medium was selected based on the solubility of the active compounds: a 2% nonionic surfactant in phosphate buffer was used for hydrolyzed collagen. To prevent back-penetration and maintain sink conditions, the concentration of the active compound in the receptor solution was ensured to remain below 10% of its solubility. A control experiment was conducted in parallel using 1.5% solutions of hydrolyzed collagen in phosphate buffer to compare the diffusion profile of free versus encapsulated actives.

3.7. Statistical Analysis

All experiments were performed in at least three replicates, and results are presented as mean ± standard deviation (SD). Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test to assess differences between groups. Differences were considered statistically significant when the p-value was less than 0.05 (p < 0.05).

4. Conclusions

In this study, ethosomal formulations were successfully developed for the encapsulation of hydrolyzed collagen peptides. The formulations were prepared using synthetic phosphatidylcholine (Lipoid P75), ethanol, vitamin E, and water, and stable vesicles were obtained through high-pressure homogenization, resulting in effective vesicle size reduction. After optimization, hydrolyzed collagen peptides were efficiently incorporated into the ethosomes, achieving high encapsulation efficiencies of not less than 85%. Comprehensive physicochemical characterization including particle size, zeta potential, pH, conductivity, density, and cryo-SEM imaging demonstrated that the selected formulations were uniform, structurally intact, and physically stable over 180 days. Morphological analysis confirmed spherical vesicles, with hydrolyzed collagen peptide loading slightly increasing the aspect ratio while maintaining narrow size distributions. Cell viability assays further confirmed the biocompatibility of the ethosomal carriers at low concentrations.

The physical stability of the ethosomal formulations was evaluated by monitoring particle size and encapsulation efficiency over 180 days (long-term stability), while zeta potential was measured over 30 days to assess short-term stability.

Validated analytical methods, developed in accordance with ICH guidelines, enabled reliable quantification of encapsulated hydrolyzed collagen and evaluation of release behavior. The in vitro release studies clearly indicated that ethosomal encapsulation provides a sustained and controlled release profile compared to simple aqueous solutions of the active ingredient. Diffusion experiments performed using cellulose acetate and Strat-M synthetic membranes served as comparative, non-biological models to elucidate formulation-dependent release behavior rather than to assess transdermal penetration.

To the best of our knowledge, this work represents the first systematic investigation of hydrolyzed collagen peptide encapsulation within ethosomal vesicles, addressing an important gap in the formulation of peptide-based anti-aging cosmetic ingredients. In conclusion, the results highlight ethosomes as effective and biocompatible nanocarriers capable of encapsulating hydrolyzed collagen peptides with high efficiency while maintaining long-term physical stability and controlled in vitro release behavior.