PTS-loaded mPEG-b-PLGA nano drug delivery system and preparation method thereof

Abstract

The invention provides a PTS-loaded mPEG-b-PLGA nano drug delivery system which is prepared by taking PTS as a drug loading object and mPEG-b-PLGA as a carrier material through a multiple emulsion evaporation method. According to the invention, mPEG-b-PLGA is taken as a carrier material, PTS is loaded, and the prepared nano drug delivery system has better stability and bioavailability.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to an mPEG-b-PLGA nano drug delivery system loaded with PTS and a preparation method thereof. Background Art

[0002] The modernization of traditional Chinese medicine (TCM) preparations is an important way and method to achieve the modernization of TCM, and is a key link connecting TCM research and development, production and clinical practice. With the progress of science and technology in various fields, the research and application of TCM dosage forms have developed from simple mixing, granulation and tableting to highly complex new drug delivery systems, among which nano drug delivery is an important development direction for the improvement of the pharmaceutics of active ingredients of TCM.

[0003] Most of the active ingredients in TCM extracts, such as terpenoids, flavonoids, tannins, etc., have high water solubility. However, due to their inability to cross the cell lipid membrane and their large molecular weight, their intestinal absorption is poor and their bioavailability is low, so it is difficult to ensure the drug efficacy. Using a variety of nano drug delivery technologies including polymer NPs is a good solution to these problems. Such NPs are a colloidal system made of biodegradable and biocompatible polymers, and are used as drug carriers to control the release and targeting of active ingredients.

[0004] The amphiphilic block copolymer mPEG-b-PLGA is polymerized from the high molecular weight polymer PLGA, which is safe, non-toxic, biocompatible, has good film-forming ability and good drug encapsulation ability, and mPEG capped with methoxy. Due to the addition of the strongly hydrophilic mPEG molecule, mPEG-b-PLGA becomes an amphiphilic molecule with hydrophilic and hydrophobic ends at both ends, thereby increasing the drug loading capacity for water-soluble drugs, and having the advantages of high safety, long in vivo circulation time, inhibiting P-gp efflux function, avoiding RES clearance, etc. It is an ideal polymer nano carrier and has good industrialization potential.

[0005] Previous oral absorption studies on PTS mainly focused on its monomer components, concentrating on ginsenoside Rg1, followed by notoginsenoside R1. Relevant literature shows that PTS has good water solubility, and the solubility and dissolution rate of each component are less affected by other factors. However, ginsenoside Rg1 and notoginsenoside R1 will be destroyed by gastric acid and are relatively stable in the weakly alkaline environment of the intestine. After oral administration, each component is unstable in the stomach and large intestine but basically stable in the small intestine, indicating that the small intestine participates in the uptake of each component as the main absorption site; each component can be absorbed in the form of passive diffusion, with poor membrane permeability, not affected by the metabolic enzymes and efflux carriers in the intestinal mucosa, and shows a first-order kinetic process within a certain range, that is, the absorption rate is linearly related to the dosage, and at the same time, it further verifies that the absorption of ginsenoside Rg1 and notoginsenoside R1 shows a passive diffusion effect. Small intestinal P-glycoprotein (P-gp) is mainly present in the small intestinal mucosal epithelial cells and participates in the absorption of ginsenoside Rg1 and notoginsenoside R1. As a substrate of P-gp, it participates in extracellular efflux, pumping the two components inside the cell back into the intestinal lumen, reducing the absorption amount, which is one of the important reasons for the low oral bioavailability of PTS.

[0006] By preparing NPs loaded with PTS, its membrane transport mechanism can be changed, the transmembrane absorption of the drug can be enhanced, the drug can be protected from the efflux of P-gp, and the local retention of the drug may also be enhanced to achieve a sustained-release effect. However, although the triol-type ginsenoside components represented by ginsenoside Rg1 have certain solubility in both the aqueous and oil phases, their oil-water partition coefficients are relatively low, and it is difficult to form capsules or films for preparing drug delivery systems. For example, it is difficult to ensure the encapsulation efficiency when preparing liposomes, and there are great difficulties in the drug utilization efficiency. Therefore, there is currently no research report on NDDS targeting PTS. As a representative drug of the extract of effective parts of traditional Chinese medicine, the main pharmacodynamic components of PTS are clear and its purity can be further improved. How to improve its oral bioavailability by using nanodrug delivery technology is a technical problem that needs to be solved urgently. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide an mPEG-b-PLGA nanodrug delivery system loaded with PTS and its preparation method, which improves the loading amount of PTS and the prepared nanodrug delivery system has higher bioavailability.

[0008] The present invention provides an mPEG-b-PLGA nanodrug delivery system loaded with PTS, using PTS as the drug-loading object and mPEG-b-PLGA as the carrier material, which is prepared by the double emulsion evaporation method.

[0009] To solve the problems of difficult PTS loading and low loading amount, the present invention performs the following pretreatment on PTS:

[0010] Purify PTS through a weakly basic ion exchange resin.

[0011] Preferably, the weakly basic ion exchange resin is selected from D301 resin.

[0012] In some specific embodiments of the present invention, the D301 resin is D301T resin or D301R resin.

[0013] The pretreatment is preferably specifically to dissolve PTS powder in pure water, add it into a resin column, and elute it with acidic water having a pH of 2 - 6, preferably pH = 4. The dosage of the acidic water is preferably 1 - 3 times the column volume, more preferably 2 times the column volume.

[0014] The present invention has no special limitation on the above acidic water, and it can be an aqueous solution whose pH value is adjusted by a general acidic compound. The acidic compound includes but is not limited to hydrochloric acid, etc.

[0015] The present invention further refines PTS by adopting ion exchange resin technology on the basis of existing PTS. PTS is a protopanaxatriol - type dammarane tetracyclic triterpenoid saponin, mostly neutral or weakly acidic, without charge or with a weak positive charge. The pH range of the weakly basic anion exchange resin is 1 - 9. If the pH environment for its adsorption and elution is set to weakly acidic or neutral, the protopanaxatriol - type saponins will be eluted due to no charge or weak positive charge, while other components such as protopanaxadiol saponins, flavonoids, polysaccharides, amino acids, etc. will be adsorbed due to the anionic groups contained in their molecules.

[0016] The purified PTS components include ginsenoside Rg1, ginsenoside Re, and ginsenoside R1.

[0017] Optionally, the purified PTS components include: 60wt% - 75wt% of ginsenoside Rg1, 5wt% - 10wt% of ginsenoside Re, and 5wt% - 12wt% of ginsenoside R1.

[0018] Detected according to the PTS content determination method included in the Chinese Pharmacopoeia (2020 Edition), the sum of the contents of the three components after elution is 91.11%, among which the content of ginsenoside Rg1 is 72.04%, the content of ginsenoside Re is 9.59%, and the content of notoginsenoside R1 is 9.48%.

[0019] The present invention improves the loading amount of PTS by pretreating PTS. Calculated with ginsenoside Rg1 as the index component, the loading amount of ginsenoside Rg1 can reach 66.92% ± 0.94%.

[0020] The present invention also provides a preparation method of the above - mentioned mPEG - b - PLGA nano - drug delivery system loaded with PTS, including the following steps:

[0021] S1) Pretreatment of PTS

[0022] S2) Prepare the mPEG-b-PLGA nano drug delivery system loaded with PTS by the double emulsion-solvent evaporation method.

[0023] Preferably, the step S2) is specifically as follows:

[0024] A) Mix the pretreated aqueous solution of PTS and the mPEG-b-PLGA solution, and perform ultrasonic emulsification with an ultrasonic cell disruptor under an ice-water bath condition to obtain a W / O type primary emulsion.

[0025] B) After the above primary emulsion is stabilized, add it to the aqueous solution of the emulsifier for ultrasonic emulsification, and separate to obtain the mPEG-b-PLGA nano drug delivery system loaded with PTS.

[0026] In the present invention, the pretreated aqueous solution of PTS is used as the inner aqueous phase. In the aqueous solution, the concentration of the pretreated PTS is preferably 25-100 mg / mL. In some specific embodiments of the present invention, the concentration of PTS is 50 mg / mL.

[0027] The mPEG-b-PLGA solution is used as the oil phase. The solvent of the mPEG-b-PLGA solution is preferably dichloromethane, and the concentration of mPEG-b-PLGA is preferably 25-100 mg / mL. In some specific embodiments of the present invention, the concentration of mPEG-b-PLGA is 50 mg / mL.

[0028] The volume ratio of the pretreated aqueous solution of PTS to the mPEG-b-PLGA solution is preferably 1:(3-8). In some specific embodiments of the present invention, the volume ratio is 1:5.

[0029] After mixing evenly, perform ultrasonic emulsification with an ultrasonic cell disruptor under an ice-water bath condition to obtain a W / O type primary emulsion. The power of the ultrasonic cell disruptor is preferably 100-200 W. In some specific embodiments of the present invention, the power is 150 W. The time of ultrasonic emulsification is preferably 1-2 min. In some specific embodiments of the present invention, the time is 1 min.

[0030] After the above primary emulsion is stabilized, use the aqueous solution of the emulsifier as the outer aqueous phase, and add it to the aqueous solution of the emulsifier for ultrasonic emulsification.

[0031] The emulsifier is preferably polyvinyl alcohol (PVA).

[0032] The concentration of the aqueous solution of the emulsifier is preferably 1%-3% (w / v). In some specific embodiments of the present invention, the concentration is 2% (w / v).

[0033] The power of the phacoemulsification is preferably 40 - 80 W. In some specific embodiments of the present invention, the power is 60 W; the time of the phacoemulsification is preferably 1 - 2 min. In some specific embodiments of the present invention, the time is 1 min.

[0034] After the phacoemulsification, it is preferably further included to stir and volatilize the obtained multiple emulsion in an aqueous solution of an emulsifier to remove the solvent, so that the polymer carrier is solidified to form a PTS-NPs suspension.

[0035] The emulsifier is preferably polyvinyl alcohol (PVA).

[0036] The concentration of the aqueous solution of the emulsifier is preferably 0.1% - 0.3% (w / v). In some specific embodiments of the present invention, the concentration is 0.2% (w / v).

[0037] Then, separation is carried out to obtain the mPEG-b-PLGA nano drug delivery system loaded with PTS.

[0038] Compared with the prior art, the present invention provides an mPEG-b-PLGA nano drug delivery system loaded with PTS, with PTS as the drug-loading object and mPEG-b-PLGA as the carrier material, which is prepared by the multiple emulsion evaporation method. The present invention uses mPEG-b-PLGA as the carrier material to load PTS, and the prepared nano drug delivery system has better stability and bioavailability. Description of the Drawings

[0039] Figure 1 For the FT-IR (a) and 1 1H-NMR (b) characterization results of the copolymer mPEG-b-PLGA;

[0040] Figure 2 For the HPLC detection spectrum of the eluate of D301T resin in Example 2;

[0041] Figure 3 For the HPLC detection spectrum of the eluate of D392 resin in Example 2;

[0042] Figure 4 For the HPLC detection spectrum of the eluate of D296R resin in Example 2;

[0043] Figure 5 For the HPLC detection spectrum of the eluate of D301R resin in Example 2;

[0044] Figure 6 For the elution curve of D301T resin in Example 2;

[0045] Figure 7 For the elution curve of D296R resin in Example 2;

[0046] Figure 8 It is the elution curve of D301R resin in Example 2;

[0047] Figure 9 It is the elution curve of D301T resin in Example 3;

[0048] Figure 10 It is the elution curve of D301R resin in Example 3;

[0049] Figure 11 It is the characterization result of PTS-NPs. (a) is the scanning electron microscopy image, (b) is the XRD characteristic spectrum, (c) is the infrared characteristic spectrum, (d) is the particle size distribution diagram, and (e) is the potential test spectrum;

[0050] Figure 12 It is the in vitro drug release curve of PTS-NPs;

[0051] Figure 13 It is the specificity investigation spectrum of the LC-MS detection method. a is the blank plasma spectrum of rats, b is the blank plasma spectrum with the addition of the reference substance and internal standard, and c is the spectrum of the reference substance alone;

[0052] Figure 14 It is the average plasma drug concentration-time curve of PTS-NPs and PTS after intragastric administration in rats (n = 6). Detailed implementation manners

[0053] To further illustrate the present invention, the PTS-loaded mPEG-b-PLGA nano drug delivery system provided by the present invention and its preparation method are described in detail below in combination with examples.

[0054] Using the synthesized mPEG-b-PLGA as the carrier material and the purified PTS as the drug-loading object, the PTS-loaded mPEG-b-PLGA nano drug delivery system is prepared by the double emulsion evaporation method, and its appearance morphology is observed by scanning electron microscopy, the particle size distribution and polydispersity are investigated by a laser diffraction particle size analyzer, the drug loading amount and encapsulation efficiency are detected by HPLC, and its in vitro drug release is investigated.

[0055] Experimental materials and instruments

[0056] 1. Experimental materials

[0057] The main chemical reagents and related material information are shown in Table 1.

[0058] Table 1 Experimental reagents and consumables

[0059]

[0060]

[0061] 2. Main Instruments

[0062] The information of main instruments is shown in Table 2 below.

[0063] Table 2 Experimental Instruments

[0064]

[0065] Synthesis and Characterization of mPEG-b-PLGA in Example 1

[0066] The amphiphilic polymer mPEG-b-PLGA was synthesized by covalently coupling poly(L-lactide-co-glycolide)-Carboxylic acids (PLGA-COOH) with bifunctional PEG. The synthesis route is as follows:

[0067]

[0068] Step 1) DCC / NHS activation reaction of PLGA-COOH (PLGA with a molecular weight of 15000 Da): First, weigh 1.0 g of PLGA-COOH powder and dissolve it in 4 mL of dichloromethane (DCM). After complete dissolution, add 0.1100 g (i.e., the molar ratio of PLGA to DCC is 1:8) of 1,3-Dicyclohexylcarbodiimide (DCC) and 0.0614 g (i.e., the molar ratio of PLGA to NHS is 1:8) of N-hydroxysuccinimide (NHS) for room temperature activation for 24 h. This reaction is carried out under magnetic stirring and nitrogen protection. After the reaction, DCM is removed by low-temperature vacuum distillation to obtain a small amount of concentrated solution. The concentrated solution is dropped into ice-cold anhydrous ether for precipitation, and the obtained product is washed 3 times with an ice-cold methanol-ether mixed solution ((30%:70%, v / v)), and then dried under low-temperature vacuum to obtain PLGA-NHS powder.

[0069] Step 2) Coupling reaction of adding mPEG (PEG molecular weight is 5000 Da) into the above system: Weigh 1.0 g of PLGA-NHS powder and dissolve it in 5 mL of chloroform. After complete dissolution, add 0.4 g of mPEG (i.e., the molar ratio of PLGA to PEG is 1:1.2) and 0.230 g (i.e., 0.2 mmol) of N,N-diisopropylethylamine (DIPEA) for activation at room temperature for 24 h. This reaction is carried out under magnetic stirring and nitrogen protection. After the reaction, remove chloroform by low-temperature vacuum distillation to obtain a small amount of concentrated solution. Drop the concentrated solution into ice-cold anhydrous ether for precipitation. The obtained product is then washed 3 times with an ice-cold methanol-ether mixed solution (30%:70%, v / v) to remove unreacted PEG and other by-products. Freeze-dry the finally obtained mPEG-b-PLGA polymer micelles for 48 h, and store the lyophilized powder at -20 °C.

[0070] Structural characterization:

[0071] Fourier transform infrared spectroscopy (FT-IR) analysis: Take a certain amount of mPEG-b-PLGA lyophilized powder sample, mix it with potassium bromide powder, grind and press it into a tablet, and use an infrared spectrometer to qualitatively analyze the infrared spectrum of the sample in the wavelength range of 500 cm -1 -4000 cm -1 Wavelength range.

[0072] The infrared characteristic spectrum of the copolymer mPEG-b-PLGA is as shown in Figure 1 Figure a in it. The absorption peaks at 2883 cm -1 and 1452 cm -1 indicate the stretching vibration of methyl C-H. The absorption peak at 1747 cm -1 is the stretching vibration of ester carbonyl C=O. The two absorption peaks at 1275 cm -1 and 1180 cm -1 correspond to the asymmetric stretching vibration and symmetric stretching vibration of ester C-O-C respectively, while the absorption peak at 1086 cm -1 represents the stretching vibration of ether bond C-O-C. The analysis result of the infrared spectrum is consistent with the expected structure of mPEG-b-PLGA, indicating that the synthesized product is the required copolymer.

[0073] Proton nuclear magnetic resonance spectroscopy ( 1 1H-NMR) analysis: Take a certain amount of mPEG-b-PLGA lyophilized powder sample in a nuclear magnetic tube, use dimethyl sulfoxide (DMSO) as the deuterated solvent and tetramethylsilane (TMS) as the internal standard, and measure it with a nuclear magnetic spectrometer (400 MHz) at 25 °C, and observe and analyze the 1 1H-NMR spectrum of the sample.

[0074] Of the copolymer mPEG-b-PLGA 1 The 1H-NMR spectrum is as Figure 1 shown in Figure b therein. The absorption peaks at chemical shifts of 5.19 and 1.47 are the methine proton peak and methyl peak of the lactic acid (LA segment) unit, and the chemical shift of 4.86 is the methylene proton absorption peak of the glycolic acid moiety (GA segment); while the strong absorption peak at the chemical shift of 3.51 is the characteristic absorption peak of the methylene group of the repeating unit in the ethylene glycol moiety; the chemical shifts of 3.33 and 2.5 are the two methyl proton peaks and the impurity water absorption peak on the solvent DMSO, respectively.

[0075] Example 2 Pretreatment of PTS

[0076] Resins D301T and D301R were respectively used for the pretreatment of PTS, and the specific methods are as follows:

[0077] 0.3769 g of PTS powder was ultrasonically dissolved in 7.5 mL of pure water and divided into 2 portions, each portion being 3.75 mL. Different pretreated resin columns D301T and D301R (the volume of each resin column was 12.4 mL) were added. At the end of the sample loading, the effluent was collected, and the content of notoginsenoside triol in the effluent was measured to observe whether there was any leakage of notoginsenoside triol during the sample loading process. 90 mL of acid water with pH = 4 was used for elution, and the eluate was collected in fractions to determine whether there were Rg1, Re, and R1 components in the eluate, and the elution curves of the three components of different resins were respectively plotted.

[0078] The experimental results showed that: at the end of the sample loading, no notoginsenoside triol was detected in the effluent, indicating that there was no leakage of the active ingredient during the sample loading process. The elution results and elution curves of different resins for PTS are shown in Table 3 and Figures 2 - 5 .

[0079] Comparative Examples 1-2

[0080] Using the method of Example 2, the resin was replaced with D392 and D296R, and other conditions remained unchanged for the pretreatment of PTS.

[0081] The experimental results showed that: at the end of the sample loading, no notoginsenoside triol was detected in the effluent, indicating that there was no leakage of the active ingredient during the sample loading process. The elution results and elution curves of different resins for PTS are shown in Table 3 and Figures 2 - 5 .

[0082] Table 3 Detection results (peak area) of the eluates of 4 resins

[0083]

[0084] Note: Elution section 1 represents the residue

[0085] FromFigures 2 - 5 As can be seen from Table 3, Resin No. 2 (D392) only adsorbed the sample solution and failed to effectively elute the components.

[0086] Elution curves were plotted for the peak areas of the elution segments of the remaining 3 resins (D301T, D296R, D301R), and the results are as Figures 6 - 8 shown. The results showed that the elution curve of notoginsenoside R1 of Resin No. 3 (D296R) was inconsistent with those of the other two components.

[0087] Therefore, in the present invention, D301T and D301R resins were used for the pretreatment of PTS.

[0088] Example 3 Pretreatment of PTS

[0089] Weigh 0.19 g of PTS powder and dissolve it ultrasonically in 3.5 mL of pure water. Add the pretreated different resin columns (D301T, D301R, two parallel for each resin). At the end of the sample loading, collect the effluent and measure the content of nototriol saponins in the effluent to observe whether there is any leakage of nototriol saponins during the sample loading process. Elute with 90 mL of acid water with pH = 4, collect the eluate in fractions, measure the contents of Rg1, Re, and R1 in each fraction of the eluate, plot the elution curves of the three components of different resins respectively, and calculate the transfer rate.

[0090] The experimental results showed that: at the end of the sample loading, none of the three active components were detected in the effluent, indicating that there was no leakage of active components during the sample loading process. The elution results and elution curves of different resins for PTS are shown in Table 4 below and Figures 9 - 10 :

[0091] Table 4 Transfer rates of three components of different resins at different elution volumes

[0092]

[0093] According to the above test results, it was shown that Resin D301T was better than D301R, with a higher transfer rate of active components, an obvious inflection point in the elution curve, and the components were mostly eluted at 2 times the column volume.

[0094] Therefore, the optimal pretreatment condition of the present invention is to use D301T resin for the refining and purification of PTS, and the elution volume is 2 times the column volume.

[0095] Example 4 Preparation and Characterization of PTS-NPs

[0096] mPEG-b-PLGA nanocapsules loaded with PTS were prepared by the double emulsion-solvent evaporation method

[0097] The operation process is as follows:

[0098] ①Accurately weigh the PTS purified by D301T resin in Example 3, and prepare an aqueous solution with a concentration of 50 mg / mL in 1 mL (inner aqueous phase: W1). Slowly add it to 5 mL of mPEG-b-PLGA DCM solution with a concentration of 50 mg / mL (oil phase: O). After mixing evenly, perform ultrasonic emulsification with an ultrasonic cell disruptor under an ice-water bath condition, with a power of 150 W and a time of 1 min to obtain a W / O primary emulsion. In this step of operation, deionized pure water is selected as W1, and the obtained nanocapsules are blank capsules, which are used as blank controls. The subsequent operations are the same as the preparation method of drug-loaded nanocapsules.

[0099] ②After the primary emulsion prepared in the above step is stabilized, quickly drop it into 10 mL of an aqueous solution containing 2% (w / v) emulsifier polyvinyl alcohol (PVA) (outer aqueous phase: W2) for ultrasonic emulsification, with a power of 60 W and a time of 1 min.

[0100] ③Quickly pour the above-prepared multiple emulsion into 20 mL of an aqueous solution containing 0.2% (w / v) PVA, and stir magnetically at 300 rpm / min at room temperature for 4 h to volatilize and remove DCM, thereby solidifying the polymer carrier to form a PTS-NPs suspension.

[0101] ④Centrifuge at 12000 rpm / min at low temperature for 20 min to separate the nanocapsules, wash them 3 times with deionized water, and freeze-dry for 24 h. The freeze-dried powder is stored at 4 °C at low temperature.

[0102] Take a certain amount of mPEG-b-PLGA freeze-dried powder, PTS, and PTS-NPs samples respectively, mix them with potassium bromide powder, grind and press them into tablets, and use an infrared spectrometer to perform qualitative analysis on the FT-IR spectra of the samples in the wavelength range of 400 cm -1 -4000 cm -1 At the same time, use an X-ray diffractometer (Cu K λ = 1.5418 nm) to measure and analyze the components of the three samples. The 2θ scanning range is from 5° to 90°, and the scanning step is 0.02° / s.

[0103] The FT-IR characteristic spectrum of PTS-NPs is as shown in Figure 11 Figure c in the middle. It can be seen that in PTS-NPs, in addition to the characteristic peaks of mPEG-b-PLGA, the infrared characteristics of PTS represented by ginsenoside Rg1 are also reflected. For example, the absorption peak at 2931 cm -1 represents the stretching vibration of C-H on the aglycone, the absorption peak at 1426 cm -1 represents the rocking vibration of C-H on the sugar ring and the bending vibration of O-H of the hydroxyl group, the absorption peak at 3308 cm -1 represents the stretching vibration of O-H of the hydroxyl group, and it reflects 3030 - 3660 cm-1 The broad peak shape between them is due to the aggregation state of hydroxyl groups caused by molecular overlap phenomenon.

[0104] The XRD characteristic spectrum of PTS-NPs is as Figure 11 shown in Figure b in the middle. It can be seen that PTS shows a diffuse diffraction peak of amorphous state, while obvious crystal diffraction peaks appear at 19.3° and 23.5° for mPEG-b-PLGA. Generally speaking, the spectral curves of PTS-NPs and PTS are generally quite similar. The crystal diffraction peak at 19.3° of the material becomes the peak of the diffuse diffraction peak, and a smaller crystal diffraction peak appears at the back end of the slope peak of the diffuse diffraction peak (i.e., at 23.5°), and no other obvious diffraction peaks appear.

[0105] Observation of the surface morphology of PTS-NPs

[0106] Prepare an aqueous solution of PTS-NPs with a concentration of 0.5 mg / mL. Take a drop and add it to a copper mesh. Blot off the excess liquid with filter paper. After air-drying at room temperature, stain it negatively with 2% phosphotungstic acid solution for 3 minutes. After drying at room temperature, observe its surface morphology using a transmission electron microscope (TEM).

[0107] Determination of the average particle size distribution and Zeta potential of PTS-NPs

[0108] Add 1 mL of an aqueous solution of PTS-NPs with a concentration of 1.0 mg / mL to a particle size cell, and use a Malvern dynamic light scattering particle size analyzer (DLS) to detect the average particle size and polydispersity index (PDI) of the nanocapsules; take another 1 mL of the aqueous solution of the nanocapsules and place it in a potential cell, and use DLS to measure the Zeta potential of the nanoparticles. The test temperature is 25°C for all, and each sample is tested three times. The test results are expressed as mean ± standard deviation.

[0109] The scanning electron microscopy determination of PTS-NPs is as Figure 11 shown in Figure a in the middle. The scanning electron microscopy morphology of PTS-NPs after freeze-drying shows a spherical structure with a relatively regular and smooth surface, arranged tightly, with relatively uniform size, no obvious broken spheres or fusion phenomena, and the particle size is basically consistent with the DLS measurement results. The particle size distribution results and potential test results of PTS-NPs are as follows Figure 11 shown in Figure d in the middle and Figure e in Figure 11. The particle size of this drug-loaded nanocapsule is mainly distributed in the range of 170 - 400 nm, and the average particle size (Z-average Size) is 281.81 ± 13.71 nm, and the PDI is 0.1693 ± 0.0228, indicating that the nanocapsules are normally distributed and have good dispersion. The average Zeta potential is -46 ± 0.31 mV, indicating that the prepared nanocapsules carry a relatively low negative charge and are less affected by the adhesion of intestinal mucus.

[0110] Detection of Drug Loading Capacity and Encapsulation Efficiency of PTS-NPs

[0111] Accurately weigh 5.0 mg of PTS-NPs freeze-dried powder, dissolve it in 4 mL of DCM, stir to completely dissolve it, then add 10 mL of PBS with pH 4, and magnetically stir and mix for 20 min to allow PBS to fully extract PTS in DCM. Let it stand at room temperature for 1 h to separate PBS from DCM, and collect the aqueous phase PBS. Repeat the extraction of the remaining DCM according to the above operation once, combine the aqueous phase solutions obtained twice, and centrifuge to take the supernatant. Detect by HPLC, and the method refers to the method for determining the content of PTS in the 2020 edition of the Chinese Pharmacopoeia:

[0112] Accurately pipette 3 control solution with 5 concentration gradients respectively, with two parallel samples for each concentration, and use methanol as the blank for determination. Inject samples according to the content determination method, perform linear regression processing, and obtain the standard curve regression equation as follows: For ginsenoside Rg1, y = 3235.6x - 0.1667 (R 2 = 0.9999); for notoginsenoside R1, y = 2889.5x + 2.867 (R 2 = 0.9999); for ginsenoside Re, y = 2968.4x - 1.4919 (R 2 = 0.9999). The correlation coefficients of all equations are greater than 0.995, and the standard curve has feasibility and linear relationship.

[0113] Calculate the contents of ginsenoside Rg1, ginsenoside Re and notoginsenoside R1 in the supernatant according to the pre-obtained standard curve. Repeat the experiment three times (recorded as experimental groups 1, 2, and 3 respectively), and use the calculation formulas of drug loading capacity (DLC) and encapsulation efficiency (EE) to calculate the percentage content and encapsulation efficiency of each component in PTS in the drug-loaded nanocapsules:

[0114] DLC(%) = Content of drug in nanocapsules / Mass of nanocapsules × 100%

[0115] EE(%) = Content of drug in nanocapsules / Dosage of drug × 100%

[0116] The results are shown in Table 5. The drug loading capacity of PTS-NPs is 66.92 ± 0.94, and the encapsulation efficiency is 93.75 ± 0.75.

[0117] Table 5 Detection results of drug loading capacity and encapsulation efficiency of PTS-NPs (%)

[0118]

[0119] Investigation of in vitro drug release behavior

[0120] The release behavior of PTS from nanocapsules was investigated by the dynamic dialysis method. PTS and PTS-NPs freeze-dried powders were accurately weighed separately and diluted with PBS solution at pH 4 to prepare a sample solution with a concentration of 2 mg / mL. 3 mL of the sample solution was transferred into a sealed dialysis bag (MWCO: 8k - 14k) and placed in a centrifuge tube containing 30 mL of PBS buffer solution at pH 4. The mixture was shaken at a constant temperature of 37 °C in an air shaker at a rate of 150 r / min. Then, samples were taken for detection at the set time points of 2, 4, 6, 8, 12, 24, 36, and 48 h. Each time samples were taken, 1.0 mL of solution was taken from two centrifuge tubes, and 10 mL of the corresponding fresh PBS buffer solution was added simultaneously to maintain the total volume constant. After sampling in sequence, the samples were filtered through a 0.45 μm microporous membrane, and the subsequent filtrate was taken for HPLC detection. The drug release amount (%) at each time point was calculated according to the known standard curve, and the cumulative release amount (%) mean ± standard deviation at this time point was calculated by supplementing 1 / 30 of the drug release amount at the previous time point, as well as the release rate (%) per time period -1 ). The experiment was repeated 3 times, and the in vitro drug release curve of the drug-loaded nanocapsules was plotted with the drug cumulative release amount as a parameter.

[0121] The results are as Figure 12 shown in and Table 6. The cumulative release degree of PTS-NPs can reach up to 54.35%. The initial burst release can reach nearly 50% in the first 4 - 8 h, and then the drug in the nanocapsules is continuously and steadily released at a uniform speed for at least 24 h, generally showing a certain sustained-release performance. Compared with free PTS, PTS-NPs have a similar release curve, and both can show a drug burst release phenomenon in the initial 4 to 8 h, and then release slowly. Comparing the standard deviations of the three test data reflects that the release status of PTS-NPs is more stable; comparing the slopes of the release curves, it can be seen that the release curve is relatively flatter starting from 4 h, and the downward trend appears later and the decrease amplitude is more gentle after 8 h; the cumulative release rates of free PTS and PTS-NPs start to decrease at the time points of 24 h and 36 h respectively, indicating that neither of them releases drugs or the drug release efficiency is lower than the natural loss of the drug in the buffer solution between 12 - 24 h and 24 - 36 h respectively, and the drug release time of PTS-NPs is longer.

[0122] Table 6 In vitro drug release test results of PTS-NPs (n = 3)

[0123]

[0124] Note: The release efficiencies of free PTS and PTS-NPs become negative values in the time periods from 12 to 24 h and from 24 to 36 h respectively, so the subsequent data are not shown.

[0125] Example 5 Non-clinical pharmacokinetic evaluation of the nanodrug delivery system

[0126] Test Materials and Instruments

[0127] 1. Test Materials

[0128] The information of chemical reagents and related materials is shown in Table 7 below.

[0129] Table 7 Reagents and Consumables for Pharmacokinetics Experiments

[0130]

[0131]

[0132] 2. Main Instruments

[0133] The information of main instruments is shown in Table 8 below.

[0134] Table 8 Instruments for Pharmacokinetics Experiments

[0135]

[0136] 3. Test Animals and Venue

[0137] SD rats, SPF grade, half male and half female, with a body weight of (220±20) g, were purchased from Chengdu Dashuo Biotechnology Co., Ltd., and the production license number of the test animals is SCXK(Sichuan)2019-031.

[0138] 4. Test Methods

[0139] 4.1 Sample Preparation Method

[0140] (1) Preparation of Test Article Solutions for Animal Experiments

[0141] Accurately weigh PTS and the PTS-NPs freeze-dried powder prepared in Example 2, and use normal saline to prepare test article solutions of 10 mg / ml (with a PTS content of 5 mg / ml) and 5 mg / ml (with a PTS content of 5 mg / ml) respectively, and store them at 4°C for later use.

[0142] (2) Preparation of Plasma Standard Curve Reference Substances

[0143] Accurately weigh each reference substance, and use chromatographic methanol to prepare reference substance stock solutions containing 1 μg of ginsenoside Rg1, notoginsenoside R1, ginsenoside Re and the internal standard rutin per mL. Accurately pipette the stock solutions of the first three reference substances, and dilute them with methanol to prepare solutions with concentrations of 1000 ng·mL -1 , 500 ng·mL -1 , 250 ng·mL -1 , 125 ng·mL -1 , 62 ng·mL -1, 32 ng·mL -1 , 16 ng·mL -1 , 8 ng·mL -1 , 4 ng·mL -1 of the mixed reference solution. Subsequently, 100 μL of blank rat plasma was taken and 100 μL of the mixed reference solution was added to each. Then, after adding 20 μL of methanol for dilution, the concentration became 200 ng·mL -1 of the internal standard solution, so that the internal standard concentration reached 40 ng·mL -1 . After vortexing for 30 s, 300 μL of methanol was added to precipitate proteins. After vortexing for 5 min, centrifugation was performed at 12000 rpm for 10 min. The supernatant was taken, dried under nitrogen at 30 °C, redissolved in 100 μL of acetonitrile-water (15:85, V / V) solution, vortexed for 5 min, centrifuged at 12000 rpm for 10 min, and the supernatant was transferred to a brown injection vial and stored in the dark for testing.

[0144] (3) Preparation of the test article of rat plasma containing the drug

[0145] 100 μL of rat plasma containing the drug was taken, and starting from the addition of the internal standard solution, sample preparation was carried out according to the treatment operation of the reference biological sample.

[0146] 4.2, Detection method

[0147] (1) Detection conditions

[0148] Chromatographic conditions: Mobile phase A is pure water, B is acetonitrile, and gradient elution is used. The elution program is as follows: 0 - 3 min, B is 15%; 3 - 8 min, B is 15% - 55%; 8 - 12 min, B is 55% - 80%; 12 - 15 min, B is 80%; 15 - 15.01 min, B is 15%; 15.01 - 18 min, B is 15%. The flow rate is 0.2 mL·min -1 , and the column temperature is 35 °C, and the injection volume is 3 μL.

[0149] Mass spectrometry conditions: ESI ion source is used, the acquisition mode is positive ion mode, the scanning range is from 100 - 1300 Da, and the default scanning time is 0.14 s. The main working parameters are: capillary voltage 3.2 KV, desolvation temperature 400 °C, cone voltage 35 V, extraction cone voltage 4 V, ion source temperature 120 °C, cone gas flow rate 50 L·h -1 , desolvation gas flow rate 800 L·h -1 , collision energy 3 eV, collision gas pressure 2.8×10 3 mbar. Under the MS conditions, the m / z of Rg1 detected was 823.34, the m / z of Re was 969.41, the m / z of R1 was 955.48, and the m / z of rutin was 1131.54.

[0150] (2) Methodological verification

[0151] ① Specificity investigation

[0152] Prepare the test sample solution of rat blank plasma without adding the reference substance and internal standard solution according to the above method. According to the above detection conditions, compare the blank plasma and the reference substance of the plasma standard curve.

[0153] ② Standard curve and lower limit of quantitation investigation

[0154] Take the ratio of the peak area of the reference substance solution at different concentrations to the peak area of the internal standard as the ordinate (Y), and calculate the standard curve equation and linear coefficient R of the component. 2 , Take the signal-to-noise ratio S / N = 10 of the chromatographic peak as the lower limit of quantitation, and measure the lowest detection concentration.

[0155] ③ Intra-day and inter-day precision investigation

[0156] Prepare three kinds of mixed plasma samples of reference substances with concentrations of low (8 ng·mL -1 ), medium (32 ng·mL -1 ), and high (250 ng·mL -1 ). Then add 20 μL of rutin internal standard with a concentration of 200 ng·mL -1 to make the internal standard concentration 40 ng·mL -1 . After vortexing for 30 s, precipitate the protein with 300 μL of methanol, vortex for 5 min, centrifuge at 12000 rpm for 10 min, take the supernatant, dry it with nitrogen at 30 °C, re-dissolve it with 100 μL of acetonitrile-water (15:85, V / V) solution, vortex for 5 min and then centrifuge at 12000 rpm for 10 min. Transfer the supernatant to a brown injection vial, and the injection volume is 3 μL. Inject samples in parallel 3 times at 0, 2, 4, 6, 8, 10, and 12 h respectively, and inject samples in parallel 3 times continuously for 7 days. Record the peak areas of the reference substances and the internal standard at each concentration, and calculate the ratio of the peak area of each component to the internal standard. Use the ratio of the peak areas of 6 parallel samples to investigate the intra-day precision and the inter-day precision for 7 consecutive days. The results are expressed as relative standard deviation RSD (%).

[0157] ④ Accuracy investigation

[0158] Inject the samples processed according to the precision investigation method continuously for 6 times, record the peak areas of the reference substances and the internal standard at each concentration, and calculate the ratio of the peak area of each component to the internal standard. Compare the value calculated through the standard curve with the actual concentration value when preparing the standard curve to investigate the accuracy. The results are expressed as relative error RE (%).

[0159] ⑤ Recovery investigation

[0160] The samples processed according to the precision investigation method were subjected to 6 parallel experiments for each concentration. Calculate the ratio of the peak areas of the reference substance and the internal standard, Ai, at each concentration. The blank plasma samples of rats without adding the standard substance and the internal standard solution were reconstituted with mixed reference substance methanol solutions at low (8 ng / mL), medium (32 ng / mL), and high (250 ng / mL) concentrations (containing 40 ng / mL internal standard) respectively. Six replicates were prepared for each concentration, and the ratio of the peak areas of the reference substance and the internal standard, Bi, at each concentration was calculated. The recovery rate % = Ai / Bi * 100% was obtained by calculating the ratio of the corresponding components Ai and Bi, so as to obtain the recovery rates of the reference substance and the internal standard during the plasma treatment process.

[0161] ⑥ Stability investigation

[0162] The samples processed according to the precision investigation method were subjected to 6 parallel experiments for each concentration. The stability of the plasma samples at room temperature for 12 h, at 4 °C for 24 h, long-term stability at -80 °C for 7 days, and stability after 3 freeze-thaw cycles were determined. The results were expressed as relative standard deviation RSD (%).

[0163] 4.3 Animal grouping and experimental operation

[0164] After the rats were fed for 3 days to adapt to the environment, they were fasted for 12 h without water deprivation before the experiment. After weighing, they were randomly divided into the PTS group and the PTS-NPs group according to body weight stratification, with 6 animals in each group. Based on the weight of PTS, gavage administration was carried out at a dose of 50 mg of the drug per kg of animal body weight. Blood was collected from the orbital vein of each rat at the time points of 3 min, 5 min, 10 min, 20 min, 40 min, 1 h, 2 h, 4 h, 6 h, 10 h, 12 h, 16 h, 24 h, 48 h, and 72 h before and after administration. The single blood collection volume was 0.3 mL, which was placed in a heparinized centrifuge tube and centrifuged at 3500 rpm for 10 min. The supernatant plasma was taken and stored at -80 °C. The test sample solutions of serum biological samples at each time point were prepared according to the above sample treatment method.

[0165] 4.4 Data analysis

[0166] According to the established UPLC-MS detection method, the processed plasma samples were analyzed. The peak areas of the main pharmacodynamic component ginsenoside Rg1 at different time points were measured using the established method. The blood drug concentrations of the effect components at different time points were calculated by the internal standard method through the ratio of the peak areas. The average blood drug concentration-time curve was fitted using Winnolin (version 6.3) pharmacokinetic software, and non-compartmental model calculation was used to obtain the maximum concentration (Cmax), time to peak concentration (Tmax), area under the concentration-time curve (AUC0-∞), half-life (T1 / 2), apparent volume of distribution (V / F), clearance rate (CL / F), and mean residence time (MRT).

[0167] 5. Test Results

[0168] 5.1 Method Validation for Detection

[0169] (1) Specificity

[0170] As Figure 13 shown, no interference from endogenous substances was found within the analysis time of 18 min. Under the experimental conditions, chromatographic peaks with high responses were obtained for the three reference substances and the internal standard. The retention times of ginsenoside Rg1, notoginsenoside R1, ginsenoside Re, and the internal standard were approximately 8.40 min, 9.8 min, 8.38 min, and 14.46 min, respectively. Although the separation effect of the total ion current chromatograms of the three compounds was not ideal, detection could be achieved by extracting the accurate molecular weights.

[0171] (2) Precision, Accuracy, and Recovery

[0172] As shown in Table 9, the intra-day and inter-day precision RSDs of the samples at each concentration were less than 10.0%, the accuracy was between 85.71% and 110.37%, and the recoveries at each concentration were greater than 80% (85.74% for the internal standard), indicating that the method was accurate, reliable, and had good reproducibility.

[0173] (3) Stability

[0174] As shown in Table 10, the samples at each concentration had good long-term stability (7 d) at -80 °C, short-term stability (24 h) at 4 °C, stability at room temperature (12 h), and freeze-thaw stability after three cycles at -80 °C, indicating that the method was stable and reliable and suitable for the analysis of plasma samples.

[0175] (4) Standard Curve

[0176] As shown in Table 11, the correlation coefficients of the standard curve regression equations for all reference substances were greater than 0.99, and the linear ranges were all between 4 and 1000 ng·mL -1 , indicating that the standard curve had feasibility and a linear relationship.

[0177] Table 9 Results of Precision, Accuracy, and Recovery of the LC-MS Detection Method

[0178]

[0179] Table 10 Results of Stability Investigation of the LC-MS Detection Method

[0180]

[0181] Table 11 Standard Curve and Quantification Limit of the LC-MS Detection Method

[0182]

[0183] 5.2 Pharmacokinetic analysis results

[0184] Similar to the in vitro release results, after detecting the samples, it was found that notoginsenoside R1 and ginsenoside Re could not obtain effective detection data in the samples at most time points due to their low contents. Therefore, only ginsenoside Rg1 was analyzed.

[0185] The results are as Figure 14 shown in Table 12. After normal rats were orally administered with PTS and its nano - preparation by gavage, they both conformed to the absorption characteristics of components with active transport. There was no obvious difference in Cmax among different dosage forms; from the peak time Tmax, NPs had a certain sustained - release effect, and Tmax was slightly prolonged, but there was no significant difference. There was no obvious difference in the in - vivo absorption amount AUC0 - t and AUC0 - ∞ of each component.

[0186] Table 12 Pharmacokinetic data of PTS - NPs and PTS in rats by gavage (n = 6)

[0187]

[0188] The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A PTS-loaded mPEG-b-PLGA nano drug delivery system, characterized in that, It is prepared by the double emulsion evaporation method with PTS as the drug-loading object and mPEG-b-PLGA as the carrier material.

2. The mPEG-b-PLGA nano drug delivery system loaded with PTS according to claim 1, characterized in that, The PTS is pretreated as follows: The PTS is purified by passing through a weakly basic ion exchange resin.

3. The PTS-loaded mPEG-b-PLGA nanodelivery system according to claim 2, wherein The weakly basic ion exchange resin is selected from D301 resin.

4. The mPEG-b-PLGA nano drug delivery system loaded with PTS according to claim 2, wherein, The purified PTS components include ginsenoside Rg1, ginsenoside Re, and ginsenoside R1.

5. The mPEG-b-PLGA nano drug delivery system loaded with PTS according to claim 4, wherein, The purified PTS components include 60wt%-75wt% ginsenoside Rg1, 5wt%-10wt% ginsenoside Re, and 5wt%-12wt% ginsenoside R1.

6. The mPEG-b-PLGA nanodelivery system loaded with PTS according to claim 4, wherein, The loading amount of ginsenoside Rg1 is 66.92% ± 0.94%.

7. A preparation method of an mPEG-b-PLGA nano drug delivery system loaded with PTS, comprising the following steps: S1) Pretreatment of PTS S2) Prepare an mPEG-b-PLGA nano drug delivery system loaded with PTS by the double emulsion-solvent evaporation method.

8. The preparation method according to claim 7, characterized in that, The specific steps of step S2) are as follows: A) Mix the aqueous solution of pretreated PTS and the mPEG-b-PLGA solution, and perform ultrasonic emulsification with an ultrasonic cell disruptor under an ice-water bath condition to obtain a W / O type primary emulsion; B) After the above primary emulsion is stabilized, add it to the aqueous solution of the emulsifier for ultrasonic emulsification, and separate to obtain an mPEG-b-PLGA nano drug delivery system loaded with PTS.

9. The preparation method according to claim 8, characterized in that, In step A), the power of the ultrasonic cell disruptor is 100-200W, and the ultrasonic emulsification time is 1-2 min.

10. The preparation method according to claim 8, characterized in that, In step B), the emulsifier is selected from polyvinyl alcohol.

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