CN116392442A - Skin administration preparation prepared by microfluid and having coexistence of lipid vesicles and micelles and preparation method thereof - Google Patents

Abstract

The invention provides a novel skin drug delivery preparation prepared by a micro-fluid technology and a preparation method thereof, wherein a micro-fluid device is utilized to prepare a coexisting system [ PNS@ (V-M) -CSs ] of vesicles and mixed micelles formed by phospholipids and PNS, wherein the physicochemical properties of the coexisting system are controllable. The special transdermal drug delivery system plays unique advantages of two carriers in the delivery process, so that better transdermal delivery capacity is shown, the capacity of the two carriers to synergistically promote transdermal is exerted, the bioavailability of the drug is improved, the treatment period is shortened, and the toxic and side effects of the drug are reduced; the novel skin administration preparation prepared by the microfluidics technology comprises the following components: 50-200 parts of total saponins of panax notoginseng, 250-350 parts of phospholipids, 30-60 parts of cholesterol, 4-7 parts of vitamin E, 15000-30000 parts of phosphate buffer solution and 1500-3000 parts of absolute ethyl alcohol.

Description

A preparation for skin drug delivery in which lipid vesicles and micelles coexist and are prepared by microfluidics Preparation

technical field

The invention belongs to the skin external preparation in the field of pharmaceutical preparations, and in particular relates to a novel skin drug preparation prepared by microfluidic technology and a preparation method thereof.

Background technique

Lipid vesicles refer to a class of supramolecular aggregates formed by the self-assembly of amphiphilic molecules (usually natural or synthetic phospholipids) in aqueous phase, because they can promote drug transdermal, exert slow and controlled release, and It can reduce the toxic and side effects of drugs, and has been widely used in transdermal drug delivery systems in recent years. Traditional lipid vesicle liposomes (Liposomes, LPSs) are mainly composed of amphipathic components phospholipids and cholesterol, and its main component phospholipids have large packing parameters, so they mainly exist in the form of vesicles after self-assembly, which makes them have Large rigidity and good physical stability are suitable for the entrapment of hydrophilic and lipophilic drugs, but because they do not have the ability to deform, they cannot penetrate the skin in a complete form. In order to enhance its transdermal properties, it is usually necessary to add a single chain surfactant (single chain surfactant, SCS) (such as Tween, Span and cholate, etc.) to the liposome formulation. Because SCS often has smaller packing parameters, and their chain length is the optimal chain length for insertion into phospholipid bilayers, it can improve the elasticity and deformability of lipid vesicles to promote their transdermal properties. When they were used in combination with phospholipids, the type of aggregates obtained changed with the proportion of SCS in the mixed system when the total lipid concentration was appropriate. When the proportion of SCS is high, mixed micelles (MMLs) are obtained, and it has been reported that this carrier can be used to increase the oral absorption of poorly soluble drugs. When the proportion of SCS is low, bilayer vesicles are obtained, and SCS monomers are distributed in the lipid bilayer until the distribution reaches saturation. There is no significant change in the shape of the vesicles during this process, but it will significantly affect its transdermal performance; in addition, this process will also affect the flexibility of the bilayer membrane, and the resulting flexible liposomes are called transfersomes (TFSs), which can penetrate deep into the skin or penetrate intact human skin, so they have been widely studied Used as a carrier for transdermal drug absorption. When the ratio of SCS is between the above two, the obtained aggregates are vesicle-micelle coexistence systems (V-M)-CSs), which have both kinds of aggregation At the same time, these two aggregates may undergo dynamic mutual transformation with the action of organisms after administration, so they may play unique advantages in various routes of administration. For example, in transdermal administration, the vesicles and micelles in the coexistence system are expected to play an ideal role in promoting drug transdermal due to their large elasticity and small particle size. Because the coexistence system of lipid vesicles and micelles requires precise control of the ratio of SCS, a controllable and stable microenvironment is crucial for the success of its preparation. However, due to the heterogeneity of the microenvironment involved in the two-phase mixing, the traditional preparation process will show obvious inhomogeneity and uncontrollability. Therefore, it is necessary to find a suitable method to prepare (V-M)-CSs.

Radix Notoginseng, warm in nature, sweet in taste, slightly bitter, returns to liver and stomach meridian, is the dry root and rhizome of Radix Notoginseng (Panax notoginseng (Burk.) F.H.Chen) of Araliaceae. The key medicine for the syndrome of bleeding, bruises, stasis, swelling and pain. Panax notoginseng saponins (PNS) is its main effective part. Clinically, PNS is widely used in soft tissue injury, fracture healing, bone and joint injury, recovery of limb function and softening of scars. At present, the commercially available dosage forms of PNS are mainly oral and intravenous infusion preparations, but the drug concentration at the local injury site after administration is low.

Contents of the invention

The purpose of the present invention is to address the deficiencies of existing preparations, to provide a new type of skin drug delivery preparation in which lipid vesicles and micelles co-exist, which are prepared by microfluidics, so as to exert the synergistic ability of the two carriers to promote transdermal penetration and improve the bioavailability of drugs degree, shorten the treatment cycle, and reduce the toxic and side effects of the drug; another object of the present invention is to provide a method for preparing the above-mentioned novel skin drug delivery preparation.

The novel skin drug delivery preparation in which lipid vesicles and micelles co-exist prepared by microfluidics is characterized in that it consists of the following components by weight:

160-200 parts of Panax notoginseng total saponins, 250-350 parts of phospholipids, 30-60 parts of cholesterol, 4-7 parts of vitamin E, 15000-30000 parts of buffer solution, 1500-3000 parts of absolute ethanol.

The novel skin drug delivery preparation in which lipid vesicles and micelles co-exist prepared by microfluidics is characterized in that it consists of the following components by weight:

180 parts of Panax notoginseng total saponins, 300 parts of phospholipids, 45 parts of cholesterol, 5 parts of vitamin E, 23000 parts of buffer solution, 2000 parts of absolute ethanol.

The novel skin drug preparation in which lipid vesicles and micelles co-exist prepared by microfluidics is characterized in that the phosphate buffer solution is 1/15mol/L potassium dihydrogen phosphate solution and 1/15mol/L L disodium hydrogen phosphate solution is a mixed solution with a volume ratio of 53.4:46.6.

The novel skin drug preparation in which lipid vesicles and micelles coexist, which is prepared by microfluidics, is characterized in that the total saponins of notoginseng are not only single-chain surfactants in prescriptions, but also therapeutic active ingredients.

The preparation method of a novel skin drug delivery preparation in which lipid vesicles and micelles co-exist prepared by microfluidics is characterized in that it comprises the following process steps:

1) Weigh the total saponins of Panax notoginseng, phospholipids, cholesterol and vitamin E in the formula, dissolve them in absolute ethanol, mix them evenly, and obtain the organic phase;

2) Measure the phosphate buffer saline solution of formula quantity as water phase;

3) Under the condition of controlling the mixing temperature at 30°C, the microfluidic experimental conditions were designed, and the two solutions were pushed by the syringe pump according to the flow rate ratio of the water phase and the alcohol phase at 10:1, and the total flow rate of the water-alcohol two-phase was 198 μL/min (Wherein, the flow rate of the aqueous phase is 180 μL/min, and the flow rate of the alcohol phase is 18 μL/min) are transported to the microfluidic mixer for mixing by an airtight syringe.

4) Take the sample at the outlet of the tube and collect it in a vial. Three samples were collected consecutively for each preparation condition. A novel skin drug delivery preparation in which lipid vesicles and micelles coexist is obtained.

The preparation method of a novel skin drug delivery preparation in which lipid vesicles and micelles co-exist prepared by microfluidics is characterized in that the weight part of total saponins of Panax notoginseng and the weight part of phospholipids in the step 1) The ratio ranges from 0 to 1:1, preferably 0.6:1.

The preparation method of a novel skin drug delivery preparation in which lipid vesicles and micelles co-exist prepared by microfluidics is characterized in that the range of the ratio of the weight part of cholesterol to the weight part of phospholipid in the step 1) 0 to 0.3:1, preferably 0.15:1.

The preparation method of a novel skin drug delivery preparation in which lipid vesicles and micelles co-exist prepared by microfluidics is characterized in that the preparation process of microfluidic mixing technology is mainly based on microfluidic preparations prepared by 3-D printing technology. Cross-shaped microfluidic device with separate mixing chambers for hydrodynamic focusing.

The preparation method of a new type of skin drug preparation in which lipid vesicles and micelles are prepared by microfluidics is characterized in that the microfluidic mixing temperature in step 3) ranges from 25°C to 55°C, Preferably it is 30°C.

The preparation method of a novel skin drug delivery preparation in which lipid vesicles and micelles co-exist prepared by microfluidics is characterized in that in the step 3) when the fixed center alcohol phase flow rate is 20 μl/min, water The flow rate ratio of the phase and the alcohol phase is set to 2.5:1, 5:1, 10:1, 15:1, 20:1, 25:1 and 30:1, preferably the flow rate ratio of the water-alcohol phase is 10:1.

The preparation method of a novel skin drug delivery preparation in which lipid vesicles and micelles co-exist prepared by microfluidics is characterized in that in the step 3) when the fixed water-alcohol phase flow rate ratio is 10:1, the water The total flow rate of the alcohol phase is set to 33, 66, 132, 198, 264, 330 and 660 μl/min, preferably the total flow rate of the water-alcohol phase is 198 μl/min; when the fixed water-alcohol phase flow rate ratio is 20:1, the total water-alcohol phase The flow rate is set to 42, 84, 105, 168, 210, 420 and 630 μl/min, preferably the total flow rate of the water-alcohol phase is 210 μl/min; when the fixed water-alcohol phase flow rate ratio is 30:1, the total flow rate of the water-alcohol phase is set The total flow rate of the hydroalcoholic phase is preferably 248 μl/min.

The microfluidic device used in the present invention is a cross-shaped microfluidic device with independent mixing chambers based on microfluidic hydrodynamic focusing method designed by computer simulation and prepared by 3-D printing technology.

The total saponins of Panax notoginseng in the present invention are the effective parts of traditional Chinese medicines extracted from the traditional Chinese medicine Panax notoginseng, which meet the standards of the national pharmacopoeia, and there are commercially available products.

In the present invention, natural phospholipids and synthetic phospholipids can be used as phospholipids for forming the coexistence system of lipid vesicles and micelles. Natural phospholipids include phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylinositol, egg yolk lecithin, hydrogenated egg yolk lecithin, egg phosphatidylglycerol, egg yolk phosphatidylserine, egg yolk phosphatidylinositol, soy lecithin, hydrogenated Soy lecithin, hydrogenated lecithin, lecithinyl glycerol, lecithinylserine and lecithinositol, etc. Synthetic phospholipids are dioleoylphosphatidylcholine, distearylphosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, dilauroylphosphatidylcholine, distearoylphosphatidylcholine Glycerol, dipalmitoylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dilauroylphosphatidylglycerol, and polyethylene glycol derivatized phospholipids such as distearoylphosphatidylethanolamine-polyethylene glycol 2000 (DSPE-mPEG2000) ; Dipalmitoylcholine-polyethylene glycol 2000 (DPPG-mPEG2000); Hydrogenated soybean phosphatidylcholine-polyethylene glycol 2000 (HSPC-mPEG2000); Dioleoylphosphatidylcholine-polyethylene glycol 2000 (DOPC-mPEG2000) and the like.

The inventors have found through research that commercially available egg yolk lecithin with a PC content of 60% to 95% is particularly suitable as a basic phospholipid membrane material to form a high-quality coexistence system of lipid vesicles and micelles, and the PC content of egg yolk lecithin is preferred. 80% to 90%.

In the invention, the cholesterol plays the role of regulating the fluidity of the membrane, and can improve the stability of the lipid bilayer membrane and the encapsulation rate of the medicine. The cholesterol used complies with the Pharmacopoeia 2010 standard.

In the present invention, the total saponins of Panax notoginseng are selected as the edge activator, and the active ingredients of traditional Chinese medicine are used as the edge activator relative to other surface active agent edge activators, so that the safety of the formed lipid vesicles and mixed micelles is greatly improved, and at the same time , Panax notoginseng saponins can be used as therapeutic active ingredients, and have the pharmacological effects of promoting blood circulation, analgesia, anti-inflammation and promoting fracture healing.

In the present invention, vitamin E acts as an antioxidant to prevent phospholipids from being oxidized.

Microfluidic devices can be used to manipulate liquid flow in channels ranging in size from tens of microns to hundreds of microns. Microfluidic channels can be used for high-throughput experiments, and have the characteristics of low reagent consumption, fast reaction and continuous production, which are difficult to achieve with conventional methods. Among them, microfluidic hydrodynamic focusing (MHF, microfluidic hydrodynamic focusing) may be the preferred method for preparing lipid vesicles at the nanoscale. In addition, the advantages of MHF include: the preparation of nano-scale controllable lipid vesicles; preparation without post-processing steps; in situ monitoring of liposome formation process; continuous production; and scale-up of production. Therefore, it can provide a good way to prepare (V-M)-CSs with controllable physicochemical properties.

Drug skin application can increase the drug concentration in the local tissue under the skin of the drug site and effectively reduce the adverse reactions of the drug. Therefore, transdermal administration can be regarded as an ideal route of administration of PNS for treating local soft tissue injuries in the body. As a saponin component of traditional Chinese medicine, PNS has a lipophilic core and a hydrophilic sugar chain, so it has the structural characteristics of a surfactant, and can self-assemble in aqueous solution to form micelles or vesicles. At the same time, PNS has the above-mentioned good therapeutic activity, so that the obtained carrier has good potential clinical therapeutic value. Therefore, the present invention intends to use PNS as a model single-chain surfactant to study the formation principle of the lipid vesicle-micelle coexistence system in the micromixer. The PNS in the system is not only a component of the carrier, but also a therapeutic active agent. Element.

Research has found that the present invention provides a stable and controllable mixing environment by using a microfluidic mixing device through a reasonable ratio of total saponins of notoginseng, phospholipids, cholesterol and vitamin E, and can prepare total saponins of notoginseng lipid vesicles with controllable physical and chemical properties The coexistence system with mixed micelles has better transdermal properties, smaller particle size, lower toxicity and higher bioavailability than simple vesicles.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those skilled in the art, other drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 The three-dimensional design drawing (A) and physical drawing (B) of the 3D printed micromixer.

Fig. 2 TEM image of PNS@(V-M)-CSs under optimal formulation process.

Fig. 3 is the in vitro skin penetration curve of the lipid vesicle-micelle coexistence system of each embodiment and the mixed micelles of the reference preparation.

Figure 4 CLSM images of various fluorescence distributions in skin samples of PNS@(V-M)-CSs-1 (A), PNSLs (B) and PNSMs (C) after transdermal administration for 2 hours (1: R6G; 2: C153; 3: DAPI; 4: fluorescence pooled).

Fig. 5 CLSM images of various fluorescence distributions in skin samples of PNS@(V-M)-CSs-1 (A), PNSLs (B) and PNSMs (C) after transdermal administration for 8 hours (1: R6G; 2: C153; 3: DAPI; 4: fluorescence pooled).

Fig. 6 CLSM diagrams of various fluorescence distributions in skin samples of PNS@(VM)-CSs-1 (A), PNSLs (B) and PNSMs (C) after transdermal administration for 12 hours (1: R6G; 2: C153; 3: DAPI; 4: fluorescence pooled). Figure 7 Eff _FRET (%) of three R6G and C153 co-labeled preparations at different time points (n=3).

Detailed ways

The present invention will be further elaborated below in conjunction with specific examples. The methods are conventional methods unless otherwise specified, and the raw materials can be obtained from open commercial channels unless otherwise specified.

The particle size and particle size distribution, morphology, encapsulation efficiency, and elasticity of the lipid vesicle-micelle coexistence system [PNS@(V-M)-CSs] of Panax notoginseng saponins (PNS) described in the examples of the present invention , skin permeability and other indicators are evaluated as follows:

1. Particle size and distribution evaluation method

Take the above PNS@(V-M)-CSs, and use a laser particle size analyzer to measure the particle size and distribution of PNS@(V-M)-CSs.

2. Morphological observation

Take an appropriate amount of the above PNS@(V-M)-CSs suspension, add a small amount dropwise to the copper grid, negatively stain with 2% phosphotungstic acid solution for 3 minutes, take out the copper grid, absorb the excess negative dye solution with filter paper, and place the copper grid on the front side Place it upwards in a glass dish, and observe its morphology under a transmission electron microscope after it dries naturally.

3. Determination of Encapsulation Efficiency

Micro-centrifugation method was used for measurement, and 0.2 mL of PNS@(V-M)-CSs suspension was accurately measured, added to the top of the micro-gel column, centrifuged at 3000 r/min for 3 min, the centrifuged liquid was collected, and 0.3 mL of distilled water was added to the gel Centrifuge the top of the column at 3000r/min for 1min, collect the centrifugate from the first three tubes, add 0.4ml of methanol for ultrasonic demulsification, then dilute to a 10mL volumetric flask with methanol, and measure the encapsulated drug in 0.2mL of the sample by HPLC concentration. Precisely measure 0.2mL of PNS@(V-M)-CSs suspension, add 0.4ml of methanol, ultrasonically break the emulsion, dilute the methanol to a 10mL volumetric flask, measure the total drug concentration without micro-column centrifugation by HPLC, encapsulate The rate (Encapsulation Efficiency, EE) was calculated according to the formula, and three batches of experiments were repeated according to this method.

EE＝C ₁ /C ₀ ×100%

In the formula, C ₁ is the drug concentration (μl/ml) encapsulated in the 0.2mL sample, and C ₀ is the total drug concentration (μl/ml) without microcolumn centrifugation.

4. Elasticity measurement method

The elasticity of PNS@(V-M)-CSs was measured by the constant pressure extrusion method. A certain amount of PNS@(V-M)-CSs was placed in a high-pressure film extruder and squeezed through 0.05 under nitrogen pressure (0.05MPa). μm polycarbonate film, record the extruded volume within 5 minutes and measure the particle size of PNS@(V-M)-CSs after extrusion, the elastic index (deformability index, DI) was calculated according to the formula, and each sample was measured in triplicate. (References for this method: Chaudhary H, Kohli K, Kumar V. Nano-transfersomes as novel carrier for transdermal delivery [J]. Int J Pharm, 2013, 454(1): 367-380.)

DI＝J×(r _v /r _p ) ²

where J is the volume of PNS@(VM)-CSs extruded through the membrane within 5 min, r _v is the particle size of PNS@(VM)-CSs after extrusion, and r _p is the pore size of the polycarbonate track-etched membrane.

5. Examination method of in vitro skin penetration characteristics

SD rats were taken, killed by cervical dislocation, and placed on a fixed board. The hair from the clavicle to the abdomen of the rat was cut off with surgical scissors, the skin of the abdomen was cut off, and the subcutaneous tissue was carefully removed to ensure the integrity of the cuticle. Rinse with salt water, flatten and absorb the attached water with filter paper, sandwich in aluminum foil, freeze at -80°C, and set aside. During the experiment, the skin was first thawed at room temperature, cut to a suitable size and fixed between the supply pool and the receiver pool (the effective skin area was 0.785cm ² , and the volume of the receiver pool was 5mL), with the epidermis facing the supply pool, and the receiver solution (0.9 % physiological saline-ethanol (4:1) mixture), make the liquid surface contact with the inner layer of the skin, exhaust the air bubbles in the receiving pool, control the temperature of the water bath in the receiving pool at 32°C, and the magnetic stirring speed in the pool is 400r/min, balance After 30 minutes, a new receiving solution was replaced, and 180 μL of the PNS@(VM)-CSs sample to be tested was given. 200 μL samples were taken at several time points in 24 hours, and an equal volume of fresh receiving solution was added in time, and 20 μL samples were taken for HPLC analysis to calculate the cumulative drug penetration.

In the formula, 0.785 is the percutaneous penetration effective area (cm ² ), 5 is the receiving pool volume (mL), 0.2 is the percutaneous penetration test effective volume (mL), and Cn is the drug concentration (mg/mL) measured at the nth sampling point. mL), Ci is the measured concentration (mg/mL) of each point before the sampling point.

Calculation of the steady-state transdermal penetration rate: take the cumulative permeation amount of each sampling point as the ordinate, and the time as the abscissa, draw the percutaneous penetration curve, and perform linear regression on the steady-state penetration section (straight line part) in the curve to obtain a straight line The slope of is the steady-state transdermal penetration rate J (mg/cm ² ·h).

Example 1

The lipid vesicle-micelle coexistence system [PNS@(V-M)-CSs-1] of PNS is composed of the following components by weight:

Specifications and sources of main components: Panax notoginseng total saponins (in line with medicinal standards), Yunnan Botanical Pharmaceutical Co., Ltd.; egg yolk lecithin (80% PC content, injection grade), Shanghai Aiweite Pharmaceutical Technology Co., Ltd.; cholesterol (according to the Chinese Pharmacopoeia 2015 standard), Shanghai Aiweite Pharmaceutical Technology Co., Ltd.; vitamin E (conforming to the Chinese Pharmacopoeia 2015 standard), Sigma company; phosphate buffer solution is 1/15mol/L potassium dihydrogen phosphate solution and 1/15mol/ L disodium hydrogen phosphate solution is a mixed solution with a volume ratio of 53.4:46.6; absolute ethanol, Tianjin Kemiou Chemical Reagent Co., Ltd.

The preparation method of the lipid vesicle and micelle coexistence system of PNS is:

A 3D printed microfluidic mixer is used for preparation, and the method comprises the following steps:

1) Weigh the total saponins of Panax notoginseng, phospholipids, cholesterol and vitamin E in the formula, dissolve them in absolute ethanol, mix them evenly, and obtain the organic phase;

2) Measure the phosphate buffer saline solution of formula quantity as water phase;

3) Under the condition of controlling the mixing temperature at 30°C, the microfluidic experimental conditions were designed, and the two solutions were pushed by the syringe pump according to the flow rate ratio of the water phase and the alcohol phase at 10:1, and the total flow rate of the water-alcohol two-phase was 198 μL/min (Wherein, the flow rate of the aqueous phase is 180 μL/min, and the flow rate of the alcohol phase is 18 μL/min) are transported to the microfluidic mixer for mixing by an airtight syringe.

4) Take the sample at the outlet of the tube and collect it in a vial. Three samples were collected consecutively for each preparation condition. A novel skin drug delivery preparation in which lipid vesicles and micelles coexist is obtained.

Example 2

The lipid vesicle-micelle coexistence system [PNS@(V-M)-CSs-2] of PNS consists of the following components by weight:

Specifications and sources of main components: Panax notoginseng total saponins (in line with medicinal standards), Yunnan Botanical Pharmaceutical Co., Ltd.; egg yolk lecithin (80% PC content, injection grade), Shanghai Aiweite Pharmaceutical Technology Co., Ltd.; cholesterol (according to the Chinese Pharmacopoeia 2015 standard), Shanghai Aiweite Pharmaceutical Technology Co., Ltd.; vitamin E (conforming to the Chinese Pharmacopoeia 2015 standard), Sigma company; phosphate buffer solution is 1/15mol/L potassium dihydrogen phosphate solution and 1/15mol/ L disodium hydrogen phosphate solution is a mixed solution with a volume ratio of 53.4:46.6; absolute ethanol, Tianjin Kemiou Chemical Reagent Co., Ltd.

The preparation method of the lipid vesicle and micelle coexistence system of PNS is:

A 3-D printed microfluidic mixer is used for preparation, and the method comprises the following steps:

1) Weigh the total saponins of Panax notoginseng, phospholipids, cholesterol and vitamin E in the formula, dissolve them in absolute ethanol, mix them evenly, and obtain the organic phase;

2) Measure the phosphate buffer saline solution of formula quantity as water phase;

3) Under the condition of controlling the mixing temperature at 35°C, the microfluidic experimental conditions were designed, and the two solutions were pushed by the syringe pump according to the flow rate ratio of the water phase and the alcohol phase at 10:1, and the total flow rate of the water-alcohol two-phase was 198 μL/min (Wherein, the flow rate of the aqueous phase is 180 μL/min, and the flow rate of the alcohol phase is 18 μL/min) are transported to the microfluidic mixer for mixing by an airtight syringe.

4) Take the sample at the outlet of the tube and collect it in a vial. Three samples were collected consecutively for each preparation condition. A novel skin drug delivery preparation in which lipid vesicles and micelles coexist is obtained.

Example 3

The lipid vesicle-micelle coexistence system [PNS@(V-M)-CSs-3] of PNS is composed of the following components by weight:

Specifications and sources of main components: Panax notoginseng total saponins (in line with medicinal standards), Yunnan Botanical Pharmaceutical Co., Ltd.; egg yolk lecithin (80% PC content, injection grade), Shanghai Aiweite Pharmaceutical Technology Co., Ltd.; cholesterol (according to the Chinese Pharmacopoeia 2015 standard), Shanghai Aiweite Pharmaceutical Technology Co., Ltd.; vitamin E (conforming to the Chinese Pharmacopoeia 2015 standard), Sigma company; phosphate buffer solution is 1/15mol/L potassium dihydrogen phosphate solution and 1/15mol/ L disodium hydrogen phosphate solution is a mixed solution with a volume ratio of 53.4:46.6; absolute ethanol, Tianjin Kemiou Chemical Reagent Co., Ltd.

The preparation method of the lipid vesicle and micelle coexistence system of PNS is:

A 3-D printed microfluidic mixer is used for preparation, and the method comprises the following steps:

1) Weigh the total saponins of Panax notoginseng, phospholipids, cholesterol and vitamin E in the formula, dissolve them in absolute ethanol, mix them evenly, and obtain the organic phase;

2) Measure the phosphate buffer saline solution of formula quantity as water phase;

3) Under the condition of controlling the mixing temperature at 40°C, the microfluidic experimental conditions were designed, and the two solutions were pushed by the syringe pump according to the flow rate ratio of the water phase and the alcohol phase at 20:1, and the total flow rate of the water-alcohol two-phase was 210 μL/min (Wherein, the flow rate of the aqueous phase is 200 μL/min, and the flow rate of the alcoholic phase is 10 μL/min) are transported into the microfluidic mixer by an airtight syringe for mixing.

4) Take the sample at the outlet of the tube and collect it in a vial. Three samples were collected consecutively for each preparation condition. A novel skin drug delivery preparation in which lipid vesicles and micelles coexist is obtained.

Example 4

The lipid vesicle-micelle coexistence system [PNS@(V-M)-CSs-4] of PNS is composed of the following components by weight:

Specifications and sources of main components: Panax notoginseng total saponins (in line with medicinal standards), Yunnan Botanical Pharmaceutical Co., Ltd.; egg yolk lecithin (80% PC content, injection grade), Shanghai Aiweite Pharmaceutical Technology Co., Ltd.; cholesterol (according to the Chinese Pharmacopoeia 2015 standard), Shanghai Aiweite Pharmaceutical Technology Co., Ltd.; vitamin E (conforming to the Chinese Pharmacopoeia 2015 standard), Sigma company; phosphate buffer solution is 1/15mol/L potassium dihydrogen phosphate solution and 1/15mol/ L disodium hydrogen phosphate solution is a mixed solution with a volume ratio of 53.4:46.6; absolute ethanol, Tianjin Kemiou Chemical Reagent Co., Ltd.

The preparation method of the lipid vesicle and micelle coexistence system of PNS is:

A 3-D printed microfluidic mixer is used for preparation, and the method comprises the following steps:

1) Weigh the total saponins of Panax notoginseng, phospholipids, cholesterol and vitamin E in the formula, dissolve them in absolute ethanol, mix them evenly, and obtain the organic phase;

2) Measure the phosphate buffer saline solution of formula quantity as water phase;

3) Under the condition of controlling the mixing temperature at 50°C, the microfluidic experimental conditions were designed, and the two solutions were driven by a syringe pump at a flow rate ratio of 30:1 between the water phase and the alcohol phase, and the total flow rate of the water-alcohol two-phase was 248 μL/min (Wherein, the flow rate of the aqueous phase is 240 μL/min, and the flow rate of the alcohol phase is 8 μL/min) is transported to the microfluidic mixer for mixing by an airtight syringe.

4) Take the sample at the outlet of the tube and collect it in a vial. Three samples were collected consecutively for each preparation condition. A novel skin drug delivery preparation in which lipid vesicles and micelles coexist is obtained.

Preparation of the reference preparation

The micellar PNSMs of the reference preparation 1PNS consists of the following ingredients in parts by weight: [no phospholipids and cholesterol]

Specifications and sources of main components: Panax notoginseng saponins (in line with medicinal standards), Yunnan Plant Pharmaceutical Co., Ltd.; vitamin E (in line with Chinese Pharmacopoeia 2015 standards), Sigma Company; phosphate buffer solution is 1/15mol/L phosphoric acid Potassium dihydrogen solution and 1/15 mol/L disodium hydrogen phosphate solution mixed at a volume ratio of 53.4:46.6; absolute ethanol, Tianjin Kemiou Chemical Reagent Co., Ltd.

The preparation method of the lipid vesicle and micelle coexistence system of PNS is:

A 3-D printed microfluidic mixer is used for preparation, and the method comprises the following steps:

1) Weigh the total panax notoginseng saponins and vitamin E of the formula amount, dissolve in absolute ethanol, mix evenly, and obtain the organic phase;

2) Measure the phosphate buffer saline solution of formula quantity as water phase;

3) Under the condition of controlling the mixing temperature at 30°C, the microfluidic experimental conditions were designed, and the two solutions were pushed by the syringe pump according to the flow rate ratio of the water phase and the alcohol phase at 10:1, and the total flow rate of the water-alcohol two-phase was 198 μL/min (Wherein, the flow rate of the aqueous phase is 180 μL/min, and the flow rate of the alcohol phase is 18 μL/min) are transported to the microfluidic mixer for mixing by an airtight syringe.

4) Take the sample at the outlet of the tube and collect it in a vial. Three samples were collected consecutively for each preparation condition. A novel skin drug delivery preparation in which lipid vesicles and micelles coexist is obtained.

Reference preparation 2PNS liposome (PNSLs) is made up of following components by weight:

Specifications and sources of main components: Panax notoginseng total saponins (in line with medicinal standards), Yunnan Botanical Pharmaceutical Co., Ltd.; egg yolk lecithin (80% PC content, injection grade), Shanghai Aiweite Pharmaceutical Technology Co., Ltd.; cholesterol (according to the Chinese Pharmacopoeia 2015 standard), Shanghai Aiweite Pharmaceutical Technology Co., Ltd.; vitamin E (conforming to the Chinese Pharmacopoeia 2015 standard), Sigma company; phosphate buffer solution is 1/15mol/L potassium dihydrogen phosphate solution and 1/15mol/ L disodium hydrogen phosphate solution is a mixed solution with a volume ratio of 53.4:46.6; absolute ethanol, Tianjin Kemiou Chemical Reagent Co., Ltd.

The preparation method of PNS liposome is:

Adopt ethanol injection method to prepare, and this method comprises the following steps:

1) Weigh the formulated amount of phospholipids, cholesterol and vitamin E, and ultrasonically treat them for 5 minutes under the conditions of 40KHZ and 250W, so that they are completely dissolved in 2mL of absolute ethanol to obtain the organic phase;

2) Weigh the PNS of the formula amount, dissolve it in the phosphate buffer saline, and obtain the water phase;

3) Under the conditions of 25°C and magnetic stirring (500r/min), inject the organic phase into the water phase slowly at a uniform speed. After the injection, continue to stir for 10 minutes until the ethanol evaporates. After standing at room temperature for 2 hours, under a certain nitrogen pressure Pass through polycarbonate membranes with pore diameters of 0.10 and 0.05mm in turn (3 times for 0.10mm pore diameter membranes, 2 times for 0.05mm pore diameter membranes; extrusion pressures are 0.05 and 0.30MPa respectively) to obtain PNSLs.

The lipid vesicle-micelle coexistence system of PNS, PNS micelles and PNS liposomes prepared in the above examples were evaluated for elasticity, particle size and encapsulation efficiency determination, morphology observation and two main components in PNS The penetration characteristics of Rg ₁ and Rb ₁ in isolated skin were investigated, and the results obtained are shown in Table 1, Table 2, and Figure 3. The results show that there are two kinds of nanoparticles with different particle sizes in the lipid vesicle-micelle coexistence system of PNS, which is also confirmed by transmission electron microscopy, among which the larger volume is lipid vesicles, and the smaller volume is They are mixed micelles, both of which are round or quasi-round in shape, with uneven size distribution and no obvious aggregation. The lipid vesicle-micelle coexistence system of PNS has good elasticity and transdermal penetration rate.

Table 1 The particle size, elasticity and encapsulation efficiency of the lipid vesicle-micelle coexistence system of PNS

The steady-state transdermal penetration rate of the lipid vesicle-micelle coexistence system of table 2PNS

Experimental study on the percutaneous penetration mechanism of the preparation of the present invention:

1. Materials and methods

1.1 Drugs:

① Panax notoginseng total saponins (in line with medicinal standards), Yunnan Phytopharmaceutical Co., Ltd. ② Egg yolk lecithin (80% PC content, injection grade), Shanghai Aiweite Pharmaceutical Technology Co., Ltd. ③Cholesterol (according to the Chinese Pharmacopoeia 2015 standard), Shanghai Evert Pharmaceutical Technology Co., Ltd. ④Vitamin E (according to the Chinese Pharmacopoeia 2015 standard), Sigma company. ⑤ Rhodamine 6G (R6G), Shanghai Aladdin Biochemical Technology Co., Ltd. ⑥ Coumarin 153 (C153), Xiamen Senkesi Technology Co., Ltd. ⑦4',6-diamidino-2-phenylindole (DAPI), Hangzhou Chengrui Biotechnology Co., Ltd.

1.2 Preparation of lipid vesicle-micelle coexistence system of fluorescently labeled PNS

According to the in vitro transdermal evaluation results, the lipid vesicle-micelle coexistence system of fluorescently labeled PNS was prepared with the formulation and process of PNS@(V-M)-CSs-1 with the best transdermal performance. The preparation process is as follows: Weigh the prescribed amount of VE, EPC, CH, C153, PNS, dissolve in 2mL of absolute ethanol to obtain a lipid solution; weigh the prescribed amount of R6G, dissolve it in 23mL of PBS buffer solution with a pH value of 7 as an aqueous solution. According to the flow rate ratio of the water phase and the alcohol phase at 10:1, the total flow rate of the water and alcohol phase is 198 μL/min (the flow rate of the water phase is 180 μL/min, and the flow rate of the alcohol phase is 18 μL/min) is delivered to the microfluidic mixing chamber by an airtight syringe. Mix in a mixer. Both the mixer and the pipeline connected to the mixer were submerged in a water bath to control the mixing temperature to 30°C, and collected at the outlet of the pipeline. The lipid vesicle-micelle coexistence system of PNS co-labeled with R6G and C153 was obtained. The sample bottle was wrapped in tinfoil and placed in a refrigerator at 4°C for standby. The entire preparation process was protected from light.

1.3 Grouping and administration

SD rats were taken, killed by cervical dislocation, and placed on a fixed board. The hair from the clavicle to the abdomen of the rat was cut off with surgical scissors, the skin of the abdomen was cut off, and the subcutaneous tissue was carefully removed to ensure the integrity of the cuticle. Rinse with salt water, flatten and absorb the attached water with filter paper, sandwich in aluminum foil, freeze at -80°C, and set aside. During the experiment, the skin was first thawed at room temperature, cut to a suitable size and fixed between the supply pool and the receiver pool (the effective skin area was 0.785cm ² , and the volume of the receiver pool was 5mL), with the epidermis facing the supply pool, and the receiver solution (0.9 % physiological saline-ethanol (4:1) mixture), make the liquid surface contact with the inner layer of the skin, exhaust the air bubbles in the receiving pool, control the temperature of the water bath in the receiving pool at 32°C, and the magnetic stirring speed in the pool is 400r/min, balance After 30 minutes, a new receiving solution was replaced, and 180 μL of R6G and C153 co-labeled PNS lipid vesicle-micelle coexistence system (test group), PNS micelle group (reference preparation group 1) were added to the supply pool. ), PNS liposome group (reference preparation group 2), a total of three groups, each group of 10 skin samples, record this time as 0h. The skins were removed at 2, 8, and 12 hours respectively. Immediately scrub each skin sample with saline cotton balls and alternately rinse three times with saline to remove residues from the skin surface. The whole process was strictly protected from light at room temperature.

1.4 Preparation of skin frozen sections

Before using the cryostat, pre-cool the machine to -20°C. Put the skin tissue to be sliced on the sample holder, and after freezing and hardening, trim the tissue into long strips for easy embedding. Add an appropriate amount of embedding agent dropwise on the sample holder to form a circular shape, put it in the freezer to freeze and semi-solidify, put the tissue to be sliced longitudinally, continue to add embedding agent dropwise, put it in the freezer and continue to freeze and harden. Adjust the section thickness to 7 μm, fix the sample holder with skin tissue, slice vertically, trim to the desired tissue section, and start formal sectioning. Samples were nuclei-stained with DAPI, mounted in anhydrous glycerol, covered with a coverslip, and capped.

1.5 Confocal microscopy (CLSM) observation of skin frozen sections

The distribution of fluorescence in skin tissue sections at different time points was observed using a confocal microscope. The parameters of the confocal microscope were kept consistent in scanning at different time points, and the objective lens was adjusted to 10 times. The excitation wavelength of R6G is 570nm, excited by krypton laser, and the emission of fluorescence is red light. The excitation wavelength of C153 is 408nm, excited by helium laser, and the emitted fluorescence is green. The excitation wavelength of DAPI is 461nm, excited by helium laser, and the emitted fluorescence is blue. R6G and C153 are mainly used to explore the transdermal depth of the three carriers and the change of FRET efficiency during the transdermal process, so as to explore the transdermal mechanism of the three preparations; DAPI is a fluorescent dye that can strongly bind to DNA. The DNA binding inside is mainly used for skin cell positioning, which is convenient for judging the depth of the carrier reaching the skin. The entire procedure was performed in the dark to avoid the influence of ambient light.

1.6 Fluorescence resonance energy transfer to explore the penetration mechanism of PNS@(V-M)-CSs

Fluorescence resonance energy transfer (FRET) technology was used to investigate the transdermal absorption mechanism of the system. R6G was selected as the acceptor because of its strong hydrophilicity, which can exist in the polar region (inner and outer aqueous phase) of the formulation; C153 was chosen as the donor because of its strong hydrophobicity. Under the condition that the excitation light wavelength is 408nm, scan the fluorescence emission spectra of PNS@(V-M)-CSs-1, PNSLs and PNSMs within 500-600nm, and calculate the fluorescence intensity of fluorescent labels at each time according to the fluorescence intensity of the spectrum according to the following formula Changes in the FRET efficiency of the three preparations (references for this method: Mandal S, Kuchlyan J, Banik D, et al.Ultrafast FRET to Study Spontaneous Micelle-to-Vesicle Transitions in an Aqueous Mixed Surface-Active Ionic-Liquid System[J ]. Chem Phys Chem, 2014, 15(16): 3544-3553.).

Eff _FRET (%)＝IR/(IG+IR)

In the formula, Eff _FRET is the FRET efficiency; IR is the fluorescence intensity of C153 at 408nm; IG is the fluorescence intensity of R6G at 570nm.

1.7 Statistical methods

SPSS 24.0 software was used for statistical analysis of the experimental data, and the comparison of the means between the two groups of data was performed by the t test method, and all data were expressed as mean ± standard deviation (x ± s). When p<0.05, the difference is significant, and when p<0.01, the difference is extremely significant.

2. Experimental results

2.1 Results of permeation experiments of fluorescently labeled preparations

The CLSM images of the three R6G and C153 co-labeled preparations at different time points are shown in Figures 4, 5, and 6. It can be seen from the figures that after 2 hours of transdermal administration, PNS@(V-M)-CSs can be observed in the skin R6G and C153 in -1 have obvious fluorescence intensity, while the other two preparations have almost no fluorescence; after 8h, the R6G transdermal delivery in PNSMs is obvious, and the C153 in it is still mostly isolated outside the stratum corneum, PNS lipid Both R6G and C153 had little penetration in the body group, but the fluorescence intensity and penetration depth of the two fluorescent substances in PNS@(V-M)-CSs-1 were still higher than the other two; after 12 hours of transdermal administration, the It was observed that the fluorescent substances in PNSLs were retained on the surface of the skin, while in PNSMs only R6G was partially penetrated, and C153 was very weak. preparations. CLSM observation results showed that PNS@(V-M)-CSs-1 had stronger transdermal delivery ability to strongly hydrophilic R6G and strongly lipophilic C153 at the three time points of transdermal administration: 2h, 8h and 12h. PNSLs group and PNSMs group at the time point.

2.2 Experimental results of investigation on the penetration mechanism of fluorescently labeled preparations

The FRET results of PNS@(VM)-CSs-1 group, PNSLs group and PNSMs at 2h, 8h, and 12h time points are shown in Figure 7, Table 3, as shown in the chart, PNS@(VM)-CSs-1 group At 2h, 8h, and 12h, they were (55.23±2.833)%, (28.34±2.815)% and (48.62±11.02)%, showing a process of first decreasing and then rising; the PNSLs group was (40.78 ±12.67)%, (48.77±11.94)% and (58.86±1.881)%, showing a rising process; while the PNSMs group were (67.50±0.637)%, (64.70±2.350) at 2h, 8h, and 12h % and (11.21±1.315)%, showed a relatively stable Eff _FRET change in the first 8 hours, but then decreased significantly in 12 hours. By calculating the Eff _FRET changes of the three preparations, it can be deduced that the mixed micelles and lipid vesicles in PNS@(VM)-CSs-1 cannot penetrate the skin synchronously, and the mixed micelles and strong fat-soluble components will The lipid vesicles and water-soluble components penetrate first, and then the lipid vesicles and water-soluble components will penetrate the stratum corneum into the deep skin and accumulate deep in the skin. As for the PNSLs group and PNSMs group, both of them cannot penetrate the skin in a complete form, and cannot reach the deep part of the skin, and can only accumulate on the surface of the skin.

Table 3 Eff _FRET (%) of R6G and C153 co-labeled preparations at different time points (x±s, n=3)

3. Experimental conclusion

In this experiment, a 3D printed micromixer was used to prepare a lipid vesicle-micelle hybrid system of PNS with controllable physical and chemical properties by using its good microenvironment controllability. Through the investigation of skin penetration experiments and the study of the mechanism of transdermal penetration, it has been fully verified that the lipid vesicle-micelle hybrid system of PNS has good transdermal penetration properties, indicating that the lipid vesicle-micelle hybrid system is a new type of transdermal preparation. , can exert excellent transdermal potential.

In addition, it should be noted that the names of specific reagents and raw materials described in this specification may be different. All equivalent or simple changes made according to the principles and embodiments described in the patent concept of the present invention are included in the protection scope of the patent of the present invention. Those skilled in the art to which the present invention belongs can make various modifications or supplements to the described specific embodiments or adopt similar methods to replace them, as long as they do not deviate from the structure of the present invention or exceed the scope defined in the claims. All should belong to the protection scope of the present invention.

Claims (9)

1. A microfluidically prepared skin formulation for the co-existence of lipid vesicles and micelles, the skin formulation comprising the following components:

50-200 parts of total saponins of panax notoginseng, 250-350 parts of phospholipids, 30-60 parts of cholesterol, 4-7 parts of vitamin E, 15000-30000 parts of phosphate buffer solution and 1500-3000 parts of absolute ethyl alcohol.

2. The microfluidic preparation of claim 1, wherein the phosphate buffer solution is a mixture of 1/15mol/L potassium dihydrogen phosphate solution and 1/15mol/L disodium hydrogen phosphate solution at a volume ratio of 53.4:46.6.

3. The microfluidics-prepared lipid vesicle and micelle coexisting skin drug delivery formulation according to claim 1, wherein the skin drug delivery formulation comprises the following components:

180 parts of total saponins of panax notoginseng, 300 parts of phospholipids, 45 parts of cholesterol, 5 parts of vitamin E, 23000 parts of phosphate buffer solution and 2000 parts of absolute ethyl alcohol.

4. A method for preparing a microfluid of a skin administration preparation in which lipid vesicles and micelles coexist, comprising the following preparation steps:

weighing a certain amount of total saponins of panax notoginseng, phospholipid, cholesterol and vitamin E, dissolving in absolute ethyl alcohol, and uniformly mixing to obtain an alcohol phase, wherein 50-200 parts of total saponins of panax notoginseng, 250-350 parts of phospholipid, 30-60 parts of cholesterol, 4-7 parts of vitamin E and 1500-3000 parts of absolute ethyl alcohol;

step two, taking 15000-30000 parts of phosphate buffer solution as a water phase;

step three, designing micro-fluid experimental conditions at the mixing temperature of 25-55 ℃, controlling the ratio of the aqueous phase to the alcohol phase and the total flow rate of the aqueous phase to the alcohol phase under the pushing of a syringe pump, and conveying the two solutions into a micro-fluid mixer for mixing by an airtight syringe;

and step four, obtaining a sample at the outlet of the pipeline, and collecting the sample in a tube-type bottle to obtain the novel skin administration preparation in which the lipid vesicles and the micelles coexist.

5. The method for preparing a skin preparation for co-existence of lipid vesicles and micelles as in claim 4, wherein in the first step, the ratio of total saponins of pseudo-ginseng to phospholipids is 0-1:1; the ratio of parts by weight of cholesterol to parts by weight of phospholipids is in the range of 0 to 0.3:1.

6. The method for preparing a microfluidics preparation of a lipid vesicle and micelle coexisting skin drug delivery preparation according to claim 5, wherein the ratio of the weight parts of total saponins of pseudo-ginseng to the weight parts of phospholipids is 0.6:1; the ratio of parts by weight of cholesterol to parts by weight of phospholipids was 0.15:1.

7. The method for preparing the microfluidic device of the skin drug delivery preparation with the coexistence of the lipid vesicles and the micelles as in claim 4, wherein the microfluidic device used in the third step is a cross-shaped microfluidic device with independent mixing chambers, which is prepared by using a 3D printing technology and is based on a microfluidic hydrodynamic focusing method.

8. The method for preparing a microfluidics preparation of a lipid vesicle and micelle coexisting skin drug delivery preparation according to claim 4, wherein in the third step, when the flow rate of the fixed center alcohol phase is 20 μl/min, the flow rate ratio of the aqueous phase to the alcohol phase is set to 2.5:1 or 5:1 or 10:1 or 15:1 or 20:1 or 25:1 or 30:1;

when the fixed aqueous-alcoholic phase flow rate ratio was 10:1, the total aqueous-alcoholic phase flow rates were set to 33, 66, 132, 198, 264, 330 and 660. Mu.l/min,

when the fixed water-alcohol phase flow speed ratio is 20:1, the total flow rate of the water-alcohol phase is set to be 42, 84, 105, 168, 210, 420 and 630 mu l/min;

when the fixed aqueous-alcoholic phase flow ratio was 30:1, the total aqueous-alcoholic phase flow rates were set to 31, 62, 93, 155, 248, 310 and 620 μl/min.

9. The method for preparing the skin drug delivery preparation by coexistence of lipid vesicles and micelles as in claim 7, wherein the cross-shaped microfluidic device is provided with three inlet pipes at the upper end and a cross-shaped outlet pipe at the lower end.

Images