CN113521304B - Dual-curative-effect anti-tumor drug based on nanocellulose load and preparation method thereof - Google Patents
Dual-curative-effect anti-tumor drug based on nanocellulose load and preparation method thereof Download PDFInfo
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- CN113521304B CN113521304B CN202110778777.0A CN202110778777A CN113521304B CN 113521304 B CN113521304 B CN 113521304B CN 202110778777 A CN202110778777 A CN 202110778777A CN 113521304 B CN113521304 B CN 113521304B
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Abstract
The invention belongs to the field of antitumor drugs, and discloses preparation and application of a dual-curative-effect antitumor drug based on nanocellulose-loaded nanogold and curcumin. The drug carrier of the invention adopts nanocellulose, and after proper treatment, gold nanoparticles and cyclodextrin are loaded on the carrier, and the cyclodextrin is used for containing the antitumor drug. The problem that the existing natural antitumor drugs are indissolvable is solved. The AuNP loaded on the nanoparticles achieves a hyperthermia effect under near infrared irradiation, and the thermal effect promotes curcumin heat release, thereby enhancing the efficacy of chemotherapy. The heat resistance and the stability of the antitumor drug curcumin are obviously improved, which is beneficial to pretreatment and also beneficial to improving the bioavailability of the curcumin. The water solubility of curcumin is obviously improved, and compared with the natural solubility of curcumin, the curcumin is improved by nearly 150 times and reaches 131.7 mug/mL. It is expected that the dual therapeutic antitumor drug will become a powerful candidate for tumor treatment.
Description
Technical Field
The invention belongs to the field of antitumor drugs, and in particular relates to a dual-curative-effect antitumor drug based on a nanocellulose carrier and a preparation method thereof.
Background
Tumors are one of the major diseases that jeopardize human health. With higher demands on therapeutic effects, therapeutic procedures, side effects, etc., drugs with multiple therapeutic effects and natural antitumor drugs are increasingly favored by patients and researchers. However, many natural antitumor drugs are poorly soluble in water, greatly limiting the application of such drugs. Taking curcumin as an example, curcumin has good inhibition effect on formation, proliferation and metastasis of tumor cells. Curcumin is inexpensive, widely available and has a variety of medical properties and is therefore of increasing interest to researchers. However, it is difficult to have various therapeutic effects at the same time because of poor solubility, low loading capacity and weak releasing capacity of curcumin, resulting in poor chemotherapeutic effect. The drugs developed based on curcumin are still not efficient enough. Improving the efficacy of curcumin drugs is very important for curing tumors.
In order to solve the problems of insoluble drugs in application, some nano drug delivery systems have been developed at home and abroad, mainly comprising liposome, nanoparticle, micelle, gel, suspension, nanoemulsion, phospholipid complex, cyclodextrin embedding, polymer system and the like. These delivery systems improve drug availability primarily through two aspects: improving the water solubility of the medicine or improving the blood concentration of the medicine during in vivo medicine delivery. Current research focuses on improving drug availability, while exhibiting good efficacy for certain tumors, is limited to chemotherapy for tumors and is lacking in multi-means combined therapies. Development of antitumor drugs with multiple therapeutic effects is still worth further study.
In recent years, development of novel light-related nanomedicines is receiving attention. Some preparations having dual therapeutic effects of hyperthermia and chemotherapy have been developed using the photothermal effect of nanoparticles. However, these formulations are often structurally complex and disadvantageous for production and transportation. Such as microcapsules, liposomes and multi-layered core-shell structures, leaked drug may mask the gold nanoparticles, and may not smoothly warm up to release the encapsulated drug under light irradiation, thereby losing dual therapeutic effects.
Thus, there is still a need to improve the water solubility and release capacity of antitumor drugs to enhance the effect of chemotherapy, and there is still a challenge in making antitumor drugs have dual therapeutic effects of chemotherapy and physiotherapy.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the primary aim of the invention is to provide a preparation method of a dual-curative-effect anti-tumor medicament based on a nanocellulose carrier.
The invention also aims to provide the dual-curative-effect anti-tumor medicament based on the nanocellulose carrier.
The invention also aims to provide the application of the dual-curative-effect antitumor drug based on the nanocellulose carrier to the insoluble drug curcumin.
The aim of the invention is achieved by the following scheme:
the preparation method of the dual-curative-effect anti-tumor medicament based on the nanocellulose carrier comprises the following steps:
(1) Preparation of CNC (cellulose nanocrystalline) carrier: crushing the cotton fiber pulp plate by a crushing grinder to form fluffy cotton fibers, then adding the fluffy cotton fibers into ammonium persulfate solution for heating reaction, purifying the obtained reaction solution after the reaction is finished, and obtaining supernatant which is CNC (computer numerical control), and preserving for later use;
(2) Preparation of AuNP (nano gold particles): mixing chloroauric acid solution with glutathione solution under stirring, heating for reaction, and purifying the obtained reaction solution after the reaction is finished, wherein the obtained precipitate is AuNP;
(3) Load of AuNP: loading the CNC prepared in the step (1) into an open container, adjusting the pH to 6-8, adding NHS and EDC into the open container under the stirring condition, adding L-cysteine after uniformly stirring, continuously stirring for more than 12 hours, and then dialyzing and purifying to obtain CNC-SH; dispersing the AuNP prepared in the step (2) in water, mixing with CNC-SH, and stirring for more than 20min to complete grafting of the nano gold particles, thereby obtaining the AuNP-CNC;
(4) Grafting of βcd (β -cyclodextrin): dissolving beta-cyclodextrin in AuNP-CNC, adding epoxy chloropropane under stirring to react, purifying the reaction solution after the reaction is ended to obtain AuNP-CNC-beta CD grafted with beta-cyclodextrin, and preserving for later use;
(5) Loading of antitumor drug: dissolving the medicine with volatile solvent, dropping medicine solution into AuNP-CNC-beta CD dropwise until insoluble medicine particles appear, removing volatile solvent, centrifuging the obtained mixture to remove unencapsulated medicine, collecting upper clear transparent liquid, and obtaining the dual therapeutic antitumor medicine based on nanocellulose carrier.
The dosages of the cotton fiber pulp board and the ammonium persulfate solution in the step (1) are as follows: per 1g of cotton fiber pulp board, not less than 0.1mol of ammonium persulfate is correspondingly used.
The heating reaction in the step (1) is carried out at 45-80 ℃ for 4-20 h.
The purification in step (1) specifically comprises the following steps: after the reaction is finished, centrifuging the obtained reaction solution, washing the precipitate by water until the supernatant is light blue, retaining all the precipitate and the light blue supernatant obtained by the last centrifugation, dispersing the precipitate by ultrasonic dispersion after dilution, then filling the precipitate into a dialysis bag, dialyzing by water until the dialysate does not show acidity, taking out the jelly in the dialysis bag, centrifuging after ultrasonic dispersion, discarding the precipitate, retaining the supernatant, namely CNC (computer numerical control), and storing the supernatant in a refrigerator at 4 ℃ for later use;
preferably, in the purification process of the step (1), the centrifugal speed is about 5000-10000 revolutions, and the single centrifugal time is 10-15 minutes; taking out the jelly in the dialysis bag, performing ultrasonic dispersion and centrifugation to obtain solution, re-dispersing the jelly formed by agglomeration, and performing centrifugation to obtain sediment which is completely unreacted large fibers and impurities.
The heating reaction in the step (2) means a heating reaction at 90 to 100℃for 30 to 40 minutes, preferably at 95℃for 35 minutes.
The dosage of the chloroauric acid solution and the glutathione solution in the step (2) is as follows: the concentration of the chloroauric acid solution is 0.5-2 mol/L, the concentration of the glutathione solution is 3-6 mmol/L, and the volume ratio of the chloroauric acid solution to the glutathione solution is 100-200 mu L: 10-150 mL; preferably, the amounts of chloroauric acid solution and glutathione solution in step (2) are as follows: the concentration of the chloroauric acid solution is 1mol/L, the concentration of the glutathione solution is 4.8mmol/L, and the volume ratio of the chloroauric acid solution to the glutathione solution is 150 mu L:50mL.
The separation and purification in the step (2) means that the obtained reaction liquid is cooled to room temperature, the centrifugation (centrifugation under the centrifugal force of 11000-15000 g for 4-20 min) is carried out to remove large aggregates, the pH value of the solution is adjusted to 3-4, ethanol is added into the solution, the centrifugation (centrifugation under the centrifugal force of 3500-5000 g for 4-10 min) is carried out to further purify the supernatant, and the obtained precipitate is AuNP; the amount of ethanol added is more than 0.5 times of the volume of water in the solution.
The CNC, NHS, EDC and L-cysteine amounts in step (3) satisfy the molar ratio: the-COOH in EDC/CNC is more than or equal to 1.5; EDC/nhs=3 to 5, preferably 4; the-COOH in the L-cysteine/CNC is more than or equal to 1.2; the carboxyl content of CNC needs to be measured in advance, for example, as can be measured by acid-base titration or conductivity titration.
The amounts of AuNP and CNC-SH described in step (3) satisfy: the molar amount of AuNP is not lower than the carboxyl content of the CNC prior to the sulfhydryl group (i.e., CNC as starting material in step (3)).
The dosage of the epichlorohydrin in the step (4) is 1% -2% of the total volume of the reaction system in the step (4); the dosage relation of the beta-cyclodextrin and the AuNP-CNC meets the following conditions: n (beta-cyclodextrin)/n (carboxyl in the raw material CNC of AuNP-CNC) is more than or equal to 3;
the reaction in the step (4) is carried out for more than half an hour at the temperature of between room temperature and 60 ℃;
the purification in the step (4) means that the reaction liquid is cooled to room temperature, the excessive cyclodextrin and epichlorohydrin are removed by centrifugation (5000-10000 turns, 10-15 min), the precipitate is taken for dialysis and purification, the obtained AuNP-CNC-beta CD is dispersed by ultrasonic, and the obtained AuNP-CNC-beta CD is stored in a refrigerator at the temperature of 4 ℃ for standby;
the molecular weight cut-off of the dialysis bag used in the dialysis in the steps (1) to (4) is 12-14 kDa;
the volatile solvent in step (5) should be selected according to the dissolving capacity of the drug and the compatibility of the solvent with water, for example, acetone, methanol, etc.;
the removal of the volatile solvent described in step (5) may be accomplished by vigorous stirring to allow the solvent to evaporate rapidly, followed by spin evaporation to remove the volatile solvent.
The step (5) of centrifuging to remove the unencapsulated drug means centrifuging for a plurality of times at 3000-10000 rpm, and selecting the rotation speed according to the operation conditions;
the medicine in the step (5) is at least one of curcumin, quercetin, catechin, gallic acid, paclitaxel and camptothecine, preferably curcumin.
The unspecified temperatures in the steps (1) to (5) are all carried out at room temperature, which in the present invention means 20.+ -. 5 ℃.
The stirring in the steps (1) to (5) is to make the raw materials fully contacted, and the stirring speed conventional in the art can achieve the technical effect of the invention, so that the stirring speed is not limited;
the dual-curative-effect anti-tumor medicine based on the nanocellulose carrier, which is prepared by the method, especially the dual-curative-effect anti-tumor medicine based on nanocellulose-loaded nanogold and curcumin, which is prepared by the method.
The dual-curative-effect antitumor drug based on the nanocellulose carrier is applied to the insoluble drug curcumin.
The dual-therapeutic curcumin antitumor drug based on the nanocellulose carrier is prepared by the preparation method of the dual-therapeutic antitumor drug based on the nanocellulose carrier, and specifically comprises the following steps of:
(1) According to the preparation method steps (1) to (4) of the dual-curative-effect anti-tumor medicament based on the nanocellulose carrier;
(2) Load of CUR (curcumin): and (3) dropwise adding an acetone solution (with the concentration of preferably 1.4 mg/mL) of curcumin into the AuNP-CNC-beta CD until insoluble CUR particles appear, stirring to volatilize the acetone, further removing the acetone by rotary evaporation (with the temperature of preferably 45 ℃), centrifuging the obtained mixture at 6000rpm for 30 minutes, removing unencapsulated CUR, collecting the upper clear and transparent yellow liquid, and obtaining the dual-curative curcumin antitumor drug based on the nanocellulose carrier.
The mechanism of the invention is as follows:
the current indissolvable antitumor drug delivery system is difficult to have both chemical curative effect and thermal therapeutic effect. According to the invention, nanocellulose (CNC) is used as a carrier, sulfhydryl groups are grafted to the surface of CNC through Schiff base reaction, then CNC nanoparticles are loaded with gold nanoparticles (AuNP) and beta-cyclodextrin (beta CD), and insoluble drugs can be embedded into hydrophobic cavities of beta CD, so that the water solubility of the insoluble drugs is increased. In the preparation process, along with gradual reduction of the volatile solvent, the indissolvable drug is gradually separated out from the solvent, the cavity of the cyclodextrin in the liquid phase is hydrophobic, and according to the similar principle of miscibility, the hydrophobic drug is transferred into the cyclodextrin from the liquid phase, so that the embedding of the indissolvable drug by the cyclodextrin is realized. When insoluble drug particles begin to appear in the system, the capacity of AuNP-CNC- βcd is shown to reach an upper limit. In application, the photothermal effect of AuNP is utilized to achieve the hyperthermia effect, and the photothermal effect can enhance the thermal movement of the embedded drug molecules, so that the release capacity of the drug is improved and the chemotherapy effect is improved.
Compared with the prior art, the invention has the following advantages:
(1) The double-effect antitumor drug based on the nanocellulose carrier has a simple structure, has chemotherapeutic and hyperthermia effects, and the heat effect can promote the release of the drug to improve the chemotherapeutic effect. The carrier has simple structure, is much simpler than the multi-layer structure of other double-effect medicaments, and is more convenient for production, transportation and use.
(2) The double-effect antitumor drug based on the nanocellulose carrier, which is prepared by the invention, has better heat resistance, and the common sterilization process at 121 ℃ can not influence the heat stability of the product nano drug. The curcumin-loaded medicine starts thermal decomposition at 230 ℃, so that the heat resistance of the curcumin is obviously improved.
(3) The double-effect antitumor drug based on the nanocellulose carrier has higher drug loading capacity, and the water solubility of the drug is obviously improved. The loading amount of CUR measured in the curcumin drug product is 31.4 mug/mg, the CUR dissolution capacity reaches 131.7 mug/mL, and the solubility is improved by nearly 150 times, which is higher than that of other carrier curcuminoid delivery systems.
Drawings
FIG. 1 is a process diagram of preparing a dual-effect antitumor drug based on a nanocellulose carrier from raw materials and exerting curative effect.
Fig. 2 is a specific reaction sequence diagram of the dual-effect curcumin anti-tumor drug based on the nanocellulose carrier in the invention.
FIG. 3 is a graph of the microtopography of several nanoparticles produced in example 1 of the present invention.
FIG. 4 is a graph showing the IR analysis of the product at various stages in the preparation of example 1 according to the present invention.
FIG. 5 shows DSC curves of the products at different stages during the preparation of example 1 of the present invention.
FIG. 6 is a thermogravimetric analysis curve and a first order differential curve of the main product of example 1 of the present invention.
FIG. 7 shows the ultraviolet absorption spectrum (a) and fluorescence emission spectrum (b) of curcumin, nanogold and final products in example 1 of the invention.
FIG. 8 shows XPS spectrum of the product of example 1 of the invention, wherein (a) is the full spectrum and (b) is the fine spectrum of Au.
FIG. 9 is a photograph of fluorescence of the product of example 1 of the present invention taken under a laser confocal microscope.
FIG. 10 is a photograph of the starting material, the major intermediate product and the final product of example 1 of the present invention.
FIG. 11 is a graph showing the stability of the product of example 1 of the present invention in water and PBS buffer, and a comparison of the stability with the results reported in the literature.
Fig. 12 shows the release profile and the comparison of the release profiles before and after release of the drug of the product of example 1 according to the present invention under different conditions, wherein (a) is release at different pH at 37 ℃, (b) is release at different temperature at ph=6.8, (c) is a comparison of release rates reported in other literature, and (d) is a comparison of physical photographs before and after release.
FIG. 13 is a thermal effect diagram of example 1 of the present invention, (a) is a thermal effect test schematic, (b) is AuNP-CNC- βCD/CUR at 0.8W/cm 2 An infrared thermal imaging photograph under the irradiation of laser, (c) is AuNP-CNC-beta CD/CUR at 0.8W/cm 2 And 0.5W/cm 2 CNC at 0.8W/cm 2 Temperature rise curve under laser irradiation.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
The reagents used in the examples are commercially available as usual unless otherwise specified. L-glutathione (purity 99%, biotechnological grade, shanghai microphone); l-cysteine (purity 98%, shanghai Boao organism); chloroauric acid (99.9% pure, U.S. Fisher Scientific); NHS (N-hydroxysuccinimide, purity 98%, shanghai Michelin); EDC (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, purity 98.5%, shanghai microphone Lin)
In the embodiment, a multi mode 8 Atomic Force Microscope (AFM) of Bruker corporation, germany is adopted to observe the microscopic morphology of the nano particles; the gold nanoparticles were observed by a Transmission Electron Microscope (TEM) of Jeol company 2100; the microscopic morphology of the CNC particles was observed by a German Zeiss company EVO18 Scanning Electron Microscope (SEM); the functional groups of the analysis substances are tested by a Tensor27/Hyperion Fourier transform infrared spectrometer (FTIR) of Bruker company, germany; the TA instruments Q200 differential calorimeter scanner (DSC) in the united states tests the change in the endothermic peak; the thermal decomposition temperature was measured by a Netzsch company TG209F3 thermogravimetric analyzer (TG); the spectrum and curcumin absorption intensity were measured by a UV-1900 ultraviolet visible spectrophotometer of Shimadzu corporation of Japan; fluorescent emission spectra were measured using a fluorescent Max-4 fluorescence spectrometer from Horiba, U.S.A.; photoelectron spectroscopy was tested using the Axis Ultra DLD X-ray photoelectron spectrometer (XPS) of KratOs company, uk; the fluorescence of the sample was tested by the TCS-SP5 laser confocal microscope (CLSM) of Leica company, germany; the U.S. Fluke Ti400 infrared thermal imaging camera recorded the temperature and took a photograph of the thermal effect.
The process of preparing the dual-effect antitumor drug from the raw materials and exerting the curative effect in the embodiment is shown in figure 1. CNC and AuNP (a-c in FIG. 1) were first prepared. Then grafting sulfhydryl to the surface of CNC through Schiff base reaction, mixing and stirring AuNP and CNC-SH, so that the AuNP is loaded on CNC. The βcd was then joined to AuNP-CNC by Epichlorohydrin (ECH) to produce AuNP-CNC- βcd (d in fig. 1). The AuNP is connected first and then ECH is connected to beta CD to avoid damage to mercapto group, so that the AuNP is loaded to protect mercapto group. After this, curcumin was loaded, thereby obtaining a dual effect antitumor drug (g in fig. 1). The hyperthermia effect and the chemotherapy effect (h in fig. 1) are obtained simultaneously under near infrared light irradiation. In fig. 1, i shows a schematic preparation process, CNC is used as a carrier to simultaneously load AuNP and beta CD, and then beta CD is used for accommodating CUR, so that the dual-effect antitumor drug AuNP-CNC-beta CD/CUR is obtained. Specifically, a detailed reaction procedure during the preparation is shown in fig. 2.
Example 1: preparation of double-effect curcumin antitumor drug based on nanocellulose carrier
(1) Preparation of CNC (cellulose nanocrystalline) carrier: first, a cotton fiber pulp sheet was crushed by a crushing mill to become fluffy cotton fibers. The prepared 2mol/L ammonium persulfate solution was heated to 60℃by 500mL and stirred continuously, 5g of cotton fiber was added thereto, and after 16 hours of reaction, the reaction solution was taken out and centrifuged, and the precipitate was washed with ultrapure water. The rotation speed is 10000 revolutions per minute, and the centrifugation is carried out for 15 minutes each time until the supernatant liquid is light blue. All the precipitate and the light blue supernatant from the last centrifugation are retained, and after proper dilution, the supernatant is subjected to ultrasonic dispersion to disperse the massive precipitate. Putting into a dialysis bag (with a molecular weight cut-off of 14 kDa), dialyzing with ultrapure water for 3-4 days, and changing the dialysis water every 4 hours. Finally the dialysate should not show acidity. The gum in the dialysis bag was removed, sonicated 3 times for 10 minutes each, and cooled 5 minutes each. The pellet was discarded and the supernatant was retained, again centrifuged at 10000rpm for 10 minutes. Precipitating to obtain unreacted large fiber and impurities, collecting supernatant as CNC, and storing in a refrigerator at 4deg.C.
(2) Preparation of AuNP (nano gold particles): all glassware was soaked with aqua regia (hydrochloric acid: nitric acid=3:1, volume ratio) for at least 12 hours, and then repeatedly washed with ethanol and ultra-pure water. 150. Mu.L of 1M chloroauric acid solution was mixed with 50mL of 4.8mM glutathione solution under vigorous stirring. The mixture was heated at 95℃for 35 minutes. The resulting reaction solution was cooled to room temperature and centrifuged at 12840g centrifugal force for 15 minutes to remove large aggregates. The pH of the solution was adjusted to 3-4, and ethanol (water: ethanol=2:1, volume ratio) was added to the solution, and the supernatant was further purified. Finally, the solution was centrifuged at 4000g for 5 minutes to obtain a precipitate, namely AuNP, and 0.2g of AuNP was prepared repeatedly for further use.
(3) Load of AuNP: putting 500mL of CNC prepared in the step (1) into a 1L conical flask, placing the conical flask on a magnetic stirrer, and adjusting the pH to about 7.5. The CNC concentration is measured in advance to be 0.3% of solid content, and the carboxyl content is measured by a conductivity titration method to be 526.67 mmol/(kgCNC). To this was added 0.075g of NHS and 0.5g of EDC at room temperature, stirred for half an hour to activate the carboxyl group, and then 0.2g of L-cysteine was added. Stirring is continued for 24 hours at normal temperature, and the bottle mouth is not sealed so as to discharge the gas generated in the process. And (5) dialyzing and purifying. After dialysis is complete, the nanoparticles are dispersed by ultrasound. Stored in a refrigerator at 4℃for further use, designated CNC-SH. And (3) dispersing 0.16g of the AuNP prepared in the step (2) in 50mL of water, mixing with all CNC-SH, and stirring for 30 minutes to complete grafting of the nano gold particles, thereby obtaining the AuNP-CNC. The AuNP-CNC was found to have a solids content of 0.25% and a total volume of 670mL.
(4) Grafting of βcd (β -cyclodextrin): to all of the AuNP-CNC solution obtained in the step (3), 5g of beta-cyclodextrin was dissolved, heated to 45℃and about 10mL of epichlorohydrin was added dropwise with vigorous stirring, and the reaction was continued for 2 hours. After the reaction was terminated, the reaction solution was cooled to room temperature and centrifuged at 10000rpm for 10 minutes to remove excess cyclodextrin and epichlorohydrin. And taking the precipitate for further purification after dialysis for 3-4 days. And (3) performing ultrasonic dispersion on the obtained AuNP-CNC-beta CD, and storing the AuNP-CNC-beta CD in a refrigerator at the temperature of 4 ℃ for standby.
(5) Load of antitumor drug CUR (curcumin): a certain amount of curcumin powder is dissolved in acetone to prepare a solution with the concentration of about 1.4 mg/mL. An acetone solution of curcumin was dropwise added to 250mL of AuNP-CNC- βcd until insoluble curcumin particles appeared. During this process vigorous stirring was maintained to volatilize the acetone. The acetone was further removed by rotary evaporation at 45 ℃. The rotary evaporation is carried out under negative pressure to ensure complete acetone removal. The resulting mixture was centrifuged at 6000rpm for 30 minutes to remove unencapsulated curcumin and the upper clear transparent yellow liquid was collected and designated as AuNP-CNC-. Beta.CD/CUR. The obtained curcumin antitumor drug with dual curative effects based on the nanocellulose carrier.
Performance test:
in order to avoid the influence of nano-gold on performance detection and characterization, a batch of nano-drugs without grafting nano-gold was additionally prepared, namely, steps (2) and (3) in example 1 were removed, the AuNP-CNC in step (4) was replaced by the CNC in step (1), and the obtained product was designated CNC- βCD/CUR. In order to facilitate the investigation of the loading of nanocellulose and the effect of cyclodextrin entrapment, a batch of cyclodextrin-entrapped curcumin was prepared, i.e. steps (1) - (4) of example 1 were deleted, and the AuNP-CNC- βcd in step (5) was replaced with βcd, and the resulting product was designated βcd/CUR.
(1) Microscopic topography analysis
The carrier and the groups on the surface of the carrier have a great influence on the drug loading. The microscopic morphology of the nanoparticles was observed by various electron microscopy means. The microtopography of the various nanoparticles prepared in example 1 is shown in fig. 3. In fig. 3 a is an AFM image of curcumin powder, which is difficult to disperse well and agglomerate together into a flocculent shape. As can be seen from the TEM image, auNP is a spherical particle with a diameter of about 5-8 nm (b in FIG. 3). Both c (SEM images) and d (AFM images) in fig. 3 can be seen to produce CNCs having needle-like morphology. The prepared CNC has a length of about 150-300 nm and a width of about 10-20 nm. Such a diameter is sufficient to support the produced nano-gold particles. After a series of treatments, the particles of AuNP-CNC-. Beta.CD/CUR became short rods. According to e, f and g in fig. 3, the structure of CNC appears to vary slightly due to the graft modification. The length is slightly reduced to about 150-200 nm and the diameter is increased to about 40nm. The reduction in length may be due to the CNC being partially degraded during the thiolation process or to the loss of a portion of the too short fibers during the purification process. The AuNP-CNC- βCD/CUR diameter resulting from the modification is larger than CNC, which may be due to a number of factors. On the one hand, schiff base and epichlorohydrin increase the chain length of the branched chain where the sulfhydryl group and beta CD are located respectively; on the other hand, curcumin may be partially adsorbed on the surface of CNC in addition to the cavities of βcd. In addition, CNCs may also swell throughout the graft modification process. Under the combined action of these factors, the diameter of the AuNP-CNC-beta CD/CUR particles is obviously increased, and the length-diameter ratio (L/D) is reduced from 15 to about 4-5. The black dots on the short rod fibers in g of fig. 3 are CNC loaded AuNP. In transmission electron microscopy, electrons can penetrate relatively easily through fibers of relatively soft texture, but it is difficult to penetrate harder texture gold nanoparticles. The nanogold appears very clearly on the nearly translucent fiber image. As can be seen from the graph, the grafting rate of gold is not very high, and only less than 10 nano gold particles are grafted on a single fiber, and some fibers only load one or two nano gold particles, and even a small number of fibers do not successfully load AuNP. According to principle analysis, the photothermal effect of the nano gold particles can be shown under the near infrared irradiation only by loading nano gold on most of fibers, so that the expected purpose is achieved. Therefore, the grafting of AuNP achieves the aim of loading nano gold.
(2) Infrared spectroscopic analysis
The raw materials used were attached to the support by chemical grafting, as determined by infrared spectroscopy, rather than physical adsorption. The FTIR spectrum of the nanoparticle prepared in example 1 is shown in fig. 4.
CUR and beta CD/CUR at 1630cm -1 、1603cm -1 And 1515cm -1 The peak at which corresponds to the benzene ring vibration of curcumin. Except for the two curcumin-free samples βCD and CNC, the remainder were at 1515cm -1 A sharp peak was observed at the location, which indicated that the final finished AuNP-CNC- βcd/CUR did contain curcumin. 1630cm in a sample of simple mixture of beta CD and CUR -1 And 1603cm -1 The vibration peak of the two benzene ring skeletons is located at 1635cm by beta CD -1 Is masked by the peak of (a). In the beta CD/CUR, not only the vibration peak of the benzene ring but also 1718cm- 1 A new peak appears at the same time 1630cm -1 And 1603cm -1 The peak shapes at the two positions are slightly changed, and 1718cm -1 The peak at this point was also detected in the final finished product. Indicating that cyclodextrin encapsulates curcumin not only physically loaded but also accompanying chemical changes, 1718cm -1 The new peak at this point is most likely caused by the interaction of the two. Curcumin was also demonstrated to bind successfully to CNC-beta CD. 1650 to 1610cm can be seen on the CNC-SH curve -1 There is a broad peak corresponding to c=o stretching vibration and c=n of schiff base conjugation, so the peak shape is widened. The epichlorohydrin used in grafting beta-cyclodextrin opens the C=N double bond to a single bond, breaks the conjugation, and the peak shape of C=O narrows and shifts to 1632cm -1 . This indicates that grafting of the thiol groups was successful and that subsequent steps did not break the thiol-containing branches.
(3) Thermal performance testing
The thermal performance test was to further confirm the manner in which CUR was bound to CNC and cyclodextrin.
Thermodynamic interactions between the various components can be explored by DSC measuring the endothermic and exothermic behavior during the warming process, to determine if curcumin is successfully intercalated into the cavities of the beta-cyclodextrin. The DSC curves of the various particles prepared in example 1 are shown in FIG. 5. The βcd and CUR show a single endothermic peak at 148 ℃ and 179 ℃, respectively, and 179 ℃ should be the melting point of the curcumin used. The endothermic peak of the mixture of the two is also at the two temperatures, and the simple mixing is simply superposition of the two, so that the embedding purpose cannot be achieved. The curve shape of the inclusion compound beta CD/CUR is different from the three, and a wider endothermic peak appears at 130 ℃ and a sharp endothermic peak appears at 220 ℃. These two endothermic peaks indicate thermodynamic interactions between cyclodextrin and curcumin molecules, altering the endothermic temperature, confirming that curcumin is encapsulated within the cavities of βcd. CNC showed two endothermic peaks at 130℃and 200℃and the mixture of CNC and. Beta. CD/CUR showed endothermic peaks at the same temperature, except that the peak shape was widened. The CNC-beta CD/CUR only has one endothermic peak at 124 ℃, which indicates that the thermodynamic property of the grafted product is changed; the disappearance of the peak at about 200 ℃ is the strong thermal stability, and the endothermic peak generated by the decomposition and the heat absorption is transferred to more than 300 ℃ and exceeds the detection capability of the instrument.
The TG and DTG curves of the particles prepared in example 1 are shown in fig. 6. At 100deg.C, the water loss amounts of CNC, βCD/CUR and CNC- βCD/CUR are 2.57%, 5.27% and 3.15%, respectively. The inclusion compound beta CD/CUR has the highest water content, which indicates that the overall inclusion compound is highly hydrophilic, and the hydrophilicity of curcumin is greatly improved. Compared to CNC, CNC- βCD/CUR surface has more hydrophilic groups, so the grafted βCD product is more hydrophilic than ungrafted CNC. Comparing the curves of TG, it was found that the initial decomposition temperatures of CNC and βcd/CUR were 167 ℃ and 196 ℃, respectively, and the grafted product increased to 230 ℃. There is only one stage of thermal decomposition of βcd/CUR, indicating thermodynamic interactions between cyclodextrin and curcumin after inclusion, and no thermal decomposition of βcd and CUR alone. The thermal decomposition of both CNC and CNC-. Beta.CD/CUR has two stages, corresponding to the two peaks on the DTG curve. The maximum degradation rates for CNC occur at 204.7 ℃and 258.6 ℃and CNC-. Beta.CD/CUR at 314.8 ℃and 330.8 ℃. Obviously, the heat resistance of the grafted product is obviously improved. The first thermal decomposition stage, at a lower temperature, corresponds to the decomposition of the defective portion and amorphous region of the fiber, and the second stage, at a higher temperature, corresponds to the decomposition of the remaining portion. The defective sites are less well aligned, more easily broken, and more easily grafted at these locations. The disorder degree of the modified region is reduced, and the heat resistance is improved. Furthermore, 314.8 ℃is also the temperature corresponding to the maximum decomposition rate of βCD/CUR, which suggests that the first stage of CNC- βCD/CUR decomposition is dominated by βCD/CUR decomposition. In view of the greatly increased decomposition temperature of the nanofibers after grafting, grafting should occur mainly at defective sites and amorphous areas of CNC, where the cellulose chains are broken and cyclodextrin is grafted at these sites.
The result of comprehensive thermal analysis shows that the common sterilization process at 121 ℃ does not influence the thermal stability of the product nano-drug.
(4) Optical Properties of curcumin
Maintaining the optical properties of curcumin during synthesis is an important prerequisite for ensuring the efficacy of curcumin. Curcumin is a drug with poor stability, and maintaining its structure unchanged is a necessary concern in curcumin drug utilization. When the structure changes, the optical characteristics change. Conversely, no change in optical properties means no change in curcumin structure. The optical properties of CUR, auNP, auNP-CNC-. Beta.CD/CUR were compared by UV-visible and fluorescence spectra.
The optical properties of the product prepared in example 1 are shown in fig. 7, where a in fig. 7 is the result of ultraviolet-visible absorption spectrum and b in fig. 7 is the fluorescence emission spectrum. The result is very obvious, the ultraviolet absorption characteristic and the fluorescence characteristic of the curcumin are not changed obviously, and the effectiveness of the curcumin is well reserved in the whole preparation process. The ultraviolet absorption peak of curcumin in water and ethanol is 425nm, and the peak of fluorescence emission spectrum under irradiation of 425nm excitation wavelength is 533nm. In the ultraviolet absorption spectrum and the fluorescence emission spectrum, the peak position of the curcumin is not changed, which means that the optical characteristics of the curcumin are reserved in the preparation process of the medicine, the structure of the curcumin is not changed, and the curcumin loaded on the AuNP-CNC-beta CD/CUR has the same biochemical function and medicinal value as the free curcumin.
In addition, as can be seen from FIG. 7 a, auNP has a gentle absorption peak at 283nm, and after grafting onto CNC, a new absorption peak appears at 804nm in addition to the absorption peak at 283 nm. There may be a portion of the AuNP that aggregates during grafting with CNC, with the agglomerated and dispersed aunps having different optical properties, resulting in this variation. 804nm is in the near infrared region where absorption provides conditions for the application of photothermal therapy. The UV absorption around 283nm indicated that the particle size of the AuNP produced was about 5nm, consistent with TEM results.
(5) XPS analysis
To explore the interactions between nanofibers and gold nanoparticles, X-ray photoelectron spectroscopy analysis was performed on AuNP-CNC- βcd/CUR. The full spectrum results in the range of 0 to 1100eV are shown in a of FIG. 8, and a spectrum peak of O, C, S, au appears. Fig. 8 b shows the fine energy spectrum of Au. 83.7eV and 87.5eV correspond to Au 4f7/2 and Au 4f 5/2, respectively, due to spin-orbit splitting of the Au 4f energy level. The characteristic peaks of 0-valent metal Au are at 84.0eV and 87.7eV, and the actually measured values are negatively shifted compared with the values, which are caused by the transfer of electrons from the fiber to the AuNP, indicating that Au in the sample is-1 valent and Au-S bonds exist in the sample.
(6) Fluorescent photograph
In order to make the fluorescence effect of the AuNP-CNC-beta CD/CUR nano-composite drug more visual, the fluorescence emitted by the AuNP-CNC-beta CD/CUR nano-particles was photographed by a laser confocal microscope, and the result is shown in FIG. 9. Fluorescence can not be detected in a bright field environment with weak natural light, and the AuNP-CNC-beta CD/CUR nano-composite drug can be irradiated with 408nm laser in a dark field to see very bright green fluorescence. After the sample is sufficiently diluted, only weak fluorescence can be observed in the field of view, and only a few green bright spots can be seen. These bright spots are fluorescent light emitted by small amounts of agglomerated nanoparticles. The photo clearly shows the bright fluorescence of the nano-drug, which provides convenience for the subsequent cell experiment and can be imaged without additionally introducing fluorescent substances.
(7) Enhancement of curcumin water solubility and analysis of curcumin loading
Physical photographs of the results of key steps in the preparation process in example 1 are shown in fig. 10 a to e. From the figure, it can be seen that curcumin powder is almost completely insoluble in water, can be dispersed in water only in a granular state after shaking for a plurality of days, and has extremely low solubility. After embedding curcumin, βcd/CUR shows good water solubility. This is because the hydroxyl groups at the C6 position of the glucose molecules constituting the beta-cyclodextrin are all arranged on the outer wall, making the cyclodextrin highly hydrophilic, exhibiting a strong polarity; the polar cyclodextrin encapsulates the non-polar curcumin after encapsulation such that the non-polar character of the curcumin is masked, which alters the non-polar state of the curcumin to some extent. The beta CD/CUR prepared by the validation experiment of cyclodextrin embedded curcumin proves that the embedding can obviously improve the water solubility of the curcumin, and the aqueous solution is clear and transparent. After the CNC is connected with the AuNP and the beta CD in sequence, the appearance is not changed obviously, and the CNC is still light blue liquid. Finally, the curcumin becomes yellow liquid after being loaded, and the curcumin becomes clear as beta CD/CUR, but still has higher transparency.
Curcumin loadings of the product prepared in example 1 are shown in table 1 below. In order to measure the loading of curcumin, the AuNP-CNC-beta CD/CUR is diluted by methanol and ethanol respectively, so that all the curcumin is dissolved out, and after the nano particles are removed by centrifugation, the content of the curcumin is measured by an ultraviolet spectrophotometer. After dilution with methanol or ethanol, the nanoparticles need to be removed by centrifugation or by passing through a membrane with a syringe, avoiding that the ultraviolet light absorbed by the nanoparticles is erroneously calculated as ultraviolet light absorption by curcumin. The curcumin content was found to be substantially uniform after sufficient dilution with both solvents, and was found to be 43.9 μg/mL. The concentration of the sample was 1.4mg/mL (solid content about 0.14% (w/w)), and the loading of curcumin was calculated to be 31.4. Mu.g/mg.
TABLE 1 enhancement of curcumin Water solubility
The final concentrations of curcumin of the products prepared in example 1, as compared to other carrier-based systems, are shown in table 2 below. Part of the sample is steamed at normal temperature, no solid is separated out when the total volume of AuNP-CNC-beta CD/CUR liquid is reduced by two thirds, and the load of AuNP-CNC-beta CD to CUR is stable. The concentration of curcumin in the concentrated system is 131.7 mug/mL, which is higher than other carrier systems.
TABLE 2 comparison of curcumin solubility with results reported in the literature
Among them, references 1, 2, 3, 4 are as follows:
【1】Liandong Hu,Kong Dongqian,Hu Qiaofeng,et al.Preparation and optimization of a novel microbead formulation to improve solubility and stability of curcumin[J].Particulate Science and Technology,2017,35(4):448-454.
【2】Umesh Kannamangalam Vijayan,Shah Nirali-Nitin,Muley Abhijeet-Bhimrao,et al.Complexation of curcumin using proteins to enhance aqueous solubility and bioaccessibility:Pea protein vis-à-vis whey protein[J].Journal of Food Engineering,2021,292110258.
【3】Gautier-M-A-Ndong Ntoutoume,Granet Robert,Mbakidi Jean-Pierre,et al.Development of curcumin–cyclodextrin/cellulose nanocrystals complexes:New anticancer drug delivery systems[J].Bioorganic&Medicinal Chemistry Letters,2016,26(3):941-945.
【4】SDey,Sreenivasan K.Conjugation of curcumin onto alginate enhances aqueous solubility and stability of curcumin[J].Carbohydr Polym,2014,99499-507.
(8) Stability of the formulation and release of curcumin
The stability profile of curcumin In water and PBS of the product prepared In example 1 and other documents (document 5 (Ngwabebhoh-F Asabawa, ilke Erdagi-S, YIldiz U.Pickering emulsions stabilized nanocellulosic-based nanoparticles for coumarin and curcumin nanoencapsulations: in vitro release, anticancer and antimicrobial activities [ J ]. Carbohydro Polym,2018,201317-328 ]), document 6 (Rohan-V Tikekar, pan Yuanjie, nitin N.Fate of curcumin encapsulated In silica nanoparticle stabilized Pickering emulsion during storage and simulated digestion [ J ]. Food Research International,2013,51 (1): 370-377 ]), document 7 (Bakht-Ramin Shah, li Yan, ji Weiping, et al preparation and optimization of Pickering emulsion stabilized by chitosan-tripolyphosphate nanoparticles for curcumin encapsulation [ J ]. Food Hydrocolloids,2016,52369-377 ]), document 8 (Surya-Prasah Singh, alvi-Baseard, syradju Bj-triggered liposome gold nanoparticles entrapping curcumin as In situ adjuvant for photothermal treatment of skin cancer, U.745) are shown by NIR. No. TVs, J.8211. The AuNP-CNC-beta CD/CUR is diluted by 10 times by water and 0.1M PBS buffer solution with the pH of 6.8, and the stability under the condition of light shielding and normal temperature is better. The curcumin content in the PBS buffer was slightly reduced but about 96% remained after 48 hours, indicating that there was little curcumin precipitation or degradation denaturation in the weakly acidic PBS buffer. The stability of the present invention is better than that of other reports (document 5, document 6, document 7, document 8). At the same time, it is also demonstrated that dilution within a certain range does not reduce the stability of nanoparticle-supported curcumin.
thereleasecomparisoncurvesandthepre-releaseandpost-releasecomparisondiagramsofcurcuminoftheproductpreparedinexample1underdifferentconditionsareshowninfig.12,wherein(a)releaseatdifferentpHat37℃,(b)releaseatdifferenttemperatureatph=6.8,and(c)releaseatdifferenttemperaturearereportedinliterature(Gautier-M-a-ndongntoutoume,granetrobert,mbakidijean-pierre,etaldevelopmentofcurcumin-cycloodextrin/cellulosenanocrystalscomplexes:Newanticancerdrugdeliverysystems[J].bioorganic&MedicinalChemistryLetters,2016,26(3):941-945],document9(XingyiLi,nanKaihui,LilingiFatemeh,AlizadehEffat,etalLysine-embeddedcellulose-basednanosystemforefficientdual-deliveryofchemotherapeuticsincombinationcancertherapy[J].CarbohydratePolymers,2020,250116861]),document12(nLi,KoneckeStephanie,Weiel-A,Biboy-A,J].35,35[J].35,35]ofthecompany,J., 2013,98 (1): 1108-1116.), reference 13 (ChJ Pan, tang J-J, weng Y-J, et al preparation, characterization and anticoagulation of curcumin-eluting controlled biodegradable coating stents [ J ]. J Control Release,2006,116 (1): 42-49 ]), and (d) is a graphical comparison of the physical photographs before and after release. Curcumin release was studied in phosphate buffered saline containing 15% ethanol. Some ethanol was added to the PBS to meet the sink conditions. In vitro release experiments, higher temperatures and lower pH favor curcumin release. At higher temperatures, the thermal movement of the molecules is enhanced, promoting the release of curcumin. The highest point at pH 5.5 exceeds 80% at 37 ℃; the highest point at 44 ℃ is close to 77% when the experiment is carried out at pH 6.8, the release rate is relatively high, and the curcumin release is relatively complete. From the graph, it can be seen that the release profile gradually decreased after reaching the peak within about 10 hours, by about 3.2% to 4.5% compared to the maximum release, and by about 4% in the same range as the 48 hour decrease in the stability test. Thus, the decrease in the second half of the release profile can be attributed to the effect of PBS buffer on curcumin dissolved therein. The color change of the drug-loaded nanoparticles AuNP-CNC- βCD/CUR before and after release can be seen more intuitively from FIG. 12 d. After release, curcumin is dispersed in a dissolution medium to form a dilute solution with very low concentration, and yellow color of curcumin is difficult to see. The nanofiber in the dialysis bag has yellow fading, only has light faint yellow residues, and also intuitively shows that the curcumin release is complete. The release rate of the present invention is higher than other reports.
(9) Photo-thermal effect
To evaluate the photothermal conversion efficiency, 0.5W/cm was used 2 And 0.8W/cm 2 The 808nm near infrared laser with two power densities irradiates AuNP-CNC-beta CD/CUR liquid drops. The test schematic is shown by a of fig. 13. As can be seen very intuitively in FIG. 13 b, the drop is 0.8W/cm 2 Exhibits a relatively pronounced thermal effect after laser irradiation. As can be seen from the comparison of the curves in c of FIG. 13, CNC with the same solids content was measured at 0.8W/cm 2 The temperature after 10 minutes of laser irradiation was only raised from 17.4 ℃ to 26.7 ℃ which is well below the photothermal effect of AuNP-CNC- βcd/CUR. The supported nano gold shows higher photo-thermal conversion efficiency. In combination with the release behavior of curcumin, it was confirmed that near infrared hyperthermia can promote the release of CUR. Obviously, with the extension of the laser irradiation duration, the curcumin release rate of AuNP-CNC-beta CD/CUR is further improved. This suggests that the release of CUR loaded on AuNP-CNC- βcd/CUR is controllable to some extent, which is beneficial for increasing local drug concentration and improving bioavailability of curcumin. The nano-drug can have dual therapeutic effects of heat treatment and chemotherapy.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (3)
1. The preparation method of the dual-therapeutic curcumin antitumor drug based on the nanocellulose carrier is characterized by comprising the following steps of:
(1) Preparation of CNC carrier: crushing the cotton fiber pulp plate by a crushing grinder to form fluffy cotton fibers, heating 500mL of prepared 2mol/L ammonium persulfate solution to 60 ℃ and continuously stirring, adding 5g of fluffy cotton fibers into the mixture, reacting for 16 hours, purifying the obtained reaction liquid after the reaction is finished, and obtaining supernatant which is CNC (computerized numerical control), and preserving for later use;
(2) Preparation of AuNP: mixing 150 mu L of 1M chloroauric acid solution with 50mL of 4.8mM glutathione solution under stirring, heating the mixture at 95 ℃ for reaction for 35 minutes, and purifying the obtained reaction liquid after the reaction is finished, wherein the obtained precipitate is AuNP;
(3) Load of AuNP: putting 500mL of CNC prepared in the step (1) into a 1L conical flask, placing the conical flask on a magnetic stirrer, adjusting the pH to 7.5, measuring the concentration of CNC to be 0.3% of solid content in advance, and measuring the carboxyl content to be 526.67mmol/kg of CNC by a conductivity titration method; to this, 0.075g of NHS and 0.5g of EDC were added at normal temperature, and the mixture was stirred for half an hour to activate the carboxyl group, and then 0.2g of L-cysteine was added; stirring is continued for 24 hours at normal temperature, and the bottle mouth is not sealed so as to discharge the gas generated in the process; dialyzing and purifying; after the dialysis is completed, the nano particles are dispersed by ultrasonic wave; storing in a refrigerator at 4 ℃ for standby, and recording as CNC-SH; dispersing 0.16g of AuNP prepared in the step (2) in 50mL of water, mixing with all CNC-SH, and stirring for 30 minutes to complete grafting of nano gold particles, thereby obtaining AuNP-CNC, wherein the solid content of the AuNP-CNC is measured to be 0.25%;
(4) Grafting of beta CD: dissolving 5g of beta-cyclodextrin in all AuNP-CNC solution obtained in the step (3), heating to 45 ℃, dropwise adding 10mL epoxy chloropropane under intense stirring, continuously reacting for 2 hours, purifying the reaction solution after the reaction is ended to obtain the AuNP-CNC-beta CD grafted with the beta-cyclodextrin, and preserving for later use;
(5) Loading of curcumin: dripping acetone solution of curcumin into AuNP-CNC-beta CD dropwise until insoluble curcumin particles appear, stirring to volatilize acetone, further removing acetone by spin evaporation, centrifuging the obtained mixture at 6000rpm for 30 minutes, removing unencapsulated curcumin, collecting upper clear transparent yellow liquid, and obtaining the dual-effect curcumin antitumor drug based on nanocellulose carrier.
2. The method for preparing the dual-therapeutic curcumin antitumor drug based on the nanocellulose carrier as claimed in claim 1, wherein the method is characterized by comprising the following steps:
the molecular weight cut-off of the dialysis bag used for dialysis is 12-14 kDa.
3. The dual therapeutic curcumin antitumor drug based on nanocellulose carrier prepared by the method of claim 1 or 2.
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Patent Citations (3)
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| CN106074451A (en) * | 2016-07-05 | 2016-11-09 | 同济大学 | Reduction stimuli responsive pharmaceutical carrier containing nano cages and preparation method and application |
| CN111803629A (en) * | 2020-06-02 | 2020-10-23 | 南京邮电大学 | Organic-inorganic hybrid multifunctional biological material based on nano cellulose crystals and preparation method and application thereof |
| CN112059204A (en) * | 2020-09-15 | 2020-12-11 | 北京师范大学 | Preparation method of gold nanoclusters |
Non-Patent Citations (2)
| Title |
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| Gautier M.A. Ndong Ntoutoume等."Development of curcumin–cyclodextrin/cellulose nanocrystals complexes: New anticancer drug delivery systems".Bioorganic & Medicinal Chemistry Letters.2016,第26卷(第3期),摘要、正文第1页右栏第1段、第3页左栏第4段至第4页右栏第3段以及图1. * |
| Surya Prakash Singh等."NIR triggered liposome gold nanoparticles entrapping curcumin as in situ adjuvant for photothermal treatment of skin cancer".International Journal of Biological Macromolecules.2018,第110卷摘要、第376页左栏第1段. * |
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