CN111643679A - Preparation method and application of chitosan oligosaccharide modified betulinic acid drug delivery system - Google Patents
Preparation method and application of chitosan oligosaccharide modified betulinic acid drug delivery system Download PDFInfo
- Publication number
- CN111643679A CN111643679A CN202010569071.9A CN202010569071A CN111643679A CN 111643679 A CN111643679 A CN 111643679A CN 202010569071 A CN202010569071 A CN 202010569071A CN 111643679 A CN111643679 A CN 111643679A
- Authority
- CN
- China
- Prior art keywords
- cos
- betulinic acid
- chitosan oligosaccharide
- drug
- delivery system
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
- A61K47/6907—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a microemulsion, nanoemulsion or micelle
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
Abstract
The invention discloses a preparation method and application of a betulinic acid drug delivery system modified by chitosan oligosaccharide, which modifies betulinic acid on the basis of active chitosan oligosaccharide to prepare amphiphilic active unit molecules, so that on one hand, the anticancer activities of chitosan oligosaccharide and betulinic acid can be combined, and the aim of safely and efficiently and synergistically enhancing the anticancer can be realized; on the other hand, the purpose of drug transportation can be realized in a micelle or self-assembly form by virtue of the amphipathy of the molecules. As a drug-carrying system, on one hand, the traditional chemotherapeutic drug can be carried to realize the purpose of multiple chemotherapies, and the anticancer effect is enhanced; on the other hand, the photosensitizer can be carried, so that the purposes of triple synergistic enhancement of anticancer curative effect are achieved on the basis of safe chemotherapy and combined with phototherapy, and clinical cancer treatment is expected to be overcome and solved.
Description
Technical Field
The invention belongs to the field of biomedical materials, and relates to a preparation method of a natural micromolecule nano-carrier based on active chitosan oligosaccharide bridging and a synergistic anticancer application thereof.
Background
Currently, cancer is one of the malignant diseases that seriously threaten the life safety of human beings. Tumor treatment modalities based on a combination of chemotherapy, phototherapy, hyperthermia, immunotherapy have been widely explored and used for clinical cancer treatment. However, the traditional chemotherapy drugs (such as paclitaxel, doxorubicin hydrochloride and the like) have the defects of low bioavailability, large toxic and side effects and the like, and even the combination therapy based on the traditional chemotherapy drugs also has certain side effects. Therefore, the safe and efficient chemotherapy drug is searched, and the significant significance of realizing the cancer cure is realized by combining with phototherapy or thermotherapy.
Chitosan Oligosaccharides (COS) as cationic basic amino oligosaccharides with only positive charge in nature have excellent biological activity, such as antioxidant activity, anti-inflammatory activity, cholesterol reduction activity, immunity enhancing activity, and antitumor activity. In addition, the good biocompatibility, the non-toxicity and the non-sensitization characteristic to living organs of the nano-material lead the nano-material to be a potential drug transport carrier and a tissue scaffold in nano-engineering. However, its high water solubility characteristics also greatly limit its preparation as a nano-drug delivery system. Therefore, it is necessary to modify the structure and introduce new reactive groups.
The natural product is from plant or animal, so it has good biocompatibility and easy degradation ability. Among them, Betulinic Acid (BA), a hydrophobic small molecule natural product, has been widely reported as an anticancer agent because of its excellent anticancer activity and its natural biocompatibility.
Disclosure of Invention
The invention aims to provide a preparation method and application of a chitosan oligosaccharide modified betulinic acid drug delivery system. According to the invention, the betulinic acid is modified on the basis of the active chitosan oligosaccharide to prepare an amphiphilic active unit molecule, so that on one hand, the anticancer activities of the chitosan oligosaccharide and the betulinic acid can be combined, and the purpose of safely and efficiently enhancing the anticancer effect in a synergistic manner is realized; on the other hand, the purpose of drug transportation can be realized in a micelle or self-assembly form by virtue of the amphipathy of the molecules. As a drug-carrying system, on one hand, the compound can carry traditional chemotherapeutic drugs (such as paclitaxel, camptothecin and doxorubicin hydrochloride) to realize the purpose of multiple chemotherapies and enhance the anticancer effect; on the other hand, the photosensitizer (such as chlorin e6, Ce6 for short) can be carried, so that the purpose of triple synergistic enhancement of anticancer curative effect is achieved on the basis of safe chemotherapy and combined phototherapy, and clinical cancer treatment is expected to be overcome and solved.
The purpose of the invention is realized by the following technical scheme:
a preparation method of a chitosan oligosaccharide modified betulinic acid drug delivery system comprises the following steps:
dissolving Betulinic Acid (BA), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in an organic reagent acetone, DMF or DMSO, and controlling the molar ratio of BA, EDC and NHS to be 1: 1-4: 0.5-4;
step two, slowly dropwise adding a chitosan oligosaccharide (COS, polymerization degree of 2-6) water solution into the organic reagent obtained in the step one, controlling the mass ratio of BA to COS to be 1: 2-5, and then stirring and reacting for 20-30 hours at room temperature in an ice bath;
step three, after the reaction is finished, petroleum ether: acetone ═ 5:1(v/v) removed unreacted betulinic acid and intermediate by-products, and the reaction mixture was purified by petroleum ether: separating COS-BA by using acetone as a solvent in a ratio of 3:1 (v/v);
step four, dissolving COS-BA in dimethyl sulfoxide (DMSO) to prepare COS-BA mother liquor with the concentration of 0.1-35 mM;
and step five, quickly dropwise adding the COS-BA mother liquor into secondary distilled water under the ultrasonic condition, controlling the volume ratio of DMSO to water to be 0.1-1: 10 (namely 0.1-100 mu l of DMSO to 1mL of water), and performing ultrasonic treatment (1-25 min) and centrifugation to obtain COS-BANPs.
A method for preparing chemotherapeutic drug with a delivery system of betulinic acid drug modified by chitosan oligosaccharide as a carrier comprises the following steps:
dissolving Betulinic Acid (BA), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in an organic reagent acetone, DMF or DMSO, and controlling the molar ratio of BA, EDC and NHS to be 1: 1-4: 0.5-4;
step two, slowly dropwise adding a chitosan oligosaccharide (COS, polymerization degree of 2-6) water solution into the organic reagent obtained in the step one, controlling the mass ratio of BA to COS to be 1: 2-5, and then stirring and reacting for 20-30 hours at room temperature in an ice bath;
step three, after the reaction is finished, petroleum ether: acetone ═ 5:1(v/v) removed unreacted betulinic acid and intermediate by-products, and the reaction mixture was purified by petroleum ether: separating COS-BA by using acetone as a solvent in a ratio of 3:1 (v/v);
step four, preparing a chemotherapeutic drug solution with the concentration of 0.1-25 mM and COS-BA mother liquor;
step five, uniformly mixing the COS-BA mother liquor and the chemotherapeutic drug solution, adding the mixture into water under the condition of stirring, and controlling the volume ratio of COS-BA to chemotherapeutic drug to be 1-8: 1; and (3) continuously stirring for 4-8 h on a magnetic stirrer, transferring into a dialysis bag for dialysis for 12-48 h to remove organic reagents, and centrifuging to obtain COS-BA @ chemotherapeutic drug NPs.
A preparation method of a synergistic photosensitive drug taking a betulinic acid drug delivery system modified by chitosan oligosaccharide as a carrier comprises the following steps:
dissolving Betulinic Acid (BA), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in an organic reagent acetone, DMF or DMSO, and controlling the molar ratio of BA, EDC and NHS to be 1: 1-4: 0.5-4;
step two, slowly dropwise adding a chitosan oligosaccharide (COS, polymerization degree of 2-6) water solution into the organic reagent obtained in the step one, controlling the mass ratio of BA to COS to be 1: 2-5, and then stirring and reacting for 20-30 hours at room temperature in an ice bath;
step three, after the reaction is finished, petroleum ether: acetone ═ 5:1(v/v) removed unreacted betulinic acid and intermediate by-products, and the reaction mixture was purified by petroleum ether: separating COS-BA by using acetone as a solvent in a ratio of 3:1 (v/v);
step four, dissolving COS-BA in DMSO to prepare COS-BA mother liquor with the concentration of 0.1-35 mM;
and step five, uniformly mixing the COS-BA mother liquor and the photosensitizer (Ce6), adding the mixture into secondary distilled water containing NaOH, controlling the volume ratio of COS-BA to the photosensitizer to be 1-8: 1-2 and the molar concentration ratio of NaOH to the photosensitizer to be 1: 1-3, and performing ultrasonic treatment (1-25 min) and centrifugation to obtain COS-BA @ photosensitizer NPs.
Compared with the prior art, the invention has the following advantages:
1. the invention provides a novel, safe and effective chitosan oligosaccharide-betulinic acid (COS-BA) chemotherapeutic drug, and an active nano-drug for constructing multiple chemotherapies or combining chemotherapies and photodynamic therapy with anticancer by using the drug as a carrier.
2. On the basis of active chitosan oligosaccharide, anticancer natural micromolecule betulinic acid is modified, an amphiphilic dual chemotherapy drug carrier is constructed, and a chemotherapy drug taxol is carried to realize multiple chemotherapy; or carrying a photosensitizer Ce6, achieving the purpose of synergistic enhancement of anticancer while realizing photodynamic therapy.
3. The invention solves the problems of low bioavailability and the like caused by poor water solubility and easy aggregation in a physiological environment of the traditional chemotherapeutic drug or photosensitizer.
4. The chitosan oligosaccharide-betulinic acid (COS-BA) which is the novel chemotherapy medicament based on the chitosan oligosaccharide can be used as a safe and effective chemotherapy reagent for treating tumors, and can also be used as an active medicament transportation carrier to load the traditional chemotherapy medicament or photosensitizer, so that the aim of synergistic combined anticancer of dual chemotherapy and photodynamic therapy is fulfilled. Most importantly, the chitosan oligosaccharide-betulinic acid has excellent biocompatibility and easy biodegradability, and is expected to become a safe and efficient tumor treatment reagent and a drug transport carrier.
Drawings
FIG. 1 shows the synthesis of chitooligosaccharide-betulinic acid (COS-BA);
FIG. 2 is the nuclear magnetic resonance hydrogen spectrum of COS-BA: (1H-NMR)；
FIG. 3 is an SEM image of COS-BANPs and the synergistic anti-tumor drug COS-BA @ PTXNPs assembled with the chemotherapeutic drug PTX, and the synergistic photosensitive drug COS-BA @ Ce6NPs co-assembled with the photosensitizer Ce6, (a) COS-BANPs, (b) COS-BA @ PTXNPs, (c) COS-BA @ Ce6 NPs;
FIG. 4 is an in vitro anticancer activity assessment of COS-BA @ PTX NPs;
FIG. 5 is a graph of cell viability under COS-BA @ Ce6NPs treatment;
FIG. 6 is a graph of tumor volume changes in female Balb-c mice treated with COS-BA @ Ce6 NPs;
FIG. 7 is a graph of body weight change of tumor-bearing mice treated with COS-BA @ Ce6 NPs.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings, but not limited thereto, and any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be covered by the protection scope of the present invention.
Example 1
Synthesis of compound Chitosan oligosaccharide-Betulinic acid (COS-BA):
the synthesis of the chitosan oligosaccharide-betulinic acid (COS-BA) is mainly prepared by a one-pot method under the catalysis of a condensing agent EDC/NHS, and the specific synthesis method is explored as follows: 40mg of betulinic acid BA, 20mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 21mg of N-hydroxysuccinimide (NHS) were dissolved in acetone (4mL) which is an organic reagent, and then 1mL of an aqueous solution of chitosan oligosaccharide (COS, polymerization degree of 2-6, 96mg) was slowly dropped into the organic reagent, followed by stirring and reacting at room temperature for 24 hours in an ice bath. Detecting the reaction condition by thin-layer chromatography; after the reaction is finished, petroleum ether: acetone ═ 5:1 removed unreacted betulinic acid and intermediate by-products, and the reaction mixture was purified by petroleum ether: and separating the point COS-BA with the highest content by using acetone as a ratio of 3: 1.
The synthetic process of the chitosan oligosaccharide-betulinic acid (COS-BA) is shown in figure 1, wherein n is 2-6.
And (3) detecting the nuclear magnetic hydrogen spectrum of the COS-BA by using deuterated chloroform as a solvent and containing internal standard TMS (tetramethylsilane). As shown in fig. 2, the two singlet peaks appearing at 4.60ppm and 4.73ppm are the olefin peaks on betulinic acid; methyl peaks on BA at 0.76ppm, 0.83 ppm. The peak at 8.98ppm is a newly generated hydrogen peak on an amido bond, and a plurality of multiple peaks appearing at 3.0-4.0 ppm are characteristic peaks of hydrogen on carbon on a parent ring of the chitosan oligosaccharide. These newly appearing peaks confirm the successful grafting of the chitosan oligosaccharide onto betulinic acid compared to free BA.
Example 2
Construction of drug delivery system (i.e. preparation of COS-BANPs):
a certain amount of COS-BA was dissolved in DMSO to prepare a mother solution of COS-BA at a concentration of 33.5 mM. A40. mu.l DMSO sample was quickly added dropwise to 1M redistilled water under ultrasonic conditions, followed by ultrasonic for 10min and centrifugation at 13500rpm to obtain COS-BANPs.
Example 3
Carrying chemotherapeutic drugs: take the drug paclitaxel PTX as an example.
(1) COS-BA @ PTX NPs are prepared by adopting a coprecipitation method.
Firstly, preparing 20mM PTX and COS-BA DMSO mother liquor, taking 50 mu L COS-BA solution and 10 mu LPTX solution, fully and uniformly mixing, and slowly dripping the solution into 2mL water under the condition of stirring; stirring was continued for 4h on a magnetic stirrer, and the mixture was transferred into a dialysis bag (MW 3000) and dialyzed for 24h to remove the organic reagent, and centrifuged at 13500rpm to obtain COS-BA @ PTXNPs.
As can be seen in FIG. 3, the particle morphology also exhibited a tendency to sphere when COS-BA was assembled with the chemotherapeutic drug PTX, but exhibited smaller particle diameters with an average diameter of about 84nm, further illustrating that PTX in combination with COS-BA can change the nanoparticle size and the PTX loading was successful.
(2) PTX content determination in COS-BA @ PTX NPs.
Subsequent in vitro cell experiments were performed with a volume ratio of COS-BA/PTX DMSO of 8: 1. Namely, 80 mu L of COS-BA solution and 10 mu L of LPTX solution are added into 2mL of secondary distilled water to obtain COS-BA @ PTX NPs, parallel to 4 groups, the nano-drug is destroyed by using methanol as a cracking system to release PTX, and the drug loading capacity of the PTX is detected by adopting a high performance liquid chromatography. The concrete conditions are as follows: mobile phase: column temperature 25 ℃, mobile phase: acetonitrile/water 65/35 (volume ratio), flow rate: 1mL/min, detection wavelength: 227 nm. Finally, the drug loading of Ce6 was determined to be 1.3%, and the encapsulation efficiency was determined to be 26.3%.
Example 4
Carrying photosensitizer drug: ce6 is taken as an example.
(1) Preparation of COS-BA @ Ce6NPs co-assembly medicine
The COS-BA and Ce6 co-assembled nano composite medicine is prepared by adopting a coprecipitation method, namely: COS-BA and Ce6 DMSO mother liquor was first prepared at a concentration of 33.5 mM. COS-BA and Ce6(Ce6 with a volume of 5 μ l) with different volume ratios are respectively taken and uniformly mixed, and then added into 1mL of secondary distilled water containing 10 μ l of NaOH, and then the mixture is subjected to ultrasonic treatment for 10min under the ultrasonic condition and centrifuged at 13500rpm to obtain COS-BA @ Ce6 NPs.
As can be seen from FIG. 3, free COS-BA itself can be assembled to form a uniform spherical nano-drug with an average DLS diameter of 254nm, which indicates that the single synthesized COS-BA itself can be used as a dual anticancer active drug for nano-tumor anticancer. When co-assembled with the photosensitizer Ce6, spherical nanoparticles still appeared, and there was a tendency for particle size reduction (about 187nm) despite the reduced particle uniformity, further indicating the successful assembly of COS-BA and Ce6 drugs for multiple synergistic anticancer studies.
(2) Determination of content of photosensitizer Ce6 in COS-BA @ Ce6NPs
The subsequent experiment was carried out with a volume ratio of COS-BA/Ce6 DMSO of 8:1, i.e., 5. mu.l of Ce6 and 40. mu.l of COS-BADMSO solution were added to 1mL of redistilled water containing 10. mu.l of NaOH, and obtained by ultrasonic reaction. In parallel with 4 experiments, COS-BA @ Ce6NPs obtained by direct dissolution and centrifugation are dissolved by using dimethyl sulfoxide as a solvent, the volume is fixed to a certain volume, and then the Ce6 drug-loading rate is detected by adopting a high performance liquid chromatography. The concrete conditions are as follows: mobile phase: column temperature 30 ℃, mobile phase: 0.2% aqueous phosphoric acid/acetonitrile 40/60 (vol), flow rate: 1mL/min, detection wavelength: 402 nm. Finally, the Ce6 drug loading rate is determined to be 8.1%, and the encapsulation efficiency is 96%.
Example 5
Chemotherapeutic Activity of COS-BA @ PTXNPs
The murine 4T1 breast cancer cells are taken as cell strains, and the chemotherapeutic activity of COS-BA @ PTX NPs is detected by adopting a standard MTT method. The specific process is as follows: 4T1 cells are inoculated by adopting a 96-well plate, after the cells adhere to the wall, COS-BA @ PTX NPs medicines of PTX with different equivalent weight are added, and after the cells are continuously cultured for 24h, the MTT method is used for detecting the cell activity.
FIG. 4 is a graph showing the cell viability of 4T1 cells cultured for 48h with the synergistic chemotherapeutic agent COS-BA @ PTX NPs. As can be seen from FIG. 4, PTX and COS-BA NPs exhibited concentration-dependent chemotherapeutic activity at equivalent amounts of PTX. Further indicates that the betulinic acid modified by the chitosan oligosaccharide has obvious chemotherapeutic activity and is expected to realize the purpose of multiple synergistic chemotherapy. And COS-BA @ PTX NPs are higher than PTX and COS-BANPs alone at the specified concentration, which shows that the aim of combined anticancer is achieved, and the anticancer activity is more prominent under 1ug/mL of PTX, thereby further confirming the aim of synergistic anticancer of multiple chemotherapies.
Example 6
In vitro chemotherapy and photodynamic anti-cancer activity evaluation of COS-BA @ Ce6 NPs:
the murine 4T1 breast cancer cells are taken as cell strains, and the standard MTT method is adopted to detect the chemotherapeutic activity of COS-BANPs and the photodynamic anti-cancer activity of COS-BA @ Ce6 NPs. The specific process is as follows: 4T1 cells are inoculated by adopting a 96-well plate, after the cells adhere to the wall, COS-BA @ Ce6NPs medicines of Ce6 with different equivalent weight are added, and the cells do not need to be irradiated for evaluating the chemotherapy activity; for evaluating the photodynamic therapy activity of the drug, after the drug is added, the cell survival rate is detected by an MTT method after the culture is continued for 4h and the illumination is carried out for 10min under 675 +/-10 nm infrared light.
FIG. 5 is a graph showing the cell viability of the synergistic anticancer agent COS-BA @ Ce6NPs under the condition of light. As can be seen from FIG. 5, under the condition of no light, the free Ce6 does not present any cytotoxicity, but COS-BA @ Ce6NPs present concentration-related anticancer activity, which fully embodies the excellent anticancer activity of COS-BA, and further proves that the betulinic acid modified by chitosan oligosaccharide has obvious chemotherapeutic activity and is expected to realize drug-loading synergistic anticancer therapy. On the other hand, under the illumination condition, COS-BA @ Ce6NPs show obviously enhanced cytotoxicity compared with the equivalent concentration of Ce6, which further proves that chemotherapy and photodynamic therapy are synergistic in anticancer. In addition, when the equivalent low-concentration Ce6(1 mu g/mL) is adopted, COS-BA @ Ce6 shows 79% of cell lethality rate, further explaining the remarkable anticancer activity of COS-BA @ Ce6, and the method is expected to be used for clinical anticancer tumor research.
Example 7
COS-BA @ Ce6NPs are taken as an example to carry out specific in vivo anticancer activity evaluation.
Magnetic Balb-c mice bearing 4T1 breast cancer tumor are used as research objects, and the chemotherapy characteristics of the drug COS-BA @ Ce6NPs under the condition of no light and the anti-cancer activity of the combination of the photodynamic therapy and the chemotherapy under the condition of light are researched in a tail vein injection administration mode. The specific experiment was divided into 5 groups of 5 mice each: group 1) blank group was 5% glucose solution; group 2) Ce6 light group 3.5 mg/Kg; group 3) Ce6 no light group; group 4) COS-BA @ Ce6NPs (equivalent Ce63.5 mg/kg) chemoactive group, i.e., tumor non-light group); group 5) COS-BA @ Ce6NPs phototherapy and chemotherapy combination treatment group, i.e. after 6h of administration, rats were anesthetized and tumors were red-lighted for 15 min. The treatment frequency is three times, the administration treatment is respectively carried out on the 0 th, 2 th and 4 th days, the treatment period is 2 weeks, and important parameters such as the volume of the tumor, the body weight of the mouse and the like are recorded in the period. Tumor volumes were recorded according to the following formula:
V＝(L×W2)/2；
in the formula, V represents the tumor volume, and L and W represent the length and width of the tumor, respectively.
From the trend of tumor volume shown in fig. 6, as with the results described in the cell experiments, the absence of light exposure to free Ce6 did not cause significant inhibition of tumor volume. In contrast, under the condition of no light, COS-BA @ Ce6NPs show moderate tumor inhibition, and the tumor volume inhibition rate reaches 40%, which is caused by the chemotherapeutic activity of COS-BA, and further proves that the betulinic acid modified by the chitosan oligosaccharide shows certain antitumor activity. On the other hand, under the condition of illumination, COS-BA @ Ce6NPs show an obvious tumor inhibition trend, the tumor volume inhibition rate reaches 76 percent and is obviously higher than that of free Ce6(36 percent) in an illumination group, which shows that the increase of the anticancer activity is the combination of COS-BA chemotherapy and Ce6 photodynamic therapy, and the obvious synergistic anticancer effect is shown.
As can be seen from FIG. 7, the weight average of the rats of each group of the tumor-bearing mice treated by the synergistic drug COS-BA @ Ce6NPs did not show significant change. Although the mice treated with COS-BA @ Ce6NPs under light showed a tendency to lose weight during the early treatment period (0-4 days), the tumor volume quickly returned to normal during the latter period, indicating that the synergistic agent was biologically safe. Further indicates that the chitosan oligosaccharide-betulinic acid has excellent biocompatibility and easy biodegradability, and is expected to become a safe and efficient tumor treatment reagent and a drug transport carrier.
Claims (10)
1. A preparation method of a chitosan oligosaccharide modified betulinic acid drug delivery system is characterized by comprising the following steps:
dissolving betulinic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in an organic reagent, and controlling the molar ratio of BA, EDC and NHS to be 1: 1-4: 0.5-4;
slowly dropwise adding a chitosan oligosaccharide aqueous solution into the organic reagent obtained in the step one, controlling the mass ratio of BA to COS to be 1: 2-5, and then stirring and reacting for 20-30 hours at room temperature in an ice bath;
step three, after the reaction is finished, petroleum ether: acetone ═ 5:1(v/v) removed unreacted betulinic acid and intermediate by-products, and the reaction mixture was purified by petroleum ether: COS-BA was isolated with acetone 3:1 (v/v).
2. The method for preparing the chitosan oligosaccharide modified betulinic acid drug delivery system according to claim 1, wherein the degree of polymerization of the chitosan oligosaccharide is 2-6, and the organic reagent is acetone, DMF or DMSO.
3. The method for preparing a chitosan oligosaccharide modified betulinic acid drug delivery system according to claim 1, characterized in that the method further comprises the steps of:
step four, dissolving COS-BA in DMSO to prepare COS-BA mother liquor with the concentration of 0.1-35 mM;
and step five, quickly dropwise adding the COS-BA mother liquor into secondary distilled water under the ultrasonic condition, controlling the volume ratio of DMSO to water to be 0.1-1: 10, and performing ultrasonic treatment and centrifugation to obtain COS-BA NPs.
4. Use of the chitosan oligosaccharide modified betulinic acid drug delivery system prepared by the method of any one of claims 1-3 as an active drug delivery vehicle for loading a photosensitizer or a chemotherapeutic drug.
5. Use of a chitosan oligosaccharide modified betulinic acid drug delivery system prepared by the method of any one of claims 1-3 in a tumor treatment agent.
6. A preparation method of a synergistic photosensitive drug taking a betulinic acid drug delivery system modified by chitosan oligosaccharide as a carrier is characterized by comprising the following steps:
dissolving betulinic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in an organic reagent, and controlling the molar ratio of BA, EDC and NHS to be 1: 1-4: 0.5-4;
slowly dropwise adding a chitosan oligosaccharide aqueous solution into the organic reagent obtained in the step one, controlling the mass ratio of BA to COS to be 1: 2-5, and then stirring and reacting for 20-30 hours at room temperature in an ice bath;
step three, after the reaction is finished, petroleum ether: acetone ═ 5:1(v/v) removed unreacted betulinic acid and intermediate by-products, and the reaction mixture was purified by petroleum ether: separating COS-BA by using acetone as a solvent in a ratio of 3:1 (v/v);
step four, dissolving COS-BA in DMSO to prepare COS-BA mother liquor with the concentration of 0.1-35 mM;
and step five, uniformly mixing the COS-BA mother liquor and the photosensitizer, adding the mixture into secondary distilled water containing NaOH, controlling the volume ratio of COS-BA to the photosensitizer to be 1-8: 1-2, controlling the molar concentration ratio of NaOH to Ce6 to be 1: 1-3, and carrying out ultrasonic treatment and centrifugation to obtain COS-BA @ photosensitizer NPs.
7. The method for preparing the synergic photoactivated drug with the transportation system of betulinic acid drug modified with chitosan oligosaccharide as the carrier of claim 6, wherein the polymerization degree of the chitosan oligosaccharide is 2-6, and the organic reagent is acetone, DMF or DMSO.
8. A preparation method of a chemotherapeutic drug taking a betulinic acid drug delivery system modified by chitosan oligosaccharide as a carrier is characterized by comprising the following steps:
dissolving betulinic acid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in an organic reagent, and controlling the molar ratio of BA, EDC and NHS to be 1: 1-4: 0.5-4;
slowly dropwise adding a chitosan oligosaccharide aqueous solution into the organic reagent obtained in the step one, controlling the mass ratio of BA to COS to be 1: 2-5, and then stirring and reacting for 20-30 hours at room temperature in an ice bath;
step three, after the reaction is finished, petroleum ether: acetone ═ 5:1(v/v) removed unreacted betulinic acid and intermediate by-products, and the reaction mixture was purified by petroleum ether: separating COS-BA by using acetone as a solvent in a ratio of 3:1 (v/v);
step four, preparing a chemotherapeutic drug solution with the concentration of 0.1-25 mM and COS-BA mother liquor;
step five, uniformly mixing the COS-BA mother liquor and the chemotherapeutic drug solution, adding the mixture into water under the condition of stirring, and controlling the volume ratio of COS-BA to chemotherapeutic drug to be 1-8: 1; and (3) continuously stirring for 4-8 h on a magnetic stirrer, transferring into a dialysis bag for dialysis for 12-48 h to remove organic reagents, and centrifuging to obtain COS-BA @ chemotherapeutic drug NPs.
9. The method for preparing a chemotherapeutic drug using a chitosan oligosaccharide modified betulinic acid drug delivery system as a carrier according to claim 8, wherein the polymerization degree of the chitosan oligosaccharide is 2-6, and the organic reagent is acetone, DMF or DMSO.
10. Use of a chemotherapeutic drug with a chitosan oligosaccharide modified betulinic acid drug delivery system as a carrier, prepared by the method of any one of claims 8 to 9, in a tumor treatment agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010569071.9A CN111643679B (en) | 2020-06-19 | 2020-06-19 | Preparation method and application of chitosan oligosaccharide modified betulinic acid drug delivery system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010569071.9A CN111643679B (en) | 2020-06-19 | 2020-06-19 | Preparation method and application of chitosan oligosaccharide modified betulinic acid drug delivery system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111643679A true CN111643679A (en) | 2020-09-11 |
| CN111643679B CN111643679B (en) | 2022-09-16 |
Family
ID=72351685
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010569071.9A Active CN111643679B (en) | 2020-06-19 | 2020-06-19 | Preparation method and application of chitosan oligosaccharide modified betulinic acid drug delivery system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111643679B (en) |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040138152A1 (en) * | 2001-08-18 | 2004-07-15 | Kwon Ick Chan | Anticancer drug-chitosan complex forming self-aggregates and preparation method thereof |
| WO2007086651A1 (en) * | 2006-01-26 | 2007-08-02 | Jae-Woon Nah | Anti-cancer agent loaded hydrophobic bile acid conjugated hydrophilic chitosan oligosaccharide nanoparticles and preparation method thereof |
| CN101011579A (en) * | 2006-12-29 | 2007-08-08 | 浙江大学 | Application of cytoplasm-targeting chitosan oligosaccharide-aliphatic acid grafting medicinal-vector colloidal cluster |
| US20080008755A1 (en) * | 2005-01-14 | 2008-01-10 | Korea Institute Of Science And Technology | Pharmaceutical formulation of cholanic acid-chitosan complex incorporated with hydrophobic anticancer drugs and preparation method thereof |
| CN101314622A (en) * | 2008-06-26 | 2008-12-03 | 浙江大学 | Salicylic-g-chitosan oligosaccharide grafts and synthesizing process |
| CN101711873A (en) * | 2008-10-06 | 2010-05-26 | 中国科学院大连化学物理研究所 | Method for preparing amphiphilic chitosan nanometer medicament carrier |
| CN103536926A (en) * | 2013-09-20 | 2014-01-29 | 河北联合大学 | Preparation method of chitosan oligosaccharide nanoparticle |
| CN103705939A (en) * | 2013-12-30 | 2014-04-09 | 中国药科大学 | Preparation of amphiphilic ursolic acid-polysaccharide coupled substance and application thereof in treating tumors |
| CN105085711A (en) * | 2015-08-21 | 2015-11-25 | 哈尔滨工业大学 | Preparation method and application of chitobiose |
| CN107158404A (en) * | 2017-05-26 | 2017-09-15 | 中国医学科学院生物医学工程研究所 | It is a kind of applied to Liver targeting pH sensitivity nanoparticles delivery systems of chemotherapy of hepatocellular carcinoma administering drug combinations and preparation method thereof |
| CN109464676A (en) * | 2018-10-25 | 2019-03-15 | 浙江中医药大学 | A kind of preparation method and product of the photosensitive targeted nano granule of chitosan oligosaccharide |
| CN110327311A (en) * | 2019-08-22 | 2019-10-15 | 哈尔滨工业大学 | A kind of preparation method of oleanolic acid slow release nanometer microcapsules |
| CN110354100A (en) * | 2019-08-17 | 2019-10-22 | 哈尔滨工业大学 | A kind of amphiphilic polymer preparation method of novel deoxycholic acid modified chitosan oligosaccharide |
-
2020
- 2020-06-19 CN CN202010569071.9A patent/CN111643679B/en active Active
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040138152A1 (en) * | 2001-08-18 | 2004-07-15 | Kwon Ick Chan | Anticancer drug-chitosan complex forming self-aggregates and preparation method thereof |
| US20080008755A1 (en) * | 2005-01-14 | 2008-01-10 | Korea Institute Of Science And Technology | Pharmaceutical formulation of cholanic acid-chitosan complex incorporated with hydrophobic anticancer drugs and preparation method thereof |
| WO2007086651A1 (en) * | 2006-01-26 | 2007-08-02 | Jae-Woon Nah | Anti-cancer agent loaded hydrophobic bile acid conjugated hydrophilic chitosan oligosaccharide nanoparticles and preparation method thereof |
| CN101011579A (en) * | 2006-12-29 | 2007-08-08 | 浙江大学 | Application of cytoplasm-targeting chitosan oligosaccharide-aliphatic acid grafting medicinal-vector colloidal cluster |
| CN101314622A (en) * | 2008-06-26 | 2008-12-03 | 浙江大学 | Salicylic-g-chitosan oligosaccharide grafts and synthesizing process |
| CN101711873A (en) * | 2008-10-06 | 2010-05-26 | 中国科学院大连化学物理研究所 | Method for preparing amphiphilic chitosan nanometer medicament carrier |
| CN103536926A (en) * | 2013-09-20 | 2014-01-29 | 河北联合大学 | Preparation method of chitosan oligosaccharide nanoparticle |
| CN103705939A (en) * | 2013-12-30 | 2014-04-09 | 中国药科大学 | Preparation of amphiphilic ursolic acid-polysaccharide coupled substance and application thereof in treating tumors |
| CN105085711A (en) * | 2015-08-21 | 2015-11-25 | 哈尔滨工业大学 | Preparation method and application of chitobiose |
| CN107158404A (en) * | 2017-05-26 | 2017-09-15 | 中国医学科学院生物医学工程研究所 | It is a kind of applied to Liver targeting pH sensitivity nanoparticles delivery systems of chemotherapy of hepatocellular carcinoma administering drug combinations and preparation method thereof |
| CN109464676A (en) * | 2018-10-25 | 2019-03-15 | 浙江中医药大学 | A kind of preparation method and product of the photosensitive targeted nano granule of chitosan oligosaccharide |
| CN110354100A (en) * | 2019-08-17 | 2019-10-22 | 哈尔滨工业大学 | A kind of amphiphilic polymer preparation method of novel deoxycholic acid modified chitosan oligosaccharide |
| CN110327311A (en) * | 2019-08-22 | 2019-10-15 | 哈尔滨工业大学 | A kind of preparation method of oleanolic acid slow release nanometer microcapsules |
Non-Patent Citations (4)
| Title |
|---|
| JIANJUN CHENG等: "Photosensitive pro-drug nanoassemblies harboring a chemotherapeutic dormancy function potentiates cancer immunotherapy", 《ACTA PHARMACEUTICA SINICA B》 * |
| 任国飞: "壳寡糖-脂肪酸嫁接物给药系统的药物控释研究", 《万方学位论文》 * |
| 刘秀: "壳寡糖衍生物纳米载药胶束体系的构建及体外抗肿瘤研究", 《万方学位论文》 * |
| 杨苑健等: "水杨酸-g-壳寡糖作为抗癌性药物紫杉醇载体的研究", 《医药论坛杂志》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111643679B (en) | 2022-09-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108619511B (en) | Preparation method and application of metal-organic framework drug delivery system based on cytarabine small-molecule prodrug | |
| CN108409756B (en) | Camptothecin-based heterodimer multifunctional prodrug and preparation method and application thereof | |
| CN112604002A (en) | Disulfide-bond bridged docetaxel-fatty acid prodrug and self-assembled nanoparticles thereof | |
| CN1840193A (en) | Nanometer capsule of anthracene nucleus anticancer antibiotic with polyethylene glycol-phospholipid | |
| CN102429870A (en) | Novel tumor-targeting arboraceous polymer nano carrier of camptothecin drug | |
| CN113663079B (en) | Carrier-free self-assembly nano particle and preparation method and application thereof | |
| CN113350503B (en) | Carrier-free hybrid nano assembly and preparation method and application thereof | |
| CN105997943B (en) | A kind of nano particle and its preparation method and application of human serum albumins load camptothecine | |
| CN109846857B (en) | Preparation method and application of active natural supramolecular photosensitizer | |
| CN112933052A (en) | Nano drug delivery system for improving tumor hypoxia microenvironment and enhancing immunotherapy | |
| CN111617246A (en) | Self-assembled nanoparticles of pure photosensitizer and preparation and application thereof | |
| CN111135299A (en) | Construction of photosensitizer-hypoxia activated prodrug integrated prodrug self-assembled nanoparticles | |
| WO2022052413A1 (en) | Drug-loaded polymer vesicle having asymmetric membrane structure, preparation method therefor, and application thereof in preparation of drugs for treating acute myeloid leukemia | |
| CN108853512A (en) | The preparation and antitumor application thereof of double Types of Medicine cis-platinum/adriamycin-poly-dopamine prodrug nanoparticles | |
| CN114099674B (en) | Copper or zinc carrier for carrying diethyl dithiocarbamic acid prodrug and preparation and application thereof | |
| CN1927203A (en) | Nano micelle preparation of Catharanthus roseus alkaloids antineoplastic drugs with coating of phospholipid derived from polyethylene glycol | |
| CN109464676B (en) | Preparation method and product of chitosan oligosaccharide photosensitive targeting nanoparticles | |
| CN111643664B (en) | Preparation method and application of active natural small molecule mediated co-assembled photosensitive drug | |
| CN113648401A (en) | Hybrid nano assembly for protease inhibition sensitization photodynamic therapy and preparation and application thereof | |
| CN111643679B (en) | Preparation method and application of chitosan oligosaccharide modified betulinic acid drug delivery system | |
| CN109481418B (en) | Anti-tumor nano-particles and preparation method and application thereof | |
| CN113633784B (en) | Hybrid nano-assembly for heat shock protein inhibition sensitization photothermal therapy and preparation and application thereof | |
| CN115581707A (en) | Preparation method of chitosan oligosaccharide-curcumin nano complex | |
| CN111700862B (en) | Bispecific nano micelle based on folic acid targeting and Cherenkov radiation response and preparation method and application thereof | |
| CN115105606A (en) | Hyaluronic acid-mangiferin-methotrexate anti-tumor coupling drug and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |