CN108912324A - A kind of inner membrance is the polyester peptide vesica and the preparation method and application thereof of positive electricity - Google Patents

A kind of inner membrance is the polyester peptide vesica and the preparation method and application thereof of positive electricity Download PDF

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CN108912324A
CN108912324A CN201810825480.3A CN201810825480A CN108912324A CN 108912324 A CN108912324 A CN 108912324A CN 201810825480 A CN201810825480 A CN 201810825480A CN 108912324 A CN108912324 A CN 108912324A
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polyester
peptide
cpp33
vesica
clp
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CN108912324B (en
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邓超
邱敏
钟志远
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Suzhou University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/14—Lactams
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/14—Lactams
    • C08G69/16—Preparatory processes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/48—Polymers modified by chemical after-treatment

Abstract

The invention discloses the polyester peptide vesicas and the preparation method and application thereof that a kind of inner membrance is positive electricity, belong to high molecular material and applied chemistry technical field.The polyester peptide vesica (CLP) with asymmetric membrane structure that inner membrance is positive electricity is prepared firstly, PEG-b-PAPA-b-PLL triblock copolymer is simply prepared by NCA ring-opening polymerisation and deprotection in the present invention；And efficiently siRNA is loaded into the hydrophilic inner cavity of vesica by the electrostatic interaction of siRNA and PLL, the firm wall film of vesica can prevent siRNA to be degraded；Vesica (CPP33-CLP) that load siPLK1, lung carcinoma cell selectivity cell-penetrating peptide CPP33 modification has that good targeting, CPP33 can help siRNA quickly to flee from endosome, into cytoplasm simultaneously to A549 lung carcinoma cell, significant sequence specific gene silencing efficiency is generated, shows the tumor inhibitory effect significantly increased in the A549 lung cancer in mice body of lotus original position.Simple, the stable multifunctional nano vesica platform shows very big potentiality in terms of the gene therapy of cancer.

Description

A kind of inner membrance is the polyester peptide vesica and the preparation method and application thereof of positive electricity
Technical field
The present invention relates to the polyester peptide vesicas and the preparation method and application thereof that a kind of inner membrance is positive electricity, belong to high molecular material With applied chemistry technical field.
Background technique
The RNA interference therapy mediated by siRNA (siRNA) is shown in the treatment of the various diseases including cancer Show very huge potentiality.However, that there are circulation time in vivo is short, be easy to be degraded by enzymes, cell permeability and interior by siRNA Contain the disadvantages of body escape performance is poor.Therefore, how siRNA is delivered to lesions position safe and efficiently is that RNA interferes this base Because Therapy converts faced biggest obstacle and challenge.In order to solve this problem, scientists develop various nanometers Carrier system is used for the delivering of siRNA.Currently, having several polymer siRNA composite nano-granules enters clinical investigation phase, But that there are stability is poor for these nanometer systems, the defects of easily dissociation in vivo.And polymer vesicle be it is a kind of have than The nano-carrier of micella and the higher stability of other nano complexes, huge hydrophilic inner cavity can be used to load siRNA, and SiRNA can then be isolated from the outside by its firm wall film, play the role of protecting siRNA.
Summary of the invention
To solve the above problems, present invention design has synthesized a kind of asymmetric three block polyester peptide and has been prepared by it One inner membrance be negative electricity the polyester peptide vesica with asymmetric membrane structure be used for siRNA drug it is safe and stable, efficiently contain And targeted delivery.
The first purpose of the invention is to provide a kind of polyester fret peptide, the polyester fret peptide is poly- for polyethylene glycol-b- (2- aminohexadecanoic acid)-b- poly (L-lysine) (PEG-b-PAPA-b-PLL), structural formula is as follows：
Wherein, the value range that the value range that the value range of x is 40-2300, y is 30-800, z is 1-500.
In one embodiment of the invention, the polyester fret peptide further includes repairing in the end of hydrophilic section polyethylene glycol Adorn the polyester fret peptide with targeting that selectively targeted molecule obtains.
In one embodiment of the invention, the selectively targeted molecule is small peptide, small molecule targeted molecular, resists Body, polysaccharide or monosaccharide.
In one embodiment of the invention, the antibody further includes antibody fragment.
In one embodiment of the invention, the small peptide is cRGD, the sequence cNGQGEQc that sequence is cRGDfC CNGQ, sequence be CSNIDARAC CC-9 or sequence be RLWMRWYSPRTRAYGC CPP33.
In one embodiment of the invention, the small molecule targeted molecular is folic acid or anisamide.
A second object of the present invention is to provide the preparation methods of above-mentioned polyester fret peptide, include the following steps：With poly- second Glycol-amino (PEG-NH2) it is macromole evocating agent, successively cause 2- aminohexadecanoic acid N- carboxyl inner-acid anhydride (APA-NCA) It is poly- that polyethylene glycol-b- is prepared with ε-carbobenzoxy-L-lysine N- carboxyl inner-acid anhydride monomer (ZLL-NCA) ring-opening polymerisation Poly- (ε-carbobenzoxy-L-lysine) (PEG-b-PAPA-b-PZLL) triblock copolymer of (2- aminohexadecanoic acid)-b-, so Carbobenzoxy is sloughed by being deprotected afterwards, obtains the polyester fret peptide.
In one embodiment of the invention, the preferred 11KDa of the molecular weight of the preferred 5KDa of the molecular weight of PEG, PAPA, The preferred 2KDa of the molecular weight of PAsp.
In one embodiment of the invention, the method the specific steps are：
(1) under a nitrogen atmosphere, the DMF solution of 2- aminohexadecanoic acid-N- carboxyl inner-acid anhydride monomer is added to poly- second It is reacted in glycol-amino DMF solution；
(2) ε-carbobenzoxy-L-lysine N- carboxyl inner-acid anhydride monomer is added into the reaction solution of step (1) to continue instead It answers, is then precipitated using ice ether, obtain the poly- poly- (ε-benzene oxygen of (2- aminohexadecanoic acid)-b- of polyethylene glycol-b- after dry Carbonyl-L-lysine)；
(3) by deprotection method by poly- (2- the aminohexadecanoic acid)-b- of polyethylene glycol-b- it is poly- (ε-carbobenzoxy-L- rely Propylhomoserin) in benzyloxycarbonyl group slough, precipitated using ice ether, obtain poly- (the 2- amino hexadecane of polyethylene glycol-b- after dry Acid)-b- poly (L-lysine).
In one embodiment of the invention, above-mentioned preparation method can be expressed as follows：
In one embodiment of the invention, in step (1), reaction condition be 30-40 DEG C reaction 60-80 hours.
In one embodiment of the invention, in step (2), reaction condition be 30-40 DEG C reaction 60-80 hours.
Third object of the present invention is to provide application of the polyester fret peptide in genomic medicine delivering.
In one embodiment of the invention, the genomic medicine include but is not limited to siRNA, microRNA, mRNA, shRNA、DNA、CRISPR-Cas9。
In one embodiment of the invention, the application be the polyester fret peptide is prepared into polyester peptide vesica into Row genomic medicine, which is sent, to be passed.
Fourth object of the present invention is to provide a kind of polyester peptide vesica, is prepared by the polyester fret peptide, including By being prepared without the polyester fret peptide of targeted molecular, be prepared by the polyester fret peptide containing targeted molecular, or by The polyester peptide vesicle surface modification targeted molecular that polyester fret peptide without targeted molecular is prepared obtains.
Fifth object of the present invention is to provide application of the polyester peptide vesica in genomic medicine delivering.
Beneficial effects of the present invention：
1, poly- (2- the aminohexadecanoic acid)-b- poly (L-lysine) of polyethylene glycol-b- prepared by the present invention, can be by gradually Ring-opening polymerisation and subsequent deprotection reaction obtain.Whole preparation process is simple directly, controllability is good.
2, polyester peptide vesica disclosed by the invention has the characteristics that size is small, stability is good, the siRNA amount of containing is high.
3, preparation method of the present invention is simple, raw materials used from a wealth of sources, reproducible, has a good application prospect.
Detailed description of the invention
Fig. 1 is the nucleus magnetic hydrogen spectrum figure of the PEG-b-PAPA-b-PZLL in embodiment 1；
Fig. 2 is the nucleus magnetic hydrogen spectrum figure of the PEG-b-PAPA-b-PLL in embodiment 1；
Fig. 3 is the Mal-PEG-b-PAPA nucleus magnetic hydrogen spectrum figure in embodiment 2；
Fig. 4 is the nucleus magnetic hydrogen spectrum figure of the CPP33-PEG-b-PAPA in embodiment 2；
Fig. 5 is the transmission electron microscope of the particles size and distribution figure (a) of CPP33-CLP, CPP33-CLP in embodiment 3 Picture (b), agarose gel electrophoresis figure (c)；
Fig. 6 is cell of the CPP33-CLP (CPP33-CLP-Cy5) of Cy5 label in embodiment 4 in A549 lung carcinoma cell Streaming result figure (a), different cells are handled to the endocytosis result figure (b) of CPP33-CLP-Cy5, through different cell endocytic inhibitor A549 cell to the endocytosis result figure (c) of CPP33-CLP-Cy5 and load the CPP33-CLP of Cy5-siPLK1 in A549 lung cancer Endosome in cell escapes result figure (d)；
Fig. 7 is the real-time quantitative Polymerase Chain Reaction knot that siPLK1-CPP33-CLP is intracellular in A549 in embodiment 4 Fruit schemes (a) and western blot result figure (b)；
Fig. 8 is the cRGD-CLP of Cy5 label in embodiment 5 in the intracorporal blood circulation result of study figure (a) of healthy mice And its in the intracorporal bio distribution result figure (b) of the original position lotus A549-Luc lung cancer in mice；
Fig. 9 is that load siPLK1-CPP33-CLP is intracorporal antitumor in the original position lotus A549-Luc lung cancer in mice in embodiment 6 Activity Results figure, wherein a is the growth curve chart of tumour, and b is the survivorship curve of mouse, and c is the changes of weight figure of mouse.
Specific embodiment
The technology contents of invention are described in detail below by embodiment for the essence invented in order to better understand.
Embodiment 1：Poly- (2- the aminohexadecanoic acid)-b- poly (L-lysine) (PEG-b-PAPA-b- of polyethylene glycol-b- PLL) the synthesis of triblock copolymer
The synthesis of PEG-b-PAPA-b-PLL mainly includes two steps.Firstly, under a nitrogen atmosphere, in DMF solution, with PEG-NH2Cause 2- aminohexadecanoic acid N- carboxyl inner-acid anhydride monomer (APA-NCA) and ε-benzyloxy for macromole evocating agent sequence PEG-b-PAPA-b-PZLL three block is prepared in carbonyl-L-lysine N- carboxyl inner-acid anhydride monomer (ZLL-NCA) ring-opening polymerisation Copolymer.Specific synthesis step is as follows：In a nitrogen environment, the DMF of the APA-NCA of 15.0mL (1.04g, 3.52mmol) is molten Liquid is added rapidly to PEG-NH2In DMF (4.0mL) solution of (0.4g, 0.08mmol)；After being reacted 72 hours at 35 DEG C, it is added Second comonomer ZLL-NCA (0.44g, 1.44mmol), the reaction was continued later 72 hours.After reaction, by reaction solution at 20 times It is repeatedly precipitated in excessive ice ether, last vacuum drying obtains white solid product.Yield：90%.1H NMR (600MHz,CDCl3/CF3COOH (9/1, v/v), Fig. 1, δ):7.29(5H,-C6H5),5.07(2H,C6H5CH2-),4.48 (1H,-COCHNH-),3.73(4H,-OCH2CH2O-),3.48(3H,-OCH3),3.07(2H,-OCONHCH2-),1.66(4H,- CH(NH)CH2CH2-),1.24(28H,-CH2(CH2)12CH3&-CH(NH)CH2CH2CH2-),0.87(3H,-CH2CH3).
Then, the PEG-b-PAPA-b-PLL triblock copolymer that PEG-b-PAPA-b-PZLL is deprotected in HBr Object.Specific step is as follows：PEG-b-PAPA-b-PZLL is added in HBr (33wt.%in HOAc, 0.19mL, 1.0mmol) In trifluoroacetic acid (3mL) solution of (0.3g, 0.015mmol), reacted 2 hours in 0 DEG C.After reaction, with excessive ice The repeated multiple times precipitating of ether, last vacuum drying obtain white solid product.Yield：83%.1H NMR(600MHz, CDCl3/CF3COOH (30/1, v/v), Fig. 2, δ):4.49(1H,-COCHNH-),3.74(4H,-OCH2CH2O-),3.49(3H,- OCH3),3.18(-CH2NH2),1.69(2H,-CH(NH)CH2CH2-),1.47(-CH2CH2CH2CH2NH2),1.23(26H,-CH2 (CH2)12CH3&-CH(NH)CH2CH2CH2-),0.85(3H,-CH2CH3).
From the nuclear magnetic spectrogram (Fig. 1) of PEG-b-PAPA-b-PZLL, it is clear that PEG (δ 3.73,3.48), The characteristic peak of PAPA (δ 1.66,1.24,0.87) and PZLL (δ 7.29,5.07,3.07), also, by comparing the methylene on PEG The peak area between the methylene (δ 5.07) on methyl (δ 0.87), PZLL on base (δ 3.73) and PAPA, can be calculated The degree of polymerization (DP) of PAPA and PZLL is respectively 40 and 15, and then it is 5.0-10.8-3.9kg/mol that its molecular weight, which is calculated, (table 1).Later, the benzyloxycarbonyl group on PEG-b-PAPA-b-PZLL is sloughed using HBr/HOAc and obtains PEG-b-PAPA-b-PLL. From the nuclear magnetic spectrogram (Fig. 2) of PEG-b-PAPA-b-PLL, it can be seen that benzyloxycarbonyl group characteristic peak at δ 7.29 and 5.07 disappears It loses, it was demonstrated that it is sloughed completely.Moreover, the DP and PEG-b-PAPA-b- of the PAPA and PLL being calculated from its nuclear magnetic spectrogram PZLL is close, illustrates, poly- polypeptide chain will not be broken during deprotection.
Embodiment 2：The synthesis of CPP33-PEG-b-PAPA copolymer
The preparation of CPP33-PEG-b-PAPA polymer is divided into two steps.Firstly, with Mal-PEG-NH2For macromolecular initiation Agent causes APA-NCA ring-opening polymerisation, Mal-PEG-b-PAPA is prepared；From its nuclear magnetic spectrogram (Fig. 3), it can calculate It is 6.0-11.0kg/mol to its molecular weight；Then, it is reacted by the sulfydryl on CPP33 with Mal and CPP33-PEG- is prepared b-PAPA.From the nuclear magnetic spectrogram (Fig. 4) of CPP33-PEG-b-PAPA, it can be found that the characteristic peak of CPP33 (δ 6.97), passes through The grafting rate that 9,10- phenanthrenequione methods measure to obtain CPP33 is 87%.
The characterization of 1. polymer of table
aBy1H NMR is calculated.
Embodiment 3：Load the system of the polyester peptide vesica (siPLK1-CPP33-CLP) with asymmetric membrane structure of siRNA It is standby
Vesica (CPP33-CLP) that load siRNA, CPP33 modification is prepared by solvent displacement.Its letter Want that steps are as follows：0.1mL concentration is contained into 20mol.%CPP33-PEG-b-PAPA and 80mol.%mPEG-b- for 5mg/mL The mixed solution of PAPA-b-PLL and the siRNA solution of 0.1mL mix, and are added drop-wise to the HEPES buffer solution of 0.8mL dropwise later In medium (5mM, pH6.8), the bag filter for being later 350k with molecular cut off, dialysis removes unloaded in HEPES medium The siRNA to enter.The useful load (SLC) and efficiency of loading (SLE) of siRNA can be surveyed by NanoDrop ultraviolet specrophotometer ?.SLC and SLE can be calculated by the following formula：
SLC (wt.%)=(being loaded in quality/polymer of the siRNA in vesica and the adduction of siRNA mass) × 100
SLE (%)=(quality for the siRNA being loaded in vesica/quality for the siRNA being initially added) × 100
Dynamic laser light scattering is the results show that the partial size of CPP33-CLP is about 90 nanometers (Fig. 5 a).Transmission electron microscope picture is aobvious Show, CPP33-CLP imitated vesicle structure (Fig. 5 b) spherical in shape.CPP33-CLP can efficiently contain siRNA, and encapsulation rate is greater than 95% (table 2).Agarose gel electrophoresis results show that siRNA can be firmly encapsulated into inside CPP33-CLP, even and through 10%FBS remains to maintain good stability (Fig. 5 c) after handling 17 hours, further proves that the firm wall film of vesica can have The protection siRNA of effect, it is prevented to be degraded.
Table 2. loads the characterization of the vesica of siRNA
A is measured by NanoDrop ultraviolet-uisible spectrophotometer.
bThe partial size and particle diameter distribution of vesica are measured by dynamic laser light scattering.
cSurface potential is measured in the PB of pH 7.4 by electrophoresis.
Embodiment 4：The In vitro cell experiment of siPLK1-CPP33-CLP
We study CPP33-CLP's by flow cytometer (FACS) and laser confocal fluorescence microscope (CLSM) Cell endocytic and release behavior intracellular.Firstly, we study it using the CPP33-CLP (CPP33-CLP-Cy5) that Cy5 is marked To the targeting ability of A549 cell.As shown in Figure 6 a, surface modification CPP33 can significantly enhance the energy that CLP enters A549 cell Power, the fluorescence intensity of the A549 handled through CPP33-CLP-Cy5 intracellular Cy5 are 1.7 times of no targeting group CLP-Cy5.It connects , we further study CPP33-CLP to the specificity of A549 cell.Streaming the experimental results showed that, A549 cell pair The intake of CPP33-CLP is significantly higher than other cancer cells, such as MDA-MB-231 breast cancer cell, SMMC-7721 liver cancer cells With mouse brain colloid endothelial cell (Fig. 6 b), CPP33 is further demonstrated to the tomour specific penetrability of A549 cell.
In order to further study the cellular endocytosis machinery of CPP33-CLP, we use various cell endocytic inhibitor in advance, such as (big pinocytosis inhibits for Dynasore (dynein inhibitor), chlorpromazine hydrochloride (clathrin inhibitor), amiloride hydrochloride Agent) and methyl beta-cyclodextrin (caveolin inhibitor) A549 cell is handled.Streaming experimental result shows, dynasore It can significantly inhibit A549 cell to CPP33-CLP endocytosis, and other several inhibitor will not influence A549 cell substantially To the endocytosis (Fig. 6 c) of CPP33-CLP.The above result shows that CPP33-CLP be by dynein mediate endocytosis mode into Enter A549 lung carcinoma cell.
Endosome retention is that siRNA preparation carries out the most stern challenge faced in clinical conversion process.Therefore, Wo Menyong CLSM studies endosome flight behavior of the Cy5-siPLK1-CPP33-CLP in A549 cell.From CLSM picture (Fig. 6 d) It can be found that the cell handled through Cy5-siPLK1-CPP33-CLP, the fluorescence intensity of Cy5 is apparently higher than no target in cytoplasm To Cy5-siPLK1-CLP group.Also, Cy5-siPLK1-CPP33-CLP can effectively flee from endosome, it was demonstrated that CPP33 Can play the role of that siRNA is helped to flee from endosome.
Then, we use real-time quantitative Polymerase Chain Reaction (qRT-PCR) and western blot (Western blot) Test the Gene silencing efficacy to evaluate the CPP33-CLP (siPLK1-CPP33-CLP) for loading siPLK1 in A549 cell. As shown in Figure 7a, siPLK1-CPP33-CLP is substantially better than without targeting siPLK1- the specific silencing efficiency of PLK1 gene CLP, when siPLK1 is 200nM, their gene silencing efficiency is respectively 74% and 59%.And control group siScramble- CPP33-CLP is almost without any Gene silencing efficacy.Protein blot experiment result further demonstrates siPLK1-CPP33- CLP can effectively lower the expression (Fig. 7 b) of PLK1 albumen.SiPLK1-CPP33-CLP excellent Gene silencing efficacy is mainly returned Function is in its good tumor-targeting and endosome fugacity.
Embodiment 5：Internal pharmacokinetics, the bio distribution of siPLK1-CPP33-CLP
All zoopery operations meet University Of Suzhou's Experimental Animal Center relevant regulations.Firstly, we are by Cy5- SiPLK1-CPP33-CLP, Cy5-siPLK1-CLP and Cy5-siPLK1 pass through tail vein injection to health Balb/c Mice Body It is interior, to study its internal pharmacokinetics.The results show that Cy5-siPLK1-CPP33-CLP and Cy5-siPLK1-CLP can be Long circulating, their elimination half-life period (t are carried out in vivo1/2,β) it is respectively 3.4 and 2.9 hours (Fig. 8 a).And Cy5-siPLK1 is then It is rapidly cleared out of in vivo, circulation time is only 0.14 hour.The above result shows that CPP33-CLP can effectively extend The blood circulation time of Cy5-siPLK1.When noticeable, according to the literature, many cationic polymer-siRNA's is compound Nanoparticle is in vivo easy to dissociate afterwards entering, and then is rapidly cleared in humans out in vivo, so that their circulation time is lower than 0.5 hour.Then, we grind the bio distribution in Cy5-siPLK1-CPP33-CLP in situ A549 lung cancer in mice Study carefully.The result shows that after injection 6 hours Cy5-siPLK1-CPP33-CLP tumor locus enriching quantity be 3.14%ID/g, It is 1.86 times (Fig. 8 b) of no targeting group Cy5-siPLK1-CLP.The above result shows that CPP33-CLP can be effectively targeted to A549 tumour and can tumor locus for a long time be detained.
Embodiment 6：The internal antitumor activity evaluation of siPLK1-CPP33-CLP
We are using A549-Luc lung cancer in situ as model, to evaluate the internal antitumous effect of SAP-cRGD-CLP.By lotus Tumor mouse is randomly divided into 3 groups, and every group of 6 mouse (wherein 1 are only used as histologic analysis, remaining 5 is served only for observation survival rate and body Change again).We measure mouse lung with IVIS II small animal living body imaging system (Caliper Life Sciences) Bioluminescence intensity, thus indirectly calculate tumour size.Mouse needs the D- fluorescein that 100 μ L are first injected intraperitoneally before imaging Sylvite (15mg/mL is dissolved in PBS).Measurement in mouse weight every two days is primary, and the variation of mouse relative body weight is to pass through m/m0(m0For 0th day mouse weight) be calculated.By tail intravenous administration approach by siPLK1-CPP33-CLP, siPLK1-CLP and SiScramble-CPP33-CLP is injected into Mice Body, and injection dosage is 2.0mg siPLK1equiv./kg, is administered within every two days Once, altogether to 4 medicines.The growth of tumour is monitored by the bioluminescence imaging of tumour.Mouse lung tumor fluorescence semidefinite The results show that at the 12nd day for the treatment of, the fluorescence intensity of the mouse lung through siPLK1-CPP33-CLP treatment is obvious for amount analysis It is weaker (Fig. 9 a) than siPLK1-CLP group and siScramble-CPP33-CLP group, it was demonstrated that its good tumor inhibitory effect.From In lung's picture of mouse, it has also been discovered that, the mouse lung shape treated through siPLK1-CPP33-CLP is more normal. The median survival of siPLK1-CPP33-CLP treatment group mouse be 45 days, be significantly higher than siPLK1-CLP group (36 days) and SiScramble-CPP33-CLP group (26 days) (Fig. 9 b).Also, the weight of siPLK1-CPP33-CLP treatment group mouse is without bright Aobvious variation, it was demonstrated that it is with good biocompatibility and tolerance (Fig. 9 c).Result above proves, siPLK1-CPP33-CLP Can be safe and efficient by siRNA targeted delivery to A549 tumor locus, show good antitumous effect.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention It encloses without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in the present invention Protection scope within.Protection scope of the present invention is subject to claims.

Claims (10)

1. a kind of polyester fret peptide, which is characterized in that the polyester fret peptide is poly- (the 2- amino hexadecane of polyethylene glycol-b- Acid)-b- poly (L-lysine), structural formula is as follows：
Wherein, the value range that the value range that the value range of x is 40-2300, y is 30-800, z is 1-500.
2. polyester fret peptide according to claim 1, which is characterized in that the polyester fret peptide is in hydrophilic section polyethylene glycol End modified have selectively targeted molecule.
3. polyester fret peptide according to claim 2, which is characterized in that the selectively targeted molecule is small peptide, small Molecular targeted molecule, antibody, polysaccharide or monosaccharide.
4. polyester fret peptide according to claim 3, which is characterized in that the small peptide be sequence be cRGDfC cRGD, The CPP33 that the CC-9 or sequence that cNGQ that sequence is cNGQGEQc, sequence are CSNIDARAC are RLWMRWYSPRTRAYGC.
5. polyester fret peptide according to claim 3, which is characterized in that the small molecule targeted molecular is folic acid or fennel Amide.
6. a kind of preparation method of the described in any item polyester fret peptides of Claims 1 to 5, which is characterized in that including walking as follows Suddenly：Using polyethylene glycol-amino as macromole evocating agent, successively cause 2- aminohexadecanoic acid N- carboxyl inner-acid anhydride and ε-carbonyl phenoxy It is poly- that poly- (2- the aminohexadecanoic acid)-b- of polyethylene glycol-b- is prepared in base-L-lysine N- carboxyl inner-acid anhydride monomer ring-opening polymerisation Then carbobenzoxy is sloughed by being deprotected, obtains the polyester peptide by (ε-carbobenzoxy-L-lysine) triblock copolymer Material.
7. application of the described in any item polyester fret peptides of Claims 1 to 5 in genomic medicine delivering.
8. application according to claim 7, which is characterized in that the application is that the polyester fret peptide is prepared into polyester Peptide vesica progress genomic medicine, which is sent, to be passed.
9. a kind of polyester peptide vesica, which is characterized in that be prepared, wrapped by any one of Claims 1 to 5 polyester fret peptide It includes and is prepared by the polyester fret peptide without targeted molecular, is prepared by the polyester fret peptide containing targeted molecular, Huo Zhe The polyester peptide vesicle surface modification targeted molecular being prepared by the polyester fret peptide without targeted molecular obtains.
10. application of the polyester peptide vesica as claimed in claim 9 in genomic medicine delivering.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060094673A1 (en) * 2002-04-23 2006-05-04 Benjamin Chu Polymer surfactants for gene therapy applications
CN103755953A (en) * 2013-12-12 2014-04-30 深圳先进技术研究院 Intelligent polycation nano-carrier, and preparation method and application thereof
CN107266384A (en) * 2017-06-29 2017-10-20 苏州大学 N carboxyl inner-acid anhydride monomers and polyaminoacid based on 2 aminohexadecanoic acids and preparation method thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060094673A1 (en) * 2002-04-23 2006-05-04 Benjamin Chu Polymer surfactants for gene therapy applications
CN103755953A (en) * 2013-12-12 2014-04-30 深圳先进技术研究院 Intelligent polycation nano-carrier, and preparation method and application thereof
CN107266384A (en) * 2017-06-29 2017-10-20 苏州大学 N carboxyl inner-acid anhydride monomers and polyaminoacid based on 2 aminohexadecanoic acids and preparation method thereof

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