CN106620699A - Targeted photothermal black phosphorus nano-preparation as well as preparation method and application thereof - Google Patents
Targeted photothermal black phosphorus nano-preparation as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a targeted photothermal black phosphorus nano-preparation, which is prepared from a black phosphorus nano-slice, polyethylene glycol absorbed on the surface of the black phosphorus nano-slice by means of electrostatic attraction, and folic acid connected to the polyethylene glycol by means of an amide bond, wherein one end of the polyethylene glycol is amino, and the other end of the polyethylene glycol is imino; nitrogen atom of the imino and carbonyl carbon atom of the folic acid are connected with each other so as to form the amide bond; the folic acid is exposed at the outermost layer of the nano-preparation. The targeted photothermal black phosphorus nano-preparation is high in stability, good in target recognition ability and excellent in light-heat property, is capable of effectively killing cancer cells, and can be used for performing targeted photothermal treatment on cancers at the cellular level. The invention also provides a preparation method and application of the targeted photothermal black phosphorus nano-preparation.
Description
Technical field
The present invention relates to Nano biomedical material technical field, more particularly to a kind of targeting photo-thermal black phosphorus nanometer formulation
And its preparation method and application.
Background technology
As one of the great malignant disease for threatening human health, the treatment of cancer either chemotherapy, operative treatment
Or radiotherapy is all have very big side effect to body, and after it there is pernicious transfer, no matter using above-mentioned which kind of side
Formula is all difficult to thoroughly healing.Although existing substantial amounts of human and material resources, financial resources have been put into the research of cancer, progress is very
Limited and its effectively treatment remains the very big test that the mankind face.
There are some researches show, nanometer technology is applied to into cancer diagnosis therapy field, have broad prospects and clinical value.
The fuel factor that photo-thermal therapy is produced as a kind of new Cancer Treatment Regimens, using photothermal deformation is to kill cancer cell, to swollen
Knurl position carries out thermal ablation therapy, and the exciting light of its irradiation typically adopts near infrared light, is that a kind of cancer of Noninvasive is controlled
Treatment mode, can effectively penetrate human normal tissue and reach cancer location, and the infringement of normal tissue is reduced to a great extent.
Recent studies have found that, black phosphorus two-dimensional material due to hypotoxicity, high extinction coefficient and high photo-thermal conversion efficiency,
The photo-thermal therapy aspect of bio-medical field, especially cancer has great application potential.However, list prepared by existing mode
, when the photo-thermal therapy of cancer is directly applied to, it is poor in physiological status stability inferior, and blood for black phosphorus nano material
Circulation time is not long, and to be improved in the concentration effect of cancer location.
The content of the invention
In consideration of it, first aspect present invention provides a kind of targeting photo-thermal black phosphorus nanometer formulation, the targeting photo-thermal black phosphorus is received
Rice preparation stability is high, targets identification ability is good, light thermal property is excellent, can effectively kill cancer cell, can enter in cell aspect
The targeting photo-thermal therapy of row cancer.
In a first aspect, the invention provides a kind of targeting photo-thermal black phosphorus nanometer formulation, including black phosphorus nano flake, by quiet
Electric attraction absorption is connected on the polyethylene glycol in the polyethylene glycol on the black phosphorus nano flake surface and by amido link
Folic acid, one end of the polyethylene glycol is amino, and the other end is imino group, in the nitrogen-atoms of the imino group and the folic acid
Carbonylic carbon atom be connected and constitute the amido link, the folic acid is exposed to the outermost layer of the nanometer formulation.
The present invention is repaiied by being modified black phosphorus nano flake using polyethylene glycol while carrying out targeting using folic acid
Decorations, wherein, polyethylene glycol can be prevented effectively from black phosphorus nano flake to be assembled, and polyethylene glycol has water-wet behavior and pole concurrently
Property, immune identification can be significantly inhibited, the phagocytosis of monokaryon-mononuclear phagocyte system is reduced, and effectively extend nano flake
The blood long circulating time, so as to strengthen the bioavilability of nanometer formulation and the enrichment of cancer location.And folic acid is exposed to institute
State nanometer formulation outermost layer and targeting ligand is provided, the targeting ligand can specifically bind with cancer cell, improve nanometer formulation
Targeting specific to cancer cell, further enhances aggregation of the nanometer formulation in cancer location, significantly improves photo-thermal therapy effect.
Alternatively, the targeting photo-thermal black phosphorus nanometer formulation is by by black phosphorus nano flake, double ammonia ends polyethylene glycol, folic acid
In mass ratio (1～3): (5～12): (2～6) proportioning is obtained.
In the present invention, the size of the black phosphorus nano flake is 50nm-200nm.
In the present invention, the thickness of the black phosphorus nano flake is 2nm-3nm.
In the present invention, the weight average molecular weight of described pair of ammonia end polyethylene glycol is 550-30000.
The targeting photo-thermal black phosphorus nanometer formulation that first aspect present invention is provided, stability is high, targets identification ability is good,
Light thermal property is excellent, can effectively kill cancer cell, and the targeting photo-thermal therapy of cancer can be carried out in cell aspect, so as to can be notable
Reduce the internal removing of black phosphorus nano flake；And the targeting photo-thermal black phosphorus nanometer formulation has good biocompatibility, can lead to
Cross biodegradable or normal physiological pathway to excrete, organism is had no toxic side effect, biological safety it is high.
Second aspect, the invention provides a kind of preparation method of targeting photo-thermal black phosphorus nanometer formulation, comprises the following steps：
Black phosphorus nano flake is provided；
Hydroxysuccinimide-activated folic acid is added in the dimethyl sulphoxide solution of double ammonia ends polyethylene glycol, and is added
Proper catalyst, obtains mixed solution, and the mixed solution is stirred reaction 16-32 hours under the conditions of room temperature, lucifuge, instead
Filter after the completion of answering, obtain the polyethylene glycol of modified with folic acid, the folic acid is connected on polyethylene glycol by amido link, it is described poly-
One end of ethylene glycol is amino, and the other end is imino group, the carbonylic carbon atom in the nitrogen-atoms of the imino group and the folic acid
It is connected and constitutes the amido link；
Successively by the way of probe sonication, magnetic agitation, in the Surface coating modified with folic acid of the black phosphorus nano flake
Polyethylene glycol, obtain targetting photo-thermal black phosphorus nanometer formulation, the polyethylene glycol of the modified with folic acid is existed by electrostatic attraction absorption
The black phosphorus nano flake surface, the folic acid is exposed to the outermost layer of the targeting photo-thermal black phosphorus nanometer formulation.
In the present invention, alternatively, the black phosphorus nano flake, double ammonia ends polyethylene glycol, folic acid (1～3) in mass ratio: (5
～12): (2～6) feed intake.
In the present invention, the catalyst can be triethylamine.The catalyst is 1 with the volume ratio of reaction system:75-
85。
In the present invention, alternatively, the time of the probe sonication is 20-40 minutes, during the probe sonication, is continued
Ultrasound -1 hour 45 seconds, and -1 hour 15 seconds is waited for a cycle, power amplifier is 12%-20%.
In the present invention, alternatively, the rotating speed of the magnetic agitation can be 800rpm-1200rpm, and the duration is 3-4
Hour.
In the present invention, the black phosphorus nano flake is peeled off bonding probes ultrasonic method and is prepared using solution, and adopts centrifuge tube
Centrifugation is collected, purifying, and the ultrasonic time of the probe sonication is 12-18 hours, during the probe sonication, is persistently surpassed
Sound -1 hour 45 seconds, and -1 hour 15 seconds is waited for a cycle, power amplifier is 20%-30%, and the speed of the centrifugation is
1000rpm-2000rpm, the time is 6-15 minutes, and temperature is 4 DEG C.
In the present invention, the hydroxysuccinimide-activated folic acid is prepared in the following way：Folic acid is dissolved in anhydrous
In dimethyl sulfoxide (DMSO) (DMSO), add HOSu NHS and N, N'- Dicyclohexylcarbodiimide obtains mixed liquor, it is described
Mixed liquor is stirred reaction 16-32 hours under room temperature, lucifuge and triethylamine catalytic condition, filters after the completion of reaction, obtains
The hydroxysuccinimide-activated folic acid.
In the present invention, the preparation method of the targeting photo-thermal black phosphorus nanometer formulation is further included using milipore filter centrifugation
Mode is purified at least one times, screened to gained targeting photo-thermal black phosphorus nanometer formulation, obtains optimizing the pure target of particle size
To photo-thermal black phosphorus nanometer formulation, the speed of the milipore filter centrifugation is 2000-4000rpm, and the time is 30-60 minutes, and temperature is
4-8℃。
The preparation method of the targeting photo-thermal black phosphorus nanometer formulation that second aspect present invention is provided, by by black phosphorus nano flake
Combination is coated by way of Electrostatic Absorption with the polyethylene glycol of modified with folic acid, targeting photo-thermal black phosphorus nanometer formulation is obtained, prepared
Process is simple to operation.
The third aspect, the invention provides a kind of above-mentioned targeting photo-thermal black phosphorus nanometer formulation is preparing cancer light heating ablation
Application in medicine.The targeting photo-thermal black phosphorus nanometer formulation is applied to the targeting light thermal ablation therapy of cancer.The light
Thermal ablation therapy passes through 808nm near infrared band laser excitations.There is the 808nm near infrared bands laser very strong skin to wear
Thoroughly ability, can effectively reach deep tissues and cancer location.Meanwhile, the targeting photo-thermal black phosphorus nanometer formulation has good
Biocompatibility, have no toxic side effect.
In sum, beneficial effect of the present invention includes the following aspects：
(1) targeting photo-thermal black phosphorus nanometer formulation of the present invention can effectively prevent the aggregation of black phosphorus nano flake, carry
The stability of high black phosphorus nano flake, extends blood circulation time, strengthens the Targeting Effect of black phosphorus nano flake；
(2) the inventive method preparation process is simple to operation, and the targeting photo-thermal black phosphorus nanometer formulation of preparation possesses excellent life
Thing compatibility, have no toxic side effect；
(3) the targeting photo-thermal black phosphorus nanometer formulation prepared by the present invention can apply to the targeting photo-thermal therapy of cancer.
Description of the drawings
Fig. 1 is the structural representation of targeting photo-thermal black phosphorus nanometer formulation (BP-PEG-FA) prepared by the embodiment of the present invention 1；
Fig. 2 is the scanning transmission electronic display of targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA prepared by the embodiment of the present invention 1
Micro mirror (STEM) and X-ray energy dispersion spectrum (EDS mapping)；
Fig. 3 is the grain size distribution of targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA prepared by the embodiment of the present invention 1；
Fig. 4 is that the embodiment of the present invention 1 targets photo-thermal black phosphorus nanometer formulation BP-PEG-FA Zeta potentials in preparation process
Variation diagram；
Fig. 5 is the FTIR spectrum of targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA prepared by the embodiment of the present invention 1
Figure (FT-IR)；
Fig. 6 exists for targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA prepared by the embodiment of the present invention 2 of variable concentrations
Time dependent photo-thermal curve spectrum under the irradiation of 808nm near-infrared lasers；
Fig. 7 is that targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA prepared by the embodiment of the present invention 2 is closely red in multiple 808nm
Photo and thermal stability under outer laser irradiation, wherein black phosphorus concentration are 100 μ g/mL；
Fig. 8 is the targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA of the preparation of the embodiment of the present invention 2 under different preparation times
Photo-thermal curve spectrum, wherein black phosphorus concentration be 100 μ g/mL；
Fig. 9 is that targeting photo-thermal prepared by laser confocal scanning electron microscope (CLSM) the observation embodiment of the present invention 1 is black
The Hela cellular uptake pictures of phosphorus nanometer formulation BP-PEG-FA；
Figure 10 is the targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA of the preparation of the embodiment of the present invention 1 in various cancers cell
In toxicity data；
Figure 11 is the targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA of the preparation of the embodiment of the present invention 1 in Hela cells
Targeting photo-thermal therapy effect.
Specific embodiment
Described below is the preferred embodiment of the embodiment of the present invention, it is noted that for the common skill of the art
For art personnel, on the premise of without departing from embodiment of the present invention principle, some improvements and modifications can also be made, these improvement
With the protection domain that retouching is also considered as the embodiment of the present invention.
Embodiments provide a kind of targeting photo-thermal black phosphorus nanometer formulation, including black phosphorus nano flake, by electrostatic
Gravitation absorption is connected on the polyethylene glycol in the polyethylene glycol on the black phosphorus nano flake surface and by amido link
Folic acid, one end of the polyethylene glycol is amino, and the other end is imino group, in the nitrogen-atoms of the imino group and the folic acid
Carbonylic carbon atom is connected and constitutes the amido link, and the folic acid is exposed to the outermost layer of the nanometer formulation.
Alternatively, the targeting photo-thermal black phosphorus nanometer formulation is by by black phosphorus nano flake, double ammonia ends polyethylene glycol, folic acid
In mass ratio (1～3): (5～12): (2～6) proportioning is obtained.Still optionally further, the targeting photo-thermal black phosphorus nanometer formulation leads to
Cross and be obtained black phosphorus nano flake, double ammonia ends polyethylene glycol, folic acid 1: 4.2: 2.1 proportioning in mass ratio.Suitable black phosphorus nanometer
Thin slice, double ammonia ends polyethylene glycol, folic acid mass ratio, advantageously form constitutionally stable targeting photo-thermal black phosphorus nanometer formulation, can have
Effect avoids the aggregation of black phosphorus nano flake, improves the stability and Targeting Effect of nanometer light heating agent.
In the present invention, the black phosphorus nano flake surface is negatively charged, therefore can be gathered with amino-terminated by electrostatic attraction
Ethylene glycol combines, and polyethylene glycol provides hydrophilic surface to target photo-thermal black phosphorus nanometer formulation.
In the present invention, alternatively, the size of the black phosphorus nano flake is 50nm-200nm.Suitable black phosphorus nano flake
Size, can effectively strengthen the EPR effects of nanometer formulation, increase enrichment of the nanometer formulation in cancer location, at the same ensure its
It is difficult to be removed by human body reticuloendothellium during blood circulation.Alternatively, the size of black phosphorus nano flake is 50nm-
150nm、100nm-150nm、120nm-180nm、160nm-200nm。
In the present invention, the thickness of the black phosphorus nano flake is 2nm-3nm.The suitable thickness of black phosphorus nano flake can show
Write the surface area-mass ratio for improving nanometer formulation, the photo-thermal therapy effect of reinforced nano preparation.Thickness is thinner, surface area-quality
It is bigger than ratio.The black phosphorus nano flake preparation of phase homogenous quantities, specific surface is bigger, and photo-thermal effect is better；Conversely, under photo-thermal effect
Drop.
In the present invention, the black phosphorus nano flake has very high surface area-mass ratio, on the one hand can be to greatest extent
Ground absorbs near-infrared excitation light, produces excellent light thermal property；Another aspect black phosphorus nano flake surface is full of negative electrical charge, can
Strengthen and the Electrostatic Absorption ability between polyethylene glycol, strengthen the polyethylene glycol area load amount of modified with folic acid, strengthen targeting light
The stability and Targeting Effect of hot black phosphorus nanometer formulation.
In the present invention, the weight average molecular weight of described pair of ammonia end polyethylene glycol is 550-30000.
The targeting photo-thermal black phosphorus nanometer formulation that first aspect present invention is provided, stability is high, targets identification ability is good,
Light thermal property is excellent, can effectively kill cancer cell, and the targeting photo-thermal therapy of cancer can be carried out in cell aspect, so as to can be notable
Reduce the internal removing of black phosphorus nano flake；And the targeting photo-thermal black phosphorus nanometer formulation has good biocompatibility, can lead to
Cross biodegradable or normal physiological pathway to excrete, organism is had no toxic side effect, biological safety it is high.
Correspondingly, the embodiment of the present invention additionally provide it is a kind of targeting photo-thermal black phosphorus nanometer formulation preparation method, including with
Lower step：
(1) black phosphorus nano flake is provided；
(2) hydroxysuccinimide-activated folic acid is added in the dimethyl sulphoxide solution of double ammonia ends polyethylene glycol, and
Proper catalyst is added, mixed solution is obtained, it is little that the mixed solution is stirred reaction 16-32 under the conditions of room temperature, lucifuge
When, filter after the completion of reaction, the polyethylene glycol of modified with folic acid is obtained, the folic acid is connected on polyethylene glycol by amido link,
One end of the polyethylene glycol is amino, and the other end is imino group, the carbonyl in the nitrogen-atoms of the imino group and the folic acid
Carbon atom is connected and constitutes the amido link；
(3) successively by the way of probe sonication, magnetic agitation, repair in the Surface coating folic acid of the black phosphorus nano flake
The polyethylene glycol of decorations, obtains targetting photo-thermal black phosphorus nanometer formulation, and the polyethylene glycol of the modified with folic acid is adsorbed by electrostatic attraction
On the black phosphorus nano flake surface, the folic acid is exposed to the outermost layer of the targeting photo-thermal black phosphorus nanometer formulation.
In embodiment of the present invention step (1), alternatively, the size of the black phosphorus nano flake is 50nm-200nm.It is adapted to
Black phosphorus nano flake size, can effectively strengthen the EPR effects of nanometer formulation, increase richness of the nanometer formulation in cancer location
Collection, while ensureing that it is difficult to be removed by human body reticuloendothellium during blood circulation.Alternatively, black phosphorus nano flake
Size is 50nm-150nm, 100nm-150nm, 120nm-180nm, 160nm-200nm.Alternatively, the black phosphorus nano flake
Thickness be 2nm-3nm.The suitable thickness of black phosphorus nano flake can significantly improve the surface area-mass ratio of nanometer formulation, by force
Change the photo-thermal therapy effect of nanometer formulation.
In embodiment of the present invention step (1), the black phosphorus nano flake can peel off bonding probes ultrasound legal system using solution
It is standby, and collected using centrifuge tube centrifugation, purified, the ultrasonic time of the probe sonication is 12-18 hours, and the probe surpasses
During sound, continual ultrasonic -1 hour 45 seconds, and wait -1 hour 15 seconds be a cycle, power amplifier is 20%-30%, it is described from
The speed of the heart is 1000rpm-2000rpm, and the time is 6-15 minutes, and temperature is 4 DEG C.Centrifugally operated be in order to precipitate and separate not by
The block black phosphorus of stripping, with purification ＆ isolation black phosphorus nano flake.
In embodiment of the present invention step (2), alternatively, the weight average molecular weight of described pair of ammonia end polyethylene glycol is 550-
30000.Alternatively, the catalyst can be triethylamine.The catalyst is 1 with the volume ratio of reaction system:75-85.Institute
It is 300-600rpm to state the mixing speed during stirring reaction.
In embodiment of the present invention step (2), the hydroxysuccinimide-activated folic acid can be prepared in the following way：Will
Folic acid is dissolved in anhydrous dimethyl sulphoxide (DMSO), adds HOSu NHS (NHS), triethylamine and the ring of N, N'- bis-
Hexyl carbimide (DCC) obtains mixed liquor, and the mixed liquor is stirred reaction 16-32 hours under the conditions of room temperature, lucifuge,
Filter after the completion of reaction, obtain the hydroxysuccinimide-activated folic acid.
In step (2) of the present invention, the filter operation after the completion of reaction can be specifically：Using bag filter (MWCO:3000)
Dialysis removes unreacted hydroxysuccinimide-activated folic acid.
The polyethylene glycol of gained modified with folic acid has stronger affinity to cell membrane in the embodiment of the present invention, can be well
Promote nanometer formulation to enter intracellular, be conducive to improving the follow-up photo-thermal therapy of cancer effect of targeting photo-thermal black phosphorus nanometer formulation.
In the present invention, alternatively, the black phosphorus nano flake, double ammonia ends polyethylene glycol, folic acid (1～3) in mass ratio: (5
～12): (2～6) feed intake.Still optionally further, the black phosphorus nano flake, double ammonia ends polyethylene glycol, folic acid in mass ratio 1:
Feed intake at 4.2: 2.1.
In step (3) of the present invention, first pre-coated is carried out to the black phosphorus nano flake using probe sonication mode, then adopted
Magnetic agitation mode is further coated.Alternatively, the time of the probe sonication be 20-40 minutes, the probe sonication
During, continual ultrasonic -1 hour 45 seconds, and -1 hour 15 seconds is waited for a cycle, power amplifier is 12%-20%.The magnetic force
The rotating speed of stirring can be 800rpm-1200rpm, and the duration is 3-4 hours.
In step (3) of the present invention, further include to target photo-thermal black phosphorus nanometer to gained by the way of milipore filter is centrifuged
Preparation is purified at least one times, screened, and obtains optimizing the pure targeting photo-thermal black phosphorus nanometer formulation of particle size, the ultrafiltration
The speed of film centrifugation is 2000-4000rpm, and the time is 30-60 minutes, and temperature is 4-8 DEG C.
Still optionally further, the preparation method of the targeting photo-thermal black phosphorus nanometer formulation includes the multiple ultrafiltration
Film centrifugally operated, specially：It is black with the targeting photo-thermal on the resuspended milipore filter of physiological saline after every time milipore filter centrifugally operated terminates
Phosphorus nanometer formulation, then carries out the milipore filter centrifugally operated next time.
The parameter of each index such as ultrasound and centrifugation duration, cycle, rotating speed and power amplifier in above-mentioned each step of the present invention
Setting is not limited to above-mentioned restriction.
The preparation method of the targeting photo-thermal black phosphorus nanometer formulation that second aspect present invention is provided, by by black phosphorus nano flake
Combination is coated by way of Electrostatic Absorption with the polyethylene glycol of modified with folic acid, targeting photo-thermal black phosphorus nanometer formulation is obtained, prepared
Process is simple to operation.
The third aspect, the invention provides a kind of above-mentioned targeting photo-thermal black phosphorus nanometer formulation is preparing cancer light heating ablation
Application in medicine.The targeting photo-thermal black phosphorus nanometer formulation is applied to the targeting light thermal ablation therapy of cancer.The light
Thermal ablation therapy passes through 808nm near infrared band laser excitations.There is the 808nm near infrared bands laser very strong skin to wear
Thoroughly ability, can effectively reach deep tissues and cancer location.Meanwhile, the targeting photo-thermal black phosphorus nanometer formulation has good
Biocompatibility, have no toxic side effect.
Multiple embodiments are divided to be further detailed the embodiment of the present invention below.Wherein, the embodiment of the present invention is not limited
Due to following specific embodiment.In the range of constant principal right, can be appropriate carry out change enforcement.
Embodiment 1
A kind of preparation method of targeting photo-thermal black phosphorus nanometer formulation, comprises the following steps：
(1) bonding probes ultrasonic method is peeled off using solution and prepares black phosphorus nano flake：50mg bulk black phosphorus materials are taken, is placed in
In 100mL ultra-pure waters, probe sonication is carried out, the ultrasonic time of the probe sonication method is 12 hours, and process is continual ultrasonic 45
Second, 15 seconds are waited for a cycle, power amplifier is 20%；After ultrasound terminates, take brownish black stripper and be placed in centrifuge tube,
1000rpm is centrifuged 10 minutes, and temperature is 4 DEG C, precipitates the block black phosphorus that is not peeled off, and the block black phosphorus of careful separation precipitation and
Black phosphorus nano flake in supernatant, with further purification ＆ isolation black phosphorus nano flake；
(2) triethylamine (0.5mL), NHS are added in the anhydrous DMSO of the 40mL of folic acid (1.0g, 2.27mmol) is dissolved with
(0.52g, 5.00mmol) and DCC (0.50g, 2.50mmol), obtains mixed liquor, and the mixed liquor is under the conditions of room temperature, lucifuge
After magnetic agitation reaction 24h, insoluble accessory substance and unreacted raw material is filtered to remove, obtains NHS activation folic acid.In triethylamine
On the premise of presence, the NHS activation folic acid of 2.5mL is added to into the anhydrous DMSO at double ammonia ends polyethylene glycol (molecular weight is 3000)
In solution, both (NHS activation after folic acid：Both-end ammonia polyethylene glycol) mol ratio be 1:2, obtain mixed solution.The mixed solution
The magnetic agitation reaction 24h under the conditions of room temperature, lucifuge, by coupling ammonia end copolymer, synthesizes the polyethylene glycol FA- of modified with folic acid
PEG-NH2, dialyse (MWCO in distilled water:1000), to remove unreacted folic acid, to FA-PEG-NH2Purified；
(3) by the way of probe sonication is in combination with magnetic agitation, in the Surface coating folic acid of black phosphorus nano flake
The polyethylene glycol of modification prepares targeting photo-thermal black phosphorus nanometer formulation；The wherein polyethylene glycol of black phosphorus nano flake and modified with folic acid
Mass ratio is 1:6.3.Specifically, it is first pre- to the polyethylene glycol that black phosphorus nano flake carries out modified with folic acid using probe sonication mode
Cladding, probe sonication process is carried out in the two suspension, and the time of probe sonication is 40 minutes, and process is continual ultrasonic 45 seconds,
15 seconds are waited for a cycle, power amplifier is 15%.Then black phosphorus nano flake is entered by the way of magnetic agitation at room temperature
The polyethylene glycol of row modified with folic acid is further coated, and magnetic agitation rotating speed is 800rpm, and the duration is 4 hours；Simple poly- second
Black phosphorus nanometer light heating agent (without modified with folic acid) of glycol cladding can be prepared in the same way, and preparing raw material is repaiied by folic acid
The ammonia end polyethylene glycol of decorations replaces with literalness ammonia end polyethylene glycol；
(4) purified by the way of milipore filter centrifugation, screen step (3) gained targeting photo-thermal black phosphorus nanometer formulation, obtained
The pure targeting photo-thermal black phosphorus nanometer formulation of optimization particle size：Take reaction mixture in step (3) and be added to super filter tube (MWCO
100kDa；Millipore centrifugation in), the polyethylene glycol for separating uncoated modified with folic acid is black with the targeting photo-thermal for preparing
Phosphorus nanometer formulation；The speed of milipore filter centrifugation is 4000rpm, and the time is 30 minutes, and temperature is 4 DEG C.After milipore filter centrifugation, with life
Targeting photo-thermal black phosphorus nanometer formulation on the resuspended filter membrane of reason salt solution, repeats milipore filter centrifugation and physiological saline is resuspended 3 times, you can
To pure targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA.
Fig. 1 is the structural representation of targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA prepared by the embodiment of the present invention 1, is wrapped
Include black phosphorus nano flake 10, adsorbed in the polyethylene glycol 20 on the surface of black phosphorus nano flake 10 and by amido link by electrostatic attraction
The folic acid 30 being connected on polyethylene glycol 20；One end of polyethylene glycol 20 is amino, and the other end is imino group, the imino group
Nitrogen-atoms is connected with the carbonylic carbon atom in folic acid 30 and constitutes the amido link, and the folic acid 30 is exposed to the outermost of nanometer formulation
Layer.
Fig. 2 (a) and Fig. 2 (b) are respectively the targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA of the preparation of the embodiment of the present invention 1
Scanning transmission electron microscope (STEM) and X-ray energy dispersion spectrum (EDS mapping), it is seen that this enforcement
The obtained BP-PEG-FA particle diameters of example are about 100nm or so, and particle size range is adapted to produce preferable EPR effects, and can obtain
The element for knowing BP-PEG-FA obtained in the present embodiment is configured to carbon, nitrogen, oxygen, four kinds of elements of phosphorus, preliminary identification targeting photo-thermal black phosphorus
Nanometer formulation BP-PEG-FA is successfully prepared.
Fig. 3 is the grain size distribution of targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA prepared by the embodiment of the present invention 1, with
Result has good uniformity in Fig. 2.
Fig. 4 is targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA Zeta in preparation process prepared by the embodiment of the present invention 1
The variation diagram of current potential., in -14mV or so, surface negative electricity absolute value is higher, can for the Zeta potential of black phosphorus nano flake BP NSs
Effectively interact with ammonia end polyethylene glycol, carry out coated with polyethylene glycol.Black phosphorus photo-thermal nanometer after coated with polyethylene glycol
Preparation (including without modified with folic acid BP-PEG NSs and have modified with folic acid BP-PEG-FA NSs) Zeta potential -10mV～-
Between 18mV, higher absolute value is maintained, repulsive interaction is stronger between nanometer formulation, thus height is steady in dispersion phase
It is fixed.
Fig. 5 is the FTIR spectrum of targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA prepared by the embodiment of the present invention 1
Figure (FT-IR).It can be seen that in～2900cm-1Absworption peak is in targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA
The CH vibration peaks and C-O vibration peaks of polyalkylene glycol moiety ,～1637 to～1653cm-1Absworption peak from folic acid moieties acyl
Amine is combined, and is further demonstrated targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA and is successfully prepared.
Embodiment 2
A kind of preparation method of targeting photo-thermal black phosphorus nanometer formulation, comprises the following steps：
(1) bonding probes ultrasonic method is peeled off using solution and prepares black phosphorus nano flake：50mg bulk black phosphorus materials are taken, is placed in
In 100mL ultra-pure waters, probe sonication is carried out, the ultrasonic time of the probe sonication method is 18 hours, and process is that continual ultrasonic 1 is little
When, 1 hour is waited for a cycle, power amplifier is 20%.After ultrasound terminates, take brownish black stripper and be placed in centrifuge tube,
2000rpm is centrifuged 6 minutes, and temperature is 4 DEG C, precipitates the block black phosphorus that is not peeled off, and the block black phosphorus of careful separation precipitation and
Black phosphorus nano flake in supernatant, with further purification ＆ isolation black phosphorus nano flake；
(2) triethylamine (0.5mL), NHS are added in the anhydrous DMSO of the 40mL of folic acid (1.0g, 2.27mmol) is dissolved with
(0.52g, 5.00mmol) and DCC (0.50g, 2.50mmol), obtains mixed liquor, and the mixed liquor is under the conditions of room temperature, lucifuge
After magnetic agitation reaction 24h, insoluble accessory substance and unreacted raw material is filtered to remove, obtains NHS activation folic acid.In triethylamine
On the premise of presence, the NHS activation folic acid of 2.5mL is added to into the DMSO solution at double ammonia ends polyethylene glycol (molecular weight is 5000)
In, both equimolar ratios obtain mixed solution.Mixed solution magnetic agitation reaction 24h under the conditions of room temperature, lucifuge, passes through
Coupling ammonia end copolymer, synthesizes the polyethylene glycol FA-PEG-NH of modified with folic acid2, dialyse (MWCO in distilled water:3000), with
Unreacted folic acid is removed, to FA-PEG-NH2Purified；
(3) by the way of probe sonication is in combination with magnetic agitation, in the Surface coating folic acid of black phosphorus nano flake
The polyethylene glycol preparation targeting photo-thermal black phosphorus nanometer formulation of modification, the wherein polyethylene glycol of black phosphorus nano flake and modified with folic acid
Mass ratio is 1:5.Specifically, first the ammonia end polyethylene glycol of modified with folic acid carried out to black phosphorus nano flake using probe sonication mode
Pre-coated is carried out, probe sonication process is carried out in the two suspension, the time of probe sonication, power amplifier was to continue 30 minutes
12%；By the way of magnetic agitation the further of the polyethylene glycol of modified with folic acid is carried out to black phosphorus nano flake at room temperature again
Cladding, magnetic agitation rotating speed is 1000rpm, and the duration is 4 hours；The black phosphorus nanometer light heating agent of simple coated with polyethylene glycol
(without modified with folic acid) can be prepared in the same way, and preparing raw material replaces with nothing and repaiies by the ammonia end polyethylene glycol of modified with folic acid
The ammonia end polyethylene glycol of decorations；
(4) purified by the way of milipore filter centrifugation, screen step (3) gained targeting photo-thermal black phosphorus nanometer formulation, obtained
The pure targeting photo-thermal black phosphorus nanometer formulation of optimization particle size：Take reaction mixture in step (3) and be added to super filter tube (MWCO
100kDa；Millipore centrifugation in), the polyethylene glycol for separating uncoated modified with folic acid is black with the targeting photo-thermal for preparing
Phosphorus nanometer formulation；The speed of milipore filter centrifugation is 4000rpm, and the time is 30 minutes, and temperature is 4 DEG C.After milipore filter centrifugation, with life
Targeting photo-thermal black phosphorus nanometer formulation on the resuspended filter membrane of reason salt solution, repeats milipore filter centrifugation and physiological saline is resuspended 4 times, you can
To pure targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA.
Fig. 6 is that targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA prepared by the embodiment of the present invention 2 swashs in 808nm near-infrareds
Time dependent photo-thermal curve spectrum under light irradiation.It can be seen that embodiment of the present invention targeting photo-thermal black phosphorus nanometer
Preparation BP-PEG-FA has good Photothermal characterisation, is 1.0W/cm in intensity2808nm laser irradiation under, concentration be 100 μ
The temperature of the targeting photo-thermal black phosphorus nanometer formulation of g/mL rose about 26 DEG C in 10 minutes.
Fig. 7 is that targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA prepared by the embodiment of the present invention 2 is closely red in multiple 808nm
Photo and thermal stability under outer laser irradiation.It can be seen that embodiment of the present invention targeting photo-thermal black phosphorus nanometer formulation BP-
PEG-FA temperature changes under the irradiation of multiple 808nm near-infrared lasers are stablized, the good light stability that near-infrared laser is excited.
Fig. 8 is the targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA of the preparation of the embodiment of the present invention 2 under different preparation times
Photo-thermal curve spectrum.It can be seen that preparing the targeting photo-thermal black phosphorus nanometer formulation BP- that a Zhou Houyu has just been prepared
PEG-FA near-infrared lasers under the same conditions excite rear temperature change consistent, it is therefore shown that prepared by the embodiment of the present invention
Targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA can lay in and used after placement.
Embodiment 3
A kind of preparation method of targeting photo-thermal black phosphorus nanometer formulation, comprises the following steps：
(1) bonding probes ultrasonic method is peeled off using solution and prepares black phosphorus nano flake, take 50mg bulk black phosphorus materials, be placed in
In 100mL ultra-pure waters, probe sonication is carried out, the ultrasonic time of the probe sonication method is 16 hours, and process is that continual ultrasonic 1 is little
When, 1 hour is waited for a cycle, power amplifier is 25%.After ultrasound terminates, take brownish black stripper and be placed in centrifuge tube,
1000rpm is centrifuged 15 minutes, and temperature is 4 DEG C, precipitates the block black phosphorus that is not peeled off, and the block black phosphorus of careful separation precipitation and
Black phosphorus nano flake in supernatant, with further purification ＆ isolation black phosphorus nano flake；
(2) triethylamine (0.5mL), NHS are added in the anhydrous DMSO of the 40mL of folic acid (1.0g, 2.27mmol) is dissolved with
(0.52g, 5.00mmol) and DCC (0.50g, 2.50mmol), obtains mixed liquor, and the mixed liquor is under the conditions of room temperature, lucifuge
After magnetic agitation reaction 24h, insoluble accessory substance and unreacted raw material is filtered to remove, obtains NHS activation folic acid.In triethylamine
On the premise of presence, the NHS activation folic acid of 2.5mL is added to into the DMSO solution at double ammonia ends polyethylene glycol (molecular weight is 550)
In, both equimolar ratios obtain mixed solution.Mixed solution magnetic agitation reaction 24h under the conditions of room temperature, lucifuge, passes through
Coupling ammonia end copolymer, synthesizes the polyethylene glycol FA-PEG-NH of modified with folic acid2, dialyse (MWCO in distilled water:500), removing
Unreacted folic acid is removed, to FA-PEG-NH2Purified；
(3) by the way of probe sonication is in combination with magnetic agitation, in the Surface coating folic acid of black phosphorus nano flake
The polyethylene glycol preparation targeting photo-thermal black phosphorus nanometer formulation of modification, the wherein polyethylene glycol of black phosphorus nano flake and modified with folic acid
Mass ratio is 1:8.Specifically, first the ammonia end polyethylene glycol of modified with folic acid carried out to black phosphorus nano flake using probe sonication mode
Pre-coated is carried out, probe sonication process is carried out in the two suspension, the time of probe sonication, power amplifier was to continue 30 minutes
15%；By the way of magnetic agitation the further of the polyethylene glycol of modified with folic acid is carried out to black phosphorus nano flake at room temperature again
Cladding, magnetic agitation rotating speed is 850rpm, and the duration is 4 hours；The black phosphorus nanometer light heating agent of simple coated with polyethylene glycol
(without modified with folic acid) can be prepared in the same way, and preparing raw material is replaced with without leaf by the ammonia end polyethylene glycol of modified with folic acid
The ammonia end polyethylene glycol of acid modification；
(4) purified by the way of milipore filter centrifugation, screen step (3) gained targeting photo-thermal black phosphorus nanometer formulation, obtained
The pure targeting photo-thermal black phosphorus nanometer formulation of optimization particle size：Take reaction mixture in step (3) and be added to super filter tube (MWCO
100kDa；Millipore centrifugation in), the polyethylene glycol for separating uncoated modified with folic acid is black with the targeting photo-thermal for preparing
Phosphorus nanometer formulation；The speed of milipore filter centrifugation is 4000rpm, and the time is 40 minutes, and temperature is 4 DEG C.After milipore filter centrifugation, with life
Targeting photo-thermal black phosphorus nanometer formulation on the resuspended filter membrane of reason salt solution, repeats milipore filter centrifugation and physiological saline is resuspended 5 times, you can
To pure targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA.
Embodiment 4
A kind of preparation method of targeting photo-thermal black phosphorus nanometer formulation, comprises the following steps：
(1) bonding probes ultrasonic method is peeled off using solution and prepares black phosphorus nano flake, take 50mg bulk black phosphorus materials, be placed in
In 100mL ultra-pure waters, probe sonication is carried out, the ultrasonic time of the probe sonication method is 14 hours, and process is continual ultrasonic 0.5
Hour, 0.5 hour is waited for a cycle, power amplifier is 30%.After ultrasound terminates, take brownish black stripper and be placed in centrifuge tube,
2000rpm is centrifuged 10 minutes, and temperature is 4 DEG C, precipitates the block black phosphorus that is not peeled off, and the block black phosphorus of careful separation precipitation and
Black phosphorus nano flake in supernatant, with further purification ＆ isolation black phosphorus nano flake；
(2) triethylamine (0.5mL), NHS are added in the anhydrous DMSO of the 40mL of folic acid (1.0g, 2.27mmol) is dissolved with
(0.52g, 5.00mmol) and DCC (0.50g, 2.50mmol), obtains mixed liquor, and the mixed liquor is under the conditions of room temperature, lucifuge
After magnetic agitation reaction 24h, insoluble accessory substance and unreacted raw material is filtered to remove, obtains NHS activation folic acid.In triethylamine
On the premise of presence, the DMSO that the NHS activation folic acid of 2.5mL is added to into double ammonia ends polyethylene glycol (molecular weight is 30000) is molten
In liquid, both equimolar ratios obtain mixed solution.Mixed solution magnetic agitation reaction 24h under the conditions of room temperature, lucifuge, leads to
Overcoupling ammonia end copolymer, synthesizes the polyethylene glycol FA-PEG-NH of modified with folic acid2, dialyse (MWCO in distilled water:3000),
To remove unreacted folic acid, to FA-PEG-NH2Purified；
(3) by the way of probe sonication is in combination with magnetic agitation, in the Surface coating folic acid of black phosphorus nano flake
The polyethylene glycol preparation targeting photo-thermal black phosphorus nanometer formulation of modification, the wherein polyethylene glycol of black phosphorus nano flake and modified with folic acid
Mass ratio is 1:12.Specifically, the poly- second two in ammonia end for first modified with folic acid being carried out to black phosphorus nano flake using probe sonication mode
Alcohol carries out pre-coated, and probe sonication process is carried out in the two suspension, and the time of probe sonication, power amplifier was to continue 20 minutes
20%；By the way of magnetic agitation the further of the polyethylene glycol of modified with folic acid is carried out to black phosphorus nano flake at room temperature again
Cladding, magnetic agitation rotating speed is 1200rpm, and the duration is 3 hours；The black phosphorus nanometer light heating agent of simple coated with polyethylene glycol
(without modified with folic acid) can be prepared in the same way, and preparing raw material replaces with nothing and repaiies by the ammonia end polyethylene glycol of modified with folic acid
The ammonia end polyethylene glycol of decorations；
(4) purified by the way of milipore filter centrifugation, screen step (3) gained targeting photo-thermal black phosphorus nanometer formulation, obtained
The pure targeting photo-thermal black phosphorus nanometer formulation of optimization particle size：Take reaction mixture in step (3) and be added to super filter tube (MWCO
100kDa；Millipore centrifugation in), the polyethylene glycol for separating uncoated modified with folic acid is black with the targeting photo-thermal for preparing
Phosphorus nanometer formulation；The speed of milipore filter centrifugation is 4000rpm, and the time is 40 minutes, and temperature is 4 DEG C.After milipore filter centrifugation, with life
Targeting photo-thermal black phosphorus nanometer formulation on the resuspended filter membrane of reason salt solution, repeats milipore filter centrifugation and physiological saline is resuspended 4 times, you can
To pure targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA.
Application Example
Using the high sugar nutrient solution culture Hela cells of the DMEM containing 10% hyclone in 37 DEG C, 5%CO2Incubator
In.After cell is paved with Tissue Culture Flask bottom, outmoded culture medium is discarded, rinsed once with PBS, add the pancreas of 1mL 0.25%
Protease, 37 DEG C of digestion 1-2 minutes, adds the complete medium containing serum on a small quantity, and gently being blown and beaten with elbow straw makes cell
Departing from bottle wall becomes single cell suspension.This single cell suspension is uniformly inoculated in 6 porocyte culture plates, 1mL cultures are added
Base, 37 DEG C, 5%CO2Cultivate 24 hours in incubator.Targeting photo-thermal black phosphorus prepared by the embodiment of the present invention 1 is added to receive in cell
Metric system agent BP-PEG-FA, continues to cultivate 4 hours.Flushed three times with ice-cold PBS, add methyl alcohol to fix cell 20min, discarded
Methyl alcohol, adds DAPI dye liquors incubation 5min, then is flushed three times with PBS, the observation of cell under FV-1000 laser confocal microscopes
Intake situation.The a length of 340nm of excitation light wave used by observation DAPI, by Bright passages observation targeting photo-thermal black phosphorus nanometer formulation
The intake situation of BP-PEG-FA.
Treat that the Hela cells being normally incubated in the high sugar nutrient solutions of the DMEM of 10% hyclone are paved with cell culture bottom of bottle
Behind portion, outmoded culture medium is discarded, rinsed once with PBS, add the trypsase of 1mL 0.25%, 37 DEG C digest 1-2 minutes, plus
Enter a small amount of complete medium containing serum, gently piping and druming makes cell detachment bottle wall become single cell suspension.This suspension is carried out
It is inoculated in 96 porocyte culture plates by the even density of 5000 cells/wells after cell count, dilution, in 37 DEG C, 5%CO2
Incubator in continue cultivate make cell attachment within 24 hours.Disperse the targeting light of the preparation of the embodiment of the present invention 1 with complete medium
Hot black phosphorus nanometer formulation BP-PEG-FA (concentration is respectively 10,25,50 μ g/mL) is used as testing sample；Without targeting photo-thermal black phosphorus
The Hela cells of the complete medium culture of nanometer formulation BP-PEG-FA are as a control group.It is to be seeded in 96 porocyte culture plates
In Hela cell culture 24h it is adherent after, discard outmoded culture medium, with PBS rinse once, add variable concentrations experimental group and
Control group is cultivated respectively 4 hours, is subsequently 1.0W/cm in intensity2808nm laser irradiate 10 minutes, discard outmoded culture medium,
After being rinsed once with PBS, complete medium is added, after 37 DEG C of overnight incubations, by mtt assay (each group is respectively provided with 5 multiple holes)
The cytoactive of different groups is determined to determine the external photo-thermal therapy of cancer effects of targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA.
Fig. 9 is that targeting photo-thermal prepared by laser confocal scanning electron microscope (CLSM) the observation embodiment of the present invention 1 is black
The Hela cellular uptake pictures of phosphorus nanometer formulation BP-PEG-FA.Specifically Fig. 9 (a), Fig. 9 (b), Fig. 9 (c) are respectively nucleus
Blueness is dyed with DAPI, the image of DAPI passages, Bright passages and hybrid channel observation is passed sequentially through.Can from figure
Go out, the targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA of the embodiment of the present invention can be efficiently entering cancer cell, and then can be
There is photo-thermal therapy effect in cell interior.
Figure 10 is the targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA of the preparation of the embodiment of the present invention 1 in various cancers cell
Toxicity data in (Hela, MCF-7, HepG2 and PC3).It can be seen that the targeting photo-thermal black phosphorus of the embodiment of the present invention
Nanometer formulation BP-PEG-FA does not possess cytotoxicity under without laser radiation situation, and biological safety is good, have no toxic side effect.
Figure 11 exists for targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA prepared by the embodiment of the present invention 1 of variable concentrations
Targeting photo-thermal therapy effect in Hela cells.Abscissa black phosphorus concentration is referred to and contained in black phosphorus targeting light heating agent in Figure 11
There is the concentration of black phosphorus.Control refers to and does not do any process cell, used as blank reference.It can be seen that being in intensity
1.0W/cm2808nm laser irradiation under, with the black phosphorus nano flake (BP NSs) and black phosphorus photo-thermal nanometer without targeting modification
Preparation (BP-PEG NSs) is compared, and targeting photo-thermal black phosphorus nanometer formulation BP-PEG-FA has more excellent targeting photo-thermal
Therapeutic effect, can effectively kill cancer cell, and only in the irradiated site generation therapeutic effect of near-infrared laser, so as to very big
The injury to normal cell, tissue and organ is reduced in degree, with specific aim.
It should be noted that according to the above description the announcement of book and and illustrate, those skilled in the art in the invention are also
Above-mentioned embodiment can be changed and changed.Therefore, the invention is not limited in concrete reality disclosed and described above
Mode is applied, some equivalent modifications and change to the present invention should also be as within the scope of the claims of the present invention.This
Outward, although some specific terms used in this specification, these terms merely for convenience of description, not to the present invention
Constitute any restriction.
Claims (13)
1. it is a kind of to target photo-thermal black phosphorus nanometer formulation, it is characterised in that to exist including black phosphorus nano flake, by electrostatic attraction absorption
The polyethylene glycol on the black phosphorus nano flake surface and the folic acid on the polyethylene glycol is connected to by amido link, it is described
One end of polyethylene glycol is amino, and the other end is imino group, and the nitrogen-atoms of the imino group is former with the carbonyl carbon in the folic acid
Son is connected and constitutes the amido link, and the folic acid is exposed to the outermost layer of the nanometer formulation.
2. it is as claimed in claim 1 to target photo-thermal black phosphorus nanometer formulation, it is characterised in that the targeting photo-thermal black phosphorus nanometer system
Agent is by by black phosphorus nano flake, double ammonia ends polyethylene glycol, folic acid (1～3) in mass ratio: (5～12): (2～6) proportioning system
.
3. it is as claimed in claim 1 to target photo-thermal black phosphorus nanometer formulation, it is characterised in that the length and width of the black phosphorus nano flake
Size is 100nm-200nm；The thickness of the black phosphorus nano flake is 2nm-3nm.
4. it is as claimed in claim 2 to target photo-thermal black phosphorus nanometer formulation, it is characterised in that the weight of described pair of ammonia end polyethylene glycol
Average molecular weight is 550-30000.
5. it is a kind of targeting photo-thermal black phosphorus nanometer formulation preparation method, it is characterised in that comprise the following steps：
Black phosphorus nano flake is provided；
Hydroxysuccinimide-activated folic acid is added in the dimethyl sulphoxide solution of double ammonia ends polyethylene glycol, and is added appropriate
Catalyst, obtains mixed solution, and the mixed solution is stirred reaction 16-32 hours under the conditions of room temperature, lucifuge, has reacted
Into rear filtration, the polyethylene glycol of modified with folic acid is obtained, the folic acid is connected on polyethylene glycol by amido link, the poly- second two
One end of alcohol is amino, and the other end is imino group, and the nitrogen-atoms of the imino group is connected with the carbonylic carbon atom in the folic acid
Constitute the amido link；
Successively by the way of probe sonication, magnetic agitation, the black phosphorus nano flake Surface coating modified with folic acid it is poly-
Ethylene glycol, obtains targetting photo-thermal black phosphorus nanometer formulation, and the polyethylene glycol of the modified with folic acid is adsorbed described by electrostatic attraction
Black phosphorus nano flake surface, the folic acid is exposed to the outermost layer of the targeting photo-thermal black phosphorus nanometer formulation.
6. the preparation method of photo-thermal black phosphorus nanometer formulation is targetted as claimed in claim 5, it is characterised in that the black phosphorus nanometer
Thin slice, double ammonia ends polyethylene glycol, folic acid (1～3) in mass ratio: (5～12): (2～6) feed intake.
7. the preparation method of photo-thermal black phosphorus nanometer formulation is targetted as claimed in claim 5, it is characterised in that the catalyst bag
Triethylamine is included, the catalyst is 1 with the volume ratio of reaction system:75-85.
8. the preparation method of photo-thermal black phosphorus nanometer formulation is targetted as claimed in claim 5, it is characterised in that the probe sonication
Time be 20-40 minutes, during the probe sonication, continual ultrasonic -1 hour 45 seconds, and wait -1 hour 15 seconds be one
Individual cycle, power amplifier is 12%-20%.
9. the preparation method of photo-thermal black phosphorus nanometer formulation is targetted as claimed in claim 5, it is characterised in that the magnetic agitation
Rotating speed be 800rpm-1200rpm, the duration be 3-4 hours.
10. the preparation method of photo-thermal black phosphorus nanometer formulation is targetted as claimed in claim 5, it is characterised in that the black phosphorus is received
Rice thin slice is peeled off bonding probes ultrasonic methods and is prepared using solution, and using being collected by centrifugation, purify, the probe sonication it is ultrasonic when
Between be 12-18 hours, during the probe sonication, continual ultrasonic -1 hour 45 seconds, and wait -1 hour 15 seconds be a week
Phase, power amplifier is 20%-30%, and the speed of the centrifugation is 1000rpm-2000rpm, and the time is 6-15 minutes, and temperature is 4 DEG C.
The preparation method of 11. targeting photo-thermal black phosphorus nanometer formulations as claimed in claim 5, it is described hydroxysuccinimide-activated
Folic acid is prepared in the following way：Folic acid is dissolved in anhydrous dimethyl sulphoxide, HOSu NHS and N, N'- is added
Dicyclohexylcarbodiimide obtains mixed liquor, and the mixed liquor is stirred instead under room temperature, lucifuge and triethylamine catalytic condition
16-32 hours are answered, is filtered after the completion of reaction, obtain the hydroxysuccinimide-activated folic acid.
The preparation method of 12. targeting photo-thermal black phosphorus nanometer formulations as claimed in claim 5, it is characterised in that further include
By the way of milipore filter centrifugation gained targeting photo-thermal black phosphorus nanometer formulation is purified at least one times, screened, optimized
The pure targeting photo-thermal black phosphorus nanometer formulation of particle size, the speed of milipore filter centrifugation is 2000-4000rpm, and the time is
30-60 minutes, temperature is 4-8 DEG C.
The 13. targeting photo-thermal black phosphorus nanometer formulations as described in any one of claim 1-4 are preparing cancer light thermal ablation therapy medicine
Application in thing.
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| PCT/CN2017/088021 WO2018095017A1 (en) | 2016-11-25 | 2017-06-13 | Targeted photothermal black phosphorus nano preparation and preparation method and application thereof |
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