CN105561325B - A kind of antitumor slow releasing pharmaceutical material and the preparation method and application thereof - Google Patents
A kind of antitumor slow releasing pharmaceutical material and the preparation method and application thereof Download PDFInfo
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- CN105561325B CN105561325B CN201510927181.7A CN201510927181A CN105561325B CN 105561325 B CN105561325 B CN 105561325B CN 201510927181 A CN201510927181 A CN 201510927181A CN 105561325 B CN105561325 B CN 105561325B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0002—Galenical forms characterised by the drug release technique; Application systems commanded by energy
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- 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
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
- C08B37/0012—Cyclodextrin [CD], e.g. cycle with 6 units (alpha), with 7 units (beta) and with 8 units (gamma), large-ring cyclodextrin or cycloamylose with 9 units or more; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
- C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
- C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
- C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
Abstract
The present invention discloses a kind of antitumor slow releasing pharmaceutical material and the preparation method and application thereof.The antitumor slow releasing pharmaceutical material applies a kind of extraordinary chitosan derivatives of water solubility, improve the water solubility of HCPT, by the host-guest interaction of cyclodextrin and adamantane hydroxycamptothecin and water-soluble tetraethylenepentamine chitosan are combined together, solubilising HCPT.HCPT is connected on material with ehter bond, drug difficulty or ease fall off, and consumingly extend the half-life period of drug.Based on the C=N structure contained in material structure can restore in acid condition disconnection, the cyclodextrin in material structure and adamantane host-guest interaction and also open, therefore material plays the role of dual control sustained release.The material has good biocompatibility, does not have apparent inhibiting effect to the growth of normal cell；And fixed point long-acting slow-release, the inhibiting effect to tumour cell is enhanced, and extend administration time, greatly improves the availability of drug.
Description
Technical field
The invention belongs to antitumor slow releasing pharmaceutical technical fields, and in particular to a kind of antitumor slow releasing pharmaceutical material and its system
Preparation Method and application.
Background technique
Cancer is the common name of one group of more than 100 kinds of disease that can influence any position of body, is to jeopardize the mankind in the world today
One of the major disease of health.In numerous anticancer drugs, camptothecine is that a kind of one kind extracted from camplotheca acuminata is micro
Alkaloid and its derivative, wherein 10-hydroxycamptothecine (10-Hydroxy camptothecin, HCPT) be clinical application compared with
A kind of good broad-spectrum anti-cancer drug, effect is than 10 times of camptothecine, to gastric cancer, liver cancer, the carcinoma of the rectum (including bladder cancer etc.), head
Neck cancer and leukemia treating are effective.
However 10-hydroxycamptothecine series antineoplastic medicament generally deposits tumor tissues and normal cell almost non-selectivity
The problems such as curative effect is low, toxicity is big, transfer stove is uncontrollable, patient medication poor compliance.Therefore, anti-tumor drug transmission system
Have become the research emphasis and hot spot of art of pharmacy.Wherein the gentle controlled release delivery system of targeted delivery systems has been demonstrated can have
Effect reduces the adverse reaction of anti-tumor drug, improves the compliance of clinical efficacy and patient medication.Chitosan and its derivative because
To be made into microballoon as the carrier of anti-tumor drug with good biocompatibility, micro-capsule, nanoparticle, hydrogel, burying
The different dosage forms such as agent, polymer micelle are planted, can reach targeting and the effect of sustained release in vivo.In addition, chitosan and its derivative
Carrier of the object as anti-tumor drug, the research of the anti-tumor drug kind contained to it is also very extensive, studied as
5 FU 5 fluorouracil, Anthraquinone, cis-platinum class, vitamins, steroids, chemo-immunity class drug, crude drug species (including purple
The pure and mild camptothecin of China fir), biological species drug (cell factor, gene and vaccine) etc., illustrate chitosan and its derivative conduct
Drug carrier system has good compatibility and huge potentiality.
Cyclodextrin (CD) is the nontoxic macrocyclic oligo sugar being made of 6 or 6 or more glucose units, common
Tetra- kinds of cyclodextrin of α, β, γ and δ contain 6,7,8 and 9 glucose units respectively.Because of the hydrophobic cavity suitable size and warp of β-CD
It helps the reasons such as cost is relatively low, is often used as eliminating smell agent, stabilizer, emulsifier in food, is used as drug in a medicament and includes
Material and auxiliary material etc. are especially widely used in pharmaceutical preparation and sustained release field.But the solubility very little of β-CD, this
Just limit its application to a certain extent.In recent years, researcher has synthesized a series of biggish β-cdderivatives of solubility
Include drug.Cyclodextrine derivatives good water solubility and low toxicity, being formed with hydrophobic guest drug molecule can be big after inclusion compound
The solubility of big raising drug molecule in water, is added to the drugloading rate that drug can be improved in slow-release material.Also someone is logical
It crosses freeze-drying method and prepares a kind of biodegradable matrix bracket-chitosan-g- beta-cyclodextrin (CS-g- β-CD), use
Come the notch filled up in extracellular matrix agglutination, while can load and slow releasing pharmaceutical；The form of bracket, swelliong power and
Medicine-releasing performance depends on the degree of bracket crosslink density；CS-g- β-CD bracket, which is shown, discharges ketone Lip river than chitosan stent
It is fragrant more slow, and cytotoxicity experiment (MTT) experiment shows that the bracket does not have apparent cytotoxicity, bracket is to surrounding tissue
Close friend simultaneously can improve its surrounding tissue power of regeneration；The bracket is likely to become a potential biodegradable medicine controlled release and carries
The packing material of body and wound healing.
Research using the inclusion complexation of β-CD and adamantane effect (host-guest interaction) slow releasing pharmaceutical is also a lot of.Someone will
1- adamantanecarboxylic acid (Ad-COOH) and adriamycin (Dox) obtain Ad-Dox after N '-carbonyl base diimidazole (CDI) coupling by N,
It is poly- that self assembly host and guest precursor reactant generation PEI-CD/Ad-Dox supermolecule nano is carried out with cyclodextrin-polyethyleneimine (PEI-CD)
Close object.Adriamycin successfully combines adamantyl and forms supramolecular complex PEI-CD/Ad- with PEI-CD by Subjective and Objective connection
After Dox, confirm that PEI-CD/Ad-Dox can compress simultaneously connexus when N/P ratio is less than 3 completely using gel electrophoresis retardation experiment
Grain DNA.And tumour cell can be improved in vivo and in vitro to drug susceptibility, preferable synergistic function is embodied, to change
The use in conjunction for treating drug and genomic medicine lays the foundation.Also someone has synthesized mPEG-Ad/ by polyethylene glycol (PEG)
BAC-CD (BAC:N, N '-bis- acryloyl cystines) micella passes through the stability protection camptothecine of micella for loading camptothecine
Lactone ring structure, to reduce the usage amount and side effect of drug.
Outstanding ability based on chitosan and its derivative to the good biocompatibility of biological tissue and in load medicine field,
The problem of numerous researchers are explored and studied to it, however there is also poorly water-solubles, grinds anti-tumor drug
Study carefully aspect, poor, targeting hardly possible and slow release effect difference problem that there is also safeties.
Summary of the invention
In order to overcome the disadvantages and deficiencies of the prior art, the primary purpose of the present invention is that providing a kind of antitumor slow releasing medicinal
Object material.The material applies a kind of extraordinary chitosan derivatives of water solubility, improves 10-hydroxycamptothecine (10-
Hydroxy camptothecin, HCPT) water solubility, by the host-guest interaction of cyclodextrin and adamantane by hydroxycamptothecin
It is combined together with water-soluble tetraethylenepentamine chitosan, solubilising HCPT.The main problem of HCPT clinical application is not medicine
Effect, but frequent drug administration patient is difficult to endure drug side-effect, the half-life period for extending HCPT seems extremely important.By HCPT with ether
On material, drug difficulty or ease fall off key connection, consumingly extend the half-life period of drug.Based on the C=contained in material structure
N structure can restore in acid condition disconnection, the cyclodextrin in material structure and adamantane host-guest interaction and also beat
It opens, therefore material plays the role of dual control sustained release.
Another object of the present invention is to provide the preparation methods of above-mentioned antitumor slow releasing pharmaceutical material.
It is yet a further object of the present invention to provide the applications of above-mentioned antitumor slow releasing pharmaceutical material.
The purpose of the invention is achieved by the following technical solution:
A kind of antitumor slow releasing pharmaceutical material, structure is as shown in formula I:
A kind of preparation method of antitumor slow releasing pharmaceutical material, comprises the following steps:
(1) HCPT-COOH is weighed, is dissolved in solvent, is stirred at room temperature, Tanabe Seiyoku is added, crosslinking agent, room temperature is added
Reaction；Low-temperature aqueous solution is added, low temperature is static, and solid is precipitated；Centrifugation goes out solid, HCPT-CD obtained by drying；
(2) synthesis of tetraethylenepentamine chitosan (TEPACS), can be divided into three steps；A: weighing chitosan, dissolution；Slowly plus
Enter methanol, continue to stir, stop stirring, obtains solution A, for use；B: tetraethylenepentamine is dissolved, and it is molten to obtain tetraethylenepentamine
Liquid；By dissolution of benzaldehyde, it is then added in tetraethylenepentamine solution, is heated to reflux；Epichlorohydrin reaction is slowly added dropwise, obtains
Obtain B solution；C: B solution is added in solution A, is stirred at room temperature；PH is adjusted, is precipitated, is filtered, washing is dried in vacuo up to four
Five amine chitosan (TEPACS) of ethylene；
(3) TEPACS for obtaining step (2) dissolves, and obtains TEPACS solution；Weigh Ad-CHO (1- adamantanecarboxylic acid-
4- benzaldehyde ester) dissolution, it is then added into TEPACS solution, reacts at room temperature；Methanol is rotated out, is washed, it is so dry that produce
Product TEPACS-Ad；
(4) product that step (1) and (3) obtain is combined together by nesting method.Nesting is exactly in material structure
Adamantane mixed with cyclodextrin, theoretically adamantane is nested in cyclodextrin.Weigh step (3) resulting TEPACS-
HCPT-CD is added in Ad, dissolution, dissolves and make the sufficiently nested adamantane of cyclodextrin, dry, obtains the antitumor slow releasing pharmaceutical material of product
Expect TEPACS-Ad/HCPT-CD.
HCPT-COOH structure described in step (1) is as shown in formula II:
Formula II；
Tanabe Seiyoku (CD-NH described in step (1)2) structure as shown in formula III:
Formula III；
The structure of HCPT-CD described in step (1) is as shown in formula IV:
Formula IV；
HCPT-COOH described in step (1) is referring to bibliography (Wani et al, 1980) synthesis；
Solvent described in step (1) be DMSO, THF and water equal solvent in any one or at least two, it is described
Solvent be preferably DMSO；
The crosslinking agent is any one or two kinds of EDCHCl and NHS；
Final concentration of 3.75mmol/L~10mmol/L of HCPT-COOH described in step (1)；
The mixing time being stirred at room temperature described in step (1) is preferably 1~5h；
Final concentration of 3.75mmol/L~10mmol/L of Tanabe Seiyoku is added described in step (1)；
The reaction time of room temperature reaction described in step (1) is preferably 6~for 24 hours；
Low-temperature aqueous solution described in step (1) is preferably 0~4 DEG C of aqueous solution；
It is dissolved as being dissolved in described in step (2) A in 2% acetic acid aqueous solution；
Final concentration of 0.05mol/L~0.25mol/L of the chitosan in solution A described in step (2) A；
The time of dissolution described in step (2) A is preferably 1~3d；
The time for continuing stirring described in step (2) A is preferably 3~6h；
Final concentration of 1~5mol/L of the tetraethylenepentamine in B solution described in step (2) B；
Final concentration of 1~5mol/L of the benzaldehyde in B solution described in step (2) B；
The condition being heated to reflux described in step (2) B be preferably 40~80 DEG C reflux 12~for 24 hours.
Final concentration of 1~5mol/L of the epoxychloropropane in B solution described in step (2) B；
The condition of reaction described in step (2) B be preferably 40~60 DEG C reaction 12~for 24 hours；
The churned mechanically time described in step (2) C is preferably 4~5d；
PH described in step (2) C is preferably 5~6；
Ad-CHO described in step (3) (1- adamantanecarboxylic acid -4- benzaldehyde ester) structure is as shown in formula V:
Formula V；
TEPACS-Ad described in step (3) is referring to bibliography (Kulkarni et al, 2012) synthesis；
The molar ratio of chitosan monomer and Ad-CHO in TEPACS described in step (3) is preferably (2~5): 1；
The time of reaction described in step (3) is preferably 6~12h；
Drying described in step (3) and (4) is preferably freeze-dried；
The molar ratio of chitosan monomer and HCPT-CD in TEPACS-Ad described in step (4) is preferably (4~15):
1。
Application of the antitumor slow releasing pharmaceutical material in antitumor slow releasing pharmaceutical field.
The principle of the present invention is: the present inventor is first with the tetraethylenepentamine containing a large amount of amino to natural good high score
Sub- material chitosan is modified, the good tetraethylenepentamine chitosan (TEPACS) of obtained water solubility；By adamantane acyl chlorides
The good tetraethylenepentamine shell of water solubility that a series of C=N structure by forming pH sensitivity after modifications is connected to synthesis is poly-
On sugar, TEPACS-Ad is obtained.Separately it is different from others and is connected to the 20-OH of camptothecine to biological tissue's tool by PEG with ester bond
Have on the β-CD of good safety, but the 10-OH of HCPT is connected to β-CD by linking arm in the form of more stable ehter bond
On, obtain HCPT-CD.It is acted on by the inclusion complexation of β-CD and adamantane, finally prepares a kind of nanometre glue for being sustained HCPT
Beam TEPACS-Ad/HCPT-CD.
The present invention compared with the existing technology, have following advantages and effects
(1) antitumor slow releasing pharmaceutical material prepared by the present invention has double controlled-release functions.
(2) antitumor slow releasing pharmaceutical material prepared by the present invention has good biocompatibility, the life to normal cell
Long no apparent inhibiting effect.
(3) antitumor slow releasing pharmaceutical material prepared by the present invention pinpoints long-acting slow-release, enhances the inhibition to tumour cell
Effect, and administration time is extended, greatly improve the availability of drug.
Detailed description of the invention
Fig. 1 be the resulting antitumor slow releasing pharmaceutical of embodiment 1 nucleus magnetic hydrogen spectrum (1H NMR)；Wherein, Fig. 1 a is HCPT-
The nucleus magnetic hydrogen spectrum of COOH；Fig. 1 b is the nucleus magnetic hydrogen spectrum of HCPT-CD.
Fig. 2 be the resulting antitumor slow releasing pharmaceutical of embodiment 1 nucleus magnetic hydrogen spectrum (1H NMR)；Wherein, Fig. 2 c is TEPACS
Nucleus magnetic hydrogen spectrum；Fig. 2 d is the nucleus magnetic hydrogen spectrum of Ad-CHO；Fig. 2 e is the nucleus magnetic hydrogen spectrum of TEPACS-Ad；Fig. 2 f is TEPACS-Ad/
The nucleus magnetic hydrogen spectrum of HCPT-CD.
Fig. 3 is the sustained release analysis of experimental results figure of the resulting antitumor slow releasing pharmaceutical material of embodiment 1.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1
(1) referring initially to document (Wani et al, 1980) synthesis HCPT-COOH, 0.1mmol HCPT- is then weighed again
COOH is dissolved in 10mL DMSO, and 1h is stirred at room temperature, and 0.1mmol Tanabe Seiyoku is added, a small amount of EDCHCl, room temperature is added
React 6h.4 DEG C of aqueous solutions are added, low temperature is static, and solid is precipitated.Centrifugation goes out solid, HCPT-CD obtained by drying；
(2) synthesis of tetraethylenepentamine chitosan (TEPACS), can be divided into three steps.A: the addition of 0.01mol chitosan is weighed
In beaker, the acetic acid aqueous solution of 100mL 2% is added, dissolves 1d.It is slowly added into 100mL methanol, continues to stir 3h, stops stirring
It mixes, obtains solution A, for use.B: 0.1mol tetraethylenepentamine is dissolved in 50mL isopropanol in three-necked flask, in beaker
By 0.1mol dissolution of benzaldehyde in 50mL isopropanol, also it is added in three-necked flask later, 40 DEG C are heated to reflux 12h.Slowly drop
Add 0.1mol epoxychloropropane, 12h is reacted at 40 DEG C, obtains B solution.C: B solution is added in solution A, machinery stirs at room temperature
Mix 4d.It is slowly added in dilute hydrochloric acid solution, adjusting pH is 5, a large amount of acetone are added, until precipitating is completely, filtering, then with anhydrous second
Alcohol washing, is dried in vacuo up to tetraethylenepentamine chitosan (TEPACS)；
(3) TEPACS-Ad is synthesized, it need to be referring initially to document (Kulkarni et al, 2012) synthesis Ad-CHO (1- adamantane
Formic acid -4- benzaldehyde ester), it weighs above-mentioned resulting TEPACS a certain amount of (containing about 5mmol chitosan monomer) and round-bottomed flask is added,
It is dissolved in water.It weighs 1mmol Ad-CHO to be dissolved in 100mL methanol, be added in round-bottomed flask later, react at room temperature 6h.It rotates out
Methanol washes out unreacted Ad-CHO, is freeze-dried to obtain product TEPACS-Ad；
(4) product that step (1) and (3) obtain is combined together by nesting method.Nesting is exactly in material structure
Adamantane mixed with cyclodextrin, theoretically adamantane is nested in cyclodextrin.Weigh TEPACS-Ad obtained by step (3)
A certain amount of (containing about 1mmol chitosan monomer), is dissolved in the water, and 0.1mmol HCPT-CD is added, dissolves and keeps cyclodextrin abundant
Nested adamantane, it is rear to be freeze-dried, obtain the antitumor slow releasing pharmaceutical material TEPACS-Ad/HCPT-CD of product.By attached drawing 1 and attached drawing
2 nucleus magnetic hydrogen spectrums are available as a result, successfully synthesizing target product.
Embodiment 2
(1) referring initially to document (Wani et al, 1980) synthesis HCPT-COOH, 0.5mmol HCPT- is then weighed again
COOH is dissolved in 100mL DMSO, and 5h is stirred at room temperature, and 0.5mmol Tanabe Seiyoku is added, a small amount of EDCHCl, room is added
Temperature reaction is for 24 hours.4 DEG C of aqueous solutions are added, low temperature is static, and solid is precipitated.Centrifugation goes out solid, HCPT-CD obtained by drying；
(2) synthesis of tetraethylenepentamine chitosan (TEPACS), can be divided into three steps.A: the addition of 0.05mol chitosan is weighed
In beaker, the acetic acid aqueous solution of 100mL 2% is added, dissolves 3d.It is slowly added into 100mL methanol, continues to stir 4h, stops stirring
It mixes, obtains solution A, for use.B: 0.5mol tetraethylenepentamine is dissolved in 50mL isopropanol in three-necked flask, in beaker
By 0.5mol dissolution of benzaldehyde in 50mL isopropanol, also it is added in three-necked flask later, 80 DEG C are heated to reflux for 24 hours.Slowly drop
Add 0.5mol epoxychloropropane, is reacted at 60 DEG C for 24 hours, obtain B solution.C: B solution is added in solution A, machinery stirs at room temperature
Mix 5d.It is slowly added in dilute hydrochloric acid solution, adjusting pH is 6, a large amount of acetone are added, until precipitating is completely, filtering, then with anhydrous second
Alcohol washing, is dried in vacuo up to tetraethylenepentamine chitosan (TEPACS)；
(3) TEPACS-Ad is synthesized, it need to be referring initially to document (Kulkarni et al, 2012) synthesis Ad-CHO (1- adamantane
Formic acid -4- benzaldehyde ester), it weighs above-mentioned resulting TEPACS a certain amount of (containing about 10mmol chitosan monomer) and round bottom burning is added
Bottle, is dissolved in water.It weighs 5mmol Ad-CHO to be dissolved in 500mL methanol, be added in round-bottomed flask later, react at room temperature 12h.Rotation
Methanol is steamed, unreacted Ad-CHO is washed out, is freeze-dried to obtain product TEPACS-Ad；
(4) product that step (1) and (3) obtain is combined together by nesting method.Nesting is exactly in material structure
Adamantane mixed with cyclodextrin, theoretically adamantane is nested in cyclodextrin.Weigh TEPACS-Ad obtained by step (3)
A certain amount of (containing about 2mmol chitosan monomer), is dissolved in the water, and 0.5mmol HCPT-CD is added, dissolves and keeps cyclodextrin abundant
Nested adamantane, it is rear to be freeze-dried, obtain the antitumor slow releasing pharmaceutical material TEPACS-Ad/HCPT-CD of product.
Embodiment 3
(1) referring initially to document (Wani et al, 1980) synthesis HCPT-COOH, 0.3mmol HCPT- is then weighed again
COOH is dissolved in 50mL DMSO, and 3h is stirred at room temperature, and 0.3mmol Tanabe Seiyoku is added, a small amount of EDCHCl, room temperature is added
React 12h.4 DEG C of aqueous solutions are added, low temperature is static, and solid is precipitated.Centrifugation goes out solid, HCPT-CD obtained by drying；
(2) synthesis of tetraethylenepentamine chitosan (TEPACS), can be divided into three steps.A: the addition of 0.03mol chitosan is weighed
In beaker, the acetic acid aqueous solution of 100mL 2% is added, dissolves 2d.It is slowly added into 100mL methanol, continues to stir 6h, stops stirring
It mixes, obtains solution A, for use.B: 0.3mol tetraethylenepentamine is dissolved in 50mL isopropanol in three-necked flask, in beaker
By 0.3mol dissolution of benzaldehyde in 50mL isopropanol, also it is added in three-necked flask later, 60 DEG C are heated to reflux 18h.Slowly drop
Add 0.3mol epoxychloropropane, 18h is reacted at 50 DEG C, obtains B solution.C: B solution is added in solution A, machinery stirs at room temperature
Mix 4.5d.It is slowly added in dilute hydrochloric acid solution, adjusting pH is 5.5, a large amount of acetone are added, until precipitating is completely, filtering, then use nothing
Water-ethanol washing, is dried in vacuo up to tetraethylenepentamine chitosan (TEPACS)；
(3) TEPACS-Ad is synthesized, it need to be referring initially to document (Kulkarni et al, 2012) synthesis Ad-CHO (1- adamantane
Formic acid -4- benzaldehyde ester), it weighs above-mentioned resulting TEPACS a certain amount of (containing about 8mmol chitosan monomer) and round-bottomed flask is added,
It is dissolved in water.It weighs 3mmol Ad-CHO to be dissolved in 300mL methanol, be added in round-bottomed flask later, react at room temperature 10h.Revolving
Methanol out washes out unreacted Ad-CHO, is freeze-dried to obtain product TEPACS-Ad；
(4) product that step (1) and (3) obtain is combined together by nesting method.Nesting is exactly in material structure
Adamantane mixed with cyclodextrin, theoretically adamantane is nested in cyclodextrin.Weigh TEPACS-Ad obtained by step (3)
A certain amount of (containing about 3mmol chitosan monomer), is dissolved in the water, and 0.2mmol HCPT-CD is added, dissolves and fills cyclodextrin
Divide nested adamantane, it is rear to be freeze-dried, obtain the antitumor slow releasing pharmaceutical material TEPACS-Ad/HCPT-CD of product.
Embodiment 4
(1) referring initially to document (Wani et al, 1980) synthesis HCPT-COOH, 0.3mmol HCPT- is then weighed again
COOH is dissolved in 50mL THF and 30mL water, and 3h is stirred at room temperature, and 0.3mmol Tanabe Seiyoku is added, a small amount of EDC is added
HCl and NHS reacts at room temperature 12h.4 DEG C of aqueous solutions are added, low temperature is static, and solid is precipitated.Centrifugation goes out solid, obtained by drying
HCPT-CD；
(2) synthesis of tetraethylenepentamine chitosan (TEPACS), can be divided into three steps.A: the addition of 0.03mol chitosan is weighed
In beaker, the acetic acid aqueous solution of 100mL 2% is added, dissolves 2d.It is slowly added into 200mL methanol, continues to stir 3h, stops stirring
It mixes, obtains solution A, for use.B: 0.3mol tetraethylenepentamine is dissolved in 50mL isopropanol in three-necked flask, in beaker
By 0.3mol dissolution of benzaldehyde in 50mL isopropanol, also it is added in three-necked flask later, 60 DEG C are heated to reflux 18h.Slowly drop
Add 0.3mol epoxychloropropane, 18h is reacted at 50 DEG C, obtains B solution.C: B solution is added in solution A, machinery stirs at room temperature
Mix 4.5d.It is slowly added in dilute hydrochloric acid solution, adjusting pH is 5.5, a large amount of acetone are added, until precipitating is completely, filtering, then use nothing
Water-ethanol washing, is dried in vacuo up to tetraethylenepentamine chitosan (TEPACS)；
(3) TEPACS-Ad is synthesized, it need to be referring initially to document (Kulkarni et al, 2012) synthesis Ad-CHO (1- adamantane
Formic acid -4- benzaldehyde ester), it weighs above-mentioned resulting TEPACS a certain amount of (containing about 8mmol chitosan monomer) and round-bottomed flask is added,
It is dissolved in water.It weighs 3mmol Ad-CHO to be dissolved in 300mL methanol, be added in round-bottomed flask later, react at room temperature 10h.Revolving
Methanol out washes out unreacted Ad-CHO, is freeze-dried to obtain product TEPACS-Ad；
(4) product that step (1) and (3) obtain is combined together by nesting method.Nesting is exactly in material structure
Adamantane mixed with cyclodextrin, theoretically adamantane is nested in cyclodextrin.Weigh TEPACS-Ad obtained by step (3)
A certain amount of (containing about 3mmol chitosan monomer), is dissolved in the water, and 0.2mmol HCPT-CD is added, dissolves and keeps cyclodextrin abundant
Nested adamantane, it is rear to be freeze-dried, obtain the antitumor slow releasing pharmaceutical material TEPACS-Ad/HCPT-CD of product.
The experiment of 5 medicament slow release of embodiment
The antitumor slow releasing pharmaceutical material prepared according to the method for embodiment 1, in three different pH=4.5,7.4,9.4
It is lower and two 37 DEG C and 0 DEG C of different temperatures at probe into slow release, one 0 DEG C of control group is arranged in when pH=7.4.
Tetra- parts of TEPACS-Ad/HCPT-CD of the preparation of 60.0mg embodiment 1 are weighed, 40mL pH=4.5 is added separately to,
7.4,7.4,9.4 volume is about during the phosphate buffer of 30% methanol is molten, and starts timing, is quickly packed into the dialysis activated
In bag (MWCO:3500Da), quickly clips clip and be respectively put into the above-mentioned conical flask equipped with the identical pH buffer of 160mL.
By pH=4.5,7.4,9.4 conical flask seals lid and is quickly placed into that have mixed up temperature be 37 DEG C of constant-temperature table
It is middle that sink condition is kept to be incubated for the revolving speed of 80rpm, it is respectively labeled as 1. number, 2. number and 3. number conical flask.By the another of pH=7.4
An outer conical flask seals lid and is quickly placed into that have mixed up temperature be to keep sink condition with 80rpm in 0 DEG C of constant-temperature table
Revolving speed be incubated in, be denoted as 4. number conical flask.Residual buffer liquid is also placed at corresponding identical temperature, for mending sample.
From starting timing, successively in 1h, 2h, 4h, 6h, 12h, for 24 hours, 36h, 48h, 60h, 72h when, to 4 conical flasks
2mL is respectively sampled outside bag filter to be fitted into 5mL centrifuge tube, is supplemented synthermal equivalent medium after sampling immediately, is protected total volume
It holds constant.So each conical flask samples 10, totally 40 samples.
Using high performance liquid chromatography and ultraviolet-visible spectrophotometer test sample data, slow release is probed into.As a result as attached
Shown in Fig. 3, slow release effect is good, it can be seen that preceding 8 hour drugs have biggish release rate, and in 2d, drug can be basic
Reach maximum burst size.Alkalinity helps to discharge, and acidity is unfavorable for discharging；Low temperature makes to discharge slack-off, is unfavorable for drug release.
6 cytotoxicity experiment of embodiment
Using CCK-8 kit (being purchased from the enzyme-linked Biotechnology Co., Ltd in Shanghai) measure Ad-CHO, TEPACS and
Inhibiting rate of the TEPACS-Ad to mouse embryonic fibroblasts (MEF cell is purchased from Shanghai Ge Fan Biotechnology Co., Ltd).
First with cell culture fluid (+10% fetal calf serum of 90%RPMI-1640 culture medium it is+1% dual anti-(200mM L-Glutamine,
10mg/mL streptomysin and 10000U penicillin)) the test substance solution of configuration series of concentrations, then cell Proliferation-toxicity detection
Experimental method is according to following operating procedure:
(1) cell suspension of 100 μ L is configured in 96 orifice plates.By culture plate incubator preculture for 24 hours (37 DEG C, 5%
CO2), so that every hole is about 5000 cells.
(2) test substance of 10 μ L various concentrations is added to culture plate.
(3) culture plate is incubated for 48h in incubator.
(4) 10 μ L CCK solution are added to every hole.
(5) culture plate is incubated for 4h in incubator.
(6) absorbance at 450nm is measured with microplate reader.
Using inhibiting rate=1- (measurement group-blank group)/(control group-blank group), calculated using SPSS software various
Material compares the inhibition concentration of corresponding cell.
Using CCK-8 kit measurement Ad-CHO, TEPACS, TEPACS-Ad, HCPT-COOH, HCPT-CD and TEPACS-
Inhibiting rate of the Ad/HCPT-CD to Hep G2 liver cancer cells (being purchased from Shanghai Ge Fan Biotechnology Co., Ltd).It is trained first with cell
Nutrient solution (+10% fetal calf serum of 90%RPMI-1640 culture medium+1% dual anti-(200mM L-Glutamine, 10mg/mL streptomysin
With 10000U penicillin)) configuration series of concentrations test substance solution, then cell Proliferation-toxicity detection experimental method is same as
The above operating procedure.It obtains as a result, the survival rate of normal cell is apparently higher than the survival rate of Hep G2 liver cancer cells, preparation resists
The kill tumour cell of the tumour slow releasing pharmaceutical material property of can choose.
A kind of antitumor slow releasing pharmaceutical material of specific example preparation of the present invention, carries out nuclear-magnetism identification, medicament slow release to it
Test, toxicity assessment etc. obtain good experimental result.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of preparation method of antitumor slow releasing pharmaceutical material, characterized by comprising the steps of:
(1) HCPT-COOH is weighed, is dissolved in solvent, is stirred at room temperature, Tanabe Seiyoku is added, crosslinking agent is added, room temperature is anti-
It answers；Low-temperature aqueous solution is added, low temperature is static, and solid is precipitated；Centrifugation goes out solid, HCPT-CD obtained by drying；
(2) synthesis of TEPACS is divided into three steps；A: weighing chitosan, dissolution；It is slowly added into methanol, continues to stir, stops stirring
It mixes, obtains solution A, for use；B: tetraethylenepentamine is dissolved, and obtains tetraethylenepentamine solution；By dissolution of benzaldehyde, then it is added
Into tetraethylenepentamine solution, it is heated to reflux；Epichlorohydrin reaction is slowly added dropwise, obtains B solution；C: it is molten that A is added in B solution
In liquid, stir at room temperature；PH is adjusted, is precipitated, is filtered, washing is dried in vacuo up to TEPACS；
(3) TEPACS for obtaining step (2) dissolves, and obtains TEPACS solution；Ad-CHO dissolution is weighed, is then added into
In TEPACS solution, room temperature reaction；Methanol is rotated out, is washed, dry product TEPACS-Ad；The Ad-CHO is 1- gold
Rigid alkane formic acid -4- benzaldehyde ester；
(4) step (3) resulting TEPACS-Ad is weighed, is dissolved, HCPT-CD is added, dissolve and makes the sufficiently nested Buddha's warrior attendant of cyclodextrin
Alkane, it is dry, obtain the antitumor slow releasing pharmaceutical material TEPACS-Ad/HCPT-CD of product；
Crosslinking agent described in step (1) is any one or two kinds of EDCHCl and NHS.
2. the preparation method of antitumor slow releasing pharmaceutical material according to claim 1, it is characterised in that:
Solvent described in step (1) is any one or at least two in DMSO, THF and water.
3. the preparation method of antitumor slow releasing pharmaceutical material according to claim 1, it is characterised in that:
Final concentration of 3.75mmol/L~10mmol/L of HCPT-COOH described in step (1)；
Final concentration of 3.75mmol/L~10mmol/L of Tanabe Seiyoku is added described in step (1).
4. the preparation method of antitumor slow releasing pharmaceutical material according to claim 1, it is characterised in that:
The mixing time being stirred at room temperature described in step (1) is 1~5h；
The reaction time of room temperature reaction described in step (1) be 6~for 24 hours.
5. the preparation method of antitumor slow releasing pharmaceutical material according to claim 1, it is characterised in that:
Final concentration of 0.05mol/L~0.25mol/L of the chitosan in solution A described in step (2) A；
The time of dissolution described in step (2) A is 1~3d；
The time for continuing stirring described in step (2) A is 3~6h.
6. the preparation method of antitumor slow releasing pharmaceutical material according to claim 1, it is characterised in that:
Final concentration of 1~5mol/L of the tetraethylenepentamine in B solution described in step (2) B；
Final concentration of 1~5mol/L of the benzaldehyde in B solution described in step (2) B；
Final concentration of 1~5mol/L of the epoxychloropropane in B solution described in step (2) B.
7. the preparation method of antitumor slow releasing pharmaceutical material according to claim 1, it is characterised in that:
The condition being heated to reflux described in step (2) B be 40~80 DEG C reflux 12~for 24 hours；
The condition of reaction described in step (2) B be 40~60 DEG C reaction 12~for 24 hours；
The time stirred at room temperature described in step (2) C is 4~5d；
PH described in step (2) C is 5~6.
8. the preparation method of antitumor slow releasing pharmaceutical material according to claim 1, it is characterised in that:
The molar ratio of chitosan monomer and Ad-CHO in TEPACS described in step (3) is (2~5): 1；Institute in step (3)
The time for the reaction stated is 6~12h；
The molar ratio of chitosan monomer and HCPT-CD in TEPACS-Ad described in step (4) is (4~15): 1.
9. a kind of antitumor slow releasing pharmaceutical material, it is characterised in that pass through preparation method system according to any one of claims 1 to 8
It is standby to obtain.
10. antitumor slow releasing pharmaceutical material as claimed in claim 9 is preparing the application in antitumor slow releasing pharmaceutical.
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