2.2. Mucoadhesive Force of the aNPs

The mucoadhesion fracture strengths of the produced aNPs were measured against the small intestines of mice, pigs, and sheep using a texture analyzer. The obtained results are presented in Figure 3 (mice), Figure 4 (pigs), and Figure 5 (sheep).

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Figure 3

Mucoadhesion fracture strength of the produced aNPs against the small intestines of mice for (A) an applied force of 20 mN and (B) 200 mN. The ANOVA test was conducted to ascertain the statistical significance of the measured mucoadhesion fracture strength compared to A. platensis NPs. The values represent the mean ± SD of n = 4, where statistical significances are denoted as ** p < 0.01, **** p < 0.0001, and ns—nonsignificant.

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Figure 4

Mucoadhesion fracture strength of the produced aNPs against the small intestines of pigs for (A) an applied force of 20 mN and (B) 200 mN. The ANOVA test was conducted to ascertain the statistical significance of the measured mucoadhesion fracture strength compared to A. platensis NPs. The values represent the mean ± SD of n = 4, where statistical significances are denoted as * p < 0.05, ** p < 0.01, *** p < 0.001, and ns—nonsignificant.

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Figure 5

Mucoadhesion fracture strength of the produced aNPs against the small intestines of sheep for (A) an applied force of 20 mN and (B) 200 mN. The ANOVA test was conducted to ascertain the statistical significance of the measured mucoadhesion fracture strength compared to A. platensis NPs. The data represent the mean ± SD of n = 4, where statistical significances are denoted as * p < 0.05, ** p < 0.01, and ns—nonsignificant.

As predicted and shown in Figure 3, A. platensis, NPs exhibited the highest mucoadhesion fracture strength of 1786 ± 81 and 3127 ± 272 µN/mm² for 20 mN and 200 mN, respectively. These forces were 2–10-fold stronger than the other tested aNPs. L. japonica NPs also exhibited high mucoadhesion of 2691 ± 509 µN/mm² at the applied force of 200 mN (statistically similar to A. platensis NPs). On the other hand, U. pinnatifida (170 ± 47 µN/mm²) and G. gracilis (338 ± 42 µN/mm²) aNPs exhibited the lowest mucoadhesion fracture strength towards the intestines of mice for both applied forces.

Generally, the measured mucoadhesive fracture strength increased when the applied force rose from 20 mN to 200 mN (Figure 3B vs. Figure 3A). We hypothesize that the increased applied force may augment the contact surface area by the disentanglement of mucins, thus increasing the number of surface interactions between the aNPs and the intestinal epithelial layer [30]. Also, since there is no clear standard regarding which animal model should be used for mucoadhesion measurements, the intestines of pigs were examined, as can be seen in Figure 4.

As shown in Figure 4 for pig intestines, A. platensis NPs exhibited the highest mucoadhesion fracture strength of 1539 ± 112 and 1901 ± 100 µN/mm² for 20 mN and 200 mN, respectively. These forces were 2–5-fold stronger than observed for the tested aNPs. P. palmata and G. skottsbergii (1800 ± 83 and 1609 ± 158 µN/mm², respectively) NPs also exhibited high mucoadhesion at the applied force of 200 mN (statistically similar to A. platensis NPs). On the other hand, S. muticum NPs showed the lowest mucoadhesion towards the intestines of pigs for both applied forces: 295 ± 52 and 427 ± 109 µN/mm² for 20 mN and 200 mN, respectively.

As observed for mice intestines, a similar trend was detected herein for pig intestines. Enhancing the applied force from 20 mN to 200 mN (Figure 4B vs. Figure 4A) increased the measured mucoadhesive force. This observation further supports the hypothesis of an increase in contact area when the applied force increases. Next, the intestines of sheep were used to measure the mucoadhesion fracture strength of the composed from aNPs, as seen in Figure 5 below.

As shown in Figure 5 for sheep intestines, A. platensis NPs exhibited the highest mucoadhesion fracture strength towards the intestines of sheep, with 828 ± 66 and 1386 ± 46 µN/mm² for 20 mN and 200 mN, respectively. These forces were 1.5–3.5-fold stronger than the rest of the tested aNPs. Notably, L. japonica and H. pluvialis (1008 ± 201 and 822 ± 230 µN/mm², respectively) aNPs also exhibited high mucoadhesion at the applied force of 200 mN (statistically similar to A. platensis NPs). Conversely, S. muticum (243 ± 37 µN/mm²) and U. pinnatifida (335 ± 52 µN/mm²) aNPs exhibited the lowest mucoadhesion fracture strengths towards the intestines of sheep for both applied forces. Finally—as observed with mice and pig intestines—for all the tested aNPs, the measured mucoadhesion fracture strength was also increased when the applied force was increased from 20 mN to 200 mN.

To further analyze and compare the three ex vivo intestinal animal models, we chose three aNPs with the highest mucoadhesion—A. platensis, L. japonica, and L. digitata —and three with the lowest—S. muticum, G. gracilis, and C. chamissoi. Note that the obtained mucoadhesion fracture strengths are categorized and color-coded by the applied forces of 20 (blue bars) and 200 mN (red bars) and presented in Figure 6 below.

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Figure 6

Mucoadhesive fracture strengths of six different aNPs with applied forces (20 mN—blue bars and 200 mN—red bars) in (A) three aNPs with the highest mucoadhesion and (B) three with the lowest. ANOVA test was conducted to ascertain the statistical significance of the measured mucoadhesion fracture strength. The data represent the mean ± SD of n = 4, where statistical significances are denoted as ns—nonsignificant, * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 for multiple-variable comparisons.

Figure 6 illustrates notable variations in mucoadhesion observed among the ex vivo intestinal models for the highest and lowest aNPs. Figure 6A highlights the algae with the most robust mucoadhesive properties, with the mouse intestinal model yielding the highest results at 200 mN, followed by pig and sheep intestines. At 20 mN, no significant difference was observed between mucoadhesion in mice and pigs, but both outperformed sheep intestines. In contrast, the findings in Figure 6B are not as definite regarding aNPs with weaker mucoadhesive properties. A. platensis NPs consistently exhibited superior mucoadhesion, particularly at 200 mN (often at 20 mN).

2.3. Protein BLAST of Mucin2

Comparing the sequence of human Mucin2 (a protein consisting of 5130 amino acids (aa)) to that of the tested ex vivo animal models —mice, pig, and sheep intestines—provides valuable insights into the correlation between animal models and humans. Thus, to evaluate which intestinal animal model is the most suited to predict mucoadhesion in humans, we compared the sequences of the glycoprotein Mucin2, which is the main component of mucus [31]. The comparison was carried out via the Protein BLAST tool, and the results are presented in Table 2.

First, it is important to define the specific sequence alignment metrics of the Identities and Positives parameters mentioned in Table 2. Identities refer to the count of positions where the amino acids in compared sequences match precisely. Positives denote positions where the aligned amino acids exhibit similar properties (i.e., both are hydrophobic/acidic, etc.). As shown in Table 2, the mouse Mucin2 sequence displayed more Identities and Positives than the sheep and pig sequences, indicating the high resemblance of Mucin2 from a mouse to that from a human.

Moreover, Insertions cause “gaps” in the alignment, resulting in missing or incomplete information known as insertion–deletion mutations (indels). The mouse sequence of Mucin2 had no gaps, whereas the sheep and pig sequences displayed 3% gaps, indicating slight structural divergence in those regions. Additionally, query cover refers to the proportion of the query sequence (human Mucin2) that aligns with the compared sequence. Higher coverage implies a more significant overlap, potentially highlighting functional regions. Here, the sheep sequence exhibited the highest coverage (87%), the pig sequence had 60%, and the mouse sequence had the lowest coverage (43%).

The final score presented in Table 2 considers all the parameters and quantifies the extent of similarity between the compared sequences. The mouse sequence exhibited the highest score (2532), showing the highest likeness to human Mucin2. The sheep and pig sequences had similar final scores (1495 and 1474, respectively), signifying relatively lower but noteworthy similarity to human Mucin2.

Mucoadhesion results from interaction with mucus composed of mucins. Thus, comparing mucins could serve as an indication of mucoadhesion in humans. Since mice showed the highest similarity to human Mucin2, it could be suggested as the most suited animal model for studying mucoadhesive properties for oral drug delivery. Further supporting this conclusion is that mice were preferred for measuring mass transport across the mucosal layer of the intestines since their intestinal resident time and mucus thickness align well with human conditions [27].

However, many other parameters, such as intestinal pH and morphology (i.e., surface area), may affect mucoadhesion. Thus, we continued to compare mice intestines to human intestines. For example, the pH range in the human intestine is 6.0–7.4 [32], and for mice it is 6.3–8.1 [33]. Additionally, Stanford et al. have demonstrated marked similarities in developmental trends over time between murine and human systems [34]. A detailed comparison of the two intestines has revealed more similarities than differences [35]. Though these observations are insufficient to prove complete similarity, they contribute to the conclusion that mice intestines are an appropriate model for assessing mucoadhesion and oral delivery efficiency in humans.

4.2. Preparation of aNPs

To prepare aNPs, each algae powder (See Table 6) was weighed and dispersed in 80 mL double-distilled water. Subsequently, the algae solution was insonated (Q700, by Qsonica) in an ice bath using a Ø 1.3 cm transducer at 60% amplitude, 50% duty cycle (DC) for two minutes for cell lysis. After sonication, the solution was transferred to a centrifuge tube (Megafuge 16R, by Thermo Scientific, Kiryat Shemona, Israel) and centrifuged at 3200× g for 5 min at 4 °C. Then, the supernatant containing the lysate was subjected to another sonication cycle and centrifugation at the same conditions for downsizing. The supernatant containing aNPs was centrifuged at 10,000× g for 60 min at 4 °C. Later, the supernatant was gently poured over 2 mL of 60% sucrose followed by ultracentrifugation (Sorvall wX, by Thermo Scientific, Kiryat Shemona, Israel) at 200,000× g for 30 min at 4 °C. Then, 600 µL of solution was carefully removed from the layer above the 60% sucrose solution [8]. After production, the NPs were stored in a refrigerator at 4 °C.

4.3. Characterization of aNPs

The size, zeta potential, and concentration of aNPs were measured in water by a DLS instrument (Zetasizer Ultra Red, by Malvern Worcestershire, UK). The aNP yield was defined as the number of aNPs obtained divided by the initial algae mass. Additionally, protein content in aNPs was assayed via the Bradford method [39]. The sample was analyzed using an ELISA reader (Infinite M200 Tecan) at 595 nm. When the concentration fell outside the linear range, the sample was diluted to ensure accurate measurement within the linear range (i.e., 0–2 mg/mL). The concentration of BSA was calculated using the calibration curve (Figure S4).

TEM at cryogenic temperatures (Cryo-TEM) was used to image the produced aNPs. Vitrified specimens were prepared on a copper grid coated with a perforated lacey carbon 300 mesh (Ted Pella Inc. CA, USA). A 2.5 µL drop from the solution was applied to the grid and blotted with filter paper to form a thin liquid film. The blotted sample was immediately plunged into liquid ethane (−183 °C). This procedure was performed automatically in the Plunger (Leica EM GP). The vitrified specimens were then transferred into liquid nitrogen for storage. The samples were studied using the FEI Talos F200C TEM at 200 kV maintained at −180 °C, and images were recorded on an FEI Ceta 16M camera (4k × 4k CMOS sensor) at low-dose conditions to minimize electron beam radiation damage. The first example involved A. platensis NPs after production. In preparing the second sample, the same NPs were taken, and the sample underwent lyophilization, which involved freezing and subsequent water removal under vacuum conditions (FreeZone 2.5 L −84 °C Labconco Kansas City, MO, USA). The resulting lyophilized powder was resuspended in 1 mL of distilled water. Then, a mild sonication (30% amplitude, 50% duty cycle for 1 min) was applied in an ice bath.

The FTIR analysis method involved the preparation of a sample tablet containing 1 mg of A. platensis NPs after lyophilization and 99 mg of KBr. The components were thoroughly mixed using a mortar and pestle until a uniform powder was achieved. Subsequently, the powder was carefully compressed into tablets. The prepared tablet was placed in an FTIR instrument (FTIR-4600-type A, Jasco, Tokyo, Japan) using a standard light source, TGS detector, 32 accumulations, and at 4 cm⁻¹ resolution.

4.4. Mucoadhesion Measurement of aNPs

Mucoadhesion measurements were conducted using a texture analyzer instrument (EZ-SX, by Shimadzu, Kyoto, Japan). Initially, 50 µL of the NPs solution was spread on a glass plate and dried for 30 min. Then, the small intestines of mice, pigs, or sheep were washed in PBS and carefully placed on top of a flat pin head (A = 40.7 mm²) so the lumen side faced outwards. The pin was mounted on the texture analyzer probe (capacity of 5 N, by Shimadzu), which was then lowered at a speed of 1.0 mm/s until the tissue came in contact with the aNPs with an applied force of 20 mN and 200 mN for 420 s [40]. The contact forces were chosen according to a study conducted to measure the peristaltic forces inside the intestines of a lamb. These forces were measured via an encapsulated prototype with a force sensor, where it was found that the range of peristaltic forces in the small intestine was between 0–180 mN [41]. As mentioned, the mucoadhesive fracture strength was derived from the peak force required to separate two layers [40]. In addition, the mucoadhesion forces of A. platensis NPs were measured at three pH values characteristic of different parts of the small intestine, and the zeta potential was tested for each pH. Everything was tested against pig intestinal tissue and is shown in Table S1.

4.5. Encapsulation of Fluorescent Molecule in aNPs

For these experiments, six algae were selected—three with the most significant mucoadhesive forces and three exhibiting the lowest mucoadhesive forces. To evaluate the cellular uptake in Caco-2 cells, FD40 was encapsulated in the chosen aNPs. First, 2 mL of the aNPs was mixed with 0.1 mL of 0.5 mg/mL FD40 and 7.4 mL of PBS. Then, the mixture was placed in an ice bath and sonicated using a microtip (Ø 0.3 cm) ultrasound transducer at 60% amplitude and 75% DC for two minutes. After sonication, the solution was refrigerated for 10 min. The sonication and refrigeration were repeated one more time. For the measurement of encapsulation efficiency, first, 200 µL of the (final) supernatant was transferred into a 96-well flat black plate (by Greiner, Monroe, NC, USA) to determine the amount of free FD40 and analyzed using a fluorometer (Infinite M200 Tecan) at excitation and emission wavelengths of 490 nm and 525 nm, respectively. The concentration of FD40 in the supernatant was calculated using the calibration curve (Figure S5).

4.6. Release profile of FD40 from A. platensis NPs

To assess the release profile from aNP, 12 mL of a solution containing aNPs from A. platensis encapsulating FD40 in PBS was placed in a 20 mL scintillation vial. The vial was covered with aluminum foil to minimize photobleaching. For nine consecutive days, at 24 h intervals, the mixture was ultracentrifuged at 200,000× g for 10 min at 4 °C. After each centrifugation, 200 µL of the supernatant was collected and replenished with an equivalent volume of PBS. The concentration of FD40 in the supernatant was calculated using the calibration curve (Figure S5).

4.7. Protein BLAST Analysis

The Mucin2 glycoprotein serves as the primary constituent of the outer mucus layer and imparts viscoelastic properties to mucus [42]. The human Mucin2 glycoprotein sequence was retrieved from the Entrez repository with the accession number AZL49145.1 [43] in FASTA format. The mouse, pig, and sheep sequences were obtained from the Protein Basic Local Alignment Search Tool (BLAST) (accessions are shown in Table 2). Later, the Protein BLAST [44] was used to search for similarities against the sequences of Mucin2 from mice, pigs, and sheep. The presented score value was used to assess the degree of similarity between the different mucin sequences.

4.8. Caco-2 Uptake of aNPs

To prepare the cell culture medium, 450 mL of DMEM was thoroughly mixed with 50 mL of FBS, 5 mL of L-Glu, and 5 mL of P/S. The cellular uptake of the chosen aNPs was evaluated with Caco-2 cells—the prominent GI in vitro model. First, Caco-2 cells were seeded in 24-well plates (Greiner) and incubated at a concentration of 2 × 10⁵ cells/mL for 48 h at 37 °C in 1 mL of cell medium. Then, the cells were counted using an automated cell counter (A₂S, b EVE™, Waltham, MA, USA), and the number of NPs was determined using the DLS instrument. Following these measurements, the ratios 1:1 and 100:1 (aNPs/Caco-2 cells) were used for each tested alga. Caco-2 cells were incubated with free FD40 at an equivalent amount as encapsulated in the aNPs and used as a control. After three hours of incubation, the wells were carefully washed twice with PBS to remove free FD40 and aNPs. Subsequently, 300 µL of trypsin was added to each well and incubated for five minutes in the incubator to detach the cells. Then, the cell suspension was transferred to an Eppendorf test tube with 1 mL of medium. Cells were obtained by centrifugation at 500× g for 12 min and were resuspended in a solution of PBS containing 0.5% BSA. Later, the samples were transferred to 96-well microplates and analyzed under the FACS instrument (CytoFLEX Beckman Coulter, Indianapolis, IN, USA) to test the degree of internalization of Caco-2 cells to algae NPs.