This study was aimed at the preparation and evaluation of walnut meal derived elastase inhibitory peptide (FFVPF) 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. And 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.
Research Article
Chitosan/tripolyphosphate nanoparticle as elastase inhibitory peptide carrier: Characterization and its in vitro release study
https://doi.org/10.21203/rs.3.rs-1604530/v1
This work is licensed under a CC BY 4.0 License
Version 1
posted
You are reading this latest preprint version
chitosan
sodium tripolyphosphate
nanoparticles
peptide
release profile
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 and modification, and good biocompatibility [1, 2]. Walnut (Juglans regia) is globally popular and high-valued 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]. And 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 a proteolytic enzyme in the human body, which degrades elastin in the body, leading to skin aging and sagging, lung diseases, rheumatoid arthritis and so on. Inhibiting the activity of elastin prevents it from being over-hydrolyzed, thus preventing skin aging and promoting health [7]. 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 (IC50) of 0.469 ± 0.010 mg/mL. 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 [8, 9], the low ability to penetrate intestinal epithelial cells and poor sensory taste [10, 11]. Many of these limitations can be solved by applying a suitable delivery system for the continuous release of peptides. The nano encapsulation 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 [12].
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 non-toxic, generally obtained by deacetylation of chitin in shrimp and crab shell [13] and has the advantages of biocompatibility and biodegradability [14]. Sodium tripolyphosphate (TPP) is the most commonly used crosslinking agent for ionic crosslinking of chitosan hydrogel. The intramolecular and intramolecular linkage between TPP phosphate group and amino group of chitosan forms NPs [15] through the electrostatic interaction with polycation chitosan or negative polyanion TPP, promoting the bioactivity (i.e. peptide and protein) entrapment [16, 17]. 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, 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.
2.1 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. CS was obtained from Macklin Biochemical Technology Co., Ltd, Shanghai, China. TPP was purchased from Yinuo Biotechnology Co., Ltd, Zhejiang, China. Acetonitrile is chromatographic pure, purchased from Fisher Chemical Co., Ltd, America. Trifluoroacetic acid with chromatographic purity was supplied by Merida Technology Co., Ltd, Beijing, China. Potassium and bromide was purchased from Institute of Photorefined Fine Chemicals Co., Ltd, Tianjin, China. All other chemicals and solvents were of analytically pure bought from Beijing Chemical Works Co., Ltd.
2.2 Preparation of chitosan-sodium tripolyphosphate nanoparticles (CS-TPP NPs)
CS-TPP NPs were prepared using the ionic gelling techniques as described by Fan Wen [18]. 1 mg/mL CS solution was prepared by acetic acid. The pH was adjusted to 4.0 with 1mol/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 mg, 5 mg, 7.5 mg 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 and freeze-dried to obtain NPs powder.
2.3 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. FFVPF were measured at 220 nm. All measurements were done in triplicates. EE and loading capacity percentages were calculated according to the following equation:
Wt—the total amount of peptide;
Wf—the amount of free peptide in supernatant;
Wn—the total weight of lyophilised NPs.
2.4 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.
2.5 Physiochemical characterization of CS-TPP NPs
2.5.1 Fourier transform infrared (FTIR) spectroscopy
A small quantity of samples (1 mg) were 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.
2.5.2 X-ray diffraction (XRD) analysis
XRD measurements were recorded on a 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 freeze-dried 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.
2.5.3 Transmission electron microscope (TEM) microscopic observation
Morphology of the particles were 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, a drop from the aqueous solution of CS-TPP FFVPF NPs was dispensed directly onto a carbon coated copper grid and allowed to dry spontaneously.
2.6 Stability analysis
2.6.1 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, respectively. 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 2.4. The control group was NPs without pH adjustment after preparation. The calculation formula of short peptide retention is as follows:
m0—the quality of encapsulated short peptide in the control group (mg);
m1—the quality of free short peptide in the treated sample (mg).
2.6.2 Temperature stability analysis
According to the method of Yang et al. [19] 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, respectively. The retention rate of FFVPF in NPs was determined after cooling to room temperature. The determination and calculation method of retention rate are the same as that in 2.7.1. The control group was the sample before heating.
2.6.3 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 2.7.1., and the samples without UV treatment were set as the control group.
2.7 Study on release characteristics in vitro
The study on release characteristics was performed according to Shah et al. (2016) with some modifications. The suspension of CS-TPP FFVPF NPs were 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). 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. The concentration of FFVPF was evaluated using RP-HPLC. The calculation formula of release rate is as follows:
c—short peptide concentration in sample;
V—system volume;
M0—initial content of short peptides in NPs.
2.8 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.
3.1 Determination of EE and EC
As shown in Fig. 1, with the addition of FFVPF in the system increasing from 2.5 mg to 10 mg, EE of FFVPF in CS-TPP NPs increased first and then decreased. When the addition levels of short peptides were 5 mg and 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 peptide-loaded NPs with EE higher than 94% were obtained. In the commercialization of food, high encapsulation rate also means lower transportation cost of functional factors [20]. The increasing peptide content makes EE increase continuously, in accordance with previous studies [21, 22]. The electrostatic interaction between polypeptide and CS-TPP carrier may lead to high EE. 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. [23].
3.2 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 [24]. 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 [25, 26]. 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 mg to 10 mg, the particle size gradually decreased from 175.67 ± 1.36 nm to 146.33 ± 3.59 nm. The causes of this phenomenon are as follows: with the increase of the amount of core material, the relative concentration of CS in the wall material decreases, and the number of - NH3+ on the surface of NPs decreases, which may lead to the weakening of intramolecular repulsion force, so as to reduce the particle size of generated NPs [27]. 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. [28], which is possibly due to the surface adsorption.
The suitable NPs have uniform particle size distribution [29]. 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 [30]. 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 mV 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. Therefore, CS-TPP is suitable as a peptide delivery system. The addition amount of FFVPF was set to 5 mg in the following experiments.
3.3 Phisyochemical characterization of CS-TPP NPs
3.3.1 FTIR analysis results
Fig. 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.
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 [31, 32]. 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 [33]. The spectrum of peptide loaded CS-TPP NPs (Fig. 2A (c) shows some differences from CS-TPP NPs, which means the formation of peptide NPs complex. Compared with (b) and (c), except that the positions of O-H stretching vibration peaks are different at about 3400 cm-1, the number and position of peaks in the infrared spectrum of NPs loaded with short peptides have no significant change compared with blank NPs, and no new vibration peaks are generated, but at 1577.70 cm-1, 1413.76 cm-1 in the functional group area and 1043.44 cm-1, 1014.51 cm-1 in the fingerprint area The peak type at 649.98 cm-1 is similar and the peak intensity is enhanced, which means that the embedded short peptide does not produce new chemical groups in NPs, but increases the number of some existing groups. Therefore, the main force of CS-TPP NPs embedding short peptide FFVPF is hydrogen bond. The short peptide characterized by Fig. 2A (d) has a peak at 1670.28 cm-1, which belongs to the amide I band, and it has double peaks at 746.42 cm-1 and 700.13 cm-1 in the fingerprint area, which represents the phenylpropane in the peptide. The monosubstituted benzene ring on the amino acid R group, but these peaks did not appear in the NPs loaded with short peptides. The difference in infrared spectra between (c) and (d) indicates that the short peptides were successfully embedded in the NPs.
3.3.2 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(b) shows two broad steamed bread-like peaks at 7.62° and 19.88°, indicating that it belongs to amorphous form. Comparing with (a) and (c), the peak of CS at 19.96° decreased after cross-linking with TPP. Both (c) and (d) showed sharp and narrow peaks, the XRD patterns of the samples showed sharp peaks at 2θ = 8.86, 17.8, 26.82, 30.96 and 36.04 angles (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 [34].
3.3.3 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. Fig. 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 [35]. Fig. 3 (b-c) enlarges the scale to 500 nm and 200 nm respectively, 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.
3.4 Nanoparticles stability
3.4.1 PH stability
Fig. 4 (a) shows the retention rate of FFVPF loaded CS-TPP NPs of stability testing under different pH conditions. In this chart, it can be seen that the retention rate of FFVPF is almost 100% at pH 4 - 6, 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, the retention rate continued to decrease and the precipitation continued to increase. In a highly acidic environment with a pH of 2 to 3, 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 [36]. Conversely, an alkaline environment will reduce the protonation of CS and destroy the cross-linking between CS and TPP, leading to core material leakage [18]. 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 can not only be applied to food systems with a pH of 4 to 6, but also help to improve the stability of FFVPF in the gastrointestinal tract.
3.4.2 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%, the CS-TPP system loaded FFVPF stability at 65℃ and below. Temperature sensitivity is one of the most important characteristics of drug delivery technology [37]. When researchers used thermogravimetric analysis to characterize the thermal stability of CS-TPP encapsulated peptides, they found that the encapsulated peptides had higher thermal stability compared with the free peptides. The cross-linking of CS and TPP also made CS thermal stability be improved [38]. Danish et al. [39] 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℃.
3.4.3 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%. The longer UV irradiation time, the lower retention rate of FFVPF. It has been reported that curcumin-loaded zein CS-TPP NPs are sensitive to UV, and its retention rate will decrease with the increase of UV exposure time [19], which is consistent with the results of this experiment. This result is attributed to the fact that the peptide contains aromatic amino acid residues and double bonds that can absorb part of UV light [40], resulting in its degradation and isomerization when subjected to UV irradiation. 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.
3.5 Characteristics of in vitro release
Peptide release from the NPs was mesured at different time intervals at 37℃ to evaluate the peptide retention of NPs. As shown in Fig. 5, we observed a 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. Time for 50% peptide release was round 60 min. These behaviors were also observed in adriamycin and BSA loaded CS NPs. In contrast, insulin loaded 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. [41] on the in vitro release of CS-TPP NPs. It indicated that the CS-TPP NPs had a good controlled release performance. In section 3.4.1, the stability of CS-TPP NPs at different pH values was investigated, 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. [39] compared the in vitro release characteristics of CS-TPP NPs loaded with two tripeptides LKP and IPP respectively. 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.
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. And its stability was evaluated, 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.
Funding Di-Feng Ren acknowledges the financial supported by the National Key R&D Program of China (2019YFD1002400) and National promotion project of scientific and technological achievements in forestry and grassland (2020133135).
Conflict of Interest Authors declare no conflict of interest.
Authors' contributions YX was a major contributor in writing the manuscript and visualization. LC performed the investigation and formal analysis. X-Y W performed the investigation. Y-H S provided the experimental resources. CL performed the methodology. D-F R was a major contributor in project administration, editing and supervision. All authors read and approved the final manuscript.
Availability of data and material The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
Ethical Approval Not applicable.
Consent to Participate Not applicable.
Consent for Publication Not applicable.
- N. Poorinmohammad, H. Mohabatkar, Int. J. Pept. Res. Ther. 21(1), 57–62 (2015). https://doi.org/10.1007/s10989-014-9432-x
- K.C. Mulder, L.A. Lima, V.J. Miranda, S.C. Dias, O.L. Franco, Front. Microbiol. 4, 1–23 (2013). https://doi.org/10.3389/fmicb.2013.00321
- D. Liu, Y. Guo, P. Wu, Y. Wang, M.K. Golly, H. Ma, Food Chem. 311<bi>,</bi> 125960 (2020). https://doi.org/10.1016/j.foodchem.2019.125960
- N. Chen, H. Yang, Y. Sun, J. Niu, S. Liu, Peptides. 38(2), 344–349 (2012). https://doi.org/10.1016/j.peptides.2012.09.017
- H. Chen, M. Zhao, L. Lin, J. Wang, D. Sun-Waterhouse, Y. Dong, M. Zhuang, G. Su, Food Res. Int. 78, 216–223 (2012). https://doi.org/10.1016/j.foodres.2015.10.008
- P. Praxedes-Garcia, I. Cruz-Silva, A.J. Gozzo, V.A. Nunes, R.J. Torquato, A.S. Tanaka, A.S.R.C. Figueiredo-Ribeiro, M.S. Araújo, Sci. World J. 2012, 562715 (2012). https://doi.org/10.1100/2012/562715
- B. Donarska, K.Z. Łączkowski, Future Med. Chem. 12(20), 1809–1813 (2020). https://doi.org/10.4155/fmc-2020-0163
- N.R. Jabir, S. Tabrez, G.M. Ashraf, S. Shakil, G.A. Damanhouri, M.A. Kamal, Int. J. Nanomed. 7(1), 4391–4408 (2012). https://doi.org/10.2147/IJN.S33838
- A.M. Piras, G. Maisetta, S. Sandreschi, M. Gazzarri, C. Bartoli, L. Grassi, S. Esin, F. Chiellini, G. Batoni, Front. Microbiol. 6, 372 (2015). https://doi.org/10.3389/fmicb.2015.00372
- P. Lundquist, P. Artursson, Adv. Drug Deliver Rev. 106(Part B), 256–276 (2016). https://doi.org/10.1016/j.addr.2016.07.007
- A. Mohan, S.R.C.K. Rajendran, Q.S. He, L. Bazinet, C.C. Udenigwe, RSC Adv. 5(97), 79270–79278 (2015). https://doi.org/10.1039/c5ra13419f
- I.J. Joye, D.J. McClements, Curr. Opin. Colloid In. 19(5), 417–427 (2014). https://doi.org/10.1016/j.cocis.2014.07.002
- A. Yamamoto, H. Ukai, M. Morishita, H. Katsumi, Pharmacol. Therapeut 211<bi>,</bi> 107537 (2020). https://doi.org/10.1016/j.pharmthera.2020.107537
- D.S. Duttagupta, V.M. Jadhav, V.J. Kadam, Curr. Drug Deliv 12(4), 369–381 (2015). https://doi.org/10.2174/1567201812666150310151657
- A. Rampino, M. Borgogna, P. Blasi, B. Bellich, A. Cesàro, Int. J. Pharmaceut 455(1–2), 219–228 (2013). https://doi.org/10.1016/j.ijpharm.2013.07.034
- Z. Sang, J. Qian, J. Han, X. Deng, J. Shen, G. Li, Y. Xie, Carbohyd Polym. 230<bi>,</bi> 115577 (2020). https://doi.org/10.1016/j.carbpol.2019.115577
- A.B. Kayitmazer, D. Seeman, B.B. Minsky, P.L. Dubin, Y. Xu, Soft Matter. 9(9), 2553–2583 (2013). https://doi.org/10.1039/c2sm27002a
- W. Fan, W. Yan, Z. Xu, H. Ni, Colloid Surf. B 90(1), 21–27 (2012). https://doi.org/10.1016/j.colsurfb.2011.09.042
- S. Yang, L. Liu, H. Chen, Z. Li, F. Chen, Y. Gao, Food Hydrocoll. 112, 106258 (2021). https://doi.org/10.1016/j.foodhyd.2020.106258
- A. Mohan, D.J. McClements, C.C. Udenigwe, Food Chem. 213, 143–148(2016). https://doi.org/10.1016/j.foodchem.2016.06.075
- J.A.J. Nesalin, A.S.A.G. Dhas, Asian J. Pharm. Sci. 7(1), 80–84 (2012).
- O.V. Rotar, K. Tenedja, A.D. Arkhelyuk, V.I. Rotar, I.S. Davidencko, V.I. Fediv, Int. J. Adv. Eng. Nano Tech. 1(6), 19–23 (2014).
- S.F. Hosseini, Z. Nahvi, M. Zandi, Food Hydrocolloid. 89(1), 637–648 (2018). https://doi.org/10.1016/j.foodhyd.2018.11.033
- M. Fathi, M.R. Mozafari, M. Mohebbi, Trends Food Sci. Tech. 23(1), 13–27 (2012). https://doi.org/10.1016/j.tifs.2011.08.003
- G. Sonavane, K. Tomoda, K. Makino, Colloid Surf. B 66(2), 274–280 (2009). https://doi.org/10.1016/j.colsurfb.2008.07.004
- J. Kim, H.S. Kim, N. Lee, T. Kim, H. Kim, T. Yu, I.C. Song, W.K. Moon, T. Hyeon, Angew Chem. Int. Edit 47(44), 8438 (2008). https://doi.org/10.1002/anie.200890224
- Z. Du, J. Liu, T. Zhang, S. Wei, L. Ding, B. Liu, Food Chem. 286<bi>,</bi> 530–536 (2019). https://doi.org/10.1016/j.foodchem.2019.02.012
- N. Hasheminejad, F. Khodaiyan, M. Safari, Food Chem. 275, 113–122 (2019). https://doi.org/10.1016/j.foodchem.2018.09.085
- Q. Gan, T. Wang, Colloid Surf. B 59(1), 24–34 (2007). https://doi.org/10.1016/j.colsurfb.2007.04.009
- R.H. Müller, C. Jacobs, O. Kayser, Adv. Drug Deliver Rev. 47(1), 3–19 (2001).
- C. Bulmer, A. Margaritis, A. Xenocostas, Biochem. Eng. J. 68(1), 61–69 (2012). https://doi.org/10.1016/j.bej.2012.07.007
- I. Nallamuthu, A. Devi, F. Khanum, Asian J. Pharm. Sci. 10(3), 203–211 (2015). https://doi.org/10.1016/j.ajps.2014.09.005
- J. Kaliaperumal, N. Hari, P. Pavankumar, N. Elangovan, Appl. Nanosci. 6(5), 615–627 (2016). https://doi.org/10.1007/s13204-015-0470-2
- N.S. Ayumi, S. Sahudin, Z. Hussain, M. Hussain, N.H.A. Samah, Drug Deliv. Transl Re. 9(2), 482–496 (2019). https://doi.org/10.1007/s13346-018-0508-6
- B.R. Shah, Y. Li, W. Jin, Y. An, L. He, Z. Li, W. Xu, B. Li, Food Hydrocolloid. 52(1), 369–377 (2016). https://doi.org/10.1016/j.foodhyd.2015.07.015
- A. Esmaeili, A. Asgari, Int. J. Biol. Macromol. 81, 283–290 (2015). https://doi.org/10.1016/j.ijbiomac.2015.08.010
- T. Shirakura, T.J. Kelson, A. Ray, A.E. Malyarenko, R. Kopelmanet, ACS Macro Lett. 3(7), 602–606 (2014). https://doi.org/10.1021/mz500231e
- R. Nasri, M. Hamdi, S. Touir, S. Li, C.M. Karra, M. Nasri, Int. J. Biol. Macromol. 167, 1445–1451 (2020). https://doi.org/10.1016/j.ijbiomac.2020.11.098
- M.K. Danish, G. Vozza, H.J. Byrne, J.M. Frias, S.M. Ryan, J. Food Sci. 82(9), 2094–2104 (2017). https://doi.org/10.1111/1750-3841.13824
- L. Dai, C. Sun, R. Li, L. Mao, F. Liu, Y. Gao, Food Chem. 237, 1163–1171 (2017). https://doi.org/10.1016/j.foodchem.2017.05.134
- Y. Luo, B. Zhang, W. Cheng, Q. Wang, Carbohyd Polym. 82(3), 942–951 (2010). https://doi.org/10.1016/j.carbpol.2010.06.029
Table 1 The particle size, PDI and zeta potential of CS-TPP FFVPF NPs with different amount of FFVPF.
|
Amount of FFVPF |
Particle size (nm) |
Polydispersity index (PDI) |
Zeta potential (mV) |
|
0 |
151.17±3.50 c |
0.20±0.009 c |
35.6±2.82 a |
|
2.5 |
175.67±1.36 a |
0.24±0.004 a |
34.43±3.12 a |
|
5 |
167.53±1.45 b |
0.23±0.009 ab |
33.33±1.84 a |
|
7.5 |
148.87±0.25 cd |
0.23±0.011 ab |
32.8±2.15 a |
|
10 |
146.33±3.59 d |
0.22±0.013 bc |
33.07±2.78 a |
The results are shown as means ± SD, and different letters indicate significantly different values (n = 3, P < 0.05).
No competing interests reported.