CN115581768A - ROCKI oral nano preparation for resisting intestinal fibrosis and preparation method thereof - Google Patents
ROCKI oral nano preparation for resisting intestinal fibrosis and preparation method thereof Download PDFInfo
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- A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
- A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
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Abstract
The invention discloses an anti-intestinal fibrosis ROCKi oral nano preparation and a preparation method thereof. The preparation method of the ROCKi oral nano preparation takes a dipeptide chain X-Ala-pro-Gly (X-ARG) as a connecting arm; and grafting a ROCK inhibitor to a hyaluronic acid side chain to form ROCKi-ARG-HA polymer, and then utilizing a ROS-responsive cross-linking agent to enable the ROCKi-ARG-HA polymer to be self-assembled and cross-linked to form nanoparticles, namely the ROCKi oral nano preparation for resisting intestinal fibrosis. The ROCKi oral nano preparation is promoted by ROS at an intestinal fibrosis lesion site, identifies intestinal myofibroblasts in a targeted manner and is activated by the intestinal myofibroblasts, so that secondary response is realized, the drug effect is increased, the adverse reaction is reduced, and the clinical application of ROCKi in intestinal fibrosis resistance becomes possible.
Description
(I) the technical field
The invention relates to an ROCKi oral nano preparation for resisting intestinal fibrosis and a preparation method thereof.
(II) background of the invention
Crohn's Disease (CD) is a chronic, recurrent intestinal nonspecific inflammatory disease. Over half of the patients with CD develop intestinal fibrosis and progress to intestinal stenosis and obstruction in their disease course. Patients with ileus have to undergo surgery many times, which seriously affects the quality of life. At present, no effective treatment medicine exists for intestinal fibrosis. While control of CD gut inflammation alone has proven to be unable to alter disease progression of gut fibrosis. Therefore, there is an urgent need to develop an effective drug system to reduce or even reverse intestinal fibrosis, prevent intestinal stenosis, and improve the quality of life of CD patients. Intestinal fibrosis in CD patients is characterized primarily pathologically by proliferation and activation of intestinal myofibroblasts, secreting large amounts of extracellular matrix (ECM). ECM is excessively deposited, resulting in thickening of the intestinal wall and ultimately in narrowing of the intestinal lumen. Therefore, the enteromyofibroblasts are the central link of the disease course of CD fibrosis, and the direct inhibition of the proliferation and secretion functions of the enteromyofibroblasts is the most effective way for inhibiting the generation and development of intestinal fibrosis. Research finds that Rho/ROCK pathway activation can lead to intestinal myofibroblast activation and proliferation, increase ECM secretion and play an important role in the process of intestinal fibrosis. ROCK inhibitors (ROCK i) can successfully inhibit intestinal myofibroblast activation and ECM secretion, and prevent or even reverse CD-induced intestinal fibrosis. However, rho/ROCK signals are commonly found in various types of cells, and systemic administration of ROCK i can widely affect various cells in different organs, resulting in serious adverse reactions in systemic application, including reduction of vascular smooth muscle contraction, systemic hypotension and the like, and even life threatening in serious cases, and limiting clinical development. Therefore, precise delivery of ROCKi to intestinal myofibroblasts in fibrotic diseased tissues, reduced distribution in healthy tissues, is a key problem for the clinical transformation of ROCKi.
Disclosure of the invention
The invention aims to provide an anti-intestinal fibrosis ROCKi oral nano preparation which is released only at intestinal fibrosis disease parts, is specifically absorbed by intestinal fibroblasts and fully exerts the drug treatment effect, and a preparation method thereof.
The technical scheme adopted by the invention is as follows:
an anti-intestinal fibrosis ROCKi oral nano preparation is prepared by the following steps: taking a dipeptide chain X-Ala-pro-Gly (X-ARG) as a connecting arm, wherein X is benzyl bicarbonate or a homologue thereof; and grafting a ROCK inhibitor to a hyaluronic acid side chain to form ROCKi-ARG-HA polymer, and then utilizing a ROS-responsive cross-linking agent to enable the ROCKi-ARG-HA polymer to be self-assembled and cross-linked to form nanoparticles, namely the ROCKi oral nano preparation for resisting intestinal fibrosis.
The research finds that the Fibroblast Activation Protein (FAP) is specifically and highly expressed on the surface of intestinal myofibroblasts, and the dipeptide connecting chain of the X-ARG containing proline can specifically recognize and combine with the FAP. In addition, FAP is also a serine hydrolase which can specifically hydrolyze the dipeptide fragment, and based on the above, the invention creatively provides a method for grafting ROCKi to a hyaluronic acid side chain by taking a dipeptide chain X-ARG as a connecting arm to form a ROCKi-ARG-HA polymer. The dipeptide chain is used for identifying FAP, so that the function of targeting intestinal muscle fibroblasts is achieved; and then, after FAP is contacted, the hydrolyzability of the dipeptide chain is utilized to release ROCKi, so that the Rho/ROCK pathway of intestinal myofibroblasts is blocked, and the anti-fibrosis effect is achieved. In addition, due to the fact that ROS in the fibrosis tissue is highly expressed, the ROCKi-ARG-HA polymer is subjected to self-assembly crosslinking to form ROS sensitive nanoparticles by using a crosslinking agent with ROS response fragmentation capability. Finally, a secondary response ROCKi oral targeting nano delivery system with ROS promotion in intestinal fibrosis microenvironment, specific recognition and activation of intestinal myofibroblasts is achieved.
At present, a secondary response ROCki oral targeting nano delivery system for treating intestinal fibrosis is not seen in the market, and research reports of similar pharmaceutical preparations are not seen. Therefore, the development of the medicine has important clinical significance and wide market prospect.
The invention also relates to a preparation method of the ROCKi oral nano preparation for resisting intestinal fibrosis, which comprises the following steps:
(1) Synthesis of ROCki-ARG: dissolving a proper amount of X-ARG and ROCK inhibitor in anhydrous formamide, reacting at room temperature for 18-24H under the catalysis of EDC and DMAP, and adding catalysts Pd/C, H 2 Continuing to react for 18-24 h at room temperature under protection, dialyzing the solution with pure water after the reaction is finished, and freeze-drying to obtain ROCKi-ARG compound;
(2) Synthesis of ROCKI-ARG-HA Polymer: dispersing appropriate amount of hyaluronic acid in anhydrous formamide, and adding appropriate amount of ROCKi-ARG, N under catalysis of EDC and DMAP 2 Stirring the mixture for reaction in a dark place under protection, and dialyzing the obtained solution with distilled water after the reaction is finished to obtain ROCKi-ARG-HA;
(3) Preparation of nanoparticles: and (3) adding the purified ROCKi-ARG-HA into a buffer solution with the pH value of 8-8.5 for complete dissolution, slowly dropwise adding an ROS sensitive cross-linking agent, stirring at a low temperature for reaction, dialyzing after the reaction is finished to remove unreacted impurities, and obtaining drug-loaded nanoparticles, namely the anti-intestinal fibrosis ROCKi oral nano preparation. The grain diameter of the prepared drug-loaded nanoparticles is 100 nm-500 nm.
The ratio of the amounts of said X-ARG and ROCKi species in step (1) is 1:1 to 3.
The ratio of the amounts of hyaluronic acid and ROCKi-ARG substance in step (2) is 1:5 to 30. The hyaluronic acid is preferably aminated hyaluronic acid with a molecular weight range of 10-200 kDa.
Specifically, in the step (1), the ROCK inhibitor is Y2763 or Fasudil (Fasudil).
Specifically, in the step (1), the X-ARG is X-Ala-pro-gly, and X is benzyl bicarbonate or a homologue thereof.
Specifically, the ROS sensitive cross-linking agent in the step (3) is diethyl bis (4-nitrophenyl) -C, C' -dimethylthiodicarbonate.
The invention has the following beneficial effects:
(1) According to the invention, the crosslinking agent sensitive to intestinal fibrous tissues is utilized to enable ROCKi-ARG-HA to be crosslinked and self-assembled to form oral nanoparticles, and the crosslinking agent is degraded in response at the fibrous tissues, so that targeted positioning release of intestinal fibrosis parts is realized, adverse reactions caused by systemic drug delivery are avoided, and the clinical application of ROCKi to intestinal fibrosis resistance becomes possible;
(2) The medicine is connected to hyaluronic acid by adopting the proline-containing X-Ala-pro-gly dipeptide connecting chain, and intestinal myofibroblasts can be specifically identified by utilizing the high affinity of FAP and proline dipeptide, so that the aggregation of the medicine in the myofibroblasts is increased.
(3) The proline-containing X-Ala-pro-gly dipeptide connecting chain is adopted to connect the medicament to the hyaluronic acid, and the hydrolysis capacity of FAP to proline dipeptide is utilized to ensure that the medicament is activated by hydrolysis after the polymer is combined with the proline dipeptide, so that the targeting and uptake capacity of the medicament to intestinal fibroblast cells is improved, the medicament effect is improved, and the side effect is reduced;
(IV) description of the drawings
FIG. 1 is the synthesis pathway of ROCKi-ARG-HA;
FIG. 2 is the hydrolysis capacity of FAP on YRS NP nanoparticles;
FIG. 3 is the targeting ability of YRS NPs to enterocyte fibroblasts;
FIG. 4 is the in vitro anti-fibrotic capacity of YRS NPs.
Fig. 5 is the ability of FAP to hydrolyze FRS NP nanoparticles.
FIG. 6 is the ability of FRS NP to target gut myofibroblasts;
FIG. 7 is the in vitro anti-fibrotic capacity of FRS NP.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1: preparation of anti-intestinal fibrosis Y27632-ARG-HA oral nanoparticles
Synthesis of Y27632-ARG-HA and Y27632-ARG-HA-FITC: dissolving X-Ala-pro-gly (5 mmoL) (wherein X is benzyl bicarbonate) and Y27632 (5 mmoL) in 20mL of anhydrous formamide, and reacting at room temperature for 24h under the catalysis of EDC and DMAP. The catalyst Pd/C (10%), H was added 2 The reaction was continued at room temperature for 24h under protection. After the reaction, the solution is dialyzed by pure water and freeze-dried to obtainY27632-ARG compounds. HA or FITC-HA (0.05 mmoL) with molecular weight of 50,000 is dispersed in 40mL of anhydrous formamide, and proper amount (0.5 mmoL) of Y27632-ARG is added into the solution under the catalysis of EDC and DMAP. N is a radical of 2 The reaction was stirred under protection from light for 24h, and the resulting solution was dialyzed against distilled water (MW =3500 Da) to give Y27632-ARG-HA or Y27632-ARG-HA-FITC. (FIG. 1).
2. Preparation of nanoparticles: taking a certain amount of purified Y27632-ARG-HA (5 mmoL) polymer or Y27632-ARG-HA-FITC polymer, adding phosphate buffer solution with pH of 8.5 for complete dissolution, adding ROS responsive (50 mmoL) cross-linking agent (bis (4-nitrophenyl) -C, C' -dimethyl diethyl thiodicarbonate) for solidification under stirring, and dialyzing with pure water to remove unreacted impurities to obtain the Y27632-ARG-HA oral nano delivery system (YRS NP or FITC-YRS NP).
The Y27632-AHG-HA oral nano delivery system (YHS NP or FITC-YHS NP) is prepared by using a dipeptide chain X-Ala-phe-gly (X-AHG) without proline (wherein X is benzyl bicarbonate) linking agent as a control group to prepare the nano particles in the same way as the preparation method.
3. 1mL of the prepared nanoparticle was taken, and the particle size and zeta potential were characterized by using a Zetasizer. The results are shown in the table below, and the particle size and potential of the responsive nanoparticles and the non-responsive nanoparticles are not significantly different.
| Particle size (nm) | Electric potential (mv) | |
| YRS NP | 280.4±9.1 | -13.7±1.4 |
| YHS NP | 259.4±13.1 | -12.1±3.1 |
Hydrolysis of nanoparticles by fap: 1mL of YRS NP and YHS NP solutions were each added with 100nM H 2 O 2 After the solution and 200ng/mL FAP solution are incubated for 4h at 37 ℃, 0.5mL solution is taken to centrifuge for 15min under 3000g, the drug concentration of Y27632 in the supernatant is analyzed by HPLC, and the hydrolysis capacity of FAP on the nanoparticles is inspected, and the result is shown in figure 2.
As can be seen from FIG. 2, FAP has good hydrolytic capacity for YRS NP, and the drug is released. The YHS NP is not hydrolyzed, the drug is not released, and the polymer is still present, so that conditions are provided for the release of the drug specificity in intestinal fibroblasts.
5. Targeting ability of intestinal myofibroblasts: and (3) inoculating the intestinal fibroblasts into a 6-well plate, adding normal saline into a control group, pretreating the model group by using TGF-beta 1 solution to activate the fibroblasts into intestinal muscle fibroblasts, and constructing an intestinal fibrosis model. After that, 10.0. Mu.M solutions of FITC-YRS NP and FITC-YHS NP were added, respectively, and after incubation for 1 hour, the cells were collected, and the uptake of FITC-YRS NP and FITC-YHS NP by different cells was examined using an efflux cytometer, and the results are shown in FIG. 3.
As shown in the flow cytometry detection of FIG. 3, the uptake of FITC-YRS NP by the cells after TGF-beta 1 activation is obviously higher than that of FITC-YHS NP and is obviously higher than that of a control group which is not treated by TGF-beta 1. The results show that the uptake of the two nanoparticles is not obvious in a non-fibrotic cell model of a control group, while in an intestinal fibrotic cell model, the uptake of FITC-YRS NP with targeted recognition and specific affinity for FAP is obviously higher than that of FITC-YHS NP, so that the uptake of intestinal myofibroblasts for drugs can be increased, and a theoretical basis is provided for the targeted delivery of anti-fibrotic drugs.
6. Examination of anti-fibrosis capability in vitro: taking intestinal fibroblasts, inoculating the intestinal fibroblasts into a 6-well plate, adding physiological saline into a control group, pretreating a model group by using TGF-beta 1, and respectively adding different oral nano preparations. mRNA of Col I in different administration groups was detected by qRT-PCR, and the results are shown in FIG. 4. As can be seen from fig. 4, after the treatment with the free drugs Y27632 and YRS NP, the mRNA expression of enterocyte Col I is significantly reduced, indicating that the free drugs Y27632 and YRS NP have significant anti-fibrosis effects in vitro. YHS NP is not able to respond to FAP protein of myofibroblast and is unable to release free drug, so it has weak anti-fibrosis effect.
Example 2: oral nanoparticles of Fasudil-ARG-HA for resisting intestinal fibrosis
Synthesis of Fasudil-ARG-HA and Fasudil-ARG-HA-FITC: X-Ala-pro-gly (5 mmoL) and Fasudl (5 mmoL) were dissolved in 20mL of anhydrous formamide and reacted at room temperature for 24h under the catalysis of EDC and DMAP. The catalyst Pd/C (10%), H was added 2 The reaction was continued at room temperature for 24h under protection. After the reaction, the solution was dialyzed with purified water, and lyophilized to obtain Fasudil-ARG compound. HA or FITC-HA (0.05 mmoL) with molecular weight of 50,000 is dispersed in 40mL of anhydrous formamide, and appropriate amount (0.5 mmoL) Farudil-ARG is added to the solution under the catalysis of EDC and DMAP. N is a radical of 2 The reaction was stirred under protection from light for 24h, and the resulting solution was dialyzed against distilled water (MW =3500 Da) to obtain Fasudil-ARG-HA or Fasudil-ARG-HA-FITC.
2. Preparation of nanoparticles: taking a certain amount of purified Fasudil-ARG-HA (5 mmoL) polymer or Fasudil-ARG-HA-FITC polymer, adding phosphate buffer solution with pH of 8.5 for complete dissolution, adding ROS responsive (50 mmoL) cross-linking agent (di (4-nitrophenyl) -C, C' -dimethyl diethyl thiodicarbonate) for solidification under stirring, and dialyzing with pure water to remove unreacted impurities to obtain the Fasudil-ARG-HA oral nano delivery system (FRS NP or FITC-FRS NP).
The dipeptide chain X-Ala-phe-gly (X-AHG) linking agent without proline is used as a control group to prepare the nano-particles, and the preparation method is the same as that of the nano-particles, so that the Fasudil-AHG-HA oral nano-delivery system (FHS NP or FITC-FHS NP) is obtained.
3. 1mL of the prepared nanoparticles are taken, and the particle size and the zeta potential are characterized by using a Zetasizer. The results are shown in the following table, and the particle size and the potential of the responsive nanoparticles and the non-responsive nanoparticles have no significant difference.
| Particle size (nm) | Electric potential (mv) | |
| FRS NP | 240.2±5.2 | -15.7±0.8 |
| FHS NP | 246.3±7.3 | -14.1±0.6 |
Hydrolysis of nanoparticles by fap: 1mL each of FRS NP and FHS NP solutions was added with 100nM H 2 O 2 After the solution and 200ng/mL FAP solution are incubated for 4h at 37 ℃, 0.5mL solution is taken to centrifuge for 15min at 3000g, the drug concentration of Fasudil in the supernatant is analyzed by HPLC, and the hydrolysis capacity of FAP on the nanoparticles is examined, and the result is shown in figure 5.
As can be seen from fig. 5, FAP has good hydrolytic capacity for FRS NP and drug release. The FHS NP has no hydrolysis capacity, the medicine is not released and still exists in a polymer form, and conditions are provided for the specific release of the medicine in intestinal myofibroblasts.
5. Targeting ability of intestinal myofibroblasts: and (3) inoculating the intestinal fibroblasts into a 6-well plate, adding normal saline into a control group, and pretreating the model group by using a TGF-beta 1 solution to activate the fibroblasts into the intestinal muscle fibroblasts so as to construct an intestinal fibrosis model. After that, 10.0. Mu.M FITC-FRS NP and FITC-FHS NP solutions were added, and after incubation for 1h, the cells were collected and examined for uptake of FITC-FRS NP and FITC-FHS NP by different cells using an efflux cytometer, and the results are shown in FIG. 6.
As can be seen from the flow cytometry in FIG. 6, the uptake of FITC-FRS NP by cells after TGF-beta 1 activation is significantly higher than that of FITC-FHS NP, and significantly higher than that of a control group which is not treated with TGF-beta 1. The results show that the uptake of the two nanoparticles is not obvious in a non-fibrotic cell model of a control group, while in an intestinal fibrotic cell model, the uptake of FITC-FRS NP with targeted recognition and specific affinity for FAP is obviously higher than that of FITC-FHS NP, so that the uptake of intestinal myofibroblasts to drugs can be increased, and a theoretical basis is provided for the targeted delivery of anti-fibrotic drugs.
6. In vitro anti-fibrosis ability examination: taking intestinal fibroblasts, inoculating the intestinal fibroblasts into a 6-well plate, adding physiological saline into a control group, pretreating a model group by using TGF-beta 1, and respectively adding different oral nano preparations. mRNA of Col I in different administration groups was detected by qRT-PCR, and the results are shown in FIG. 7. As can be seen from FIG. 7, the mRNA expression of the enterocyte Col I was significantly decreased after the treatment with the free drugs Fasudil and FRS NP, indicating that the free drugs Fasudil and FRS NP had significant anti-fibrotic effect in vitro. FHS NP cannot respond to FAP protein of myofibroblast and cannot release free drugs, so that the anti-fibrosis effect is weak.
While the preferred embodiments and principles of this invention have been described in detail, it will be appreciated by those skilled in the art that variations may be made in these embodiments without departing from the spirit of the invention, and these variations are to be considered within the scope of the invention.
Claims (8)
1. An anti-intestinal fibrosis ROCKi oral nano preparation is prepared by the following steps: the method comprises the steps of taking a dipeptide chain X-Ala-pro-Gly as a connecting arm, wherein X is benzyl bicarbonate or a homologue thereof, grafting a ROCK inhibitor to a hyaluronic acid side chain to form a ROCKi-ARG-HA polymer, and then utilizing an ROS-responsive cross-linking agent to enable the ROCKi-ARG-HA polymer to be self-assembled and cross-linked to form nanoparticles, namely the ROCKi oral nano preparation for resisting intestinal fibrosis.
2. A method of preparing an oral nanoformulation of ROCKi against intestinal fibrosis, the method comprising:
(1) Synthesis of ROCki-ARG: dissolving a proper amount of X-ARG and ROCK inhibitor in anhydrous formamide, reacting at room temperature for 18-24H under the catalysis of EDC and DMAP, and adding catalysts Pd/C, H 2 Continuing to react for 18-24 h at room temperature under protection, dialyzing the solution with pure water after the reaction is finished, and freeze-drying to obtain an ROCKi-ARG compound;
(2) Synthesis of ROCKI-ARG-HA Polymer: dispersing appropriate amount of hyaluronic acid in anhydrous formamide, and adding appropriate amount of ROCKi-ARG, N under catalysis of EDC and DMAP 2 Stirring the mixture for reaction in a dark place under protection, and dialyzing the obtained solution with distilled water after the reaction is finished to obtain ROCKi-ARG-HA;
(3) Preparing monoclonal antibody nanoparticles: and adding the purified ROCki-ARG-HA into a buffer solution with the pH value of 8-8.5 for complete dissolution, slowly dropwise adding a ROS sensitive cross-linking agent, stirring at a low temperature for reaction, dialyzing after the reaction is finished to remove unreacted impurities, and obtaining drug-loaded nanoparticles, namely the ROCki oral nano preparation for resisting intestinal fibrosis.
3. The method of claim 2, wherein in step (1) the ratio of the amounts of X-ARG and ROCKi species is 1:1 to 3.
4. The method of claim 2, wherein the amount of hyaluronic acid and the ROCKi-ARG substance in step (2) is in a ratio of 1:5 to 30.
5. The method according to claim 2, wherein the hyaluronic acid in step (2) is an aminated hyaluronic acid having a molecular weight in the range of 10-200 kDa.
6. A method according to claim 2, wherein in step (1) the ROCK inhibitor is Y27632 or fasudil.
7. The method of claim 2, wherein in step (1) the X-ARG is X-Ala-pro-gly and X is benzyl bicarbonate or a homologue thereof.
8. The method of claim 2, wherein the ROS sensitive crosslinker in step (3) is diethyl bis (4-nitrophenyl) -C, C' -dimethylthiodicarbonate.
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