CN115414492B - Nanometer preparation for treating pancreatic fibrosis and preparation method thereof - Google Patents

Nanometer preparation for treating pancreatic fibrosis and preparation method thereof Download PDF

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CN115414492B
CN115414492B CN202211198098.7A CN202211198098A CN115414492B CN 115414492 B CN115414492 B CN 115414492B CN 202211198098 A CN202211198098 A CN 202211198098A CN 115414492 B CN115414492 B CN 115414492B
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collagen
collagenase
pancreatic fibrosis
fibrosis
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CN115414492A (en
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姜虎林
李玲
祁靓
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China Pharmaceutical University
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/203—Retinoic acids ; Salts thereof
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes

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Abstract

The invention discloses a nanometer preparation for treating pancreatic fibrosis and a preparation method thereof, wherein the nanometer preparation comprises lipid nanoparticles, and collagen targeting peptide and/or collagenase are connected to the surfaces of the lipid nanoparticles; the lipid nanoparticle is prepared from a block copolymer X-PEG-Mal, phospholipid and cholesterol B. The collagen targeting peptide and the collagenase modified nano preparation can target a pancreatic fibrosis region where collagen is deposited, and the collagenase modified nano preparation can ablate and penetrate a collagen barrier formed in the course of pancreatic fibrosis, so that a novel approach is provided for efficient delivery of chemical drugs for pancreatic fibrosis.

Description

Nanometer preparation for treating pancreatic fibrosis and preparation method thereof
Technical Field
The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a nano preparation for treating pancreatic fibrosis and a preparation method thereof.
Background
Pancreatic fibrosis is an important pathological feature of diseases such as chronic pancreatitis and pancreatic cancer. When pancreas is stimulated by inflammation and is damaged by the outside, the pancreas stellate cells are continuously activated under the stimulation of inflammatory factors and secrete a large amount of collagen fibers, when the collagen fibers are excessively produced and degradation is blocked, a large amount of fibrotic collagen is abnormally deposited, and finally, pancreas fibrosis is caused.
Activation of pancreatic stellate cells plays a key role in the development of pancreatic fibrosis, and in recent years, attention has been paid in part to drugs that inhibit pancreatic stellate cell activation. But due to the small pancreas volume, the location is hidden, and drug targeting and bioavailability are both challenged. Meanwhile, a great deal of collagen fibers deposited in the fibrosis process form a pathological barrier, so that the drug is further hindered from being delivered into the pancreas, and the therapeutic effect of the drug is seriously affected.
At present, delivery of hydrophilic/hydrophobic drugs using liposomes is one of the common methods, and there have been many studies on targeting, ablation and delivery of pancreatic fibrosis drugs using collagenase-modified nano-formulations by enhancing collagen degradation, promoting drug penetration into tumor interiors, but no collagenase-modified liposomes have been used.
Disclosure of Invention
The invention aims to provide a nano-preparation for treating pancreatic fibrosis, which can realize targeted delivery of an anti-fibrosis drug to pancreatic fibrosis tissues.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A nano-preparation for pancreatic fibrosis treatment, comprising lipid nanoparticles, wherein the surface of the lipid nanoparticles is connected with collagen targeting peptides and/or collagenase for targeting type I collagen;
The lipid nanoparticle is prepared from a block copolymer X-PEG-Mal, phospholipid and cholesterol B, wherein X is DSPE, DPPE, DMPE or DOPE, the molecular weight of PEG is 1000-5000, and MAL is maleimide group.
Further, the phospholipid is selected from egg yolk lecithin, hydrogenated soybean lecithin, soybean lecithin or synthetic phospholipid (such as DPPD,DOPS,DEPE,DMPE,DSPE,DPPE,DOPE,DOPG,EPG,POPG,DPPG,DSPG,DMPG,DPPA,DEPC,DOPC,DMPC,POPC,DSPC,DPPC）.
Further, the collagenase is collagenase I or collagenase III.
Further, the lipid nanoparticle is coated with an anti-fibrosis drug. The anti-fibrosis drug is hydrophilic anti-fibrosis drug or hydrophobic anti-fibrosis drug, such as all-trans retinoic acid, tocotrienol, polyphenol, curcumin, rhein, resveratrol, ammonium tetrathiomolybdate, etc. In one embodiment of the invention, both free all-trans retinoic acid and ammonium tetrathiomolybdate are entrapped.
The preparation method of the nano preparation comprises the following steps:
Step 1, preparing lipid nanoparticles;
and 2, mixing the lipid nanoparticle with sulfhydryl collagenase and/or collagen targeting peptide to prepare the nano preparation.
The application of the nano preparation in preparing a medicine for treating diseases related to pancreatic fibrosis.
Compared with the prior art, the anti-pancreatic fibrosis drug nano-preparation provided by the invention has the following advantages:
The lipid nanoparticle prepared from the block copolymer X-PEG-Mal and the phospholipid can well realize the loading of hydrophilic and/or hydrophobic drugs, collagen targeting peptide on the carrier can target the pancreatic fibrosis collagen deposition site, collagenase on the carrier can degrade collagen (I, III type collagen) which is abnormally and excessively deposited in the pancreatic fibrosis process, PEG in the carrier can prolong the blood circulation time of the carrier, and the availability of the drugs can be greatly improved through the carrier.
The collagen targeting peptide and the collagenase modified nano preparation can target a pancreatic fibrosis region where collagen is deposited, and the collagenase modified nano preparation can ablate and penetrate a collagen barrier formed in the course of pancreatic fibrosis, so that a novel approach is provided for efficient delivery of chemical drugs for pancreatic fibrosis.
Drawings
FIG. 1 is a schematic flow chart of the preparation of the nano-formulations of the present invention.
FIG. 2 is a graph of particle size distribution of nano-formulation drug/CC.
Fig. 3 is a transmission electron microscope image of the nano-formulation drug/CC.
Figure 4 is the stability of the nano-formulation drug/CC in each solution.
FIG. 5 shows cytotoxicity of mPSCs and 266-6 cells of the nanocarriers prepared according to the present invention.
Figure 6 is a flow cytometer quantitative analysis of the uptake of the entrapped coumarin 6 nm preparation by mPSCs cells in the presence of a collagen barrier in vitro.
Fig. 7 is a photograph of a living animal showing accumulation of various formulations of entrapped fluorescent Dye (DiR) in pancreas of pancreatic fibrosis mice in each experimental group.
Fig. 8 is an analysis of the effect of different nanoformulations on reversing pancreatic fibrosis at the in vivo level.
Detailed Description
According to the invention, the collagenase and collagen targeting peptide modified lipid nanoparticle is utilized, the loading of the hydrophobic anti-fibrosis drug is realized through the hydrophobic tail, the loading of the hydrophilic anti-fibrosis drug is realized through the hydrophilic inner core, the long circulation of the nanoparticle in the body is realized through PEG, the pancreas is specifically targeted and fibrosed through the modified collagen targeting peptide, and the fibrosis pathological collagen barrier is overcome through the collagenase, so that the efficient targeted delivery of the anti-fibrosis chemical drug is realized.
The invention will now be described in further detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention. The experimental procedures and reagents not shown in the formulation of the examples were all in accordance with the conventional conditions in the art.
Example 1
Synthesis and preparation of nano preparation components
1. Preparation of lipid nanoparticle for encapsulating medicine
The nanometer preparation loaded with all-trans retinoic acid, ammonium tetrathiomolybdate or fluorescent dye (used as a model for substituting medicines for preparation tracking) can be prepared by adopting a film dispersion method, an injection method or an anti-solvent method, and the principle is shown in figure 1.
The present embodiment preferably employs a thin film dispersion method to prepare a nano-preparation loaded with an anti-pancreatic fibrosis drug. The preparation method comprises the following steps:
30mg of phospholipid, 5mg of DSPE-PEG 2000 -Mal,5mg of cholesterol and 1.5mg of all-trans-retinoic acid were weighed and dissolved in 50mL of methylene chloride, the organic solvent was removed from the round bottom flask by rotary evaporation to form a homogeneous film, hydration was performed using PBS solution in which 1mg/mL of ammonium tetrathiomolybdate was dissolved, liposome with uniform particle size was prepared by a sonicator, and free carrier material and unencapsulated drug were removed by centrifugation and G50 purification column, respectively.
2. Sulfhydrylation collagenase I
Weighing 100 mg I type collagenase and 2.75 mg 2-iminothiolane hydrochloride, dissolving in 5mL 0.01M phosphate buffer solution, reacting for 1h at room temperature, desalting and purifying by a sephadex column (G-25), and detecting the protein concentration of the purified solution by ultraviolet (280 nm) for later use.
3. Lipid nanoparticle preparation with collagen targeting peptide and collagenase
Incubating the drug-loaded lipid nanoparticle with collagen targeting peptide (LRELHLNNNC) and thiolated collagenase I together according to a mass ratio of 1:2, and reacting in 0.01M phosphate buffer solution at room temperature overnight; the pellet was collected by centrifugation using ultra high speed centrifugation (20 w×g) and resuspended in an appropriate amount of PBS to obtain the final preparation (drug/CC) with free collagen targeting peptide and thiolated collagenase I removed.
Simultaneously, the lipid nanoparticle loaded with the drug and collagen targeting peptide (LRELHLNNNC) are incubated together (1 mg/mL), and the reaction is carried out in 0.01M phosphate buffer solution at room temperature for overnight; the pellet was collected by centrifugation using ultra high speed centrifugation (20 w×g) and resuspended in an appropriate amount of PBS to obtain a preparation (drug/CBP) with free collagen targeting peptide removed.
In addition, the drug-loaded lipid nanoparticle was co-incubated with thiolated collagenase I (2 mg/mL), and reacted overnight in 0.01M phosphate buffer at room temperature; the pellet was collected by centrifugation using ultra high speed centrifugation (20 w×g) and resuspended in an appropriate amount of PBS to obtain a preparation (drug/Col I) with free collagenase removed.
The medicine/CBP and the medicine/Col I prepared by the method have the medicine loading rate of 2-3 percent and the grain size of 100-200 nm.
The particle size distribution, transmission electron microscope image and nanometer preparation stability of the nanometer preparation medicine/CC are shown in figures 2, 3 and 4, the nanometer preparation has uniform particle size distribution and good shape; has good stability in PBS solution, physiological buffer salt solution and DME/F2 medium containing 10% serum.
Example 2
Investigation of the safety of the nano-preparation Carrier Material
Lipid nanoparticles (Lip) without drug entrapped, lipid nanoparticles (Lip-CBP) with collagen targeting peptide alone, lipid nanoparticles (Lip-Col I) with collagenase I alone, lipid nanoparticles (Lip-CC) with targeting and collagenase I were prepared as in example 1, and cytotoxicity of empty vector to various pancreatic cells (mPSCs, 266-6) was measured by MTT method.
The specific operation steps are as follows: first, cells in the logarithmic phase were individually added to 96-well plates, and the plating density per well was 1X10 4/200. Mu.L. The empty vector was then diluted to a series of concentration gradients using FBS-free medium and cells without material were used as control groups, the drug concentrations given to the cells were 5,10, 20, 50, 100 μg/mL, respectively, medium in 96-well plates was aspirated, and 100 μl of samples of different concentration gradients were added per well, 5 wells being repeated. After the administration, the culture was continued for 24 or 48 hours, 20. Mu.L of MTT was added under dark conditions, and the culture was continued in a cell incubator for 4 hours. The 96-well plate was taken out, the supernatant was aspirated by a 1mL syringe, 150. Mu.L of DMSO was added, the wells were shaken in a shaker at 37℃for 10-15min, the OD value of each well was measured at 490nm using an ELISA reader, and the cell viability was calculated.
The cytotoxicity data measured in this example are shown in FIG. 5, and the empty carriers Lip, lip-CBP, lip-Col I and Lip-CC are within 100 mug/mL, and have no toxic or side effect on mPSCs and 266-6 cells, thus proving that the carrier used in the invention has good safety.
Example 3
MPSCs uptake of coumarin 6 nano preparation-entrapped flow cytometer quantitative analysis
Coumarin 6-loaded lipid nanoparticles (lip@C6), single-targeting coumarin 6-loaded lipid nanoparticles (Lip-CBP@C6), single-targeting collagenase coumarin 6-loaded lipid nanoparticles (Lip-Col I@C6), and targeting and collagenase coumarin 6-loaded lipid nanoparticles (Lip-CC@C6) were prepared as described in example 1. mPSCs was inoculated into 24-well plates at 8X 10 4/mL, grown in a 5% CO 2 cell incubator at 37℃for 24 hours, the medium was aspirated, and 500. Mu.L of serum-free medium solutions of lip@C6, lip-CBP@C6, lip-Col I@C6 and Lip-CC@C6 were added, respectively, and three auxiliary wells were provided for each group, with a concentration of coumarin 6 contained of 0.025mg/mL as a unified standard. Culturing for 6h, washing with PBS three times, digesting with pancreatin, centrifuging at 2000rpm to precipitate cells, re-suspending the cells with serum-free medium, and quantitatively detecting the ingested coumarin 6 by using a flow cytometer.
As shown in FIG. 6, the cell uptake data of this example is shown that in mPSCs with more secreted collagen, the uptake of coumarin/Col and coumarin/CC is significantly higher than that of other groups, demonstrating that collagenase grafting can increase the cell uptake rate by degrading collagen.
Example 4
Fluorescent Dye (DiR) -entrapped nanoformulation of pancreatic accumulation in pancreatic fibrosis mice
DiR-carrying lipid nanoparticles (lip@DiR), single-targeting DiR-carrying lipid nanoparticles (Lip-CBP@DiR), single-targeting DiR-carrying lipid nanoparticles (Lip-Col I@DiR), targeting and collagenase-carrying lipid nanoparticles (Lip-CC@DiR) were prepared as described in example 1.
Experiments were performed using 4-6 week old C57 male black mice, and a model of chronic pancreatitis was constructed using ranpirin intraperitoneal injection for 6 weeks (5 mg/kg,6 times/day, 3 times/week). The mice of the blank group and the fibrosis modeling group are randomly distributed into 5 groups, 3 mice of each group are subjected to living imaging by a small animal living imaging instrument through tail vein injection of free DiR, lip@DiR, lip-CBP@DiR, lip-Col I@DiR and Lip-CC@DiR nano preparations after tail vein injection for 1h, 3h, 6d, 9d, 12d and 24d respectively.
The fluorescent Dye (DiR) -entrapped nano-preparation prepared in this example was used for imaging pictures (shown in fig. 7A) and quantitative analysis (shown in fig. 7B) of animals living in pancreas accumulation in pancreas-fibrotic mice. In the fibrotic group, the extent of accumulation of the nanoformulation in the pancreas is as follows: lip-CC@DiR > Lip-CBP@DiR > Lip-Col I@DiR > Lip@DiR > free DiR, and it is proved that modification of collagen targeting peptide and collagenase is beneficial to deposition of nano preparation on fibrotic pancreas sites.
Experiments were performed using 4-6 week old C57 male black mice, and a model of chronic pancreatitis was constructed using ranpirin intraperitoneal injection for 6 weeks (5 mg/kg,6 times/day, 3 times/week). The mice of the fibrosis model group were randomly assigned to 6 groups of 8 mice each, treated by tail vein injection of PBS, free all-trans retinoic acid (a) and ammonium tetrathiomolybdate (T), double drug loaded lipid nanoparticles (AT), single target-loaded double drug loaded lipid nanoparticles (AT-CBP), single collagenase-loaded double drug lipid nanoparticles (AT-Col I), target-loaded double drug lipid nanoparticles (AT-CC) and collagenase (3 times per week, 3 weeks), respectively. Dissecting after treatment was completed, and the degree of improvement in fibrosis was assessed by H & E staining.
The histological changes of pancreas after treating pancreatic fibrosis with the two drug-entrapped different nano-formulations prepared in this example are shown in fig. 8. It was demonstrated that various degrees of fibrotic repair occurred in pancreatic tissue following treatment, with the drug/CC nanoformulation treated group showing optimal fibrotic repair.
The functionalized phospholipid comprises DSPE and DPPE; natural phospholipids including egg yolk lecithin, hydrogenated soybean lecithin, soybean lecithin and various synthetic phospholipids are common hydrophobic blocks with good biocompatibility, are often used as hydrophobic cores in amphiphilic block copolymers, and have better affinity for most drugs with certain hydrophobicity. In the above examples, the DPPE-PEG-MAL block copolymer was used instead of the DSPE-PEG-MAL block copolymer, and the yolk lecithin and soybean lecithin were used instead of soybean lecithin to carry the drugs having a certain hydrophobicity as well, so that the purpose of forming polymer nanoparticles was clear to those skilled in the art.
The nano preparation can be loaded with a plurality of anti-pancreatic fibrosis drugs at the same time, firstly, the collagen targeting peptide is used for targeting pancreatic tissues deposited in a large amount by pancreatic fibrosis collagen, and then, the collagen barrier is penetrated through the degradation of collagenase ablation, so that the anti-pancreatic fibrosis drugs are delivered in a high-efficiency targeting manner, and the purpose of reversing the pancreatic fibrosis in a high-efficiency manner is achieved. The invention overcomes the difficult problem of targeting accumulation of the medicine at the pancreas part by constructing a targeting, ablating and delivering integrated carrier platform for the first time, promotes permeation and accumulation of potential therapeutic medicines, and provides a new way and strategy for high-efficiency delivery of the medicine for resisting pancreatic fibrosis.
The foregoing is only a preferred embodiment of the invention, it being noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the invention.

Claims (2)

1. A nano-preparation for treating pancreatic fibrosis, which is characterized by comprising lipid nanoparticles, wherein the surface of the lipid nanoparticles is connected with collagen targeting peptide for targeting type I collagen and collagenase;
The lipid nanoparticle is prepared from phospholipid, a block copolymer DSPE-PEG2000-Mal and cholesterol, wherein the mass ratio of the phospholipid to the block copolymer DSPE-PEG2000-Mal to the cholesterol is 30:5:5, a step of;
the lipid nanoparticle is coated with anti-fibrosis drugs of all-trans retinoic acid and ammonium tetrathiomolybdate;
The sequence of the collagen targeting peptide targeting the type I collagen is LRELHLNNNC;
The collagenase is collagenase I;
The preparation method of the nano preparation comprises the following steps:
Step 1, preparing lipid nanoparticles;
And 2, mixing the lipid nanoparticles with thiolated collagenase and collagen targeting peptide, wherein the mass ratio of the lipid nanoparticles to the collagen targeting peptide to the thiolated collagenase is 1:2, and preparing the nano preparation.
2. Use of the nano-formulation of claim 1 for the preparation of a medicament for the treatment of pancreatic fibrosis.
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