CN109316604A - A kind of preparation method and application of functionality erythrocyte membrane - Google Patents
A kind of preparation method and application of functionality erythrocyte membrane Download PDFInfo
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- 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
- A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
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- 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
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
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Abstract
The invention discloses a kind of preparation methods of functional erythrocyte membrane, it is characterised in that: erythrocyte membrane 0.6mg is dissolved into deionized water 1ml or phosphate buffer, the phosphate buffer is NaH2PO4&Na2HPO4, pH 7.4,100mM；Coating solution and DSPE-PEG-RGD are subjected to ultrasonic wave or stirring, functionalization can be obtained and obtain erythrocyte membrane.Its is easy to operate, final functional membrane can be used as carrier material transport drug or biomolecule carries out medicine and beauty and be wrapped in other materials surface as modification camouflage material, it is modified and is pretended, to improve nano material or active biomolecule in the accumulation of privileged site, enhancing treatment and using effect.
Description
Technical field
The invention belongs to biological medicine and cosmetic fields, more specifically it what is involved is a kind of functional red blood cells
The preparation method and application of film.
Background technique
Red blood cell (RBC) is a kind of haemocyte that quantity is most in blood, it has natural biocompatibility, biology
Degradability, and there is no biological immune.Erythrocyte membrane can preferably protect the activity for being carried substance, have it is longer and
The more controllable lifetime.These performances make " Carrier erythrocytes " to become a kind of valuable transport agent.
In recent years, Surface of Erythrocytes modification technique had obtained significant progress, these novel erythrocyte membrane camouflages are received
Rice grain can also design a variety of Nano medications, such as vaccine other than it may be used as medicament transport carrier.
RGD peptide (arginine-glycine-aspartic acid) is the first choice for being applied to be related to being integrated to cell cortex protein
Sequence, the ability with efficient identification cell improve transfection efficiency.
It is not found the technical report that RGD peptide and red blood cell (RBC) are carried out to biocompatibility processing now.
Summary of the invention
The first object of the present invention is in place of overcoming above-mentioned the deficiencies in the prior art, and provides a kind of with high transfection
The preparation method of the functional erythrocyte membrane of efficiency.There is original egg on erythrocyte membrane using the cell membrane that this method obtains
It is white, thus with erythrocyte membrane Examples of natural biocompatible, by nexus be wrapped in nano material or medical surfaces to its into
Row camouflage and transport, RGD then assigns cell membrane high transfection efficiency, so that the accumulation of nano material or functional molecular is improved,
Enhance disease treatment or cosmetics using effect.
The second object of the present invention is the application of above-mentioned functional erythrocyte membrane.
To reach above-mentioned first invention purpose, the technical solution adopted by the present invention are as follows:
A kind of preparation method of functionality erythrocyte membrane: it is characterized by: erythrocyte membrane 0.6mg is dissolved into deionization
In water 1ml or phosphate buffer, the phosphate buffer is NaH2PO4&Na2HPO4, pH 7.4,100mM, by coating solution with
DSPE-PEG-RGD (phosphatide PEG polypeptide, active targeting polypeptide) carries out ultrasonic wave or stirring, and functionalization can be obtained and obtain
Erythrocyte membrane.
In the above-mentioned technical solutions, the erythrocyte membrane solution and DSPE-PEG-RGD press 1:10；1:1 or 10:1 ratio
Carry out ultrasound or stirring.
In the above-mentioned technical solutions, ultrasonic wave and the temperature of stirring are 15-45 DEG C, time 10s-2h；Ultrasonic power is
10-500W；Mixing speed is 50r/min-1000r/min；
In the above-mentioned technical solutions, by functional erythrocyte membrane and gold nano grain (50 μ g mL-1), with ultrasonic power
For 200W, at 37 DEG C, ultrasound 10 minutes, 10000rpm, which is centrifuged 5 minutes, removes extra erythrocyte membrane, obtains functional red thin
The nanogold of after birth package.
In the above-mentioned technical solutions, by hyaluronic acid (the 10050 μ g mL of functional erythrocyte membrane and FAM fluorescent marker-1) ultrasonic power is 200W, at 37 DEG C, ultrasound 10 minutes, 10000rpm, which is centrifuged 5 minutes, removes extra erythrocyte membrane, obtains
The hyaluronic acid of functional erythrocyte membrane package.
The application of functional erythrocyte membrane, it is characterised in that: the functionality erythrocyte membrane is answered in nano material
With；Drug is transported as carrier material or biomolecule carries out therapeutic treatment and cosmetic field application；Pretend material as modification
Material is wrapped in other materials surface, is modified other materials and is pretended.
The present invention has the advantage that erythrocyte membrane has good biocompatibility and lasting transport capacity, body is escaped
Immunocapture；RGD then assigns film good cell transfecting efficiency；The present invention overcomes micro-nano transport agents not can avoid
The defect removed by immunity of organism；Its is easy to operate, final film can be used as carrier material transport drug or biomolecule into
Row therapeutic treatment and cosmetic applications and as modification camouflage material be wrapped in other function molecular surface, to other materials
It is modified and is pretended, to improve nano material or drug in the accumulation of lesion, enhancing treatment or using effect.
Functionality erythrocyte membrane of the invention mainly organizes the various albumen as erythrocyte membrane and cell membrane surface and wears film
Peptide RGD.
Detailed description of the invention
Fig. 1 SDA-PAGE gel electrophoresis figure.
The transmission electron microscope figure of the nanogold of Fig. 2 function film package.
The photo-thermal therapy of the nanogold melanoma of Fig. 3 .1 and Fig. 3 .2 function film package.
Fig. 3 function film wraps up fluorescein-labeled hyaluronic acid epidermal cell transfection experiment.
Fig. 4 functionality erythrocyte membrane and sodium hyaluronate rice grain transfect comparison diagram.
1. label, 2. erythrocyte membranes in figure.
Specific embodiment
The performance that the invention will now be described in detail with reference to the accompanying drawings, but they and do not constitute a limitation of the invention, only
It is for example, while by illustrating that advantages of the present invention will become clearer and be readily appreciated that.
Refering to known to attached drawing: it is red thin that Fig. 1 shows that the erythrocyte membrane that the polyphenyl alkene gel electrophoresis of denaturation proves remains
Characteristic protein such as CD47 on born of the same parents etc., the presence of these albumen can prevent the identification of macrophage, escape immune clearance, mention
The circulation time of high film lapping in vivo；Wherein KDa: kilomol quality.
Fig. 2 shows that nanogold particle is successfully wrapped up by functional cell film, and multiple nano particles can be wrapped simultaneously
It wraps up in, is conducive to the biocompatibility for improving nano particle；Multiple nano particles, which are simultaneously included, can increase package efficiency, mention
High photothermal conversion effect, black spherical shape is nanogold particle in Fig. 2, and package is cell membrane.
Mouse (A) of Fig. 3 .1 relative to unused nano material photo-thermal therapy, rapid growth is presented in tumour, under mouse weight
Drop；
The growth of mouse (B) tumour of the nanogold photo-thermal therapy of Fig. 3 .2 functionality package is inhibited well, and 2 weeks
Afterwards, tumor disappearance, mouse body weight increase；Illustrate this nano material good therapeutic effect.
Strong fluorescence shows that the sodium hyaluronate rice grain of functional erythrocyte membrane and FAM fluorescent marker can in Fig. 4 figure
High-efficiency transfection (B) is carried out to NHDF.Relative to the hyaluronic acid not wrapped up, its transfection efficiency greatly improves (A), in medicament transport
And cosmetic field has broad application prospects.
A kind of preparation method of functional erythrocyte membrane of the present invention: erythrocyte membrane 0.6mg is dissolved into deionized water 1ml
Or in phosphate buffer, the phosphate buffer is NaH2PO4&Na2HPO4, 7.4,100 mM of pH, by coating solution and DSPE-
PEG-RGD (phosphatide PEG polypeptide, active targeting polypeptide) carries out ultrasonic wave or stirring, and functionalization can be obtained and obtain red blood cell
Film.
The erythrocyte membrane solution and DSPE-PEG-RGD carry out ultrasound in 1:10-10:1 ratio or stir.
Ultrasonic wave and the temperature of stirring are 15-45 DEG C, time 10s-2h；Ultrasonic power is 10-500W；Mixing speed
For 50r/min-1000r/min；
The application of functional erythrocyte membrane, the functionality erythrocyte membrane are applied in nano material；As carrier
Transport of materials drug or biomolecule carry out therapeutic treatment and cosmetic field application；It is wrapped in as modification camouflage material
Its material surface, modifies other materials and is pretended.
Embodiment 1
The EDTA (ethylenediamine tetra-acetic acid) of 1.5mg, at 4 DEG C, 800g are added in the fresh whole blood of every mL male ICR mouse
Centrifugation removes blood plasma and buffy coat in 5 minutes.Cold PBS (the NaH of obtained erythrocyte membrane2PO4And Na2HPO4,pH
7.4,100mM) be centrifuged and wash 3 times.The erythrocyte membrane of generation is dissolved in (NaH2PO4And Na2HPO4, pH 7.4,25mM) in, ice bath
30 minutes.15000rpm, which is centrifuged 5 minutes, removes hemoglobin, further uses PBS (NaH2PO4And Na2HPO4,pH 7.4,
100mM) centrifugation is washed 2 times.Erythrocyte membrane (0.6mg) will be obtained to be dissolved into deionized water (1ml) or phosphate buffer
(NaH2PO4And Na2HPO4, pH 7.4,100mM), by coating solution and DSPE-PEG-RGD (0.06 mg, 0.6mg or 6mg), with
Ultrasonic power is 200W, and at 37 DEG C, ultrasound 30 minutes obtains functional erythrocyte membrane.
Embodiment 2
It will be from functionality erythrocyte membrane obtained in 1ml whole blood and gold nano grain (50 μ g mL-1), with ultrasonic power
For 200W, at 37 DEG C, ultrasound 10 minutes, 10000rpm, which is centrifuged 5 minutes, removes extra erythrocyte membrane, obtains functional red thin
The nanogold of after birth package.
B16-F10 melanoma cells are grafted in 6 months big BALB/c nude mice by subcutaneous, by the nanogold of functionality package
With 5mg/kg tail vein injections 4 as a child after, with infrared laser irradiate 10 minutes (1064nm, 1W/cm2), to melanin
Tumor carries out photo-thermal therapy.
Embodiment 3
By hyaluronic acid (the 100 50 μ g mL of functionality erythrocyte membrane and FAM fluorescent marker obtained in the 1ml whole blood-1) ultrasonic power is 200W, at 37 DEG C, ultrasound 10 minutes, 10000rpm, which is centrifuged 5 minutes, removes extra erythrocyte membrane, obtains
The hyaluronic acid of functional erythrocyte membrane package.
Function erythrocyte membrane is wrapped up into hyaluronic acid, NHDF (104) (normal human skin fiber female patch parietal cell) be copolymerized
After cultivating 12 hours in burnt culture dish, it is small that culture solution changes Han Yong function erythrocyte membrane package hyaluronic acid (50nM) culture 4 into
When, nucleus Hoechst 33342, obtained cell is observed with co-focusing imaging.
It is other it is unspecified be the prior art.
Claims (8)
1. a kind of preparation method of functionality erythrocyte membrane, it is characterised in that: erythrocyte membrane 0.6mg is dissolved into deionized water
In 1ml or phosphate buffer, the phosphate buffer is NaH2PO4&Na2HPO4, pH7.4,100mM；By coating solution with
DSPE-PEG-RGD carries out ultrasonic wave or stirring, and functionalization can be obtained and obtain erythrocyte membrane.
2. a kind of preparation method of functional erythrocyte membrane according to claim 1, it is characterised in that: the erythrocyte membrane
Solution and DSPE-PEG-RGD are in 1:10-10:1 ratio progress ultrasonic wave or stirring.
3. a kind of preparation method of functional erythrocyte membrane according to claim 2, it is characterised in that: the erythrocyte membrane
Solution and DSPE-PEG-RGD press 1:10；Or 1:1；Or 10:1 ratio carries out ultrasonic wave or stirring.
4. a kind of preparation method of functional erythrocyte membrane according to claim 1 or 2, it is characterised in that: the ultrasound
Temperature is 15-45 DEG C when wave and stirring, time 10s-2h；Ultrasonic power is 10-500W；Mixing speed is 50r/min-
1000r/min。
5. a kind of preparation method of functional erythrocyte membrane according to claim 1, it is characterised in that: use erythrocyte membrane
Cold PBS (NaH2PO4And Na2HPO4, pH 7.4,100mM) and it is centrifuged and washes 3 times；The erythrocyte membrane of generation is dissolved in (NaH2PO4With
Na2HPO4, pH 7.4,25mM) in, ice bath 30 minutes；15000rpm, which is centrifuged 5 minutes, removes hemoglobin, further uses PBS
(NaH2PO4And Na2HPO4, pH 7.4,100mM) and it is centrifuged and washes 2 times；It will obtain erythrocyte membrane 0.6mg and be dissolved into deionized water 1ml
Or (NaH in phosphate buffer2PO4And Na2HPO4, pH 7.4,100mM), by coating solution and DSPE-PEG-RGD (0.06mg
Or 0.6mg or 6mg), it is 200W with ultrasonic power, at 37 DEG C, ultrasound 30 minutes obtains functional erythrocyte membrane.
6. a kind of preparation method of functional erythrocyte membrane according to claim 5, it is characterised in that: will be functional red thin
After birth and gold nano grain (50 μ g mL-1), it is being 200W with ultrasonic power, at 37 DEG C, ultrasound 10 minutes, 10000rpm centrifugation 5
Minute removes extra erythrocyte membrane, obtains the nanogold of functional erythrocyte membrane package.
7. a kind of preparation method of functional erythrocyte membrane according to claim 5, it is characterised in that: will be functional red thin
Hyaluronic acid (the 10050 μ g mL of after birth and FAM fluorescent marker-1) ultrasonic power is 200W, at 37 DEG C, ultrasound 10 minutes,
10000rpm, which is centrifuged 5 minutes, removes extra erythrocyte membrane, obtains the hyaluronic acid of functional erythrocyte membrane package.
8. the application for the functional erythrocyte membrane that in the claims 1-7 prepared by any claim the method, feature
Be: the functionality erythrocyte membrane is applied in nano material；As carrier material transport drug or biomolecule into
Row therapeutic treatment and cosmetic field application；It is wrapped in other materials surface as modification camouflage material, other materials are carried out
Modification and camouflage.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109718380A (en) * | 2019-03-06 | 2019-05-07 | 苏州大学 | Tumor antigen presentation system and its application in preparation of anti-tumor drugs, immune system activation agent and anti-tumor compositions |
| CN109966266A (en) * | 2019-02-26 | 2019-07-05 | 南华大学 | The polymer matrix medicament-carried nano compound system and preparation method thereof of erythrocyte membrane package |
| CN110090309A (en) * | 2019-05-28 | 2019-08-06 | 上海大学 | The preparation method of functionalizing erythrocyte membrane |
| CN111471100A (en) * | 2020-01-27 | 2020-07-31 | 江苏帆博生物制品有限公司 | Novel erythrocyte membrane monoclonal antibody production method and processing device thereof |
| CN112138158A (en) * | 2020-10-20 | 2020-12-29 | 苏州大学 | Application of red blood cells in preparation of photothermal conversion material |
| CN112402626A (en) * | 2020-11-26 | 2021-02-26 | 中国人民解放军陆军军医大学第二附属医院 | Biological camouflage nano drug delivery system for targeting tumors and preparation method thereof |
| CN112402617A (en) * | 2020-11-20 | 2021-02-26 | 汕头大学 | Taxol and its preparation method and application |
| CN112791061A (en) * | 2021-01-05 | 2021-05-14 | 石河子大学 | Preparation method of multi-stage bionic nano-drug carrier with targeting long circulation |
| CN113648288A (en) * | 2021-08-19 | 2021-11-16 | 中国药科大学 | Nano compound of erythrocyte membrane coated functional molecule, preparation method and application |
| CN115192774A (en) * | 2022-07-20 | 2022-10-18 | 四川大学 | Wear-resistant multifunctional cell membrane coating and preparation method thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109966266A (en) * | 2019-02-26 | 2019-07-05 | 南华大学 | The polymer matrix medicament-carried nano compound system and preparation method thereof of erythrocyte membrane package |
| CN109718380A (en) * | 2019-03-06 | 2019-05-07 | 苏州大学 | Tumor antigen presentation system and its application in preparation of anti-tumor drugs, immune system activation agent and anti-tumor compositions |
| CN109718380B (en) * | 2019-03-06 | 2022-03-22 | 苏州大学 | Tumor antigen presentation system and application thereof in preparation of antitumor drugs, immune system activator and antitumor composition |
| CN110090309A (en) * | 2019-05-28 | 2019-08-06 | 上海大学 | The preparation method of functionalizing erythrocyte membrane |
| CN111471100A (en) * | 2020-01-27 | 2020-07-31 | 江苏帆博生物制品有限公司 | Novel erythrocyte membrane monoclonal antibody production method and processing device thereof |
| CN112138158A (en) * | 2020-10-20 | 2020-12-29 | 苏州大学 | Application of red blood cells in preparation of photothermal conversion material |
| CN112402617A (en) * | 2020-11-20 | 2021-02-26 | 汕头大学 | Taxol and its preparation method and application |
| CN112402626A (en) * | 2020-11-26 | 2021-02-26 | 中国人民解放军陆军军医大学第二附属医院 | Biological camouflage nano drug delivery system for targeting tumors and preparation method thereof |
| CN112791061A (en) * | 2021-01-05 | 2021-05-14 | 石河子大学 | Preparation method of multi-stage bionic nano-drug carrier with targeting long circulation |
| CN112791061B (en) * | 2021-01-05 | 2022-08-05 | 石河子大学 | Preparation method of multi-stage bionic nano-drug carrier with targeting long circulation |
| CN113648288A (en) * | 2021-08-19 | 2021-11-16 | 中国药科大学 | Nano compound of erythrocyte membrane coated functional molecule, preparation method and application |
| CN115192774A (en) * | 2022-07-20 | 2022-10-18 | 四川大学 | Wear-resistant multifunctional cell membrane coating and preparation method thereof |
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