CN111116826B - Preparation method of double-responsiveness polymer and nanoparticles and application of double-responsiveness polymer and nanoparticles in procyanidine - Google Patents
Preparation method of double-responsiveness polymer and nanoparticles and application of double-responsiveness polymer and nanoparticles in procyanidine Download PDFInfo
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- CN111116826B CN111116826B CN201911386725.8A CN201911386725A CN111116826B CN 111116826 B CN111116826 B CN 111116826B CN 201911386725 A CN201911386725 A CN 201911386725A CN 111116826 B CN111116826 B CN 111116826B
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- hyaluronic acid
- histidine
- polymer
- nanoparticles
- deionized water
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- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
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Abstract
The invention discloses a synthetic method of a dual-responsiveness polymer, which comprises the following steps: 1) performing amide reaction on carboxyl of hyaluronic acid and amino of histidine to obtain a hyaluronic acid-histidine graft; dissolving a hyaluronic acid-histidine graft in deionized water, adding potassium persulfate serving as an initiator and N' N-cysteamine serving as a cross-linking agent, reacting under the protection of inert gas, dialyzing the reaction product in the deionized water, and freeze-drying, wherein the obtained cystamine-hyaluronic acid-histidine polymer is a double-responsive polymer. The double-response polymer can be used for preparing nanoparticles loaded with procyanidine (OPC).
Description
Technical Field
The invention belongs to the field of food engineering; in particular to a preparation method of a double-responsiveness polymer and application thereof in procyanidine nanoparticles.
Background
Procyanidins are present in pollen, nuts, fruits, bark and seeds of various plants as defense against biotic and abiotic stresses; the essential components include catechin and epicatechin.
Procyanidins, an oligomeric flavonoid found in a variety of plant foods, have a variety of biological activities, such as antioxidant, free radical scavenging, anticancer, antimicrobial, antiviral and neuroprotective capabilities. The intake of procyanidins in human diet is related to the reduction of the incidence rate of various chronic diseases, has great potential in the aspects of reducing myocardial infarction and the death possibility of cardiovascular diseases, and has certain treatment and prevention effects on colon cancer, liver cancer and breast cancer, thereby attracting great attention of people. However, the extremely strong biological activity of procyanidins is sensitive to pH, temperature and light, which easily causes procyanidins to be oxidized.
Hyaluronic acid has good biocompatibility as a natural polysaccharide, but unmodified hyaluronic acid is difficult to cope with adverse environments such as acid, alkali, heat and the like.
Disclosure of Invention
The invention aims to solve the technical problem of providing a preparation method of a double-response polymer and application of the double-response polymer in procyanidine nanoparticles.
In order to solve the above technical problems, the present invention provides a method for synthesizing a dual-responsive polymer, comprising the steps of:
1) synthesis of hyaluronic acid-histidine graft (HA-His):
performing amide reaction between carboxyl of hyaluronic acid and amino of histidine (L-histidine) to obtain hyaluronic acid-histidine graft (HA-His);
2) and synthesis of cystamine-hyaluronic acid-histidine (BAC-HA-His):
dissolving hyaluronic acid-histidine graft (HA-His) in deionized water, adding potassium persulfate as initiator and N' N-cysteamine as cross-linking agent, and reacting at 50 + -5 deg.C (1 + -0.2) for 1 + -0.2 h under the protection of inert gas (such as nitrogen);
putting the obtained reaction product into a dialysis bag (Mr is 8,000-14,000D), dialyzing in deionized water for 22-26 h, and freeze-drying the trapped fluid in the dialysis bag (drying for 24h at-50 ℃) to obtain a cystamine-hyaluronic acid-histidine polymer (BAC-HA-His), wherein the cystamine-hyaluronic acid-histidine polymer is a double-response polymer;
potassium persulfate: hyaluronic acid-histidine graft ═ 5 ± 0.5: 1 in mass ratio;
n' N-cysteamine bisacryloyl: hyaluronic acid-histidine graft ═ 1.2 ± 0.1: 1, in a mass ratio of the components.
Description of the drawings: the purpose of dialysis in deionized water was: thereby removing other impurities such as unreacted monomers (i.e., unreacted potassium persulfate, N' N-cysteamine).
As an improvement of the method for synthesizing the double-responsive polymer of the present invention: the step 1) comprises the following steps:
s1.1, dissolving hyaluronic acid (MW 90000) in deionized water, adding EDC and NHS, and magnetically stirring and mixing for 1 +/-0.2 hours to activate carboxyl of the hyaluronic acid;
hyaluronic acid: the molar ratio of EDC and NHS is 1 (1.2 +/-0.1) to (1.2 +/-0.1);
EDC, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; NHS, N-hydroxysuccinimide;
s1.2, according to histidine (L-histidine): adding histidine to the product obtained in S1.1 at a mass ratio of 0.5-4: 1 (preferably 3: 1), magnetically stirring and mixing (2 +/-0.2) h, and then carrying out ultrasonic treatment at 20-60 ℃ (preferably 50 ℃) and 200-400W (preferably 250W) for 20-60 min (preferably 40 min);
and (3) filling the obtained reaction solution into a dialysis bag (Mr is 8000-14000D), dialyzing in deionized water for 22-26 h, and freeze-drying the trapped fluid in the dialysis bag (drying at-50 ℃ for 24h) to obtain the hyaluronic acid-histidine graft (HA-His).
Description of the drawings: in the ultrasonic treatment, the ultrasonic probe was inserted 2cm below the liquid surface, and dialyzed against deionized water to remove unreacted histidine, EDC, and NHS.
As a further improvement of the method for synthesizing the double-responsive polymer of the present invention:
in the step S1.1, every 0.32mmol of hyaluronic acid is mixed with (75 +/-25) ml of deionized water;
in the step 2), 20mL of deionized water is added for every 20mg of hyaluronic acid-histidine graft (HA-His).
As a further improvement of the method for synthesizing the double-responsive polymer of the present invention: in step S1.2, histidine: hyaluronic acid in a mass ratio of 3: 1; ultrasonic treatment is carried out for 40min at 50 ℃ and 250W.
The invention also discloses a preparation method of the double-responsiveness polymer nanoparticles, which comprises the following steps:
dissolving cystamine-hyaluronic acid-histidine polymer (BAC-HA-His) in deionized water to prepare a solution with the concentration of (1 +/-0.2) mg/mL;
magnetically stirring the solution (about 20 ml-100 ml) at (25 + -5) deg.C for (8 + -2) hours to fully swell the cystamine-hyaluronic acid-histidine polymer; then placing the mixture at the temperature of (4 +/-1) ℃ for 10-12 hours; and then carrying out ultrasonic treatment for 6-15 min (preferably 10min) at 140-200W (preferably 180W) of ultrasonic power under the condition of ice bath to obtain the double-response polymer nanoparticles. Namely, the cystamine-hyaluronic acid-histidine (BAC-HA-His) self-assembled nanoparticles are obtained.
The invention also provides the application of the double-response polymer: preparing nanoparticles for loading procyanidine (OPC).
As an improvement of the use of the present invention, it comprises the steps of:
1) dissolving procyanidine in a phosphoric acid buffer solution with the pH value of 6.5 to prepare (1 +/-0.2) mg/mL procyanidine solution;
2) adding 4mg of cystamine-hyaluronic acid-histidine (BAC-HA-His) polymer into (4 +/-1) mL of deionized water, magnetically stirring for (8 +/-2) hours, then adding 80-150 mu L (preferably 130 mu L) of procyanidin solution, and carrying out W ultrasonic treatment for (10 +/-2) min under an ice bath condition (180 +/-20) to obtain the ultrasonic nanoparticles embedded with OPC.
Because the procyanidin is easily damaged by the external environment, the procyanidin is embedded and protected by preparing the nano carrier, so that the procyanidin enters the organism to achieve the aim of targeted release, and the biological activity and the utilization rate of the procyanidin are improved. The invention prepares polymer nanoparticles with double responsiveness by modifying hyaluronic acid to carry out load protection on procyanidine.
The invention has the following technical advantages:
1. the hyaluronic acid is used as a macromolecular carrier, the pH response characteristic is endowed by modifying the hyaluronic acid by histidine through an amide reaction, and the grafting degree of the hyaluronic acid is improved by adopting an optimal process.
2. The self-assembled polymer nanoparticles with thermal stability, gastrointestinal stability and biocompatibility are prepared.
3. The preparation method has the advantages that the nano particles are used for carrying out load protection on OPC, the encapsulation rate of the OPC is high, the OPC can be delayed due to the protection effect of the nano particles on the OPC, the reduction of the antioxidation performance of the OPC in the heat treatment and storage processes can be delayed, the OPC can be prevented from being damaged by environments such as food preservatives and the like to a certain extent, and the stability of the OPC is improved so as to ensure the bioactivity of the OPC.
4. The preparation method is simple, no organic solvent or toxic solvent is added, the procyanidin-loaded nanoparticles are prepared by an ultrasonic dispersion method, the core material is conveyed to a specific part through the receptor-mediated targeting effect, and the active targeting property of the nanoparticles can be further improved by utilizing the responsiveness of the carrier.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is an IR spectrum of HA, His, HA-His;
FIG. 2 is an infrared spectrum of HA, BAC, and BAC-HA-His;
FIG. 3 is a thermogravimetric analysis curve of HA, HA-His, and BAC-HA-His;
FIG. 4 is a graph of the effect of ultrasound power on HA-His grafting;
FIG. 5 is a graph showing the effect of reaction time on the degree of HA-His grafting;
FIG. 6 is the effect of mass ratio on the degree of HA-His grafting;
FIG. 7 is a graph showing the effect of reaction temperature on the degree of HA-His grafting;
FIG. 8 is a comparison of turbidity tests to verify the reductive degradability of polymers;
FIG. 9 is an acid-base titration curve of hyaluronic acid-histidine, cystamine-hyaluronic acid-histidine complex, sodium chloride, hyaluronic acid;
FIG. 10 shows the particle size distribution of nanoparticles before and after sonication;
FIG. 11 is a graph of the effect of ultrasonic power on nanoparticle average particle size and PDI;
FIG. 12 effect of ultrasound time on nanoparticle average particle size and PDI;
FIG. 13 is a graph showing the effect of pH on the average particle size (A) and potential (B) of nanoparticles;
FIG. 14 is a graph of the effect of GSH concentration on nanoparticle average particle size and PDI;
FIG. 15 is a graph of the effect of temperature on the average particle size and PDI of nanoparticles;
FIG. 16 shows the particle size distribution of nanoparticles in simulated gastric and intestinal fluids;
FIG. 17 shows the survival rates of CMT93 cells after 24h, 48h and 72h treatment with BAC-HA-His nanoparticles
FIG. 18 is a graph of the effect of OPC addition on encapsulation efficiency and loading;
fig. 19 is a study of antioxidant activity of loaded OPC nanoparticles at different heating temperatures;
FIG. 20 is a graph of the effect of food additives on retention of loaded OPC nanoparticles;
fig. 21 is an in vitro release study of loaded OPC nanoparticles;
fig. 22 shows the survival rates of CMT93 cells after free OPC and loaded OPC nanoparticle treatment for 24h (a), 48h (b), and 72h (c).
Detailed Description
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1, a method of synthesis of a dual-responsive polymer, sequentially performing the following steps:
synthesis of hyaluronic acid-histidine graft (HA-His):
performing amide reaction on carboxyl of Hyaluronic Acid (HA) and amino of histidine (L-histidine, His) to obtain hyaluronic acid-histidine graft; the method comprises the following specific steps:
s1.1, dissolving 0.32mmol of hyaluronic acid (MW 90000, about 250mg) in 75ml of deionized water, adding 0.384mmol of EDC and 0.384mmol of NHS, and mixing for 1h with magnetic stirring to activate the carboxyl group of hyaluronic acid;
namely, hyaluronic acid: EDC, NHS ═ 1:1.2:1.2 molar ratio.
EDC, i.e., 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride;
NHS, i.e., N-hydroxysuccinimide;
s1.2, according to histidine (i.e., L-histidine): adding histidine into the product obtained in S1.1 at a mass ratio of 3:1, magnetically stirring and mixing for 2h, inserting an ultrasonic probe into the liquid surface for 2cm, and performing ultrasonic treatment at 50 ℃ and 250W for 40 min;
putting the obtained reaction solution into a dialysis bag (Mr is 8000-14000D), and dialyzing in deionized water for 24h to remove unreacted histidine, EDC and NHS; the retentate in the dialysis bag was freeze-dried (24 h at-50 ℃) to obtain hyaluronic acid-histidine graft (HA-His).
Detecting HA, His, and HA-His with Fourier infrared spectrometer, wherein the infrared pattern is shown in FIG. 1, hyaluronic acid is at 3415cm-1Shows a broad peak associated with stretching vibration of hydroxyl group and amino group, and further, at 1615cm-1A spike appeared, expressed as N-acetyl (-COCH)3) And a symmetric stretching vibration absorption peak of a carbonyl (C ═ O) group contained in a carboxylate (-COO-) group. At 1411cm-1The peak appearing there is an asymmetric stretching vibration absorption peak of the carboxylate (-COO-) group. HA-His graft at 1642cm-1The absorption peak is the stretching vibration of the secondary amide C ═ O group, and the secondary amide absorption peak is generated because the carboxyl of hyaluronic acid and the amino of histidine are subjected to amide reaction to generate an amide bond. 1570cm-1The peak is the stretching vibration peak of the amide II with N-H, and is 1463cm-1The absorption peak is C-N extension of amide III band, HA-His graft is 1079cm-1And 1042cm-1Two absorption peaks appear at the position of the histidine imidazole ring, namely N-H and C-H stretching vibration peaks. Histidine at 1095cm-1And 1075cm-1Two peaks at are primary amine-NH2Is characterized byAbsorption peak, primary amine is changed into secondary amine after reacting with carboxyl of hyaluronic acid, and double peak is changed into sharp single peak.
② the synthesis of cystamine-hyaluronic acid-histidine (BAC-HA-His):
weighing 20mg of hyaluronic acid-histidine graft (HA-His) and dissolving in 20mL of deionized water, adding 0.1g of potassium persulfate serving as an initiator and 24mg of N' N-cysteamine serving as a cross-linking agent, and reacting at 50 ℃ for 1h under the protection of inert gas (such as nitrogen);
putting the reaction product into a dialysis bag (Mr is 8,000-14,000D), dialyzing in deionized water for 24h to remove other impurities such as unreacted monomers (namely, unreacted potassium persulfate and N' N-bisacryloyl cystamine), and freeze-drying the trapped fluid in the dialysis bag (drying at-50 ℃ for 24h) to obtain a cystamine-hyaluronic acid-histidine polymer (BAC-HA-His); storing in a dryer for later use.
Namely, potassium persulfate: hyaluronic acid-histidine graft ═ 5: 1 in mass ratio;
n' N-cysteamine bisacryloyl: hyaluronic acid-histidine graft ═ 1.2:1, mass ratio.
Detecting HA, BAC (N' N-cysteamine), BAC-HA-His with Fourier infrared spectrometer, the infrared chart is shown in FIG. 2;
as can be seen from FIG. 2, BAC was found to be 3251cm-1The peak is the C-H stretching vibration peak of olefin at 1657cm-1、1555cm-1The absorption peaks at (A) are characteristic absorption peaks of the primary amide group and the secondary amide group, respectively. BAC-HA-His was at 3413cm due to stretching vibration of-OH and-NH groups of hyaluronic acid-1Shows a broad absorption peak and the stretching vibration peak is shifted to low frequencies, the hydrogen bonding interaction between HA and BAC makes this peak slightly broader than the peak in HA. Furthermore, BAC-HA-His was found at 3079cm-1And 2927cm-1The new absorption peak is respectively SP in imidazole ring2Olefins (═ C-H) and SP in the main chain3Stretching vibration of (-C-H-) bond at 2080cm-1A relatively blunt absorption peak appears, which is a characteristic infrared absorption peak of disulfide bonds. It can be seen that BAC-HThe A-His synthetic substance contains histidine and disulfide bond. BAC-HA-His at 1735cm-1A new absorption peak appears, probably due to C ═ O telescopic absorption peak of BAC amido bond cleavage, and in addition, 1298cm-1A sharp absorption peak is related to the in-plane bending vibration of C-H bonds in imidazole ring interior and hyaluronic acid glycosidic bonds, and analysis results show that BAC containing disulfide bonds is successfully introduced into the polymer under the catalysis of potassium persulfate.
In addition, BAC-HA-His is at 2080cm-1A relatively blunt absorption peak appears, which is a characteristic infrared absorption peak of disulfide bonds.
FIG. 3 is a thermogravimetric analysis curve of HA, HA-His, and BAC-HA-His;
as can be seen from fig. 3: the first stage, from a temperature of 25 ℃ to 150 ℃, weight loss of HA, HA-His and BAC-HA-His was about 12.1%, 3.1%, 2.5%, respectively, which is mainly the weight loss due to loss of bound water. Hyaluronic acid exhibits a distinct thermal decomposition phase with an initial decomposition temperature of 230 ℃ and a constant weight reduction with increasing temperature, with a weight loss of about 52% at 300 ℃ and about 66% at 600 ℃. The thermal degradation of the HA-His and the BAC-HA-His is mainly divided into three stages, the first stage of the thermal degradation of the HA-His graft is 25-223 ℃, the weight loss curve of the stage is relatively flat, and the weight loss is mainly caused by the separation of bound water and volatile monomers. The second stage is 223-367 ℃, the weight loss rate is accelerated in the second stage, the initial decomposition temperature is lower than that of the hyaluronic acid, the crystallinity of the hyaluronic acid is damaged due to the grafting of histidine, an amorphous region is converted into an amorphous state, the amorphous region is increased, and the stability is slightly reduced due to the fact that the amido bond generated by the reaction is weaker than the ether bond of the hyaluronic acid. The third stage is 367 to 600 ℃, and the stage is degradation and carbonization of the hyaluronic acid main chain. The first stage of thermal degradation of the BAC-HA-His synthetic substance is 25-217 ℃, and the first stage is the loss of bound water and unreacted and volatile monomers, and the degradation rate of the first stage is less than that of the original hyaluronic acid. The second stage is 217-343 ℃, and the weight loss in the stage is mainly the separation of the oxygen-containing group from the system and the degradation of the side chain. The third stage is 343-600 ℃, and when the thermal decomposition is basically in an equilibrium state, the remaining substances can be some ash components.
Namely, thermogravimetric analysis shows that the polymer has good thermal stability below 220 ℃ and does not influence the specific practical application of the polymer. XRD results show that the original repeating unit structure of hyaluronic acid is not broken by the access of histidine and BAC.
Comparative example 1-1, the ultrasonic power in step S1.2 of example 1 was changed from 250W to 200W, 300W, 350W and 400W, respectively, and the rest was equivalent to the step (r) of example 1.
The resulting ratio of the degree of grafting of the hyaluronic acid-histidine graft (HA-His) is described in FIG. 4; the degree of grafting is at a maximum at a power of 250W.
Description of the drawings: the degree of grafting can be determined by conventional OPA detection methods.
In comparison with the comparative example 1-2, the reaction time in the step S1.2 of the example 1 is respectively changed from 40min to 20min, 30min, 50min and 60min, and the rest is equal to the step (r) of the example 1.
The comparison of the degree of grafting of the resulting hyaluronic acid-histidine grafts (HA-His) is illustrated in FIG. 5; the degree of grafting reached a maximum when the reaction time reached 40 min.
Comparative examples 1 to 3, the mass ratio of histidine to hyaluronic acid in step S1.2 of example 1 was changed to 3:1, respectively: 1:2, 1:1, 2:1, 4: 1; the rest is equivalent to the step (r) of example 1.
The comparison of the degree of grafting of the resulting hyaluronic acid-histidine grafts (HA-His) is illustrated in FIG. 6; the degree of grafting is maximized when the mass ratio of histidine to hyaluronic acid is 3: 1.
Comparative examples 1 to 4, the reaction temperature in step S1.2 of example 1 was changed from 50 ℃ to 20 ℃, 30 ℃, 40 ℃ and 60 ℃, respectively; the rest is equivalent to the step (r) of example 1.
The resulting comparison of the degree of grafting of the hyaluronic acid-histidine grafts (HA-His) is illustrated in FIG. 7; the grafting degree of the graft is at its highest when the temperature reaches 50 ℃.
Description of the drawings: the high grafting degree represents that the amount of the accessed histidine is large, so that the pH response characteristic of the graft is endowed, and the change of the pH in a living body can be responded, so that the aim of targeted release of the procyanidine is fulfilled.
The cystamine-hyaluronic acid-histidine (BAC-HA-His) prepared in example 1 was subjected to the following performance experiment:
Weighing 15mg of cystamine-hyaluronic acid-histidine polymer, dissolving the cystamine-hyaluronic acid-histidine polymer in 30mL of 10mmol/mL glutathione solution, uniformly dispersing the polymer by ultrasonic, taking out 2mL of solution at the timing of 0.5h, 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h and 12h respectively, measuring the absorbance of the solution at 500nm by using an ultraviolet visible spectrophotometer, carrying out three parallels on each group of samples, recording the absorbance of the polymer at 0h as turbidity 100%, and calculating the relative turbidity; thereby obtaining the degree of reduction of the polymer under the action of the reduced glutathione.
As shown in FIG. 8, it is understood from FIG. 8 that the cystamine-hyaluronic acid-histidine synthetic substance is reduced and degraded by glutathione to destroy the structure thereof, thereby exhibiting glutathione sensitivity.
Description of the invention: the glutathione sensitivity of the polymer can respond to the change of the glutathione content in pathological cells, thereby achieving the aim of targeted release of procyanidine.
The proton buffering capacity of hyaluronic acid-histidine, cystamine-hyaluronic acid-histidine complex, sodium chloride and hyaluronic acid under different pH conditions is detected by an acid-base titration method, and a pH meter is used for testing. Four samples of sodium chloride, hyaluronic acid, HA-His and BAC-HA-His were prepared as solutions with a concentration of 2 mg/mL. Each set of samples was adjusted to pH 10.0 with 1mol/L NaOH solution, followed by slowly adding 0.01mol/L HCl to the solution in an amount of 20. mu.L per drop, and after the addition, the samples were allowed to stand for 2min to measure the pH until the pH of the sample decreased to 3.0.
As shown in FIG. 9, it is understood from FIG. 9 that NaCl has no proton buffering capacity and the pH value is rapidly decreased from 10.0 to 3.0 as the amount of hydrochloric acid added is increased. Compared with hyaluronic acid, the HA-His and BAC-HA-His synthetic substances have certain proton buffering capacity, and obvious buffering platforms appear in the range of pH 5.0-7.5, and the obvious buffering platforms show stronger buffering capacity.
Description of the drawings: the stronger buffering capacity indicates that the polymer has better pH response characteristics and can respond to the change of pH in organisms.
Example 2 preparation of BAC-HA-His nanoparticles
20mg of cystamine-hyaluronic acid-histidine synthetic substance (i.e., cystamine-hyaluronic acid-histidine polymer, BAC-HA-His) was weighed and dissolved in deionized water to prepare a solution with a concentration of 1 mg/mL. Placing the prepared solution in a 25 ℃ magnetic stirrer, magnetically stirring for 8 hours to fully swell the solution, and placing the solution in a 4 ℃ refrigerator for overnight (10-12 hours); standing overnight, and then under the condition of ice bath, setting the ultrasonic power at 180W and the ultrasonic time at 10min to obtain the cystamine-hyaluronic acid-histidine (BAC-HA-His) self-assembled nanoparticles.
The particle size distribution diagram of the cystamine-hyaluronic acid-histidine composition and the cystamine-hyaluronic acid-histidine (BAC-HA-His) self-assembled nanoparticles is shown in fig. 10, that is, the particle size distribution diagram of the cystamine-hyaluronic acid-histidine polymer nanoparticles before and after the ultrasound is shown in fig. 10.
As can be seen from FIG. 10, the BAC-HA-His polymer had a broad particle size distribution before sonication, with many distinct small peaks above 1 micron and a large average particle size. After the ultrasonic action, the diffusion of the solvent in the system can be accelerated, and the cavitation effect generated on the solid-liquid surface can disperse a plurality of large particles in the solution, so that the size is reduced, the structure of the polymer becomes more regular, and the distribution becomes more uniform.
Comparative example 2-1 Effect of different ultrasonic powers on BAC-HA-His nanoparticles
The ultrasonic power in the embodiment 2 is changed from 180W to 140W, 160W and 200W respectively, and the rest is equal to the embodiment 2. The average particle diameter and polydispersity of the obtained BAC-HA-His nanoparticles were measured with a Malvern particle sizer.
The results obtained are depicted in FIG. 11; it can be seen from FIG. 11 that the particle size distribution is relatively uniform at the power of 180W and 200W, and the average particle size is reduced to a different extent compared to the condition of 140W and 160W ultrasonic power. When the ultrasonic power is 140W, the average particle size is 202nm, and the PDI is 0.109 larger than that under the condition that the power is 180W, which indicates that when the ultrasonic power is smaller, the impact between particles is not severe enough, and the distribution of nanoparticles is not uniform enough. When the ultrasonic power is too high, cavitation causes violent interaction between particles, and breaks up irregular structures extending on the surfaces of the particles. Therefore, the optimal ultrasonic power of the experiment is 180W, the average particle size is 162nm under the condition, the PDI value is 0.254, and the formed self-assembled nanoparticles have smaller particle size and are most uniformly distributed.
Comparative example 2-2 Effect of different sonication times on BAC-HA-His nanoparticles
The ultrasonic time in the embodiment 2 is changed from 10min to 6min, 8min and 15min respectively, and the rest is equal to the embodiment 2. The average particle diameter and polydispersity of the obtained BAC-HA-His nanoparticles were measured with a Malvern particle sizer.
The results obtained are depicted in FIG. 12; as can be seen in fig. 12: the cavitation of ultrasonic waves and the generated heat energy can promote the nanoparticles to be uniformly dispersed in the solution, so that the stability of the nanoparticles is improved, but the ultrasonic time is too long, so that the nanoparticles are polymerized, aggregates can be generated in the system, the particle size distribution is widened, and the stability of the nanoparticles is reduced. When the ultrasonic power is low and the ultrasonic time is too short, the nanoparticle particles cannot be dispersed to be small.
The deionized water in example 2 was changed to phosphoric acid buffer solutions of different pH values (pH 5.0, pH 6.0, pH 7.0, pH 7.5, pH 8.0); the rest is equivalent to example 2. The average particle size of the obtained BAC-HA-His nanoparticles was measured with a Malvern particle sizer, and the Zeta potential was detected with a nanoparticle sizer (Zetasizer NanoZS-90 type);
pH affects the average particle size of nanoparticles as shown in fig. 13 (a) and potential affects as shown in fig. 13 (B), and thus it can be known that: the nanoparticles have pH responsiveness.
The cystamine-hyaluronic acid-histidine (BAC-HA-His) self-assembled nanoparticles obtained in example 2 were filtered through a 0.45 μ M filter membrane, 6mL of the obtained filtrate was taken out and placed in a beaker, GSH (glutathione) was added to the 6mL of filtrate until the GSH concentration was 0 μ M, 10 μ M, 5mM, 10mM, and the effect of the GSH concentration on the average particle size and polydispersity of the BAC-HA-His nanoparticles was investigated with a Malvern particle sizer after magnetically stirring for 24 h.
The results obtained are depicted in FIG. 14; therefore, it can be seen that: the nanoparticle has GSH responsiveness.
The cystamine-hyaluronic acid-histidine (BAC-HA-His) self-assembled nanoparticles obtained in example 2 were incubated in a thermostatic water bath at 4 deg.C, 25 deg.C, 40 deg.C, 60 deg.C, and 80 deg.C for 10min, and then the average particle size and polydispersity of BAC-HA-His nanoparticles were measured using a Malvern particle sizer.
The results obtained are depicted in FIG. 15; therefore, it can be seen that: the nanoparticle has good thermal stability.
Preparing simulated gastric juice: 7mL of concentrated hydrochloric acid, 3.2g of pepsin and 2.0g of sodium chloride are dissolved in 250mL of deionized water, the pH of the solution is adjusted to 1.2 by 1mol/L of HCL, and the volume is adjusted to 1000mL by a volumetric flask.
Preparing simulated intestinal juice: weighing 6.8g of monopotassium phosphate, dissolving the monopotassium phosphate with 250mL of deionized water, adding 0.2mol/L NaOH190mL, adding 10g of trypsin, fully and uniformly mixing, adjusting the pH of the solution to 7.0 with 0.2mol/L NaOH, and then using a volumetric flask to fix the volume to 1000 mL. After the solution is fully dissolved, the solution is centrifuged for 15min at a high speed of 4000rpm in a centrifuge, and the supernatant is simulated intestinal fluid.
The cystamine-hyaluronic acid-histidine (BAC-HA-His) self-assembled nanoparticles obtained in example 2 were mixed with simulated gastric fluid and simulated intestinal fluid at a volume of 1:1, respectively, and then the average particle size and polydispersity were measured using a malvern particle sizer.
The results obtained are depicted in FIG. 16; therefore, it can be seen that: the nanoparticle has good gastrointestinal stability.
Taking CMT93 cells in logarithmic growth phase, digesting with 0.25% pancreatin, diluting the cell suspension to 5.0 × 104and/mL, inoculating the cells on a 96-well cell culture plate, adding 100 mu L of cell suspension into each well, placing the culture plate in a carbon dioxide incubator for culturing for 24 hours, respectively adding DMEM culture solution containing BAC-HA-His nanoparticles with different concentrations, setting the culture solution without the cells as blank wells, setting the control wells as cell wells normally cultured by the solution without the nanoparticles, setting 5 parallel wells in each group, and culturing in 5% CO2And continuously incubating for 24h, 48h and 72h in the incubator at 37 ℃.
The results are shown in FIG. 17: after the BAC-HA-His nanoparticles with higher concentration of 1mg/mL are used for treating cells for 72 hours, the survival rate of CMT93 cells can still reach 85%, and the nanoparticles are basically nontoxic and have good biocompatibility probably because hyaluronic acid and histidine have good biodegradability.
Example 3, a method for preparing procyanidin loaded (OPC) nanoparticles using the cystamine-hyaluronic acid-histidine polymer prepared in example 1, sequentially performing the following steps:
1) and 5mg of procyanidine is weighed and dissolved in a phosphate buffer solution with the pH value of 6.5 to prepare a procyanidine solution with the concentration of 1 mg/mL.
2) Weighing 4mg of cystamine-hyaluronic acid-histidine (BAC-HA-His) polymer into a beaker according to the feed-liquid ratio of 1mg/1mL, adding 4mL of deionized water, then placing the beaker on a magnetic stirrer, and magnetically stirring for 8 hours to fully swell the polymer; then adding 130 mu L of procyanidine solution, and carrying out ultrasonic treatment for 10min at 180W under the ice bath condition to obtain the ultrasonic nanoparticles embedded with OPC.
The ultrasonic nanoparticles embedded with OPC obtained in example 3 were tested as follows:
1. determination of OPC embedding Rate
4mL of the ultrasonic nanoparticles embedded with OPC obtained in example 3 was taken and put into a 15mL ultrafiltration tube (Millipore, USA, with a cut-off of 3KD), and then put into a centrifuge for high-speed centrifugation at 6000rpm at 4 ℃ for 10 min. Finally, the absorbance of free OPC in the supernatant at 301nm was measured using a UNICO uv-visible spectrophotometer.
(W) load capacity (entrapment rate)2-W1)/W3×100％
Encapsulation efficiency ═ W2-W1)/W2×100％
W2: the total OPC mass; w is a group of1: the quality of OPC in the supernatant; w is a group of3: total mass of nanoparticles.
The results obtained are shown in FIG. 18.
Comparative example 3, the procyanidin solution in example 3 is changed from 130 μ L to 80 μ L, 90 μ L, 100 μ L, 110 μ L, 120 μ L, 140 μ L and 150 μ L respectively, and the rest is the same as example 3. The resulting product encapsulation efficiency comparison is shown in figure 18. As can be seen from fig. 18: when the addition amount of the procyanidin is 130 mu L, the loading amount is 10.8 percent, and the encapsulation rate is 88.5 percent; this is the best condition, and the encapsulation rate of the nano-particles is high and the loading capacity is better.
2. Fig. 19 shows that the DPPH clearance of the loaded OPC nanoparticles does not change significantly in the temperature range of 40-80 ℃, which indicates that the nanoparticle carrier has a good protection effect on OPC, and the oxidation resistance and the heat resistance stability of OPC are improved to a certain extent. Namely, the nanoparticle carrier can well protect the OPC from contacting the outside at the temperature of 40-80 ℃, and the oxidation resistance of the OPC is improved.
3. Retarding the destruction of OPC by food preservatives:
2 sample bottles with the same volume are taken, 4mL of the nanoparticle solution loaded with OPC (OLEC-embedded ultrasonic nanoparticles obtained in example 3) is added, and then 0.1% of food-grade preservatives, namely sodium benzoate and potassium sorbate, are respectively added. Storing at 4 deg.C in dark, measuring OPC content of BAC-HA-His nanocapsule every 5 days, and calculating retention rate according to formula.
Measuring the residual OPC content in the nanoparticles/the OPC content in the original nanoparticles after the retention rate is 5 days;
the results are shown in FIG. 20: the retention rate is 71% and 70% respectively by day 30, and the nanoparticles can still maintain a relatively high retention rate after one month in the presence of food preservatives, which indicates that the nanoparticles have a protective effect on the core material.
4. Sustained release effect
4mL of the nanoparticle solution loaded with OPC is filled into a dialysis bag (MWCO: 8000-; the mixture was placed in a beaker containing 50mL of PBS buffer solutions containing different GSH concentrations (0mM, 10. mu.M, 10mM) and different pH values (pH 5.8, 7.4), dialyzed and released (shaking speed 100rpm) in a constant temperature shaking incubator at 37 ℃, 4mL of the solution was periodically withdrawn at 1h, 2h, 4h, 8h, 12h, 16h, 24h, 36h, and 48h and supplemented with the same volume of PBS buffer solution, and the release rate of OPC was further calculated by measuring the absorbance of the withdrawn solution.
C: the concentration of OPC released in the solution at time t;
v: the volume of the mixed solution at time t;
m: total amount of OPC in the nanocapsules at the initial moment.
The results are shown in FIG. 21: under the conditions that the pH value is 5.8 and the GSH concentration is 10mM, the release rate of OPC reaches the maximum, and the accumulative release rate of OPC reaches 78.4% after 30h, so that the nanocapsule synthesized by the invention can be used as a carrier with pH and GSH response and can be applied to the embedding of nutrients.
5. Improving the activity of colon cancer resisting cells
Taking CMT93 cells in logarithmic growth phase, digesting with 0.25% pancreatin, diluting the cell suspension to 5.0 × 104and/mL, inoculating the cells on a 96-well cell culture plate, adding 100 mu L of cell suspension into each well, placing the culture plate in a carbon dioxide incubator for culturing for 24 hours, respectively adding a series of DMEM culture solutions containing OPC solutions with different drug concentrations and OPC-loaded nanoparticle solutions, and setting a cell-free culture solutionBlank wells and negative control wells are wells of cells cultured normally without drug, each set of 5 parallel wells in 5% CO2After incubation at 37 ℃ for 24 hours, 48 hours, and 72 hours, the 96-well plate was removed and placed on a clean bench with 20. mu.L of MTT solution added to each well, and further incubation was carried out for 4 hours. Then, the solution in each well was carefully aspirated, 150. mu.L of DMSO was added thereto, and after shaking for ten minutes at a low speed to completely dissolve the purple crystals at the bottom, the OD at 490nm of each well was measured using a microplate reader, and the cell viability was calculated according to the following formula.
Cell survival (%) ═ (OD)Experiment hole-ODBlank hole)/(ODControl well-ODBlank hole)×100％；
The results obtained are shown in FIG. 22: compared with free OPC, the toxicity of the loaded OPC nanoparticles to cells is relatively high, which may be attributed to that glycoproteins on cell membranes have a certain immune function to prevent free OPC from entering cells, and the concentration of OPC in the cells is relatively low and the activity of the cells is relatively high. For the loaded OPC nanoparticles, as a large amount of receptors CD44 of hyaluronic acid are arranged on the surfaces of CMT93 cells, the nanoparticles modified by hyaluronic acid can be quickly and specifically combined with CD44 receptors, so that the loaded OPC is absorbed by CMT93 cells, endosomes of the loaded OPC nanoparticles in the cells are aggregated, the acidic environment and the reduction environment of disulfide bond response in the endosomes can cause the structural damage of a carrier, and the OPC is greatly and quickly released and acts in the cells, so that the survival rate of the cells is reduced, and the cell activity is expressed as anti-colon cancer cell activity.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (7)
1. A method for synthesizing a dual-responsive polymer, characterized by comprising the steps of:
1) synthesis of hyaluronic acid-histidine graft:
performing amide reaction on carboxyl of hyaluronic acid and amino of histidine to obtain a hyaluronic acid-histidine graft;
2) synthesis of cystamine-hyaluronic acid-histidine:
dissolving a hyaluronic acid-histidine graft in deionized water, adding potassium persulfate serving as an initiator and N' N-cysteamine serving as a cross-linking agent, and reacting at 50 +/-5 ℃ for 1 +/-0.2 h under the protection of inert gas;
putting the obtained reaction product into a dialysis bag with the Mr of 8,000-14,000D, dialyzing in deionized water for 22-26 h, and freeze-drying trapped fluid in the dialysis bag to obtain a cystamine-hyaluronic acid-histidine polymer, wherein the cystamine-hyaluronic acid-histidine polymer is a double-responsive polymer;
potassium persulfate: hyaluronic acid-histidine graft = (5 ± 0.5): 1 in mass ratio;
n' N-cysteamine bisacryloyl: hyaluronic acid-histidine graft = (1.2 ± 0.1): 1, mass ratio.
2. A method of synthesizing a bi-responsive polymer according to claim 1, characterized in that: the step 1) comprises the following steps:
s1.1, dissolving hyaluronic acid in deionized water, adding EDC and NHS, and then magnetically stirring and mixing for 1 +/-0.2 h to activate carboxyl of hyaluronic acid;
hyaluronic acid: EDC and NHS = 1 (1.2 plus or minus 0.1): (1.2 plus or minus 0.1) molar ratio;
EDC: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride;
NHS: n-hydroxysuccinimide;
s1.2, according to histidine: hyaluronic acid = 0.5-4: 1, histidine is added into the product obtained in S1.1, the mixture is stirred and mixed by magnetic force for 2 +/-0.2 h, and then ultrasonic treatment is carried out for 20-60 min at 20-60 ℃ under 200-400W;
and (3) filling the obtained reaction solution into a dialysis bag with the Mr of 8000-14000D, dialyzing in deionized water for 22-26 h, and freeze-drying the trapped fluid to obtain the hyaluronic acid-histidine graft.
3. A method of synthesizing a bi-responsive polymer according to claim 2, characterized in that:
in the step S1.1, 75 plus or minus 25ml of deionized water is added for every 0.32mmol of hyaluronic acid;
in the step 2), 20 plus or minus 5mL of deionized water is added for every 20mg of hyaluronic acid-histidine graft.
4. A method for synthesizing a bi-responsive polymer according to any one of claims 1 to 3, wherein in step S1.2,
histidine: hyaluronic acid =3:1 mass ratio;
ultrasonic treatment is carried out for 40min at 50 ℃ and 250W.
5. The preparation method of the double-responsiveness polymer nanoparticles is characterized by comprising the following steps:
dissolving the cystamine-hyaluronic acid-histidine polymer prepared according to claim 1 in deionized water to prepare a solution with a concentration of 1 ± 0.2 mg/mL;
magnetically stirring the solution at 25 +/-5 ℃ for 8 +/-2 hours, so that the cystamine-hyaluronic acid-histidine polymer is fully swelled; and then carrying out ultrasonic treatment for 6-15 min at the ultrasonic power of 140-200W under the ice bath condition to obtain the double-responsiveness polymer nanoparticles.
6. Use of a dual-responsive polymer synthesized according to any one of claims 1 to 4, wherein: preparing the nanoparticles for loading the procyanidine.
7. Use according to claim 6, characterized in that it comprises the following steps:
1) dissolving procyanidine in a phosphoric acid buffer solution with the pH value of 6.5 to prepare a procyanidine solution with the concentration of 1 +/-0.2 mg/mL;
2) adding 4mg of cystamine-hyaluronic acid-histidine polymer into 4 +/-1 mL of deionized water, magnetically stirring for 8 +/-2 hours, adding 80-150 mu L of procyanidine solution, and carrying out ultrasonic treatment at 180 +/-20W for 10 +/-2 minutes under an ice bath condition to obtain the ultrasonic nanoparticles embedded with OPC.
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