CN112370530A - Lactoferrin-modified pegylated graphene oxide-loaded puerarin nano platform as well as preparation method and application thereof - Google Patents
Lactoferrin-modified pegylated graphene oxide-loaded puerarin nano platform as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a lactoferrin-modified pegylated graphene oxide-loaded puerarin nano platform as well as a preparation method and application thereof. The nano platform comprises graphene oxide, polyethylene glycol, puerarin and lactoferrin. The polyethylene glycol modified graphene oxide improves the dispersibility and stability of the graphene oxide; the puerarin is loaded by the polyethylene glycol graphene oxide, so that the solubility of the puerarin is improved, the bioavailability of the puerarin is improved, and the loading rate of the puerarin is enhanced; finally, the biocompatibility of the graphene oxide is improved by modifying the lactoferrin, and the graphene oxide has obvious brain targeting property and is expected to be applied to the biomedical field in the aspect of brain diseases. In addition, the preparation method of the lactoferrin modified pegylated graphene oxide loaded puerarin nano platform provided by the invention has the advantages of simple production process, high yield, environmental friendliness and capability of realizing low-cost large-scale production.
Description
Technical Field
The invention belongs to the technical field of nano-drug carriers, and particularly relates to a lactoferrin-modified pegylated graphene oxide-loaded puerarin nano-platform as well as a preparation method and application thereof.
Background
Clinically common neurodegenerative diseases are Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease (HD), Alzheimer's Disease (AD), and the like. The nervous system degenerative diseases are diseases with slow onset, progressive disease course and poor prognosis, and an effective radical treatment method is not available so far.
Puerarin (Pue) is used as effective component extracted from dried root of Pueraria lobata Ohwi of Leguminosae, and has multiple pharmacological activities, antiinflammatory, antioxidant and anti-apoptosis, and obvious neuroprotective effect. However, puerarin belongs to BCS IV compounds in a biological pharmaceutical classification system, has the characteristics of poor solubility and poor permeability, further causes low bioavailability of the puerarin and limits the puerarin to play the maximum pharmacological activity role. Therefore, the invention is necessary to overcome the solubility of puerarin and improve the bioavailability thereof.
On the other hand, the failure of a drug to effectively pass through the Blood-brain barrier (BBB) is a major constraint factor in the development of new drugs for central nervous system diseases, and how to make the drug pass through the BBB or increase the transmittance becomes a problem for people to treat the central nervous system diseases.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention aims to provide a preparation method of a lactoferrin modified pegylated graphene oxide loaded puerarin nano platform.
The invention also aims to provide the lactoferrin modified pegylated graphene oxide loaded puerarin nano platform prepared by the preparation method.
The invention also aims to provide application of the lactoferrin modified pegylated graphene oxide loaded puerarin nano platform.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a lactoferrin-modified pegylated graphene oxide-loaded puerarin nano platform comprises the following steps:
s1, completely dispersing graphene oxide in an aqueous solution through ultrasonic crushing, adding sodium hydroxide and chloroacetic acid into the aqueous solution, and reacting; neutralizing and purifying the reaction solution to obtain surface carboxylated graphene oxide; then adding EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) for reaction; finally adding carboxyl polyethylene glycol amino (HOOC-PEG-NH)2) Reacting; washing and dialyzing the reaction solution to obtain polyethylene glycol graphene oxide;
s2, dissolving puerarin in an organic solvent to obtain a puerarin solution, adding the puerarin solution into the water solution of the polyethylene glycol graphene oxide obtained in the step S1, stirring and reacting; washing and dialyzing the reaction solution to obtain a puerarin nano platform loaded by the pegylated graphene oxide;
s3, amino-polyethylene glycol-lactoferrin (NH)2-PEG-Lactoferrin) is added to the aqueous solution of the pegylated graphene oxide loaded puerarin nano platform obtained in step S2, stirred and reacted; washing and dialyzing the reaction solution to obtain the lactoferrin modified pegylated graphene oxide loaded puerarin nano platform.
In step S1, the ultrasonication conditions preferably include power of 600W and time of 2-4 h.
In step S1, the mass ratio of the sodium hydroxide to the chloroacetic acid is preferably 1-2: 1-2.
In step S1, the reaction conditions after adding sodium hydroxide and chloroacetic acid are preferably stirring reaction at 40-43 ℃ for 12-24 h.
In the step S1, the mass ratio of EDC to NHS is preferably 2-3: 1; more preferably 5: 3.
In step S1, the reaction conditions after adding EDC and NHS are preferably stirred for 2-4 hours.
In step S1, the weight average molecular weight (Mw) of the carboxyl polyethylene glycol amino is preferably 1000 to 5000; more preferably 2000.
In step S1, the amount of the carboxyl polyethylene glycol amino group is preferably calculated according to a mass ratio of the carboxyl polyethylene glycol amino group to graphene oxide of 4-6: 1; more preferably 5: 1.
In the step S1, the reaction conditions after the carboxyl polyethylene glycol amino group is added are preferably stirring reaction at 40-43 ℃ for 12-24 h.
In step S1, the dialysis conditions are preferably dialysis bags with a molecular weight of 3500KD for 24 hours.
In step S2, the organic solvent is preferably dimethyl sulfoxide (DMSO).
In the step S2, the concentration of the puerarin solution is preferably 8-12 mg/mL; more preferably 10 mg/mL.
In the step S2, the concentration of the aqueous solution of the pegylated graphene oxide is preferably 0.05 to 0.2 mg/mL; more preferably 0.1 mg/mL.
In step S2, the volume ratio of the aqueous solution of the pegylated graphene oxide to the puerarin solution is preferably 8-10: 1; more preferably 9: 1.
In step S2, the stirring time is preferably 12 to 24 hours.
In step S2, the dialysis conditions are preferably dialysis bags with a molecular weight of 3500KD for 24 hours.
In step S3, the weight average molecular weight of the amino-polyethylene glycol-lactoferrin is preferably 1000 to 5000; more preferably 2000.
In step S3, the amount of the amino-polyethylene glycol-lactoferrin is preferably as follows: the mass-to-volume ratio of the aqueous solution of the pegylated graphene oxide loaded puerarin nano platform is 1-2: 1-2; more preferably 1: 1.
In the step S3, the aqueous solution of the pegylated graphene oxide loaded puerarin nano platform is preferably prepared according to the puerarin concentration in the system of 8-12 mg/mL; more preferably 10 mg/mL.
In the step S3, the reaction condition is preferably continuous stirring reaction at room temperature for 12-24 h.
In step S3, the dialysis conditions are preferably dialysis bags with a molecular weight of 3500KD for 24 hours.
A lactoferrin-modified pegylated graphene oxide-loaded puerarin nano platform is prepared by the preparation method.
The lactoferrin modified pegylated graphene oxide loaded puerarin nano platform is applied to preparation of neuroprotective drugs.
The neuroprotective drug is preferably an anti-Parkinson drug.
Graphene Oxide (GO), as a relatively mature two-dimensional material, is in a lamellar structure and may consist of a single-layer or multi-layer film-like structure. GO has distinct advantages over other inorganic materials including mass producibility and low cost, having a large surface area and thus a strong drug loading capacity, and having excellent mechanical, electronic, optical, thermal and chemical properties. In addition, the GO surface has various active functional groups (such as hydroxyl, carboxyl and epoxy groups), and surface functional modification is easy to perform, so that the biocompatibility of GO and the targeting property of organ tissues are further improved, and the GO is further applied to multi-directional biomedical research.
The GO has active functional groups on the surface, so that different functional modifications can be carried out according to self research requirements, and the GO has certain specific properties. The polyethylene glycol (PEG) oxidized graphene can be used for improving the dispersity, stability and biocompatibility of GO.
Lactoferrin (Lf) is a mammalian cationic iron-binding serum glycoprotein belonging to the transferrin family. The iron-carrying biological carrier has multiple biological functions of iron transfer, anti-inflammation, antibiosis, anti-tumor, immunoregulation and the like. The main physiological function of the drug delivery system is that Lf can be specifically combined with a lactoferrin receptor on brain microvascular endothelial cells in a blood brain barrier, so that the drug delivery system overcomes the blood brain barrier and delivers the drug into the brain through a receptor-mediated transcytosis mode. Relevant researches show that the Lf modified nano-drug delivery system using the brain targeting molecule has obvious brain targeting property in vivo and has good in vivo and in vitro biocompatibility.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, firstly, the graphene oxide is pegylated, so that the dispersibility and biocompatibility of the graphene oxide are greatly improved. And the puerarin is loaded by the polyethylene glycol graphene oxide, so that the problem of poor solubility of the puerarin is greatly improved, the drug loading rate of the puerarin is also improved, and a basis and a direction are provided for the application of the traditional Chinese medicine in the field of multidisciplinary biomedicine. Finally, the puerarin is loaded on the pegylated graphene oxide modified by lactoferrin, so that the biocompatibility of the whole carrier is further improved, and the brain targeting property is obvious. Meanwhile, the main obstacle of treating brain diseases is overcome, and a new idea is provided for designing a brain-targeted drug delivery carrier.
The lactoferrin modified pegylated graphene oxide loaded puerarin nanometer platform provided by the invention can improve the dispersibility and biocompatibility of graphene oxide, improve the solubility of puerarin, improve the bioavailability of puerarin and overcome the blood brain barrier to achieve obvious brain targeting.
The preparation method provided by the invention has the advantages of simple production process, high yield, no toxicity and environmental friendliness, and can realize low-cost large-scale production, and the final product solvent is water.
The lactoferrin-modified pegylated graphene oxide-loaded puerarin nano platform provided by the invention is favorable for being used as a drug delivery strategy in the field of multidirectional biomedicines, such as brain tumors, Parkinson's disease, Alzheimer's disease and other brain diseases.
Drawings
FIG. 1 is a transmission electron micrograph of fresh samples from examples 1 to 4, respectively; wherein, a is example 1, B is example 2, C is example 3, and D is example 4.
FIG. 2 is a scanning electron micrograph of fresh samples prepared according to examples 1 to 4, respectively; wherein, a is example 1, B is example 2, C is example 3, and D is example 4.
FIG. 3 is a Zeta potential diagram of fresh samples from examples 1-4, respectively.
FIG. 4 is a Fourier infrared spectrum of each fresh sample prepared in examples 1-4.
FIG. 5 is a graph of the results of an in vitro release experiment for Lf-GO-Pue of the present invention.
FIG. 6 is a graph showing the results of the cytotoxicity experiments with Lf-GO-Pue of the present invention.
FIG. 7 is a graph showing the results of in vitro neuroprotective experiments with Lf-GO-Pue of the present invention.
FIG. 8 is a graph of the results of in vitro blood brain barrier transport measurements of Lf-GO-Pue of the present invention.
FIG. 9 is a graph showing the results of hemolysis experiments with Lf-GO-Pue of the present invention.
FIG. 10 is a graph of experimental results demonstrating the behavioral effects of Lf-GO-Pue of the present invention on animals; wherein, A is a graph of the experiment result of rotating the rod on the seventh day after the MPTP mold is made, B is a graph of the experiment result of climbing the rod on the seventh day after the MPTP mold is made, and C is a graph of the experiment result of open field on the eighth day after the MPTP mold is made.
FIG. 11 is a graph showing the results of experiments to study the effect of Lf-GO-Pue of the present invention on the number of TH positive neurons in the substantia nigra pars compacta of the brain.
Detailed Description
The idea of the present invention and the results thereof will be described in detail below with reference to the embodiments and the accompanying drawings. The described embodiments are only used for illustrating the present invention, and other embodiments obtained by persons skilled in the art without creative efforts shall fall within the protection scope of the present invention based on the embodiments of the present invention.
Materials referred to in the following examples:
graphite powder: purchased from Qingdao Baichuan graphite, Inc.
Carboxylic acid polyEthylene glycol amino (HOOC-PEG-NH)2Mw 2000) from alatin biotechnology.
Puerarin: purchased from Nantong biological medicine company, with a purity of more than or equal to 99 percent.
Amino-polyethylene glycol-lactoferrin (NH)2PEG-Lactoferrin, Mw 2000), available from sienna ruixi biotechnology company under the product designation R-1018-2K.
Human neuroblastoma cell strain SH-SY5Y, mouse brain microvascular endothelial cell strain bEnd.3: purchased from wuhan punuosai.
Example 1
Step 1: preparation of Graphene Oxide (GO)
4g of graphite powder was placed in 100mL of H in an ice bath2SO4(98%) while stirring was continued. Then, 25g KMnO was slowly added during stirring4And the mixture was kept at a temperature of 35 ℃ for 1 h. Subsequently, 200mL of deionized water was carefully added and the temperature was maintained at 98 ℃. After 30 minutes, 400mL deionized water and 8mL H were added2O2(30%). The reaction mixture was filtered and washed with aqueous HCl (V (HCl): V (water) ═ 1:10) to remove metal ions. Finally, the mixture was filtered and rinsed with deionized water until the pH reached 7, then dried under vacuum at 60 ℃.
Step 2: preparation of pegylated graphene oxide (PEG-GO)
The aqueous graphene oxide suspension obtained in step 1 (10mL, 4mg/mL) was sonicated at 600W for about 4 hours to obtain a homogeneous solution. Then, 1g of sodium hydroxide and 0.95g of chloroacetic acid were added to the graphene oxide suspension, and stirred at 45 ± 3 ℃ for about 24 hours. Finally, the resulting graphene oxide carboxylated suspension is neutralized and purified by repeated washing and filtration. The graphene oxide carboxylated suspension was then transferred to a 50mL round bottom flask, 40mg EDC and 24mg NHS were added to the flask, and the mixture was stirred for about 3 hours. Subsequently, 200mg HOOC-PEG-NH was added to the flask2And stirred at 40 + -3 deg.C for 24 hours. Washing the final product of the polyethylene glycol graphene oxide with double distilled water for multiple times, and dialyzing with a dialysis bag with a molecular weight of 3500KDFor 24 hours, stored in a refrigerator at 4 ℃ for further use.
And step 3: preparation of pegylated graphene oxide loaded puerarin (GO-Pue)
The puerarin solution (1mL, 10mg/mL, dissolved in dimethyl sulfoxide) was added rapidly to the aqueous solution of pegylated graphene oxide (9mL, 0.1mg/mL) obtained in step 2 at 800rpm and stirred for 24 hours. The sample was repeatedly rinsed with ultrapure water several times and dialyzed for 24 hours with a dialysis bag having a molecular weight of 3500 KD. The obtained pegylated graphene oxide loaded puerarin is stored at 4 ℃, or lyophilized for later use.
And 4, step 4: preparation of lactoferrin modified pegylated graphene oxide loaded puerarin (Lf-GO-Pue)
Adding 10mg of NH2Adding PEG-Lactoferrin into the solution (10mg, the concentration of puerarin is 1mg/mL) of the pegylated graphene oxide loaded puerarin obtained in the step 3, and continuously stirring for 24 hours at room temperature. The sample was rinsed with ultrapure water and dialyzed for 24 hours using a dialysis bag with a molecular weight of 3500 KD. Finally, freeze-drying for later use.
And 5: characterization of
And (4) testing the particle size distribution of the respective fresh samples in the steps 1-4. The results are that the particle size of the sample in the step 1 is 233.1nm, the particle size of the sample in the step 2 is 237.3nm, the particle size of the sample in the step 3 is 185.3nm, and the particle size of the sample in the step 4 is 236.1 nm.
And (4) testing the PDI values of the respective fresh samples in the steps 1-4. The results were 0.265 PDI for the step 1 sample, 0.34 for the step 2 sample, 0.258 for the step 3 sample, and 0.235 for the step 4 sample, respectively.
And (4) carrying out transmission electron microscope test on the forms of the respective fresh samples in the steps 1-4, wherein the results are shown in figure 1. Transmission electron microscope images show that the shapes of the samples in the steps 1-4 are all composed of one or more layers of nano sheets, and the shapes of the samples in the steps 2-4 are slightly changed from those in the embodiment 1.
And (4) performing scanning electron microscope testing on the appearances of the respective fresh samples in the steps 1-4, wherein the results are shown in FIG. 2. Scanning electron microscopy images show that the step 1 sample is composed of aggregated, shrunken porous carbon, closely associated with each other. Step 2 samples showed stacking and aggregation of the microparticles formed after pegylation. Step 3 the sample showed a flat plate structure with corrugations. Finally, the step 4 sample clearly showed relatively high coarseness and softness due to the higher dispersibility of the lactoferrin modified.
And (3) testing the Zeta potential values of the fresh samples in the steps 1-4, wherein the results are shown in figure 3. The results are that the Zeta potential value of the sample in the step 1 is-32.13 mV, the Zeta potential value of the sample in the step 2 is-41.43 mV, the Zeta potential value of the sample in the step 3 is-12.65 mV, and the Zeta potential value of the sample in the step 4 is-24.71 mV.
Fourier infrared spectrum tests are carried out on the surface functional groups of the respective fresh samples in the steps 1-4, and the results are shown in figure 4. All of the most characteristic functional groups in the Fourier Infrared Spectrum of the samples from Steps 1-4 (e.g., 1718 cm)-1Carbonyl group of (2) < CHEM > 1572 cm-1Or 2873cm-1The hydroxyl group at (c).
The initial concentration of synthesized GO is 4mg/mL, Pue is loaded after PEGylation, the concentration (0.1mg/mL) of the contained GO is greatly reduced, and the reduction of the GO concentration is found in the experimental exploration process, so that the dispersibility and the stability of the whole carrier system are obviously enhanced.
The method is characterized in that puerarin is selected finally, during the experiment exploration process, the selected drugs comprise icariin, paeoniflorin, osthole, resveratrol and quercetin, and finally the puerarin is selected due to the highest loading rate, stable solution system and transparency; the other drugs have low loading rate and are obviously separated out.
An organic solvent DMSO is finally selected to dissolve puerarin, the selected organic solvent comprises (absolute ethyl alcohol, acetone and methanol) in the experiment exploration process, and finally the stability of the whole system is ensured as the puerarin dissolved in the DMSO is added into the PEG-GO solution; other organic solvents dissolve puerarin and can be quickly separated out and turbid when added into the PEG-GO solution.
We finally selected a puerarin solution: the volume ratio of the PEG-GO solution is 1: during the experimental exploration, the volume ratios screened included (1: 4, 1: 5, 2: 3), finally due to the puerarin solution: the volume ratio of the PEG-GO solution is 1: when 9 hours, the puerarin drug loading rate is highest, and the whole GO-Pue system is bright and stable; other volume ratios have lower drug loading rates and poor solution dispersibility.
We finally selected amino-polyethylene glycol-lactoferrin (NH)2PEG-Lactoferrin), screening Lactoferrin during experimental exploration, finally due to NH2PEG2000-Lactoferri is added into the GO-Pue solution, and the whole system is transparent and stable after stirring; other lactoferrin added to GO-Pue solution was unstable and precipitated black agglomerates.
An Lf-GO-Pue nano platform, wherein lactoferrin is firstly connected with a carrier construction design, and then a drug is loaded (Pue); in the experimental exploration process, the organic solvent influences the lactoferrin, so that the effect of puerarin or lactoferrin is not influenced in the mode of carrying the medicine first and then connecting the medicine to the lactoferrin in the final use, and the in-vivo safety and the environmental friendliness of the whole system are greatly enhanced.
EXAMPLE 2 in vitro drug Release assay
The release behavior of Lf-GO-Pue in vitro was studied by dialysis. 1mL of Lf-GO-Pue was placed in a dialysis bag (MWCO 3.0kDa) and then the dialysis bag was placed in 500mL of PBS (pH 7.4) at 400 rpm. At various time points (0h, 0.5h, 1h, 2h, 4h, 8h, 12h), 2.0mL of the solution outside the dialysis bag was collected and the absorbance at 270nm was measured with a uv spectrophotometer to determine the concentration of released Pue. The results are shown in FIG. 5. Compared with Lf-GO-Pue and pure Pue, pure Pue belongs to a drug with poor water solubility, and the result shows that the solubility of Pue is obviously improved by utilizing Lf-GO-Pue, and the in vitro release amount of Pue is further obviously improved.
Example 3 in vitro neuroprotective Effect test
Human neuroblastoma (SH-SY5Y) cells were cultured in DMEM with 10% FBS, 1% penicillin (100U/mL) and streptomycin (100. mu.g/mL). The cells were left at 37 ℃ with 5% CO2And 95% relative humidity. Changing a fresh culture medium every 24 hours, and observing the cell growth to 70-80% under a microscope for subculture.
Safety evaluation of Lf-GO-Pue at SH-SY5Y cell level:
to demonstrate the safety of Lf-GO-Pue at the SH-SY5Y cell level, its cell viability was determined by MTT assay. Will be 5X 103The cells/well were seeded in 96-well culture plates by first incubating the cells in the well plates for 24 hours, removing the medium, then setting different groups (free Pue group and Lf-GO-Pue group, respectively) to incubate into the well plates, setting different concentrations in the range of 0 (control), 1, 5, 10, 20, 50, 100, 200 μmol/L. After an additional 24 hours of incubation, 10. mu.L of MTT (5mg/mL in PBS) was added to each well, and after 4 hours, 150. mu.L of DMSO was added to each well in place of the medium. The absorbance value at 570nm was measured using a microplate reader (A570 nm). Cell viability was expressed as a percentage of the administered group at a570nm relative to the control group. The results are shown in FIG. 6. In the concentration range of 1-200 mu moL/L, no matter the Lf-GO-Pue group is compared with Pue group or a control group, the cell activity is not obviously different.
The MTT method is used for detecting the neuroprotective effect of Lf-GO-Pue on SH-SY5Y cell level:
will be 5X 103The cells/well were seeded in 96-well culture plates by first incubating the cells in the well plates for 24 hours, removing the medium, then setting different groups (free Pue group and Lf-GO-Pue group, respectively) to incubate into the well plates at different concentrations of 1, 5, 10, 20, 50 μmol/L, respectively, and pre-dosing for 4 hours. Then 2mmol/L MPP is used+After a further 20 hours incubation (1 methyl 4 phenylpyridinium), 10. mu.L of MTT (5mg/mL in PBS) was added to each well and after 4 hours, 150. mu.L of DMSO was added to each well in place of the medium. The absorbance value at 570nm was measured using a microplate reader (A570 nm). Cell viability was expressed as a percentage of the administered group at a570nm relative to the control group. The results are shown in FIG. 7. In the concentration range of 1-50 mu mol/L, the Lf-GO-Pue group shows more excellent in-vitro neuroprotective effect (p) compared with the Pue group<0.05)。
From the above results, it can be seen that Lf-GO-Pue exhibits a more pronounced neuroprotective effect without significant toxicity than free Pue.
Example 4 in vitro blood brain barrier transport measurements
bEnd.3 cells (1X 10)5One/well) were cultured in the upper chamber of a 12-well plate in a Transwell system. After about 7 days, a monolayer tight junction similar in structure to the blood-brain barrier is formed. Cells were then treated with Pue and Lf-GO-Pue, respectively, to assess blood brain barrier transport in vitro. The formation of a monolayer of cell tight junctions was monitored by measuring the resistance value (TEER) when it reached about 200. omega. cm2The next experiment was performed. 500 μ L of DMEM containing different sets of solutions (same Pue concentration) was added to the upper chamber of the Transwell and incubated for 4 h. The Pue concentration level in the lower chamber was evaluated at 270nm according to a standard curve of UV-spectrophotometry. In vitro blood brain barrier transport measurements were calculated using the following equation:
co and Cs represent the concentrations before and after incubation Pue in the chamber of the Transwell system, respectively. The results are shown in fig. 8, and the results of simulating the in vitro blood brain barrier transport capacity at the level of mouse brain microvascular endothelial cells (bned.3) in vitro show that the drug permeability of Lf-GO-Pue in the Transwell system is obviously higher than that of pure Pue group (p < 0.05).
Example 5 hemolysis experiment
The 4% mouse erythrocyte suspension is centrifuged at 1500rpm and washed three times by PBS solution to obtain pure erythrocytes. Then, 0.1mL of 4% red blood cells (v/v) were mixed with 0.1mL of water (as a positive control group), physiological saline (as a negative control group) and Lf-GO-Pue solution, respectively, and incubated at 37 ℃ for 3 hours. And centrifuging, collecting supernatant, and measuring absorbance at 540 nm with a microplate reader. Percent hemolysis was calculated using the following equation:
wherein A istAbsorbance of different sets of solutions, AncAbsorbance of negative control group, ApcThe absorbance of the positive control group was obtained.A percent (%) hemolysis of greater than 5% is when hemolysis occurs. Three tests were performed for each sample. The results are shown in FIG. 9. The hemolysis results of the negative group (normal saline) and the positive group (water) of the Lf-GO-Pue show that the hemolysis scores of the Lf-GO-Pue and the negative group<5%, showing good blood compatibility.
Example 6 animal experiments
Mice (C57BL/6, guangdong province medical laboratory animal center, 8 weeks old, body weight 22 to 25g) were housed under specific pathogen-free experimental conditions for 12h light: the water was fed daily with standard feed under standard laboratory conditions of 12h dark growth cycle, 25 + -2 deg.C temperature and 55 + -5% relative humidity.
In vivo targeting verification experiment: mice are randomly divided into a puerarin (Pue) group and a lactoferrin modified graphene oxide loaded puerarin (Lf-GO-Pue) group. Separately, tail-injected with 100 μ L of the above solution, anesthetized at three time points of 4h, 6h and 10h, brain was collected, homogenized, centrifuged, and the supernatant was measured by absorbance at 270nm in an ultraviolet spectrophotometer. The results show that 4h after dosing, the brain Pue uptake was significantly higher in the Lf-GO-Pue group than in the other two groups, and this significant trend continued until 6h after dosing, and finally, the overall concentration decreased in each group 10h after dosing, but the Lf-GO-Pue group remained the highest. After 10h, the clearance of the drug in the body is accelerated considering that the drug circulates in the body for too long. This result also clearly shows the significant brain targeting produced by lactoferrin modification.
In vivo pharmacodynamic experiments: mice were randomly divided into 6 groups: a control group, a Model (MPTP) group, a positive (levodopa) group, and an Lf-GO-Pue group. MPTP (prepared with 0.9% physiological saline) was injected intraperitoneally with 18mg/kg, administered every 2 hours, 4 times a day. Groups of Lf-GO-Pue were administered tail intravenous for a total of 7 days (at a dose of 5 mg/kg). The positive (levodopa) group was intraperitoneally injected with 25mg/kg mice for seven days and further evaluated for behavioral testing in order to evaluate the neuroprotective effect of Lf-GO-Pue on PD mice.
Rod turning experiment: exercise balance ability was tested on day seven after MPTP administration. In a 2 minute test, mice were placed in a rotating rod (7 cm diameter) at a fixed speed of 20 r/min. The time each mouse dropped on the rod for the first time (latency) and the number of times the mouse dropped within 2 minutes were measured. Three replicates of each mouse were performed to obtain data. The results are shown in FIG. 10A.
Pole climbing experiment: the test for bradykinesia was performed on day seven after MPTP administration. The mouse head was placed down on top of a vertical rod (1 cm diameter, 50cm height) with a rough surface. Each mouse was raised near the top of the rod and the turn time was recorded and the total time required to reach the bottom of the rod was recorded. The results are shown in FIG. 10B.
Open field experiment: open field experiments were performed on day eight after MPTP dosing. Open field tests were performed in an empty box (60 cm. times.60 cm. times.40 cm) and divided into 16 squares (15 cm. times.15 cm). Prior to testing, each mouse was placed in the center of an empty box and free-sought within 10 minutes. The total distance moved and the average speed of movement were then recorded in a 20 minute test. The results are shown in FIG. 10C.
The results of a rod rotation experiment and a rod climbing experiment show that the behavioral defects of the Parkinson mice are obviously improved by the Lf-GO-Pue and the levodopa, and the treatment effect of the Lf-GO-Pue is slightly lower than that of the levodopa. However, the results of open field experiments in the eighth day after MPTP molding show that the total moving distance and the average moving speed of the mice are obviously increased after the Lf-GO-Pue and levodopa are treated, and the treatment effect of the Lf-GO-Pue is equivalent to that of the levodopa. The increase and decrease of the number of TH positive neurons in the substantia nigra pars compacta of the brain caused by Lf-GO-Pue after MPTP modeling was also examined on the level of the in vivo C57BL/6 mice, and the result is shown in FIG. 11. The results show that the number of TH positive neurons is obviously enhanced after the Lf-GO-Pue and levodopa are treated, and the treatment effect of the Lf-GO-Pue and levodopa is almost the same.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. A preparation method of a lactoferrin-modified pegylated graphene oxide-loaded puerarin nano platform is characterized by comprising the following steps of: the method comprises the following steps:
s1, completely dispersing graphene oxide in an aqueous solution through ultrasonic crushing, adding sodium hydroxide and chloroacetic acid into the aqueous solution, and reacting; neutralizing and purifying the reaction solution to obtain surface carboxylated graphene oxide; adding EDC and NHS for reaction; finally adding carboxyl polyethylene glycol amino for reaction; washing and dialyzing the reaction solution to obtain polyethylene glycol graphene oxide;
s2, dissolving puerarin in an organic solvent to obtain a puerarin solution, adding the puerarin solution into the water solution of the polyethylene glycol graphene oxide obtained in the step S1, stirring and reacting; washing and dialyzing the reaction solution to obtain a puerarin nano platform loaded by the pegylated graphene oxide;
s3, adding amino-polyethylene glycol-lactoferrin into the aqueous solution of the pegylated graphene oxide loaded puerarin nano platform obtained in the step S2, stirring and reacting; washing and dialyzing the reaction solution to obtain the lactoferrin modified pegylated graphene oxide loaded puerarin nano platform.
2. The preparation method of the lactoferrin modified pegylated graphene oxide loaded with puerarin nanoplatform of claim 1, wherein:
in the step S1, the mass ratio of the sodium hydroxide to the chloroacetic acid is 1-2: 1-2;
in the step S1, the reaction conditions after adding the sodium hydroxide and the chloroacetic acid are that the mixture is stirred and reacts for 12-24 h at the temperature of 40-43 ℃;
in the step S1, the mass ratio of EDC to NHS is 2-3: 1;
in the step S1, adding EDC and NHS, and reacting for 2-4 h under stirring;
in the step S1, the molecular weight of the carboxyl polyethylene glycol amino is 1000-5000;
in step S1, the amount of the carboxyl polyethylene glycol amino group is calculated according to a mass ratio of the carboxyl polyethylene glycol amino group to graphene oxide of 4-6: 1;
in the step S1, the reaction conditions after the carboxyl polyethylene glycol amino group is added are that the mixture is stirred and reacts for 12-24 hours at the temperature of 40-43 ℃.
3. The preparation method of the lactoferrin modified pegylated graphene oxide loaded with puerarin nanoplatform of claim 2, wherein:
the mass ratio of EDC to NHS is 5: 3;
the molecular weight of the polyethylene glycol is 2000;
the dosage of the polyethylene glycol is calculated according to the mass ratio of 5:1 of the polyethylene glycol to the graphene oxide.
4. The preparation method of the lactoferrin modified pegylated graphene oxide loaded with puerarin nanoplatform of claim 1, wherein:
in step S2, the organic solvent is dimethyl sulfoxide;
in the step S2, the concentration of the puerarin solution is 8-12 mg/mL;
in the step S2, the concentration of the water solution of the pegylated graphene oxide is 0.05-0.2 mg/mL;
in the step S2, the volume ratio of the water solution of the pegylated graphene oxide to the puerarin solution is 8-10: 1.
5. The preparation method of the lactoferrin modified pegylated graphene oxide loaded with puerarin nanoplatform of claim 1, wherein:
the concentration of the puerarin solution is 10 mg/mL;
the concentration of the water solution of the polyethylene glycol graphene oxide is 0.1 mg/mL;
the volume ratio of the water solution of the polyethylene glycol graphene oxide to the puerarin solution is 9: 1.
6. The preparation method of the lactoferrin modified pegylated graphene oxide loaded with puerarin nanoplatform of claim 1, wherein:
in the step S3, the weight average molecular weight of the amino-polyethylene glycol-lactoferrin is 1000-5000;
in step S3, the dosage of the amino-polyethylene glycol-lactoferrin is as follows: the mass-to-volume ratio of the aqueous solution of the pegylated graphene oxide loaded puerarin nano platform is 1-2: 1-2;
in the step S3, preparing the aqueous solution of the pegylated graphene oxide loaded puerarin nano platform according to the concentration of puerarin in the system of 8-12 mg/mL;
in the step S3, the reaction condition is that the reaction is continuously stirred at room temperature for 12-24 hours.
7. The preparation method of the lactoferrin modified pegylated graphene oxide loaded with puerarin nanoplatform of claim 1, wherein:
in the step S1, the ultrasonic crushing conditions are that the power is 600W and the time is 2-4 h;
in steps S1, S2 and S3, the dialysis conditions are all dialysis for 24 hours by using a dialysis bag with a molecular weight of 3500 KD.
8. A lactoferrin-modified pegylated graphene oxide-loaded puerarin nano platform obtained by the preparation method of any one of claims 1 to 7.
9. The use of the lactoferrin modified pegylated graphene oxide-loaded puerarin nanoplatform of claim 8 in the preparation of a neuroprotective drug.
10. Use according to claim 9, characterized in that:
the nerve protection drug is an anti-Parkinson drug.
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