CN112843240A - Beta-cyclodextrin modified PAMAM targeted nano drug delivery carrier and preparation method thereof - Google Patents
Beta-cyclodextrin modified PAMAM targeted nano drug delivery carrier and preparation method thereof Download PDFInfo
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- CN112843240A CN112843240A CN202110312742.8A CN202110312742A CN112843240A CN 112843240 A CN112843240 A CN 112843240A CN 202110312742 A CN202110312742 A CN 202110312742A CN 112843240 A CN112843240 A CN 112843240A
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- pamam
- peg
- beta
- cyclodextrin
- drug delivery
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- A61K47/56—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
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Abstract
The invention discloses a PAMAM targeting nano drug delivery carrier based on beta-cyclodextrin modification, which has the following general formulaFormula (II): T-PEGm‑SS‑PAMAM‑(CD)nThe preparation process comprises the following steps: the PAMAM and carboxymethyl-beta-cyclodextrin sodium salt react to prepare the PAMAM-CD by taking a phosphate buffer solution as a solvent and N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride as catalysts; using methanol as solvent, and reacting PAMAM-CD, 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester and PEG (HS-PEG-OCH) with one-end sulfhydrylation3) Reacting to obtain PEG-SS-PAMAM-CD; reacting the carboxylated PEG-SS-PAMAM-CD with a tumor targeting ligand, ultrafiltering, purifying, and freeze-drying to obtain T-PEGm‑SS‑PAMAM‑(CD)n。
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a preparation method of a high-efficiency pH redox double-sensitive PAMAM targeted nano drug delivery carrier based on beta-cyclodextrin modification.
Background
Cancer has become the strongest killer severely threatening human life health for the last fifty-six decades. According to statistics, it is expected that by 2020, new tumor cases will reach 2000 ten thousand and death cases will reach 1200 ten thousand worldwide. National tumor registration center data shows a more obvious trend of malignant tumor youthfulness, which is closely related to life style of people. Therefore, the prevention and treatment of cancer is of great importance at present. Today, the clinical approaches to cancer treatment mainly include radiotherapy, chemotherapy and surgery, however, these traditional treatment methods have certain limitations. In addition, the limitations of solubility, bioavailability and drug targeting greatly affect the efficacy and application of the drug. Researchers related to medical disciplines try to apply the nano-carrier to an anti-tumor drug delivery system so as to improve the targeting property of the drug, increase the storage amount of the drug at tumor parts and achieve the effect of reducing the toxic and side effects of the drug. At present, the research on a wider novel drug delivery system mainly comprises liposomes, dendrimers, nanoemulsions, polymer micelles and the like, wherein the polyamidoamine dendrimers (PAMAM) are more and more paid attention and paid attention to by researchers due to the uniform nano-scale particle size, the unique hyperbranched 3D structure and the effective drug carrying space, and a multifunctional anti-tumor drug delivery system which is based on the PAMAM and is easy for drug loading, targeted delivery and drug release is developed.
As a main group of macromolecules in dendrimers, polyamidoamine dendrimers (PAMAM) are the most extensively studied dendrimers to date. PAMAM is mainly composed of three parts: a centrally located single atom or group of atoms comprising an initiating nucleus (usually ethylenediamine or ammonia), a surface functional group, and a branched structure linking the two. The branched segments produce a series of concentric layers, known as generations (G), by repeated Micheal addition reactions and amidation reactions. From the fourth generation, the PAMAM molecule has a certain base number of surface functional groups, so that the PAMAM molecule has a highly branched, symmetrical, radial and closed three-dimensional spherical structure. In the gaps of the dendritic molecules, hydrophobic flexible spaces are formed, an effective container-shaped carrier is provided for hydrophobic drugs, and the surface structure of the carrier is hydrophilic, so that the solubility of the drugs can be increased. Meanwhile, the PAMAM has good reactivity and tolerance, can introduce a large number of functional groups which are easy to react with primary amino groups on the surface of a molecule, and can also be bonded with drugs, targeting groups and various modifying groups.
However, previous studies found that high generation PAMAMs with NH2 terminal groups were associated with cytotoxicity, hemolysis and hepatotoxicity, which limited their biomedical applications. To improve the biocompatibility and reduce toxicity of PAMAM- (NH2) n, different methods have been proposed to passivate the NH2 end groups, including acetylation and pegylation. Pegylation has been studied more extensively because, in addition to reduced cytotoxicity, pegylation also spatially masks the opsonization of nanocarriers, thereby increasing their length of circulation in the bloodstream, thereby enhancing tumor accumulation of therapeutic agents in vivo via the EPR effect.
The super-strong reproductive capacity of cancer cells prevents the tumor part from supplying corresponding nutrition, energy and oxygen, so that the cancer cells cannot complete aerobic respiration, and glycolysis in an anaerobic environment can generate a large amount of lactic acid, so that the pH value (5.0) of the tumor part is lower than that (7.4) of a normal cell. Aiming at the huge difference of the survival environment, namely the acidic environment with the pH value of 5.0 and the neutral environment with the pH value of 7.4 of normal cells, researchers design and synthesize a nano drug delivery system based on pH value sensitivity, and deliver the wrapped antitumor drug to a specific tumor part through a carrier, so that the antitumor effect can be realized. For example, the polyamide-amine dendrimer contains a plurality of primary amino groups at the periphery, so protonation is easy to occur to generate a 'proton sponge effect', the swelling of lysosomes in cells is triggered, and the medicine can be released into the cells more quickly. The difference between the survival microenvironment of cancer cells and normal cells is not only shown in pH sensitivity, but also in the content of Glutathione (GSH) in cytoplasm of the cancer cells is about 4-5 times of the content of the normal cells, so that a drug delivery system with reduction sensitivity can be designed by utilizing the concentration difference between the cancer cells and the glutathione in a blood passage. For example; the PAMAM dendrimer is indirectly bonded with PEG through disulfide bonds (-S-S-) to synthesize PAMAM-S-S-PEG (PSSP).
Another common limitation associated with dendrimers (such as PAMAMs) is their relatively small size making it difficult to package large quantities of payload. Meanwhile, Cyclodextrins (CDs) have been extensively studied in the field of controlled drug delivery. Cyclodextrins are cyclic oligosaccharides composed of alpha-D-glucose units linked by (1 → 4) bonds, forming truncated cones. Because of this unique structure, cyclodextrins have a conical cavity that is essentially hydrophobic in nature. The cavity provides a hydrophobic microenvironment to attract hydrophobic drug molecules with proper sizes, and the drug is stabilized through the formation of the inclusion compound, so that the drug loading rate is increased, and the anti-tumor effect is improved.
Since the specific microenvironment at the tumor site can enhance the in vivo tumor accumulation of therapeutic agents through EPR effects, however, the degree of EPR effect alone through passive tumor targeting is generally limited. If the pegylated nanocarrier modified by the active tumor targeting ligand specific to the receptor overexpressed in the cancer cells is used, the in vivo tumor accumulation of the nanocarrier can be further enhanced, and thus more accurate targeted therapy can be realized. The T7 peptide (sequence HAIYPRH) can specifically recognize transferrin receptor on the surface of cancer cells, so the T7 modified drug delivery system can realize high accumulation of anticancer drugs and improve the cellular uptake efficiency.
Disclosure of Invention
The invention aims to provide a PAMAM targeting nano drug delivery carrier based on beta-cyclodextrin modification and a preparation method thereof.
The invention has a technical scheme that:
a PAMAM targeting nano drug delivery carrier based on beta-cyclodextrin modification comprises the following general formula: T-PEGm-SS-PAMAM-(CD)n,
Wherein the content of the first and second substances,
t is a tumor targeting ligand;
PEG is polyethylene glycol;
-SS-is a bridged disulfide bond;
PAMAM is a 0-10 th generation cationic polyamidoamine dendrimer;
CD is carboxymethyl-beta-cyclodextrin;
m is 1 to 4096;
n is 1 to 4096.
Furthermore, the general formula of the beta-cyclodextrin modified PAMAM targeting nano drug delivery carrier carrying the drug is T-PEGm-SS-PAMAM- (CD) n/X,
wherein the content of the first and second substances,
t is a tumor targeting ligand;
PEG is polyethylene glycol;
-SS-is a bridged disulfide bond;
PAMAM is a 0-10 th generation cationic polyamidoamine dendrimer;
-CD is carboxymethyl- β -cyclodextrin;
x is an anti-tumor drug;
m is 1 to 4096;
n is 1 to 4096.
Further, the tumor targeting ligand is T7 peptide.
Further, the anti-tumor drug is adriamycin.
Further, m is 30.
Further, n is 70.
Further, the cationic polyamidoamine dendrimer is a 5 th generation polyamidoamine dendrimer taking ethylenediamine as a core.
The other technical scheme of the invention is as follows:
a preparation process of a PAMAM targeting nano drug delivery carrier based on beta-cyclodextrin modification comprises the following steps:
1) phosphate Buffered Saline (PBS) pH7.4 is used as a solvent, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are used as catalysts, and the molar ratio of the PAMAM to the carboxymethyl-beta-cyclodextrin sodium salt is 1: 70, reacting to obtain PAMAM-CD;
2) using methanol as solvent to make the PAMAM-CD, 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester and PEG (HS-PEG-OCH) with one end sulfhydrylated3) In a molar ratio of 1: 30: 30 reacting to obtain PEG-SS-PAMAM-CD;
3) and (3) carboxylating the PEG-SS-PAMAM-CD and then mixing the carboxylated PEG-SS-PAMAM-CD with a tumor targeting ligand in a molar ratio of 1: reacting for 24 hours for 30 hours, ultrafiltering, purifying, and freeze drying to obtain T-PEGm-SS-PAMAM-(CD)n。
Further, a preparation process of the PAMAM targeting nano drug delivery carrier carrying drugs and based on beta-cyclodextrin modification is characterized by comprising the following steps:
1) weighing X, dissolving in a DMSO solution, adding triethylamine, stirring at room temperature in a dark place for 4h to neutralize hydrochloric acid, so that X becomes a hydrophobic drug;
2) DMSO and distilled water are used as solvents, and the volume ratio of the DMSO to the distilled water is 1: according to X and T-PEGm-SS-PAMAM-(CD)nIn a molar ratio of 40:1, stirring and reacting for 24 hours under the protection of nitrogen and at the temperature of 30 ℃ in a dark condition;
3) transferring the reaction solution into an ultrafiltration tube after the reaction is finished, centrifuging for 20min at 5000rpm to remove unencapsulated X, taking the supernatant, and freeze-drying to obtain T-PEGm-SS-PAMAM-(CD)n/X。
The invention provides a PAMAM targeting nano drug delivery carrier based on beta-cyclodextrin modification and a preparation method thereof, and the PAMAM targeting nano drug delivery carrier has the advantages that:
(1) the modification of beta-CD can improve the drug-loading rate of the nano-carrier and increase the anti-tumor effect;
(2) the PEG is connected to the surface of the PAMAM through a disulfide bond, so that on one hand, the hemolytic toxicity of the PAMAM can be obviously reduced, and the circulation time of the polymer in blood is prolonged; on the other hand, after the compound enters tumor cells, the disulfide bonds are broken in a high-reducibility cytoplasmic environment, so that the rapid release of the drug carried by the compound is promoted, and the aim of dual-sensitive drug release of reduction and pH is fulfilled;
(3) in order to increase the uptake of the polymer by tumor cells and increase the active targeting effect of the polymer, the surface of a polymer drug delivery system is modified by T7 peptide, and the TfR specificity which is highly expressed on the surface of T7, the surface of the tumor cells and the surface of the tumor cells is identified to promote the uptake of the cells, play the active targeting effect and increase the anti-tumor effect.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein the content of the first and second substances,
FIG. 1 shows the results of PAMAM (A), PEG-SS-PAMAM-CD (B) and T7-PEG-SS-PAMAM-CD (C)1H-NMR chart;
FIG. 2 is a graph of the in vitro release of PEG-SS-PAMAM-CD/DOX (abbreviated as PSSCD/DOX in the figure) and PEG-PAMAM-CD/DOX (abbreviated as PPCD/DOX in the figure);
FIG. 3 is a graph of the effect of in vitro anti-MCF-7 cell experiments with PEG-SS-PAMAM-CD/DOX (PSSCD/DOX), PEG-PAMAM-CD/DOX (PPCD/DOX) and DOX;
FIG. 4 is a graph showing the effect of in vitro cell uptake assays for PEG-SS-PAMAM-CD/DOX (PSSPCD/DOX) and T7-PEG-SS-PAMAM-CD/DOX (T7-PSSPCD).
Detailed Description
G5 PAMAM dendrimer (128 amino groups at the end, relative molecular mass 28826Da, Dendritech, USA); carboxymethyl-beta-cyclodextrin (CM-beta-CD, Shanghai Turke chemical Co., Ltd.); doxorubicin hydrochloride (purity not less than 98%, beijing huafeng bibco technologies ltd); n-hydroxysuccinimide (NHS, sahn chemical technology (shanghai) ltd); potassium chloride, disodium hydrogen phosphate, sodium chloride, potassium dihydrogen phosphate, triethylamine (national drug group chemical reagent limited); 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC-HCl, Tokyo chemical Co., Ltd.); dialysis bag(molecular weight cut-off is 8K-14 KDa, 3.5KDa, Solibao); NHS-PEG5000-OCH3(Beijing Kekai science and technology Co., Ltd.); t7 peptide (HAIYPRH, Shanghai Qianyi Biotech, Inc.); SPDP (Protech corporation); millipore Amicon Uitra-4 ultrafiltration tube (Millipore corporation, USA)
The structure of the polymer was confirmed with a superconducting nuclear magnetic resonance spectrometer (Unity Inova 400MHz), the particle size and potential of the polymer were characterized with a Nicomp 380ZLS type laser nanosized particle/potentiostat, and the cellular uptake was investigated with a flow cytometer (FC500, Beckman Coulter, usa).
The cationic polyamidoamine dendrimers of generations 0-10 have the following structures:
in the general formula T-PEGm-SS-PAMAM-(CD)nIn the/X, "/" means that the nano-carrier is loaded with X, wherein X is a hydrophobic anti-tumor drug.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are further described below. The invention is not limited to the embodiments listed but also comprises any other known variations within the scope of the invention as claimed.
First, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The present invention is described in detail by using the schematic structural diagrams, etc., and for convenience of illustration, the schematic diagrams are not enlarged partially according to the general scale when describing the embodiments of the present invention, and the schematic diagrams are only examples, which should not limit the scope of the present invention. In addition, the actual fabrication process should include three-dimensional space of length, width and depth.
Example 1
Respectively taking the reaction molar ratio as 1: 70: 70: 70G 5 PAMAM, beta-cyclodextrin, EDC and NHS as starting materials to synthesize a PCD (mol/mol, PAMAM/beta-CD 1/30) polymer. The specific operation method comprises the following steps: precisely weighing a certain mass of beta-cyclodextrin by using an electronic analytical balance, dissolving the beta-cyclodextrin in 5ml of phosphate buffer solution (pH7.4), dissolving EDC and NHS in the same molar ratio in the mixed solution, stirring the solution for 8 hours in a dark place at room temperature to activate carboxyl of the beta-cyclodextrin, sucking G5 PAMAM, adding the mixture into a reaction bottle, stirring the mixture for reaction for 24 hours at room temperature under the protection of nitrogen, finally dialyzing the mixture for 2 days by using deionized water, and freeze-drying the mixture for 2 days to obtain the final product PAMAM-CD.
Adding the synthesized product PAMAM-CD and SPDP into methanol solution at a molar ratio of 1:30 for neutralization, adding triethylamine, stirring at 30 ℃ in the dark for 5h under the protection of nitrogen, and then adding a certain amount of HS-PEG-OCH3Stirring for 24h, dialyzing with deionized water, and lyophilizing to obtain PEG-SS-PAMAM-CD (PSSPCD).
Dissolving PEG-SS-PAMAM-CD in 2ml PBS (0.1M, pH7.4), adding 30 times molar amount of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), activating carboxyl for 1h, adding 30 times molar amount of T7 peptide, protecting with nitrogen, reacting in dark for 24h, dialyzing with deionized water, and lyophilizing to obtain T7-PEG-SS-PAMAM-CD (T7-PSSPCD).
The PAMAM-CD and NHS-PEG prepared in example 1 were precisely weighed5000-OCH3Dissolving the mixture in a phosphate buffer (0.1, pH8.2) according to the reaction molar ratio of 1:30, and stirring the mixture for reaction for 24 hours in a dark place. After the reaction is finished, the reaction mixture is transferred into a dialysis bag (MWCO ═ 10KDa), deionized water is dialyzed for 2 days to remove free PEG, liquid in the dialysis bag is dialyzed by a mobile phone, and the white solid is PEG-PAMAM-CD (PPCD) after freeze drying.
Nuclear magnetic analysis
The results of nuclear magnetic analysis of PAMAM, PAMAM-CD of the product synthesized in example 1 and PEG-SS-PAMAM-CD are shown in fig. 1, and the nuclear magnetic analysis of the synthesized product by nuclear magnetic hydrogen spectroscopy shows that characteristic proton characteristic peaks are assigned as follows: of which there are four PAMAM characteristicsProton peaks, which are δ 2.45, 2.61, 2.81 and 3.28ppm, respectively; characteristic proton peaks of β -CD, which are δ 5.16, 3.76 and 3.51ppm, respectively; simultaneous HS-PEG5000-OCH3Has two obvious characteristic proton peak signals, delta is 3.35ppm (OCH)3),δ=3.67ppm(CH2CH2O), the characteristic proton peaks of the T7 peptide were δ 7.04 and 7.73ppm, respectively. The nuclear magnetic hydrogen spectrum of the product T7-PSSPCD can find that the product has PAMAM, beta-CD and HS-PEG at the same time5000Characteristic proton peaks of-COOH and T7 peptides, indicating that β -CD, PEG, and T7 peptides have successfully covalently bound to PAMAM surface. The number of H of beta-CD at delta-5.16 is 7 by peak area normalization method, and the number of H is 7 in HS-PEG5000The number of H at δ — 3.67 in COOH was 455, the number of H at δ — 7.73 in T7 peptide was 2, and the number of H at δ — 2.45 in PAMAM was 504, and the number of β -CD, PEG and T7 peptide linkages was calculated according to the following formula:
measurement of Polymer particle diameter and Zeta potential
Dissolving the synthesized polymers PAMAM-CD, PEG-SS-PAMAM-CD and T7-PEG-SS-PAMAM-CD in ultrapure water respectively to a final concentration of 3mg/ml, performing ultrasonic treatment for 5min, filtering with a 0.22 μm filter, discarding the primary filtrate, collecting the subsequent filtrate and a measuring cell, measuring the particle size with a laser particle size analyzer, and measuring each sample for 3 times in parallel. Zeta potential measurements were carried out using the same instrument and samples were measured in the same concentration and procedure as described above, with 3 runs of each sample in parallel.
The particle size of the T7-PEG-SS-PAMAM-CD polymer synthesized in example 1 was found to be 49.12. + -. 0.29nm, and the Zate potential was found to be 2.47. + -. 0.18 mV.
The PEG-SS-PAMAM-CD polymer synthesized in example 1 has a particle size of 49.10 + -0.17 nm and a Zate potential of 2.90 + -0.36 mV.
The PEG-PAMAM-CD polymer has a particle size of 53.14 + -0.21 nm and a Zate potential of 5.28 + -0.16 mV.
The three synthesized polymers PEG-PAMAM-CD, PEG-SS-PAMAM-CD and T7-PEG-SS-PAMAM-CD have larger grain diameter than PAMAM due to the introduction of PEG chain, reduce Zeta potential, remarkably improve stability and reduce toxicity.
Preparation of drug-loaded complexes
Accurately weighing a certain amount of DOX & HCl, dissolving in a methanol solution, adding a proper amount of triethylamine to neutralize hydrochloric acid, and dissociating DOX. Followed by reaction with DOX at a molar ratio of 40:1 between the synthetic polymers PEG-SS-PAMAM-CD and PEG-PAMAM-CD as described above. Weighing a certain amount of PEG-SS-PAMAM-CD polymer, adding the PEG-SS-PAMAM-CD polymer into a methanol solution, stirring for 24h in the dark at 30 ℃, performing rotary evaporation to remove the methanol solution, adding a proper amount of distilled water to dissolve the compound, centrifuging at 5000rpm for 10min to remove unencapsulated DOX, taking supernatant, and performing freeze drying to obtain the drug-loaded compound PSSPCD/DOX and PPCD/DOX of the synthetic polymer.
Precisely weighing a certain amount of DOX & HCl, dissolving in DMSO solution, adding a proper amount of triethylamine, stirring at room temperature in a dark place for 4h, and neutralizing hydrochloric acid to free DOX. Followed by reaction with DOX at a molar ratio of 40:1 between the synthetic polymers PAMAM-CD and T7-PEG-SS-PAMAM-CD described above. Weighing a certain amount of PAMAM-CD polymer, dissolving the PAMAM-CD polymer in 1ml of distilled water, adding the PAMAM-CD polymer into the DMSO solution continuously stirring one drop by one drop, protecting with nitrogen, and stirring for 24 hours at 30 ℃ in a dark place. Transferring the liquid into an ultrafiltration tube after the reaction is finished, centrifuging at 5000rpm for 10min to remove unencapsulated DOX, taking the supernatant, and freeze-drying to obtain the drug-loaded complexes PAMAM-CD and T7-PEG-SS-PAMAM-CD of the synthetic polymers.
In vitro release study of Carrier Complex
The reduction and pH sensitivity of each drug-loaded compound are inspected under different release conditions by adopting a dialysis bag method, wherein the release conditions are as follows: (1) a phosphate buffer at ph7.4 containing 10mM Glutathione (GSH), (2) a phosphate buffer at ph7.4 containing no 10mM Glutathione (GSH), (3) a phosphate buffer at ph5.0 containing 10mM Glutathione (GSH), (4) a phosphate buffer at ph5.0 containing 10mM Glutathione (GSH). Wherein pH7.4 is normal tissue non-acidic pH physiological condition, pH5.0 is acidic pH condition in tumor cell, and GSH 10mM is high reducing condition in tumor cell. Precisely weighing each complex with the same molar weight, dissolving the complex in 2mL of buffer solution, placing the complex in a dialysis bag (MWCO is 3500Da), fastening two ends of the buffer solution, then putting the complex into a conical flask containing 20mL of different release media, shaking the conical flask at 37 ℃ and 100rpm in a constant temperature shaking table, sampling 41mL of the release media in 0.5, 1, 2, 4, 6, 8, 12 and 24 hours respectively, and simultaneously supplementing fresh release media with the same volume and the same temperature. The concentration of DOX in each medium was determined using an ultraviolet spectrophotometer and the cumulative percent release was calculated according to the following formula. Wherein Er is DOX cumulative release percentage; ve is the displacement volume of phosphate buffer; v0 is the total volume of the release medium; mDOX is the content of DOX in the compound (namely 200 mug); ci is the concentration of drug released at the time of the ith displacement sample.
From FIG. 2, it can be seen that the release of DOX from PSSPCD/DOX and PPCD/DOX drug-loaded complex is significantly pH sensitive, and the release rate and cumulative release rate of DOX at pH5.0 are significantly higher than those at pH 7.4. The pH drug release behavior is mainly due to the fact that PAMAM has pH sensitivity, under the condition of low pH, the conformation of PAMAM can be changed from a 'compact center' to a 'compact shell' at a high pH value, the external branch chains are shrunk, the internal cavity is opened, and DOX is rapidly released from the compound.
The release of DOX from PSSPCD/DOX complexes all had significant reduction sensitivity. Under the same pH condition of the release medium, the pH value of the release medium is 5.0, and the cumulative release rate of the compound in the presence of high-concentration GSH is about 10 percent higher than that of the compound without GSH. Similarly, the release media were all 7.4 under the same pH conditions, and the cumulative release rate of the complex in the presence of GSH was about 10% higher than that in the absence of GSH. The main reason may be that under the stimulation of high concentration GSH, the disulfide bond between PAMAM and PEG is broken, so that PEG is detached from the surface of PAMAM, and the steric hindrance caused by the release of DOX from the inner core of PAMAM to the external medium is eliminated.
In vitro antitumor assay
To examine the in vitro anti-tumor effects of the drug-loaded complexes PCD, PPCD/DOX, PSSPCD/DOX, and T7-PSSPCD, cells in logarithmic growth phase 4T1 were taken, digested with 0.25% pancreatin and formed into a single cell suspension, seeded in 96-well plates at a cell concentration of 1 × 104cells/mL, 100 μ L per well, and a negative group and a blank control group were set. After putting the 96-well plate in an incubator for 24h, discarding the old culture solution, washing twice with PBS, respectively adding 100 μ L of drug-loaded complex solution containing PCD, PPCD/DOX, PSSPCD/DOX and T7-PSSPCD with serial concentrations and free DOX solution, each group having three multiple wells, and placing in a CO2 incubator for 48 h. The culture medium in the plate was discarded, washed twice with PBS, and 100. mu.L of MTT solution (0.5mg/mL) was added to each well. After placing in the incubator for another 4h, the MTT solution was discarded, 100. mu.L DMSO was added to each well, and vortexed for 10min until the crystals dissolved. Color comparison: the absorbance value (OD) at 490nm of each well was measured with a microplate reader, and the cell viability (cell viability) was calculated.
As can be seen from FIG. 3, each complex had a certain antitumor effect on 4T1 cells. With increasing concentrations of DOX, the cytotoxicity of cancer cells increased in dependence of the different complexes. Under the same administration dose, PSSPCD/DOX drug-loaded complex shows the strongest cytotoxicity, which indicates that the existence of disulfide bond can remarkably enhance the cytotoxicity of the complex. The results show that the disulfide bonds contribute to the release of the drug, thereby exerting a significant antitumor effect.
In vitro cell uptake assay
To examine the active targeting effect of the complex linked to the T7 peptide on 4T1 cells, 4T1 in the logarithmic growth phase was taken and digested with 0.25% pancreatin to form a single cell suspension at 1 × 105cell/mL is inoculated in a 6-well plate with the cell concentration of 2mL per well, the incubation is carried out for 24h, after the cells grow adherent to the wall, the culture solution is discarded, PBS is washed for 3 times, and 2mL PSSPCD (platelet-rich plasma cell plasma) diluted by serum-free culture solution is addedDOX and T7-PSSPCD complex (final doxorubicin concentration 5. mu.g/ml) were incubated for 2 h. After the incubation was completed, the old culture solution was discarded, washed with cold PBS 3 times, 500 μ L of pancreatin was added to each well, digestion was terminated, cells were collected, centrifuged, the supernatant was discarded, 500 μ L of PBS was added to disperse the cells, and the fluorescence intensity of intracellular DOX was detected by flow cytometry.
As can be seen from FIG. 4, the T7 peptide targeted modification enables the uptake of T7-PSSPCD/DOX nanoparticles by cells to be significantly increased, which is 1.8 times of the PSSPCD/DOX nanoparticles which are not connected with T7, which indicates that the introduction of T7 can significantly increase the active targeting property of the nanoparticles and increase the uptake of the cells, thereby improving the effective concentration of the drug in the cells and further enhancing the in vitro anti-tumor effect of the drug.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (9)
1. The beta-cyclodextrin modified PAMAM targeted nano drug delivery carrier is characterized by comprising the following general formula: T-PEGm-SS-PAMAM-(CD)n,
Wherein the content of the first and second substances,
t is a tumor targeting ligand;
PEG is polyethylene glycol;
-SS-is a bridged disulfide bond;
PAMAM is a 0-10 th generation cationic polyamidoamine dendrimer;
CD is carboxymethyl-beta-cyclodextrin;
m is 1 to 4096;
n is 1 to 4096.
2. The PAMAM targeting nano drug delivery carrier based on beta-cyclodextrin modification of claim 1, which is characterized in that: the general formula of the beta-cyclodextrin modified PAMAM targeting nano drug delivery carrier carrying drugs is T-PEGm-SS-PAMAM- (CD) n/X,
wherein the content of the first and second substances,
t is a tumor targeting ligand;
PEG is polyethylene glycol;
-SS-is a bridged disulfide bond;
PAMAM is a 0-10 th generation cationic polyamidoamine dendrimer;
-CD is carboxymethyl- β -cyclodextrin;
x is an anti-tumor drug;
m is 1 to 4096;
n is 1 to 4096.
3. The PAMAM targeting nano drug delivery carrier based on beta-cyclodextrin modification as claimed in claim 2, wherein: the tumor targeting ligand is T7 peptide.
4. The PAMAM targeting nano drug delivery carrier based on beta-cyclodextrin modification as claimed in claim 2, wherein: the anti-tumor drug is adriamycin.
5. The PAMAM targeting nano drug delivery carrier based on beta-cyclodextrin modification as claimed in claim 2, wherein: and m is 30.
6. The PAMAM targeting nano drug delivery carrier based on beta-cyclodextrin modification as claimed in claim 2, wherein: the n is 70.
7. The PAMAM targeting nano drug delivery carrier based on beta-cyclodextrin modification as claimed in claim 2, wherein: the cationic polyamidoamine dendrimer is a 5 th generation polyamidoamine dendrimer taking ethylenediamine as a core.
8. A preparation process of a PAMAM targeting nano drug delivery carrier based on beta-cyclodextrin modification is characterized by comprising the following steps:
1) phosphate Buffered Saline (PBS) pH7.4 is used as a solvent, N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride are used as catalysts, and the molar ratio of the PAMAM to the carboxymethyl-beta-cyclodextrin sodium salt is 1: 70, reacting to obtain PAMAM-CD;
2) using methanol as solvent to make the PAMAM-CD, 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester and PEG (HS-PEG-OCH) with one end sulfhydrylated3) In a molar ratio of 1: 30: 30 reacting to obtain PEG-SS-PAMAM-CD;
3) and (3) carboxylating the PEG-SS-PAMAM-CD and then mixing the carboxylated PEG-SS-PAMAM-CD with a tumor targeting ligand in a molar ratio of 1: reacting for 24 hours for 30 hours, ultrafiltering, purifying, and freeze drying to obtain T-PEGm-SS-PAMAM-(CD)n。
9. A preparation process of a PAMAM targeting nano drug delivery carrier carrying drugs and modified based on beta-cyclodextrin is characterized by comprising the following steps:
1) weighing X, dissolving in a DMSO solution, adding triethylamine, stirring at room temperature in a dark place for 4h to neutralize hydrochloric acid, so that X becomes a hydrophobic drug;
2) DMSO and distilled water are used as solvents, and the volume ratio of the DMSO to the distilled water is 1: according to X and T-PEGm-SS-PAMAM-(CD)nIn a molar ratio of 40:1, stirring and reacting for 24 hours under the protection of nitrogen and at the temperature of 30 ℃ in a dark condition;
3) transferring the reaction solution into an ultrafiltration tube after the reaction is finished, centrifuging for 20min at 5000rpm to remove unencapsulated X, taking the supernatant, and freeze-drying to obtain T-PEGm-SS-PAMAM-(CD)n/X。
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