WO2019137005A1 - Drug-loaded nanoparticle based on tannic acid and preparation method therefor and use thereof - Google Patents

Abstract

Disclosed are a drug-loaded nanoparticle based on tannic acid and a preparation method therefor. The preparation method comprises the following steps: S1. formulating a mixed organic solution of a hydrophobic micromolecular drug and tannic acid, and a polyvinylpyrrolidone or vitamin E polyethylene glycol succinate aqueous solution respectively; S2. under stirring conditions, mixing the mixed organic solution of the hydrophobic micromolecular drug and tannic acid, and the polyvinylpyrrolidone or vitamin E polyethylene glycol succinate aqueous solution in S1 uniformly to form a nanoparticle solution, and then removing the organic solvent to prepare a drug-loaded nanoparticle aqueous solution into which a lyoprotectant can be further added to prepare a lyophilized preparation of drug-loaded nanoparticles. The drug-loaded nanoparticles of the present invention have properties of small particle size, narrow dispersion, pH-responsive drug release, etc., and have greater application prospects in drug delivery, especially in oral administration.

Description

Drug-loaded nano particle based on tannic acid and preparation method and application thereof Technical field

The invention belongs to the technical field of biomedicine, and more particularly relates to a drug-loaded nanoparticle based on tannic acid and a preparation method and application thereof.

Background technique

Oral administration is simple and convenient, and the patient has high compliance. The patient can take it by himself, which greatly saves time and medical resources. After oral administration, the drug gradually enters the body through the absorption of the gastrointestinal tract, which can avoid the excessive blood concentration in the body, prolong the half-life of the drug, and improve the therapeutic effect of the drug. However, many hydrophobic small molecule drugs such as paclitaxel, curcumin, etc., have low efficiency across the small intestinal epithelial cells and have low oral bioavailability.

In order to solve the above problems, the preparation of a hydrophobic drug into an oral nano drug delivery system using a carrier material has attracted wide interest. Nanoparticles have special physicochemical properties. The use of nanosystems to transport drugs can improve stability, enhance targeting, and improve bioavailability. Based on this, polymer nanoparticles, liposomes, micelles, inorganic particles, etc. have been developed. Different nano delivery systems are used for drug delivery. Some nano-drugs such as doxil liposome (Doxil) and paclitaxel-albumin-bound nanoparticle (Abraxane) have been approved by the FDA for clinical application in cancer therapy. However, most of the anti-tumor drugs currently used in clinical practice are administered intravenously. This mode of administration causes a sharp increase in the concentration of blood drugs in the body, which greatly exceeds the concentration of the drug treatment window and causes serious side effects on the human body. Moreover, cancer patients need frequent injections during chronic treatment, which brings great inconvenience and risk of infection to patients. Therefore, the development of anti-tumor nano drugs based on oral drug delivery systems has potential applications.

The preparation technology of nanoparticles is crucial for the clinical transformation of nanomedicine. Nanoparticles conventionally obtained by intermittent preparation methods such as emulsion/solvent evaporation, bulk mixing, and stepwise dropping are generally uncontrollable in particle size and dispersibility, and poor in batch repeatability. In recent years, the use of the developed rapid nanoprecipitation technology to prepare nanoparticles has the advantages of controllable particle size, uniform size, and batch repeatability. Its main mechanism is to rely on high turbulence mixer device (for example, coaxial turbulent mixer, four-channel vortex mixer, etc.) to achieve rapid exchange of solvent (drug-containing) and non-solvent (including stabilizer), by regulating solute nucleation The growth rate controls the particle size and dispersibility of the nanoparticles.

Tannic acid is an FDA-recognized natural polyphenol with a pKa of about 8.5. It has many biological activities such as anti-oxidation, anti-bacterial, anti-tumor, free radical capture, inhibition of Pgp activity and the like. High levels of tannic acid and other plant polyphenols in food can also reduce cardiovascular disease. Polyvinylpyrrolidone and vitamin E polyethylene glycol succinate are also a class of biocompatible polymeric materials commonly used as pharmaceutical excipients. At present, there have been no reports on the preparation of drug-loaded nanoparticles for oral delivery systems using tannic acid and polyvinylpyrrolidone or vitamin E polyethylene glycol succinate as carriers.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the defects and deficiencies in the oral administration of the existing hydrophobic small molecule drugs, and to provide a carrier of tannic acid and polyvinylpyrrolidone or vitamin E polyethylene glycol succinate. A drug-loaded nanoparticle preparation loaded with a hydrophobic drug, the drug-loaded nanoparticle having small particle size, narrow dispersion, pH-responsive drug release and the like, and having a large application in drug delivery, especially in oral administration prospect.

A first object of the present invention is to provide a method for preparing a drug-loaded nanoparticle based on tannic acid.

A second object of the present invention is to provide a drug-loaded nanoparticle prepared by the above preparation method.

A third object of the invention is to provide the use of the drug-loaded nanoparticles.

The above object of the present invention is achieved by the following technical solutions:

A method for preparing drug-loaded nanoparticles based on tannic acid, which is an organic mixed solution of hydrophobic small molecule drug and tannic acid with polyvinylpyrrolidone or vitamin E polyethylene glycol amber under stirring The aqueous solution of the acid ester is mixed to form a nanoparticle solution, and then the organic solvent is removed to prepare an aqueous solution of the drug-loaded nanoparticle.

Tannic acid is a strong hydrogen bond donor material that can be used to form multiple layers of membranes, capsules and microspheres with multiple hydrogen bonds between polyvinylpyrrolidone. The present invention utilizes a stepwise dropping method, a pouring method or a rapid nanoprecipitation by interaction between a hydrophobic small molecule drug and a hydrophobic and/or hydrogen bond between tannic acid, polyvinylpyrrolidone or vitamin E polyethylene glycol succinate. The method prepares nanoparticles loaded with different drugs.

Preferably, the method of mixing is a rapid nanoprecipitation method, in particular, a four-channel eddy current is separately obtained by separately mixing a hydrophobic small molecule drug with an organic mixed solution of tannic acid and a polyvinylpyrrolidone or a vitamin E polyethylene glycol succinate aqueous solution. The first, second, and third and fourth channels of the mixer achieve high turbulent mixing to rapidly form a nanoparticle solution;

More preferably, the flow rate of the hydrophobic small molecule drug and the tannic acid organic mixed solution is from 1 to 100 mL/min (preferably 20 mL/min).

More preferably, the flow rate of the polyvinylpyrrolidone or vitamin E polyethylene glycol succinate aqueous solution is from 1 to 100 mL/min (preferably 50 mL/min).

Preferably, the organic solvent is ethanol, acetone, methanol, acetonitrile or the like (preferably ethanol or acetone).

Preferably, the method of removing the organic solvent is water dialysis or rotary evaporation.

Preferably, the tannic acid concentration is from 0.1 to 10 mg/mL (preferably 0.5 mg/mL).

Preferably, the polyvinylpyrrolidone concentration is from 0.1 to 20 mg/mL (preferably 0.7 mg/mL).

Preferably, the vitamin E polyethylene glycol succinate concentration is from 0.1 to 10 mg/mL (preferably 1.0 mg/mL).

Preferably, the polyvinylpyrrolidone has a molecular weight (M _w ) of from 1 kDa to 400 kDa.

More preferably, the polyvinylpyrrolidone has a weight average molecular weight of from 1 kDa to 10 kDa, from 10 kDa to 40 kDa (preferably 10 kDa or 40 kDa), from 40 kDa to 100 kDa, and from 100 kDa to 400 kDa.

Preferably, the hydrophobic small molecule drug is paclitaxel, curcumin, testosterone or docetaxel.

More preferably, the paclitaxel concentration is from 0.1 to 6 mg/mL (preferably 0.5 mg/mL).

More preferably, the curcumin concentration is from 0.1 to 10 mg/mL (preferably 0.5 mg/mL).

More preferably, the testosterone concentration is from 0.1 to 8 mg/mL (preferably 1.5 mg/mL).

More preferably, the docetaxel concentration is from 0.1 to 5 mg/mL (preferably 0.5 mg/mL).

The present invention also claims a tannic acid-based drug-loaded nanoparticle prepared by any of the above methods.

Preferably, the drug-loaded nanoparticles are paclitaxel nanoparticles having a particle diameter of 30 to 150 nm (preferably 55 nm), a dispersion degree of 0.04 to 0.3, an encapsulation efficiency of 70 to 85%, and a drug loading amount of 14 to 18 %.

Preferably, the drug-loaded nanoparticles are curcumin nanoparticles having a particle size of 50 to 70 nm, a dispersion of 0.09 to 0.3, an encapsulation efficiency of 90 to 96%, and a drug loading of 16 to 22%.

Preferably, the drug-loaded nanoparticles are testosterone nanoparticles having a particle diameter of 40 to 100 nm, a dispersion degree of 0.07 to 0.11, an encapsulation efficiency of 38 to 48%, and a drug loading amount of 13 to 18%.

Preferably, the drug-loaded nanoparticles are docetaxel nanoparticles having a particle size of 50-100 nm, a dispersion of 0.1-0.3, an encapsulation efficiency of about 79%, and a drug loading of 9%.

In addition, the present invention also claims the use of the tannic acid-based drug-loaded nanoparticles for the preparation of an oral hydrophobic small molecule pharmaceutical formulation.

An oral hydrophobic small molecule pharmaceutical preparation comprising the above-described tannic acid-based drug-loaded nanoparticles.

Preferably, the pharmaceutical preparation is a lyophilized preparation, in particular, a lyophilized protective agent is added to the nanoparticle solution prepared by the invention, and the lyophilized nano preparation is obtained by freezing and drying.

Preferably, the lyoprotectant is mannitol, xylitol, trehalose, sorbitol or a combination thereof.

More preferably, the lyoprotectant is a mannitol/xylitol composition wherein the ratio of mannitol mass/xylitol mass/drug-loaded nanoparticle aqueous solution volume is from 0 to 5 g/0.5 to 5 g/100 mL.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention utilizes a stepwise dropping method, a pouring method or a rapid nanoprecipitation by interaction between a hydrophobic small molecule drug and a hydrophobic and/or hydrogen bond between tannic acid, polyvinylpyrrolidone or vitamin E polyethylene glycol succinate. The method prepares nanoparticles loaded with different drugs. The drug-loaded nanoparticles obtained by the invention not only have the properties of small particle size, narrow dispersion, pH-responsive drug release, but also have a drug protection function, and only a small amount of drug is released in the gastric acid medium (pH 2), and the oral administration process can be avoided. In the small intestine environment (pH 6.8) or physiological environment (pH 7.4), the nanoparticles can release the drug in a small intestinal environment (pH 6.8) or physiological environment (pH 7.4), which is beneficial to further absorption of the drug. Compared with the traditional injection solution, the drug-loaded nanoparticles of the invention have better biocompatibility and the therapeutic effect is comparable to or better than the injection solution; the tannin-based drug-loaded nanoparticles prepared by the invention are in the drug Delivery, especially in oral administration, has great application prospects.

DRAWINGS

Figure 1 is a schematic representation of the preparation of paclitaxel nanoparticles by a four-channel vortex mixer.

Figure 2 shows the relevant parameters affecting the particle size and dispersion of paclitaxel nanoparticles. (A) fluid flow rate (Reynolds number), (B) ethanol/water ratio, (C) paclitaxel concentration, (D) carrier tannic acid concentration, (E) carrier polyvinylpyrrolidone concentration, (F) carrier aggregation Molecular weight of vinylpyrrolidone.

Figure 3 shows the relevant parameters affecting the particle size and dispersion of curcumin nanoparticles. (A) ethanol/water ratio, (B) curcumin concentration, (C) carrier tannic acid concentration, and (D) carrier polyvinylpyrrolidone concentration.

Figure 4 shows the in vitro stability of paclitaxel nanoparticles. (A) Nanoparticle 1 and Nanoparticle 2 were allowed to stand at room temperature in the dark for one week, and (B) Nanoparticle 2 was allowed to stand in a PBS buffer solution of pH 7.4 for 12 hours.

Figure 5 is the pH sensitivity of paclitaxel nanoparticles. (A) The particle size and transmittance of paclitaxel nanoparticles change with time under different pH conditions; (B) The particle size distribution of paclitaxel nanoparticles under different pH conditions. (C) Transmission electron micrograph of the initially prepared nanoparticle 2, (D) transmission electron micrograph of the nanoparticle 2 under pH 2.0, (E) transmission electron micrograph of the nanoparticle 2 under pH 6.8, (F) Transmission electron micrograph of Nanoparticle 2 under pH 7.4 conditions.

Figure 6 is a graph showing cumulative drug release profiles of paclitaxel nanoparticles under simulated pH conditions in the gastrointestinal tract.

Figure 7 shows the in vitro toxicity of polyvinylpyrrolidone, tannic acid, paclitaxel injection (Taxol) and paclitaxel nanoparticles to MCF-7 (A), HeLa (B) and HepG2 cells (C). And in vitro toxicity of polyoxyethylene castor oil/ethanol solvent corresponding to paclitaxel content to MCF-7 cells (D).

Figure 8 shows the in vitro uptake of Caco2 (A) and MCF-7 cells (B) of paclitaxel nanoparticles.

Figure 9 is a comparison of paclitaxel cumulative penetration (A) and apparent permeability coefficient (B) of paclitaxel injection (Taxol) and paclitaxel nanoparticles.

Figure 10 is a comparison of the pharmacokinetic curves of rat oral paclitaxel injection (Taxol) and paclitaxel nanoparticles.

Figure 11 is a nude mouse tumor suppression test.

Detailed ways

The invention is further described in the following with reference to the drawings and specific examples, but the examples are not intended to limit the invention. Unless otherwise indicated, the reagents, methods, and devices employed in the present invention are routine reagents, methods, and devices in the art.

The reagents and materials used in the following examples are commercially available unless otherwise stated.

Example 1 Preparation of paclitaxel-loaded tannic acid/polyvinylpyrrolidone nanoparticles (paclitaxel nanoparticles)

1, method

(1) 10 mg of paclitaxel (PTX) and 10 mg of tannic acid (TA) were weighed and dissolved in 20 mL of ethanol, and 25 mg of polyvinylpyrrolidone (PVP) was dissolved in 50 mL of deionized water. The paclitaxel/tannic acid ethanol solution was added to the aqueous solution of polyvinylpyrrolidone by a stepwise dropping or pouring method under stirring, and after stirring for 30 minutes, the prepared nanoparticle solution was dialyzed against water using a dialysis bag (molecular weight cut off, 3.5 kDa). An aqueous solution of paclitaxel nanoparticles is obtained.

(2) The particle size and dispersion of the nanoparticles were characterized by Malvern particle size analyzer; the encapsulation efficiency and drug loading of paclitaxel were determined by high performance liquid chromatography (HPLC): the detection wavelength was 227 nm, and the mobile phase was acetonitrile/water. (volume ratio: 40/60), the flow rate was 1 mL/min. The encapsulation efficiency and drug loading amount of the drug-loaded nanoparticles are calculated as follows: encapsulation efficiency (%) = drug quality of drug-loaded nanoparticles / total drug quality × 100%; drug loading (%) = drug loading The mass of the drug contained in the nanoparticles / the mass of the drug-loaded nanoparticles × 100%.

2, the results

As shown in Table 1, the paclitaxel nanoparticles prepared by the stepwise dropping method had a particle diameter of 39 nm, an encapsulation efficiency of 66.8%, and a drug loading of 14.8%. The paclitaxel nanoparticles prepared by the pouring method had a particle diameter of 47 nm, an encapsulation efficiency of 73.9%, and a drug loading of 16.4%. As shown in the results of Table 1, the dispersion of the paclitaxel nanoparticles prepared by the stepwise dropping or pouring method is usually large.

Table 1 Comparison of Preparation of Paclitaxel Nanoparticles by Stepwise Addition and Pour Method

Example 2 Preparation of paclitaxel-loaded tannic acid/polyvinylpyrrolidone nanoparticles

Figure 1 shows a four-channel vortex mixer structure for the preparation of drug-loaded tannic acid/polyvinylpyrrolidone nanoparticles by rapid nanoprecipitation, wherein the device detailed parameters are described in the inventor's prior application number PCT/US2017/014080 in.

1, method

(1) 10 mg of paclitaxel and 10 mg of tannic acid were weighed and dissolved in 20 mL of ethanol, and 25 mg of polyvinylpyrrolidone was dissolved in 50 mL of deionized water. The paclitaxel/tannic acid ethanol solution was separately injected into the first and second channels, and the polyvinylpyrrolidone aqueous solution was injected into the third and fourth channels. The flow rate of each channel fluid was controlled by the thrust of the syringe pump, wherein the fluid velocity of the first and second channels was 20 mL/min, and the fluid velocity of the third and fourth channels was 50 mL/min. The prepared drug-loaded nanoparticle solution was collected and dialyzed against water (molecular weight cut off, 3.5 kDa) using a dialysis bag to obtain an aqueous solution of paclitaxel nanoparticles.

2, the results

As shown in Table 2, when the concentration of tannic acid and polyvinylpyrrolidone was 0.5 mg/mL, respectively, the particle diameter of the blank nanoparticles was 36 nm. When paclitaxel having an initial concentration of 0.5 mg/mL was used, the obtained paclitaxel nanoparticles had a particle diameter of 35 nm, and the encapsulation efficiency and drug loading amount were 74.5% and 16.6%, respectively. When the polyvinylpyrrolidone concentration was increased to 0.7 mg/mL, the obtained paclitaxel nanoparticles had a particle diameter of 55 nm, and the encapsulation efficiency and drug loading amount were 80.0% and 14.7%, respectively. In addition, changing the fluid flow rate (Reynolds number), ethanol/water ratio, drug concentration, carrier tannic acid concentration, carrier polyvinylpyrrolidone concentration or molecular weight can regulate the particle size and dispersion of paclitaxel nanoparticles.

Figure 2A shows that the fluid flow rate (Reynolds number) has less effect on the particle size of the drug-loaded nanoparticles, but at high Reynolds numbers, the drug-loaded nanoparticles have a smaller dispersion.

Figure 2B shows that when the ethanol/water ratio was adjusted from 5:5 to 2:5, the particle size of the obtained paclitaxel nanoparticles was slightly reduced, but the dispersion did not change significantly.

Figures 2C, D and E respectively examine the effects of paclitaxel concentration, carrier tannic acid and polyvinylpyrrolidone concentration on the particle size and dispersion of paclitaxel nanoparticles, and the results show an increase in paclitaxel, tannic acid, polyvinylpyrrolidone concentration, paclitaxel nanometers. The particle size of the particles will increase, but the dispersion is small.

Figure 2F examines the effect of polyvinylpyrrolidone molecular weight on paclitaxel nanoparticles. The results show that the paclitaxel nanoparticles have a smaller particle size and dispersion when the molecular weight of the polyvinylpyrrolidone is 10 kDa or 40 kDa.

Table 2 Preparation of different components of paclitaxel nanoparticles by rapid nanoprecipitation

Example 3 Preparation of Curcumin-Containing Tannic Acid/Polyvinyl Pyrrolidone Nanoparticles

1, method

10 mg of curcumin (Cur) and 10 mg of tannic acid (TA) were weighed and dissolved in 20 mL of ethanol, and 25 mg of polyvinylpyrrolidone (PVP) was dissolved in 50 mL of deionized water. The first and second channels of the four-channel vortex mixer shown in Fig. 1 were injected with a curcumin/tannic acid ethanol solution, and the third and fourth channels were injected with an aqueous solution of polyvinylpyrrolidone. The fluid velocity of the first and second channels was controlled to be 20 mL/min, and the fluid velocity of the third and fourth channels was 50 mL/min. The collected drug-loaded nanoparticle solution was dialyzed against water through a dialysis bag (molecular weight cutoff, 3.5 kDa) to obtain an aqueous solution of curcumin nanoparticles.

2, the results

As shown in Table 3, the curcumin drug and the carrier tannic acid and polyvinylpyrrolidone can form curcumin nanoparticles by rapid nanoprecipitation. The particle size of the drug-loaded nanoparticles is 50-70 nm, and the dispersion is less than 0.17. The rate is higher than 90% and the drug loading is higher than 16%.

The results in Figure 3 show that changing the ethanol/water ratio, curcumin concentration, carrier tannic acid and polyvinylpyrrolidone concentration during particle preparation can modulate the particle size and dispersion of curcumin nanoparticles.

Table 3 Preparation of different compositions of curcumin nanoparticles by rapid nanoprecipitation

Example 4 Preparation of testosterone-loaded tannic acid/polyvinylpyrrolidone nanoparticles (testosterone nanoparticles)

1, method

30 mg of testosterone (Tes) and 30 mg of tannic acid (TA) were co-dissolved in 20 mL of ethanol, and 35 mg of polyvinylpyrrolidone (PVP) was dissolved in 50 mL of deionized water. The first channel of the four-channel vortex mixer shown in Figure 1 is a testosterone/tannic acid ethanol solution, and the second, third and fourth channels are aqueous solutions of polyvinylpyrrolidone, and the fluid velocity of the first channel is adjusted to 10 mL/min, the second The 3 and 4 channels have a fluid velocity of 10 mL/min. The prepared drug-loaded nanoparticle solution was collected and dialyzed against water using a dialysis bag (molecular weight cutoff, 3.5 kDa) to obtain an aqueous testosterone nanoparticle solution.

2, the results

The results in Table 4 show that the fixed testosterone concentration is 1.5 mg/mL, the carrier tannic acid concentration is changed to 1.5-2.5 mg/mL, the carrier polyvinylpyrrolidone concentration is 0.7-1.0 mg/mL, and the prepared testosterone nanoparticles are prepared. The range is from 40 to 53 nm, the dispersion is less than 0.11, the encapsulation efficiency of the drug-loaded nanoparticles is 38 to 48%, and the drug loading is 13 to 18%.

Table 4 Preparation of different components of testosterone nanoparticles by rapid nanoprecipitation

Example 5 Preparation of Docetaxel-Containing Tannic Acid/Vitamin E Polyethylene Glycol Succinate Nanoparticles

1, method

10 mg of docetaxel (DTX) and 20 mg of tannic acid (TA) were weighed into 20 mL of ethanol, and 50 mg of Vitamin E polyethylene glycol succinate (TPGS) was dissolved in 50 mL of deionized water. The first channel of the four-channel vortex mixer shown in Fig. 1 was injected with a docetaxel/tannic acid ethanol solution, and the second, third, and fourth channels were filled with a vitamin E polyethylene glycol succinate aqueous solution. The fluid velocity of channels 1, 2, 3 and 4 was controlled to be 20 mL/min. The collected nanoparticle solution was dialyzed against water through a dialysis bag (molecular weight cutoff, 3.5 kDa) to obtain an aqueous solution of docetaxel nanoparticles.

2, the results

As shown in Table 5, docetaxel and carrier tannic acid, vitamin E polyethylene glycol succinate (TPGS) can form docetaxel nanoparticles and particle size of drug-loaded nanoparticles by rapid nanoprecipitation. About 72 nm, the dispersion is less than 0.1, the encapsulation efficiency is about 79%, and the drug loading is 9%.

Table 5 Docetaxel nanoparticles prepared by rapid nanoprecipitation

Example 6 Lyophilized preparation of paclitaxel nanoparticles

1, method

Adding mannitol, xylitol, trehalose, sorbitol or different compositions thereof to the paclitaxel nanoparticle aqueous solution, mixing and homogenizing, freezing with liquid nitrogen for 10 min, then at -30 ° C, 0.37 bar vacuum Drying for 48 hours gave a lyophilized nanoformulation.

2, the results

After experimental screening, the optimal lyoprotectant for aqueous solution of paclitaxel nanoparticles is a mannitol/xylitol composition. The optimum ratio of mannitol mass/xylitol mass/loaded drug nanoparticle aqueous solution volume is 2g/2g/100mL. Table 6 shows the performance comparison results of the nanoparticle 1 or the nanoparticle 2 in Table 2 before and after reconstitution of the lyophilized preparation under the above optimal lyophilization conditions, compared with the lyophilized pre-loaded nanoparticle, freeze-dried complex The particle size and dispersion of the dissolved nanoparticles 1 or 2 were slightly increased.

Table 6 freeze-drying conditions of mannitol mass / xylitol mass / drug-loaded nanoparticle aqueous solution volume ratio of 2g / 2g / 100mL, the comparison of particle size and dispersion of the obtained paclitaxel nanoparticles before and after lyophilization

Example 7 In vitro stability of paclitaxel nanoparticles

1, method

The nanoparticles 1 and 2 prepared in Table 2 were each allowed to stand at room temperature in the dark for one week, or the nanoparticles 2 prepared in Table 2 were placed in a PBS buffer solution of pH 7.4 for 12 hours. In the set time, the particle size change of paclitaxel nanoparticles was studied.

2, the results

The results of Figures 4A and B show that the paclitaxel nanoparticles have no significant change in particle size after standing for a period of time in a PBS buffer solution protected from light or pH 7.4 in vitro, and thus the paclitaxel nanoparticles have good in vitro stability.

Example 8 pH Sensitivity of Paclitaxel Nanoparticles

1, method

The particle size and transmittance (UV-Vis spectrometer, 500 nm) of the nanoparticles 2 prepared in Table 2 were examined as a function of time under different pH conditions. After 10 mL of paclitaxel nanoparticles were adjusted to pH 2.0 with a hydrochloric acid solution, the change in particle size and transmittance of the drug-loaded nanoparticles was observed within 2 hours. The pH was then adjusted to 6.8 with a NaOH solution, and the change in particle size and transmittance of the drug-loaded nanoparticles within 5 hours was further observed. Finally, the pH was adjusted to 7.4 with a NaOH solution, and it was further observed that the particle size and transmittance of the drug-loaded nanoparticles changed with time within 5 hours. The morphology of paclitaxel nanoparticles at different pH conditions was observed by transmission electron microscopy.

2, the results

As shown in FIG. 5A, paclitaxel nanoparticles (Nanoparticle 2 prepared in Table 2) had an initial particle diameter of 55 nm, and when pH = 2.0, the transmittance of the paclitaxel nanoparticle solution was decreased, and the particle diameter was increased to about 2 μm. When pH = 6.8, the transmittance of the paclitaxel nanoparticle solution increased sharply, and the particle size decreased to about 80 nm. When the pH was further increased to 7.4, the transmittance of the paclitaxel nanoparticle solution increased slightly, and the particle size decreased to about 65 nm. The above results indicate that the prepared paclitaxel nanoparticles have a pH-sensitive particle size change behavior. The results of dynamic light scattering (Fig. 5B) and transmission electron microscopy (Fig. 5C-F) further confirmed the pH sensitivity of the paclitaxel nanoparticle size.

Example 9 In vitro drug release of paclitaxel nanoparticles

1, method

1 mL of paclitaxel nanoparticles were added to a dialysis bag with a molecular weight cut-off of 14 kDa and dialyzed against 40 mL of different media at an oscillation rate of 100 rpm and a temperature of 37 °C. Among them, the medium for simulating gastric acid was pH 2.0 (7 mL HCl, 2.5 g SDS, 2 g NaCl volume to 1 L). The medium simulating the small intestine was pH 6.8 PBS (containing 0.1% Tween 80). The medium simulating the physiological environment was pH 7.4 PBS (containing 0.1% Tween 80). During the preset time, remove 5 mL of the release solution and add an equal amount of fresh medium. The content of paclitaxel in the release solution was measured by HPLC.

2, the results

Figure 6 shows that paclitaxel nanoparticles release only a small amount of drug at pH 2. The drug-loaded nanoparticles can release the paclitaxel drug faster at pH 7.4, with about 30% released within 2 hours and about 70% released within 24 hours. We also compared the in vitro release of paclitaxel injection (Taxol) and paclitaxel nanoparticles. At pH 2, paclitaxel injection released up to 55% in 2 hours, while paclitaxel nanoparticles released less than 10%. Subsequent changes to pH 6.8 allow paclitaxel nanoparticles to release the drug more slowly than paclitaxel injection. The above indicates that paclitaxel nanoparticles release only a small amount of drug in the gastric acid mimic medium (pH 2), and the nanoparticles have a drug protective function, which can avoid gastric damage caused by the drug itself during oral administration. In the small intestine simulated environment (pH 6.8) or physiological environment (pH 7.4), paclitaxel nanoparticles can release the drug, which is beneficial to the further absorption of the drug.

Example 10 In vitro cytotoxicity of paclitaxel nanoparticles

1, method

The in vitro cytotoxicity of drug-loaded nanoparticles was evaluated using MTT. MCF-7, HepG2 and HeLa cells were added to a 96-well plate at a cell density of 5×10 ³ cells/well. After 24 hours of cell culture, 200 μL of paclitaxel injection (Taxol) containing different amounts of paclitaxel and paclitaxel nanoparticles were obtained. The original medium was replaced with a paclitaxel amount of tannic acid and polyvinylpyrrolidone concentration, and a complete medium of 50% polyoxyethylene castor oil/ethanol solvent. After 48 hours of co-incubation, the viability of the corresponding cells was detected using MTT reagent.

2, the results

Figure 7 shows the in vitro toxicity of the carriers polyvinylpyrrolidone and tannic acid, paclitaxel injection (Taxol) and paclitaxel nanoparticles to MCF-7 (A), HeLa (B) and HepG2 cells (C), respectively, and the corresponding amount of paclitaxel. Toxicity of polyoxyethylene castor oil/ethanol solvent to MCF-7 cells (D). The results indicate that the carrier polyvinylpyrrolidone and tannic acid have better biocompatibility relative to the polyoxyethylene castor oil/ethanol contained in the paclitaxel injection (Taxol). Moreover, paclitaxel nanoparticles have a similar ability to kill tumor cells as paclitaxel injection (Taxol).

Example 11 In vitro cellular uptake of paclitaxel nanoparticles

1, method

MCF-7 and Caco2 cells were cultured in a 12-well plate at a cell density of 1×10 ⁵ cells/well. After incubation at 37 ° C for 24 h, 1 mL of paclitaxel injection (Taxol) and paclitaxel containing 10 μg/mL paclitaxel were taken. The complete medium of the granules was replaced with the original culture medium, and after incubation for 1, 2, and 4 hours, the residual drug was removed by washing three times with PBS, then trypsin digestion for 5 minutes, 0.8 mL of PBS was added to disperse the cells, and the cell density was calculated by a cell counter. 0.5 mL of the dispersion was added to 2 mL of methanol, and after centrifugation for 2 minutes, the mixture was centrifuged at 10,000 rpm for 10 minutes, and 20 μL of the supernatant was taken for HPLC to determine the drug concentration of paclitaxel.

2, the results

Figure 8 shows the drug uptake of paclitaxel injection (Taxol) and paclitaxel nanoparticles co-incubated with Caco2 (A) and MCF-7 cells (B) for different times. The results showed that the paclitaxel nanoparticles were more uptaken by cells than the paclitaxel injection (Taxol), regardless of whether they were Caco2 or MCF-7 cells at the same time. It is indicated that the nanoparticles are more favorable for the uptake of paclitaxel drugs by MCF-7 and Caco2 cells.

Example 12 Cumulative Permeability and Apparent Permeability Coefficient of Paclitaxel Nanoparticles

1, method

Caco2 cells were cultured in a 12-well Transwell chamber (pore size: 0.4 μm, inner chamber area: 1.14 cm ² ), and the cell density was 1 × 10 ⁵ /well. The culture medium was changed every two days in the first week, and then the culture medium was changed once a day. The transmembrane resistance (TEER) was measured using Millicells-2, and after 2 to 3 weeks of culture, when the TEER value of the Caco2 monolayer cell membrane exceeded 800 Ωcm ² Follow-up experiments. The Transwell chamber and substrate culture medium were replaced with 0.5 and 1.5 mL HBSS balanced salt solution, respectively. After 30 min incubation, the fresh HBSS balanced salt solution was replaced and contained 10 μg/mL paclitaxel amount of Taxol injection (Taxol) and paclitaxel nanoparticles. After 0.5, 1, 1.5, 2 and 3 hours of incubation, 0.5 mL of medium was taken from the substrate and supplemented with an equal amount of fresh medium. 0.5 mL of methanol was added to the taken-out medium, and after vortexing for 1 minute, it was centrifuged at 10,000 rpm for 10 minutes, and 20 μL of the supernatant was taken to measure the paclitaxel concentration by HPLC. The apparent permeability coefficient (Papp) is calculated according to the following formula: Papp = Q / AC ₀ t, Q is the total amount of paclitaxel accumulated in permeation, t is the permeation time, A is the osmotic membrane area of the cell culture, and C ₀ is the addition of paclitaxel. The initial concentration.

2, the results

Figure 9A shows that the cumulative penetration of paclitaxel nanoparticles is higher than that of paclitaxel injection (Taxol) at the same time, and the display of Figure 9B indicates that the apparent permeability coefficient of paclitaxel nanoparticles is higher than that of paclitaxel injection (Taxol). The above results indicate that the nanoparticles are more favorable for paclitaxel to permeate through the Caco2 monolayer cell membrane.

Example 13 Pharmacokinetic Evaluation of Oral Paclitaxel Nanoparticles in SD Rats

1, method

Male Sprague-Dawley rats (180-200 g) were randomly divided into two groups after fasting for 12 hours, with 5 rats in each group. The first group received paclitaxel injection (Taxol), the second group received paclitaxel nanoparticles, and the oral paclitaxel dose was 10 mg/kg. 0.5 mL of blood was taken from the orbital vein of the rat at 0.25, 0.5, 1, 2, 4, 6, 12, 24, and 36 hours after gavage, and placed in a centrifuge tube containing sodium heparin (10 μL, 10 mg/mL) at 3000 rpm. The plasma was obtained by centrifugation for 10 minutes. Take 125 μL of plasma, add 25 μL of internal standard docetaxel (13 μg/mL) for 1 minute, add 500 μL of methanol to vortex for 5 minutes to precipitate protein, then centrifuge at 12000 rpm for 10 minutes, and take 20 μL of supernatant to determine the concentration of paclitaxel in plasma by HPLC. .

2, the results

As shown in Fig. 10, paclitaxel nanoparticles have a higher blood concentration than paclitaxel injection (Taxol) when the paclitaxel oral dose is 10 mg/kg. The statistical results in Table 7 show that the highest plasma concentration of paclitaxel nanoparticles is about 2 times that of paclitaxel injection, and the oral bioavailability of paclitaxel nanoparticles is 5.8 times that of paclitaxel injection (Taxol).

Table 7 Comparison of pharmacokinetic correlation indices between oral administration of paclitaxel injection (Taxol) and paclitaxel nanoparticles in SD rats

parameter	Taxol	Paclitaxel nanoparticles
C max (ng/mL)	85.6±13.1	164.7±45.7
T max (h)	1	2
AUC 0-t (ng/mL/h)	519.1±116.1	2689.3±570.6
AUC 0-∞ (ng/mL/h)	549.3±121.7	3179.5±723.2
T 1/2 (h)	9.0±0.4	13.2±1.1

MRT(h)

11.6±0.1

20.0±0.8

Example 14 nude mice tumor suppression test

1, method

Nude mice inoculated with MCF-7 cells were used to evaluate in vivo anti-tumor experiments of paclitaxel nanoparticles. When the tumors of nude mice reached 100 mm ³ , they were randomly divided into 6 groups, 6 rats in each group, respectively, orally administered with normal saline, oral Taxol (10 mg/kg), oral paclitaxel nanoparticles (10 mg/kg), and oral paclitaxel nanoparticles (20 mg/kg). ), intravenous Taxol (10 mg/kg) and intravenous paclitaxel nanoparticles (10 mg/kg). Each group of nude mice was given a drug twice a day for a total of 6 times during the test period, and the tumor volume of each nude mouse was recorded.

2, the results

As shown in Fig. 11, oral paclitaxel nanoparticles (10 mg/kg) exhibited a tumor suppressing effect comparable to intravenous Taxol (10 mg/kg), and produced a significant difference from the control group and oral Taxol (10 mg/kg). When the oral administration amount of paclitaxel nanoparticles is increased to 20 mg/kg, it can exert a stronger tumor suppressing effect.

Claims (10)

1. A method for preparing drug-loaded nanoparticles based on tannic acid, characterized in that a hydrophobic small molecule drug and an organic mixed solution of tannic acid, polyvinylpyrrolidone or vitamin E polyethylene glycol succinic acid are stirred under stirring conditions The aqueous solution of the ester is mixed to form a nanoparticle solution, and then the organic solvent is removed to prepare an aqueous solution of the drug-loaded nanoparticle.
2. The method according to claim 1, wherein the hydrophobic small molecule drug concentration is 0.1 to 10 mg/mL, the tannic acid concentration is 0.1 to 10 mg/mL, and the polyvinylpyrrolidone concentration is 0.1 to 20 mg/mL. The concentration of vitamin E polyethylene glycol succinate is 0.1 to 10 mg/mL.
3. The method according to claim 1, wherein the polyvinylpyrrolidone has a molecular weight of from 1 kDa to 400 kDa.
4. The method according to claim 1, wherein the method of mixing is a stepwise dropping method, a pouring method or a rapid nanoprecipitation method.
5. The method according to claim 4, wherein the method of mixing is a rapid nanoprecipitation method, and the flow rate of the hydrophobic small molecule drug and the tannic acid organic mixed solution is 1 to 100 mL/min; The flow rate of the pyrrolidone or vitamin E polyethylene glycol succinate aqueous solution is from 1 to 100 mL/min.
6. The method of claim 1 wherein the hydrophobic small molecule drug is paclitaxel, curcumin, testosterone or docetaxel.
7. The tannic acid-based drug-loaded nanoparticle prepared by the method according to any one of claims 1 to 6.
8. An oral hydrophobic small molecule pharmaceutical preparation comprising the tannic acid-based drug-loaded nanoparticle of claim 7.
9. The oral pharmaceutical preparation according to claim 8, wherein the pharmaceutical preparation is a lyophilized preparation.
10. The oral pharmaceutical preparation according to claim 8, wherein the lyoprotectant of the preparation is a mannitol/xylitol composition, a mass volume of the mannitol/xylitol composition and the drug-loaded nanoparticle aqueous solution. The ratio is 0 to 5 g: 0.5 to 5 g: 100 mL.

Images