CN109731104A - A kind of polypeptide-rare earth material delivery system and preparation method and application - Google Patents
A kind of polypeptide-rare earth material delivery system and preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of polypeptide-rare earth material delivery system and preparation method and application, the polypeptide-rare earth material delivery system includes: nanocluster (LDN), two different anticancer peptides (BIM and PMI) and a cyclic peptide iNGR.The nanocluster is formed by lanthanide-doped nano-particles self assemble, polypeptide BIM, PMI, iNGR are bound directly by the sulfydryl of cysteine and the surface of nanocluster, polypeptide of the present invention-rare earth material delivery system enhances polypeptide drugs to the resistance of protease hydrolytic, it is decomposed in response to reducing condition present in tumor microenvironment, inhibit growth of cancer cells in vivo, there is good prospect in the targeted therapy of cancer.
Description
Technical field
The present invention relates to biomedical material and field of nanometer technology more particularly to a kind of polypeptide-rare earth material delivering systems
System and its preparation and application.
Background technique
Protein-protein interaction (PPI) is a kind of high specific contact between two or more protein, is
The result of metabolic response in nearly all life process.In in the past few decades, exist largely to proton pump inhibitor
The basic research of structure and biological function, and it was found that in 650,000PPIs in human protein's group, at least
40% is made of the key interaction between protein globular domain and the peptide or segment of another protein.Therefore,
These natural polypeptides or protein fragments can be used as the good frame of exploitation polypeptide therapy.Even better, with it is traditional small
Molecule is compared, polypeptide small toxicity, and specificity is high, is readily synthesized, therefore be sent to very big hope to the effect of insecticide.However,
Most people always selects small molecule or recombination biotherapy (such as antibody and fusion protein) rather than polypeptide is solved as them
The preferred therapeutic modality of fresh target.Therefore, polypeptide drugs only account for the sub-fraction of global drug market, and sales volume is less than entirely
The 5% of ball drug total sales volume.Nevertheless, nearly all polypeptide for being approved for clinical use is all only extracellularly acting as
With.However, overwhelming majority PPIs occurs in the cell.These protrude missing the reason of be peptide birth defect: polypeptide be film not
It permeates and biologically unstable.
In order to overcome these obstacles and improve the curative effect of polypeptide drugs, we have developed a variety of strategies, such as D- isomerization is changed
Learn modification, trunk circulation, nanometer engineering and carrier encapsulation.These methodologies are benefited from, over nearly 5 years, clinical each stage has 170
A polypeptide and preclinical phase have a polypeptide more than 200 to be in exploitation.However, as far as we know, to the end of the year 2017, only three
Kind intracellular polypeptides drug completes the first stage (Cilengitide, SAH-p53-8 and AT-406) of clinical test.These
The reason of clinical test fails may largely be because it is considered that the shortcomings that these modifications and encapsulation (such as low specificity, low
Load capacity and high side effect) be more than their own advantage.Therefore, much remains to be done, especially with regard to how
Peptide is effectively transmitted to interested position, the problem such as tumour on the cell of unhealthy tissue.
Due to the permeability and reserve effects (EPR) of enhancing and controllable surface chemical modification, nanotechnology is anticancer peptide
Transmitting provide a feasible approach.However, the clinical antineoplastic application of various Nano medications is always subjected to low tumour spy
The restriction of the opposite sex positioning and safety defect.Although some fine nanosystems may be implemented to have the targeting of biological safety anti-
Oncotherapy, but these nano-carriers have relative complex structure and ingredient mostly, and this is largely dependent upon time-consuming
The chemical process of effort.In addition, biological unsafe organic reagent is constantly present and can not keep away after these laborious synthesis
The residual exempted from, causes biocompatibility to reduce.Further, since the complicated physics and chemical property of peptide, including mysterious open up
Structure and charge characteristic are flutterred, nano-delivery system inevitably encounters the low intrinsic problem of drug load.Even worse cake
It is that the complicated chemical property (hydrophobicity, charge and redox) of peptide can also destroy the stable state after colloidal nanoparticles coupling.
Stablize nanoparticle aggregate and precipitate in physiological conditions, inevitably will lead to the unstable of internal particle circulation dynamics.
Secondly, unstability will lead to anticancer peptide and discharge too early for treatment of cancer, reticuloendothelial system intake increases, so as to cause
Transmit the outer toxicity of invalid and target.
Therefore, it is necessary to a kind of new polypeptide-rare earth material delivery system and preparation method and application be designed, on overcoming
State defect.
Summary of the invention
One of mesh of the invention is to provide a kind of polypeptide-rare earth material and preparation method and application, to overcome above-mentioned lack
It falls into.
In order to achieve the above object, the present invention provides a kind of polypeptide-rare earth material delivery system, including LaOF:Ce, Tb, anticancer
Peptide BIM, anticancer peptide PMI and cyclic peptide iNGR.
Preferably, the amino acid of the anticancer peptide PMI forms are as follows: TSFAEYWALLSP.
Preferably, the amino acid of the anticancer peptide BIM forms are as follows: IWIAQELRRIGDEFNAYYARR.
Preferably, the amino acid of the cyclic peptide iNGR forms are as follows: CRNGRGPDC.
The present invention also provides a kind of aforementioned polypeptides-rare earth material delivery system preparation methods, include the following steps:
Trifluoroacetic acid precursor is synthesized the 30%-50% cerium and 5%- of oleyl amine cladding by High Boiling Solvent by step a
It strong ligand exchange reaction with polyacrylic acid later and EDC/ occurs with arginine by 35% terbium codope fluorine lanthana occurs
NHS chemical reaction, finally obtains LaOF:Ce, Tb；
Prepared LDN is dispersed in the acetonitrile that concentration is 10%-30%, so by step b again by 0.5mg/ml concentration
After be separately added into 0.5-2mgBIM-Cys, 0.5-2mg PMI-Cys, 0.5-2mg SH-PEGn-iNGR, under 15-50 degrees Celsius
Stirring is incubated for 10-30 minutes；It is eventually adding the HS-PEG-SH solution that 1mL concentration is 0.5-2mg/ml, is stirred under 20-45 degrees Celsius
Incubation 0.5-1.5 hours is mixed, 8000-13000rpm centrifugation collects product after removing supernatant, dry to get polypeptide-rare earth
Material delivery systems.
Preferably, being to be separately added into 1mg BIM-Cys, 1mg PMI-Cys, 1mg SH-PEGn-iNGR in step b.
Preferably, being the acetonitrile that concentration is 20% in step b.
Preferably, stirring is incubated for 20 minutes under 30 degrees Celsius in step b.
Preferably, the HS-PEG-SH solution that 1mL concentration is 1mg/ml is added in step b, stirring is incubated for 1 under 30 degrees Celsius
Hour, 10000rpm centrifugation collects product after removing supernatant.
The polypeptide being prepared the present invention also provides the above method-rare earth material delivery system application, by anticancer peptide BIM
It is delivered in cancer cell with anticancer peptide PMI, leads to cancer cell-apoptosis, or inhibit the growth of cancer cell.
Compared with prior art, the present invention provides a kind of polypeptide-rare earth material delivery system and its preparation method and application,
The polypeptide-rare earth material delivery system includes: nanocluster (LDN), two different anticancer peptides (BIM and PMI) and a ring
Peptide iNGR.The nanocluster is formed by lanthanide-doped nano-particles self assemble, and polypeptide BIM, PMI, iNGR pass through half Guang
The sulfydryl of propylhomoserin and the surface of nanocluster are bound directly, and polypeptide of the present invention-rare earth material delivery system enhances polypeptide drugs
To the resistance of protease hydrolytic, it is decomposed in response to reducing condition present in tumor microenvironment, inhibits cancer cell in vivo
Growth has good prospect in the targeted therapy of cancer.
Detailed description of the invention
Fig. 1 is peptide-group of the lanthanides nano particle schematic diagram for the treatment of tumour cell targeted molecular of the invention；
Fig. 2 is the physicochemical property figure of LDN-iNGR-PMI-BIM of the invention；
In figure, the hydrodynamic diameter of (A) LDN-iNGR-PMI-BIM；(B) HRTEM of LDN-iNGR-PMI-BIM
Image；(C) the UV-His figure of LDN and LDN-iNGR-PMI-BIM；(D) the FTIR spectrum of LDN and LDN-iNGR-PMI-BIM.
(E) in the 10mm PBS buffer solution of pH7.4 LDN and LDN-iNGR-PMI-BIM ZETA current potential；
Fig. 3 is the tumour-specific accumulation and tumour cell targeting research of LDN-iNGR-PMI-BIM of the invention；
In figure, bio distribution of (A) the fluorescence microscope LDN-iNGR-PMI-BIM in tumour and major organs.
(B) relative intensity of fluorescence of the LDN-iNGR-PMI-BIM in tumour and major organs.(C) LDN-iNGR-PMI-BIM is targeted
The schematic diagram of cancer cell.(D) cellular uptake of the flow cytometry quantitatively characterizing nano particle to HCT116 and PMBC；
Fig. 4 is influence of the external LDN-iNGR-PMI-BIM of the invention implemented to tumour cell cycle and apoptosis；
In figure, (A) HCT116p53+ /+is handled for 24 hours by above-mentioned different nanoparticulate drugs, is contaminated using Annexin V-PI
Color method and flow cytometer (FACS) analyze the cell cycle.(B) with flow cytomery HCT116 and HCT116p53+ /+it is thin
Born of the same parents' apoptosis situation.Control group of the PBS as (A) and (B)；
Fig. 5 is the LDN-iNGR-PMI-BIM of the invention implemented to HCT116p53+ /+mice with tumor antitumaous effect；
In figure, (A) different disposal group mouse HCT116p53+ /+tumor growth curve.(B) average tumor after treatment in 12 days
Weight, P < 0.01 * *.H&E (C) and TUNEL (D) dyeing of tumour after treatment 12 days.All images are all tumor tissues amplifications
200 times of image.
Fig. 6 is nude mice weight and the assessment of blood relative physiologic index after administration 12 days；
In figure, (A) records the weight of nude mice daily during studying.At the end of experiment, mouse blood is acquired before putting to death mouse
Liquid.Next with conventional method detection WBC (B), the level of PTL (C), RBC (D), HB (E).
Fig. 7 is different groups of nude mice liver weights (A), AST (B), ALT (C), BUN (D), CRE (E), spleen weight (F)
Variation diagram.
Fig. 8 dyes for the representative histology H&E of the major organs of nude mice after drug-treated.
Specific embodiment
To make to have further understanding to the purpose of the present invention, construction, feature and its function, hereby cooperate embodiment detailed
It is described as follows.
Some vocabulary is used in specification and claims to censure specific element.Have in fields
Usual skill is, it is to be appreciated that manufacturer may call the same element with different nouns.This specification and right are wanted
Ask book not in such a way that the difference of title is as element is distinguished, but with the difference of element functionally as differentiation
Criterion.Mentioned " comprising " is open term throughout the specification and claims, therefore should be construed to " including but
It is not limited to ".
Polypeptide of the present invention-rare earth material delivery system and its preparation and application
Polypeptide of the present invention-rare earth material delivery system, including group of the lanthanides nanocluster LaOF:Ce, Tb (hereinafter referred to as LDN,
Lanthanide-doped fluorescent nanoparticles, or LaOF:Ce, Tb are represented with LDN), anticancer peptide
BIM, anticancer peptide PMI and cyclic peptide iNGR (LDN)., the amino acid composition of the anticancer peptide PMI are as follows: TSFAEYWALLSP.It is described
The amino acid of anticancer peptide BIM forms are as follows: IWIAQELRRIGDEFNAYYARR.The amino acid of the cyclic peptide iNGR forms are as follows:
CRNGRGPDC。
Polypeptide of the present invention-rare earth material delivery system the preparation method is as follows:
Firstly, by High Boiling Solvent by trifluoroacetic acid precursor synthesize oleyl amine cladding 30%-50% (such as 30%,
45%, 50%) cerium and 5%-35% (such as 5%, 10%, 15%, 20%, 25%, 30%, 35%) terbium codope fluorine oxidation
It strong ligand exchange reaction with polyacrylic acid (PAA) later and EDC/NHS chemical reaction occurs with arginine by lanthanum occurs,
Rare earth material LaOF:Ce is finally obtained, Tb (represents LaOF:Ce, Tb below with LDN), such as (LaOF:45%Ce, 15%
Tb), or (GdOF:35%Ce, 25%Tb), or (GdOF:30%Ce, 35%Tb), or (GdOF:50%Ce, 30%
Tb)。
Then, iNGR, PMI-Cys and BIM-Cys are obtained with FMOC method and reversed-phased high performace liquid chromatographic synthesizing and purifying.
INGR obtains SH-PEG-iNGR by SH-PEH-SH and maleimide guanidine-acetic acid N- succinimide ester Pegylation.
Finally, it is slow to disperse PBS of the 10ml containing 10%-30% (for example, 10%, 20%, 30%) acetonitrile for 5mg LDN
Fliud flushing (for example, PH7.4) is dispersed in the acetonitrile that concentration is 10%-30% again by 0.5mg/ml concentration, takes 0.5-2mg
(such as 0.5mg, 1.0mg, 2mg) PMI-Cys, 0.5-2mg (such as 0.5 mg, 1.0mg, 2mg) BIM-Cys, 0.5-2mg (example
Such as 0.5mg, 1.0mg, 2mg) SH-PEG-iNGR simultaneously be added in LDN solution.In 15-50 (such as 15,20,30,45,50)
Degree Celsius stirring 10-30 (such as 10,20,30) minutes after, additions 1ml concentration be 0.5-2mg/ml, (such as 0.5mg/ml,
1mg/ml, 1.5mg/ml, 2mg/ml) SH-PEG-SH solution, 20-45 DEG C (such as 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 45 DEG C)
Lower stirring 0.5-1.5 hours (such as 30 minutes, 40 minutes, 1 hour, 1.2 hours, 1.5 hours), 8000-13000rpm (example
Such as 8000,10000,12000,13000) product is collected by centrifugation in rpm, be freeze-dried available LDN-iNGR-PMI-BIM powder
End.
Polypeptide of the present invention-rare earth material delivery system LDN-iNGR-PMI-BIM physicochemical property
Prepared by further being measured by dynamic light scattering (DLS), high score variability transmission electron microscope (HRTEM)
Nanocrystalline morphology, size and physical structure.After peptide coupling, in PBS buffer solution (pH 7.4), LDN-iNGR-PMI-BIM
Hydrodynamic diameter increase to 9.5nm-12.5nm from the 3.4nm-6.1nm (such as 4.1nm, 4.6nm, 5.0nm) of LDN
(such as 9.5nm, 11.0nm, 12.5nm) (Fig. 2A).In addition, high resolution transmission electron microscope image (HRTEM) is shown
The LDN-iNGR-PMI-BIM uniform diameter of mono-dispersed nano structure be 6.0 ± 0.5-8.0 ± 0.5nm (such as 7.5 ±
0.5nm) (Fig. 2 B).Compared with the partial size that dynamic light scattering (DLS) measures, there is some difference for the partial size that HRTEM is measured, this
It may be since nanoparticle forms hydrated sheath in aqueous solution, illustrating LDN-iNGR-PMI-BIM in aqueous solution has very
Good dissolubility.In addition, confirming polypeptide by ultraviolet-visible spectrum (UV-vis) and Fourier Transform Infrared Spectroscopy (FTIR)
The success of functionalization.There are two typical UV-vis absorption peaks at 280nm and 214nm, are aromatic structure and polypeptide respectively
(Fig. 2 C).In addition, positioned at 3500 and 1700cm-1 two infrared spectroscopies spectrum respectively with N-H the and C=O function from polypeptide
Group is related (Fig. 2 D).The surface potential (ZETA current potential) of LDN becomes 18.2mV (Fig. 2 E) from -21.3mV after polypeptide coupling, these
The success of LDN-iNGR-PMI-BIM connection is all further demonstrated that.
The case where detecting polypeptide-rare earth material delivery system LDN-iNGR-PMI-BIM targets neoplastic cells
It is designed to verify our cancer target, we use chlorination Rhodamine 101 (a kind of red fluorescence dyestuff) to mark
The LDN-iNGR-PMI-BIM of note detects it in the bio distribution of HCT116 (colon cancer) mouse.As shown in (Fig. 3 A), infusing
Bright fluorescence is observed that in tumor locus after penetrating nanoparticle (dosage: 2 mgs/kg) 4h of 200 μ L.By into one
These results (Fig. 3 B) of sxemiquantitative are walked, although there are some LDN-iNGR-PMI-BIM distribution in kidney and liver, in tumour
For the LDN-iNGR-PMI-BIM of accumulation far more than non-Neoplastic organ, these illustrate that LDN-iNGR-PMI-BIM can be special at tumour
Opposite sex accumulation.In addition, iNGR, which is combined, can also assign nanoparticle with cancer cell specificity.It just reports as in the previous, into the cell
Changing can activate CendR motif CRNGR to trigger by the proteolysis process of cancer cell surfaces.This strategy only allows
CendR motif is activated in target cancer cell, avoids that internalization process (Fig. 3 B) occurs in other cells.To verify this
Saying, respectively with the LDN-PMI-BIM of the LDN-iNGR-PMI-BIM of dye marker and dye marker be incubated for HCT116 cell and
Peripheral blood mononuclear cells (PBMC) 4 hours.As shown in the cell phagocytosis situation of Fig. 3 D flow cytometry measure, LDN-iNGR-
The ratio that PMI-BIM is occupied by cancer cell is up to 91.3%, and almost without discovery cell to there is no the suctions of the drug of iNGR
It receives.Importantly, either LDN-iNGR-PMI-BIM or LDN-PMI-BIM, which are not shown, significantly healthy PBMC
Cellular uptake.In conclusion these results confirm that there is LDN-iNGR-PMI-BIM fabulous tumor cell specific to target
Ability.
Polypeptide of the present invention-external evoked apoptosis of tumor cells of rare earth material delivery system LDN-iNGR-PMI-BIM and period
Retardance
In order to further confirm that peptide can be transported in cancer cell by LDN-iNGR-PMI-BIM, lead to Apoptosis.We
LDN-iNGR-PMI-BIM LDN-iNGR-PMI HCT116 (wild type p53) respectively with 1 μm of concentration is incubated for, and is being trained
After supporting for 24 hours, as the result is shown: LDN-iNGR-PMI-BIM and LDN-iNGR-PMI significantly induction HCT116 cell is in the G0/G1 phase
Cycle Arrest (Fig. 4 A), this demonstrates the binding mode of PMI from mechanism.In order to further verify, we are by HCT116
(p53 wild type) and HCT116p53-/- (p53 defect) be incubated for LDN-iNGR-PMI-BIM and LDN-iNGR-PMI respectively
48h carries out Apoptosis detection.Consistent with cell cycle result, facs analysis confirms that HCT116 cell (p53 wild type) makes
Different degrees of apoptosis (Fig. 4 B) occurs after treating 48h with LDN-iNGR-PMI-BIM and LDN-iNGR-PMI.Meanwhile LDN-
The percentage of cerebral apoptosis (being approximately equal to 10% from foundation level) of iNGR-PMI-BIM group is higher than LDN-iNGR-PMI (Fig. 4 B), this
Illustrate that BIM enhances the apoptosis-induced function of PMI.Sharp contrast is formed therewith, in LDN-iNGR-PMI processing
The apoptosis induction higher than control group is not observed in HCT116p53-/- cell, in the sample of LDN-iNGR-PMI-BIM processing not
It was found that being more than 30% apoptosis (Fig. 4 B), this illustrates that LDN-iNGR-PMI-BIM mainly induces cancer cell in such a way that p53 is relied on
Apoptosis.
Vehicle evaluations of the polypeptide of the present invention-rare earth material delivery system LDN-iNGR-PMI-BIM to liver cancer treatment effect
To construct the subcutaneous liver cancer model of HCT116, female BAl BIc/c nude mice (5 week old) 30,200 μ L HCT116 (packet are taken
Containing 5 × 106 cells) suspension cell is injected into subcutaneous tissue in right hips, mouse is divided into 6 groups of (N=5): LDN-iNGR-
PMI-BIM, LDN-iNGR-PMI, PMI, PMI+BIM, DOX, PBS (blank control group).Drug is given after inoculation the 12nd day
Treatment 1 day time, 12 times, is subcutaneously injected totally according to 1.5mg/Kg, 200 μ L.The gross tumor volume of measurement in every two days.Gross tumor volume
Formula: V (mm3)=length (mm) × width (millimeter) × width (millimeter)/2.As shown in Figure 5A, compared with PBS control group,
LDN-iNGR-PMI-BIM group tumour growth is significantly suppressed (Fig. 5 A).After treatment in 12 days, mouse removes swollen after putting to death
Tumor weighing, the results show that PMI or PMI/BIM combination therapy is almost no impact tumour weight, it may be possible to due to free
Peptide cannot be assembled in tumor locus and pass through cancer cell film.In addition, the weight of LDN-iNGR-PMI-BIM treatment group tumors
The substantially less than weight (Fig. 5 B) of LDN-iNGR-PMI treatment group tumors, this supports the synergistic effect of PMI and BIM again.This
Outside, in H&E histologic analysis (Fig. 5 C) and TUNEL Apoptosis dyeing (Fig. 5 D) result, PBS-, PMI- and PMI/BIM connection
The tumour for the treatment of is closed without apoptosis, and the visible massive tumor in DOX, LDN-iNGR-PMI and LDN-iNGR-PMI-BIM treatment group
Apoptosis.It is worth noting that, the tunel positive cell of LDN-iNGR-PMI-BIM treatment tumour is controlled more than LDN-iNGR-PMI
The cell (Fig. 5 D) for the treatment of, illustrates that BIM enhances the apoptosis-induced function of PMI again, that is, realizes anticancer peptide BIM and anticancer peptide
PMI is delivered in cancer cell jointly and (penetrates love cell membrane), and aggregation promotes cancer cell-apoptosis in cancer cell.In conclusion
Our result of study for LDN-iNGR-PMI-BIM there is powerful antitumaous effect to provide a large amount of evidences in vivo.
Polypeptide of the present invention-the safety evaluatio of rare earth material delivery system LDN-iNGR-PMI-BIM in vivo
In order to evaluate the safety of LDN-iNGR-PMI-BIM in vivo, during administration in 12 days, tumor formation mouse is had recorded
Changes of weight, as the result is shown: DOX group mouse weight is substantially reduced (Fig. 6 A).In addition, under the weight of PBS control group has slightly
The trend (Fig. 6 A) of drop, it may be possible to the hypofunction due to caused by tumour growth.But pass through LDN-iNGR-PMI-BIM and
The mouse of LDN-iNGR-PMI processing weight between treatment phase almost has no change (Fig. 6 A).Simultaneously we have found that DOX causes
Leucocyte (Fig. 6 B) and blood platelet (Fig. 6 C) are seriously reduced, but both of these case is in LDN-iNGR-PMI-BIM and LDN-iNGR-
It is not observed in PMI processing mouse.In addition, the mouse of two kinds of nano particles processing does not find haemolysis (Fig. 6 C and 6D), these
As a result confirm that LDN-iNGR-PMI-BIM is nontoxic to hematological system.
Similarly, liver kidney plays a crucial role in drug metabolism, therefore we are had rated by immune and biochemical apparatus
Influence of the LDN-iNGR-PMI-BIM to two organ dysfunctions.In terms of liver, after LDN-iNGR-PMI-BIM administration, with control group phase
Than the activity of liver weight and two kinds of enzymes (AST and ALT) does not have difference, and DOX administration mouse liver weight is decreased obviously, liver
The obvious disorder of function (Fig. 7 A-C).In terms of kidney, after LDN-iNGR-PMI-BIM treatment, urea nitrogen (BUN, Fig. 7 D) in blood
Unchanged with serum creatinine (CRE, Fig. 7 E), prompting LDN-iNGR-PMI-BIM is safe to kidney.In addition, DOX is treated
It will lead to serious spleen failure, and the mouse of LDN-iNGR-PMI-BIM treatment keeps spleen healthy (Fig. 7 F).In histology
Aspect is not observed mouse device major organs lesion in LDN-iNGR-PMI-BIM group and control group, but deposits in DOX group
, including hepatonecrosis, the alveolar wall thickening (Fig. 8) of nephritis disease and lung.These the result shows that, LDN-iNGR-PMI-BIM is without obvious
Toxic side effect.
It should be strongly noted that above-described embodiment is illustrated for liver cancer to treat, but the present invention and unlimited
In liver cancer, also can be used for other cancers, such as kidney, cancer of the esophagus etc. can reach the identical effect of above-described embodiment, herein not
It is described in detail again.
To sum up, the present invention provides a kind of polypeptide-rare earth material delivery system and its preparation method and application, the polypeptides-
Rare earth material delivery system includes: nanocluster (LDN), two different anticancer peptides (BIM and PMI) and a cyclic peptide iNGR.
The nanocluster is formed by lanthanide-doped nano-particles self assemble, and polypeptide BIM, PMI, iNGR pass through cysteine
The surface of sulfydryl and nanocluster is bound directly, and polypeptide of the present invention-rare earth material delivery system enhances polypeptide drugs to albumen
The resistance of enzyme hydrolysis is decomposed in response to reducing condition present in tumor microenvironment, inhibits growth of cancer cells in vivo,
There is good prospect in the targeted therapy of cancer.
The present invention is described by above-mentioned related embodiment, however above-described embodiment is only to implement example of the invention.
It must be noted that the embodiment disclosed is not limiting as the scope of the present invention.On the contrary, do not depart from spirit of the invention and
It is changed and retouched made by range, belongs to scope of patent protection of the invention.
Claims (10)
1. a kind of polypeptide-rare earth material delivery system, it is characterised in that: including LaOF:Ce, Tb, anticancer peptide BIM, anticancer peptide
PMI and cyclic peptide iNGR.
2. a kind of polypeptide-rare earth material delivery system according to claim 1, which is characterized in that the anticancer peptide PMI's
Amino acid composition are as follows: TSFAEYWALLSP.
3. a kind of polypeptide-rare earth material delivery system according to claim 1, which is characterized in that the anticancer peptide BIM's
Amino acid composition are as follows: IWIAQELRRIGDEFNAYYARR.
4. a kind of polypeptide-rare earth material delivery system according to claim 1, which is characterized in that the cyclic peptide iNGR's
Amino acid composition are as follows: CRNGRGPDC.
5. a kind of preparation method of polypeptide-rare earth material delivery system described in a kind of claim 1, which is characterized in that including such as
Lower step:
Trifluoroacetic acid precursor is synthesized the 30%-50% cerium and 5%-35% terbium of oleyl amine cladding by High Boiling Solvent by step a
It strong ligand exchange reaction with polyacrylic acid later and EDC/NHSization occurs with arginine by codope fluorine lanthana occurs
Reaction is learned, LaOF:Ce, Tb are finally obtained；
Prepared LDN is dispersed in the acetonitrile that concentration is 10%-30% again by 0.5mg/ml concentration, is then divided by step b
Not Jia Ru 0.5-2mgBIM-Cys, 0.5-2mg PMI-Cys, 0.5-2mg SH-PEGn-iNGR, stir under 15-50 degrees Celsius and incubate
It educates 10-30 minutes；It is eventually adding the HS-PEG-SH solution that 1mL concentration is 0.5-2mg/ml, stirs and is incubated under 20-45 degrees Celsius
0.5-1.5 hours, 8000-13000rpm centrifugation collected product after removing supernatant, and drying is passed to get polypeptide-rare earth material
Send system.
6. polypeptide as claimed in claim 5-rare earth material delivery system preparation method, which is characterized in that in step b, be
It is separately added into 1mg BIM-Cys, 1mg PMI-Cys, 1mg SH-PEGn-iNGR.
7. polypeptide as claimed in claim 5-rare earth material delivery system preparation method, which is characterized in that in step b, be
The acetonitrile that concentration is 20%.
8. polypeptide as claimed in claim 5-rare earth material delivery system preparation method, which is characterized in that in step b, 30
Stirring is incubated for 20 minutes under degree Celsius.
9. polypeptide as claimed in claim 5-rare earth material delivery system preparation method, which is characterized in that in step b, add
Enter the HS-PEG-SH solution that 1mL concentration is 1mg/ml, stirring is incubated for 1 hour under 30 degrees Celsius, and 10000rpm centrifugation is removed
Product is collected after supernatant.
10. the application of the polypeptide that method as claimed in claim 5 is prepared-rare earth material delivery system, which is characterized in that will resist
Cancer peptide BIM and anticancer peptide PMI are delivered in cancer cell, lead to cancer cell-apoptosis, or inhibit the growth of cancer cell.
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