Chitosan/tripolyphosphate nanoparticle as elastase inhibitory peptide carrier: characterization and its in vitro release study

This study is aimed at the preparation and evaluation of walnut meal-derived elastase inhibitory peptide loaded in chitosan-tripolyphosphate (CS-TPP) nanoparticles (NPs). It was shown that the maximum encapsulation efficiency of FFVPF could reach 94.58 ± 0.23%. TEM microphotographs, polydispersity index, and zeta-sizer reports indicated that FFVPF-loaded CS-TPP NPs were in nanometric range and were spherical, discrete, and uniform in size with PDI less than 0.3. FTIR analysis indicated that the peptides interacted with CS-TPP NPs through strong hydrogen bonds and electrostatic interactions. The CS-TPP FFVPF NPs showed better stability with heating treatment, pH treatment, or photochemical treatment. Moreover, the in vitro release profile of peptides was identified. The release rate of encapsulated FFVPF was released explosively to 77.22 ± 2.21% and gradually slowed down. These findings highlighted the prospect of CS-TPP NPs as an oral delivery system, and the application of peptides within food and pharmaceutical products.


Introduction
Nowadays, food-derived peptides from enzymatic hydrolysis have attracted much attention due to their potential properties of human health-promoting functions and limited effects. They have the advantages of small volume, few side effects, easy synthesis 1 3 Vol:. (1234567890) and modification, and good biocompatibility [1,2]. Walnut (Juglans regia) is globally popular and highvalued for its nutritional, healthy, and pharmacological components. The by-product meal of walnut after pressing and oil production is commonly used in feed or discarded, with extremely low added value. As an excellent source of protein, walnut meal contains up to 50% of protein, which is rich in arginine, aspartic acid, and glutamic acid. It is considered to be an excellent source of bioactive peptides and can be used for nutritional and health purposes. Studies have shown that the polypeptides prepared by protease hydrolysis of plant proteins have a variety of biological activities such as antioxidant, anti-cancer, and immune regulation [3]. Researchers isolated an antioxidant tetrapeptide (AGGA) from the pepsin hydrolysate of walnut protein, and its ability to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical was equivalent to that of glutathione (GSH) [4]. Peptides extracted from walnut meal protease hydrolysate have been proved to have the potential to improve learning and memory [5]. Walnut meals were confirmed to contain significant amounts of proteases and inhibitors that take part in human biological processes [6], which attracted more interest of researchers to create high value-added products for nutritive and economic utilization.
Elastin is an essential protein composed of tropospheric elastin monomers to maintain the mechanical structure of the skin. Its degradation can be exacerbated by environmental stresses (such as ultraviolet radiation) and internal factors (such as overexpression of elastase) and consequently leads to the loss of skin structural integrity, which leads to the formation of skin wrinkles. Naturally derived elastase inhibitors have been reported to show ameliorative effects against elastase-related wrinkle formation and are considered to be effective anti-wrinkle ingredients in cosmetics [7]. Inhibition of general elastase activity also had protective effects in lungs, large blood vessels, and ligaments [8]. Our previous study isolated a pentapeptide we named FFVPF from walnut meal, which has shown elastase inhibitory activity in vitro, having a half inhibitory concentration (IC 50 ) of 0.469 ± 0.010 mg/mL [9]. Bioactive peptides usually exert their functional activity and benefit health only when they remain structurally intact when they reach their physiological action sites, but the advancement of this promising natural compound is hampered by the sensitivity of renal filtration and enzyme degradation, the risk of immune response, and the uptake of reticuloendothelial system [10,11], the low ability to penetrate intestinal epithelial cells and poor sensory taste [12,13]. Many of these limitations can be solved by applying a suitable delivery system for the continuous release of peptides. The nanoencapsulation of polymer nanoparticles (NPs) provides continuous release of encapsulated drugs by slowing down the filtration rate of the kidney and better accumulates at the physiological action site through enhanced permeability and retention effect, so as to provide a promising delivery route for peptides and proteins, protect functional factors from environmental damage and loss of activity, and improve the appearance and stability of food taste and texture [14].
Chitosan-based encapsulation system is considered to be the most attractive strategy for peptide encapsulation and delivery due to its biodegradability. Chitosan (CS) is the N-deacetylation product of chitin. It is a cationic polysaccharide with high molecular weight and nontoxic, generally obtained by deacetylation of chitin in shrimp and crab shell [15], and has the advantages of biocompatibility and biodegradability [16]. Sodium tripolyphosphate (TPP) is the most commonly used crosslinking agent for ionic crosslinking of chitosan hydrogel. The intramolecular and intramolecular linkages between TPP phosphate group and amino group of chitosan forms NPs [17] through the electrostatic interaction with polycation chitosan or negative polyanion TPP, promoting the bioactivity (i.e., peptide and protein) entrapment [18,19]. Regardless of the effective understanding of the unique physical properties of CS-TPP NPs, it is still likely to be affected by the manufacturing process, so it is necessary to study the encapsulation properties of CS-TPP NPs.
Thus, in this study, we prepared and evaluated FFVPF-loaded CS-TPP NPs by ionogel method and demonstrated how to produce CS-TPP NPs with controlled colloidal properties such as EE, LE, particle size, PDI and zeta potential, after loading with different amounts of peptides. Furthermore, physicochemical properties and system stability of NPs were measured in an attempt to design a controlled-release system by loading elastase inhibitory peptides into CS-TPP NPs. This approach has the potential to produce formulations with food-based ingredients and open up a prospect for improving commercial applications.

Reagents and materials
Hydraulic defatted walnut meal was provided by Huangjinlong Edible Oil Co., Ltd. (Hebei, China). The polypeptide was synthesized by Fmoc solid phase synthesis according to the previous separation and purification results of walnut meal polypeptide. This process was commissioned by Yuan-peptide Biotechnology Co., Ltd. (Nanjing, China). Synthetic peptide FFVPF (sequence: Phe-Phe-Val-Pro-Phe) with purity greater than 95% has been verified for its elastase inhibitory activity. Medium viscous chitosan (200-400 mPa.s) was obtained from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). TPP was purchased from Yinuo Biotechnology Co., Ltd. (Zhejiang, China). Acetonitrile is chromatographic pure and purchased from Fisher Chemical Co., Ltd. (USA). Trifluoroacetic acid with chromatographic purity was supplied by Merida Technology Co., Ltd. (Beijing, China). Potassium and bromide were purchased from Institute of Photorefined Fine Chemicals Co., Ltd. (Tianjin, China). All other chemicals and solvents were analytically pure and bought from Beijing Chemical Works Co., Ltd.
Preparation of chitosan-sodium tripolyphosphate nanoparticles (CS-TPP NPs) CS-TPP NPs were prepared using the ionic gelling techniques as described by Fan Wen [20]. CS solution 1 mg/ mL was prepared by acetic acid. The pH was adjusted to 4.0 with 1 mol/L sodium hydroxide solution through a 0.45-μm filter membrane. A solution of TPP at the concentration of 2.0 mg/mL was prepared by dissolving TPP powder in ultrapure water. Different masses of FFVPF (2.5 m, 5, 7.5, and 10 mg) were added to 10 mL CS solution (mass ratios of core material to wall material were 1:4, 1:2, 3:4 and 1:1). Then, 1.25 mL of TPP solution was dropped into FFVPF/CS solution and treated with ultrasonic probe for 4 min. The suspension of control NPs was prepared without FFVPF (CS-TPP blank NPs). CS-TPP FFVPF NPs were separated from suspension by ultrafiltration centrifuge tubes (PALL Co., MAP001, USA) with a molecular weight cut-off of 0 to 1 kDa. The freshly collected NPs were then dried under vacuum in a freeze dryer (Biosafer-12A; Nanjing Biosafer Biotechnology Co., Ltd., China) under a pressure of 0.010 mbar at − 50 ℃ for 24 h. The final product was kept in a sealed vial under dry atmosphere and in a freezer at − 20 ℃.

Determination of entrapment efficiency (EE) and loading efficiency (LE)
The peptide concentration was determined by reversed-phase high performance liquid chromatography (RP-HPLC) to measure the EE and LE of FFVPF. The mobile phase was used in gradient elution mode of solvent B (0.1% trifluoroacetic acid, TFA) in acetonitrile) into solvent A (0.1% TFA in H 2 O) and flow rate of 1 mL/min for 30 min and detection at 220 nm, and 10 µL of filtrate sample was injected onto a Won-daSil C18 Superb (4.6 × 250 mm, 5 µm) column. The analyses were performed at 40 ℃. The diluted solutions were filtered in a 0.22-µm filter membrane prior to the analyses that were performed in triplicate. EE and loading capacity percentages were calculated according to the following equation: where W t is the total amount of peptide, W f is the amount of free peptide in supernatant, and W n is the total weight of lyophilized NPs.
Particle size, zeta potential, and polydispersity index (PDI) measurement The CS-TPP NPs were dispersed in ethanol, and the measurements of particle size, PDI, and zeta potential of NPs were performed on a Zetasizer Nano-ZS (Malvern Instruments, UK) based on dynamic light scattering (DLS) techniques with a DTS1060 capillary cell. This instrument determines the particle size from intensity-time fluctuations of a laser beam. Particle sizes were presented on the basis of intensity frequency.

Fourier transform infrared (FTIR) spectroscopy
A small quantity of samples (1 mg) was mixed with 100 mg potassium bromide (KBr) and compressed into transparent pellet. The samples to be tested were CS, FFVPF, CS-TPP blank NPs, and CS-TPP FFVPF NPs (the addition of FFVPF was 5 mg, and the mass ratio of core material to wall material was 1:2). The FTIR spectra were obtained using a X70 FTIR spectrometer (Netzsch, GER). Finally, the resulting pellets were scanned in a spectral region of 4000-400 cm −1 using a resolution of 4 cm −1 and 64 scanning times.

X-ray diffraction (XRD) analysis
XRD measurements were recorded on an X-ray diffractometer (Rigaku Corporation, D/max-2550, Tokyo, Japan) to confirm the crystalline or amorphous nature of samples, CS, FFVPF, CS-TPP blank NPs, and CS-TPP FFVPF NPs. The freezedried samples were ground into powder and evenly placed on the sample pool. The data were collected in the 2θ ranges 3-55°, and the scanning speed was 4°/min.

Transmission electron microscope (TEM) microscopic observation
Morphology of the particles was examined by TEM (Jeol, JEM-1200 EX, Tokyo, Japan) at 100 kV. For TEM test, the samples were stained with 2% (w/v) phosphomolybdic acid, and a drop of the aqueous solution of CS-TPP FFVPF NPs was dispensed directly onto a carbon coated copper grid and allowed to dry spontaneously.

Stability analysis pH stability analysis
An appropriate amount of newly prepared CS-TPP FFVPF NPs was taken, and the solution pH was adjusted to 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 with 1 mol/L HCl or 1 mol/L NaOH. The appearance of the sample was observed visually, and its pH stability was explored by taking the retention rate of short peptide FFVPF in NPs as an index. The quality of free short peptide was determined by ultrafiltration centrifuge tube and RP-HPLC. The method is the same as in the "Determination of entrapment efficiency (EE) and loading efficiency (LE)" section. The control group was NPs without pH adjustment after preparation. The calculation formula of short peptide retention is as follows: where m 0 is the quality of encapsulated short peptide in the control group (mg) and m 1 is the quality of free short peptide in the treated sample (mg).

Temperature stability analysis
According to the method of Yang et al. [21] with some modifications, an appropriate amount of newly prepared CS-TPP FFVPF NPs was placed in a centrifuge tube and heated in water bath of 55, 65, 75, and 85 ℃ for 30 min. The retention rate of FFVPF in NPs was determined after cooling to room temperature. The control group was the sample before heating.

Photochemical stability
A proper amount of the newly prepared CS-TPP FFVPF NPs was placed in a small glass bottle and irradiated continuously for 0-150 min under ultraviolet (UV) light source. The retention rate of short peptide FFVPF in NPs was measured every 30 min. The calculation method of retention rate is the same as that in the "pH stability analysis" section, and the samples without UV treatment were set as the control group.
In vitro release studies The study on release characteristics was performed according to Shah et al. [22] with some modifications. The suspension of CS-TPP FFVPF NPs was placed in a dialysis membrane bag (Biotech CE tubing MW 3500 Da). The dialysis bag was then placed in 50 mL 0.2 M PBS solutions at pH 7.4. The entire system was kept in a 37 ℃ water bath shaker (100 rpm), and 1 mL of the release medium was removed at regular intervals. With every 1 mL of the release medium drawn out, 1 mL of freshly prepared PBS solution was added into the system to keep the volume constant. Free FFVPF in the system was quantified with an established standard curve at 220 nm (R 2 ≥ 0.999) recording from a UV-Vis spectrophotometer (V-1000, Aoyi Instruments (Shanghai) Co. Ltd., China). The calculation formula of release rate is as follows: where c is short peptide concentration in sample, V is system volume, and M 0 is initial content of short peptides in NPs.

Statistical analysis
Results were represented by the mean ± standard deviation (SD) (n = 3). Analysis was performed using SPSS software (SPSS 26, SPSS Inc., Chicago, IL, USA). Comparison of means was performed with Duncan's test, with the confidence level set at P < 0.05.

Determination of EE and LE
The EE and LE of FFVPF with different addition amounts are shown in Fig. 1. The EE became higher when the FFVPF amount changed from 2.5 to 7.5 mg, and after that, the EE further decreased when the amount changed from 7.5 to 10 mg (P < 0.05).
When the addition levels of FFVPF were of 5 mg and Release rate(%) = c × V M 0 × 100 7.5 mg, EE reached 94.10 ± 0.79% and 94.58 ± 0.23%, respectively, and there was no significant difference between them (P > 0.05). CS-TPP composite peptideloaded NPs with EE higher than 94% were obtained. In the commercialization of food, high encapsulation rate also means lower transportation cost of functional factors [23]. The increasing peptide content makes EE increase continuously, which is in parallel with the efficiency values described by previous studies [24,25]. The electrostatic interaction between polypeptide and CS-TPP carrier may lead to high EE [26]. With the increase of polypeptide content, the LE is higher, which is caused by the increase of the ratio of polypeptide to carrier in NPs. Subsequently, due to the limitation of the loading capacity of NPs, the chemical potential of drug diffusion to the solution increased, and the increase rate of LE gradually tended to smooth out, which was similar to the experimental results of Hosseini et al. [27].
Determination of particle size, zeta potential, and PDI Table 1 summarizes the effects of FFVPF addition on the particle size and PDI of CS-TPP NPs. The size of NPs affects the biocompatibility and activity. NPs with a small size are easier to be taken up by the cells, so they can function efficiently [28]. The NPs with the size of < (smaller than) 200 nm are difficultly filtered by spleen, while those > (larger than) 300 nm can be easily captured by phagocytic cells [29,30]. Accordingly, the small size of the NPs obtained in this study may lead to a longer circulation. As the core content increased from 2.5 to 10 mg, the particle size gradually decreased from 175.67 ± 1.36 to 146.33 ± 3.59 nm. This phenomenon could be explained as follows: with the increase of the amount of FFVPF, the relative concentration of CS in the wall material decreases. As the concentration of chitosan decreases, the intermolecular distance increases, resulting in the decrease of cross-linking between chitosan molecules while there was an increased density in cross-linking between chitosan and TPP, thus reducing the size of NPs [26]. On the other hand, the amino (-NH 3 + ) groups of CS could be involved in the interaction with increasing amounts of peptides [31,32]. In particular, the particle size of blank NPs is smaller than that of all NPs loaded with FFVPF, which was similar to the experimental results of embedding eugenol essential oil by Hasheminejad et al. [33], which is possibly due to the surface adsorption.
The suitable NPs have uniform particle size distribution [34]. PDI was used to characterize the uniformity of particle size in the system. A larger PDI value represents a large difference in material size and wide particle size distribution in the system. Generally, if the PDI is less than 0.3, it can be considered that the particle size distribution of the system is narrow. The PDI of blank NPs and peptide-loaded NPs measured by DLS ranged from 0.2 to 0.25, showing a suitable and narrow particle size distribution (PDI < 0.3), which confirmed the formation of homogeneous and monodisperse NPs and peptide-loaded NPs.
Zeta potential is an important index of aqueous nanoparticle systems. For NPs stabilized by electrostatic interaction, the absolute value of zeta potential greater than 30 mV is a necessary condition for the stability of the system [35]. The poor surface charge might cause some problems such as aggregation and lower peptide absorption into NP. The changes of zeta potential with the addition of short peptide FFVPF are shown in Table 1. CS-TPP NPs have strong positive charge, and the zeta potential values range from + 33 to + 36 mV, which are greater than 30 mV, indicating that the system is sufficiently stable. Zeta potential decreased with the increase of FFVPF addition. The results obtained by Zetasizer revealed that due to the presence of amine groups on the surface of CS NPs, the surface charge is positive. The adsorption of peptides on the particles will reduce the surface positive charge of the cationic chitosan molecules, resulting in a decrease in zeta potential, which explained the decline in the particle size. Therefore, CS-TPP is suitable as a peptide delivery system. The addition amount of FFVPF was set to 5 mg in the following experiments. Figure 2A indicates the FTIR spectra of CS, FFVPF, CS-TPP blank NPs, and CS-TPP FFVPF NPs. The NPs added with 5 mg FFVPF were selected for characterization based on their high encapsulation efficiency, good particle size, and zeta potential. Interaction of FFVPF with CS-TPP NPs led to some structural changes in FTIR spectra. Also, FTIR spectra confirmed the interaction between peptides and synthetic NPs.

FTIR analysis results
The strong broad peak at 3433.14 cm −1 in the CS spectrum is attributed to the hydrogen bond O-H stretching vibration. The peak value of C-O-C asymmetric tensile vibration is about 1100 cm −1 . The absorption band at 1604.71 cm −1 is attributed to the N-H bending mode in the chitosan primary amine 10 ( Fig. 2A (a)). In CS-TPP blank NPs, the peak at 1654.85 cm −1 (C = O stretching vibration in amide I area) and the N-H bending vibration peak at 1604.71 cm −1 of amine I at 1604.71 cm −1 shifted to 1577.70 cm −1 and 1413.76 cm −1 , respectively [36,37]. Also, the peak at 1043.44 cm −1 indicates that the tripolyphosphate group of TPP is connected to the amine group of chitosan ( Fig. 2A (b). CS is a cationic polysaccharide; its amino group is positively charged, while the phosphate group of TPP is negatively charged, and the two form NPs based on electrostatic interaction. The FTIR spectra of peptides and peptide-loaded CS NPs are shown in Fig. 2A (c,  d). In previous studies, it has been determined that the peptide binds to the chitosan hydroxyl group through its carbonyl group and forms a carboxyl group [38]. Pure walnut meal-derived peptide spectra ( Fig. 2A  (d) ; this may be attributed to the electrostatic interactions between the negative charges of peptides and positive charges of CS [31]. As mentioned earlier (see the "Determination of EE and LE" section), electrostatic interactions between both the entities were considered to play critical roles in the amount of drug loading into polymeric NPs.

XRD measurements
XRD analysis can identify the crystalline phase of the substance and judge that the substance belongs to an amorphous or crystalline structure. The characterization results of XRD are shown in Fig. 2B. The two peaks at 10.6 and 19.96° in CS (a) are the typical peaks of its crystal structure, while the short peptide FFVPF in Fig. 2B (Fig. 2B (a)), which characterized the crystalline properties of the NPs. CS-TPP NPs have strong reflection corresponding to the crystal morphology. The crystallization peaks in CS-TPP FFVPF NPs increased (Fig. 2B (d)), indicating a slight increase in the crystallization properties in NPs. This structural modification may be related to the intermolecular and/or intramolecular network structure of CS, cross-linked via TPP counterions [39].

TEM measurements
The TEM was utilized to assess the shape, size, and uniformity of the NPs. This monograph clearly shows that the nanoparticles were spherical in shape. Figure 3 shows the spherical shape of FFVPF-loaded CS-TPP NPs under TEM measurement, which were approximately 100 to 200 nm. In this case, some NPs are clustered together in a compact structure. CS and TPP were aggregated under the interaction of surface groups to form new particles of constant size and rearrange to form a denser structure [22]. Figure 3 (b,  c) enlarges the scale to 500 and 200 nm, respectively and the number of particles in the field of vision decreases, and it can be seen that the surface morphology of NPs is basically smooth with slight sharp corners. The NPs exhibited almost homogeneous morphology, which can be due to mild preparation conditions.

Nanoparticle stability pH stability
Since the formation mechanism of CS-TPP NPs was based on ion gelation, the pH solution is critical for NPs and their stability, providing a way to adjust the formulation and performance of NPs. Figure 4(a) shows the retention rate of FFVPF-loaded CS-TPP NPs in stability testing under different pH conditions. In this chart, it can be seen that the retention rate of FFVPF was almost 100% when pH increased from 4.0 to 6.0, and the stability of the NPs is high without leakage. When the pH was adjusted to 7, the retention rate decreased to 77.97 ± 1.15%. At the same time, it can be observed that the transmittance of nanoparticle suspension decreases and flocculation precipitation occurs. If the pH was continuously adjusted to 8.0, the retention rate continued to decrease and the precipitation continued to increase. In a highly acidic environment with a pH of 2.0 to 3.0, the retention rate of NPs dropped to about 80%, but the liquid could maintain high transparency without precipitation. This is due to the protonation and mutual repulsion of free amino groups on CS chain caused by high concentration of acid, resulting in the dissolution and swelling of CS NPs [40]. Conversely, an alkaline environment will reduce the protonation of CS and destroy the cross-linking between CS and TPP, leading to core material leakage [20]. In addition, the pH of the human gastric juice environment is about 2.0, and the pH of the intestinal juice environment is about 7.5. The retention rate of short peptides can reach more than 70% in this range. In conclusion, the CS-TPP FFVPF NPs cannot only be applied to food systems with a pH range from 4.0 to 6.0 but also help to improve the stability of FFVPF in the gastrointestinal tract.

Temperature stability
NPs may undergo heat treatment during food processing and storage. Studying the stability of NPs at different temperatures is conducive to finding appropriate processing methods and storage conditions. We evaluated the effects of different temperature treatments on the stability of NPs (Fig. 4(b)). It can be observed that the retention rate below 65 ℃ is close to 100%, and the CS-TPP system loaded FFVPF stability at 65 ℃ and below. Temperature sensitivity is one of the most important characteristics of drug delivery technology [41]. When researchers used thermogravimetric analysis to characterize the thermal stability of CS-TPP encapsulated peptides, they found that the encapsulated Fig. 4 The effect of different pH (a), temperatures (b), and UV exposure times (c) on retention rate of CS-TPP NPs loaded with FFVPF peptides had higher thermal stability compared with the free peptides. The cross-linking of CS and TPP also made CS thermal stability improve [42]. Danish et al. [43] used CS-TPP system to embed tripeptide LKP and explored the effect of temperature on the stability of nanoparticles through accelerated stability testing. The results showed that the system remained stable at 60 ℃, but when the temperature reached 70 ℃, the particle size and PDI of NPs changed significantly, and the phenomenon of instability will appear, which was consistent with the results of this experiment. Therefore, the CS-TPP system loaded with FFVPF can adapt to room temperature storage and processing conditions not exceeding 65 ℃.

Photochemical stability
The photochemical stability of FFVPF-loaded CS-TPP NPs is shown in Fig. 4(c). When the irradiation time increased to 60 min, the retention rate was still more than 90% and the longer UV irradiation time, the lower retention rate of FFVPF. A similar release was reported by Yang et al. [21] for curcumin release from zein CS-TPP NPs, and its retention rate was decreased with the increase of UV exposure time. This can be attributed to the fact that the peptide contains aromatic amino acid residues and double bonds that were able to absorb part of UV light, resulting in its degradation and isomerization when subjected to UV irradiation [44]. The above results show that if the CS-TPP system is used in the food industry, the product can withstand the processing process (sterilization, filling, etc.) without avoiding light within 60 min, but it needs to be packaged in dark or dark storage and transportation to avoid the leakage of functional factors.

Characteristics of in vitro release
Peptide release from the NPs was measured at different time intervals at 37 ℃ to evaluate the peptide retention of NPs. As shown in Fig. 5, we observed approximate burst peptide release by 77.22 ± 2.21% within the initial 120 min, which may be the result of the physical adsorption of peptides on the surface of NPs and the diffusion of the biomolecule from the donor to receiver solution through the concentration gradient. Time for 50% peptide release was round 60 min. These behaviors were also observed in adriamycin and BSA-loaded CS NPs. In contrast, insulinloaded CS NPs released 100% within 15 min. The phosphate group of NPs has strong peptide binding. There may be two interactions between peptides and NPs: weak surface adsorption leads to initial release and strong electrostatic contact leads to slow release.
When the time exceeded 120 min, the release rate significantly slowed down, which shows the same trend as the research results of Luo et al. [45] in the in vitro release of CS-TPP NPs. It indicated that the CS-TPP NPs had a good controlled release performance. In Sect. 3.4.1, the stability of CS-TPP NPs at different pH values was investigated, and it was found that when pH was greater than 7.0, the stability of NPs decreases and the retention rate was about 78%. The pH value of in vitro release experiment was also exactly 7.4. The initial sudden release of the core material may be related to the pH of the system, which may be because the alkaline environment will reduce the protonation degree of CS and destroy the cross-linking between CS and TPP. Previous studies have shown that the release characteristics of CS-TPP NPs are related to CS concentration and drug loading. High concentration of CS increased the cross-linking density and reduced the swelling capacity of NPs, thereby slowing down the drug release rate. In addition, the NPs with large drug loading are more prone to initial suddenly release. Danish et al. [43] compared the in vitro release characteristics of CS-TPP NPs loaded with two tripeptides, LKP and IPP. The results showed that IPP released faster than LKP in acidic environment, possibly due to the rigidity and steric hindrance of IPP limited its interaction with CS, indicating that the release characteristics of NPs were also related to the drug properties of NPs.

Conclusion
In the current study, an oral nano-scale degradable delivery system is proposed based on ionic gel method to load elastase inhibitory peptide FFVPF. CS-TPP NPs with an overall positive charge could be transported across the cell membrane and, more importantly, lead to the release of peptides in a controlled manner. In addition, we compared different additions to peptide encapsulation. The maximum encapsulation efficiency can reach 94.58 ± 0.23%. NPs with particle size of about 140-180 nm, PDI of 0.2-0.25, and surface charge of 33-36 mV were synthesized. Their stability was evaluated, and CS-TPP NPs showed good acid-base environment stability and could be stored at room temperature and processed below 65 ℃. In vitro release studies have shown that CS-NPs can be used for controlled release and sustained release of peptide drugs. These results encourage that CS-TPP can be used as an effective carrier for controlled peptide delivery and favored its useful applicability in food and pharmaceutical industries, but further in vivo release studies are needed to clarify their potential and match the preclinical physicochemical characteristics required for oral delivery.