CN102836443A - Polypeptide-modified brain-targeted nanometer device and preparation method and usage thereof - Google Patents
Polypeptide-modified brain-targeted nanometer device and preparation method and usage thereof Download PDFInfo
- Publication number
- CN102836443A CN102836443A CN2011101747371A CN201110174737A CN102836443A CN 102836443 A CN102836443 A CN 102836443A CN 2011101747371 A CN2011101747371 A CN 2011101747371A CN 201110174737 A CN201110174737 A CN 201110174737A CN 102836443 A CN102836443 A CN 102836443A
- Authority
- CN
- China
- Prior art keywords
- brain
- polypeptide
- rvg29
- sequence
- cation
- 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
Images
Abstract
The invention belongs to the field of biotechnology and relates to a polypeptide-modified brain-targeted nanometer device and a preparation method and usage thereof. The nanometer device consists of a cationic dendritic polymer material, an activated Caspase-3 specific response element, a quencher and a street virus glycoprotein derivative polypeptide RVG29 with a brain-targeted function. By taking the RVG29 to serve as a brain-targeted head group and the cationic dendritic polymer as a carrier material, the RVG29 is connected through a bi-functional bonding agent to construct a brain-targeted carrier. The polypeptide-modified brain-targeted nanometer device penetrates a blood brain barrier to reach inside of a brain by utilizing a receptor-mediated transcellular action, and the polypeptide-modified brain-targeted nanometer device can be bonded with a specific receptor on the surface of a neuron, and thereby, the nanometer device is gathered to the neuron, and the cell apoptosis condition in the neuron can be favorably detected. When the nanometer device is applied in vivo, not only is whether activated Caspase-3 exists or not confirmed but also the existence amount comparison of the activated Caspase-3 can be realized.
Description
Technical field
The invention belongs to biological technical field; Relate to a kind of peptide modified brain targeted nano device; Be specifically related to a kind of brain targeted nano device and method for preparing and purposes of deriving peptide modified, relate in particular to a kind of brain targeted nano device and method for preparing and its application in the activated form Caspase-3 imaging in brain of deriving peptide modified by rabies virus glycoprotein by rabies virus glycoprotein.
Background technology
Neurodegenerative diseases comprises that Alzheimer and parkinson disease are one type of diseases with higher incidence and mortality rate; It is long to have the course of disease; Progressivity; Lack clear and definite characteristics such as diagnostic test, and before the manifest symptom such as the dyskinesia etc. occurring, the pathological characters that has been attended by some row comprises generations such as irreversible neuronal death; Therefore, early stage detection and diagnosis are most important for treatment this type disease, especially delay and stop neuronic death.The change of brain was not easy to found by nuclear magnetic resonance (MRI) and tomoscan (CT) usually in chemical level when neurodegenerative diseases took place; In early days it is diagnosed at neurodegenerative diseases and to be full of challenge.In addition, though central nervous system's stable state has been kept in the existence of blood brain barrier on the one hand, simultaneously it has also hindered treatment and diagnostic medicine and passs through the whole body administering mode and be released in the brain, especially for macromolecular drug.
Prior art discloses the typical characteristic that relevant neuronal death is a neurodegenerative diseases; Therefore, note abnormalities with the neurocyte of apoptosis and give to treat timely and seem particularly important.Discover, play an important role by the mediated cell death in chronic neurodegenerative diseases of Caspase mediated Apoptosis approach; And the activation of Caspase-3 ubiquity in the apoptosis of neurocyte.Though cutter system is also not clear and definite as yet really to cause this type disease, the participation of Caspase-3 activated pathway has obtained extensive common recognition.Described Caspase-3 exists with the zymogen forms of nonactive 32k Dalton molecular weight in cell usually; When apoptosis took place, the protease signal at the upper reaches can be cracked into 12k and the daltonian subunit of 17k with it.There is report to prove that the male substantia nigra dopaminergic neuron ratio of activated form Caspase-3 obviously raises in parkinson disease; Therefore, realize interior detection of body of activated form Caspase-3, will be of value to the potential neuronal damage of monitoring, and in time intervene.
In order to achieve the above object, diagnostic reagent must possess the ability that strides across blood brain barrier.The tight connection that described blood brain barrier mainly is made up of brain capillary endothelial cell makes that most molecules are difficult to get in the brain from blood.At present, have higher selectivity and see through efficient because of it, pass striding in the blood brain barrier transhipment strategy of release system and more be applied to medicine by receptor-mediated endocytic pathway; Simultaneously, by deutero-29 amino acid polypeptides of rabies virus glycoprotein (RVG29), in the research in early stage, the blood brain barrier of striding that has been proved to be a kind of targeting of brain efficiently head base mediation medicine is passed and is released.
Research confirms, PLL dendrimer (DGLs) is one type of novel nano-grade size, has the polymer of degradability, and monomer whose is an aminutrin, is connected by degradable amido link, and end has abundant amino, can be by further modification.Utilize the characteristics of DGLs combine Recent study comparatively widely FRET (FRET) technology can make up the nano-device of brain targeting, realize the imaging of activated form Caspase-3 in the brain.The polypeptide of the substrate sequence (DEVD) that contains activated form Caspase-3 identification of fluorescent probe Cy5 labelling can be covalently attached to the surface of DGLs through bifunctional linking reagent; Adopt the quencher QSY21 of Cy5 simultaneously; Be connected in the DGLs surface with nonlinear mode; Through controlling synthetic ratio, can reach intermolecular efficiently cancellation, and then reduce the interference of background fluorescence signal.
Adopt the modification of RVG29 brain targeting head base, can transport, make nano-device to get in the brain and play a role through the receptor-mediated blood brain barrier of striding; But do not see the brain targeted nano device report that relevant RVG29 modifies at present as yet.
Summary of the invention
The purpose of this invention is to provide a kind of peptide modified brain targeted nano device, relate in particular to a kind of brain targeted nano device and method for preparing of deriving peptide modified by rabies virus glycoprotein.
Another object of the present invention provides the application in the activated form Caspase-3 imaging in brain of described brain targeted nano device.
Brain targeted nano device of the present invention combines initiatively targeting and FRET technology, carries out the imaging of activated form Caspase-3 in the brain, can important evidence be provided for the early diagnosis of neurodegenerative diseases.
Particularly; Peptide modified brain targeted nano device of the present invention; It is characterized in that, form by cation dendroid macromolecule material, activated form Caspase-3 specific reaction element, quencher and deutero-29 amino acid polypeptide RVG299 of rabies virus glycoprotein with brain targeting; The deutero-29 amino acid polypeptide RVG29 of described rabies virus glycoprotein are as brain targeting head base, and the cation dendrimer is a carrier material, connect RVG29 through bifunctional linking reagent and make up the brain targeting vector.
Among the present invention, described cation dendroid macromolecule material is selected from PLL dendrimer (DGLs) and polyamide-amide (polyamidoamine is called for short PAMAM);
Among the present invention, described bifunctional linking reagent is Sulfo-LC-SMPT;
Among the present invention, what described activated form Caspase-3 specific reaction element was selected from the Cy5 labelling contains the DEVD polypeptide of sequence, and its sequence is CGDEVDAPK (Cy5);
The quencher of described Cy5 fluorescence is QSY21.
Among the present invention, the deutero-29 amino acid polypeptide RVG29 of described rabies virus glycoprotein have the sequence of SEQ ID NO.1.
Among the present invention; Contain DEVD polypeptide of sequence, QSY21 and polypeptide RVG29 with cation dendroid macromolecule material, Cy5 labelling are component; The DEVD polypeptide of sequence that contains of Cy5 labelling is connected tree-shaped macromolecular carrier through bifunctional linking reagent Sulfo-LC-SMPT with covalent manner with RVG29, and QSY21 directly is covalently attached to the dendrimer carrier, makes up the brain targeting vector; Wherein
The molecular proportion that contains DEVD polypeptide of sequence and cation dendrimer material of described Cy5 labelling is 1～10: 1;
The molecular proportion that contains the DEVD polypeptide of sequence of described QSY21 and Cy5 labelling is 5～10: 1;
The molecular proportion of described RVG29 polypeptide and cation dendrimer material is 1～10: 1.
The invention provides the method for the peptide modified brain targeted nano device of preparation, it is characterized in that it comprises:
Cation dendroid macromolecule material and bifunctional linking reagent Sulfo-LC-SMPT are dissolved in the phosphate buffer of pH value 7.4 with 1: 1～70 molecular proportion; Stirring at room reaction 60～180 minutes; The amino on cation dendroid macromolecule surface and the butanimide generation specific reaction of Sulfo-LC-SMPT are removed unreacted bridging agent with the phosphate buffer dialysed overnight of the pH value 7.4 that contains EDTA; Contain DEVD polypeptide of sequence and the RVG29 polypeptide end of Cy5 labelling are connected with cysteine and introduce sulfydryl; Respectively with 1～15: 1 and 1～10: what the special group stirring at room reaction on 1 molecular proportion and the cation dendrimer material-Sulfo-LC-SMPT junctional complex generated brain target cationic dendrimer-Cy5 labelling after 12～72 hours contains DEVD polypeptide of sequence complex; Molecular exclusion chromatography is removed unreacted polypeptide, and solvent is removed in lyophilizing; In the phosphate buffer of pH value 7.4～8.5 further with the QSY21 that contains butanimide directly with the amino on cation dendroid macromolecule surface according to molecular proportion 5～100: 1 ratio is reacted, and is built into brain targeted activation type Caspase-3 in-vivo imaging nano-device.
Brain targeted activation type Caspase-3 in-vivo imaging nano-device of the present invention is applicable to that brain contains the imaging of activated form Caspase-3.
Among the present invention; Contain the DEVD polypeptide of sequence through what bifunctional linking reagent connected several Cy5 labellings on the described brain targeting vector, utilize the QSY21 of butanimide functionalization to be directly connected in the cation dendroid macromolecule surface again, through controlling the fluorescence that synthetic ratio reaches complete cancellation Cy5; Can obviously reduce the interference of background fluorescence signal; In the cell that activated form Caspase-3 exists, but the DEVD specificity is identified and cracking, discharges Cy5 fluorescence; This signal can be realized the activated form Caspase-3 imaging in the brain further by detections such as living imaging appearance.
The present invention adopts the living imaging instrument system that the cancellation reaction that QSY21 connects is monitored, and the result shows that the molecular proportion of QSY21 and Cy5 modified polypeptides is, can reach intermolecular efficiently cancellation at 1: 7 o'clock;
The present invention adopts the ultraviolet-visible light spectrophotography to measure the absorption spectrum that contains DEVD polypeptide of sequence and brain targeted activation type Caspase-3 in-vivo imaging nano-device of cation dendroid macromolecule, Cy5 labelling respectively; The result shows; The characteristic absorption of brain targeted activation type Caspase-3 in-vivo imaging nano-device comprises the characteristic peak that contains DEVD polypeptide of sequence and cation dendroid macromolecule of Cy5 labelling; The result confirms that brain targeted activation type Caspase-3 in-vivo imaging nano-device synthesizes successfully;
The present invention adopts AFM and particle diameter appearance that brain targeted activation type Caspase-3 in-vivo imaging nano-device is characterized; The result shows; Brain targeted activation type Caspase-3 in-vivo imaging nano-device is a homogeneous, spherical, and particle diameter is between the particle of 10-25nm.
The fluorescence emission spectrum of brain targeted activation type Caspase-3 in-vivo imaging nano-device of the present invention is presented under the normal physiological state, and nano-device is from the state of fluorescent quenching, and this kind state all keeps stable in the presence of Reducing agent DTT and serum; Add after the 5 activated form Caspase-3 of unit hatch, nano-device obviously absorbs in that 660nm is visible, and the result shows; Described nano-device is under the state that activated form Caspase-3 exists; Can and cut off by specific recognition, discharge the Cy5 fluorescence light segments, so to be detected.
After the present invention induces the cell that produces activated form Caspase-3 to hatch with normal HNB's cell SH-SY5Y cell with through the rotenone of variable concentrations respectively; Place under the Laser Scanning Confocal Microscope and observe, the normal cell group does not detect the Cy5 signal, and the result shows; The normal cell state; Caspase-3 exists with the form of proenzyme, but not activated form Caspase-3, nano-device keeps intermolecular cancellation state; Induce in the cell that produces activated form Caspase-3 at the low concentration rotenone; Can see the fluorescence of portion C y5; The result shows; The containing DEVD polypeptide of sequence element and can in the cell that activated form Caspase-3 exists, be identified and cut off of Cy5 labelling in the brain targeted activation type Caspase-3 in-vivo imaging nano-device, the Cy5 fluorescence signal can be observed by laser confocal microscope; Induce in the cell that produces activated form Caspase-3 at the high concentration rotenone; The ratio of activated form Caspase-3 further raises; The fluorescence signal of Cy5 also almost is present in all cells; The result shows that brain targeted activation type Caspase-3 in-vivo imaging nano-device can detect the existence of activated form Caspase-3 on cellular level, and fluorescence signal raises with the amount of activated form Caspase-3 and strengthens.
Brain targeted activation type Caspase-3 in-vivo imaging nano-device of the present invention is observed fluorescence signal variation in the rat brain that compares rotenone administration different number of days through the living imaging appearance behind the tail intravenously administrable, the result shows; In the normal rat brain, almost do not see fluorescence signal, along with the increase of rotenone administration natural law; Activated form Caspase-3 increases; Fluorescence signal increases, and in the time of 35 days, fluorescence signal descends in the rotenone administration; Because the neuron mortality takes place, activated form Caspase-3 reduces; The living imaging appearance carries out the paraformaldehyde perfusion to rat after observing and accomplishing, brain frozen section and the dyeing of neuron mark; Place under the laser confocal microscope and observe; The fluorescence signal of Cy5 mainly concentrates on around the green neuron mark green, exists morely through protein immunoblot proof activated form Caspase-3 black substance midbrain zone in rotenone administration 5 days and 10 days rat brain, and this regional signal of observation is also comparatively obvious down for laser confocal microscope; The result shows; Behind the brain targeted activation type Caspase-3 in-vivo imaging nano-device process tail intravenously administrable, can arrive in the brain, and after the activated form Caspase-3 that is existed in the cell cut-out; Demonstrate the fluorescence signal of Cy5, the amount of the power of this signal and activated form Caspase-3 has certain positive correlation.
The present invention compared with prior art has the following advantages and characteristic:
Utilize the height branch characteristic of cation dendroid macromolecule material; What connect activated form Caspase-3 specific reaction element Cy5 labelling on its surface contains the DEVD polypeptide of sequence; Further utilize QSY21 to be connected in the cation dendroid macromolecule material surface through covalent bond; Be that with the difference of general fluorescent energy resonance transfer technical application fluorogen Cy5 and being connected of quencher QSY21 are non-linear; The reaction ratio of quencher and fluorogen is regulatable, and then helps reducing the background fluorescence signal, and the specificity that improves imaging is with sensitive; Use a basic RVG29 that cation dendroid macromolecule is further modified simultaneously, make nano-device can utilize the receptor-mediated cytosis of wearing to pass in the blood brain barrier arrival brain with brain targeting; In addition,, realize the neuronotropic enrichment of nano-device, help detecting the apoptosis situation in the neuron because RVG29 also can further combine with the specific receptor on neuron surface.
When nano-device of the present invention is used in vivo; The amount height of distinguishable activated form Caspase-3; And then embody the difference of fluorescence signal intensity; The existence of not only proving conclusively activated form Caspase-3 whether, the sxemiquantitative that also can realize activated form Caspase-3 relatively is expected on the early diagnosis of neurodegenerative diseases, play an important role.
Description of drawings
Fig. 1 has shown the sign of brain targeted activation type Caspase-3 in-vivo imaging nano-device of the present invention, wherein,
A: the living imaging appearance observe different Q SY21 and cation dendroid macromolecule-Cy5 labelling contain DEVD polypeptide of sequence complex reaction ratio the time, the efficient of intermolecular fluorescent quenching;
B: uv-visible absorption spectroscopy; Green curve: brain targeted activation type Caspase-3 in-vivo imaging nano-device; Blue curve: the Cy5 labelling contain the DEVD polypeptide of sequence; Red curve: cation dendroid molecule DGLs;
C: the AFM figure of nano-device;
D: the comparison of the fluorescence emission spectrum of nano-device and Cy5 fluorescence emission spectrum under the different condition.
Fig. 2 has shown under the laser confocal microscope of the present invention fluorescence signal situation after observing brain targeted activation type Caspase-3 in-vivo imaging nano-device and HNB's cell SH-SY5Y cell hatches, wherein, and amplification: * 200 times; The painted nucleus of blue signal: DAPI; Danger signal: Cy5;
A: the DAPI nucleus dyeing fluorogram of normal SH-SY5Y cell;
B: normal SH-SY5Y cell and nano-device are hatched back Cy5 fluorogram;
The stacking chart of C:A and B;
The D:0.1uM rotenone was hatched the SH-SY5Y cell after 24 hours, DAPI nucleus dyeing fluorogram;
The E:0.1uM rotenone was hatched the SH-SY5Y cell after 24 hours, and nano-device is hatched back Cy5 fluorogram;
The stacking chart of F:D and E;
The G:0.5uM rotenone was hatched the SH-SY5Y cell after 24 hours, DAPI nucleus dyeing fluorogram;
The H:0.5uM rotenone was hatched the SH-SY5Y cell after 24 hours, and nano-device is hatched back Cy5 fluorogram;
The stacking chart of I:G and H.
Fig. 3 has shown brain targeted activation type Caspase-3 in-vivo imaging nano-device of the present invention behind intravenous administration, fluorescence distribution situation in the rat brain, wherein,
A a: left side: normal rat; In: the rat of rotenone administration after 1 day; Right: the rat of rotenone administration after 5 days;
B a: left side: normal rat; In: the rat of rotenone administration after 10 days; Right: the rat of rotenone administration after 35 days.
Fig. 4 has shown laser confocal microscope observation brain targeted activation type Caspase-3 in-vivo imaging nano-device of the present invention behind intravenous administration, each regional fluorescence distribution situation in the rat brain; Amplification: * 400 times; The painted nucleus of blue signal: DAPI; Danger signal: Cy5; Green: the neuron of Alexa Fluor 488 specific antibody labellings, wherein,
A: normal rat striatum zone fluorogram;
B: normal rat black substance zone fluorogram;
C: normal rat hippocampus zone fluorogram;
D: normal rat cortex zone fluorogram;
E: the rotenone administration is rat striatum zone fluorogram after 1 day;
F: the rotenone administration is rat substantia nigra zone fluorogram after 1 day;
G: the rotenone administration is rat hippocampus zone fluorogram after 1 day;
H: the rotenone administration is rat layer zone fluorogram after 1 day;
I: the rotenone administration is rat striatum zone fluorogram after 5 days;
J: the rotenone administration is rat substantia nigra zone fluorogram after 5 days;
K: the rotenone administration is rat hippocampus zone fluorogram after 5 days;
L: the rotenone administration is rat layer zone fluorogram after 5 days;
M: the rotenone administration is rat striatum zone fluorogram after 10 days;
N: the rotenone administration is rat substantia nigra zone fluorogram after 10 days;
O: the rotenone administration is rat hippocampus zone fluorogram after 10 days;
P: the rotenone administration is rat layer zone fluorogram after 10 days;
Q: the rotenone administration is rat striatum zone fluorogram after 35 days;
R: the rotenone administration is rat substantia nigra zone fluorogram after 35 days;
S: the rotenone administration is rat hippocampus zone fluorogram after 35 days;
T: the rotenone administration is rat layer zone fluorogram after 35 days.
The specific embodiment
DGLs (second filial generation) and bifunctional linking reagent Sulfo-LC-SMPT are dissolved in the phosphate buffer (being called for short PBS) of pH 7.4 at 1: 10 according to molecular proportion; Stirring at room reaction 2h removes unreacted bridging agent with the phosphate buffer dialysed overnight of the pH value 7.4 that contains EDTA.
In the DGLs-Sulfo-LC-SMPT complex solution of above-mentioned preparation; What add the terminal Cy5 labelling that is connected with cysteine contains DEVD polypeptide of sequence and RVG29 polypeptide; Contain DEVD polypeptide of sequence complex with what the molecular proportion of 1: 3 and 1: 2 and the special group stirring at room reaction on cation dendrimer material-Sulfo-LC-SMPT junctional complex generated brain target cationic dendrimer-Cy5 labelling after 12 hours respectively; Molecular exclusion chromatography is removed unreacted polypeptide, and solvent is removed in lyophilizing.
The DEVD polypeptide of sequence complex redissolution amino further direct with the QSY21 that contains butanimide and the DGLs surface in the phosphate buffer of pH value 7.4～8.5 that contains of the brain target cationic dendrimer-Cy5 labelling of above-mentioned preparation reacts according to 21: 1 ratio of molecular proportion, is built into brain targeted activation type Caspase-3 in-vivo imaging nano-device.
After the brain targeted activation type Caspase-3 in-vivo imaging nano-device that embodiment 3 makes up induces the cell that produces activated form Caspase-3 to hatch with normal HNB's cell SH-SY5Y cell with through the rotenone of variable concentrations respectively; Place under the Laser Scanning Confocal Microscope and observe, the normal cell group does not detect the Cy5 signal; Induce in the cell that produces activated form Caspase-3 at the low concentration rotenone; Can see the fluorescence of portion C y5; The result shows; Containing DEVD polypeptide of sequence element and can in the cell that activated form Caspase-3 exists, being identified and cutting off of Cy5 labelling in the brain targeted activation type Caspase-3 in-vivo imaging nano-device produces the Cy5 fluorescence signal; Induce in the cell that produces activated form Caspase-3 at the high concentration rotenone; The ratio of activated form Caspase-3 further raises; The fluorescence signal of Cy5 also almost is present in all cells; The result shows that brain targeted activation type Caspase-3 in-vivo imaging nano-device can detect the existence of activated form Caspase-3 on cellular level, and fluorescence signal raises with the amount of activated form Caspase-3 and strengthens.
The brain targeted activation type Caspase-3 in-vivo imaging nano-device that embodiment 3 makes up is observed fluorescence signal variation in the rat brain that compares rotenone administration different number of days through the living imaging appearance behind the tail intravenously administrable; The result shows, in the normal rat brain, does not almost see fluorescence signal, along with the increase of rotenone administration natural law; Activated form Caspase-3 increases, and fluorescence signal increases, in the rotenone administration in the time of 35 days; Fluorescence signal descends, because the neuron mortality takes place, activated form Caspase-3 reduces.
Embodiment 6
The brain targeted activation type Caspase-3 in-vivo imaging nano-device that embodiment 3 makes up is behind the tail intravenously administrable; Rat is carried out the paraformaldehyde perfusion; Brain frozen section and the dyeing of neuron mark place under the laser confocal microscope and observe, and the fluorescence signal of Cy5 mainly concentrates on around the green neuron mark green; Black substance midbrain zone exists more in the rat brain of protein immunoblot proof activated form Caspase-3 rotenone administration 5 days and 10 days; It is also comparatively obvious that laser confocal microscope is observed this regional signal down, and the result shows, behind the brain targeted activation type Caspase-3 in-vivo imaging nano-device process tail intravenously administrable; Can arrive in the brain; And after the activated form Caspase-3 that is existed in the cell cuts off, demonstrate the fluorescence signal of Cy5, the amount of the power of this signal and activated form Caspase-3 has certain positive correlation.
The second filial generation DGLs that adopts in the above embodiment of the present invention is available from COLCOM company.
SEQUENCE?LISTING
< 110>Fudan University
< 120>a kind of peptide modified brain targeted nano device and method for preparing and purposes
<130>
<160> 1
<170> PatentIn?version?3.3
<210> 1
<211> 29
<212> PRT
<213> Rabies?virus
<400> 1
Tyr?Thr?Ile?Trp?Met?Pro?Glu?Asn?Pro?Arg?Pro?Gly?Thr?Pro?Cys?Asp
1 5 10 15
Ile?Phe?Thr?Asn?Ser?Arg?Gly?Lys?Arg?Ala?Ser?Asn?Gly
20 25
Claims (8)
1. a peptide modified brain targeted nano device is characterized in that, is made up of cation dendroid macromolecule material, activated form Caspase-3 specific reaction element, quencher and rabies virus glycoprotein polypeptides derived RVG29; As brain targeting head base, the cation dendrimer is a carrier material with polypeptide RVG29, connects RVG29 through bifunctional linking reagent and is built into the brain targeting vector.
2. by the described peptide modified brain targeted nano device of claim 1; It is characterized in that; In the described brain targeting vector; Contain DEVD polypeptide of sequence, QSY21 and polypeptide RVG29 with cation dendroid macromolecule material, Cy5 labelling are component, and the DEVD polypeptide of sequence that contains of Cy5 labelling is connected tree-shaped macromolecular carrier through bifunctional linking reagent with covalent manner with RVG29, and QSY21 directly is covalently attached to the dendrimer carrier.
3. by claim 1 or 2 described peptide modified brain targeted nano devices, it is characterized in that described rabies virus glycoprotein polypeptides derived RVG29 has the sequence of SEQ ID NO.1;
Described cation dendroid macromolecule material is selected from PLL dendrimer and polyamide-amide;
Described bifunctional linking reagent is Sulfo-LC-SMPT;
Its sequence of DEVD polypeptide of sequence that contains of described Cy5 labelling is CGDEVDAPK;
Described quencher is QSY21.
4. by the described peptide modified brain targeted nano device of claim 2, it is characterized in that the molecular proportion that contains DEVD polypeptide of sequence and cation dendrimer material of described Cy5 labelling is 1～10: 1.
5. by the described peptide modified brain targeted nano device of claim 2, it is characterized in that the molecular proportion that contains the DEVD polypeptide of sequence of described QSY21 and Cy5 labelling is 5～10: 1.
6. by the described brain targeted nano of claim 2 device, it is characterized in that the molecular proportion of described RVG29 polypeptide and cation dendrimer material is 1～10: 1.
7. claim 1 or 2 peptide modified brain targeted nano preparation of devices method is characterized in that it comprises step:
Cation dendroid macromolecule material and bifunctional linking reagent Sulfo-LC-SMPT are dissolved in the phosphate buffer of pH value 7.4 with 1: 1～70 molecular proportion; Stirring at room 60～180 minutes makes the amino on cation dendroid macromolecule surface and the butanimide generation specific reaction of Sulfo-LC-SMPT;
Phosphate buffer dialysed overnight with the pH value 7.4 that contains EDTA is removed unreacted bridging agent;
Contain DEVD polypeptide of sequence and the polypeptide RVG29 end of Cy5 labelling are connected with cysteine and introduce sulfydryl; Respectively with 1～15: 1 and 1～10: the special group stirring at room reaction on 1 molecular proportion and the cation dendrimer material-Sulfo-LC-SMPT junctional complex is after 12～72 hours, and what generate brain target cationic dendrimer-Cy5 labelling contains DEVD polypeptide of sequence complex;
Molecular exclusion chromatography is removed unreacted polypeptide, and solvent is removed in lyophilizing;
In the phosphate buffer of pH value 7.4～8.5 with the QSY21 that contains butanimide directly with the amino on cation dendroid macromolecule surface in molecular proportion 5～100: 1 ratio is reacted, and is built into peptide modified brain targeted nano device.
8. claim 1 or 2 peptide modified brain targeted nano device contain the purposes in the imaging of activated form Caspase-3 at brain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101747371A CN102836443A (en) | 2011-06-26 | 2011-06-26 | Polypeptide-modified brain-targeted nanometer device and preparation method and usage thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101747371A CN102836443A (en) | 2011-06-26 | 2011-06-26 | Polypeptide-modified brain-targeted nanometer device and preparation method and usage thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102836443A true CN102836443A (en) | 2012-12-26 |
Family
ID=47364339
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101747371A Pending CN102836443A (en) | 2011-06-26 | 2011-06-26 | Polypeptide-modified brain-targeted nanometer device and preparation method and usage thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102836443A (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101772577A (en) * | 2007-08-03 | 2010-07-07 | 塞诺菲-安万特股份有限公司 | caspase imaging probes |
-
2011
- 2011-06-26 CN CN2011101747371A patent/CN102836443A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101772577A (en) * | 2007-08-03 | 2010-07-07 | 塞诺菲-安万特股份有限公司 | caspase imaging probes |
Non-Patent Citations (2)
| Title |
|---|
| YANG LIU ET AL.: "Brain-targeting gene delivery and cellular internalization mechanisms for modified rabies virus glycoprotein RVG29 nanoparticles", 《BIOMATERIALS》, vol. 30, 20 May 2009 (2009-05-20) * |
| 林居强: "细胞凋亡过程中caspase蛋白酶激活的光学成像研究", 《华中科技大学博士学位论文》, 10 March 2008 (2008-03-10) * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Zhang et al. | Peptide-based nanoprobes for molecular imaging and disease diagnostics | |
| Licha et al. | Synthesis, characterization, and biological properties of cyanine-labeled somatostatin analogues as receptor-targeted fluorescent probes | |
| Cho et al. | Development of a novel imaging agent using peptide-coated gold nanoparticles toward brain glioma stem cell marker CD133 | |
| US8841085B2 (en) | Nanoparticle sensor for measuring protease activity and method for manufacturing the same | |
| Brunetti et al. | Near-infrared quantum dots labelled with a tumor selective tetrabranched peptide for in vivo imaging | |
| KR102231672B1 (en) | Nanoparticle drug conjugates | |
| US10124077B2 (en) | Plectin-1 targeted agents for detection and treatment of pancreatic ductal adenocarcinoma | |
| Mukherjee et al. | Quantum dot as probe for disease diagnosis and monitoring | |
| Gao et al. | Stem cell tracking with optically active nanoparticles | |
| Ren et al. | Self‐assembled peptide‐based nanoprobes for disease theranostics and disease‐related molecular imaging | |
| Xiong et al. | A review on recent advances in amino acid and peptide-based fluorescence and its potential applications | |
| CN103402547B (en) | Switching mode fluorescent nanoparticle probe and use its fluorescent molecules imaging method | |
| CN104146964B (en) | Multipurpose polylysine fluorescent self-assembly nano microsphere carrier and preparation method and application thereof | |
| Liang et al. | Integration of Indocyanine Green Analogs as Near‐Infrared Fluorescent Carrier for Precise Imaging‐Guided Gene Delivery | |
| Wang et al. | A quantitative study of the intracellular fate of pH-responsive doxorubicin-polypeptide nanoparticles | |
| Naghibi et al. | AIEgen‐enhanced protein imaging: Probe design and sensing mechanisms | |
| Yang et al. | Enzyme Responsive Peptide-Based AIE Bioprobes | |
| Zhang et al. | AIEgen for cancer discrimination | |
| Liang et al. | An Activatable X‐Ray Scintillating Luminescent Nanoprobe for Early Diagnosis and Progression Monitoring of Thrombosis in Live Rat | |
| Chilakamarthi et al. | Synthesis and functional characterization of a fluorescent peptide probe for non invasive imaging of collagen in live tissues | |
| Fang et al. | In situ protease secretion visualization and metastatic lymph nodes imaging via a cell membrane-anchored upconversion nanoprobe | |
| Yoshimoto et al. | Preparation of antibody-immobilized gelatin nanospheres incorporating a molecular beacon to visualize the biological function of macrophages | |
| Zhao et al. | Research progresses on the functional polypeptides in the detection and imaging of breast cancer | |
| Liu et al. | Emerging Applications of Fluorogenic and Non‐fluorogenic Bifunctional Linkers | |
| CN102836443A (en) | Polypeptide-modified brain-targeted nanometer device and preparation method and usage thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20121226 |