WO2019148810A1 - Nanoparticle formulation for oral insulin delivery, and preparation method therefor - Google Patents

Abstract

A nanoparticle formulation for oral insulin delivery, and a preparation method therefor. Said nanoparticles have a core-shell structure, wherein said core is a nanocomposite core consisting of a chitosan quaternary ammonium salt and an insulin, and said shell is a hyaluronic acid or a thiolated hyaluronic acid coated on the surface of said core. The preparation method therefor comprises: preparing said insulin-loaded nanoparticles by applying an FNC technology, and coating the surface of said nanoparticles with said hyaluronic acid.

Description

Oral insulin nanoparticle preparation and preparation method thereof Technical field

The invention belongs to the field of biomedical technology, and more particularly to an oral insulin nanoparticle preparation and a preparation method and application thereof.

Background technique

Insulin has been the drug of choice for insulin-dependent diabetes patients since its inception. Insulin is mainly administered through the subcutaneous route and requires repeated administration over a long period of time, which causes great pain and low compliance, and also causes a series of safety problems such as allergic reactions, subcutaneous fat atrophy or hyperplasia, and tolerance. Sexuality and so on. Oral administration is considered to be a more suitable route of administration than a series of problems caused by subcutaneous injection. Oral administration can effectively reduce hyperinsulinemia, prevent the increase in body mass associated with systemic insulin therapy, and reduce the risk of hypoglycemia. At the same time, oral insulin can be directly involved in the metabolism of glucose in the liver through absorption into the portal vein through the gastrointestinal tract. Simulate the normal physiological pathways of the human body. However, due to the easy degradation and degeneration of insulin in the gastrointestinal tract, and the low absorption efficiency in the small intestine, the bioavailability of oral insulin is extremely low. Protein-loaded nanoparticle systems, such as liposomes, micelles, polymer/protein composite particles, inorganic particles, etc., can effectively improve the oral availability of insulin and are widely used for oral delivery of insulin.

The surface of the small intestine epithelium is covered with a layer of mucus that blocks and quickly removes foreign particles and harmful substances into the small intestine. After the oral drug-loaded granules reach the small intestine, they must first pass through the mucus barrier to be absorbed by the small intestine. Overcoming this barrier is mainly achieved by two strategies: mucus penetration and mucus adsorption. Studies have found that particles with hydrophilic and electrically neutral or electronegative surface have little force and can effectively pass through the mucus layer to reach intestinal epithelial cells. However, due to its "inert" surface properties, the particles are difficult to be absorbed by the intestinal epithelial cells; and due to their lack of interaction with the intestinal mucus, the retention time in the small intestine is reduced; this series of reasons lead to the bioavailability of the mucus penetration system. Low. The mucus-adsorbing material can interact with the mucus layer by means of electrostatic force, hydrogen bonding, hydrophobic force, covalent bonding, etc., increasing the efficiency of the particles entering the mucus layer and the residence time of the particles in the small intestine, and promoting the absorption of insulin in the small intestine. . However, due to the strong interaction between the mucus-adsorbing particles and the mucus, the particles are easily trapped in the mucus layer and quickly cleared by the mucus layer, which cannot reach the epithelium of the small intestine, resulting in low bioavailability of insulin. There is still a lack of an insulin delivery system that effectively overcomes the mucus barrier of the small intestine.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the existing insulin delivery system, and obtain a core-shell structure loaded with insulin by using the modified chitosan quaternary ammonium salt and thiolated hyaluronic acid. Nanoparticles, which have the function of adhering and penetrating small intestinal mucus, improve the absorption of insulin in the small intestine, have high oral bioavailability, and can provide a convenient, painless, safe and efficient treatment for type I diabetic patients. The mode of administration.

A first object of the invention is to provide an insulin-loaded nanoparticle.

A second object of the present invention is to provide a method of preparing the loaded insulin nanoparticles.

A third object of the invention is to provide the use of said loaded insulin nanoparticles.

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

An insulin-loaded nanoparticle having a core-shell structure, a core comprising a chitosan quaternary ammonium salt and a nanocomposite core composed of insulin, and the shell being hyaluronic acid or thiolated hyaluronic acid coated on the core surface .

The chitosan quaternary ammonium salt modified by quaternization in the nanoparticle of the invention has good water solubility under physiological conditions, and has better ability to open tight junction; and hyaluronic acid has good biocompatibility. The thiolated hyaluronic acid modified by thiol can form a disulfide bond with mucin in the intestinal mucus and enhance mucus adsorption.

Preferably, the chitosan quaternary ammonium salt has a molecular weight of 50 kDa to 200 kDa (preferably 50 kDa), a degree of quaternization of 10% to 70% (preferably 43%); and a molecular weight of thiolated hyaluronic acid of 3.4 kDa ～200 kDa (preferably 35 kDa), the degree of thiolation is 5% to 30% (preferably 12.3%).

Preferably, the nanoparticles have a particle size of 50 to 200 nm (preferably 100 nm).

Preferably, the nanoparticles have a PDI of from 0.1 to 0.2.

Preferably, the potential of the nanoparticles is from -5 mV to -30 mV.

Preferably, the nanoparticle has a drug loading of 25% to 60%.

The insulin-loaded nanoparticles of the invention have the function of adhering and penetrating the intestinal mucus, improving the absorption of insulin in the small intestine, and having high oral bioavailability; therefore, the present invention also claims to protect the above-mentioned nanoparticles in the preparation of oral insulin preparations. application.

An oral insulin pharmaceutical preparation comprising the above-described insulin-loaded nanoparticles.

Preferably, the pharmaceutical formulation further comprises a pharmaceutically acceptable excipient.

Preferably, the pharmaceutical preparation is a lyophilized preparation.

Preferably, the pharmaceutical preparation is a capsule.

A method of preparing a loaded insulin nanoparticle, comprising the steps of:

S1. The chitosan quaternary ammonium salt solution is passed through channels 1 and 2, and the insulin solution is passed through 3 and 4, and mixed into the vortex mixing region to obtain a nanocomposite core composed of insulin and chitosan quaternary ammonium salt; Flow, flow rate is 2mL / min ~ 50mL / min (preferably 5mL / min ~ 50mL / min, more preferably 40mL / min);

S2. The mixture obtained by S1 is passed through channels 1 and 2, hyaluronic acid or thiolated hyaluronic acid solution through channels 3 and 4, and mixed into the vortex mixing region to obtain surface coated hyaluronic acid or thiolated hyaluronic acid nanometer. Particles; four channels of liquid flow at a constant rate, flow rate of 2mL / min ~ 50mL / min (preferably 5mL / min ~ 50mL / min, more preferably 40mL / min);

Preferably, the present invention employs FNC technology to prepare loaded insulin nanoparticles in a multi-inlet vortex mixer, the multi-inlet vortex mixer (shown in Figures 1A, 1B) including a first component at the top, a central portion a second member and a third member located at a lower portion, the first member, the second member and the third member being cylinders having the same diameter; the first member being provided with a plurality of passages (preferably 4, respectively, the passage 1 Channel 2, channel 3, channel 4), the second component is provided with a vortex mixing zone and a plurality of flow guiding zones, the third component is provided with a channel; the channel of the first component is in fluid communication with the flow guiding zone of the second component; The flow guiding regions are all in fluid communication with the vortex mixing region; the vortex mixing region of the second component is in fluid communication with the passage of the third component; and has high flux and strong controllability, and the prepared nanoparticles are uniformly distributed and the particle diameter is relatively high. Small, small differences between batches; the above-mentioned techniques and devices are described in the inventor's prior application number PCT/US2017/014080.

Preferably, the pH of the insulin solution is 7-9 (preferably 8.0); specifically, the insulin is dissolved in a HCl solution of pH=2, and then the pH is adjusted to 7-9 (preferably 8.0) with a NaOH solution.

Preferably, the degree of quaternization of the chitosan quaternary ammonium salt is from 10% to 70% (preferably 43%); the degree of thiolation of the thiolated hyaluronic acid is from 5% to 30% (preferably 12.3) %).

Preferably, the concentration of the chitosan quaternary ammonium salt is 0.2 to 3 mg/mL (preferably 1 mg/mL), and the concentration of the insulin solution is 0.5 to 3 mg/mL (preferably 2 mg/mL).

Preferably, the concentration of the hyaluronic acid or thiolated hyaluronic acid is 0.25 to 1 mg/mL (preferably 1 mg/mL).

Preferably, the mass ratio of the insulin to the chitosan quaternary ammonium salt is 0.5 to 2.

Preferably, the chitosan quaternary ammonium salt is prepared by dissolving chitosan in a 2 wt% acetic acid solution, heating to 80 ° C, and then adding glycidyl trimethylammonium chloride to the solution. The aqueous solution of GTMAC) was reacted at 80 ° C for 24 h to obtain the final product chitosan quaternary ammonium salt (HTCC).

Preferably, the thiolated hyaluronic acid is prepared by dissolving hyaluronic acid (HA) in double distilled water, adding an ion exchange resin for 0.5 h, filtering off the resin, and adjusting the pH of the solution with tetrabutylammonium hydroxide. To 7.04, lyophilization gave the product HA-TBA. The product HA-TBA obtained in the first step was dissolved in DMSO, and 4-dimethylaminopyridine, dithiodipropionic acid, di-tert-butyl dicarbonate was added, and reacted at 45 ° C for 20 h. After dialysis against water, DMSO was removed, and then precipitated twice in acetone, dissolved in double distilled water, and lyophilized. The lyophilized product was dissolved in double distilled water, the pH was adjusted to 8.5, dithiothreitol was added, the reaction was carried out for 3 h, the pH was adjusted to 3.5, once precipitated in ice ethanol, reconstituted in double distilled water, and lyophilized to obtain Thiolated hyaluronic acid (HA-SH).

The invention realizes thiolated hyaluronic acid by thiolation modification of the mucus penetrating material-hyaluronic acid, and applies thiolated hyaluronic acid to the surface of the loaded insulin nanoparticle by FNC technology, so that it can be combined with the small intestine The mucus layer has a strong interaction, compared with the simple use of hyaluronic acid as a coating layer, increasing the retention time of the particles into the intestinal mucus layer and in the small intestine; meanwhile, after entering the small intestinal mucus layer, the thiolated hyaluronic acid coating It can gradually degrade and detach over time, which has better mucus permeability than the simple nano-composite core of chitosan quaternary ammonium salt, reduces the nanoparticles retained in the mucus layer, and increases the particles reaching the intestinal epithelium; During the process of the granule passing through the mucus layer to reach the intestinal epithelium, the outer thiolated hyaluronic acid coating layer is detached, and the chitosan quaternary ammonium salt having the function of opening the intestinal epithelium tightly connected is exposed, and the insulin is promoted to pass through the cell. The road is absorbed by the small intestine. The insulin nano preparation of the invention can effectively control blood sugar after oral administration, and the blood sugar change is more stable than the subcutaneous injection, and has good blood sugar lowering effect and high bioavailability. The oral insulin preparation of the invention can effectively control blood sugar, greatly reduce unnecessary pain of diabetic patients, and is safe and convenient.

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

The nanoparticle of the invention has a high encapsulation rate and/or a drug loading amount and has the function of adhering and penetrating the intestinal mucus, improving the absorption of insulin in the small intestine, and having high oral bioavailability; the insulin nanometer The preparation can effectively control blood sugar after oral administration, and the blood sugar change is more stable than the subcutaneous injection, has good blood sugar lowering effect and high bioavailability, can effectively control blood sugar, and greatly alleviate unnecessary pain of diabetic patients. Safe and convenient.

DRAWINGS

1 is a exemplarily depicted multi-inlet vortex mixer for preparing nanoparticles of the present invention; FIG. 1A is a state in which the first member, the second member, and the third member are assembled and connected to an external pipe; FIG. 1 is a bottom view of the first component; FIG. 1B-2 is a top view of the second component; FIG. 1B-3 is a top view of the third component; FIG. 1C shows the apparatus for preparing nanoparticles in Example 1, FIG. 1 shows a syringe, a high pressure pump, a plastic tube and a multi-inlet vortex mixer, and Figure 1C-2 is an enlarged view of a multi-inlet vortex mixer connected to a plastic tube.

Figure 2 is a schematic illustration of a multi-inlet vortex mixer and a schematic representation of the preparation of nanoparticles prepared in accordance with the present invention.

Figure 3 shows the nanocomposite core (NC) obtained at different flow rates in Example 3. The HTCC concentration was 1 mg/mL and the insulin concentration was 2 mg/mL.

Figure 4 shows the nanocore (NC) obtained in the ratio of different insulin to HTCC in Example 3, with a flow rate of 40 mL/min and a HTCC concentration of 1 mg/mL.

Figure 5 shows the encapsulation efficiency and drug loading of the corresponding NC under different insulin/HTCC ratios in Example 3.

Figure 6 shows NP _HA and NP _HA-SH prepared in Example 3 at a flow rate of 40 mL/min.

Figure 7 shows a transmission electron micrograph of NC, NP _HA and NP _HA-SH prepared under preferred conditions.

Figure 8 is a graph showing the insulin release profile of the simulated gastrointestinal environment in Example 5.

Figure 9 is a graph showing the insulin release profile under physiological conditions in Example 5.

Figure 10 is a DSC curve showing the lyophilized powder of the mucin, HA-SH/mucin blend and HA-SH/mucin after incubation in Example 6.

Figure 11 is a fluorescent image of NC, NP _HA and NP _HA-SH in the photobleaching process in Example 7.

Figure 12 is a graph showing the relationship between the average fluorescence intensity of the photobleaching zone in Example 7 as a function of time.

Figure 13 shows the distribution of NP _HA and NP _HA-SH in the E12 monolayer in Example 8, wherein HTCC is labeled with RITC and insulin is labeled with Cy5.

Figure 14 shows the distribution of NP _HA and NP _HA-SH in the E12 monolayer in Example 8, wherein HA and HA-SH are labeled with Rhodamine 123 and insulin is labeled with Cy5.

Figure 15 is a fluorescent image of the small intestine before and after mucus removal after treatment with three kinds of particles in Example 9.

Figure 16 is a graph showing the average fluorescence intensity of the surface of the small intestine before and after mucus removal after treatment with three kinds of particles in Example 9.

Figure 17 is a graph showing the change in transmembrane resistance of insulin-added or nanoparticle cells in the subsequent Example 10 as a function of time.

Figure 18 is a graph showing the apparent permeability coefficient of different particles transporting insulin across the caco2 monolayer in Example 10.

Figure 19 shows the tight junction of caco2 cells before and after the addition of NP _HA-SH particles in Example 10.

Figure 20 is a graph showing the absorption of insulin in the small intestine after oral administration of insulin solution or insulin-loaded granules in Example 11.

Figure 21 shows the blood glucose lowering curve after oral administration of insulin granules or subcutaneous injection of insulin solution in Example 12.

Figure 22 is a graph showing the change in plasma concentration of oral NP _HA , NP _HA-SH , insulin solution, and subcutaneous injection of insulin solution in Example 13.

Figure 23 shows NC nanoparticles obtained in Example 14 using different degrees of quaternization of HTCC.

Detailed ways

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

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

Example 1 Synthesis of Chitosan Quaternary Ammonium Salt (HTCC)

2 g of chitosan was dissolved in 100 mL of 2 wt% acetic acid solution, heated to 80 ° C, and 5 mL of aqueous solution of glycidyl trimethylammonium chloride (GTMAC) was slowly added dropwise to the solution, and reacted at 80 ° C for 24 h to obtain a solution. After cooling, it was precipitated twice in 10 volumes of acetone, and the precipitate was re-dissolved in double distilled water, dialyzed against water for 3 days, and lyophilized to obtain a final product HTCC. The GTMAC aqueous solution has a mass concentration of 20% and the HTCC quaternization degree is 43%. Further, HTCC having a degree of quaternization of 12% and 23%, respectively, can be obtained by adjusting the volume of GTMAC to 2 mL or 4 mL.

Example 2 Synthesis of thiolated hyaluronic acid (HA-SH)

1. Dissolve hyaluronic acid (HA) in double distilled water at a concentration of 5 mg/mL, add ion exchange resin (resin to hyaluronic acid mass ratio of 3:1), stir for 0.5 h, filter off the resin, and use tetrabutyl hydrogen. Ammonium Oxide The pH of the solution was adjusted to 7.04 and lyophilized to give the product HA-TBA.

2. Dissolve HA-TBA (1.5g) in DMSO at a concentration of 0.5mg/mL, and add 4-dimethylaminopyridine (0.35g), dithiodipropionic acid (2.4g), di-dicarbonate Butyl ester (0.2 mL) was reacted at 45 ° C for 20 h. After cooling to room temperature, the solution was dialyzed against double distilled water to remove DMSO, and then precipitated twice in 10 volumes of acetone, dissolved in double distilled water, dialyzed against water for 3 days, and lyophilized. The lyophilized product (0.5 g) was dissolved in double distilled water, the pH was adjusted to 8.5, dithiothreitol (0.5 g) was added, the reaction was carried out for 3 hours, the pH was adjusted to 3.5, and the solution was once precipitated in ice ethanol, and dissolved again. In double distilled water, lyophilized hyaluronic acid (HA-SH) is obtained by lyophilization. The degree of thiolation of HA-SH was 12.3%. Among them, the mass of the dithiodipropionic acid was adjusted to 1.5 g and 5.0 g, and HA-SH having a degree of thiolation of 8.4% and 32.3%, respectively, was obtained.

Example 3 Preparation of Nanoparticles Using FNC Technology

The FNC technique is a technique for preparing drug-loaded nanoparticles by electrostatic interaction in an aqueous phase (described in the inventor's prior application number PCT/US2017/014080), and the preparation process is mainly carried out in a multi-inlet vortex mixer. With high flux and strong controllability, the prepared nanoparticles are evenly distributed and have small particle size, and the difference between batches is small. Due to the absence of organic solvents in the whole process, it is especially suitable for the formulation of biological macromolecules such as proteins and nucleic acids. The multi-inlet vortex mixer (Fig. 1A, Fig. 1B) includes a first component at the upper portion, a second component at the middle portion, and a third component at the lower portion, the first component, the second component, and the third component being a cylinder having the same diameter; the first member is provided with a plurality of passages (preferably four, respectively, channel 1, channel 2, channel 3, channel 4), and the second member is provided with a vortex mixing region and a plurality of flow guiding regions, The third member is provided with a passage; the passage of the first member is in fluid communication with the flow guiding region of the second member; the flow guiding region of the second member is in fluid communication with the vortex mixing region; the vortex mixing region of the second member and the passage of the third member Fluid communication.

1. Preparation of insulin and HTCC nanocomposite core

HTCC was dissolved in water at a concentration of 1 mg/mL. The insulin was dissolved in a HCl solution of pH=2, and then the pH was adjusted to 8.0 with a NaOH solution, and the concentration of the insulin solution was 0.5 to 3 mg/mL. The HTCC solution and the insulin solution are respectively placed in four syringes, and the four syringes are respectively placed on the high pressure pump, and the injection holes of each syringe are respectively sealed and connected to the respective ends of the plastic tubes 1 to 4, and the other ends of the plastic tubes are respectively passed. The connecting member is sealingly coupled to the four passages of the first component of the multi-inlet vortex mixer. The first member, the second member and the third member of the multi-inlet vortex mixer are connected by bolt sealing, and the passage of the third member is sealingly connected to one end of the plastic tube 5 through the connecting member, and the other end of the plastic tube 5 is connected to the collecting container; 1C shows the apparatus for preparing nanoparticles according to the present embodiment, FIG. 1C-1 shows a syringe, a high pressure pump, a plastic tube and a multi-inlet vortex mixer, and FIG. 1C-2 shows a multi-inlet vortex mixer connected with a plastic tube. Enlarged image.

The high pressure pump was turned on, and the HTCC solution and the insulin solution and the insulin solution were simultaneously introduced into the multi-inlet vortex mixer through the plastic tube 1-4 at a flow rate of 25 mL/min, and mixed in the vortex mixing region of the second member; the HTCC solution was distributed at In the first and second channels, the insulin solution is distributed in the third and fourth channels, as shown in FIG. 2, the flow rates of the four channels are the same, and the flow rate of the channel is adjusted, 2 mL/min, 5 mL/min, 10 mL/min, 20 mL/min, 30 mL/min, 40 mL/min, 50 mL/min, the two solutions were mixed through four channels to the vortex mixing zone to obtain a nanocomposite core (NC) of insulin and HTCC.

Figure 3 shows the NC obtained at different flow rates. The HTCC concentration was 1 mg/mL and the insulin concentration was 2 mg/mL. When the flow rate is less than 20 mL/min, the particle size increases with decreasing flow rate, and when the flow rate is between 20 and 50 mL/min, the particle size does not change. When the flow rate was 40 mL/min, the obtained particles were most uniform. A preferred condition of a flow rate of 40 mL/min was selected.

Figure 4 shows the NC obtained from the ratio of different insulin to HTCC at a flow rate of 40 mL/min and a HTCC concentration of 1 mg/mL. When the insulin/HTCC (w/w) is greater than 2, the particle potential drops sharply and the particle size increases sharply; when the insulin/HTCC (w/w) is between 0.5 and 2, the obtained particle potential and particle size are substantially the same. .

Figure 5 shows the encapsulation efficiency and drug loading of the corresponding NC under different insulin/HTCC ratios. In order to obtain a higher drug loading amount, a preferred condition is to select an insulin concentration of 2 mg/mL and a HTCC concentration of 1 mg/mL.

2. Preparation of surface coated HA (NP _HA ) or HA-SH (NP _HA-SH ) nanoparticles

Hyaluronic acid or thiolated hyaluronic acid is dissolved in water at a concentration of 0.25 mg/mL, 0.5 mg/mL, 0.75 mg/mL, and 1 mg/mL. The NC suspension prepared in the first step is distributed in the first and the first The two-channel, HA or HA-SH aqueous solution is distributed in the third and fourth channels, as shown in Fig. 2B, the flow rates of the four channels are the same, and the flow rate of the regulating channel is 5 mL/min, 10 mL/min, 20 mL/min, 30 mL/min, 40 mL. /min, 50 mL/min, the two liquids were mixed through four channels to the vortex mixing zone to obtain nanoparticles coated with HA (NP _HA ) or HA-SH (NP _HA-SH ).

Table 1 shows NP _HA and NP _HA-SH obtained under the preferred conditions after screening.

Figure 6 shows the prepared NP _HA and NP _HA-SH at a flow rate of 40 mL/min. After the NC was coated with HA or HA-SH surface, the particle size increased and the charge reversed. For both different coatings, the particle size and potential are essentially the same. When the concentration of HA or HA-SH is gradually increased, the particle size gradually decreases while the potential gradually increases. The final HA and HA-SH concentrations were chosen to be 1 mg/mL, with the smallest particle size and polydispersity index. Table 1 shows NP _HA and NP _HA-SH prepared under preferred conditions.

Table 1 NP _HA and NP _HA-SH prepared under preferred conditions

Nanoparticle	Particle size (nm)	PDI	Ζ-potential (mV)	Encapsulation rate (%)	Drug loading(%)
NP HA	101±3	0.11±0.03	-25.3±2.0	90.5 ± 0.6	37.6±0.2
NP HA-SH	102±3	0.11±0.02	-26.2±1.0	91.1±0.4	38.0±0.1

Example 4 Characterization of particle size, potential and morphology

The particle size, polydispersity index, and surface potential of the particles were measured using a Malvern particle size meter. The morphology of the particles was characterized by transmission electron microscopy.

Figure 7 shows a transmission electron micrograph of NC, NP _HA and NP _HA-SH prepared under preferred conditions. The particle size of the three particles is consistent with the particle size obtained by the Malvern particle size analyzer. The core-shell structure of NP _HA and NP _HA-SH particles can be clearly seen from the figure.

Example 5 In vitro drug release experiment

The NC suspension was diluted 1 time for use. Take 1 mL of the double-folded NC particle suspension, 1 mL of the NP _HA suspension, and 1 mL of the NP _HA-SH particle suspension, respectively, and place them in a 1 mL dialysis tube and seal. The dialysis tube was placed in 20 mL of 10 mM PBS buffer, incubated at 37 ° C, 150 rpm shaker, and 1 mL of buffer was taken at specific time (subsequently supplemented with 1 mL of blank buffer to keep the volume of the system unchanged). The BCA assay measures insulin concentration.

(1) The pH value of the simulated granules passing through the gastrointestinal system, the pH value of the stomach is 2.5, the gastric emptying time is 2 to 3 hours, and the pH of the small intestine is 6.8 to 7.4. In order to simulate the pH environment of the gastrointestinal system, The dialysis tube was placed in a HCL solution at pH = 2.5 and sampled every 1 hour. After 2 h, the buffer was replaced with 10 mM PBS buffer at pH = 6.8 and sampled every 2 h.

Figure 8 shows the release profile of insulin. In the gastric pH environment, after 2 hours, 50% of the insulin in the NC particles was released. However, only 33% of the insulin was released from the HA and HA-SH coated particles NP _HA and NP _HA-SH , thus indicating HA. Or HA-SH coated particles have a better protective effect on insulin in the stomach.

(2) The dialysis tube was placed in 10 mM PBS buffer at pH=7.4, and sampled every 2 hours. Figure 9 shows the insulin release profile under physiological conditions. Under physiological conditions, more than 70% of insulin can be released within 24 hours.

Example 6 Measurement of the interaction between HA-SH and mucin using differential scanning calorimetry (DSC)

HA-SH and porcine mucin were co-dissolved in 10 mM PBS solution, incubated at 37 ° C for 8 hours, and then lyophilized. The endothermic curve of the lyophilized powder after incubation of mucin, HA-SH/mucin blend and HA-SH/mucin was measured by DSC. The weight ratio of HA-SH to mucin was 1:1, and the heating rate was 20 ° C / min.

It can be seen from Fig. 10 that mucin melts at 230 ° C and has an obvious endothermic peak; HA-SH/mucin blend can still observe a distinct melting peak of mucin at 230 ° C; however, HA-SH/viscose In the DSC curve of the lyophilized powder after protein incubation, this endothermic peak disappeared, demonstrating that the mucin is completely bound to HA-SH.

Example 7 Measurement of the movement of insulin-loaded nanoparticles in mucus using photobleaching

20 μL of nanoparticle suspension (insulin (INS) concentration of 0.5 mg/mL, insulin labeled with RITC) was mixed with 60 μL of intestinal mucus uniformly, incubated at 37 ° C for 0.5 h, and then placed on a fluorescent microscope cover slip, using a laser The focusing microscope exposes a specific area to 100% fluorescence intensity (excitation wavelength: 552 nm) for 60 s, and images are taken every 10 s, and the average fluorescence intensity of the exposed area is counted.

Figure 11 shows the fluorescence images of NC, NP _HA and NP _HA-SH during photobleaching. Figure 12 shows the relationship between the average fluorescence intensity of the photobleaching zone as a function of time. The fluorescence intensity of the photobleaching zone depends on the fluorescence loss of the photobleaching process and the fluorescence recovery produced by the free movement of the nanoparticles. After photobleaching, the smaller the loss of fluorescence intensity, the stronger the particle's ability to move. After 60s, NP _{HA has} only ~17% fluorescence loss and has the strongest exercise capacity. NP _HA-SH fluorescence loss is similar to NP _HA , with similar exercise capacity, NC fluorescence loss is the largest, ~ 50%, the mobility in mucus is the worst.

Example 8 Distribution of drug-loaded nanoparticles in E12 single cell layer

The E12 single cell layer was used to simulate a small intestinal epithelial system with a mucus layer. E12 cells were seeded in a chambered coverslip and incubated for 3 days. E12 cells formed a monolayer of cells. 100 μL of NP _HA and NP _HA-SH were co-cultured in E12 cells. After 2 hours of incubation, the pellets were removed, washed 3 times with PBS, cells were fixed with 4% paraformaldehyde solution, and the nuclei were stained with DAPI and then placed. Fluorescence images of different depths were observed and collected under a laser confocal microscope. Insulin was labeled with Cy5, HTCC was labeled with RITC to measure the distribution of HTCC/INS nanocomposite core; insulin was labeled with Cy5, HA and HA-SH were labeled with rhodamine 123 to measure the distribution of insulin and HA (HA-SH).

Figure 13 shows the distribution of insulin solution, NC, NP _HA and NP _HA-SH in the E12 monolayer, wherein HTCC is labeled with RITC and insulin is labeled with Cy5. The NC particles showed strong HTCC and insulin signals at 30 μm and 15 μm depth (mucus layer), and the two were co-localized. NP _HA and NP _{HA-SH could} not observe the signals of HTCC and INS at a depth of 30 μm. At a depth of 15 μm, weak HTCC and insulin signals were observed, and the two were co-localized. Strong HTCC and INS signals were observed in all three particles with a depth of 0 μm (cell layer), and the two were co-localized. It is thus proved that the positive core of HTCC and INS remain bound during the process of passing through the mucus layer, and there is no dissociation, and NP _HA and NP _HA-SH have stronger mucus penetrating ability than NC. At a depth of 15 μm, NP _HA-SH has a stronger signal than NP _HA , indicating that NP _HA-SH has a stronger interaction with the mucus layer.

Figure 14 shows the distribution of NP _HA and NP _HA-SH in the E12 monolayer in Example 8, wherein HA and HA-SH are labeled with Rhodamine 123 and insulin is labeled with Cy5. No signal of INS, HA and HA-SH was observed at a depth of 30 μm. At a depth of 15 μm, both weak insulin signals and signals of shell material (HA or HA-SH) distributed uniformly throughout the region were observed, and were not co-localized with insulin signals; at a depth of 0 μm, both Strong insulin and shell material (HA and HA-SH) signals were observed in the particles. Most of HA and HA-SH were not colocalized with insulin, demonstrating that NP _HA and NP _HA-SH particles can effectively cross mucus The layer reaches the cell, and during the passage through the mucus layer, the HA and HA-SH shells gradually fall off, and when they reach the cell layer, the positively charged core is exposed.

Example 9 Distribution of drug-loaded granules in mucus of small intestine

SD rats were fasted for 12 hours. After anesthesia, a 5 cm closed area was formed by ligation at both ends of the rat jejunum, and 0.5 ml of drug-loaded particles were injected. The insulin was labeled with Cy5, and after 2 hours, The closed area was taken out, cut in the radial direction, washed 3 times with 10 mM PBS, and then the mucus layer was fixed upward, and the imaging was observed in a small animal living imager, and then the mucus layer on the surface of the small intestine was sucked away by a vacuum pump. The imaging was again observed in a small animal living imager, and the average fluorescence intensity of the small intestine region before and after removal of the mucus layer was statistically counted.

Figure 15 is a fluorescent image of the small intestine before and after mucus removal after treatment with three kinds of particles. Figure 16 shows the statistical mean fluorescence intensity of the small intestine surface before and after removal of mucus. The NC-treated small intestine had the strongest insulin signal before mucus removal, followed by NP _HA-SH and NP _HA . After removal of the mucus layer, the small intestine treated with NP _HA-SH had the strongest insulin signal, followed by NP _HA and the worst NC. The insulin remaining after removal of mucus is insulin that successfully penetrates the mucous layer of the small intestine. It is proved that NP _HA-SH has good mucus adsorption properties and mucus penetrating properties.

Example 10 Cell Penetration Experiment

The intestinal epithelial cell layer was simulated on caco2 monolayer cells, and the 1% mucin solution mimicked the intestinal epithelial mucus layer. Cacao2 cells were seeded in transwell plates and cultured at 37 °C for 2 to 3 weeks until a monolayer of cell layers was formed in caoco2 cells and the cell transmembrane resistance was higher than 700 Ω, forming tight junctions between the cells. The medium above and below the cell membrane was replaced with HBSS before the experiment. A 1% mucin solution was added to the cell layer to simulate intestinal epithelial mucus.

(1) Cell layer transmembrane resistance: 200 μL of insulin solution, NC, NP _HA and NP _HA-SH particle suspension were incubated with caco2 cells for 2 hours, and the particles were taken out and incubation was continued for 8 hours. Cell transmembrane resistance was measured at specific times.

Figure 17 shows the change in cell transmembrane resistance over time after addition of insulin solution or nanoparticles. For the insulin solution, the cell transmembrane resistance is basically unchanged; for other particles, after the addition of the particles, the transmembrane resistance of the cells decreases; after the particles are removed, the transmembrane resistance of the cells gradually recovers, which proves that the experimental particles can reversibly open the cells tightly. link. For the mucus-free caco2 cell layer, the NC particles reduced the resistance by about 50%, with the best tight junction opening effect, and the NP _HA or NP _HA-SH particles reduced the resistance to about 40%. For mucocaco2 cell layers, NP _HA or NP _HA-SH can still reduce cell resistance by about 37%, while NC can only reduce cell resistance by 30%. The positively charged NC particles interact with the negatively charged mucin, preventing the NC particles from reaching the cell layer. The HA coating layer is electronegative and prevents NP _HA particles from interacting with mucin, so the addition of mucin has little effect on the ability to open tight junctions. For NP _HA-SH , although the sulfhydryl group of the HA-SH coating layer can form a disulfide bond with the mucin, the NP _HA-SH particle can be detached from the mucin due to the gradual peeling of the HA-SH coating layer from the particle surface. Obstruction, mucin also has little effect on the ability of the particles to open tight junctions.

(2) Apparent permeability coefficient measurement: Insulin solution and 3 different particles were incubated with caco2 cells for 4 h, and 100 μL of solution was taken from the transwell lower chamber every 1 h, and the same volume of HBSS was added to measure the fluorescence intensity. ) Calculate the Papp value.

In the formula, Papp is the apparent permeability coefficient, dQ/dt is the flow rate of the fluorescently labeled particles from the transwell upper chamber to the lower chamber, C _o is the initial fluorescence intensity of the upper chamber, and A is the transewell membrane area.

Figure 18 shows the apparent permeability coefficient of different particles transporting insulin across the caco2 monolayer. For the non-mucinous caco2 cell layer, NC has the highest apparent permeability coefficient. For the caco2 cell layer with mucus, NP _HA and NP _HA-SH have higher apparent permeability coefficients than NC. After the addition of mucin, the apparent permeability coefficient of NC decreased sharply, and the addition of mucin had almost no effect on the apparent permeability coefficient of NP _HA and NP _HA-SH . Compared with NC, NP _HA and NP _HA-SH have better mucus penetrating ability and can promote insulin absorption in the small intestine more effectively.

(3) Tight junction imaging: using a blocking protein antibody as a primary antibody, co-incubate with caco2 cells for 0.5 hour at room temperature, and fully bind to the tight junction-binding protein, then add AF488-labeled secondary antibody and incubate for 1 hour at room temperature. The second antibody is fully combined with the primary antibody. Before under a fluorescence microscope to observe the addition of NP _HA-S H particles, particles with NP _HA-SH were incubated two hours, and incubation was continued tight junctions between cells after 10 hours after the removal of particles.

Figure 19 shows the tight junction of caco2 cells before and after the addition of NP _HA-SH particles. A clear and clear ring structure was observed before the addition of the particles (0h); after adding NPHA-SH for 2 hours, the ring structure disappeared, indicating that the tight junction was opened; the particles were removed, and the incubation was continued for 10 hours, and continuous clearness was observed. The ring structure indicates that the tight connection can be restored. This demonstrates that the particles of the present invention can be reversibly opened tightly linked to facilitate absorption of insulin by the small intestine through the cell bypass.

Example 11 Absorption of Oral-Loaded Insulin Nanoparticles in the Small Intestine

SD rats were fasted for 12 h, then oral insulin solution, NC, NP _HA and NP _HA-SH particles. The insulin was labeled with RITC. After 2 hours, the rats were anesthetized, sacrificed, and the jejunum of the small intestine was removed by surgery. The small intestine tissue was fixed with 4% paraformaldehyde, and the intestinal epithelial nucleus and small intestinal mucus layer were stained with DAPI and AF-645, and imaged under a laser confocal microscope.

Figure 20 shows the absorption of insulin in the small intestine after oral insulin solution or insulin-loaded particles. Oral NP _HA-SH group had the strongest insulin absorption, and there was still a large amount of insulin in the mucus layer; oral NP _HA group had strong insulin absorption; insulin signal was observed in oral NC mucus layer, and only a small part of insulin was observed. Entering the intestinal epithelial cells, the oral absorption was poor; the insulin signal was not observed in the oral insulin solution group.

Example 12 Oral hypoglycemic effect

200-250 g SD rats were induced into type I diabetes model mice by intraperitoneal injection of 80 mg/kg streptozotocin. The model mice were divided into 6 groups of 7 each. Before the experiment, all the experimental groups were fasted for 10-12 h, group 1 was treated with deionized water, group 2 was intragastrically administered with insulin solution (75 IU/kg), group 3 was intragastrically filled with NC particle suspension (75 IU/kg), group 4 was intragastrically administered. NP _HA particle suspension (75 IU/kg), group 5 gavage NP _HA-SH particle suspension (75 IU/kg), group 6 subcutaneous injection of insulin solution (5 IU/kg). Every 1 hour, blood was taken from the tip of the tail and blood glucose was measured using a blood glucose meter.

Figure 21 shows the blood glucose lowering curve after oral administration of insulin granules or subcutaneous injection of insulin. The blood glucose of subcutaneous injection of insulin rapidly decreased to a lower level, while the blood glucose of the oral insulin nanoparticle preparation decreased steadily. NPH _A-SH has the strongest hypoglycemic effect, followed by NP _HA , and the effect of lowering blood glucose in NC is relatively poor. Oral insulin solution was used as a negative control and had no effect on blood glucose.

Example 13 Oral Bioavailability of NP _HA and NP _HA-SH

200-250 g type I diabetic mice were divided into 4 groups, and all experimental groups were fasted for 10-12 h before the experiment. Group 1 was injected subcutaneously with insulin solution (5 IU/kg), group 2 was administered with NP _HA particle suspension (75 IU/kg), group 3 was administered with NP _HA-SH particle suspension (75 IU/kg), group 4 was administered with insulin solution. (75UI/kg). At specific times, blood was taken from the eyelids, and the insulin concentration in the blood was measured using an insulin ELISA kit to obtain a curve of insulin blood concentration versus time, as shown in FIG. The subcutaneous insulin group was used as a 100% control, and the bioavailability was obtained by comparing the area under the curve of the oral granule group and the subcutaneous injection of insulin. The oral bioavailability of NP _HA and NP _HA-SH were 5.8% and 11.3%, respectively. The oral insulin solution group served as a negative control and the insulin concentration was close to zero.

Example 14 Effect of Chitosan Quaternization on Nanoparticles Loaded with Insulin

Insulin-loaded NC nanoparticles were prepared using three different degrees of quaternization of HTCC (12%, 23%, and 43%), and the other materials and methods were the same as in Example 3. By changing the degree of quaternization of chitosan, the size and surface charge of NC nanoparticles can be regulated. As shown in Fig. 23, as the degree of quaternization of chitosan decreased, the particle size of NC particles increased from 75 nm to about 263 nm, and the surface potential of the particles was reduced from +27 mV to about +17 mV.

Example 15 Preparation of hyaluronic acid sulphation degree Preparation of insulin-loaded nanoparticles

The insulin _- loaded NP _HA-SH nanoparticles were prepared by using thiolated hyaluronic acid with a degree of thiolation of 8.4%, 12.3%, and 32.3%. The other materials and preparation methods were the same as in Example 3. Since the thiolation modification of HA in this patent mainly utilizes the esterification reaction of the hydroxyl group of HA with the carboxyl group of dithiodipropionic acid, HA-SH after modification has similar chargeability to HA before modification, so different sulfhydryl groups are used. The degree of modification of hyaluronic acid is based on the surface of the NC nanoparticles after charge application. It is presumed that the insulin-loaded nanoparticles with similar particle size, dispersion, encapsulation efficiency and drug loading are obtained.

Claims (10)

1. An insulin-loaded nanoparticle, characterized in that the nanoparticle has a core-shell structure, the core is a nanocomposite core composed of a chitosan quaternary ammonium salt and insulin, and the shell is a hyaluronic acid or a sulfhydryl group coated on the core surface. Hyaluronic acid.
2. The nanoparticle according to claim 1, wherein the chitosan quaternary ammonium salt has a molecular weight of 50 kDa to 200 kDa, the degree of quaternization is 10% to 70%, and the molecular weight of the thiolated hyaluronic acid is 3.4 kDa. ~200kDa, the degree of thiolation is 5% to 30%.
3. The nanoparticle according to claim 1, wherein the nanoparticle has a particle diameter of 50 to 200 nm.
4. Use of the nanoparticle of claim 1 or 2 in the preparation of an oral insulin preparation.
5. An oral insulin pharmaceutical preparation comprising the insulin-loaded nanoparticles according to any one of claims 1 to 3.
6. A method for preparing a loaded insulin nanoparticle, comprising the steps of:

   S1. The chitosan quaternary ammonium salt solution is passed through channels 1 and 2, and the insulin solution is passed through 3 and 4, and mixed into the vortex mixing region to obtain a nanocomposite core composed of insulin and chitosan quaternary ammonium salt; Flow, flow rate is 2mL / min ~ 50mL / min;

   S2. The mixture obtained by S1 is passed through channels 1 and 2, hyaluronic acid or thiolated hyaluronic acid solution through channels 3 and 4, and mixed into the vortex mixing region to obtain surface coated hyaluronic acid or thiolated hyaluronic acid nanometer. Particles; four channels of liquid flow at a constant rate, flow rate of 2mL / min ~ 50mL / min.
7. The preparation method according to claim 6, wherein the chitosan quaternary ammonium salt has a degree of quaternization of 10% to 70%; and the degree of thiolation of the thiolated hyaluronic acid is 5% to 30%. %.
8. The preparation method according to claim 6, wherein the concentration of the chitosan quaternary ammonium salt is 0.2 to 3 mg/mL, and the concentration of the insulin solution is 0.5 to 3 mg/mL.
9. The method according to claim 6, wherein the hyaluronic acid or the thiolated hyaluronic acid has a concentration of 0.25 to 1 mg/mL.
10. The preparation method according to claim 6, wherein the mass ratio of the insulin to the chitosan quaternary ammonium salt is 0.5 to 2.

Images