CN108295265A - Cellulose nanocrystal is the acidic environment sensitivity nano prodrug system and preparation method of carrier - Google Patents
Cellulose nanocrystal is the acidic environment sensitivity nano prodrug system and preparation method of carrier Download PDFInfo
- Publication number
- CN108295265A CN108295265A CN201810101093.5A CN201810101093A CN108295265A CN 108295265 A CN108295265 A CN 108295265A CN 201810101093 A CN201810101093 A CN 201810101093A CN 108295265 A CN108295265 A CN 108295265A
- Authority
- CN
- China
- Prior art keywords
- acidic environment
- carrier
- cellulose nanocrystal
- drug
- nano
- 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.)
- Pending
Links
- 0 C*CC(C)Cl* Chemical compound C*CC(C)Cl* 0.000 description 2
Classifications
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7024—Esters of saccharides
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
- A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
- A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
Abstract
The invention discloses acidic environment sensitivity nano prodrug systems and preparation method that a kind of Cellulose nanocrystal is carrier, the prodrug system includes nanocrystalline rod-like fibre element, connection unit and drug, the nanocrystalline connection unit for carrier, acidic environment sensitivity of rod-like fibre element connects drug, has Formulas I structure；Its preparation process includes：(1) amidized Cellulose nanocrystal is prepared；(2) drug and cis-aconitic anhydride (CAA) open loop；(3) nano prodrug system for the acidic environment sensitivity that Cellulose nanocrystal is carrier is prepared for by out-phase reaction.The prodrug system prepared through the invention realizes the drug controlled release of affected area slightly acidic environment sensitivity.Cellulose nanocrystal has many advantages, such as that good biocompatibility, nontoxic and rodlike carrier are conducive to cell phagocytosis as carrier.Fluorescence probe is utilized simultaneously, detects the endocytosis behavior of nano-prodrug, has ideal cell endocytic and inhibits tumor cell proliferation ability.
Description
Technical field
The invention belongs to biological medicine technology, the application fields of nanometer medicine and recyclable materials, and in particular to rodlike
Cellulose nanocrystal is carrier, using the chemical bond of acidic environment sensitivity as the nano prodrug system of link unit and preparation method and
Using.Carrier of the recyclable organism material fiber element nanocrystal as structure nano-prodrug, the advantage is that its good life
Object compatibility is conducive to cell endocytic, and rodlike shape is conducive to enhance cell endocytic.Affected area slightly acidic environment sensitivity can
The introducing of the chemical bond of fracture can realize the drug controlled release of fixed point.
Background technology
As Global Environmental Problems are increasingly prominent, the mankind increase with the relevant behavioral activity of carcinogenicity, global tumor invasion
Rate persistently increases.Chemotherapy is still one of the main means for the treatment of tumour at present, but because self structure and physiological distribution etc. are many
Limitation limit further increasing for its curative effect, such as serious toxic side effect, poor selectivity, tumour cell easily generate it
Multidrug resistance etc..
The nano-medicament carrier (such as liposome, micella etc.) built by the methods of self assembly can make full use of entity
The Thief zone of tumor and long retention effect (EPR effects), make drug-rich in tumor tissues.But such support shapes is difficult to keep,
It is easy to rupture in blood circulation and antitumor drug is caused to discharge in advance, cannot achieve the release of drug fixed point and treatment.Simultaneously
Such carrier there is also it is inadequate to tumor tissues slightly acidic environment susceptibility the defects of, reach tumor tissues after cannot discharge in time
Antitumor drug, therapeutic effect are limited.
Cellulose nanocrystal (CNC) is to remove amorphous area in natural fiber, retains cellulose nanometer material obtained by crystalline region
Material, belonging to one of three big nano materials derived from cellulose, (nano-cellulose can be divided into according to preparation method, cellulose nanometer
Crystalline substance, microcrystalline cellulose and bacteria cellulose).Cellulose nanocrystal is bar-like nano particle, in animal body with can be in cell
Its rhabdolith structure is kept well.Computer mould fits theoretical research and shows：Compared with ball shaped nano drug, rod-like nano
Drug is conducive to enhance cell phagocytosis.
Cellulose nanocrystal body is used to prepare to the nano prodrug system of acid-sensitive, its good biofacies can be played
The form effect of capacitive, nanometer size effect and rhabdolith, enhancing cell endocytic improve therapeutic effect.
Invention content
In order to overcome drawbacks described above, it is nanocrystalline as carrier that the object of the present invention is to provide a kind of rod-like fibre elements, with right
The amido bond of tumor tissues slightly acidic environment sensitivity is the nano prodrug system of connection unit.The nano prodrug system is closed by it
Suitable particle size is enriched with using the long retention effect of Thief zone at tumor tissues in affected area passive target.While carrier
Rod-like shape is also conducive to cell endocytic, and the amido bond of acidic environment sensitivity discharges drug in time so that the nano prodrug system
Has the possibility for improving anti-swollen medication effect and reducing its toxic side effect.The present invention constructs " subacidity at lesion tissue
The nano prodrug system of environment sensitive, cell endocytic enhancing ".Nano-prodrug prepared by the present invention has good fixed point to be released in time
Put the ability of medicine, good cell endocytic and cell inhibitory effect ability.
Realizing the specific technical solution of the object of the invention is：
A kind of Cellulose nanocrystal is the acidic environment sensitivity nano prodrug system of carrier, which is characterized in that the prodrug system
System includes nanocrystalline rod-like fibre element, connection unit and drug, and rod-like fibre element is nanocrystalline for carrier, the company of acidic environment sensitivity
Order member connects drug, has formula：
Wherein, the drug is adriamycin, mitomycin, gemcitabine hydrochloride, Epi-ADM, actinomycin D, the soft ratio of pyrrole
Star；The connection unit is the connection unit of acidic environment sensitivity, specially：β-carboxyl-amido bond.
The connection unit of the acidic environment sensitivity is that the degradation of acidic environment sensitivity may be implemented under lesion microenvironment to break
Split release medicine.
A kind of Cellulose nanocrystal is the preparation method of the nano prodrug system of the acidic environment sensitivity of carrier：This method packet
Include following steps：
(1) Cellulose nanocrystal surface amination (CNC-NH2)
Cellulose nanocrystal is dispersed in the NaOH solution of 1M, cellulose dispersion liquid concentration is controlled in (0.01-0.02) g/
ML, ultrasound (30-60) min, are added dropwise epoxychloropropane (0.01-0.2) mmol/g, and temperature is controlled in (55-65) DEG C range
It is interior, stir 2h, dialysis, until the pH value of dialyzed solution is 12 or so；It is adjusted in bag filter using the NaOH solution of 50% (w/v)
Solution ph, is added concentrated ammonia liquor, and cellulose concentration is controlled in (0.05-0.2) g/mL；Stirring, dialysis, until dialyzate reaches pH
Value is freeze-dried afterwards at (7-7.4)；Deepfreeze；Obtain the Cellulose nanocrystal CNC-NH of surface amination2, with following formula
Structure；
(2) drug is reacted with connection unit
Drug is dissolved in anhydrous methylene chloride and forms solution A, concentration is controlled in (0.5-2) mg/mL, and three second are added dropwise
Amine is protected from light, nitrogen protection, stirs (2-6) h；Connection unit compound is weighed, anhydrous methylene chloride, ultrasonic (20-60) are dissolved in
Min is added dropwise in solution A, is protected from light, nitrogen protection, and (8-24) h is stirred at room temperature；Vacuum distillation removes dichloromethane, column chromatography
Separation, mobile phase are alkanolamine solution and dichloromethane；It is 98% that performance liquid chromatographic column, which detects purity, and vacuum distillation removes dichloromethane
Alkane obtains sterling CAD；Wherein, the molar ratio of drug and triethylamine is 1: 16；The molar ratio of drug and connection unit is 1: 4；Stream
Dynamic phase volume ratio is: carbinolamine: dichloromethane=1: 500；It is for use that product B is prepared；
(3) it is to carry the acidic environment sensitivity nano-prodrug stopped to prepare Cellulose nanocrystal
Product B in step (2) is dissolved in anhydrous DMSO, oxalyl chloride is added, is protected from light after 1h is stirred at room temperature, amino is added
The Cellulose nanocrystal of change, ultrasound (20-60) min, is protected from light, nitrogen protection is stirred at room temperature；High speed centrifugation, after removing supernatant, DSMO
Wash and centrifuge three times, abandon supernatant, retain seabed sediment, be freeze-dried the Cellulose nanocrystal be to carry acyclic acidic not
Border sensitivity nano-prodrug；Wherein, the molar ratio of product B and oxalyl chloride is 1: 1；Product B and amidized Cellulose nanocrystal
Mass ratio is 1: 3.
Above-mentioned Cellulose nanocrystal is to carry the acidic environment sensitivity nano prodrug system stopped in the preparation of antitumor drugs
Using.
The beneficial effects of the present invention are：The present invention prepares the nanometer for the acidic environment sensitivity that Cellulose nanocrystal is carrier
Prodrug relies on the nanometer size effect of Cellulose nanocrystal, has good passive target ability；It is real using fluorescence imaging simultaneously
When detect cell endocytic and drug distribution ability；And the sensitive degradation solution release in time medicine under intralesional microenvironment,
Has the ability of environmental response release drug；And cell endocytic and the suppression of good tumor cell proliferation of rodlike effect enhancing
Ability processed.
Description of the drawings
Fig. 1 is the nano prodrug system structural schematic diagram for the acidic environment sensitivity drug release that Cellulose nanocrystal is carrier.
Fig. 2 is CNC-CAD preparation route figures；
Fig. 3 is the mass spectrogram of CAD；
Fig. 4 is the high-efficient liquid phase chromatogram of CAD；
Fig. 5 is the X-ray diffractogram of CNC-CAD；
Fig. 6 CNC-CAD and CNC the dispersion stabilization figure in pure water；Left figure：From left to right it is followed successively by the CNC- of 1mg/mL
CAD；CNC and DIwater；Right figure is：By the left-to-right respectively CNC-CAD and CNC of 1mg/mL；
Fig. 7 CNC, CNC-NH2, the transmission electron microscope picture of CNC-CAD；
a：The length of CNC is in (200-400) nm ranges, and width is in (10-20) nm ranges；b：CNC-NH2Length be about
200nm, width 5-10nm；c：The length of CNC-CAD is about 200nm, width 5-10nm；
Fig. 8 is doxorubicin hydrochloride standard working curve figure；
Fig. 9 is the metabolic chart figures of CNC-CAD at various ph values；
Figure 10 is DOX, the KB cell proliferation inhibition assay result curve graphs of CNC-CAD, CNC；
Figure 11 is NH4After Cl preincubates, CNC-CAD acts on the laser co-focusing electron micrograph after KB cells 2h；
From left to right, picture is according to this：White light, DOX, Hoechst, superposition (white light, DOX and the superposition of Hoechst figures)；
Figure 12 is that CNC-CAD acts on the laser co-focusing electron micrograph after KB cells 2h；
From left to right, picture is according to this：White light, DOX, Hoechst, superposition (white light, DOX and Hoechst superpositions)；
Figure 13 is the endocytosis effect contrast figure of flow cytometry results, DOX and CNC-CAD on KB cell strains；
Figure 14 is that the DOX of low pH controls in the structural schematic diagram and lysosome of CNC-CAD discharges process schematic into the cell.
Specific implementation mode
In conjunction with following specific examples and attached drawing, the present invention is described in further detail.The process of the implementation present invention,
Condition, experimental method etc. are among the general principles and common general knowledge in the art, this hair in addition to the following content specially referred to
It is bright that content is not particularly limited.
The present invention is a kind of nano-prodrug to be released the drug as carrier, acidic environment sensitivity using Cellulose nanocrystal.It is natural raw
Object nano material Cellulose nanocrystal can make the nano-prodrug passive target be enriched in affected area (tumor group as carrier
It knits)；Simultaneously by the form effect of Cellulose nanocrystal good biocompatibility and Rod-like shape, cell can be significantly increased
Endocytosis, into cell after acidic environment sensitivity amido bond can be broken in lysosomal acid environment release free drug Ah
Mycin；Interfering nucleic acid replicates, blocks cell Proliferation after drug adriamycin enters nucleus.The nano-prodrug structure such as following formula：
Wherein, the structure such as following formula of the connection unit of acidic environment sensitivity：
The present invention proposes a kind of nano prodrug system for the acidic environment sensitivity that Cellulose nanocrystal is carrier simultaneously
Preparation method：It the described method comprises the following steps：(1) amido modified Cellulose nanocrystal is prepared；(2) drug adriamycin and suitable
Intermediate product CAD is obtained by the reaction in formula aconitic anhydride；(3) CAD and amidized Cellulose nanocrystal are prepared for receiving through condensation reaction
Rice prodrug CNC-CAD.
Specific implementation step is as follows：
The first step：Cellulose nanocrystal surface amination (CNC-NH2)
Cellulose nanocrystal is dispersed in the NaOH solution of 1M, epoxychloropropane is added dropwise, temperature is controlled at (55-65)
Within the scope of DEG C, after stirring, dialysis, until the pH value of dialyzed solution is 12 or so；It is adjusted using the NaOH solution of 50% (w/v) saturating
Solution ph in bag is analysed, concentrated ammonia liquor is added, is stirred, dialysis is freeze-dried until dialyzate reaches pH value at (7-7.4) afterwards；It is low
Temperature refrigeration；Obtain the Cellulose nanocrystal of surface amination.
Second step：Drug adriamycin open loop cis-aconitic anhydride prepares CAD and its characterization
With cis-aconitic anhydride ring-opening reaction occurs for No. 3 bit aminos of adriamycin, generates the β-carboxylic that can be broken in acidic environment
Base-amido bond, mass spectrum (MS) go to monitor and characterize, and as a result see figure attached drawing 3；It is prepared using high performance liquid chromatography (HPLC) pure
CAD, as a result see attached drawing 4.
Third walks：Cellulose nanometer is preparation and the characterization of the nano-prodrug of the acidic environment sensitivity of carrier
CNC-NH2Acid-sensitive prodrug is prepared by carbodiimides condensation reaction with CAD carboxyls, its load is measured with microplate reader
Dose；It is observed with transmission electron microscope (TEM) and determines its Rod-like shape；The glimmering of adriamycin is detected with inverted fluorescence microscope
Light imaging effect；Its cell endocytic is had detected with laser confocal microscope.
Comprehensive first walks to obtain the nano-prodrug knot for the acidic environment sensitivity drug release that Cellulose nanocrystal is carrier to third
Structure schematic diagram is shown in attached drawing 1.
4th step：Cellulose nanometer is degradation and the control release performance of the nano-prodrug of the acidic environment sensitivity of carrier
With microplate reader monitor Ah's nano-prodrug CNC-CAD under slightly acidic environment (pH=6.5,6.0,5.0；Simulate tumour
Tissue and intracellular lysosome microenvironment) and alkalescence environment under (pH=7.4 simulates normal blood environment) degradation drug release
Performance.
5th step：Cellulose nanometer is the Study of cytotoxicity of the nano-prodrug of the acidic environment sensitivity of carrier
Tetrazolium bromide (MTT) colorimetric determination Cellulose nanocrystal, acid-sensitive nano-prodrug CNC-CAD and DOX are in pH=7.4
With KB cytotoxicities under pH=5.0 condition of culture.
6th step：Cellulose nanometer is the cell endocytic of the nano-prodrug of the acidic environment sensitivity of carrier
MCF-7 cells are had studied in various concentration and act on the cell endocytic behavior of different time, with flow cytometer,
It sets fluorescence microscope and laser confocal microscope detects it and swallows effect.
1 drug of embodiment and cis-aconitic anhydride reactant
1.1 drug adriamycin -3-NH2CAD is prepared with cis-aconitic anhydride
Doxorubicin hydrochloride 10mg is weighed, is dissolved in 3ml anhydrous methylene chlorides (DCM), 40 μ l of triethylamine are added dropwise and are protected from light,
Nitrogen protection stirs 2h.Cis-aconitic anhydride 10mg is weighed, 3ml anhydrous methylene chlorides, ultrasonic 20min are dissolved in.By the cis- rhizome of Chinese monkshood
Anhydride solution is added dropwise in the DCM solution of adriamycin, is protected from light, and 8h is stirred at room temperature in nitrogen protection.Vacuum distillation removes dichloromethane
Alkane, column chromatography for separation, mobile phase are alkanolamine solution and dichloromethane.(mobile phase ratio is carbinolamine: dichloromethane=1:
500) it is 98% that, high performance liquid chromatography, which lives to detect purity, and vacuum distillation obtains sterling except dichloromethane.Synthetic route schematic diagram is shown in
The first step in attached drawing 2.The chemical characterization of CAD uses, mass spectrum (result is shown in the MS figures of attached drawing 3CAD), high performance liquid chromatography (knot
Fruit sees the HPLC of attached drawing 4CAD).
1.2 drug pirarubicin prepare PAD with cis-aconitic anhydride
Pirarubicin 10mg is weighed, is dissolved in 3ml anhydrous methylene chlorides, 40 μ L of triethylamine are added dropwise, are protected from light, nitrogen is protected
Shield stirs 2h.Cis-aconitic anhydride 10mg is weighed, 3ml anhydrous methylene chlorides, ultrasonic 30min are dissolved in.Cis-aconitic anhydride is molten
Liquid is added dropwise in the DCM solution of pirarubicin.It is protected from light, 10h is stirred at room temperature in nitrogen protection.Vacuum distillation removes dichloromethane,
Column chromatography for separation, mobile phase are alkanolamine solution and dichloromethane.(mobile phase ratio is carbinolamine: dichloromethane=1: 500), high
It is 97% that effect liquid phase chromatogram, which lives to detect purity, and vacuum distillation obtains sterling except dichloromethane.
1.3 Mitomycin Cs prepare MAD with cis-aconitic anhydride
Mitomycin 10mg is weighed, is dissolved in 3ml anhydrous methylene chlorides, 40 μ L of triethylamine are added dropwise, are protected from light, nitrogen is protected
Shield stirs 2h.Cis-aconitic anhydride 10mg is weighed, 3ml anhydrous methylene chlorides, ultrasonic 30min are dissolved in.Cis-aconitic anhydride is molten
Liquid is added dropwise in the dichloromethane solution of pirarubicin.It is protected from light, nitrogen protection, (8-24) h is stirred at room temperature.Vacuum distillation removes
Dichloromethane, column chromatography for separation, mobile phase are alkanolamine solution and dichloromethane.(mobile phase ratio is carbinolamine: dichloromethane=
1: 500), it is 98% that high performance liquid chromatography, which lives to detect purity, and vacuum distillation obtains sterling except dichloromethane.
1.4 gemcitabines prepare GAD with cis-aconitic anhydride
Gemcitabine 10mg is weighed, is dissolved in 3ml anhydrous methylene chlorides, 40 μ L of triethylamine are added dropwise, are protected from light, nitrogen is protected
Shield stirs 2h.Cis-aconitic anhydride 10mg is weighed, 3ml anhydrous methylene chlorides, ultrasound (20-60) min are dissolved in.By the cis- rhizome of Chinese monkshood
Anhydride solution is added dropwise in the dichloromethane solution of gemcitabine.It is protected from light, nitrogen conservation, (8-24) h is stirred at room temperature.Decompression
Distillation removes dichloromethane, column chromatography for separation, and mobile phase is alkanolamine solution and dichloromethane.(mobile phase ratio is carbinolamine: dichloro
Methane=1: 500), it is 98% that high performance liquid chromatography, which lives to detect purity, and vacuum distillation obtains sterling except dichloromethane
The amination on 2 Cellulose nanocrystal surface of embodiment
Cellulose nanocrystal (100-200) mg is weighed, NaOH solution (100-200) ml/g cellulose nanometers of 1M are dispersed in
Epoxychloropropane (6-20) mmol/g Cellulose nanocrystals are added dropwise in crystalline substance, ultrasound (30-60) min, and (55-65) DEG C stirs 2h,
(MWCO=1000-3500) dialysis dialyse until the pH value of dialyzed solution is less than 12.It will be saturating using 50% (w/vNaOH) solution
It analyses solution ph in bag to adjust to (11-13), concentrated ammonia liquor, (5-10) ml/g cellulose, (55-65) DEG C stirring (1- is added
4) h, dialysis, until the pH value of dialyzate is (7-7.4).Freeze-drying.Deepfreeze.(synthetic route schematic diagram is shown in attached drawing 2
Second step).
3 Cellulose nanocrystal of embodiment is the preparation of the acidic environment sensitivity nano-prodrug of carrier
3.1 Cellulose nanocrystals are the preparation of the acidic environment sensitivity nano-prodrug CNC-CAD of carrier
CAD 25mg are weighed to be dissolved in (5-10) ml anhydrous dimethyl sulphoxides (DMSO), oxalyl chloride (10-50) μ L are protected from light,
1h is stirred at room temperature.The amidized Cellulose nanocrystals of (100-200) mg are weighed, are scattered in the anhydrous DMSO of (3-10) ml, are surpassed
Sound (20-60) min, is added dropwise in solution of acid chloride, is protected from light, and (8-24) h is stirred at room temperature in nitrogen protection.High speed centrifugation is except upper
After clear liquid, DMSO is washed and is centrifuged three times, abandons supernatant, retains seabed sediment.Deposit is dialysed (in the PBS of pH=7.4
Dialysis, MWCO=1000--3500), freeze-drying obtains sterling.(synthetic route schematic diagram is shown in that third walks in attached drawing 2).
3.2 Cellulose nanocrystals are the preparation of the acidic environment sensitivity nano-prodrug CNC-PAD of carrier
It weighs PAD (25-50) mg to be dissolved in (5-10) ml anhydrous dimethyl sulphoxides (DMSO), oxalyl chloride (10-50) μ L are kept away
1h is stirred at room temperature in light.The amidized Cellulose nanocrystals of (100-200) mg are weighed, are scattered in the anhydrous DMSO of (3-10) ml,
Ultrasound (20-60) min, is added dropwise in solution of acid chloride, is protected from light, (8-24) h is stirred at room temperature in nitrogen protection.High speed centrifugation removes
After supernatant, DMSO is washed and is centrifuged three times, abandons supernatant, retains seabed sediment.Deposit dialysis (the PBS of pH=7.4
Middle dialysis, MWCO=1000--3500), freeze-drying obtains sterling.
3.3 Cellulose nanocrystals are the preparation of the acidic environment sensitivity nano-prodrug CNC-MAD of carrier
It weighs MAD (25-50) mg to be dissolved in (5-10) ml anhydrous dimethyl sulphoxides (DMSO), oxalyl chloride (10-50) μ L are kept away
1h is stirred at room temperature in light.The amidized Cellulose nanocrystals of (100-200) mg are weighed, are scattered in the anhydrous DMSO of (3-10) ml,
Ultrasound (20-60) min, is added dropwise in solution of acid chloride, is protected from light, (8-24) h is stirred at room temperature in nitrogen protection.High speed centrifugation removes
After supernatant, DMSO is washed and is centrifuged three times, abandons supernatant, retains seabed sediment.Deposit dialysis (the PBS of pH=7.4
Middle dialysis, MWCO=1000--3500), freeze-drying obtains sterling.
3.4 Cellulose nanocrystals are the preparation of the acidic environment sensitivity nano-prodrug CNC-GAD of carrier
It weighs GAD (25-50) mg to be dissolved in (5-10) ml anhydrous dimethyl sulphoxides (DMSO), oxalyl chloride (10-50) μ L are kept away
1h is stirred at room temperature in light.The amidized Cellulose nanocrystals of (100-200) mg are weighed, are scattered in the anhydrous DMSO of (3-10) ml,
Ultrasound (20-60) min, is added dropwise in solution of acid chloride, is protected from light, (8-24) h is stirred at room temperature in nitrogen protection.High speed centrifugation removes
After supernatant, DMSO is washed and is centrifuged three times, abandons supernatant, retains seabed sediment.Deposit dialysis (the PBS of pH=7.4
Middle dialysis, MWCO=1000--3500), freeze-drying obtains sterling.
4 dispersibility of embodiment, stability and crystal structure are investigated
CNC-CAD, CNC are dispersed in pure water, and adjusting concentration makes its dispersion concentration in 1mg/mL, stands and stores and see
It examines and takes pictures；As a result see attached drawing 6.
With X-ray powder diffraction (XRD) (Bruker D8Advance) CNC nanocrystalline to powdery cellulose, amination
Cellulose nanocrystal CNC-NH2Crystal structure determination is carried out with the rodlike adriamycin prodrug CNC-CAD of acid-sensitive.XRD results are shown in
Attached drawing 7.
5 Cellulose nanocrystal of embodiment is the measurement of the acidic environment sensitivity nano-prodrug drugloading rate of carrier
It weighs 1mg doxorubicin hydrochlorides and is dissolved in a series of solution for preparing various concentrations in 1ml PBS using gradient dilution method,
Concentration is respectively following (μ g/mL)：
0.1,0.2,0.4,0.6,0.8,1.0,1.5,2.0,4.0,8.0,10.0,20.0,30.0,40.0,50.0.
It is that the acidic environment sensitivity nano-prodrug CNC-CAD of carrier is scattered in the PBS of 1mL to take 1mg Cellulose nanocrystals,
Gradient dilution method configures a series of CNC-CAD dispersion liquids of various concentrations, and concentration is respectively following (μ g/mL)：
500.0,300.0,250.0,200.0,150.0,100.0,90.0,80.0,70.0,60.0,50.0,40.0,
30.0,20.0,10.0.
Its drugloading rate (DLC) and encapsulation rate (DLE) are measured with microplate reader quantitative fluorescence method, by the solution and nanometer of adriamycin
The PBS dispersion liquids of prodrug are added in blackboard, and per 20 μ L of hole, excitation wavelength 490nm, transmitted wave wavelength is 580nm according to fluorescence
Intensity value draws the standard working curve of DOX, and drugloading rate and encapsulation rate are respectively 0.057% and 8.0%.
Drugloading rate (DLC%)=(gross mass of quality/nano-prodrug of DOX on nano-prodrug) × 100%
Encapsulation rate (DLE%)=(quality that the DOX mass/DOX connected on nano-prodrug feeds intake) × 100%.
6 Cellulose nanocrystal of embodiment is drug release of the acidic environment sensitivity nano-prodrug of carrier in different pH medium
Behavior
Release Performance of the nano-prodrug in different pH buffer solutions is measured with microplate reader quantitative fluorescence method.Before taking 25mL nanometers
Medicine CNC-CAD (1mg/mL) is respectively placed in pH=7.4, and dialyse (MWCO=3500) for 24 hours in 6.5,5.5,5.0 buffer solutions, uses enzyme
The absorbance that instrument quantitative fluorescence method measures each group after dialysis is marked to paint according to drawn DOX standard curves (see attached drawing 8)
Metabolic chart result is made and sees attached drawing 9.
7 Cellulose nanocrystal of embodiment is the Study of cytotoxicity of the acidic environment sensitivity nano-prodrug of carrier
To study the in vitro toxicity of nano-prodrug CNC-CAD, the MTT experiment for having chosen KB cells carries out checking research.
Rodlike CNC, DOX are chosen, is control experiment group.Having investigated CNC, DOX and CNC-CAD, (KB is thin to human mouth epidermoid carcinoma cell
Born of the same parents) Proliferation Ability situation.
Cell culture processes are：By the KB cell inoculations in exponential phase in 96 orifice plates per 180 μ L of hole, constant-temperature incubation
In case after overnight incubation, every group is separately added into CNC, DOX and the CNC-CAD that 20 μ L contain a series of concentration gradients, DOX concentration
(or DOX concentration of identical equivalent) is (μ g/mL)：0.1,0.2,0.4,0.6,0.8,1,2, constant-temperature incubation case is incubated for 24 hours；It inhales
80 μ L of culture medium are removed, continues to cultivate 4h after 10 μ L MTT solution (5mg/mL) are added, it is molten that 100 μ L, tri- liquid lytic cells is added
It is retired to praise, it measures absorbance at 570nm wavelength with microplate reader and calculates cell survival rate.Cell survival rate calculation formula is as follows：
Cell survival rate (%)=(ODExperimental group-ODBlank group/ODControl group-ODBlank group) × 100%
As a result see attached drawing 10.
The cell phagocytosis behavior of embodiment 8, the acidic environment sensitivity nano-prodrug that Cellulose nanocrystal is carrier
By KB cells in culture dish (Greiner, four lattices) preincubate for 24 hours (1.0 × 105A cells/well) after, respectively
Complete medium and the NH containing 10mM is added4The culture medium preincubate 30min of Cl is separately added into containing CNC- in four lattices
The culture medium of CAD (a concentration of 250 μ g/mL) continues after being incubated 2h, discards culture medium, and PBS (pH=7.4) is washed three times,
Hoechst dyes 10min, and PBS (pH=7.4) is washed three times, and 2ml PBS are added, are placed in ice chest, laser co-focusing electron microscopic
Mirror is taken pictures.As a result see attached drawing 11 and 12.
By KB cells in six orifice plates preincubate for 24 hours (1.0 × 105A cells/well), it is separately added into normal complete medium
With the NH containing 10mM4The culture medium of Cl changes culture medium into training containing CNC-CAD (a concentration of 250 μ g/mL) after 30min
Support base.Continue after cultivating 2h, PBS (pH=7.4) washings are centrifuged and washed three times, after digestion collects cell afterwards three times, uses PBS
(pH=7.4) it is scattered in EP pipes, with flow cytomery each group cell fluorescence intensity, as a result sees attached drawing 13.
To sum up, Cellulose nanocrystal is that the cell phagocytosis behavior of the acidic environment sensitivity nano-prodrug of carrier can be by attached
Figure 14 is specifically annotated.
The protection content of the present invention includes but is not limited to above example.In the scope without departing substantially from inventive concept
Under, various changes and advantages that will be apparent to those skilled in the art are all included in the present invention, and with the attached claims
Book is protection domain.
Claims (3)
1. a kind of Cellulose nanocrystal is the acidic environment sensitivity nano prodrug system of carrier, which is characterized in that the prodrug system
Nanocrystalline, connection unit and drug including rod-like fibre element, the nanocrystalline connection for carrier, acidic environment sensitivity of rod-like fibre element
Unit connects drug, has formula：
Wherein, the drug be adriamycin, mitomycin, gemcitabine hydrochloride, Epi-ADM, actinomycin D, pirarubicin or
Other drugs with amino of person；The connection unit is the connection unit of acidic environment sensitivity, specially：β-carboxyl-amide
Key.
2. nano prodrug system according to claim 1, which is characterized in that the connection unit of the acidic environment sensitivity is
Acidic environment sensitivity degradation fracture release medicine may be implemented under lesion microenvironment.
3. a kind of Cellulose nanocrystal is the preparation method of the nano prodrug system of the acidic environment sensitivity of carrier：Its feature exists
In this approach includes the following steps：
(1) Cellulose nanocrystal surface amination (CNC-NH2)
Cellulose nanocrystal is dispersed in the NaOH solution of 1M, the control of cellulose dispersion liquid concentration surpasses in (0.01-0.02) g/mL
Sound (30-60) min, is added dropwise epoxychloropropane (0.01-0.2) mmol/g, and temperature control is stirred within the scope of (55-65) DEG C
2h is mixed, is dialysed, until the pH value of dialyzed solution is 12 or so；Solution in bag filter is adjusted using the NaOH solution of 50% (w/v)
PH value, is added concentrated ammonia liquor, and cellulose concentration is controlled in (0.05-0.2) g/mL；Stirring, dialysis, exists until dialyzate reaches pH value
(7-7.4) is freeze-dried afterwards；Deepfreeze；Obtain the Cellulose nanocrystal CNC-NH of surface amination2, with following formula knot
Structure；
(2) drug is reacted with connection unit
Drug is dissolved in anhydrous methylene chloride and forms solution A, concentration is controlled in (0.5-2) mg/mL, and triethylamine is added dropwise and keeps away
Light, nitrogen protection stir (2-6) h；Connection unit compound is weighed, is dissolved in anhydrous methylene chloride, ultrasound (20-60) min, by
It is added dropwise in solution A, is protected from light, (8-24) h is stirred at room temperature in nitrogen protection；It is evaporated under reduced pressure and removes dichloromethane, column chromatography for separation,
Mobile phase is alkanolamine solution and dichloromethane；It is 98% that performance liquid chromatographic column, which detects purity, and vacuum distillation is obtained except dichloromethane
To sterling CAD；Wherein, the molar ratio of drug and triethylamine is 1: 16；The molar ratio of drug and connection unit is 1: 4；Mobile phase
Volume ratio is：Carbinolamine: dichloromethane=1: 500；It is for use that product B is prepared；
(3) the acidic environment sensitivity nano-prodrug that Cellulose nanocrystal is carrier is prepared
Product B in step (2) is dissolved in anhydrous DMSO, oxalyl chloride is added, is protected from light after 1h is stirred at room temperature, is added amidized
Cellulose nanocrystal, ultrasound (20-60) min, is protected from light, nitrogen protection is stirred at room temperature；High speed centrifugation, except after supernatant, DSMO is washed
And centrifuge three times, abandon supernatant, retain seabed sediment, be freeze-dried the Cellulose nanocrystal be carrier acidic environment it is quick
Feel nano-prodrug；Wherein, the molar ratio of product B and oxalyl chloride is 1: 1；The quality of product B and amidized Cellulose nanocrystal
Than being 1: 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810101093.5A CN108295265A (en) | 2018-02-01 | 2018-02-01 | Cellulose nanocrystal is the acidic environment sensitivity nano prodrug system and preparation method of carrier |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810101093.5A CN108295265A (en) | 2018-02-01 | 2018-02-01 | Cellulose nanocrystal is the acidic environment sensitivity nano prodrug system and preparation method of carrier |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108295265A true CN108295265A (en) | 2018-07-20 |
Family
ID=62850869
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810101093.5A Pending CN108295265A (en) | 2018-02-01 | 2018-02-01 | Cellulose nanocrystal is the acidic environment sensitivity nano prodrug system and preparation method of carrier |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108295265A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363188A (en) * | 2019-12-17 | 2020-07-03 | 华东理工大学 | Preparation method of ultralight cellulose nanocrystal aerogel material |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107254002A (en) * | 2017-08-01 | 2017-10-17 | 张振 | Nano-cellulose containing cinnamyl functional groups and its preparation method and application |
-
2018
- 2018-02-01 CN CN201810101093.5A patent/CN108295265A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107254002A (en) * | 2017-08-01 | 2017-10-17 | 张振 | Nano-cellulose containing cinnamyl functional groups and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| KHALED A. MAHMOUD等: "Effect of Surface Charge on the Cellular Uptake and Cytotoxicity of Fluorescent Labeled Cellulose nanocrystals", 《APPLIED MATERIALS&INTERFACES》 * |
| 张建成等: "肿瘤酸度响应性聚(L-赖氨酸)-阿霉素键合药的制备与表征", 《高等学校化学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363188A (en) * | 2019-12-17 | 2020-07-03 | 华东理工大学 | Preparation method of ultralight cellulose nanocrystal aerogel material |
| CN111363188B (en) * | 2019-12-17 | 2022-11-11 | 华东理工大学 | Preparation method of ultralight cellulose nanocrystal aerogel material |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tan et al. | In vitro study on the individual and synergistic cytotoxicity of adriamycin and selenium nanoparticles against Bel7402 cells with a quartz crystal microbalance | |
| CN104520309B (en) | N- substitution second generation derivatives of antifungal antibiotic amphotericin B and its preparation method and application | |
| CN101240002B (en) | Fatty acylaminoacylcytarabine conjugate, preparation method and application thereof | |
| CN110585441B (en) | Short peptide small molecule self-assembly nano material targeting hypoxic tumor, and preparation method and application thereof | |
| Patra et al. | Cancer cell response to nanoparticles: criticality and optimality | |
| CN111544596B (en) | GSH response type nano-diamond targeted drug and preparation method and application thereof | |
| CN111848544B (en) | Amino acid derivative capable of being traced by fluorescence and preparation method and application thereof | |
| CN106916177A (en) | A kind of deuterated dipeptide boronic acid or its ester type compound and its synthetic method and purposes | |
| CN106668002A (en) | Applications of Gramine and derivatives thereof to preparation of medicaments for resisting adenovirus Type 7 | |
| CN107660210A (en) | Delivery system based on bortezomib | |
| CN108295265A (en) | Cellulose nanocrystal is the acidic environment sensitivity nano prodrug system and preparation method of carrier | |
| CN107837276A (en) | The pharmaceutical composition being locally administered | |
| CN106344539A (en) | Molecular design and preparation technique of novel multifunctional targeted nanocapsule anticancer drug | |
| CN101658484B (en) | Preparation method and application of chitosan copolymer carrier micelle modified by cholesterol formyl chloride | |
| CN104338112A (en) | Preparation method of glutathione nano sustained-release capsules | |
| CN102784397B (en) | Method for loading doxorubicine (DOX) anti-cancer medicine by laponite (LAP) clay nanoparticles | |
| Liu et al. | Spiral assembly of amphiphilic cytarabine prodrug assisted by probe sonication: Enhanced therapy index for leukemia | |
| CN102918051B (en) | N6-(methyl ferrocene) quinazoline-2,4,6-triamine (H2) and its derivative, and the prodrug being used as antiseptic-germicide, antiparasitic, protozoacide and anti-Leishmania agent | |
| CN105622704B (en) | The preparation method and applications of antitumor drug X-TOA | |
| CN105176886B (en) | The application of one plant of spherical lysine bacillus and its crystalline protein and activated product | |
| Li et al. | Preparation and pharmacokinetics of glycyrrhetinic acid and cell transmembrane peptides modified with liposomes for liver targeted-delivery | |
| CN106188236A (en) | A kind of containing glycine, L cysteine, the synthetic peptide of L phenylalanine and application thereof | |
| CN110384680A (en) | A kind of temperature/pH responsiveness is double to carry medicine composite nanoparticle and its preparation method and application | |
| CN109481438A (en) | Application of 2- amino naphtho- [1,2-d] thiazole -4,5- diketone in tumor | |
| CN104383555B (en) | Folic acid-cyclodextrin conjugate, drug delivery vehicle, Preparation method and use |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180720 |