CN107281164A - It is a kind of that the self-assembled nanometer grain of cancer therapy drug is loaded and its in the application of anti-tumor aspect based on low generation PAMAM dendrimers - Google Patents

Abstract

The invention belongs to biomedicine technical field, and in particular to a kind of preparation of administration nano-drug administration system EL@PAMAM/HA self-assembled nanometers grain and antitumor application thereof.The present invention is based on low generation dendrimer PAMAM（G0/G1）With the hyaluronic acid of tumour cell CD44 receptor targets（HA）Targeting self-assembled nanometer delivery system EL PAMAM/HA NPs are prepared using exchange of solvent method, the small molecule self-assembled nanometer drug-loading system can contain this antineoplastic of Tarceva, with pH responses, release the drug in acid condition more, and CD44 high expression tumour cells can be targetted, it can specifically convey more medicines to tumor locus and play drug effect, there is significant treatment advantage.

Description

A self-assembled nanoparticle loaded with anticancer drugs based on low-generation PAMAM dendrimers and its application in antitumor

technical field

The invention belongs to the technical field of biomedicine, and in particular relates to the preparation and anti-tumor application of a nano drug delivery system EL@PAMAM/HA self-assembled nanoparticle.

Background technique

Erlotinib, whose general trade name is Tarceva, is an anti-tumor targeted therapy drug officially approved by the US FDA in 2004, and has also been certified by my country's State Food and Drug Administration (CFDA). And it was approved to enter the Chinese market in 2007. The chemical structural formula of erlotinib (abbreviated as EL in the present invention) is

, the chemical name is N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinolineamine hydrochloride, which is a kind of epidermal growth factor receptor (Epithelial Growth Factor Receptor, EGFR) are highly selective small molecule quinazoline derivatives. EL is used for the third-line treatment of locally advanced or metastatic non-small cell lung cancer (NSCLC) after two or more chemotherapy failures, and is often used in combination with the anticancer drug gemcitabine for locally advanced or metastatic pancreatic cancer . Erlotinib is mainly cleared in the liver. In vitro cytochrome P450 analysis shows that erlotinib is mainly metabolized by CYP3A4, and a small amount is metabolized by CYP1A2 and extrahepatic isoenzyme CYP1A1. The most common adverse reactions of oral EL are rash (75%) and diarrhea (54%), both of which are unexplained adverse reactions and are relatively mild and can be managed without interruption of medication.

A large number of studies have shown that the anti-tumor effect of EL against EGFR-mutated non-small cell lung cancer (NSCLC) is reflected in its ability to inhibit the phosphorylation of intracellular tyrosine kinase (TK) associated with epidermal growth factor receptor (EGFR), thereby Blocking the signal transduction of epidermal growth factor (HER1), among them, TK is an important substance for tumor cell growth, and EL can inhibit tumor growth by inhibiting the activity of TK. In this invention, non-small cell lung cancer (NSCLC) cells represented by human lung adenocarcinoma cells (A549) are verified based on low-generation dendrimer PAMAM (G0/G1) and hyaluronic acid (HA) targeted to the CD44 receptor of tumor cells. ) to construct a self-assembled nano drug delivery system EL@PAMAM/HA NPs with targeting function.

Polyamide-amine (PAMAM) dendrimers are a new class of nano-biological materials that diverge symmetrically from the center outward and are highly branched. Molecular self-assembly to form nanoparticles of suitable size can improve the biocompatibility, bioavailability and targeting of drugs, so they can be used as delivery vehicles for anticancer drugs and imaging drugs. At present, the application of PAMAM is still mainly based on drug carriers. For example, patent CN201410271351 discloses a folic acid-PAMAM-ursolic acid nano drug with tumor targeting. The initial materials used in this nano drug are highly toxic G3 and G5 To replace polyamide-amine dendrimers (PAMAM), the amino groups on the surface of PAMAM need to be replaced with hydroxyl groups to reduce the toxicity of the material itself, and then folic acid and ursolic acid are reacted with the hydroxyl groups on the surface of PAMAM to finally synthesize the target product. The reaction process is complicated, the production cost is high, and the yield is not high, which limits its application to a certain extent. Low-generation PAMAM molecules (G0 as shown in formula I, G1 as shown in formula II) have surface amino groups of 4 and 8, are simple to synthesize, have low toxicity, and are inexpensive, and can improve the solubility and bioavailability of hydrophobic drugs, so Selected for use as the nanodelivery vehicle of the present invention.

Hyaluronic acid (Hyaluronic Acid, HA) is a natural linear glycosaminoglycan widely found in living organisms, and its chemical structure is

.

The specific receptor CD44 molecule of hyaluronic acid is highly expressed on the cell surface of various malignant tumors such as breast cancer, skin cancer and ovarian cancer. HA binds to CD44 molecules through a specific receptor-ligand mechanism, enters the cytoplasm through endocytosis, can selectively enter tumor cells, and then degrades under the action of enzymes such as hyaluronidase and acid hydrolase , to achieve targeted drug delivery, so hyaluronic acid is often used as the target of nano-drugs.

In normal tissues, the microvascular endothelial space is dense and structurally complete, and nanoparticles are not easy to penetrate. However, the tumor microenvironment is complex, with abundant tumor blood vessels, wide space between blood vessel walls, poor structural integrity, and lack of lymphatic return. Nano drug delivery system has passed, so the preparation of drugs into nano drug delivery system has become a new trend in the field of tumor treatment and has broad application prospects. Self-assembly is the occurrence of non-covalent weak interactions between molecules under equilibrium conditions, such as van der Waals forces, hydrogen bonds, hydrophobic interactions, electrostatic interactions, π-π interactions, etc., spontaneously combining to form stable aggregates with certain structures and functions A bulk or supramolecular process that transforms a disordered system into an ordered system is often used to prepare nanoparticles. Nanoparticles formed by molecular self-assembly usually have some properties that single molecules or low-level molecular aggregates do not have, such as electrical, optical, biological properties, etc. At the same time, molecular self-assembly also has a structural amplification effect, which can endow nanoparticles with Some novel properties, such as more compact and richer morphology and structure, as well as the characteristics of both functionalization and intelligent response, these advantages make the application prospects of self-assembled nano drug delivery systems increasingly broad. The present invention is based on low-generation dendritic macromolecule PAMAM (G0/G1 PAMAM) and tumor cell CD44 receptor-targeted hyaluronic acid (HA), and uses self-assembly technology to construct an EL@PAMAM/HA nano drug delivery system.

The self-assembled nano-drug delivery system constructed by the present invention can carry anti-tumor drugs such as erlotinib, and has both pH response and CD44 receptor targeting. Therefore, the nano-drug delivery system involved in the present invention innovatively utilizes The assembly technology co-produces low-generation PAMAM dendrimers, EL and HA, which is easy to operate and provides a new research direction for erlotinib anti-tumor therapy.

Contents of the invention

The purpose of the present invention is to prepare targeted self-assembled nano drug delivery system EL@PAMAM based on low-generation dendrimer PAMAM (G0/G1) and tumor cell CD44 receptor-targeted hyaluronic acid (HA) by solvent exchange method /HA NPs, so that it can target the delivery of drugs to the tumor site to exert curative effect.

The preparation steps of EL@PAMAM/HA NPs described in the present invention are as follows:

1. Preparation of EL@PAMAMEL (G0/G1)/HA

At room temperature, add 100 µL of EL good solvent solution dropwise to 700 µL of poor solvent, then add 100 µL PAMAM (G0/G1 ) aqueous solution, shake for 1 minute, then add 100 µL HA aqueous solution, ultrasonically shake for 15 minutes, at room temperature After standing still, the EL@PAMAM(G0/G1)/HA self-assembled nanocomposite was prepared, wherein the above-mentioned good solvent and poor solvent were selected as absolute ethanol and water, respectively.

2. Particle size and potential analysis and detection of EL@PAMAM(G0/G1)/HA self-assembled nanocomposites

The low-generation PAMAM dendrimers are positively charged, and HA is negatively charged. During the self-assembly process, electrostatic adsorption will occur, which will affect the morphology of the prepared nanoparticles. The Malvern particle sizer is used to measure the nanoparticles prepared by different charge ratios. Particle size distribution and Zeta potential of the composite.

Table 1 Particle size distribution of EL@PAMAM(G0)/HA nanocomposites with different charge ratios

Table 2 Particle size distribution of EL@PAMAM(G1)/HA nanocomposites with different charge ratios

EL@PAMAM(G0/G1)/HA was prepared by "solvent exchange method". The charge ratio is an important factor affecting the particle size of the EL@PAMAM(G0/G1)/HA nanocomposite, and the PDI value can reflect the particle size of the EL@PAMAM(G0/G1)/HA. Distribution, the optimal charge ratio of EL@PAMAM(G0)/HA is 4:5, and the optimal charge ratio of EL@PAMAM(G1)/HA is 3:5. When the positive and negative charge ratio is 4:5, the average particle size of EL@PAMAM(G0)/HA nanocomposite is 322.6 nm, the PDI is 0.286, and the potential is -5.72 mV; when the positive and negative charge ratio is 3:5 , the average particle size of the EL@PAMAM(G1)/HA nanocomposite is 146.1 nm, the PDI is 0.283, and the potential is -6.92 mV. In comparison, the particle size of the EL@PAMAM(G1)/HA nanocomposite is smaller, more evenly distributed.

3. Stability experiments of self-assembled nanocomposites

The particle size distribution of the nanoparticles was detected by the Malvern laser particle size analyzer, and the results are shown in Figure 6. The particle size distribution of the EL@PAMAM/HA nanocomposite did not change significantly in a continuous week, indicating that the EL@PAMAM/HA nanocomposite The complex has good stability.

4. Determination of drug loading and encapsulation efficiency

Use ultrafiltration tubes to centrifuge to obtain unloaded free erlotinib and nanocomposites, lyophilize and weigh the total nanometer mass, and use a UV spectrophotometer to measure the UV absorbance of free erlotinib, according to the standard curve of erlotinib Calculate the corresponding concentration, and then calculate the drug loading and encapsulation efficiency of the self-assembled nanocomposite.

Calculated according to the standard curve of erlotinib, when the positive and negative charge ratio was 4:5, the drug loading and encapsulation efficiency of EL@PAMAM(G0)/HA were 32.43% and 62.43%, respectively; when the positive and negative charge ratio When the ratio was 3:5, the drug loading and encapsulation efficiency of EL@PAMAM(G1)/HA were 19.22% and 76.51%, respectively.

5. Release of Erlotinib at Different pH Conditions

Move the EL@PAMAM(G0)/HA and EL@PAMAM(G1)/HA nanocomposite solutions into a dialysis bag with a molecular weight of 1000, and carry out magnetic stirring in the dialysis medium with different pH values at room temperature, and use ultraviolet Spectrophotometer measures the absorbance of samples at each time point, calculates the cumulative release amount at each time point according to the standard curve of erlotinib, and draws the release curve of erlotinib over time at different pH values , the results are shown in Figure 7, Figure 8 and Figure 9.

6. MTT Assay for Cytotoxicity Detection

Proliferation inhibitory effect of EL-loaded nano drug delivery system on cervical cancer cell HeLa and non-small cell lung cancer cell A549 detected by MTT assay

① Take a bottle of tumor cells in the logarithmic growth phase, digest and make a cell suspension of 1×10 ⁵ /ml.

② Transfer the cell suspension into a 96-well plate, 100 μL per well, and culture in a 37°C, 5% CO ₂ incubator for 24 h.

③ Remove the medium, add the self-assembled nanocomplex according to the concentration gradient, 100 μL per well, and set up the EL single-drug group and the PAMAM-G0/G1 group. After 24 h of action, remove the drug-containing medium, add 100 μL of serum-free and phenol red-free medium to each well, and then add 10 μL of MTT solution, and continue to incubate for 24 h.

④ Discard the supernatant in the plate, add 100 μL DMSO to each well, place on a shaker and shake at low speed for 10 minutes, after the crystals are fully dissolved, use a microplate reader to detect the optical absorption value (OD value) of each well, and calculate the cell proliferation Inhibition rate: cell survival rate (%) = average OD value of the medication group ÷ average OD value of the blank control group × 100%, use GraphPad Prism software for data processing, and measure the absorbance value of each well at OD 570 nm of the enzyme-linked immunosorbent assay instrument , and calculate the corresponding cell viability. The results are shown in Figure 10, Figure 11, Figure 12 and Figure 13. It can be seen from the figure that neither EL@PAMAM(G0)/HA nor EL@PAMAM(G1)/HA showed obvious cytotoxicity to HeLa, and the cytotoxicity to A549 cells was stronger than that to HeLa cells. The CD44 receptor is highly expressed in A549 cells, and Erlotinib is a targeted drug for non-small cell lung cancer A549 cells, so the toxicity to A549 cells is significantly increased.

7. Cellular Uptake Assay

The EL@PAMAM(G1)/HA nano drug delivery system was connected to FITC to characterize the uptake of the nano drug delivery system by A549 cells, and the results are shown in Figure 14. EL@PAMAM NPs without target HA will also reach the cells in a small amount, so a small amount of fluorescence can be detected in the cells, which is in obvious contrast to the low-generation dendrimers based on PAMAM (G0/G1) and The self-assembled nano-drug delivery system EL@PAMAM(G1)/HA NPs constructed from CD44 receptor-targeting hyaluronic acid (HA) significantly increased the amount of drug entering cells, indicating that it does have good targeting.

The advantages of the present invention are:

1. The EL@PAMAM(G0)/HA and EL@PAMAM(G1)/HA prepared in the present invention are prepared by solvent exchange method, which is easy to operate.

2. The small-molecule self-assembled nano-drug delivery system constructed in the present invention can carry anti-tumor drugs such as erlotinib, which has a pH response, releases more drugs under acidic conditions, and is suitable for drug release at tumor sites.

3. The small molecule self-assembled nano-drug delivery system constructed in the present invention can carry anti-tumor drugs such as erlotinib and can target tumor cells with high expression of CD44, and can specifically deliver more therapeutic drugs to tumor sites to play a role. Medicinal efficacy has significant therapeutic advantages.

4. The EL@PAMAM(G0)/HA and EL@PAMAM(G1)/HA small molecule self-assembled nano drug delivery system prepared by the present invention can not only provide a new method for tumor treatment, but also for the targeted treatment of non-small cell lung cancer. This method also points out a new way of thinking for the research of targeted drug delivery system.

Description of drawings

Figure 1. A self-assembled nano-drug delivery system EL@PAMAM (G0 )/HA nanocomposite charge ratio is the size of the nanometer when the charge ratio is 4:5;

Figure 2. The self-assembled nano-drug delivery system EL@PAMAM(G0 )/HA nanocomposite charge ratio is the size of the nano potential when the charge ratio is 4:5;

Figure 3. A self-assembled nano-drug delivery system EL@PAMAM (G1 )/HA nanocomposite charge ratio is the size of the nanometer when the charge ratio is 3:5;

Figure 4. A self-assembled nano-drug delivery system EL@PAMAM (G1 )/HA nanocomposite charge ratio is the size of the nano potential when the charge ratio is 3:5;

Figure 5. A self-assembled nano-drug delivery system EL@PAMAM (G1 )/HA nanocomposite charge ratio is 3:5 nano AFM image;

Figure 6. Self-assembled nano-drug delivery system EL@PAMAM/HA with targeting function constructed based on low-generation dendrimer PAMAM (G0/G1 PAMAM) and tumor cell CD44 receptor-targeted hyaluronic acid (HA) Stability testing;

Figure 7. Release curves of erlotinib under different pH conditions;

Figure 8. Self-assembled nano-drug delivery system EL@PAMAM-G1 with targeting function based on low-generation dendrimer PAMAM (G0/G1 PAMAM) and tumor cell CD44 receptor-targeted hyaluronic acid (HA) Release curves of /HA NPs under different pH conditions;

Figure 9. A self-assembled nano drug delivery system EL@PAMAM-G0 with targeting function based on low-generation dendrimer PAMAM (G0/G1 PAMAM) and tumor cell CD44 receptor-targeted hyaluronic acid (HA) Release curves of /HA NPs under different pH conditions;

Figure 10. The self-assembled nano-drug delivery system with targeting function based on the low-generation dendrimer PAMAM (G0) and tumor cell CD44 receptor-targeted hyaluronic acid (HA) was used to detect the toxicity of HeLa cells by MTT method;

Figure 11. A self-assembled nano-drug delivery system with targeting function based on low-generation dendrimer PAMAM (G1) and tumor cell CD44 receptor-targeted hyaluronic acid (HA) The cytotoxicity results of HeLa cells were detected by MTT method;

Figure 12. A self-assembled nano-drug delivery system with targeting function based on low-generation dendrimer PAMAM (G0) and tumor cell CD44 receptor-targeted hyaluronic acid (HA) The cytotoxicity results of A549 were detected by MTT method;

Figure 13. A self-assembled nano-drug delivery system with targeting function based on low-generation dendrimer PAMAM (G1) and tumor cell CD44 receptor-targeted hyaluronic acid (HA) The cytotoxicity results of A549 were detected by MTT method;

Figure 14. Cellular uptake of a self-assembled nano-drug delivery system with targeting function based on low-generation dendrimer PAMAM (G0/G1 PAMAM) and tumor cell CD44 receptor-targeted hyaluronic acid (HA).

detailed description

In order to make the content of the present invention easier to understand, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited thereto.

1. Preparation of EL@PAMAM(G0)/HA

At 50 °C, 100 µL of 25 µM erlotinib in absolute ethanol was quickly added to 700 µL of water with shaking (7 replicates were prepared), followed by 100 µL of 25, 50, 75, 100, 125 , 150, 175, and 200 µM PAMAM (G0) aqueous solutions were quickly added to the above solutions, and after shaking for one minute, 100 µL of 500 µM HA aqueous solutions were added to the above 7 solutions, and then shaken for 5 minutes. After the end, the samples were ultrasonically treated for 15 minutes, and then left at room temperature to prepare EL@PAMAM(G0)/HA self-assembled nanocomposites with different charge ratios.

2. Preparation of EL@PAMAM(G1)/HA

At 50 °C, 100 µL of 250 µM erlotinib in absolute ethanol was quickly added to 700 µL of water (7 parts were prepared) while shaking, and then 100 µL of 12.5, 25, 37.5, 50, 62.5, 75, 87.5, and 100 µM PAMAM (G1) aqueous solutions were quickly added to the above solutions, and after shaking for one minute, 100 µL of 500 µM HA aqueous solutions were added to the above 7 solutions, and then shaken for 5 minutes. After the end, the sample was ultrasonically treated for 15 minutes, and then left at room temperature to prepare EL@PAMAM(G1)/HA self-assembled nanocomposites with different charge ratios.

3. Determination of drug loading and encapsulation efficiency

Use ultrafiltration tubes to centrifuge to obtain unloaded free erlotinib and nanocomposites, lyophilize and weigh the total nanometer mass, and use a UV spectrophotometer to measure the UV absorbance of free erlotinib, according to the standard curve of erlotinib Calculate the corresponding concentration, and then calculate the drug loading and encapsulation efficiency of the self-assembled nanocomposite.

Calculation formula:

Drug loading = (total mass of erlotinib - mass of free erlotinib)/total mass of nanometers*100%

Encapsulation efficiency = (total mass of erlotinib - mass of free erlotinib)/total mass of erlotinib * 100%

4. Release profiles of erlotinib at different pH conditions

Transfer 1 mL of EL@PAMAM(G0)/HA and EL@PAMAM(G1)/HA nanocomposite solution into a dialysis bag (MWCO=1000), and then put it into a dialysis bag containing 49 mL of PBS (pH 7.4/pH 5.5 ) in the dialysis medium at 37 °C with constant temperature magnetic stirring, and the release experiments of erlotinib at different pH values were carried out. Samples were taken at preset time points (0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72 h), and 2 mL of dialysate was taken each time, and then 2 mL of fresh dialysis medium was added, and then Utilize the ultraviolet spectrophotometer to measure the absorbance of each time point sample, calculate the cumulative release amount of each time point according to the standard curve of erlotinib, draw the time-varying curve of erlotinib under different pH values release curve.

The present invention has a breakthrough in the application of nanometer self-assembly technology to the anticancer drug erlotinib for tumor treatment, lays a theoretical foundation for the targeted treatment of non-small cell lung cancer, and also has a certain reference for the application of nanotechnology in tumor treatment significance.

Claims (5)

1. a kind of self-assembled nanometer grain that cancer therapy drug is loaded based on low generation PAMAM dendrimers, it is characterised in that：Based on low For dendrimer PAMAM and the hyaluronic acid HA of tumour cell CD44 receptor targets, targeting is prepared certainly using exchange of solvent method Assemble nanometer delivery system EL@PAMAM/HA.

2. a kind of self-assembled nanometer that cancer therapy drug is loaded based on low generation PAMAM dendrimers according to claim 1 Grain, it is characterised in that：Wherein low generation dendrimer PAMAM is low generation dendrimer PAMAM G0 or tree-shaped big point of low generation Sub- PAMAM G1, wherein G0 structural formulas are；

G1 structural formulas are。

3. a kind of self-assembled nanometer that cancer therapy drug is loaded based on low generation PAMAM dendrimers according to claim 1 Grain, it is characterised in that：Specifically preparation method is：At room temperature, 100 μ L Tarcevas EL good-solvent solution is added dropwise Into 700 μ L poor solvents, the 100 μ L PAMAM aqueous solution are then added, vibration adds 100 μ L HA's after 1 minute The aqueous solution, ultrasonic vibration 15 minutes, is stored at room temperature, and EL@PAMAM/HA self-assembled nanometer compounds are made.

4. a kind of self-assembled nanometer that cancer therapy drug is loaded based on low generation PAMAM dendrimers according to claim 3 Grain, it is characterised in that：Wherein good solvent is absolute ethyl alcohol, and poor solvent is water.

5. a kind of a kind of self-assembled nanometer that cancer therapy drug is loaded based on low generation PAMAM dendrimers as claimed in claim 1 Grain is used for the application for preparing antineoplastic.