CN107970242B

CN107970242B - Paclitaxel/erlotinib loaded mesoporous silica-hyaluronic acid mixed targeting nanoparticles

Info

Publication number: CN107970242B
Application number: CN201711298950.7A
Authority: CN
Inventors: 邵敬伟; 王贠莞彬; 郑桂容
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2017-12-08
Filing date: 2017-12-08
Publication date: 2020-11-24
Anticipated expiration: 2037-12-08
Also published as: CN107970242A

Abstract

The invention discloses a paclitaxel/erlotinib loaded mesoporous silica-hyaluronic acid mixed targeting nanoparticle and application thereof in preparation of anti-cancer drugs. The targeted nano-particles are prepared by performing surface amination modification on mesoporous silica nano-particles, covalently coupling hyaluronic acid to the outer surface of the mesoporous silica nano-particles, respectively loading paclitaxel and erlotinib in inner pore channels of the mesoporous silica nano-particles, and mixing the two mesoporous silica nano-particles in proportion. The invention not only can improve the stability of the paclitaxel, but also can realize the targeted drug delivery of the paclitaxel/erlotinib, and the two drugs are combined to play a role in synergistic anti-tumor metastasis.

Description

Paclitaxel/erlotinib loaded mesoporous silica-hyaluronic acid mixed targeting nanoparticles

Technical Field

The invention belongs to the technical field of preparation of antitumor drugs, and particularly relates to a paclitaxel/erlotinib loaded mesoporous silica-hyaluronic acid mixed targeting nanoparticle and application thereof.

Background

In recent years, new drug delivery systems have been the focus of research in the field of medicine. The nano-drug delivery system can simultaneously transport two or more drugs, can improve the in-vivo distribution and the pharmacokinetic properties of the antitumor drugs, improves the selectivity of the drugs on specific targets, and achieves the treatment purposes of high efficiency and low toxicity. The combined use of two or more anti-tumor active ingredients has good prognosis and less adverse reaction, and can reduce the occurrence of drug resistance, but the combined use is easily influenced by the in vivo metabolic characteristics of different drugs, so that the dosage reaching the tumor part is less.

Paclitaxel (PTX for short) is named as taxol, taxol and Tesu, and the structural formula is as follows:

. Paclitaxel is a natural secondary metabolite separated and purified from the bark of the gymnosperm yew, is the most excellent natural anticancer drug discovered at present, and is widely used for treating breast cancer, ovarian cancer, partial head and neck cancer and lung cancer clinically. Paclitaxel is a diterpenoid alkaloid compound with anticancer activity, and has a novel and complex chemical structure, wide and remarkable biological activity, a completely new and unique action mechanism and a scarce natural resource, so that paclitaxel becomes an anticancer star which draws attention in the next half of the 20 th century and is the key point in research. Paclitaxel is the most popular anticancer drug in the international market in recent years, and is considered to be one of the most effective anticancer drugs in human for the next 20 years. In recent years, the global population and the cancer incidence rate have increased explosively, and the demand for paclitaxel has also increased significantly. The invention patent (CN 102579449A) discloses a combined medicine capable of improving the sensitivity of tumors to paclitaxel, wherein the combined medicine comprises praziquantel and paclitaxel in a mass ratio of 180-2200: 1. The combined medicine can improve the sensitivity of the tumor to the paclitaxel, effectively improve the killing capacity of the paclitaxel to the tumor, greatly promote the occurrence of tumor cell apoptosis, effectively improve the sensitivity of tumor cells which are insensitive to the paclitaxel or have drug resistance to the paclitaxel, and greatly reduce the drug resistance of the tumor cells to the paclitaxel. The invention patent (CN 105982888A) discloses a combined medicine which contains a first active ingredient artemisinin or a derivative thereof, a second active ingredient paclitaxel or a pharmaceutically acceptable salt and a hydrate thereof, and optional pharmaceutically acceptable auxiliary materials. The combined application of the artemisinin or the derivatives thereof and the paclitaxel can be used for treating and/or preventing melanoma, the artemisinin or the derivatives thereof and the paclitaxel have good synergistic effect, and simultaneously, the dosage of the paclitaxel is greatly reduced.

Erlotinib (Erlotinb, abbreviated as EL) is used as an EGFR (epidermal growth factor receptor) small molecule tyrosine kinase inhibitor, and the structural formula of the erlotinib is as follows:

the three-line therapy of the local advanced or metastatic non-small cell lung cancer which fails in two or more chemotherapy schemes can be tried, and the three-line therapy is simple and convenient to take orally, small in side effect, easy to accept by patients and wide in clinical application. Erlotinib is primarily cleared by hepatic metabolism and biliary secretion, so erlotinib should be used with caution in patients with liver dysfunction. The invention patent (CN 103933046A) discloses a pharmaceutical composition for resisting pancreatic cancer, which combines a therapeutically effective amount of amiloride and a therapeutically effective amount of erlotinib, an epidermal growth factor tyrosine kinase inhibitor, and can obtain a better drug effect than that of erlotinib used alone and improve the effect of clinical chemotherapy. The invention patent (CN 101300015A) discloses the combined use of suberoylanilide hydroxamic acid and erlotinib for the treatment of various cancers such as lung cancer, breast cancer, pancreatic cancer, etc.

The mesoporous silica material is a novel inorganic polymer drug carrier with ultrahigh specific surface area, large pore volume, controllable appearance and size. Compared with the traditional porous material, the material has the main characteristics that: (1) a long-range ordered structure; (2) the mesoporous aperture is uniform and adjustable; (3) the porosity is high, and the specific surface area is large; (4) better thermal stability and hydrothermal stability; (5) surface rich unsaturated groups or other groups that are easily functionalized; (6) amorphous framework composition which is easy to be doped with other components; (7) there are various morphological structures.

Hyaluronic Acid (HA) is an acidic mucopolysaccharide having the structural formula:

. Hyaluronic acid is a glycosaminoglycan widely present in the body, and plays an important role in proliferation and metastasis of cells. CD44 is the most important receptor of hyaluronic acid on cell surface, and the interaction between hyaluronic acid and its receptor makes hyaluronic acid be used as carrier of anti-malignant tumor medicine for the target treatment of such diseases. The invention patent (CN 107096034A) discloses a target nano-assembly of apigenin-carrying hyaluronic acid, and the system is prepared from hydrophilic polysaccharide hyaluronic acidThe medicine carrying system is composed of sodium alginate and hydrophobic medicine apigenin, the medicine dissolution rate is remarkably improved, and the physical stability is good; the other outstanding advantage is that the particle size is smaller, the average particle size is less than 200nm, and passive targeting can be realized by virtue of the EPR effect of a tumor part; meanwhile, the hyaluronic acid is used as a carrier, so that active targeting can be realized, the toxic and side effects are reduced, and the treatment efficiency of the antitumor drug is improved.

Disclosure of Invention

The invention aims to provide a paclitaxel/erlotinib loaded mesoporous silica-hyaluronic acid mixed drug-loaded nanoparticle with a targeted anticancer effect, which utilizes the targeting effect of hyaluronic acid on cancer cells to improve the antitumor activity of paclitaxel/erlotinib.

In order to achieve the purpose, the invention adopts the following technical scheme:

a paclitaxel/erlotinib loaded mesoporous silica-hyaluronic acid mixed targeting nanoparticle comprises the following steps:

1) dissolving Mesoporous Silica Nanoparticles (MSN) in absolute ethyl alcohol, adding 3-aminopropyltriethoxysilane with the weight of 0.4% of the weight of the nanoparticles, stirring at room temperature for 12h, centrifuging, washing with ethanol, and freeze-drying to obtain aminated modified mesoporous silica (MSN-NH)₂）；

2) Dissolving 30mg of Hyaluronic Acid (HA), 100mg of EDC and 100mg of NHS in 40mL of 0.1mol/L MES buffer solution, and stirring at room temperature for 24 hours to obtain HA-NHS; 40mg of MSN-NH₂Dispersing in 20mL ethanol and stirring for 1 hour, then adding 12mg HA-NHS, stirring for 4 hours, and synthesizing the hyaluronic acid modified mesoporous silica (MSN-HA);

3) respectively dissolving 20mg of paclitaxel and 20mg of erlotinib and 60mg of MSN-HA into 20mL of ethanol, violently stirring for 24 hours at room temperature, centrifuging to remove the solvent, washing with ethanol, and drying in a vacuum drier at-50 ℃ for 24 hours to respectively obtain mesoporous silica (PTX @ MSN-HA) loaded with paclitaxel and mesoporous silica (EL @ MSN-HA) loaded with erlotinib;

4) and uniformly mixing the PTX @ MSN-HA and the EL @ MSN-HA in a mass ratio of 1:1 to obtain the targeted nanoparticle.

The particle size of the mesoporous silica nanoparticles is 150 nm.

In view of the defects of the prior art, the invention couples hyaluronic acid to the outer surface of mesoporous silica through a covalent bond, and then loads paclitaxel/erlotinib into the inner pore canal of the mesoporous silica. This aspect improves the bioavailability of paclitaxel/erlotinib; on the other hand, the high-efficiency targeting anticancer effect of the hyaluronic acid can also improve the antitumor effect of the paclitaxel/erlotinib. Therefore, the targeted nano-particles obtained by the invention can be used for preparing targeted drugs for tumor treatment.

The invention has the advantages that:

1. according to the invention, hyaluronic acid is used as a targeting molecule, so that paclitaxel/erlotinib can be enriched around cancer cells, and the uptake rate of the cancer cells to drugs can be remarkably improved, thereby improving the anti-tumor effect of paclitaxel/erlotinib and reducing the toxic and side effects of paclitaxel/erlotinib to normal tissues;

2. the nano drug-carrying system constructed by the invention is also suitable for other anti-cancer drugs with large toxic and side effects or indissolvable properties except paclitaxel;

3. the drug-loaded system designed by the invention has simple preparation method and easily obtained materials, and is beneficial to further expanding the preparation.

Drawings

Fig. 1 is a TEM image (a) and a DLS analysis image (B) of Mesoporous Silica Nanoparticles (MSN) prepared in example 1.

FIG. 2 shows Mesoporous Silica (MSN) and mesoporous silica (MSN-NH) with aminated surface₂) And Hyaluronic Acid (HA) and hyaluronic acid modified mesoporous silica (MSN-HA).

FIG. 3 shows Mesoporous Silica (MSN) and mesoporous silica (MSN-NH) with aminated surface₂) And Zeta potential analysis chart of mesoporous silica modified by hyaluronic acid (MSN-HA).

FIG. 4 is a graph of the drug release rates of EL, PTX, EL @ MSN, PTX @ MSN and EL @ MSN-HA, PTX @ MSN-HA in PBS solution at pH5.5 (A) and pH7.4 (B).

FIG. 5 is a graph showing the results of the cell uptake assay in example 7.

FIG. 6 is a graph showing the results of the MTT test in example 8.

Detailed Description

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.

Example 1 preparation of Mesoporous Silica Nanoparticles (MSN)

After 0.1g of TEA (triethanolamine) and 20mL of deionized water were thoroughly mixed, it was transferred to a 50mL flask containing 2g of CTAC (cetyltrimethylammonium chloride), and magnetically stirred at 95 ℃ for 1 hour. 1.5mL of TEOS (tetraethyl silicate) was then added dropwise via syringe to the flask, during which time the solution gradually turned milky in color. After the addition was completed, stirring was continued for 1 hour under the same conditions, after which the solution was centrifuged at 12000rpm for 15 minutes. The resulting crude product was washed twice with 20mL of deionized water and 20mL of ethanol, then dissolved in 20mL of methanol, 2g of NaCl was added, magnetic stirring was performed at room temperature for 4 hours to remove unreacted CTAC, then the mixture was centrifuged for 15 minutes, and after the residue was washed three times with 20mL of ethanol, it was dried in a vacuum desiccator at-50 ℃ for 24 hours to obtain Mesoporous Silica Nanoparticles (MSN).

Fig. 1 is a TEM image (a) and a DLS analysis image (B) of the prepared mesoporous silica nanoparticles. As can be seen, the particle size is about 150 nm.

Example 2 MSN-NH₂Synthesis of nanoparticles

100mg of MSN was added to a 50mL flask containing 20mL of ethanol, followed by 400. mu.L of APTES (3-aminopropyltriethoxysilane) and magnetic stirring for 12 hours. After completion of the reaction, the mixture was centrifuged at 12000rpm for 15 minutes to remove the main solvent, and the residue was washed twice with 20mL of deionized water and 20mL of ethanol, respectively, to remove unreacted substances, and then dried in a-50 ℃ vacuum drier for 24 hours to obtain about 100mg of MSN-NH₂Nano meterParticles.

Example 3 Synthesis of MSN-HA nanoparticles

30mg of HA, 100mg of EDC and 100mg of NHS were dissolved in 40mL of 0.1mol/L MES buffer and stirred at room temperature for 24 hours to give HA-NHS. 40mg of MSN-NH₂After dispersing in 20mL of ethanol and stirring for 1 hour, 12mg of HA-NHS was added and stirred for 4 hours to synthesize MSN-HA.

Example 4 MSN, MSN-NH₂And characterization of MSN-HA nanoparticles

MSN, MSN-NH were measured on an FT-IR spectrometer (Bruker IFS 55, F ä llanden, Switzerland) according to the KBr precipitation technique₂Fourier transform infrared (FT-IR) spectra of HA and MSN-HA. MSN, MSN-NH were measured by Zetasizer NanoZS90 (Malvern, USA)₂And zeta potential of MSN-HA in deionized water.

FIG. 2 shows MSN and MSN-NH₂And infrared absorption spectra of HA and MSN-HA.

FIG. 3 shows MSN and MSN-NH₂And Zeta potential maps of MSN-HA. As shown in FIG. 3, the zeta potential of MSN is negative at-18.9. + -. 1.2 mV; the reverse direction is +20.2 +/-2.0 mV after amination modification; after modification of HA, the zeta potential reversed to a negative value of-26.8 ± 1.6 mV, confirming successful coupling of HA to the aminated mesoporous silica surface.

Example 5 Synthesis of EL or PTX Supported MSN-HA nanoparticles

20mg of EL and 20mg of PTX are respectively dissolved in 20mL of ethanol together with 60mg of MSN-HA, the mixture is vigorously stirred for 24 hours at room temperature, then the solvent is removed by centrifugation, and the product is obtained after washing with ethanol and drying in a vacuum drier at-50 ℃ for 24 hours, thus obtaining EL @ MSN-HA and PTX @ MSN-HA.

EXAMPLE 6 in vitro drug Release

To determine the in vitro drug release of EL and PTX from MSN-HA, 2mg of free EL, free PTX and EL @ MSN, PTX @ MSN, EL @ MSN-HA, PTX @ MSN-HA were suspended in 50mL of PBS buffer (pH 5.5 or pH 7.4) containing 40% ethanol. 1mL of the suspension was collected and centrifuged at predetermined time intervals, and the amount of PTX was measured at an ultraviolet spectrum of about 233nm and the amount of EL was measured at an ultraviolet spectrum of about 245nm, and the results are shown in FIG. 4.

As shown in fig. 4, the solubility difference between PTX and EL was not statistically significant at the same pH. Compared with the release of free PTX and EL, the MSN and MSN-HA delivery system can release PTX and EL continuously at pH7.4 and pH5.5, and the drug release efficiency of the MSN and MSN-HA delivery system at pH5.5 is faster than that at pH 7.4. PTX or EL loaded MSN-HA drug delivery systems have significantly lower percent drug release than free drug and drug loaded MSN delivery systems.

This result indicates that the HA-modified biocompatible MSN drug delivery system releases PTX or EL better in an acidic environment (such as tumor tissue) compared to neutral conditions (such as blood).

Example 7 cellular uptake

A549 cells at 2X 10⁵The density of individual cells/well was plated on 24-well plates and incubated overnight. Thereafter, the plate was washed twice with PBS, and 100. mu.g/mL of FITC @ MSN, FITC @ MSN-HA and FITC @ MSN-HA + HA were added to the cells, respectively, and cultured at 37 ℃ for 2 hours. Cells in each well were then washed three times with cold PBS and stained with Hoechst 33342 for 10 minutes. Finally, the sample was observed by confocal microscopy, and the results are shown in FIG. 5.

As can be seen in FIG. 5, FITC @ MSN-HA showed the highest fluorescence intensity, indicating the highest uptake rate by the cells. The fluorescence intensity of FITC @ MSN-HA + HA is slightly lower than that of FITC @ MSN-HA, so that HA can compete with FITC @ MSN-HA for receptors on cancer cells, and further the targeted anticancer effect of HA is proved.

Example 8 MTT assay

Separately culturing A549 cells and H1975 cells, digesting with trypsin, counting with a blood counting plate, and adjusting cell density to 1 × 10⁵Preparing cell suspension per mL; inoculating to 96-well plate at 100 μ L per well, sealing with PBS, and placing at 37 deg.C and 5% CO₂Culturing in an incubator overnight; when the cell activity reached 80%, different concentration gradients of PTX, EL, PTX + EL, PE @ MSN (EL @ MSN and PTX @ MSN mixed at a ratio of 1: 1), PE @ MSN-HA (EL @ MSN-HA and PTX @ MSN-HA mixed at a ratio of 1: 1) were added, diluted with the culture brothClosing) and culturing for 24 hours; removing culture solution, adding 100 μ L MTT solution diluted with serum-free and phenol-free medium, and culturing at 37 deg.C for 4 hr; the 96-well plate was taken out, 100. mu.L of DMSO was added after the MTT solution was aspirated, the mixture was slowly shaken on a shaker for 10 min, the OD value was measured at 570 nm with a multifunction microplate reader after shaking, and the cell proliferation inhibition rate was calculated using GraphPad Prism 5, and the results are shown in FIG. 6.

As can be seen from fig. 6, PTX and EL inhibited proliferation of a549 cells and H1975 cells in a concentration-dependent manner; when the two drugs are jointly administered, the anti-tumor effect of the two drugs can be effectively improved; compared with PTX + EL, PE @ MSN and PE @ MSN-HA can improve the anti-tumor effect of the two, and the effect of the PE @ MSN-HA is more remarkable than that of the PE @ MSN, which shows that compared with PTX or EL which is independently used, the PTX and the EL are loaded on a targeting nanoparticle carrier coupled with hyaluronic acid molecules together, and the synergistic treatment effect can be achieved.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. The mesoporous silica-hyaluronic acid mixed targeting nanoparticle loaded with paclitaxel/erlotinib is characterized in that: the preparation method comprises the following steps:

1) performing amination modification on the surface of the mesoporous silica nanoparticle;

2) coupling hyaluronic acid to the outer surface of the aminated mesoporous silica through a covalent bond.

3) Respectively loading paclitaxel and erlotinib to the inner pore channel of the mesoporous silica modified by hyaluronic acid;

4) uniformly mixing the mesoporous silica loaded with paclitaxel and the mesoporous silica loaded with erlotinib according to the mass ratio of 1: 1;

the specific operation of the step 1) is as follows: dissolving mesoporous silica nanoparticles in absolute ethyl alcohol, adding 3-aminopropyltriethoxysilane accounting for 0.4 percent of the weight of the nanoparticles, stirring at room temperature for 12 hours, centrifuging, washing with ethyl alcohol, and freeze-drying;

the specific operation of the step 2) is as follows: dissolving 30mg of hyaluronic acid, 100mg of EDC and 100mg of NHS in 40mL of 0.1mol/L MES buffer solution, and stirring at room temperature for 24 hours to obtain HA-NHS; dispersing 40mg of aminated modified mesoporous silica in 20mL of ethanol, stirring for 1 hour, adding 12mg of HA-NHS, and stirring for 4 hours to synthesize hyaluronic acid modified mesoporous silica;

the specific operation of the step 3) is as follows: respectively dissolving 20mg of paclitaxel and 20mg of erlotinib and 60mg of mesoporous silica modified by hyaluronic acid in 20mL of ethanol, violently stirring for 24 hours at room temperature, centrifuging to remove the solvent, washing with ethanol, and vacuum-drying at-50 ℃ for 24 hours to obtain mesoporous silica loaded with paclitaxel and mesoporous silica loaded with erlotinib.

2. The paclitaxel/erlotinib-loaded mesoporous silica-hyaluronic acid hybrid targeting nanoparticle according to claim 1, wherein: the particle size of the mesoporous silica nanoparticles is 150 nm.