Dietary-Polysaccharide-Modified Fish-Oil-Based Double Emulsion as a Functional Colloidal Formulation for Oral Drug Delivery

Oral delivery is the most convenient drug administration route. However, oral delivery of peptides is extremely challenging due to the physical and chemical barriers within the gastrointestinal tract. Polysaccharides are often utilized as polymeric biomaterials in drug delivery. Among these, dietary polysaccharides extracted from okra, yam, and spirulina have been reported to stimulate innate immunity with well-known nutritional benefits. In this study, we developed a dietary-polysaccharide-modified fish-oil-based emulsion for oral co-delivery of a hydrophilic PD-L1 blocking peptide and the hydrophobic small molecule simvastatin. The optimal emulsion was nano-sized and exhibited a negative surface charge, high drug encapsulation efficiency of over 97%, low viscosity, and sustained drug release manner. The formulation could significantly increase the uptake of peptides by intestinal Caco-2 cells, which demonstrated the great potential of the formulation for promoting the oral absorption of peptides. Additionally, these dietary polysaccharides could promote dendritic cell maturation and cytokine expression in macrophages, demonstrating that these nutraceutical polysaccharides had dual roles of functioning as promising colloidal delivery systems and as potential immune modulators or adjuvants. Thus, this food-based colloidal delivery system shows promise for the oral delivery of peptide drugs and lays a great platform for future applications in immunotherapy.


Introduction
Oral drug delivery is the most conventional and convenient administration route for the treatment of various diseases, as it alleviates the pain caused by injections with desirable cost-effectiveness and shows a good safety profile [1]. However, the physical and chemical barriers of the gastrointestinal tract (GIT) drastically limit the oral absorption of drugs-particularly peptide drugs, which are easily degraded by the strong acids from the stomach and the proteolysis enzymes within the GIT [2]. Therefore, to overcome the physical and biochemical barriers in the GIT, the design of a suitable delivery system for promoting oral drug absorption is crucial.
Currently, emulsion technology has attracted much attention, as it has a low cost, involves a simple fabrication process with great stability, and can be easily scaled up in manufacturing; thus, it is widely applied in food, pharmaceutical, and nutraceutical products [3]. Double-phase emulsions, which consist of two types, oil-in-water-in-oil (O 1 /W/O 2 ) and water-in-oil-in-water (W 1 /O/W 2 ) are often used in the co-delivery of hydrophilic and hydrophobic bioactive compounds [4]. Although double-phase emulsions are kinetically stable, the outer layer of an emulsion may be degraded by high temperatures, oxidation, or strong acids or bases, and it may suffer from degradation into a single-phase

Preparation of the W 1 /O/W 2 Double Emulsion
The proportions of the inner aqueous phase (W 1 ), the oil phase (O), and the external aqueous phase (W 2 ) were 1:3:12. Lecithin (1.5% w/v) was dissolved in fish oil to obtain the oil phase. Deionized water was added to the oil phase dropwise and stirred for 10 min, followed by a homogenization process for 3 min by using ultrasonic processor. The primary W 1 /O emulsion was obtained. Different polysaccharide solutions (okra, yam, and spirulina polysaccharide) with different concentrations (1.25, 2.5, 5, 10, and 20 mg/mL) were prepared. Glycerin (8% v/v) was added to each solution to form an outer aqueous phase. The primary W 1 /O emulsion was added to the outer aqueous phase, and the dispersions were homogenized for 3 min to obtain the final W 1 /O/W 2 double emulsions.

Stability Study of the W 1 /O/W 2 Emulsions
We selected 3 dietary polysaccharides that had been reported to stimulate innate immunity: okra, yam, and spirulina polysaccharide. The polysaccharide solutions acted as the external aqueous phase of the emulsions. In order to explore the optimal polysaccharide concentration to form the most stable emulsion, 5 concentrations of 1.25, 2.5, 5, 10, and 20 mg/mL were investigated for each polysaccharide. Fifteen fresh emulsions were transferred into Eppendorf (EP) tubes and stored at 25 • C (room temperature) and 37 • C (body temperature) for 21 days. The stratification was observed, and 9 emulsions with poor stabilities were eliminated. As for the forced thermal stability study [20], 6 residual emulsions were placed in a warm-water bath at 45, 65, and 85 • C, respectively. The stratification was observed to eliminate unstable emulsions. Finally, 3 optimal polysaccharide-modified emulsions with the greatest stability were obtained, with one selected from each polysaccharide category.

Morphology
Samples were placed on the glass slides and gently covered with a coverslip to prevent the emulsions from being destroyed. An optical microscope (TS2, Nikon, Japan) was Pharmaceutics 2022, 14, 2844 4 of 14 employed to observe the morphology of the emulsions at room temperature. A digital camera was used to acquire images.

Size and Zeta Potential
A Mastersizer 2000 (Malvern Instruments Ltd., Malvern, UK) was employed to determine the droplet size in the W 1 /O/W 2 emulsions. D(v, 90) was used to represent the average droplet size of the emulsions. The zeta potential [21] was determined with a Zetasizer (Nano ZS, Malvern, UK). Measurements were in triplicate. Polydispersity was expressed by the SPAN factor [22]. The equation was as follows: D(v, 10), D(v, 50), and D(v, 90) are the volume size diameters at 10%, 50%, and 90% of the cumulative volume, respectively.

Encapsulation Efficiency
The encapsulation efficiency (EE) of the drugs in the double emulsion was determined by measuring the concentrations of the drugs in the external aqueous phase (W 2 phase) [23]. The emulsions were centrifuged at 21,532× g at 4 • C for 15 min, and they were separated into creamy layers (upper, W 1 /O emulsion) and a whey layer (bottom, W 2 phase). The whey layer contained the unencapsulated drug and was collected from each centrifugal sample by using a syringe. This process was repeated 2-3 times in order to completely remove the creamy layers. The EE% of the W 1 /O/W 2 emulsions was estimated by using the equation: where m free represents the amount of the drug penetrating into the external water phase (µg), and m total represents the total amount of the drug added to the emulsion (µg).

Rheological Analysis
The viscosity of the emulsions was assessed by using a rheometer (MCR 302, Anton Paar GmbH, Austria). Shear flow tests were carried out to study the viscosity with a shear rate ranging from 0.1 to 100 s −1 at 25 • C [24].

Drug Release Study
To evaluate the drug release profile, 5 mL of free drug solution or 5 mL of drugloaded double emulsions were transferred into a dialysis bag (molecular weight cutoff: 8000-14,000 Da) and dialyzed against 100 mL of simulated intestinal fluid (enzyme-free) at 37 • C with a stirring speed of 200 rpm/min. Samples were collected at specific time intervals. The concentration of the released drug was determined with a fluorescence microplate (SpectraMax Id5, China). The accumulative release of the drug was calculated.

MTT Assay
Intestinal Caco-2 cells were seeded into 96-well plates one night in advance for cell adherence. The next day, the medium was discarded, and serum-free DMEM medium was added for cell starvation. After 8 h, the serum-free medium was discarded. W 1 /O/W 2 emulsions were added at 10% (v/v), 5% (v/v), 2.5% (v/v), 1.25% (v/v), 0.625% (v/v), and 0.3125% (v/v) and incubated for 2, 4, 6, 12, and 24 h, respectively. MTT was added into each well. When the time point was reached, the medium was discarded, and DMSO was added to fully dissolve the purple crystals. The absorbance was read at the OD of 490 nm.

In Vitro Drug Uptake Study by Caco-2 Cells via LSCM
Caco-2 cells were seeded in laser confocal cell dishes one night in advance for cell adherence. A replacement with clean culture medium took place the next day. A total of 100 µL of 0.2 mg/mL free FITC-modified peptide or peptide-loaded double emulsion was added and incubated with the cells for 2 h at 37 • C Then, the dishes were rinsed with phosphate-buffered saline (PBS) to remove residual FITC-modified peptides. Hoechst 33,258 was added and incubated for 30 min at 37 • C for nuclear staining. PBS was used to remove residual Hoechst. Laser-scanning confocal microscopy (LSCM) was employed to observe the peptide uptake by Caco-2 cells.

In Vitro Drug Uptake Study by Caco-2 Cells via Flow Cytometry
Briefly, Caco-2 cells were seeded into 48-well plate one night in advance for cell adherence. A replacement with fresh culture medium took place the next day. A total of 100 µL of 0.05 mg/mL free FITC-modified peptide or peptide-loaded double emulsion was added and incubated with the cells for 2 h at 37 • C. Then, the cells were collected and fixed, and the fluorescence intensity was measured via flow cytometry.

Effects of Polysaccharides on Dendritic Cell Maturation
This study was conducted by using bone-marrow-derived dendritic cells (BMDCs) from mice [25]. Briefly, the C57BL/6 mice were sacrificed, soaked in alcohol for 5 min, and then transferred to a sterile environment. The thigh bone was cut with scissors, and a syringe was used to flush out the bone marrow cells from the bone cavity. After filtration and centrifugation, the cell pellet was resuspended with ACK lysis buffer to lyse the red blood cells, washed with PBS twice, and cultured in a sterile culture dish with RPMI-1640 medium. GM-CSF and IL-4 were added into the medium for final concentrations of 20 and 10 ng/mL, respectively. Half of the medium was replaced daily from the third day while keeping the concentrations of GM-CSF and IL-4 unchanged. On the 6th day of induction, 2 × 10 5 cells/well were cultured, and 5 groups were set up: negative control of PBS, positive control of 1 µg/mL lipopolysaccharide (LPS), and 50 µg/mL of okra polysaccharide, yam polysaccharide, and spirulina polysaccharide. After 24 h of incubation, the cells were collected, stained with anti-CD11c-APC, anti-CD80-PerCP-efluor TM 710, and anti-CD86-PE, and analyzed via flow cytometry.

Effects of Polysaccharides on Macrophages
Murine macrophages (RAW 264.7 cells) were cultured in a 24-well plate at a density of 2 × 10 5 cells/well, and the corresponding substances were added after starvation for 8 h. A total of 5 groups were set up: negative control of PBS, positive control of 1 µg/mL LPS, and 50 µg/mL of okra polysaccharide, yam polysaccharide, and spirulina polysaccharide. Over 24 h of incubation, the cells were lysed to extract RNA and reverse transcribed into cDNA, and the mRNA levels of IL-6 and TNF-α were detected via qPCR.

Statistical Analysis
The statistical analysis was conducted with a one-tailed Student t-test for the differences between two groups. All data are shown as the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Optimization of Polysaccharide-Modified Emulsions
As shown in Figure 1A, among the okra polysaccharide emulsions, the 5, 10, and 20 mg/mL polysaccharide emulsions at 37 • C and the 10 and 20 mg/mL polysaccharide emulsions at room temperature appeared to undergo stratification. Among the yam polysaccharide emulsions, the 1.25 mg/mL polysaccharide emulsion at 37 • C and the 20 mg/mL polysaccharide emulsion at room temperature appeared to undergo stratification. Among the spirulina polysaccharide emulsions, the 2.5, 5, 10, and 20 mg/mL polysaccharide emulsions at 37 • C and the 10 and 20 mg/mL polysaccharide emulsions at room temperature appeared to undergo stratification. As a result, the relatively stable emulsions were the 1.25 and 2.5 mg/mL okra polysaccharide emulsions, the 2.5, 5, and 10 mg/mL yam polysaccharide emulsions, and the 1.25 mg/mL spirulina polysaccharide emulsion. undergo stratification over 2 h. According to the 21-day storage stability and forced thermal stability study, the 1.25 mg/mL okra polysaccharide emulsion, 2.5 mg/mL yam polysaccharide emulsion, and 1.25 mg/mL spirulina polysaccharide emulsion remained stable; thus, they were selected for further studies. Figure 1C shows the morphologies of the three optimal polysaccharide emulsions. The double-phase emulsion droplets appeared to have a round shape and the narrow size distributions without obvious aggregation, indicating the high uniformity of the droplets. In the forced thermal stability studies at 45, 65, and 85 • C, the stratification of the emulsions was observed. As shown in Figure 1B, at 85 • C, the 2.5 mg/mL okra polysaccharide emulsion and the 5 and 10 mg/mL yam polysaccharide emulsions appeared to undergo stratification over 2 h. According to the 21-day storage stability and forced thermal stability study, the 1.25 mg/mL okra polysaccharide emulsion, 2.5 mg/mL yam polysaccharide emulsion, and 1.25 mg/mL spirulina polysaccharide emulsion remained stable; thus, they were selected for further studies. Figure 1C shows the morphologies of the three optimal polysaccharide emulsions. The double-phase emulsion droplets appeared to have a round shape and the narrow size distributions without obvious aggregation, indicating the high uniformity of the droplets.

Droplet Size, Zeta Potential, SPAN Values, and Encapsulation Efficiency
The droplet sizes and zeta potentials of the emulsions were measured. As shown in Table 1, the droplet sizes of the three optimal polysaccharide emulsions were similar, ranging from 800 to 900 nm. They all showed a negative surface charge, ranging from −55 to −70 mV. SPAN is a measure of the width of the droplet size distribution. Generally speaking, the smaller the SPAN is, the narrower the droplet size distribution will be, and the larger the SPAN is, the wider the droplet size distribution will be. The three polysaccharide emulsions were also similar in terms of SPAN, which ranged from 3.30 to 3.50. The peptide was freely soluble in water, while the solubility of simvastatin in fish oil was determined to be 15 mg/mL. The EEs of these three polysaccharide emulsions for both the peptide drug and simvastatin were all over 97% ( Table 1). The high drug encapsulation efficiency is also one major advantageous characteristics of double-phase emulsions.  Figure 2 shows the drug release profiles of the free peptides and peptide-loaded polysaccharide emulsions in simulated intestinal fluid (SIF). Free peptides were rapidly dissolved and permeated over the dialysis membrane over 45 min, while the okra polysaccharide emulsion had a drug release of 23.98 ± 1.66%, the yam polysaccharide emulsion had a drug release of 34.11 ± 1.84%, and the spirulina polysaccharide emulsion had a drug release of 36.70% ± 1.17 of peptides over the same period. The sustained release profiles of the three polysaccharide-modified emulsions allowed the elongation of the drug half-life and elevation of the overall oral bioavailability of the drugs.

Drug Release Study
Forced thermal stability study. From left to right: 1.25 and 2.5 mg/mL okra polysaccharide, 2.5, 5, and 10 mg/mL yam polysaccharide, and 1.25 mg/mL spirulina polysaccharide emulsions. Here, the stratification of the emulsions at 85 °C over 2 h is shown. (C) An optical microscope was employed to observe the morphologies of the emulsions of 1.25 mg/mL okra polysaccharide, 2.5 mg/mL yam polysaccharide, and 1.25 mg/mL spirulina polysaccharide at room temperature.

Droplet Size, Zeta Potential, SPAN Values, and Encapsulation Efficiency
The droplet sizes and zeta potentials of the emulsions were measured. As shown in Table 1, the droplet sizes of the three optimal polysaccharide emulsions were similar, ranging from 800 to 900 nm. They all showed a negative surface charge, ranging from −55 to −70 mV. SPAN is a measure of the width of the droplet size distribution. Generally speaking, the smaller the SPAN is, the narrower the droplet size distribution will be, and the larger the SPAN is, the wider the droplet size distribution will be. The three polysaccharide emulsions were also similar in terms of SPAN, which ranged from 3.30 to 3.50. The peptide was freely soluble in water, while the solubility of simvastatin in fish oil was determined to be 15 mg/mL. The EEs of these three polysaccharide emulsions for both the peptide drug and simvastatin were all over 97% ( Table 1). The high drug encapsulation efficiency is also one major advantageous characteristics of double-phase emulsions.  Figure 2 shows the drug release profiles of the free peptides and peptide-loaded polysaccharide emulsions in simulated intestinal fluid (SIF). Free peptides were rapidly dissolved and permeated over the dialysis membrane over 45 min, while the okra polysaccharide emulsion had a drug release of 23.98 ± 1.66%, the yam polysaccharide emulsion had a drug release of 34.11 ± 1.84%, and the spirulina polysaccharide emulsion had a drug release of 36.70% ± 1.17 of peptides over the same period. The sustained release profiles of the three polysaccharide-modified emulsions allowed the elongation of the drug halflife and elevation of the overall oral bioavailability of the drugs.

Rheological Analysis
A flow curve is a graphical representation of how a flowing material behaves as the shear rate increases or decreases. The shape of the flow curve can distinguish the type of fluid. As shown in Figure 3, the viscosities of the three polysaccharide-modified emulsions did not increase or decrease with the increase in the shear rate from 0.1 to 100 s −1 , which was consistent with the characteristics of a Newtonian fluid. This result reflected the low viscosity of the emulsions, which could improve the mouthfeel during oral administration, reduce the adhesion to the esophageal mucosa, and be friendly to patients with dysphagia or children and the elderly. Figure 2. Drug release profile; 5 mL of free peptide solution or 5 mL of peptide-loaded double emulsion was transferred into a dialysis bag and dialyzed against 100 mL of artificial small intestine fluid (enzyme-free) at 37 °C . All data are reported as mean ± SD (n = 3).

Rheological Analysis
A flow curve is a graphical representation of how a flowing material behaves as the shear rate increases or decreases. The shape of the flow curve can distinguish the type of fluid. As shown in Figure 3, the viscosities of the three polysaccharide-modified emulsions did not increase or decrease with the increase in the shear rate from 0.1 to 100 s −1 , which was consistent with the characteristics of a Newtonian fluid. This result reflected the low viscosity of the emulsions, which could improve the mouthfeel during oral administration, reduce the adhesion to the esophageal mucosa, and be friendly to patients with dysphagia or children and the elderly.

MTT Assay
As shown in Figure 4, none of the three polysaccharide-modified emulsions affected the growth of intestinal Caco-2 cells at 2, 4, and 6 h, demonstrating the good biosafety and biocompatibility of the polysaccharide-modified emulsions with the intestinal cells in vitro. Although it was observed that the growth of Caco-2 cells was slightly affected by the emulsions at 12 and 24 h, this could be negligible, since it was not expected that the oral emulsion would have such a long retention within the gastrointestinal tract in general.

In Vitro Uptake Study
As shown in Figure 5, all three polysaccharide-modified emulsions could significantly increase the uptake of the peptide drug by Caco-2 cells compared with the uptake of free peptides, which demonstrated the ability of the delivery system to promote the intestinal permeation of drug candidates. Figure 6 shows the flow cytometry results, which indicate that all three polysaccharidemodified emulsions increased the FITC-labeled peptide uptake by Caco-2 cells compared to the uptake of free peptides. These drug uptake results that were analyzed via flow cytometry correspond to the uptake results of CLSM, which demonstrated that the polysaccharidemodified emulsions have good potential for promoting the oral absorption of peptide drugs.

MTT Assay
As shown in Figure 4, none of the three polysaccharide-modified emulsions affected the growth of intestinal Caco-2 cells at 2, 4, and 6 h, demonstrating the good biosafety and biocompatibility of the polysaccharide-modified emulsions with the intestinal cells in vitro. Although it was observed that the growth of Caco-2 cells was slightly affected by the emulsions at 12 and 24 h, this could be negligible, since it was not expected that the oral emulsion would have such a long retention within the gastrointestinal tract in general.

In Vitro Uptake Study
As shown in Figure 5, all three polysaccharide-modified emulsions could significantly increase the uptake of the peptide drug by Caco-2 cells compared with the uptake of free peptides, which demonstrated the ability of the delivery system to promote the intestinal permeation of drug candidates.  Figure 6 shows the flow cytometry results, which indicate that all three polysaccharide-modified emulsions increased the FITC-labeled peptide uptake by Caco-2 cells compared to the uptake of free peptides. These drug uptake results that were analyzed via flow cytometry correspond to the uptake results of CLSM, which demonstrated that the polysaccharide-modified emulsions have good potential for promoting the oral absorption of peptide drugs.  Figure 6 shows the flow cytometry results, which indicate that all three polysaccharide-modified emulsions increased the FITC-labeled peptide uptake by Caco-2 cells compared to the uptake of free peptides. These drug uptake results that were analyzed via flow cytometry correspond to the uptake results of CLSM, which demonstrated that the polysaccharide-modified emulsions have good potential for promoting the oral absorption of peptide drugs.

Polysaccharides Promoted Dendritic Cell Maturation
As shown in Figure 7, okra and spirulina polysaccharides significantly increased the expression of CD80, CD86, and CD40 on the surface of the BMDCs, indicating that these two polysaccharides significantly promoted the maturation of BMDCs. Although yam polysaccharide did not significantly increase the levels of CD80, CD86, and CD40, the indexes also showed a slight upward trend. Mature BMDCs have a strong antigen-presenting ability and could effectively activate naive T cells to prime an immune response [26]. cytometry was employed to observe the FITC-labeled peptide uptake by Caco-2 cells. *** p < 0.001, ns, no significance.

Polysaccharides Promoted Dendritic Cell Maturation
As shown in Figure 7, okra and spirulina polysaccharides significantly increased the expression of CD80, CD86, and CD40 on the surface of the BMDCs, indicating that these two polysaccharides significantly promoted the maturation of BMDCs. Although yam polysaccharide did not significantly increase the levels of CD80, CD86, and CD40, the indexes also showed a slight upward trend. Mature BMDCs have a strong antigen-presenting ability and could effectively activate naive T cells to prime an immune response [26].

Polysaccharides Promoted Cytokine Expression in Macrophages
The effects of the polysaccharides on macrophages were also explored. Okra, yam, and spirulina polysaccharides significantly increased the IL-6 mRNA levels in RAW 264.7 cells. Okra and yam polysaccharides also increased the TNF-α mRNA levels (Figure 8). IL-6 can induce T cell proliferation and differentiation, thus participating in the immune response [27]. TNF-α can activate neutrophils and lymphocytes, regulate other tissues' Figure 7. Effects of polysaccharides on BMDC maturation. Induced BMDCs were cultured, and five groups were set up: negative control of PBS, positive control of 1 µg/mL LPS, and 50 µg/mL of okra polysaccharide, yam polysaccharide, and spirulina polysaccharide. After 24 h of incubation, cells were collected, stained with anti-CD11c-APC, anti-CD80-PerCP-efluor TM 710, and anti-CD86-PE, and assessed via flow cytometry. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significance.

Polysaccharides Promoted Cytokine Expression in Macrophages
The effects of the polysaccharides on macrophages were also explored. Okra, yam, and spirulina polysaccharides significantly increased the IL-6 mRNA levels in RAW 264.7 cells. Okra and yam polysaccharides also increased the TNF-α mRNA levels ( Figure 8). IL-6 can induce T cell proliferation and differentiation, thus participating in the immune response [27]. TNF-α can activate neutrophils and lymphocytes, regulate other tissues' metabolic activities, and promote the release of other cytokines [28]. The results indicated that okra, yam, and spirulina polysaccharides could activate innate immunity, and they can act as immune-modulating agents in delivery systems.

Discussion
This study constructed a green oral delivery system based on dietary food. Fish oil is a well-known nutrient that reduces the risk of cardiovascular diseases and diabetes, relieves arthritis, depression, and anxiety, and has immunomodulatory and anti-tumor effects [29]. Dietary polysaccharides play a biological role in anti-tumor, anti-inflammation, anti-virus, hypoglycemic, antioxidant, anti-coagulation, and immune promotion activities, in addition to other aspects [30]. Three dietary-polysaccharide-based solutions were used as an outer aqueous phase to encapsulate a primary W1/O emulsion. The most stable formulations were obtained from a long-term storage stability study and a forced thermal stability study. The results showed that a lower concentration of the polysaccharide solution as the outer aqueous phase might be more conducive to the stability of the double emulsion. There might be a range of stability fluctuations between low-concentration polysaccharide solutions, which requires further investigation.
The optimal emulsions of the three polysaccharides had similar characteristics in terms of their nano-size, which is advantageous for oral absorption compared with microsize droplets [31]. The emulsions all showed a negative surface charge, which was due to the presence of the negatively charged outer aqueous layer of polysaccharides [32]. The EEs of the peptide and simvastatin were all over 97% for the three optimal formulations. These results corresponds well with most of the findings in the literature, indicating that a high EE is a major advantageous characteristic of double-phase emulsions in general. Another significant advantage of W1/O/W2 double emulsions is their ability to simultaneously load a water-soluble drug and an oil-soluble drug within their internal aqueous phase and oil phase [33]. The emulsions were evaluated as Newtonian fluids, which indicated that the viscous stresses arising from their flow were linearly correlated with the local strain rate at every point [34]. The results showed the low viscosity of the emulsions. Less viscous emulsions are normally better for the mouthfeel in oral administration, and this is especially beneficial for patients with dysphagia, children, and the elderly [35].
The in vitro drug release from the W1/O/W2 emulsion was evaluated by using the technique of diffusion in a dialysis bag. The drug release profile was similar to that observed from double-phase emulsions reported in the literature, which is generally well Figure 8. Effects of polysaccharides on RAW 264.7 cells. The RAW 264.7 cells were cultured, and the corresponding substances were added after starvation for 8 h. After incubation for 24 h, the cells were lysed to extract RNA and reverse transcribed into cDNA, and the mRNA levels of IL-6 and TNF-α were detected via qPCR. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, no significance.

Discussion
This study constructed a green oral delivery system based on dietary food. Fish oil is a well-known nutrient that reduces the risk of cardiovascular diseases and diabetes, relieves arthritis, depression, and anxiety, and has immunomodulatory and anti-tumor effects [29]. Dietary polysaccharides play a biological role in anti-tumor, anti-inflammation, anti-virus, hypoglycemic, antioxidant, anti-coagulation, and immune promotion activities, in addition to other aspects [30]. Three dietary-polysaccharide-based solutions were used as an outer aqueous phase to encapsulate a primary W 1 /O emulsion. The most stable formulations were obtained from a long-term storage stability study and a forced thermal stability study. The results showed that a lower concentration of the polysaccharide solution as the outer aqueous phase might be more conducive to the stability of the double emulsion. There might be a range of stability fluctuations between low-concentration polysaccharide solutions, which requires further investigation.
The optimal emulsions of the three polysaccharides had similar characteristics in terms of their nano-size, which is advantageous for oral absorption compared with micro-size droplets [31]. The emulsions all showed a negative surface charge, which was due to the presence of the negatively charged outer aqueous layer of polysaccharides [32]. The EEs of the peptide and simvastatin were all over 97% for the three optimal formulations. These results corresponds well with most of the findings in the literature, indicating that a high EE is a major advantageous characteristic of double-phase emulsions in general. Another significant advantage of W 1 /O/W 2 double emulsions is their ability to simultaneously load a water-soluble drug and an oil-soluble drug within their internal aqueous phase and oil phase [33]. The emulsions were evaluated as Newtonian fluids, which indicated that the viscous stresses arising from their flow were linearly correlated with the local strain rate at every point [34]. The results showed the low viscosity of the emulsions. Less viscous emulsions are normally better for the mouthfeel in oral administration, and this is especially beneficial for patients with dysphagia, children, and the elderly [35].
The in vitro drug release from the W 1 /O/W 2 emulsion was evaluated by using the technique of diffusion in a dialysis bag. The drug release profile was similar to that observed from double-phase emulsions reported in the literature, which is generally well correlated with pharmacokinetic results [36]. Irrespective of the nature of the sink solution, the drug release from the double-phase emulsions remained slow and incomplete as compared to that of a plain drug solution. This phenomenon was attributed to the presence of the oily internal phase, which led to a decrease in the aqueous drug gradient for membrane permeation, rendering the diffusion through the dialysis membrane the rate-limiting step in the overall kinetic process [37].
One of the main purposes of this study was to maximize the oral bioavailability of PD-L1 blocking peptides and improve the anti-tumor effects through the aid of carriers. In addition, studies have shown that PD-1/PD-L1 blockade therapy combined with simvastatin has better anti-tumor effects. Therefore, we chose simvastatin as a lipophilic drug that was loaded into the oil phase. The optimal polysaccharide-modified emulsions had high drug encapsulation efficiencies, which were very similar to those reported in the literature [38]. Additionally, the emulsions had a sustained manner of drug release, which is beneficial for the retention and absorption of drugs in the GIT, thus increasing the oral bioavailability and prolonging the half-life. As expected, the in vitro uptake experiments in Caco-2 cells demonstrated that all three polysaccharide-modified emulsions could significantly increase the uptake of peptides by Caco-2 cells compared to the uptake of free peptides, indicating the great potential of the delivery system to promote oral drug absorption and elevate the overall oral bioavailability of peptide drugs.
Finally, we evaluated the effects of okra, yam, and spirulina polysaccharides on the two major immune cells, dendritic cells and macrophages. More specifically, we investigated whether these polysaccharides could promote dendritic cell maturation and enhance cytokine expression in macrophages. The results indicated that okra and spirulina polysaccharides significantly increased the expression of CD80, CD86, and CD40 on the surface of BMDCs, indicating that these polysaccharides promoted dendritic cell maturation, which is conducive for antigen presentation and activation of naive T cells [39]. In addition, okra and yam polysaccharides also showed significant stimulation in RAW 264.7 cells to express the pro-inflammatory cytokines IL-6 and TNF-α. Therefore, this study demonstrated that these dietary polysaccharides showed the dual functions of forming promising carrier systems and being potential immune modulators. Additionally, it indicated that these dietary polysaccharides could activate natural immunity and might have a synergistic anti-tumor effect together with PD-L1 blocking peptides for immunotherapy.

Conclusions
In this study, dietary-polysaccharide-modified fish-oil-based double emulsions were successfully prepared through a simple emulsification method. The optimal emulsions were nano-sized and exhibited a negative surface charge, high drug encapsulation efficiency of over 97%, low viscosity, sustained drug release manner, and ability to improve oral drug absorption. In addition, these dietary polysaccharides could promote dendritic cell maturation and enhance cytokine expression in macrophages, which demonstrated that these dietary polysaccharides have dual roles of functioning as promising carrier systems and as potential immune modulators or adjuvants.