CN112717138A - Gamma-polyglutamic acid nano-carrier and preparation method and application thereof - Google Patents
Gamma-polyglutamic acid nano-carrier and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a gamma-polyglutamic acid nano-carrier, a preparation method and application thereof, belonging to high polymers and medical biomaterials. According to the invention, hydrophilic gamma-PGA and hydrophobic phenylalanine ethyl ester hydrochloride (L-PAE) spontaneously form nanoparticles through condensation reaction, and the preparation process is optimized by changing influencing factors in the preparation process. Secondly, the camptothecin is encapsulated by the nano material prepared by the optimal process, the structural stability of the drug-loaded nanoparticles is enhanced through the interaction of static electricity and hydrophobicity, and the drug-loaded nanoparticles are characterized by taking the encapsulation efficiency and the drug-loaded amount as main investigation indexes. The nano material has the advantages of high camptothecin encapsulation rate of 86.4 percent, higher utilization rate, less generated impurities, effective utilization of camptothecin, improvement of pharmacodynamic activity, reduction of toxic and side effects of the drug and provision of a new effective way for tumor treatment.
Description
Technical Field
The invention relates to a gamma-polyglutamic acid nano-carrier, a preparation method and application thereof, belonging to the technical field of high polymers and medical biomaterials.
Background
Gamma-polyglutamic acid (gamma-PGA) is a green environment-friendly polymeric material, has the characteristics of high molecular weight, easy dispersion, no toxicity, no harm, edible property and the like, and is applied to a plurality of fields as a biological flocculant, a drug carrier, a food additive and the like. In the field of drug carriers, high-activity carboxyl of gamma-PGA and derivatives thereof can be combined with drugs or active ingredients thereof, so that the problem that natural drugs are difficult to dissolve in water is solved, the pharmacodynamic activity is improved, and the toxic and side effects of the drugs are reduced. Camptothecin is a natural component separated from the stem of camptotheca acuminate, is the 2 nd natural product with strong anticancer activity after paclitaxel, is insoluble in water and has large toxic and side effects, the antitumor activity is obviously reduced after salt preparation, and the drug-loaded nanoparticles can reduce the toxicity of camptothecin compounds and enhance the curative effect.
Therefore, in order to improve the stability and availability of camptothecin, hydrophilic γ -PGA and hydrophobic phenylalanine ethyl ester hydrochloride (L-PAE) are first spontaneously combined into nanoparticles through a condensation reaction, and the preparation process is optimized by changing influencing factors in the preparation process and using Design-Expert software. Secondly, the camptothecin is encapsulated by the nano material prepared by the optimal process, the structural stability of the drug-loaded nanoparticles is enhanced through the interaction of static electricity and hydrophobicity, the encapsulation efficiency and the drug-loaded amount are used as main investigation indexes, the drug-loaded camptothecin nanoparticles are characterized, and reference is provided for the future pharmacodynamic evaluation of gamma-PGA.
Disclosure of Invention
The invention aims to solve the problems in the prior art, and further provides a gamma-polyglutamic acid nano-carrier, and a preparation method and application thereof.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a gamma-polyglutamic acid nano-carrier comprises the following steps:
the method comprises the following steps: preparation of small molecular gamma-polyglutamic acid by high-temperature acidolysis method
Degrading macromolecular gamma-polyglutamic acid by a high-temperature acidolysis method, placing the macromolecular gamma-polyglutamic acid in a beaker,adding ultrapure water, and then dropwise adding 1 mol/L-1NaOH solution until the gamma-polyglutamic acid is completely dissolved; slowly adding 1 mol. L to the prepared macromolecule gamma-polyglutamic acid solution-1Adjusting pH of the solution to 2.0-2.1 with HCl, and hydrolyzing in 98 deg.C water bath for 90 min; after degradation, rapidly placing on crushed ice for cooling, adjusting the pH to 7.0-7.1, centrifuging by using an ultrafiltration centrifugal tube, and freeze-drying to obtain micromolecular gamma-polyglutamic acid;
step two: preparation of gamma-polyglutamic acid nano-carrier
Adding a catalyst EDC into a small-molecular gamma-polyglutamic acid solution, adding L-PAE into the reaction system, oscillating by a table concentrator for reaction, centrifuging to remove supernatant after the reaction is finished, then re-suspending by ultrapure water, repeating for 3 times, washing off unreacted substances, and freeze-drying to obtain a product, namely the gamma-polyglutamic acid nano carrier;
in the second step, the concentration of the gamma-polyglutamic acid solution is 4 mg/mL-1EDC is added in an amount of 0.03g, L-PAE has a mass of 0.05g, the cultivation temperature is 37 ℃ during shaking of the shaking table, and the rotation speed of the shaking table is 200 r.min-1The shaking table was shaken for 24 h.
The gamma-polyglutamic acid nano material obtained by the preparation method is a nano particle with the particle size within the range of 100-300 nm.
The invention also aims to provide the gamma-polyglutamic acid nano-carrier prepared by the preparation method of the gamma-polyglutamic acid nano-carrier.
The invention also aims to provide the application of the gamma-polyglutamic acid nano-carrier in the field of medicines for treating tumors or engineering materials.
The invention has the beneficial effects that:
1. the gamma-PGA adopted by the invention is directly degraded by a macromolecular standard substance instead of a microbial synthesis method, so that the utilization rate is higher, fewer impurities are generated, and the defects of time consumption, excessive introduced reagents and complex purification caused by a microbial preparation method are overcome.
2. The particle size of the amphiphilic gamma-PGA nano-carrier material obtained by the invention is optimized and prepared by a single-factor experiment and a response surface analysis method. The nano-carrier formed by self-assembly has a certain difference in particle size, and the difference is expressed in the difference of the function and application range generated by the difference, so that the research on the reasonable application of the obtained product is particularly necessary. In the experiment, 6 influencing factors (gamma-PGA concentration, EDC quality, L-PAE quality, temperature, rotating speed and reaction time) in the preparation process of the nanoparticles are selected as evaluation factors, the particle size Y of the nanoparticles is used as a response value, each factor is respectively taken as a high level (+1) and a low level (-1), Design tests are carried out by applying Design-Expert8.0 software, the factors which obviously influence the preparation process of the nanoparticles are selected, and finally, the related research of a drug loading part is carried out after the size of the particle size is determined.
3. In order to improve the stability and availability of camptothecin, the invention firstly uses the nano material prepared by the optimal process to encapsulate camptothecin, enhances the structural stability of the drug-loaded nano particle through the interaction of static electricity and hydrophobicity, and takes encapsulation efficiency and drug-loading rate as main investigation indexes to characterize the drug-loaded camptothecin nano particle. The nano material has an entrapment rate of 86.4% for camptothecin, can effectively utilize camptothecin, solves the problem that natural medicines are difficult to dissolve in water, improves the pharmacodynamic activity, and reduces the toxic and side effects of the medicines.
4. The release test of the prepared drug-loaded camptothecin can simulate the release degree of the encapsulated camptothecin and free camptothecin, and the release rate of the encapsulated camptothecin for 24 hours is less than 25 percent, so that the gamma-PGA nano material prepared by the research can play a role in sustained release of drugs and improvement of drug release.
5. The invention firstly carries out in-vitro anti-tumor test on the prepared nano-drug, selects an oral cancer and a colon cancer, and the result shows that the drug-loaded camptothecin has the effect of inhibiting a certain solid tumor, thereby providing a new effective way for treating the tumor.
Drawings
FIG. 1 is a schematic diagram showing the effects of various concentrations of γ -PGA solutions according to the present invention on the nanoparticle size and PDI.
FIG. 2 is a schematic representation of the effect of different mass EDC solutions of the invention on nanoparticle size and PDI.
FIG. 3 is a schematic representation of the effect of different mass L-PAE solutions of the present invention on nanoparticle size and PDI.
FIG. 4 is a schematic illustration of the effect of temperature on nanoparticle size and PDI according to the present invention.
FIG. 5 is a schematic illustration of the effect of rotational speed on nanoparticle size and PDI of the present invention.
FIG. 6 is a schematic diagram showing the effect of reaction time on nanoparticle size and PDI in accordance with the present invention.
FIG. 7 is a schematic diagram showing the interaction of the change of various factors on the influence of the particle size of the nanomaterial of the present invention. Wherein (a) is an interaction schematic diagram of the influence of the concentration of gamma-PGA and the quality of L-PAE on the particle size of the nano material; (b) is an interaction schematic diagram of the influence of the concentration of gamma-PGA and the rotating speed on the grain diameter of the nano material; (c) is an interaction schematic diagram of the influence of the mass and the rotating speed of the L-PAE on the particle size of the nano material.
FIG. 8 is a schematic diagram of the particle size of the nano-material under the optimal process conditions of the present invention.
Fig. 9 is a schematic particle size diagram of the drug-loaded nanoparticles of the present invention.
Fig. 10 is a schematic ultraviolet spectrum of the drug-loaded nanoparticles of the invention.
Fig. 11 is a schematic infrared spectrum diagram of the drug-loaded nanoparticles of the invention.
Fig. 12 is a schematic view of camptothecin release curves of the drug-loaded nanoparticles of the invention.
FIG. 13 is a schematic view of the effect of the nanomaterial of the present invention and drug-loaded camptothecin on inhibiting the proliferation of human tumor cells. Wherein, (a) is the schematic view of the inhibition effect of the nano material and the drug-loaded camptothecin on the proliferation of the human oral cancer cells KB, and (b) is the schematic view of the inhibition effect of the nano material and the drug-loaded camptothecin on the proliferation of the human colon cancer cells Lovo.
Detailed Description
The invention will be described in further detail below with reference to the accompanying drawings: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation is given, but the scope of the present invention is not limited to the following embodiments.
As shown in fig. 1 to 13, the γ -polyglutamic acid nanocarriers according to the present embodiment, the preparation method and the application thereof, include:
example 1
1 materials and methods
1.1 Experimental reagents: high molecular weight polyglutamic acid (γ -PGA, japan and wako pure chemical industries co.); phenylalanine ethyl ester hydrochloride (L-PAE, Aladdin reagent, Inc.); carbodiimide hydrochloride (EDC, alatin reagent limited); camptothecin (Dalian biotechnology, Inc.), dimethylsulfoxide (DMSO, Bio-engineering, Inc.).
1.2 Experimental instruments: a freeze dryer (Labconco, USA); ultrafiltration centrifuge tubes (Millipore, NMWL:10000 kDa); nanometer particle sizer (Nicomp 380ZLS, us PSS); ultraviolet spectrophotometer (UV-2550, Shimadzu corporation, Japan); infrared spectrum analyzer (Nicolet iN10, seimer feishell scientific inc.); an incubator shaker (IS09001, shanghai wisdom city analytical instruments manufacturing ltd); high capacity high speed refrigerated centrifuge (Z36HK, Hermle, Germany).
1.3 Experimental methods
1.3.1 preparation of micromolecular gamma-PGA by high-temperature acidolysis method
And degrading the macromolecular gamma-PGA by using a high-temperature acidolysis method. Placing polymer gamma-PGA into a beaker, adding ultrapure water, and dropwise adding 1 mol. L-1NaOH solution until gamma-PGA is completely dissolved. Slowly adding 1 mol. L to the prepared macromolecule gamma-PGA solution-1Adjusting pH of the solution to about 2.0 with HCl, and hydrolyzing in 98 deg.C water bath for 90 min. After degradation, the mixture was quickly cooled on crushed ice, and the pH was adjusted to about 7.0. Centrifuging by an ultrafiltration centrifugal tube, and freeze-drying to obtain the micromolecule gamma-PGA.
1.3.2 preparation of Gamma-PGA nano material
Adding catalyst EDC into the small molecular gamma-PGA solution, adding L-PAE into the reaction system, and shaking the table to react overnight. And after the reaction is finished, centrifuging to remove the supernatant, then resuspending with ultrapure water, repeating for 3 times, washing away unreacted substances, and freeze-drying to obtain the product, namely the gamma-PGA nano material.
1.3.3 measurement of particle size with nanometer particle sizer
And (3) dispersing the gamma-PGA nano material in ultrapure water, and analyzing the particle size distribution by using a nano particle size analyzer. It is believed that particles with a particle size of 100-800nm belong to the class of nanoparticles, whereas nanoparticles in the range of 100-300nm exhibit better performance, and therefore the test will use particle size as a factor in the investigation to determine the final process parameters.
1.3.4 Single factor test
The process conditions for preparing the gamma-PGA-PAE nano material are preliminarily explored by adopting a single-factor test, and the design is shown in Table 1:
TABLE 1 Single factor test design Table
1.3.5 Plackett-Burman test design
According to the test result of a single factor, 6 influencing factors (gamma-PGA concentration, EDC quality, L-PAE quality, temperature, rotating speed and reaction time) in the preparation process of the nanoparticles are selected as evaluation factors, the particle size Y of the nanoparticles is taken as a response value, each factor is respectively taken as a high level (+1) and a low level (-1), and Design-Expert8.0 software is applied to Design a PB test, wherein the Design factors and the levels are shown in a table 2.
TABLE 2 Plackett-Burman factor level Table
1.3.6 Central Composite Design test Design
According to the PB test result, three factors (gamma-PGA concentration, L-PAE quality and rotating speed) which obviously affect the nanoparticle preparation process are selected as independent variables, the particle size Y of the nanoparticles is used as a response value, Design-expert8.0 software Design is applied to carry out CCD test, and the Design factors and the level are shown in a table 3.
TABLE 3 center combination test design factors and levels
1.3.7 validation test
And (3) preparing all factors influencing the test according to optimized conditions to obtain the gamma-PGA-PAE nano particles, and analyzing and comparing the particle size distribution by adopting a nano particle size determinator to obtain the optimal process.
1.3.8 preparation of drug-loaded nanoparticles
Dissolving gamma-PGA nano material (20.0mg) and camptothecin (10.0mg) in10 mL of phosphate buffer solution (pH 7.4), shaking for 24h in the dark to spontaneously generate camptothecin-coated nanoparticles, putting the prepared liquid into a dialysis bag (molecular weight cut-off: 8000- + 14000D), putting the dialysis bag into PBS buffer solution with a certain amount for 48h to remove unreacted substances, and then freeze-drying to obtain the camptothecin-loaded nanoparticles.
1.3.9 characterization of drug-loaded nanoparticles
1.3.9.1 particle size and potential measurements
The gamma-PGA-PAE nano material and the drug-loaded gamma-PGA-PAE nano particles are dispersed in ultrapure water, and the particle size distribution and the Zeta potential of the nano particle size analyzer are analyzed.
1.3.9.2 determination of encapsulation rate and drug loading rate of nanoparticles
An ultraviolet spectrophotometer is adopted to calculate the encapsulation efficiency and the drug-loading rate of the drug-loaded nanoparticles, and camptothecin has a characteristic absorption peak at 367 nm. The calculation formula is as follows: the encapsulation ratio ═ (total mass of camptothecin-mass of unencapsulated camptothecin)/total mass of camptothecin × 100%; drug loading ═ (total mass of camptothecin-mass of unencapsulated camptothecin)/total mass of nanoparticles × 100%.
1.3.9.3 ultraviolet spectrum scanning
An ultraviolet spectrophotometer is adopted to detect the changes of the absorption peaks of the gamma-PGA-PAE nano material and the drug-loaded nano particles in the range of 200-600 nm.
1.3.9.4 Infrared Spectrum Scan
Freeze drying the sample, oven drying, and grinding into powderPowder with specified size is measured by an infrared spectrometer at room temperature transmission method after being tabletted by KBr, and the scanning wave number range is 400--1。
1.3.10 camptothecin Release test
1mL of the contained free camptothecin and drug-loaded camptothecin solutions were added to dialysis bags (Mwco: 12000-14000). Then, both ends of the dialysis bag were clamped and placed in 50mL of PBS (pH 7.4), respectively, and shaken at 100 r.min-1 in a constant temperature shaker at 37 ℃. After 1, 2, 4, 6, 8, 10, 12, 24h, respectively, 3mL of dialysate was taken and supplemented with the same amount of release medium. The concentration of camptothecin in each sample was determined by uv-spectrophotometer.
1.3.11 MTT in vitro assay
Selecting human oral cancer cells KB and human colon cancer cells Lovo as research objects, and determining the influence of the nano materials and the nano drugs on cell growth inhibition by adopting an MTT method: inoculating tumor cells into a 96-well cell culture plate, adding samples with different concentrations, culturing in a 5% CO2 cell culture box at 37 ℃ for 48h by taking PBS as a negative control, adding MTT (methyl thiazolyl tetrazolium) for continuous culture for 3h, then absorbing the culture medium, adding DMSO, slightly oscillating, measuring the light absorption value at 490nm on an enzyme labeling instrument, and drawing a cell growth inhibition curve by taking the light absorption value as a reference.
1.3.12 statistical analysis
Design-Expert8.0 software is used for response surface test Design; SPSS 23.0 is adopted for statistical analysis; origin 75 and Excel were used for mapping.
2 results
2.1 Single factor test results
2.1.1 Effect of Gamma-PGA solution concentration on the particle size of the nanomaterial
The results of the effect of various concentrations of γ -PGA solutions on the particle size and PDI of the nanomaterials are shown in FIG. 1. As can be seen from FIG. 1, when the concentration of γ -PGA was 0.5, 1, 2 mg/mL-1When the particle size of the nano material is larger than 300nm, a large amount of raw materials in the system are not converted into the nano material probably due to the fact that the concentration of the gamma-PGA is too small; when the concentration of gamma-PGA is 6 mg/mL-1At this time, the appearance of macroscopic flocs in the solution indicatesExcessive gamma-PGA can make the system of the nano material unstable; when the concentration of gamma-PGA is 4 mg/mL-1When the particle size is 294.7nm, PDI is 0.483, both of which are optimum levels, 4 mg/mL is selected-1The subsequent experiments were performed with respect to the γ -PGA concentration.
2.1.2 Effect of EDC Mass on nanomaterial particle size
The effect of different mass of EDC on nanomaterial particle size and PDI results are shown in fig. 2. EDC has an effect of activating a carboxyl group as a catalyst for the dehydration condensation reaction in this study. The investigation of the dosage of EDC is mainly carried out from two aspects of activation effect and cost saving. As can be seen from FIG. 2, the particle size of the nanomaterial is optimum when the amount of EDC added is 0.03g, and the PDI value is slightly large but within a reasonable range; and as the dosage of the EDC is increased, the particle size of the nano material is also increased, and the activation effect is worse, so that the dosage of the EDC is selected to be 0.03g for subsequent tests in view of cost saving and activation effect.
Influence of 2.1.3L-PAE quality on particle size of nanomaterial
The results of the effect of different masses of L-PAE on the particle size and PDI of the nanomaterials are shown in FIG. 3. The side chain of the gamma-PGA micromolecule has a plurality of carboxyl groups and has hydrophilicity; in the presence of catalyst EDC, hydrophobic group L-PAE can be connected to the carboxyl terminal of the side chain thereof through condensation reaction to form amphiphilic nano material, so that the quality of L-PAE is an important factor influencing the particle size and stability of nano material. As can be seen from FIG. 3, when the amounts of L-PAE added were 0.005, 0.01 and 0.02g, the particle size of the nanomaterial exceeded 300nm, probably because the amount of L-PAE was too small to form nanoparticles in the aqueous phase; on the contrary, when the mass of the L-PAE is 0.1g, the excessive L-PAE can also influence the formation of the nano material, so that the mass of the L-PAE is 0.05g for subsequent experiments.
2.1.4 Effect of temperature on the particle size of nanomaterials
The results of the effect of temperature on the nanomaterial particle size and PDI are shown in fig. 4. The reaction temperature can affect the ionic strength and reaction rate in the solution, thereby reacting out on the properties of the nanomaterial, resulting in a change in its particle size. As can be seen from FIG. 4, the optimum temperature for the culture of the nanomaterial was 37 ℃, and this temperature was selected for the subsequent experiments.
2.1.5 Effect of rotational speed on particle size of nanomaterials
The effect of the rotation speed on the particle size of the nanomaterial is shown in fig. 5. The purpose of stirring is to bring the two raw materials into uniform contact to accelerate the reaction rate. As can be seen from FIG. 5, stopping stirring immediately after the completion of the dropwise addition of the reactants has a great influence on the synthesized nanomaterial, the particle size is already over 500nm,
however, when the rotating speed of the shaking table is too high, the rotating force is too high, which is not beneficial to the synthesis of the nanoparticles, so that the particle size of the nanoparticles is too large, and therefore, the rotating speed of the shaking table is selected to be 200 r.min-1Subsequent tests were conducted with the smallest nanometer particle size at this speed. As shown in fig. 5, the effect of rotational speed on the nanoparticle size and PDI.
2.1.6 Effect of reaction time on the particle size of nanomaterials
The effect of reaction time on the particle size of the nanomaterial is shown in fig. 6. The reaction time refers to the time for continuing stirring after the dropwise adding process is finished, and the operation can convert the raw materials which are not reacted in time in the system into products. As can be seen from FIG. 6, the reaction time is 12-32h, which has little effect on the particle size, but the reaction time is too long, which may affect aggregation between nanoparticles to increase the particle size, so that the reaction time is selected to be 24h for subsequent experiments. As shown in fig. 6, the effect of reaction time on nanoparticle size and PDI.
2.2 Plackett-Burman test results
On the basis of the single-factor test, the PB test design and the result are shown in the table 4, and the regression analysis result is shown in the table 5.
TABLE 4 Plackett-Burman test design and results
TABLE 5 Plackett-Burman test regression analysis results
Note: "+" indicates significant effect on the results (P < 0.05)
As can be seen from Table 5, the F value of the model is 18.62, the P value is 0.0028 < 0.01, and the model terms are significant, which indicates that the model has important significance. Coefficient of determination R of the model20.9572, indicating that 95.72% of the experimental data are present can be interpreted by the model, adjusted to determine the coefficient R2 adj0.9058, both are connected to 1, and the signal-to-noise ratio is 14.179 (> 4), indicating that the model has good prediction ability. The significant degree of the influence of each variable on the particle size of the nano material is E & gtA & gtC & gtD & gtF & gtB in sequence, and the data of the table 4 are analyzed by software to obtain a model fitting equation as follows: and Y is 216.09-28.46A-3.86B-20.24C +6.16D-44.11E-4.28F, and three factors of gamma-PGA concentration, L-PAE quality and rotating speed remarkably influence the particle size of the nano material according to a regression equation, so that the three factors are selected for carrying out a Central Composite Design test.
2.3 results of the Central Composite Design test
On the basis of the PB test, the results of the CCD test design level and the response value nanoparticle size are shown in Table 6, and the regression analysis is shown in Table 7.
TABLE 6 center combinatorial design test design and results
TABLE 7 regression analysis results of center combination design test
Note: "+" indicates significant effect on the results (P < 0.05)
As can be seen from Table 7, the regression model has a very significant difference (P < 0.0001), and the coefficient of determination R of the model is20.9483, the decision coefficient of adjustment R2 adj0.9017, albeit with R2Close, but decreasing values indicate that the model may contain insignificant terms, with a signal-to-noise ratio of 12.983 (> 4), indicating that the model has strong predictive power. The model mismatching item has no significant difference (P is 0.4137 is more than 0.05), which shows that the fitting degree of the model is better, thereby judging that the CCD experimental design is reliable. The data of table 6 were analyzed by software to obtain the following model fitting equation: y is 247.89-5.80A +25.38B +14.96C +5.36AB-5.81AC-15.36BC-39.04A2-23.83B2-22.44C2Wherein each variable (B, C), the interactive item (BC) and each variable are two interactive items (A)2、B2、C2) Has obvious influence on the grain size of the nanometer material (P < 0.05).
2.4 analysis of response surface interactions
The influence of various factors and interactions on the particle size of the nanomaterial is shown in fig. 7. The interaction relationship among the variables in the regression model can be seen from the 3D response surface diagram, wherein the ellipse shows that the interaction among the variables has significant influence, and the circle shows that the interaction among the variables has no significant influence. As can be seen from FIG. 7, the contour plot of the interaction between the mass and rotational speed of L-PAE is elliptical, indicating that the interaction is significant (P < 0.05).
2.5 validation test
The technological condition for predicting the optimal nano material particle size in the CCD test is that the concentration of gamma-PGA is 4 mg.mL-1The mass of the L-PAE is 0.08g, and the rotating speed is 140 r.min-1The nano-particle size is 182.783 nm. Experiments are carried out according to the obtained optimal process conditions, and the particle size of the finally obtained nano material is shown in fig. 8, the particle size is 186.4nm, the PDI is 0.575, and the particle size is not significantly different from the predicted value 182.783nm (P is more than 0.05).
2.6 characterization of drug-loaded nanoparticles
2.6.1 particle size and potential of drug-loaded nanoparticles
The experiment result is shown in fig. 9, the particle size of the nanoparticles after drug loading is in normal distribution, the average size is (147.7 +/-5.8) nm, and the PDI is 0.576, which indicates that the nanoparticles after drug loading have good dispersibility and uniform size distribution in the view of peak shape. The Zeta potential result shows that the Zeta potential of the drug-loaded nano-particle is-14.60 mV, and compared with the-3.50 mV of a nano material without drug loading, the absolute value of the Zeta potential is increased, which indicates that the nano-particle after drug loading has good stability.
2.6.2 encapsulation efficiency and drug-loading rate of drug-loaded nanoparticles
The product yield, encapsulation efficiency and drug loading during the preparation are shown in table 8. The encapsulation rate of the drug-loaded camptothecin to the drug is 86.40 percent and the drug-loaded amount of the drug-loaded camptothecin is 55.17 percent.
Table 8 shows the product yield, encapsulation efficiency and drug loading
2.6.3 ultraviolet spectrum of medicine-carrying nano-particle
The gamma-PGA nano material loads camptothecin in a wrapping mode, and after the drug is loaded successfully, the spectral absorption value of the camptothecin is reflected, so that whether the camptothecin is loaded successfully can be identified by the method. As shown in FIG. 10, the ultraviolet spectrum of the drug-loaded nanoparticles shows that compared with γ -PGA, the nanoparticles show an absorption peak around 240nm-270nm, which indicates that γ -PGA and L-PAE are successfully crosslinked. Meanwhile, the nanoparticle carrying the camptothecin has an absorption peak at 367nm, which is a characteristic absorption peak of the camptothecin, so that the successful loading of the camptothecin by the nano material can be proved.
2.6.4 Infrared Spectrum of drug-loaded nanoparticles
Compared with gamma-PGA, the nano material is in amide I belt (1689.0 cm)-1) Amide II band (1531.5 cm)-1) And amide III tape (1290.0 cm)-1) Three characteristic absorption peaks are strengthened, and simultaneously, the nano materialAt 3100cm-1The characteristic peak of amide group is shown, which shows that the amide bond in the nano material is greatly increased, and the gamma-PGA and the L-PAE form-CONH-to make the cross-linking. As shown in FIG. 11, the infrared spectrum of the drug-loaded nanoparticles and the infrared spectrum result show that the nanoparticles are formed by forming amido bond between gamma-PGA and L-PAE. Compared with camptothecin, the loaded nanomaterial simultaneously shows amide bonds and characteristic peaks of camptothecin, so that the camptothecin is successfully loaded by the nanomaterial of gamma-PGA.
2.6.5 Release results of camptothecin
As shown in fig. 12, the camptothecin release profile of drug-loaded nanoparticles, fig. 12 can show the release of free camptothecin and drug-loaded camptothecin in PBS buffer solution with pH 7.4. The release rate of free camptothecin in the control group is faster, the release rate can reach 2h, the nano material wraps the camptothecin to delay the release of the drug, and the release rate of camptothecin in 24h is shown in the specification, so that the wrapping of gamma-PGA can obviously delay the release of the camptothecin, but the curative effect is not influenced. Therefore, the drug-loaded camptothecin can play a role in drug slow release and drug release improvement.
2.6.6 in vitro MTT assay results
The MTT method is adopted to detect the influence of the nano material and the drug-loaded camptothecin on the proliferation inhibition rate of human oral cancer cells KB and human colon cancer cells Lovo. The results are shown in fig. 13, the nano material has no influence on cancer cells, which indicates that the prepared nano material has no toxic or side effect, the drug-loaded camptothecin emulsion has the function of inhibiting the proliferation of human oral cancer cells KB and human colon cancer cells Lovo, and the drug-loaded camptothecin is 5 mu g/mL-1The inhibition rate of the extract on cancer cells is about 40% at the concentration point, and the concentration is 30 mu g/mL-1In the process, the inhibition rate can reach about 80%, and a dose-inhibition rate dependence relationship exists, which shows that the drug-loaded camptothecin nanoparticles can obviously reduce the survival rate of tumor cells. As shown in FIG. 13, the nano material and the drug-loaded camptothecin (a) have KB cell and (b) have Lovo cell inhibition effect on human tumor cell proliferation.
Discussion of 3
Gamma-PGA is a macromolecule functional material synthesized by microorganisms and degradable, and is an ideal drug carrier. The side chain of the gamma-PGA has a large amount of carboxyl, so that the gamma-PGA has better hydrophilicity, therefore, in the experiment, after the macromolecule gamma-PGA is prepared into the micromolecule gamma-PGA in an acid hydrolysis mode, the carboxyl end of the micromolecule gamma-PGA is coupled with hydrophobic phenylalanine, the side chain of the modified gamma-PGA simultaneously has hydrophilic group carboxyl and hydrophobic group benzene ring, and the amphiphilic nano-particle is formed by self-assembly in a water phase. The nano-particles are formed by self-assembly, so that the particle sizes of the nano-particles have certain difference, and the difference can be shown in the difference of the functions and application ranges generated by the difference, so that the research on the reasonable application of the obtained product is particularly necessary.
The size of the nano material applied to clinical treatment is generally less than 400nm, and the size range is more than the requirement of the particles for blood circulation. The research selects 6 influencing factors which can cause the particle size change in the nano material synthesis stage, and the optimal process condition of the nano material particle size is obtained by single factor test and response surface test optimization, wherein the gamma-PGA concentration is 4 mg/mL-1The mass of the L-PAE is 0.08g, and the rotating speed is 140 r.min-1The nanometer particle size is 186.4nm, the PDI is 0.575, and the particle size is not obviously different from the predicted value of 182.783nm (P is more than 0.05), so that the particle size of the nanometer material is finally determined, and the nanometer material has important significance for being used as a medicine carrier subsequently.
The drug and the nanomaterial are linked in two ways, one can be linked by covalent bonds and the other can be linked by non-covalent bonds, such as ionic bonds, hydrogen bonds and hydrophobic interactions. The camptothecin selected in the research is coated in the nano material by hydrophobic interaction. Camptothecin exhibits spectral antitumor activity against many solid tumor tissues, but its development and utilization are limited because it is poorly soluble in water and the lactone form is unstable. The study shows that the compound formed by coupling the camptothecin and the PGA has increased water solubility and retains higher antitumor activity. In the research, after nanoparticles coated with camptothecin are prepared and spontaneously generated, the nanoparticles are characterized, and the result shows that the particle size of the nanoparticles after drug loading is in normal distribution, the average size is 147.7nm, the dispersibility of the nanoparticles after drug loading is better, the size distribution of the nanoparticles is more uniform from the aspect of peak shape, the Zeta potential of the drug-loaded nanoparticles is-14.60 mV, and the nanoparticles have good stability. The encapsulation rate of the drug-loaded camptothecin to the drug is 86.40 percent and the drug-loaded amount of the drug-loaded camptothecin is 55.17 percent. Ultraviolet spectrum and infrared spectrum prove that the camptothecin is successfully loaded by the gamma-PGA nano material. The test researches the inhibition effect of the nano material and the drug-loaded camptothecin on human tumor cells, and the cancer cells are characterized by continuous division and continuous proliferation, so whether the index has a preliminary anticancer effect can be examined. Test results prove that the nano material has no influence on cancer cells, which shows that the prepared nano material has no toxic or side effect, the drug-loaded camptothecin emulsion has the function of inhibiting the proliferation of human oral cancer cells KB and human colon cancer cells Lovo, and has a dose-inhibition rate dependence relationship, and data shows that the survival rate of tumor cells can be obviously reduced.
In conclusion, the research on the preparation of the nano-material by the gamma-PGA and the L-PAE under the optimized process conditions and the research on the application of the nano-particle in the aspect of the drug carrier have great practical significance.
The above description is only a preferred embodiment of the present invention, and these embodiments are based on different implementations of the present invention, and the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (6)
1. The preparation method of the gamma-polyglutamic acid nano-carrier is characterized by comprising the following steps of:
the method comprises the following steps: preparation of small molecular gamma-polyglutamic acid by high-temperature acidolysis method
Degrading macromolecular gamma-polyglutamic acid by high-temperature acidolysis method, placing the macromolecular gamma-polyglutamic acid in a beaker, adding ultrapure water, and dropwise adding 1 mol.L-1NaOH solution up to gamma-complete dissolution of polyglutamic acid; slowly adding 1 mol. L to the prepared macromolecule gamma-polyglutamic acid solution-1Adjusting pH of the solution to 2.0-2.1 with HCl, and hydrolyzing in 98 deg.C water bath for 90 min; after degradation, rapidly placing on crushed ice for cooling, adjusting the pH to 7.0-7.1, centrifuging by using an ultrafiltration centrifugal tube, and freeze-drying to obtain micromolecular gamma-polyglutamic acid;
step two: preparation of gamma-polyglutamic acid nano-carrier
Adding catalyst EDC into small molecular gamma-polyglutamic acid solution, adding L-PAE into the reaction system, oscillating by a table concentrator for reaction, centrifuging to remove supernatant after the reaction is finished, then re-suspending by ultrapure water, repeating for 3 times, washing off unreacted substances, and freeze-drying to obtain the product, namely the gamma-polyglutamic acid nano carrier.
2. The method for preparing a gamma-polyglutamic acid nanocarrier according to claim 1, wherein, in the second step, the concentration of the gamma-polyglutamic acid solution is 4 mg-mL-1EDC is added in an amount of 0.03g, L-PAE has a mass of 0.05g, the cultivation temperature is 37 ℃ during shaking of the shaking table, and the rotation speed of the shaking table is 200 r.min-1The shaking table was shaken for 24 h.
3. The method for preparing gamma-polyglutamic acid nano-carrier as claimed in claim 2, wherein the gamma-polyglutamic acid nano-material obtained by the preparation method is a nano-particle with a particle size in the range of 100-300 nm.
4. The gamma-polyglutamic acid nano-carrier prepared by the preparation method of the gamma-polyglutamic acid nano-carrier according to claim 1.
5. The application of the gamma-polyglutamic acid nano-carrier according to claim 4 in the medical field of tumor treatment or engineering materials.
6. The use of the gamma-polyglutamic acid nanocarrier according to claim 5 in the treatment of tumors, comprising the steps of: dissolving 20.0mg of gamma-polyglutamic acid nano-carrier and 10.0mg of camptothecin in10 mL of phosphate buffer solution, enabling the pH value of the phosphate buffer solution to be 7.4, shaking for 24 hours in a dark place, spontaneously generating camptothecin-coated nanoparticles, putting the prepared liquid into a dialysis bag, and intercepting the molecular weight: 8000- "14000D", placing the mixture into a buffer solution containing a certain amount of PBS for 48 hours; to remove unreacted materials, followed by lyophilization to obtain camptothecin-loaded nanoparticles.
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